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IL-21 mediated plasmablast differentiation is enhanced in RA. Naïve B cells were isolated from PBMC of control ( n = 12) and RA patients ( n = 8) and cultured with IL-21 (15 pg/mL or 50 ng/mL) and CD40L for 12 days. (A) Plasmablast percent was measured by staining for <t>CD38</t> + CD27 + Blimp-1 + cells following 12 days of culture with either low (15 pg/ml) or high (50 ng/ml) levels of IL-21 in addition to CD40L. (B) CD138 expression was determined on plasmablasts (CD38 + CD27 + ) from RA subjects and controls following high dose IL-21 and CD40L culture. (C) Frequency of IgM + plasmablasts (CD38 + CD27 + ) were assessed following 12 days of culture with high dose IL-21 and CD40L (left). IgM gMFI was assessed in plasmablasts by intracellular staining (right). (D) IL-6 gMFI was assessed on plasmablasts in response to low or high dose IL-21 in addition to CD40L stimulation in controls and RA subjects. (E) IL-21 expression correlates with IL-6 production following IL-21 stimulation (F) and IL-21+CD40L stimulation but not (G) PMA+Ionomycin stimulation. PBMCs from RA subjects ( n = 52) were stimulated with IL-21 (E) or IL-21+CD40L (F) for 24 h. In the last 4 h of incubation, brefeldin A and monensin were added to prevent export of IL-6. The level of IL-6 was measured by flow cytometry on total B cells. IL-6 production was measured by flow cytometry. IL-21R expression was measured at day 0 on memory B cells. Black circles are controls and gray triangles are RA patients. Significance was assessed using Mann Whitney U tests. Correlation was assessed with the Pearson correlation.
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1) Product Images from "Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis"

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01978

IL-21 mediated plasmablast differentiation is enhanced in RA. Naïve B cells were isolated from PBMC of control ( n = 12) and RA patients ( n = 8) and cultured with IL-21 (15 pg/mL or 50 ng/mL) and CD40L for 12 days. (A) Plasmablast percent was measured by staining for CD38 + CD27 + Blimp-1 + cells following 12 days of culture with either low (15 pg/ml) or high (50 ng/ml) levels of IL-21 in addition to CD40L. (B) CD138 expression was determined on plasmablasts (CD38 + CD27 + ) from RA subjects and controls following high dose IL-21 and CD40L culture. (C) Frequency of IgM + plasmablasts (CD38 + CD27 + ) were assessed following 12 days of culture with high dose IL-21 and CD40L (left). IgM gMFI was assessed in plasmablasts by intracellular staining (right). (D) IL-6 gMFI was assessed on plasmablasts in response to low or high dose IL-21 in addition to CD40L stimulation in controls and RA subjects. (E) IL-21 expression correlates with IL-6 production following IL-21 stimulation (F) and IL-21+CD40L stimulation but not (G) PMA+Ionomycin stimulation. PBMCs from RA subjects ( n = 52) were stimulated with IL-21 (E) or IL-21+CD40L (F) for 24 h. In the last 4 h of incubation, brefeldin A and monensin were added to prevent export of IL-6. The level of IL-6 was measured by flow cytometry on total B cells. IL-6 production was measured by flow cytometry. IL-21R expression was measured at day 0 on memory B cells. Black circles are controls and gray triangles are RA patients. Significance was assessed using Mann Whitney U tests. Correlation was assessed with the Pearson correlation.
Figure Legend Snippet: IL-21 mediated plasmablast differentiation is enhanced in RA. Naïve B cells were isolated from PBMC of control ( n = 12) and RA patients ( n = 8) and cultured with IL-21 (15 pg/mL or 50 ng/mL) and CD40L for 12 days. (A) Plasmablast percent was measured by staining for CD38 + CD27 + Blimp-1 + cells following 12 days of culture with either low (15 pg/ml) or high (50 ng/ml) levels of IL-21 in addition to CD40L. (B) CD138 expression was determined on plasmablasts (CD38 + CD27 + ) from RA subjects and controls following high dose IL-21 and CD40L culture. (C) Frequency of IgM + plasmablasts (CD38 + CD27 + ) were assessed following 12 days of culture with high dose IL-21 and CD40L (left). IgM gMFI was assessed in plasmablasts by intracellular staining (right). (D) IL-6 gMFI was assessed on plasmablasts in response to low or high dose IL-21 in addition to CD40L stimulation in controls and RA subjects. (E) IL-21 expression correlates with IL-6 production following IL-21 stimulation (F) and IL-21+CD40L stimulation but not (G) PMA+Ionomycin stimulation. PBMCs from RA subjects ( n = 52) were stimulated with IL-21 (E) or IL-21+CD40L (F) for 24 h. In the last 4 h of incubation, brefeldin A and monensin were added to prevent export of IL-6. The level of IL-6 was measured by flow cytometry on total B cells. IL-6 production was measured by flow cytometry. IL-21R expression was measured at day 0 on memory B cells. Black circles are controls and gray triangles are RA patients. Significance was assessed using Mann Whitney U tests. Correlation was assessed with the Pearson correlation.

Techniques Used: Isolation, Cell Culture, Staining, Expressing, Incubation, Flow Cytometry, Cytometry, MANN-WHITNEY

Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.
Figure Legend Snippet: Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Negative Control, Positive Control, MANN-WHITNEY

IL-21R expression is enhanced in total B cells in SLE subjects but unchanged in MS. IL-21R expression levels in SLE (white squares) (A) and MS (white triangles) (B) subjects compared to matched healthy controls (white circles) in total CD19 + B cells, total memory (CD20 + CD38 − CD24 hi ) and naïve (CD20 + CD38 int CD24 int ) B cells. Significance was determined using Mann Whitney U tests.
Figure Legend Snippet: IL-21R expression is enhanced in total B cells in SLE subjects but unchanged in MS. IL-21R expression levels in SLE (white squares) (A) and MS (white triangles) (B) subjects compared to matched healthy controls (white circles) in total CD19 + B cells, total memory (CD20 + CD38 − CD24 hi ) and naïve (CD20 + CD38 int CD24 int ) B cells. Significance was determined using Mann Whitney U tests.

Techniques Used: Expressing, Mass Spectrometry, MANN-WHITNEY

IL-21R expression is increased in B cells in RA. (A) (top) Gating scheme for total B, memory and naïve B cells with IL-21R expression in each population (bottom) (B) (left) IL-21R levels on CD20 + , total memory (CD20 + CD38 − CD24 hi ) and naïve (CD20 + CD38 int CD24 int ) B cells in (black circles) healthy control (n = 46) and (gray triangles) RA subjects (n = 71) by flow cytometry. (Right) Percent of IL-21R + cells (center, left) and IL-21R expression was quantified on IL-21R + cells (bottom, right) in CD20 + and memory B cells in RA and control subjects. (C) IL-21R levels in memory and naïve B cells were correlated in RA subjects. Significance was determined using a Mann Whitney U test and a Pearson correlation.
Figure Legend Snippet: IL-21R expression is increased in B cells in RA. (A) (top) Gating scheme for total B, memory and naïve B cells with IL-21R expression in each population (bottom) (B) (left) IL-21R levels on CD20 + , total memory (CD20 + CD38 − CD24 hi ) and naïve (CD20 + CD38 int CD24 int ) B cells in (black circles) healthy control (n = 46) and (gray triangles) RA subjects (n = 71) by flow cytometry. (Right) Percent of IL-21R + cells (center, left) and IL-21R expression was quantified on IL-21R + cells (bottom, right) in CD20 + and memory B cells in RA and control subjects. (C) IL-21R levels in memory and naïve B cells were correlated in RA subjects. Significance was determined using a Mann Whitney U test and a Pearson correlation.

Techniques Used: Expressing, Flow Cytometry, Cytometry, MANN-WHITNEY

2) Product Images from "Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis"

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01978

IL-21 mediated plasmablast differentiation is enhanced in RA. Naïve B cells were isolated from PBMC of control ( n = 12) and RA patients ( n = 8) and cultured with IL-21 (15 pg/mL or 50 ng/mL) and CD40L for 12 days. (A) Plasmablast percent was measured by staining for CD38 + CD27 + Blimp-1 + cells following 12 days of culture with either low (15 pg/ml) or high (50 ng/ml) levels of IL-21 in addition to CD40L. (B) CD138 expression was determined on plasmablasts (CD38 + CD27 + ) from RA subjects and controls following high dose IL-21 and CD40L culture. (C) Frequency of IgM + plasmablasts (CD38 + CD27 + ) were assessed following 12 days of culture with high dose IL-21 and CD40L (left). IgM gMFI was assessed in plasmablasts by intracellular staining (right). (D) IL-6 gMFI was assessed on plasmablasts in response to low or high dose IL-21 in addition to CD40L stimulation in controls and RA subjects. (E) IL-21 expression correlates with IL-6 production following IL-21 stimulation (F) and IL-21+CD40L stimulation but not (G) PMA+Ionomycin stimulation. PBMCs from RA subjects ( n = 52) were stimulated with IL-21 (E) or IL-21+CD40L (F) for 24 h. In the last 4 h of incubation, brefeldin A and monensin were added to prevent export of IL-6. The level of IL-6 was measured by flow cytometry on total B cells. IL-6 production was measured by flow cytometry. IL-21R expression was measured at day 0 on memory B cells. Black circles are controls and gray triangles are RA patients. Significance was assessed using Mann Whitney U tests. Correlation was assessed with the Pearson correlation.
Figure Legend Snippet: IL-21 mediated plasmablast differentiation is enhanced in RA. Naïve B cells were isolated from PBMC of control ( n = 12) and RA patients ( n = 8) and cultured with IL-21 (15 pg/mL or 50 ng/mL) and CD40L for 12 days. (A) Plasmablast percent was measured by staining for CD38 + CD27 + Blimp-1 + cells following 12 days of culture with either low (15 pg/ml) or high (50 ng/ml) levels of IL-21 in addition to CD40L. (B) CD138 expression was determined on plasmablasts (CD38 + CD27 + ) from RA subjects and controls following high dose IL-21 and CD40L culture. (C) Frequency of IgM + plasmablasts (CD38 + CD27 + ) were assessed following 12 days of culture with high dose IL-21 and CD40L (left). IgM gMFI was assessed in plasmablasts by intracellular staining (right). (D) IL-6 gMFI was assessed on plasmablasts in response to low or high dose IL-21 in addition to CD40L stimulation in controls and RA subjects. (E) IL-21 expression correlates with IL-6 production following IL-21 stimulation (F) and IL-21+CD40L stimulation but not (G) PMA+Ionomycin stimulation. PBMCs from RA subjects ( n = 52) were stimulated with IL-21 (E) or IL-21+CD40L (F) for 24 h. In the last 4 h of incubation, brefeldin A and monensin were added to prevent export of IL-6. The level of IL-6 was measured by flow cytometry on total B cells. IL-6 production was measured by flow cytometry. IL-21R expression was measured at day 0 on memory B cells. Black circles are controls and gray triangles are RA patients. Significance was assessed using Mann Whitney U tests. Correlation was assessed with the Pearson correlation.

Techniques Used: Isolation, Cell Culture, Staining, Expressing, Incubation, Flow Cytometry, Cytometry, MANN-WHITNEY

Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p
Figure Legend Snippet: Increased IL-21R expression correlates with increased SP1 binding to the IL-21R promoter in RA. (A) Total B cells were isolated from whole blood from RA patients and controls. SP1 binding to the IL21R promoter region and negative control IgG binding were determined using ChIP-qPCR analysis. The results are presented relative to input DNA. Significance was assessed using the student's t -test; n = 4 [Control (black circle), RA High (gray triangle)] n = 3 [RA Low (white square)]. ** p

Techniques Used: Expressing, Binding Assay, Isolation, Negative Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.
Figure Legend Snippet: Increased SP1 expression correlates with increased IL-21R expression in memory B cells in RA subjects (A) SP1 protein levels were assessed by flow cytometry in control ( n = 6, black circles), RA low IL-21R expressers ( n = 6; open squares) and RA high IL-21R expressers ( n = 5; gray triangles) in CD20 + and memory B cells (CD20 + CD38 − CD24 + ) (left). SP1 protein levels from left were correlated with IL-21R protein expression in total B cells in RA and control subjects ( n = 17) (right). (B) Representative histogram of mRNA levels from a no probe negative control, SP1 and positive control probe, RPL13A . (C) (left) SP1 mRNA levels were determined in total memory B cells in controls ( n = 7, black circles), RA low IL-21R expressers ( n = 7; open squares) and RA high IL-21R expressers ( n = 6; gray triangles). (right) SP1 mRNA levels from left were correlated with IL-21R protein expression in memory B cells in RA and control subjects combined ( n = 20). Significance was determined using Mann Whitney U tests (to compare RA-IL-21 high to controls and RA-IL-21 low ) and correlations were assessed with Pearson correlations.

Techniques Used: Expressing, Flow Cytometry, Cytometry, Negative Control, Positive Control, MANN-WHITNEY

IL-21R expression is enhanced in total B cells in SLE subjects but unchanged in MS. IL-21R expression levels in SLE (white squares) (A) and MS (white triangles) (B) subjects compared to matched healthy controls (white circles) in total CD19 + B cells, total memory (CD20 + CD38 − CD24 hi ) and naïve (CD20 + CD38 int CD24 int ) B cells. Significance was determined using Mann Whitney U tests.
Figure Legend Snippet: IL-21R expression is enhanced in total B cells in SLE subjects but unchanged in MS. IL-21R expression levels in SLE (white squares) (A) and MS (white triangles) (B) subjects compared to matched healthy controls (white circles) in total CD19 + B cells, total memory (CD20 + CD38 − CD24 hi ) and naïve (CD20 + CD38 int CD24 int ) B cells. Significance was determined using Mann Whitney U tests.

Techniques Used: Expressing, Mass Spectrometry, MANN-WHITNEY

IL-21R levels correlate with IL-21-mediated pSTAT3 signaling. Correlation of IL-21R expression and pSTAT3 fold change in CD20 + (A) , total memory B cells (B) , and naïve B cells (C) . pSTAT3 levels were assessed by stimulating with IL-21 for 45 min and measuring pSTAT3 gMFI with no stimulation or with stimulation. Fold change was calculated by dividing IL-21 stimulation pSTAT3 gMFI by the no stimulation pSTAT3 gMFI. As there were no differences in slope between controls and RA patients, the correlation was conducted on both controls (black circles) and RA patients (gray triangles) ( n = 116). Correlation was assessed with the Pearson correlation.
Figure Legend Snippet: IL-21R levels correlate with IL-21-mediated pSTAT3 signaling. Correlation of IL-21R expression and pSTAT3 fold change in CD20 + (A) , total memory B cells (B) , and naïve B cells (C) . pSTAT3 levels were assessed by stimulating with IL-21 for 45 min and measuring pSTAT3 gMFI with no stimulation or with stimulation. Fold change was calculated by dividing IL-21 stimulation pSTAT3 gMFI by the no stimulation pSTAT3 gMFI. As there were no differences in slope between controls and RA patients, the correlation was conducted on both controls (black circles) and RA patients (gray triangles) ( n = 116). Correlation was assessed with the Pearson correlation.

Techniques Used: Expressing

IL-21R transcript levels are increased in RA subjects with high levels of IL-21R (A) Data from Figure 1A used to show how RA high IL-21R expressers and RA low IL-21R expressers are defined. RA high IL-21R expressers in the solid black box express IL-21R at levels two times the standard deviation of the mean of the control samples. RA low IL-21R expressers in the gray dashed box are less than two times the standard deviation of the mean of the control samples. (B) Representation of the positive control probe ( RPL13A) (dark gray histogram), IL21R (black line histogram) and negative control (no probe) (light gray histogram) in CD20 + or memory B cells in a control subject. (C) IL21R mRNA levels in total and memory B cells from RA high IL-21R expressers (gray triangles, n = 7) and RA low IL-21R expressers (white squares, n = 7) Significance was determined using a Mann Whitney U test.
Figure Legend Snippet: IL-21R transcript levels are increased in RA subjects with high levels of IL-21R (A) Data from Figure 1A used to show how RA high IL-21R expressers and RA low IL-21R expressers are defined. RA high IL-21R expressers in the solid black box express IL-21R at levels two times the standard deviation of the mean of the control samples. RA low IL-21R expressers in the gray dashed box are less than two times the standard deviation of the mean of the control samples. (B) Representation of the positive control probe ( RPL13A) (dark gray histogram), IL21R (black line histogram) and negative control (no probe) (light gray histogram) in CD20 + or memory B cells in a control subject. (C) IL21R mRNA levels in total and memory B cells from RA high IL-21R expressers (gray triangles, n = 7) and RA low IL-21R expressers (white squares, n = 7) Significance was determined using a Mann Whitney U test.

Techniques Used: Standard Deviation, Positive Control, Negative Control, MANN-WHITNEY

IL-21R expression is increased in B cells in RA. (A) (top) Gating scheme for total B, memory and naïve B cells with IL-21R expression in each population (bottom) (B) (left) IL-21R levels on CD20 + , total memory (CD20 + CD38 − CD24 hi ) and naïve (CD20 + CD38 int CD24 int ) B cells in (black circles) healthy control (n = 46) and (gray triangles) RA subjects (n = 71) by flow cytometry. (Right) Percent of IL-21R + cells (center, left) and IL-21R expression was quantified on IL-21R + cells (bottom, right) in CD20 + and memory B cells in RA and control subjects. (C) IL-21R levels in memory and naïve B cells were correlated in RA subjects. Significance was determined using a Mann Whitney U test and a Pearson correlation.
Figure Legend Snippet: IL-21R expression is increased in B cells in RA. (A) (top) Gating scheme for total B, memory and naïve B cells with IL-21R expression in each population (bottom) (B) (left) IL-21R levels on CD20 + , total memory (CD20 + CD38 − CD24 hi ) and naïve (CD20 + CD38 int CD24 int ) B cells in (black circles) healthy control (n = 46) and (gray triangles) RA subjects (n = 71) by flow cytometry. (Right) Percent of IL-21R + cells (center, left) and IL-21R expression was quantified on IL-21R + cells (bottom, right) in CD20 + and memory B cells in RA and control subjects. (C) IL-21R levels in memory and naïve B cells were correlated in RA subjects. Significance was determined using a Mann Whitney U test and a Pearson correlation.

Techniques Used: Expressing, Flow Cytometry, Cytometry, MANN-WHITNEY

3) Product Images from "T Cell-Specific Knockout of STAT3 Ameliorates Dextran Sulfate Sodium-Induced Colitis by Reducing the Inflammatory Response"

Article Title: T Cell-Specific Knockout of STAT3 Ameliorates Dextran Sulfate Sodium-Induced Colitis by Reducing the Inflammatory Response

Journal: Immune Network

doi: 10.4110/in.2018.18.e30

T cell-specific STAT3 deficiency affects the production of inflammatory cytokines. (A) Representative FACS plot of a multiplex bead-based immunoassay showing the distribution of cytokine production. (B) Bar graphs for the amounts of IFN-γ, IL-6, IL-10, and IL-17A in the serum from control and DSS-treated WT and KO mice (n=5). (C, D) Bar graphs for the mRNA levels of IFN-γ, IL-6, IL-10, IL-17A, and Foxp3 in colon tissues from control and DSS-treated WT and KO mice (n=5). Relative mRNA levels were normalized to those of GAPDH. Data are presented as the means±SEM. Representative data from 3 independent experiments are shown. * p
Figure Legend Snippet: T cell-specific STAT3 deficiency affects the production of inflammatory cytokines. (A) Representative FACS plot of a multiplex bead-based immunoassay showing the distribution of cytokine production. (B) Bar graphs for the amounts of IFN-γ, IL-6, IL-10, and IL-17A in the serum from control and DSS-treated WT and KO mice (n=5). (C, D) Bar graphs for the mRNA levels of IFN-γ, IL-6, IL-10, IL-17A, and Foxp3 in colon tissues from control and DSS-treated WT and KO mice (n=5). Relative mRNA levels were normalized to those of GAPDH. Data are presented as the means±SEM. Representative data from 3 independent experiments are shown. * p

Techniques Used: FACS, Multiplex Assay, Bead-based Assay, Mouse Assay

4) Product Images from "Farrerol Relieve Lipopolysaccharide (LPS)-Induced Mastitis by Inhibiting AKT/NF-κB p65, ERK1/2 and P38 Signaling Pathway"

Article Title: Farrerol Relieve Lipopolysaccharide (LPS)-Induced Mastitis by Inhibiting AKT/NF-κB p65, ERK1/2 and P38 Signaling Pathway

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19061770

The effect of farrerol on pro-inflammatory mediator’s production in LPS-stimulated mMECs. MMECs were divided into six groups: NT group, Farrerol group, LPS group, LPS + farrerol (90, 110, 130 μM) group. The mRNA levels of TNF-α ( A ); IL-6 ( B ) and IL-1β ( C ) were measured by real-time PCR. The protein levels of iNOS ( D , E ) and COX-2 ( D , F ) were measured by Western blot. Data are presented as mean ± SEM ( n = 3). # p
Figure Legend Snippet: The effect of farrerol on pro-inflammatory mediator’s production in LPS-stimulated mMECs. MMECs were divided into six groups: NT group, Farrerol group, LPS group, LPS + farrerol (90, 110, 130 μM) group. The mRNA levels of TNF-α ( A ); IL-6 ( B ) and IL-1β ( C ) were measured by real-time PCR. The protein levels of iNOS ( D , E ) and COX-2 ( D , F ) were measured by Western blot. Data are presented as mean ± SEM ( n = 3). # p

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

The levels of TNF-α ( A ); IL-6 ( B ) and IL-1β ( C ) in the homogenate of mouse mammary glands including NT group, LPS group and LPS + farrerol (20, 30, 40 mg/kg) group; The protein levels of iNOS ( D , E ) and COX-2 ( D , F ) were measured by Western blot. Data are presented as mean ± SEM ( n = 3). # p
Figure Legend Snippet: The levels of TNF-α ( A ); IL-6 ( B ) and IL-1β ( C ) in the homogenate of mouse mammary glands including NT group, LPS group and LPS + farrerol (20, 30, 40 mg/kg) group; The protein levels of iNOS ( D , E ) and COX-2 ( D , F ) were measured by Western blot. Data are presented as mean ± SEM ( n = 3). # p

Techniques Used: Western Blot

5) Product Images from "Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells"

Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01494

Effect of macrophage migration inhibitory factor (MIF) neutralization in the expression of cytokines from primary human immunodeficiency virus-infected and uninfected monocyte-derived macrophages (MDMs). Serial dilutions of a neutralizing αMIF antibody (clone NIHlllD.9) (A) , and the MIF antagonist MIF098 (B) were used to inhibit MIF activity in infected (In, red lines) and uninfected (Un, gray lines) MDMs at a constant concentration of this cytokine (25 ng/ml). Data represent three independent experiments (donors), each evaluated in duplicate. Data represent the mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey’s post-test (intragroup analysis, In group only; **** p
Figure Legend Snippet: Effect of macrophage migration inhibitory factor (MIF) neutralization in the expression of cytokines from primary human immunodeficiency virus-infected and uninfected monocyte-derived macrophages (MDMs). Serial dilutions of a neutralizing αMIF antibody (clone NIHlllD.9) (A) , and the MIF antagonist MIF098 (B) were used to inhibit MIF activity in infected (In, red lines) and uninfected (Un, gray lines) MDMs at a constant concentration of this cytokine (25 ng/ml). Data represent three independent experiments (donors), each evaluated in duplicate. Data represent the mean ± SD. Data were analyzed by two-way ANOVA followed by Tukey’s post-test (intragroup analysis, In group only; **** p

Techniques Used: Migration, Neutralization, Expressing, Infection, Derivative Assay, Activity Assay, Concentration Assay

Expression of cytokines after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected and uninfected monocyte-derived macrophages (MDMs). (A) Expression of IL-8, IL-6, IL-1β, TNF-α, sICAM, and IL-10 in supernatants from HIV-infected (In) and uninfected (Un) MDMs obtained from one representative healthy donor. (B) Data combined from six independent experiments (donors), each evaluated in triplicate. Here, data are shown as the ratio between cytokine concentrations found under the infection condition versus the uninfected counterpart. Cells were stimulated with MIF as follows: 0, 1, 10, or 25 ng/ml. Data shown in the gray boxes depict CD74 blocking (10 ng/ml of αCD74 or the corresponding isotype control) followed by MIF stimulation (1 or 25 ng/ml as denoted). Data represent the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey’s post-test. * p
Figure Legend Snippet: Expression of cytokines after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected and uninfected monocyte-derived macrophages (MDMs). (A) Expression of IL-8, IL-6, IL-1β, TNF-α, sICAM, and IL-10 in supernatants from HIV-infected (In) and uninfected (Un) MDMs obtained from one representative healthy donor. (B) Data combined from six independent experiments (donors), each evaluated in triplicate. Here, data are shown as the ratio between cytokine concentrations found under the infection condition versus the uninfected counterpart. Cells were stimulated with MIF as follows: 0, 1, 10, or 25 ng/ml. Data shown in the gray boxes depict CD74 blocking (10 ng/ml of αCD74 or the corresponding isotype control) followed by MIF stimulation (1 or 25 ng/ml as denoted). Data represent the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey’s post-test. * p

Techniques Used: Expressing, Migration, Infection, Derivative Assay, Blocking Assay

6) Product Images from "Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells"

Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01494

Infection of monocyte-derived macrophages (MDMs) and unactivated CD4 + T-cells with T/F viruses reproduce the results obtained with the R5-tropic (BAL) and the X4-tropic (NL4-3) laboratory strains. (A) Expression of IL-1β, IL-8, IL-6, and IL-10 in supernatants from uninfected (Un) and R5-tropic T/F-infected (In) MDMs. Data represent mean ± SD from three independent donors. Data were analyzed by two-way ANOVA followed by Tukey’s post-test. * p
Figure Legend Snippet: Infection of monocyte-derived macrophages (MDMs) and unactivated CD4 + T-cells with T/F viruses reproduce the results obtained with the R5-tropic (BAL) and the X4-tropic (NL4-3) laboratory strains. (A) Expression of IL-1β, IL-8, IL-6, and IL-10 in supernatants from uninfected (Un) and R5-tropic T/F-infected (In) MDMs. Data represent mean ± SD from three independent donors. Data were analyzed by two-way ANOVA followed by Tukey’s post-test. * p

Techniques Used: Infection, Derivative Assay, Expressing

Identification of cytokines as responsible for enhancing human immunodeficiency virus type I (HIV-1) infection in unactivated CD4 + T-cells. (A) Unactivated CD4 + T-cells were stimulated with different combinations of cytokines for 72 h. Then, cells were infected and p24 antigen production was evaluated at days 4 and 7 post-infection. Each condition was compared with the corresponding RPMI condition (negative control). As a positive control, PHA stimulation was used. Percentage of living CD4 + T-cells (B) and percentage of infected (GFP + ) CD4 + T-cells (C) after stimulation with the denoted treatments are shown. Data represent mean ± SD from four independent donors evaluated in duplicate. Concentrations of cytokines used in these experiments corresponded to the average concentrations found in monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/ml macrophage migration inhibitory factor (MIF) (peak effect) as follows: 250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β. (D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with monoclonal neutralizing antibodies. Primary CD4 + T-cells were incubated with supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype control antibody conditions were tested for comparison. Also, RPMI and PHA controls were included. Viral production was evaluated at day 4 post-infection. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test (all conditions versus the corresponding RMPI control) in (A) and Tukey’s post-test in (D) . * p
Figure Legend Snippet: Identification of cytokines as responsible for enhancing human immunodeficiency virus type I (HIV-1) infection in unactivated CD4 + T-cells. (A) Unactivated CD4 + T-cells were stimulated with different combinations of cytokines for 72 h. Then, cells were infected and p24 antigen production was evaluated at days 4 and 7 post-infection. Each condition was compared with the corresponding RPMI condition (negative control). As a positive control, PHA stimulation was used. Percentage of living CD4 + T-cells (B) and percentage of infected (GFP + ) CD4 + T-cells (C) after stimulation with the denoted treatments are shown. Data represent mean ± SD from four independent donors evaluated in duplicate. Concentrations of cytokines used in these experiments corresponded to the average concentrations found in monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/ml macrophage migration inhibitory factor (MIF) (peak effect) as follows: 250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β. (D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with monoclonal neutralizing antibodies. Primary CD4 + T-cells were incubated with supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype control antibody conditions were tested for comparison. Also, RPMI and PHA controls were included. Viral production was evaluated at day 4 post-infection. Data were analyzed by one-way ANOVA followed by Dunnett’s post-test (all conditions versus the corresponding RMPI control) in (A) and Tukey’s post-test in (D) . * p

Techniques Used: Infection, Negative Control, Positive Control, Derivative Assay, Migration, Neutralization, Activity Assay, Incubation

Expression of cytokines after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected and uninfected monocyte-derived macrophages (MDMs). (A) Expression of IL-8, IL-6, IL-1β, TNF-α, sICAM, and IL-10 in supernatants from HIV-infected (In) and uninfected (Un) MDMs obtained from one representative healthy donor. (B) Data combined from six independent experiments (donors), each evaluated in triplicate. Here, data are shown as the ratio between cytokine concentrations found under the infection condition versus the uninfected counterpart. Cells were stimulated with MIF as follows: 0, 1, 10, or 25 ng/ml. Data shown in the gray boxes depict CD74 blocking (10 ng/ml of αCD74 or the corresponding isotype control) followed by MIF stimulation (1 or 25 ng/ml as denoted). Data represent the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey’s post-test. * p
Figure Legend Snippet: Expression of cytokines after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected and uninfected monocyte-derived macrophages (MDMs). (A) Expression of IL-8, IL-6, IL-1β, TNF-α, sICAM, and IL-10 in supernatants from HIV-infected (In) and uninfected (Un) MDMs obtained from one representative healthy donor. (B) Data combined from six independent experiments (donors), each evaluated in triplicate. Here, data are shown as the ratio between cytokine concentrations found under the infection condition versus the uninfected counterpart. Cells were stimulated with MIF as follows: 0, 1, 10, or 25 ng/ml. Data shown in the gray boxes depict CD74 blocking (10 ng/ml of αCD74 or the corresponding isotype control) followed by MIF stimulation (1 or 25 ng/ml as denoted). Data represent the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey’s post-test. * p

Techniques Used: Expressing, Migration, Infection, Derivative Assay, Blocking Assay

7) Product Images from "Inonotus sanghuang Polyphenols Attenuate Inflammatory Response Via Modulating the Crosstalk Between Macrophages and Adipocytes"

Article Title: Inonotus sanghuang Polyphenols Attenuate Inflammatory Response Via Modulating the Crosstalk Between Macrophages and Adipocytes

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00286

Schematic models of molecular targets affected by ISE to attenuate inflammatory signaling pathways. On the one hand, LPS- and free fatty acid (FFA)-induced inflammatory responses are regulated by both NF-κB and MAPK signaling pathways. In activated macrophages, ISE decreases pro-inflammatory cytokine production via inhibiting the ERK and p38 MAPK pathways, not NF-κB signals. On the other hand, ISE markedly suppresses the phosphorylation of STAT3 to reduce cytokines transcription. Subsequently, the lipolysis of adipocytes is suppressed and proinflammatory M1 macrophages are less recruited via the MCP-1-CCR2 pathway. And the decrease of inflammatory cytokine IL-6 further restrains the IL-6/STAT3 activation and the inflammatory mediator production. In a word, treatment with ISE reduces the levels of these pro-inflammatory mediators, thereby disrupting the crosstalk between macrophages and adipocytes in a coculture. ISE apparently exerted anti-inflammatory ability, possibly diminishing the obesity-induced inflammatory diseases.
Figure Legend Snippet: Schematic models of molecular targets affected by ISE to attenuate inflammatory signaling pathways. On the one hand, LPS- and free fatty acid (FFA)-induced inflammatory responses are regulated by both NF-κB and MAPK signaling pathways. In activated macrophages, ISE decreases pro-inflammatory cytokine production via inhibiting the ERK and p38 MAPK pathways, not NF-κB signals. On the other hand, ISE markedly suppresses the phosphorylation of STAT3 to reduce cytokines transcription. Subsequently, the lipolysis of adipocytes is suppressed and proinflammatory M1 macrophages are less recruited via the MCP-1-CCR2 pathway. And the decrease of inflammatory cytokine IL-6 further restrains the IL-6/STAT3 activation and the inflammatory mediator production. In a word, treatment with ISE reduces the levels of these pro-inflammatory mediators, thereby disrupting the crosstalk between macrophages and adipocytes in a coculture. ISE apparently exerted anti-inflammatory ability, possibly diminishing the obesity-induced inflammatory diseases.

Techniques Used: Activation Assay

Effects of ISE on RAW264.7 macrophage activation by LPS. RAW264.7 macrophages were pretreated with different concentrations of ISE for 1 h and then were incubated with LPS at 1.0 μg/mL for 24 h. Cell-free supernatants were collected to determine the concentrations of NO (A) , TNF-α (B) , IL-6 (C) , and MCP-1 (D) as described in “ Materials and Methods ” section. The values are means ± SD of 6 samples. Statistical comparisons were made with each vehicle control. * P
Figure Legend Snippet: Effects of ISE on RAW264.7 macrophage activation by LPS. RAW264.7 macrophages were pretreated with different concentrations of ISE for 1 h and then were incubated with LPS at 1.0 μg/mL for 24 h. Cell-free supernatants were collected to determine the concentrations of NO (A) , TNF-α (B) , IL-6 (C) , and MCP-1 (D) as described in “ Materials and Methods ” section. The values are means ± SD of 6 samples. Statistical comparisons were made with each vehicle control. * P

Techniques Used: Activation Assay, Incubation

Effects of ISE on inflammation in RAW264.7 macrophages activated by conditioned medium of hypertrophied 3T3-L1 adipocytes. RAW264.7 macrophages were pretreated with different concentrations of ISE for 1 h and then were incubated with conditioned medium of hypertrophied 3T3-L1 adipocytes (L1CM) for 24 h. Cell-free supernatants were collected to determine the concentrations of NO (A) , TNF-α (B) , IL-6 (C) , and MCP-1 (D) as described in “ Materials and Methods ” section. The values are means ± SD of 6 samples. Statistical comparisons were made with each vehicle control. * P
Figure Legend Snippet: Effects of ISE on inflammation in RAW264.7 macrophages activated by conditioned medium of hypertrophied 3T3-L1 adipocytes. RAW264.7 macrophages were pretreated with different concentrations of ISE for 1 h and then were incubated with conditioned medium of hypertrophied 3T3-L1 adipocytes (L1CM) for 24 h. Cell-free supernatants were collected to determine the concentrations of NO (A) , TNF-α (B) , IL-6 (C) , and MCP-1 (D) as described in “ Materials and Methods ” section. The values are means ± SD of 6 samples. Statistical comparisons were made with each vehicle control. * P

Techniques Used: Incubation

Effects of ISE on inflammation in RAW264.7 macrophages cocultured with differentiated 3T3-L1 adipocytes. RAW264.7 macrophages were pretreated with different concentrations of ISE for 1 h and then were incubated with differentiated 3T3-L1 adipocytes for 24 h. Cell-free supernatants were collected to determine the concentrations of NO (A) , TNF-α (B) , IL-6 (C) , and MCP-1 (D) as described in “ Materials and Methods ” section. The values are means ± SD of 6 samples. Statistical comparisons were made with each vehicle control. * P
Figure Legend Snippet: Effects of ISE on inflammation in RAW264.7 macrophages cocultured with differentiated 3T3-L1 adipocytes. RAW264.7 macrophages were pretreated with different concentrations of ISE for 1 h and then were incubated with differentiated 3T3-L1 adipocytes for 24 h. Cell-free supernatants were collected to determine the concentrations of NO (A) , TNF-α (B) , IL-6 (C) , and MCP-1 (D) as described in “ Materials and Methods ” section. The values are means ± SD of 6 samples. Statistical comparisons were made with each vehicle control. * P

Techniques Used: Incubation

8) Product Images from "Differential roles of TNFα-TNFR1 and TNFα-TNFR2 in the differentiation and function of CD4+Foxp3+ induced Treg cells in vitro and in vivo periphery in autoimmune diseases"

Article Title: Differential roles of TNFα-TNFR1 and TNFα-TNFR2 in the differentiation and function of CD4+Foxp3+ induced Treg cells in vitro and in vivo periphery in autoimmune diseases

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1266-6

The effect of TNFR1 and TNFR2 on Treg, Th1, Th17 cells in EAE mouse model. a EAE model were induced on WT, TNFR1 −/− , and TNFR2 −/− Foxp3-reporter mice, the clinical symptom scores were evaluated blindly. b 30 days after the first immunization, part of the brain and spinal cord were stained with H E (×100, scale bars = 50 μm). c , d , g , h The proportions of Treg cells in brain and spinal cord (SC) were detected by FACS. e , f , i , j The proportions of Th1 and Th17 cells in brain and spinal cord were detected by FACS. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments
Figure Legend Snippet: The effect of TNFR1 and TNFR2 on Treg, Th1, Th17 cells in EAE mouse model. a EAE model were induced on WT, TNFR1 −/− , and TNFR2 −/− Foxp3-reporter mice, the clinical symptom scores were evaluated blindly. b 30 days after the first immunization, part of the brain and spinal cord were stained with H E (×100, scale bars = 50 μm). c , d , g , h The proportions of Treg cells in brain and spinal cord (SC) were detected by FACS. e , f , i , j The proportions of Th1 and Th17 cells in brain and spinal cord were detected by FACS. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments

Techniques Used: Mouse Assay, Staining, FACS

rmTNFα enhances iTreg suppressive function via TNFR2 in vitro and TNFR2 mediated the suppressive capability of iTreg in vivo. a EAE was induced on WT mice. 9 days after the first immunization, iTreg derived from WT, TNFR1 −/− , and TNFR2 −/− mice were adoptively transferred into EAE model, respectively. The clinical scores were monitored. b 30 days after the first immunization, the brain and spinal cord (SC) were subjected to H E staining (×100, scale bars = 50 μm). Foxp3, IL-17A, and IFN-γ expression in brains ( c – e ) and SC ( f – h ) were detected. * P ≤ 0.05; ** P ≤ 0.01, error bars denote SD. Representative data is from six independent experiments
Figure Legend Snippet: rmTNFα enhances iTreg suppressive function via TNFR2 in vitro and TNFR2 mediated the suppressive capability of iTreg in vivo. a EAE was induced on WT mice. 9 days after the first immunization, iTreg derived from WT, TNFR1 −/− , and TNFR2 −/− mice were adoptively transferred into EAE model, respectively. The clinical scores were monitored. b 30 days after the first immunization, the brain and spinal cord (SC) were subjected to H E staining (×100, scale bars = 50 μm). Foxp3, IL-17A, and IFN-γ expression in brains ( c – e ) and SC ( f – h ) were detected. * P ≤ 0.05; ** P ≤ 0.01, error bars denote SD. Representative data is from six independent experiments

Techniques Used: In Vitro, In Vivo, Mouse Assay, Derivative Assay, Staining, Expressing

TNFR2 is a critical signaling for Treg expansion following EAE induction and in vitro. a–d We detected the proportions of Treg in spleen and LNs from naive WT, TNFR1 −/− , and TNFR2 −/− mice. e–h 30 days after immunized with MOG, the proportion of Treg in spleen and LNs from the three groups were detected. i–k Treg isolated from LNs of WT, TNFR1 −/− , and TNFR2 −/− mice were expanded in vitro. Ki-67 was detected after 3 day culture. * P ≤ 0.05; ** P ≤ 0.01, error bars denote SD. Representative data is from six independent experiments
Figure Legend Snippet: TNFR2 is a critical signaling for Treg expansion following EAE induction and in vitro. a–d We detected the proportions of Treg in spleen and LNs from naive WT, TNFR1 −/− , and TNFR2 −/− mice. e–h 30 days after immunized with MOG, the proportion of Treg in spleen and LNs from the three groups were detected. i–k Treg isolated from LNs of WT, TNFR1 −/− , and TNFR2 −/− mice were expanded in vitro. Ki-67 was detected after 3 day culture. * P ≤ 0.05; ** P ≤ 0.01, error bars denote SD. Representative data is from six independent experiments

Techniques Used: In Vitro, Mouse Assay, Isolation

rmTNFα enhances iTreg impressive function via TNFR2 in vitro and TNFR2 mediated the Suppressive capability of iTreg in vivo. a , b iTreg were induced with or without rmTNFα. After 3 days, iTreg were co-cultured with CFSE-labeled Teffs. c , d iTreg were induced without rmTNFα, and then were co-cultured with Teffs with or without rmTNFα in the culture medium. e , f iTreg induced from WT, TNFR1 −/− , and TNFR2 −/− mice were co-cultured with Teffs. ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments
Figure Legend Snippet: rmTNFα enhances iTreg impressive function via TNFR2 in vitro and TNFR2 mediated the Suppressive capability of iTreg in vivo. a , b iTreg were induced with or without rmTNFα. After 3 days, iTreg were co-cultured with CFSE-labeled Teffs. c , d iTreg were induced without rmTNFα, and then were co-cultured with Teffs with or without rmTNFα in the culture medium. e , f iTreg induced from WT, TNFR1 −/− , and TNFR2 −/− mice were co-cultured with Teffs. ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments

Techniques Used: In Vitro, In Vivo, Cell Culture, Labeling, Mouse Assay

rmTNFα increases iTreg differentiation via TNFR2 in vitro. a , b Naive CD4 + T cells were induced into iTreg with different doses of rmTNFα for three days. The percentages of Foxp3 + T cells were determined. c During iTreg induction, the same dose of rmTNFα was added on day 0, 1, 2, or 3. All the cells were harvested after 4 days. The percentages of Foxp3 + T cells were determined. d , e iTreg induced for three days and reseeded with or without rmTNFα for another three days. The percentages of Foxp3 + T cells were determined. f , g , h Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were induced to iTreg. The percentages of Foxp3 + T cells, the total cell numbers and Foxp3 + cell numbers were determined, respectively. i Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were induced to iTreg with or without rmTNFα for three days. The percentages of Foxp3 + T cells were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative result is from five independent experiments
Figure Legend Snippet: rmTNFα increases iTreg differentiation via TNFR2 in vitro. a , b Naive CD4 + T cells were induced into iTreg with different doses of rmTNFα for three days. The percentages of Foxp3 + T cells were determined. c During iTreg induction, the same dose of rmTNFα was added on day 0, 1, 2, or 3. All the cells were harvested after 4 days. The percentages of Foxp3 + T cells were determined. d , e iTreg induced for three days and reseeded with or without rmTNFα for another three days. The percentages of Foxp3 + T cells were determined. f , g , h Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were induced to iTreg. The percentages of Foxp3 + T cells, the total cell numbers and Foxp3 + cell numbers were determined, respectively. i Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were induced to iTreg with or without rmTNFα for three days. The percentages of Foxp3 + T cells were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative result is from five independent experiments

Techniques Used: In Vitro, Isolation, Mouse Assay

TNFR2 gene knockout enhanced inflammatory responses and TNFR1 gene knockout was resistant to the development of colitis. a Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were injected into Rag1 −/− mice intraperitoneally. b Weights of the recipient mice were monitored after the cell transfer. c , d The colon was subjected to staining with H E (×40, scale bars = 200 μm), and the development of colitis was evaluated blindly by two pathologists. e The morphology and size of the colon, spleen and mLNs from the recipient mice were compared. No.1 was to WT group; No.2 was to TNFR1 −/− group; No.3 was to TNFR2 −/− group. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative result is from six independent experiments
Figure Legend Snippet: TNFR2 gene knockout enhanced inflammatory responses and TNFR1 gene knockout was resistant to the development of colitis. a Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were injected into Rag1 −/− mice intraperitoneally. b Weights of the recipient mice were monitored after the cell transfer. c , d The colon was subjected to staining with H E (×40, scale bars = 200 μm), and the development of colitis was evaluated blindly by two pathologists. e The morphology and size of the colon, spleen and mLNs from the recipient mice were compared. No.1 was to WT group; No.2 was to TNFR1 −/− group; No.3 was to TNFR2 −/− group. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative result is from six independent experiments

Techniques Used: Gene Knockout, Isolation, Mouse Assay, Injection, Staining

The effect of TNFR1 and TNFR2 on iTreg stability and function in vivo. iTreg were induced from three strains (Thy1.2 + ) as above protocols. 3 days later, these cells (0.6 × 10 6 ) were i.v. injected into Rag1 −/− mice that has received WT Thy1.1 + naive CD4 + T cells (0.6 × 10 6 ) through i.p. injection on the same day. a Weights of the recipient mice were monitored after the cell transfer. b 56 days after the transfer, spleens, mLNs, and cLP were harvested and the proportions of Foxp3 + T cells gated on Thy1.2 + cells were determined. c The proportions of IL-17A + or IFN-γ + T cells gated on Thy1.1 + cells in cLP were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six similar independent experiments
Figure Legend Snippet: The effect of TNFR1 and TNFR2 on iTreg stability and function in vivo. iTreg were induced from three strains (Thy1.2 + ) as above protocols. 3 days later, these cells (0.6 × 10 6 ) were i.v. injected into Rag1 −/− mice that has received WT Thy1.1 + naive CD4 + T cells (0.6 × 10 6 ) through i.p. injection on the same day. a Weights of the recipient mice were monitored after the cell transfer. b 56 days after the transfer, spleens, mLNs, and cLP were harvested and the proportions of Foxp3 + T cells gated on Thy1.2 + cells were determined. c The proportions of IL-17A + or IFN-γ + T cells gated on Thy1.1 + cells in cLP were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six similar independent experiments

Techniques Used: In Vivo, Injection, Mouse Assay

The effect of TNFR1 and TNFR2 on iTreg, Th1, Th17 cell differentiation in vivo. a Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were injected into Rag1 −/− mice intraperitoneally. a–i 21 days and 28 days after the transfer, spleens, mLNs, and cLP were harvested and the proportions of Foxp3 + T cells were determined. j–l The proportions of IL-17A + or IFN-γ + T cells in cLP were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments
Figure Legend Snippet: The effect of TNFR1 and TNFR2 on iTreg, Th1, Th17 cell differentiation in vivo. a Naive CD4 + T cells isolated from WT, TNFR1 −/− , and TNFR2 −/− mice were injected into Rag1 −/− mice intraperitoneally. a–i 21 days and 28 days after the transfer, spleens, mLNs, and cLP were harvested and the proportions of Foxp3 + T cells were determined. j–l The proportions of IL-17A + or IFN-γ + T cells in cLP were determined. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001, error bars denote SD. Representative data is from six independent experiments

Techniques Used: Cell Differentiation, In Vivo, Isolation, Mouse Assay, Injection

9) Product Images from "NDR2 promotes the antiviral immune response via facilitating TRIM25-mediated RIG-I activation in macrophages"

Article Title: NDR2 promotes the antiviral immune response via facilitating TRIM25-mediated RIG-I activation in macrophages

Journal: Science Advances

doi: 10.1126/sciadv.aav0163

NDR2 facilitates RNA virus–induced IFN-β, IL-6, and TNF-α production via a kinase activity–independent mechanism in macrophages. ( A and B ) IFN-β, IL-6, and TNF-α expression in NDR2 f/f and Lysm + NDR2 f/f PMs infected with VSV in the indicated times was detected by enzyme-linked immunosorbent assay (ELISA) (A) and real-time polymerase chain reaction (PCR) analysis (B). ( C ) Real-time PCR analysis of IFN-β, IL-6, and TNF-α transcripts in NDR2 f/f and Lysm + NDR2 f/f PMs treated with phosphate-buffered saline (PBS) or with the infection of H1N1, SeV, RSV, EMCV, HSV-1, or MHV68 for 8 hours. ( D ) Fluorescence-activated cell sorting (FACS) analysis of enhanced green fluorescent protein (eGFP) fluorescence intensity in NDR2 f/f and Lysm + NDR2 f/f PMs infected with VSV-eGFP. ( E ) VSV-eGFP titers by TCID 50 assay in supernatants of NDR2 f/f and Lysm + NDR2 f/f PMs infected with VSV-eGFP for 6 hours. ( F ) NDR2 f/f and Lysm + NDR2 f/f PMs were transfected with empty vector (mock), NDR2, or its kinase-inactive mutants overexpressing plasmids for 36 hours and then infected with VSV, followed by real-time PCR analysis of IFN-β, IL-6, and TNF-α expression. AA, K282A/T442A. ( G ) FACS analysis of RAW264.7 cells stably overexpressing NDR2 or its kinase-inactive mutants infected with VSV-eGFP. Data are means ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. ns, no significance. * P
Figure Legend Snippet: NDR2 facilitates RNA virus–induced IFN-β, IL-6, and TNF-α production via a kinase activity–independent mechanism in macrophages. ( A and B ) IFN-β, IL-6, and TNF-α expression in NDR2 f/f and Lysm + NDR2 f/f PMs infected with VSV in the indicated times was detected by enzyme-linked immunosorbent assay (ELISA) (A) and real-time polymerase chain reaction (PCR) analysis (B). ( C ) Real-time PCR analysis of IFN-β, IL-6, and TNF-α transcripts in NDR2 f/f and Lysm + NDR2 f/f PMs treated with phosphate-buffered saline (PBS) or with the infection of H1N1, SeV, RSV, EMCV, HSV-1, or MHV68 for 8 hours. ( D ) Fluorescence-activated cell sorting (FACS) analysis of enhanced green fluorescent protein (eGFP) fluorescence intensity in NDR2 f/f and Lysm + NDR2 f/f PMs infected with VSV-eGFP. ( E ) VSV-eGFP titers by TCID 50 assay in supernatants of NDR2 f/f and Lysm + NDR2 f/f PMs infected with VSV-eGFP for 6 hours. ( F ) NDR2 f/f and Lysm + NDR2 f/f PMs were transfected with empty vector (mock), NDR2, or its kinase-inactive mutants overexpressing plasmids for 36 hours and then infected with VSV, followed by real-time PCR analysis of IFN-β, IL-6, and TNF-α expression. AA, K282A/T442A. ( G ) FACS analysis of RAW264.7 cells stably overexpressing NDR2 or its kinase-inactive mutants infected with VSV-eGFP. Data are means ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. ns, no significance. * P

Techniques Used: Activity Assay, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Fluorescence, FACS, Transfection, Plasmid Preparation, Stable Transfection

10) Product Images from "Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro"

Article Title: Allograft Inflammatory Factor-1 Governs Hematopoietic Stem Cell Differentiation Into cDC1 and Monocyte-Derived Dendritic Cells Through IRF8 and RelB in vitro

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00173

AIF1 is expressed in Lin − CD117 + SCA1 + bone marrow hematopoietic stem cells and in spleen myeloid-derived subsets. Bone marrow cells were assessed for hematopoietic subsets. Dot plots show SSC vs. FSC and Lineage (Lin) vs. CD117 (c-Kit) populations. Expression of AIF1 was assessed in Lin − CD117 + (top row) and Lin + CD117 − (bottom row) cells co-expressed with either (A) SCA1 or (B) CD34. Splenocytes were evaluated for AIF1 expression in subsets of B cells, T cells, macrophages, plasmacytoid DC, and conventional DC; cell populations were, respectively, identified by: (C) CD45R (B220) + gated from IgD + CD19 + cells; (D) CD4 + subsets gated from CD3 + TCR + cells; (E) F4/80 + subsets gated from CD11c − CD11b + cells; (F) CD11c + subsets gated on B220 + PDCA1 + ; and (G) CD8α + gated from MHC class II + CD11c + conventional dendritic cells. (H) IRF8 + cells gated from CD11c + MHC class II + subsets. (I) AIF1 expression percentage in B220 + B cells gated from CD19 + IgD + subsets, TCRβ + T cells, F4/80 + macrophages gated from CD11c − CD11b + subsets, PDCA1 + pDC, CD8a + cDC gated from CD11c + MHC class II + subsets, and IRF8 + cDC gated from CD11c + MHC class II + subsets. All gates were established using respective isotype controls. Isotype used for determining AIF1 expression is shown in all groups. Data is representative of three independent experiments.
Figure Legend Snippet: AIF1 is expressed in Lin − CD117 + SCA1 + bone marrow hematopoietic stem cells and in spleen myeloid-derived subsets. Bone marrow cells were assessed for hematopoietic subsets. Dot plots show SSC vs. FSC and Lineage (Lin) vs. CD117 (c-Kit) populations. Expression of AIF1 was assessed in Lin − CD117 + (top row) and Lin + CD117 − (bottom row) cells co-expressed with either (A) SCA1 or (B) CD34. Splenocytes were evaluated for AIF1 expression in subsets of B cells, T cells, macrophages, plasmacytoid DC, and conventional DC; cell populations were, respectively, identified by: (C) CD45R (B220) + gated from IgD + CD19 + cells; (D) CD4 + subsets gated from CD3 + TCR + cells; (E) F4/80 + subsets gated from CD11c − CD11b + cells; (F) CD11c + subsets gated on B220 + PDCA1 + ; and (G) CD8α + gated from MHC class II + CD11c + conventional dendritic cells. (H) IRF8 + cells gated from CD11c + MHC class II + subsets. (I) AIF1 expression percentage in B220 + B cells gated from CD19 + IgD + subsets, TCRβ + T cells, F4/80 + macrophages gated from CD11c − CD11b + subsets, PDCA1 + pDC, CD8a + cDC gated from CD11c + MHC class II + subsets, and IRF8 + cDC gated from CD11c + MHC class II + subsets. All gates were established using respective isotype controls. Isotype used for determining AIF1 expression is shown in all groups. Data is representative of three independent experiments.

Techniques Used: Derivative Assay, Expressing

11) Product Images from "The Human CD4+ T Cell Response against Mumps Virus Targets a Broadly Recognized Nucleoprotein Epitope"

Article Title: The Human CD4+ T Cell Response against Mumps Virus Targets a Broadly Recognized Nucleoprotein Epitope

Journal: Journal of Virology

doi: 10.1128/JVI.01883-18

GTYR-specific activation of MuTER.1 is restricted by HLA-DR*04. (A) MuTER.1 cells were stimulated with autologous BLCL pulsed with GTYR peptide in the presence of anti-HLA-DR, anti-HLA-DQ, or anti-HLA-DP blocking antibodies. T cell activation was determined by expression of CD137 and IFN-γ secretion in the supernatant, as indicated. (B) BLCL with various HLA-DRB1 types as indicated were pulsed with GTYR peptide. T cell activation by these peptide-loaded BLCL was measured by IFN-γ secretion in the supernatant. Left bar represents autologous BLCL. T cell response, measured by CD137 expression or IFN-γ secretion (A), was significantly blocked with anti-HLA-DR antibodies ( P
Figure Legend Snippet: GTYR-specific activation of MuTER.1 is restricted by HLA-DR*04. (A) MuTER.1 cells were stimulated with autologous BLCL pulsed with GTYR peptide in the presence of anti-HLA-DR, anti-HLA-DQ, or anti-HLA-DP blocking antibodies. T cell activation was determined by expression of CD137 and IFN-γ secretion in the supernatant, as indicated. (B) BLCL with various HLA-DRB1 types as indicated were pulsed with GTYR peptide. T cell activation by these peptide-loaded BLCL was measured by IFN-γ secretion in the supernatant. Left bar represents autologous BLCL. T cell response, measured by CD137 expression or IFN-γ secretion (A), was significantly blocked with anti-HLA-DR antibodies ( P

Techniques Used: Activation Assay, Blocking Assay, Expressing

The CD4 + T cell clone is specific for MuV. MuTER.1 T cells were cocultured at a 1:1, 10:1, or 100:1 effector/target (E:T) ratio, as indicated, with autologous BLCL that were pulsed with no protein, mock, recombinant N, live MuV JL, or genotype G. After 20 h, T cell responses were analyzed by measuring the levels of secreted IFN-γ in the supernatant (A) or by staining for the activation marker CD137 (B and C). Data shown are from three individual experiments, with means ± the standard deviations (SD), and flow cytometry data are from one representative experiment (C). The T cell response, measured by IFN-γ secretion or CD137 expression (A and B), was significantly higher after stimulation with recombinant N, live MuV JL, or genotype G compared to stimulation with no protein or mock at all effector/target ratios ( P
Figure Legend Snippet: The CD4 + T cell clone is specific for MuV. MuTER.1 T cells were cocultured at a 1:1, 10:1, or 100:1 effector/target (E:T) ratio, as indicated, with autologous BLCL that were pulsed with no protein, mock, recombinant N, live MuV JL, or genotype G. After 20 h, T cell responses were analyzed by measuring the levels of secreted IFN-γ in the supernatant (A) or by staining for the activation marker CD137 (B and C). Data shown are from three individual experiments, with means ± the standard deviations (SD), and flow cytometry data are from one representative experiment (C). The T cell response, measured by IFN-γ secretion or CD137 expression (A and B), was significantly higher after stimulation with recombinant N, live MuV JL, or genotype G compared to stimulation with no protein or mock at all effector/target ratios ( P

Techniques Used: Recombinant, Staining, Activation Assay, Marker, Flow Cytometry, Cytometry, Expressing

MuV-specific CD4 + T cell clone MuTER.1 display a cytotoxic/Th1 phenotype. MuTER.1 cells were stimulated by autologous BLCL that were pulsed with GTYR peptide for activation and functional analysis. (A) T cell activation to a dose range of GTYR peptide was determined by IFN-γ secretion in the supernatant, and expression of the activation marker CD137 or degranulation marker CD107a. (B) Levels of secreted inflammatory and cytotoxicity-associated molecules were determined in the supernatant. Background cytokine levels in MuTER.1 cocultures with nonpulsed BLCL were subtracted. The levels of the cytokines indicated are averages of triplicate wells. (C) BLCL were pulsed with GTYR peptide and cocultured with MuTER.1 at various E:T ratios, as indicated. Killing of BLCL was determined after 24 h by PI staining.
Figure Legend Snippet: MuV-specific CD4 + T cell clone MuTER.1 display a cytotoxic/Th1 phenotype. MuTER.1 cells were stimulated by autologous BLCL that were pulsed with GTYR peptide for activation and functional analysis. (A) T cell activation to a dose range of GTYR peptide was determined by IFN-γ secretion in the supernatant, and expression of the activation marker CD137 or degranulation marker CD107a. (B) Levels of secreted inflammatory and cytotoxicity-associated molecules were determined in the supernatant. Background cytokine levels in MuTER.1 cocultures with nonpulsed BLCL were subtracted. The levels of the cytokines indicated are averages of triplicate wells. (C) BLCL were pulsed with GTYR peptide and cocultured with MuTER.1 at various E:T ratios, as indicated. Killing of BLCL was determined after 24 h by PI staining.

Techniques Used: Activation Assay, Functional Assay, Expressing, Marker, Staining

MuTER.1 clone responds to peptides with the core sequence YRLIPNAR. MuTER.1 cells were stimulated by peptide-pulsed autologous BLCL. (A) After 6 h, T cell activation by 25 different peptide pools was determined by expression of CD137 of CD4 + T cells, in a single experiment. (B and C) BLCL were pulsed with peptides MuV-N 105–119 or MuV-N 109–123 (from pools 2, 3, and 16) or a nonstimulating control peptide MuV-N 401–415 . Clone MuTER.1 was stimulated at a 1:1, 10:1, or 100:1 ratio, as indicated, with pulsed BLCL, and T cell activation was determined from the expression of CD137 (B) or IFN-γ secretion (C). (D) MuTER.1 cells were stimulated with BLCL pulsed with 15-mer peptides representing the MuV-N 101–127 sequence with 14-mer amino acid overlap in 1:1, 10:1 or 100:1 ratio, as indicated. Peptides MuV-N 105–119 or MuV-N 109–123 are underlined. The red bars present the 15-mer peptide MuV-N 110–124 , GTYRLIPNARANLTA, that was selected for further characterization of the MuTER.1 clone. Data shown are triplicates, with means ± the SD, from one representative experiment of two to three individual experiments. The MuTER.1 clone showed significantly higher CD137 + expression upon stimulation with peptide pools 2, 3, 4, and 16 compared to medium control ( P
Figure Legend Snippet: MuTER.1 clone responds to peptides with the core sequence YRLIPNAR. MuTER.1 cells were stimulated by peptide-pulsed autologous BLCL. (A) After 6 h, T cell activation by 25 different peptide pools was determined by expression of CD137 of CD4 + T cells, in a single experiment. (B and C) BLCL were pulsed with peptides MuV-N 105–119 or MuV-N 109–123 (from pools 2, 3, and 16) or a nonstimulating control peptide MuV-N 401–415 . Clone MuTER.1 was stimulated at a 1:1, 10:1, or 100:1 ratio, as indicated, with pulsed BLCL, and T cell activation was determined from the expression of CD137 (B) or IFN-γ secretion (C). (D) MuTER.1 cells were stimulated with BLCL pulsed with 15-mer peptides representing the MuV-N 101–127 sequence with 14-mer amino acid overlap in 1:1, 10:1 or 100:1 ratio, as indicated. Peptides MuV-N 105–119 or MuV-N 109–123 are underlined. The red bars present the 15-mer peptide MuV-N 110–124 , GTYRLIPNARANLTA, that was selected for further characterization of the MuTER.1 clone. Data shown are triplicates, with means ± the SD, from one representative experiment of two to three individual experiments. The MuTER.1 clone showed significantly higher CD137 + expression upon stimulation with peptide pools 2, 3, 4, and 16 compared to medium control ( P

Techniques Used: Sequencing, Activation Assay, Expressing

GTYR-specific CD4 + T cell responses in multiple mumps cases with different HLA-DR types. PBMCs from different mumps cases (1 to 2 months after clinical onset) with known HLA-DRB1 types, as indicated, were stimulated with GTYR peptide for 12 days in the presence of IL-2 to expand GTYR-specific CD4 + T cells. After 12 days, cells were rested for 24 h and restimulated with GTYR peptide in absence or presence of anti-HLA-DR blocking antibodies. GTYR-specific CD4 + T cells were determined after 6 h by intracellular cytokine staining for IFN-γ (A) and CD107a (B) surface expression. The data are from two experiments with eight and five different mumps cases, respectively.
Figure Legend Snippet: GTYR-specific CD4 + T cell responses in multiple mumps cases with different HLA-DR types. PBMCs from different mumps cases (1 to 2 months after clinical onset) with known HLA-DRB1 types, as indicated, were stimulated with GTYR peptide for 12 days in the presence of IL-2 to expand GTYR-specific CD4 + T cells. After 12 days, cells were rested for 24 h and restimulated with GTYR peptide in absence or presence of anti-HLA-DR blocking antibodies. GTYR-specific CD4 + T cells were determined after 6 h by intracellular cytokine staining for IFN-γ (A) and CD107a (B) surface expression. The data are from two experiments with eight and five different mumps cases, respectively.

Techniques Used: Blocking Assay, Staining, Expressing

12) Product Images from "Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response"

Article Title: Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113458

Cytokine, chemokine and growth factors production. Released IL-1β ( A ), TNF-α ( B ), MCP-1 ( C ), IL-6 ( D ), IL-8 ( E ), IL-10 ( F ), VEGF ( G ), TGF-β ( H ) quantified by ELISA, indicating the secretion of pro-inflammatory cytokines/chemokines; concomitant with the secretion of IL-10 anti-inflammatory cytokine by THP-1 in contact with the build-up substrates. Mean TNF-α/mean IL-10 index ( I ), confirming a weak inflammatory profile of THP-1 in contact with the build-up substrates ( n = 10, Mann Whitney statistical test).
Figure Legend Snippet: Cytokine, chemokine and growth factors production. Released IL-1β ( A ), TNF-α ( B ), MCP-1 ( C ), IL-6 ( D ), IL-8 ( E ), IL-10 ( F ), VEGF ( G ), TGF-β ( H ) quantified by ELISA, indicating the secretion of pro-inflammatory cytokines/chemokines; concomitant with the secretion of IL-10 anti-inflammatory cytokine by THP-1 in contact with the build-up substrates. Mean TNF-α/mean IL-10 index ( I ), confirming a weak inflammatory profile of THP-1 in contact with the build-up substrates ( n = 10, Mann Whitney statistical test).

Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

13) Product Images from "Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response"

Article Title: Combining Calcium Phosphates with Polysaccharides: A Bone-Inspired Material Modulating Monocyte/Macrophage Early Inflammatory Response

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113458

Cytokine, chemokine and growth factors production. Released IL-1β ( A ), TNF-α ( B ), MCP-1 ( C ), IL-6 ( D ), IL-8 ( E ), IL-10 ( F ), VEGF ( G ), TGF-β ( H ) quantified by ELISA, indicating the secretion of pro-inflammatory cytokines/chemokines; concomitant with the secretion of IL-10 anti-inflammatory cytokine by THP-1 in contact with the build-up substrates. Mean TNF-α/mean IL-10 index ( I ), confirming a weak inflammatory profile of THP-1 in contact with the build-up substrates ( n = 10, Mann Whitney statistical test).
Figure Legend Snippet: Cytokine, chemokine and growth factors production. Released IL-1β ( A ), TNF-α ( B ), MCP-1 ( C ), IL-6 ( D ), IL-8 ( E ), IL-10 ( F ), VEGF ( G ), TGF-β ( H ) quantified by ELISA, indicating the secretion of pro-inflammatory cytokines/chemokines; concomitant with the secretion of IL-10 anti-inflammatory cytokine by THP-1 in contact with the build-up substrates. Mean TNF-α/mean IL-10 index ( I ), confirming a weak inflammatory profile of THP-1 in contact with the build-up substrates ( n = 10, Mann Whitney statistical test).

Techniques Used: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

14) Product Images from "Distinct characteristics of Tregs of newborns of healthy and allergic mothers"

Article Title: Distinct characteristics of Tregs of newborns of healthy and allergic mothers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0207998

Expression of surface markers of Treg function in Tregs from cord blood of children of allergic and healthy mothers. Samples of cord blood of children of healthy (H, n = 112) and allergic (A, n = 98) mothers were stained and analysed by flow cytometry. (A) Flow cytometry analysis showing the proportion of PD-1 + cells among CD4 + CD25 + CD127 low Tregs in cord blood (p = 0.0382). (B) Flow cytometry analysis showing the proportion of CTLA-4 + cells among CD4 + CD25 + CD127 low Tregs in cord blood. (C) Flow cytometry analysis showing the proportion of GITR + cells among CD4 + CD25 + CD127 low Tregs in cord blood. p values were calculated using unpaired Student’s t-test.
Figure Legend Snippet: Expression of surface markers of Treg function in Tregs from cord blood of children of allergic and healthy mothers. Samples of cord blood of children of healthy (H, n = 112) and allergic (A, n = 98) mothers were stained and analysed by flow cytometry. (A) Flow cytometry analysis showing the proportion of PD-1 + cells among CD4 + CD25 + CD127 low Tregs in cord blood (p = 0.0382). (B) Flow cytometry analysis showing the proportion of CTLA-4 + cells among CD4 + CD25 + CD127 low Tregs in cord blood. (C) Flow cytometry analysis showing the proportion of GITR + cells among CD4 + CD25 + CD127 low Tregs in cord blood. p values were calculated using unpaired Student’s t-test.

Techniques Used: Expressing, Staining, Flow Cytometry, Cytometry

15) Product Images from "CD180 Ligation Inhibits TLR7- and TLR9-Mediated Activation of Macrophages and Dendritic Cells Through the Lyn-SHP-1/2 Axis in Murine Lupus"

Article Title: CD180 Ligation Inhibits TLR7- and TLR9-Mediated Activation of Macrophages and Dendritic Cells Through the Lyn-SHP-1/2 Axis in Murine Lupus

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02643

Lyn-SHP-1/2 axis is required for the inhibitory effect of CD180 on TLR7- and TLR9-mediated activation of macrophages and DCs. (A–F) BMDMs and BMDCs were pretreated with NSC87877 (1 μM) for 1 h followed by stimulation with anti-CD180 Ab (0.2 μg/ml). Then the cells were stimulated with R837 (1 μg/ml) and CpG 1826 (0.5 μM). Flow cytometric analysis of CD86 expression on BMDMs (A,B) and BMDCs (C,D) at 24 h. ELISA analysis of the levels of IL-6 and IL-12 secreted by BMDMs (E) and BMDCs (F) at 24 h. (G,H) BMDMs (G) , or BMDCs (H) were stimulated with anti-CD180 antibody (0.2 μg/ml) for different times and western blot analysis of the phosphorylation levels of SHP-1 and SHP-2. (I,J) BMDMs (I) or BMDCs (J) were stimulated with anti-CD180 antibody (0.2 μg/ml) for different times and western blot analysis of the phosphorylation levels of Lyn. (K,L) BMDMs (K) and BMDCs (L) , which have been infected with lentivirus expressing negative control-RNAi (NC-RNAi-LV) or Lyn-specific RNAi (Lyn-RNAi-LV), were stimulated with anti-CD180 antibody (0.2 μg/ml) for different times and western blot analysis of the phosphorylation levels of SHP-1 and SHP-2. The data shown represent the means of three independent experiments and the error bars represent the S.E.M. * p
Figure Legend Snippet: Lyn-SHP-1/2 axis is required for the inhibitory effect of CD180 on TLR7- and TLR9-mediated activation of macrophages and DCs. (A–F) BMDMs and BMDCs were pretreated with NSC87877 (1 μM) for 1 h followed by stimulation with anti-CD180 Ab (0.2 μg/ml). Then the cells were stimulated with R837 (1 μg/ml) and CpG 1826 (0.5 μM). Flow cytometric analysis of CD86 expression on BMDMs (A,B) and BMDCs (C,D) at 24 h. ELISA analysis of the levels of IL-6 and IL-12 secreted by BMDMs (E) and BMDCs (F) at 24 h. (G,H) BMDMs (G) , or BMDCs (H) were stimulated with anti-CD180 antibody (0.2 μg/ml) for different times and western blot analysis of the phosphorylation levels of SHP-1 and SHP-2. (I,J) BMDMs (I) or BMDCs (J) were stimulated with anti-CD180 antibody (0.2 μg/ml) for different times and western blot analysis of the phosphorylation levels of Lyn. (K,L) BMDMs (K) and BMDCs (L) , which have been infected with lentivirus expressing negative control-RNAi (NC-RNAi-LV) or Lyn-specific RNAi (Lyn-RNAi-LV), were stimulated with anti-CD180 antibody (0.2 μg/ml) for different times and western blot analysis of the phosphorylation levels of SHP-1 and SHP-2. The data shown represent the means of three independent experiments and the error bars represent the S.E.M. * p

Techniques Used: Activation Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Infection, Negative Control

Treatment effect of anti-CD180 Ab on lupus-symptoms of MRL/ lpr mice. (A) Proteinuria was collected weekly and determined by ELISA. (B) Representative images of marked splenomegaly in all groups of mice. (C,D) ELISA analysis of the anti-dsDNA antibody (C) and anti-RNA antibody (D) in serum. (E) H E staining of the kidney sections from all groups of mice. (F) Immunofluorescence staining to detect IgG deposit in the kidney sections. (G) Immunofluorescence staining to detect IgM deposit in the kidney sections. (H–J) Flow cytometric analysis of CD86 expression on peritoneal macrophages (H) , splenic macrophages (I) , and DCs (J) in all groups of mice. The data are shown as the means ± SEM ( n = 6 mice/group). ** p
Figure Legend Snippet: Treatment effect of anti-CD180 Ab on lupus-symptoms of MRL/ lpr mice. (A) Proteinuria was collected weekly and determined by ELISA. (B) Representative images of marked splenomegaly in all groups of mice. (C,D) ELISA analysis of the anti-dsDNA antibody (C) and anti-RNA antibody (D) in serum. (E) H E staining of the kidney sections from all groups of mice. (F) Immunofluorescence staining to detect IgG deposit in the kidney sections. (G) Immunofluorescence staining to detect IgM deposit in the kidney sections. (H–J) Flow cytometric analysis of CD86 expression on peritoneal macrophages (H) , splenic macrophages (I) , and DCs (J) in all groups of mice. The data are shown as the means ± SEM ( n = 6 mice/group). ** p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Flow Cytometry, Expressing

Increased percentages CD180-negative macrophages and DCs in spleens from MRL/ lpr mice. (A,B) Flow cytometric analysis of CD86 expression on F4/80 + macrophages in spleens from 18-week MRL/ lpr mice ( n = 16) and control C57BL/6 mice ( n = 16). (C,D) Flow cytometric analysis of CD86 expression on CD11c + DCs in spleens from 18-week MRL/ lpr mice ( n = 16) and control C57BL/6 mice ( n = 16). (E) Flow cytometric analysis of CD86 expression on CD180-negative and CD180-positive macrophages population in spleens from 18-week MRL/ lpr mice ( n = 16) and control C57BL/6 mice ( n = 16). (F) Flow cytometric analysis of CD86 expression on CD180-negative and CD180-positive DCs in spleens from 18-week MRL/ lpr mice ( n = 16) and control C57BL/6 mice ( n = 16). Error bars represent S.E.M. ** p
Figure Legend Snippet: Increased percentages CD180-negative macrophages and DCs in spleens from MRL/ lpr mice. (A,B) Flow cytometric analysis of CD86 expression on F4/80 + macrophages in spleens from 18-week MRL/ lpr mice ( n = 16) and control C57BL/6 mice ( n = 16). (C,D) Flow cytometric analysis of CD86 expression on CD11c + DCs in spleens from 18-week MRL/ lpr mice ( n = 16) and control C57BL/6 mice ( n = 16). (E) Flow cytometric analysis of CD86 expression on CD180-negative and CD180-positive macrophages population in spleens from 18-week MRL/ lpr mice ( n = 16) and control C57BL/6 mice ( n = 16). (F) Flow cytometric analysis of CD86 expression on CD180-negative and CD180-positive DCs in spleens from 18-week MRL/ lpr mice ( n = 16) and control C57BL/6 mice ( n = 16). Error bars represent S.E.M. ** p

Techniques Used: Mouse Assay, Flow Cytometry, Expressing

Ligation of CD180 inhibits TLR7- and TLR9-mediated activation of macrophages. BMDMs were pretreated with anti-CD180 antibody (0.2 μg/ml) or IgG2b antibody (0.2 μg/ml) followed by stimulation of R837 (1 μg/ml) and CpG1826 (0.5 μM). (A,B) Flow cytometric analysis of CD40 expression at 24 h. (C,D) Flow cytometric analysis of CD86 expression at 24 h. (E) ELISA analysis of secretions of IL-6, IL-12, TNF-α and IL-1β in cell culture at 24 h. (F) Q-PCR analysis of the mRNA levels of IL-6, IL-12, TNF-α, and IL-1β at 6 h. The axis labels means the fold differences over control. (G) Western blot analysis of the phosphorylation levels of p38, Erk, JNK, and p65 at 1 h. The data shown represent the means of three independent experiments and the error bars represent the S.E.M. * p
Figure Legend Snippet: Ligation of CD180 inhibits TLR7- and TLR9-mediated activation of macrophages. BMDMs were pretreated with anti-CD180 antibody (0.2 μg/ml) or IgG2b antibody (0.2 μg/ml) followed by stimulation of R837 (1 μg/ml) and CpG1826 (0.5 μM). (A,B) Flow cytometric analysis of CD40 expression at 24 h. (C,D) Flow cytometric analysis of CD86 expression at 24 h. (E) ELISA analysis of secretions of IL-6, IL-12, TNF-α and IL-1β in cell culture at 24 h. (F) Q-PCR analysis of the mRNA levels of IL-6, IL-12, TNF-α, and IL-1β at 6 h. The axis labels means the fold differences over control. (G) Western blot analysis of the phosphorylation levels of p38, Erk, JNK, and p65 at 1 h. The data shown represent the means of three independent experiments and the error bars represent the S.E.M. * p

Techniques Used: Ligation, Activation Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Polymerase Chain Reaction, Western Blot

Ligation of CD180 inhibits TLR7- and TLR9-mediated activation of DCs. BMDCs were pretreated with anti-CD180 antibody (0.2 μg/ml) followed by stimulation of R837 (1 μg/ml) and CpG 1826 (0.5 μM). (A,B) Flow cytometric analysis of CD40 expression at 24 h. (C,D) Flow cytometric analysis of CD86 expression at 24 h. (E) ELISA analysis of secretions of IL-6, IL-12, TNF-α, and IL-1β in cell culture at 24 h. (F) Q-PCR analysis of the mRNA levels of IL-6, IL-12, TNF-α, and IL-1β at 6 h. The axis label means the fold differences over control. (G) Western blot analysis of the phosphorylation levels of p38, Erk, JNK, and p65 at 1 h. The data shown represent the means of three independent experiments and the error bars represent the S.E.M. * p
Figure Legend Snippet: Ligation of CD180 inhibits TLR7- and TLR9-mediated activation of DCs. BMDCs were pretreated with anti-CD180 antibody (0.2 μg/ml) followed by stimulation of R837 (1 μg/ml) and CpG 1826 (0.5 μM). (A,B) Flow cytometric analysis of CD40 expression at 24 h. (C,D) Flow cytometric analysis of CD86 expression at 24 h. (E) ELISA analysis of secretions of IL-6, IL-12, TNF-α, and IL-1β in cell culture at 24 h. (F) Q-PCR analysis of the mRNA levels of IL-6, IL-12, TNF-α, and IL-1β at 6 h. The axis label means the fold differences over control. (G) Western blot analysis of the phosphorylation levels of p38, Erk, JNK, and p65 at 1 h. The data shown represent the means of three independent experiments and the error bars represent the S.E.M. * p

Techniques Used: Ligation, Activation Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Polymerase Chain Reaction, Western Blot

Treatment effect of anti-CD180 Ab on lupus-symptoms of IMQ-treated mice. (A) Treatment schematic of anti-CD180 Ab and IgG2b injection to mice following epicutaneous application of the TLR7 agonist imiquimod. (B) Proteinuria was collected weekly and determined by ELISA. (C) Representative images of marked splenomegaly in all groups of mice. (D,E) ELISA analysis of the anti-dsDNA antibody (D) and anti-RNA antibody (E) in serum. (F) H E staining of the kidney sections from all groups of mice. (G) Immunofluorescence staining to detect IgG deposit in the kidney sections. (H) Immunofluorescence staining to detect IgM deposit in the kidney sections. (I–K) Flow cytometric analysis of CD86 expression on peritoneal macrophages (I) , splenic macrophages (J) , and DCs (K) in all groups of mice. The data are shown as the means ± SEM ( n = 7 mice/group). * p
Figure Legend Snippet: Treatment effect of anti-CD180 Ab on lupus-symptoms of IMQ-treated mice. (A) Treatment schematic of anti-CD180 Ab and IgG2b injection to mice following epicutaneous application of the TLR7 agonist imiquimod. (B) Proteinuria was collected weekly and determined by ELISA. (C) Representative images of marked splenomegaly in all groups of mice. (D,E) ELISA analysis of the anti-dsDNA antibody (D) and anti-RNA antibody (E) in serum. (F) H E staining of the kidney sections from all groups of mice. (G) Immunofluorescence staining to detect IgG deposit in the kidney sections. (H) Immunofluorescence staining to detect IgM deposit in the kidney sections. (I–K) Flow cytometric analysis of CD86 expression on peritoneal macrophages (I) , splenic macrophages (J) , and DCs (K) in all groups of mice. The data are shown as the means ± SEM ( n = 7 mice/group). * p

Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Flow Cytometry, Expressing

Increased percentages of CD180-negative monocytes and DCs in PBMCs from SLE patients. (A,B) Flow cytometric analysis of CD180 expression on CD14 + monocytes in PBMCs from SLE patients ( n = 19) and healthy donors ( n = 19). (C,D) Flow cytometric analysis of CD180 expression on CD11c + DCs in PBMCs from SLE patients ( n = 19) and healthy donors ( n = 19). (E) Flow cytometric analysis of CD86 expression on CD180-negative and CD180-positive monocytes population in PBMCs from SLE patients ( n = 19) and healthy donors ( n = 19). (F) Flow cytometric analysis of CD86 expression on CD180-negative and CD180-positive DCs population in PBMCs from SLE patients ( n = 19) and healthy donors ( n = 19). Error bars represent S.E.M. *** p
Figure Legend Snippet: Increased percentages of CD180-negative monocytes and DCs in PBMCs from SLE patients. (A,B) Flow cytometric analysis of CD180 expression on CD14 + monocytes in PBMCs from SLE patients ( n = 19) and healthy donors ( n = 19). (C,D) Flow cytometric analysis of CD180 expression on CD11c + DCs in PBMCs from SLE patients ( n = 19) and healthy donors ( n = 19). (E) Flow cytometric analysis of CD86 expression on CD180-negative and CD180-positive monocytes population in PBMCs from SLE patients ( n = 19) and healthy donors ( n = 19). (F) Flow cytometric analysis of CD86 expression on CD180-negative and CD180-positive DCs population in PBMCs from SLE patients ( n = 19) and healthy donors ( n = 19). Error bars represent S.E.M. *** p

Techniques Used: Flow Cytometry, Expressing

16) Product Images from "Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway"

Article Title: Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway

Journal: Nature Communications

doi: 10.1038/s41467-018-07859-7

UroA/UAS03 prevent TNBS-induced colitis and sustain beneficial barrier activities. a Pre-TNBS treatment. Male C57BL/6 mice ( n = 5 per group at 7–8 week old age) were given orally vehicle (Veh; 0.25% sodium carboxymethylcellulose) or UroA or UAS03 (20 mg/kg/bodyweight) daily for one week followed by rectal administration of TNBS to induce colitis. These mice did not receive any treatment post-TNBS administration. Mice were euthanized 72 h post-TNBS administration and characterized. b Post-TNBS treatment. Another set group of C57BL/6 mice ( n = 5 per group at 7–8 week old age) received Veh or UroA or UAS03 (20 mg/kg) 24, 48, and 72 h post-TNBS. c Percent body weight loss was recorded after TNBS-administration. (No TNBS- Solid black line; Veh + TNBS- Solid red line; Pre-TNBS + UroA- Solid blue line; Pre-TNBS + UAS03- solid purple line; Post-TNBS + UroA- dashed blue line; Post-TNBS + UAS03- dashed purple line). d Representative colon images of control (no TNBS) along with vehicle/UroA/UAS03 treated mice from pre- and post-treatment groups. e Ratio of colon weight/length, f intestinal permeability was evaluated using FITC-dextran leakage assay. g Serum levels of IL-6 and TNF-α were measured using standard ELISA methods. Statistical analysis was performed (unpaired t -test) using Graphpad Prism software. Error bars, ±SEM *** p
Figure Legend Snippet: UroA/UAS03 prevent TNBS-induced colitis and sustain beneficial barrier activities. a Pre-TNBS treatment. Male C57BL/6 mice ( n = 5 per group at 7–8 week old age) were given orally vehicle (Veh; 0.25% sodium carboxymethylcellulose) or UroA or UAS03 (20 mg/kg/bodyweight) daily for one week followed by rectal administration of TNBS to induce colitis. These mice did not receive any treatment post-TNBS administration. Mice were euthanized 72 h post-TNBS administration and characterized. b Post-TNBS treatment. Another set group of C57BL/6 mice ( n = 5 per group at 7–8 week old age) received Veh or UroA or UAS03 (20 mg/kg) 24, 48, and 72 h post-TNBS. c Percent body weight loss was recorded after TNBS-administration. (No TNBS- Solid black line; Veh + TNBS- Solid red line; Pre-TNBS + UroA- Solid blue line; Pre-TNBS + UAS03- solid purple line; Post-TNBS + UroA- dashed blue line; Post-TNBS + UAS03- dashed purple line). d Representative colon images of control (no TNBS) along with vehicle/UroA/UAS03 treated mice from pre- and post-treatment groups. e Ratio of colon weight/length, f intestinal permeability was evaluated using FITC-dextran leakage assay. g Serum levels of IL-6 and TNF-α were measured using standard ELISA methods. Statistical analysis was performed (unpaired t -test) using Graphpad Prism software. Error bars, ±SEM *** p

Techniques Used: Mouse Assay, Permeability, Enzyme-linked Immunosorbent Assay, Software

UroA/UAS03 exert beneficial activities through AhR-dependent pathways. a – e Colitis was induced using TNBS in C57BL/6 (WT) and AhR −/− mice ( n = 4/group 7–8 week old age). Mice were treated with Veh or UroA/UAS03 (20 mg/kg bodyweight) every 12 h post TNBS administration and mice were euthanized at post 60 h TNBS administration. a TNBS-induced colitis experimental design and treatment regimen. b Percent body weight loss (No TNBS- Solid black line; Veh + TNBS- Solid red line; UroA + TNBS- Solid blue line; UAS03 + TNBS- Solid purple line), c representative colon images, d colon lengths, e gut permeability, f serum levels of IL-6 and TNF-α were determined. Statistical analysis was performed (unpaired t -test) using Graphpad Prism software. Error bars, ± SEM *** p
Figure Legend Snippet: UroA/UAS03 exert beneficial activities through AhR-dependent pathways. a – e Colitis was induced using TNBS in C57BL/6 (WT) and AhR −/− mice ( n = 4/group 7–8 week old age). Mice were treated with Veh or UroA/UAS03 (20 mg/kg bodyweight) every 12 h post TNBS administration and mice were euthanized at post 60 h TNBS administration. a TNBS-induced colitis experimental design and treatment regimen. b Percent body weight loss (No TNBS- Solid black line; Veh + TNBS- Solid red line; UroA + TNBS- Solid blue line; UAS03 + TNBS- Solid purple line), c representative colon images, d colon lengths, e gut permeability, f serum levels of IL-6 and TNF-α were determined. Statistical analysis was performed (unpaired t -test) using Graphpad Prism software. Error bars, ± SEM *** p

Techniques Used: Mouse Assay, Permeability, Software

Treatment with UroA/UAS03 mitigate DSS-induced chronic colitis. a C57BL/6 mice (7–8 week age old) were treated with four cycles of DSS (2%) with 7 days/cycle with an interval of 14 days with regular water. Control group of mice ( n = 5) received the regular water without DSS. UroA/UAS03 (20 mg/kg/day/body weight) that was resuspended in 0.25% sodium carboxymethylcellulose (CMC) solution ( n = 9) or vehicle (CMC) ( n = 9) was administered on 4th and 6th day of each DSS cycle and one treatment while on regular water. n = 5/control; n = 9/veh and UroA; n = 8/UAS03 group) Mice were euthanized at day 89 and the colitis phenotype was characterized. b Intestinal permeability using FITC-dextran was evaluated. c Representative colon images d colon lengths, e ratios of colon weight/length are shown. f Serum levels of IL-6, IL-1β, and TNF-α were measured using ELISA methods. g MPO levels were determined in colon tissues. h Cldn4 expression in the colons of these mice ( n = 3) was measured by immunoblots. Statistics performed using unpaired t -test using Graphpad Prism software. Error bars, ±SEM *** p
Figure Legend Snippet: Treatment with UroA/UAS03 mitigate DSS-induced chronic colitis. a C57BL/6 mice (7–8 week age old) were treated with four cycles of DSS (2%) with 7 days/cycle with an interval of 14 days with regular water. Control group of mice ( n = 5) received the regular water without DSS. UroA/UAS03 (20 mg/kg/day/body weight) that was resuspended in 0.25% sodium carboxymethylcellulose (CMC) solution ( n = 9) or vehicle (CMC) ( n = 9) was administered on 4th and 6th day of each DSS cycle and one treatment while on regular water. n = 5/control; n = 9/veh and UroA; n = 8/UAS03 group) Mice were euthanized at day 89 and the colitis phenotype was characterized. b Intestinal permeability using FITC-dextran was evaluated. c Representative colon images d colon lengths, e ratios of colon weight/length are shown. f Serum levels of IL-6, IL-1β, and TNF-α were measured using ELISA methods. g MPO levels were determined in colon tissues. h Cldn4 expression in the colons of these mice ( n = 3) was measured by immunoblots. Statistics performed using unpaired t -test using Graphpad Prism software. Error bars, ±SEM *** p

Techniques Used: Mouse Assay, Permeability, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Software

UroA/UAS03 utilize Nrf2 pathways to mitigate colitis. a–e Colitis was induced using TNBS in C57BL/6 (WT) and Nrf2 −/− mice ( n = 4–5/group 7–8 week old age). Mice were treated with Veh or UroA/UAS03 (20 mg/kg bodyweight) every 12 h post TNBS administration ending at 72 h. Representative data from two independent experiments is shown. a TNBS-induced colitis experimental design and treatment regimen. b Percent body weight loss (No TNBS- Solid black line; Veh + TNBS- Solid red line; UroA + TNBS- Solid blue line; UAS03 + TNBS- Solid purple line), c representative colon images, d colon lengths, e gut permeability, f serum levels of IL-6 and TNF-α were determined. Statistical analysis was performed (unpaired t -test) using Graphpad Prism software. Error bars, ±SEM *** p
Figure Legend Snippet: UroA/UAS03 utilize Nrf2 pathways to mitigate colitis. a–e Colitis was induced using TNBS in C57BL/6 (WT) and Nrf2 −/− mice ( n = 4–5/group 7–8 week old age). Mice were treated with Veh or UroA/UAS03 (20 mg/kg bodyweight) every 12 h post TNBS administration ending at 72 h. Representative data from two independent experiments is shown. a TNBS-induced colitis experimental design and treatment regimen. b Percent body weight loss (No TNBS- Solid black line; Veh + TNBS- Solid red line; UroA + TNBS- Solid blue line; UAS03 + TNBS- Solid purple line), c representative colon images, d colon lengths, e gut permeability, f serum levels of IL-6 and TNF-α were determined. Statistical analysis was performed (unpaired t -test) using Graphpad Prism software. Error bars, ±SEM *** p

Techniques Used: Mouse Assay, Permeability, Software

UAS03 is a potent anti-inflammatory structural analogue of UroA and induces tight junction proteins. a Chemical structures of UroA, UAS03. UroA/UAS03 stability was examined in the presence of gastric pH 2.0 and digestive enzymes. UroA and UAS03 (0.2 mg/ml) were incubated with digestive enzymes (esterases and proteases, 100 U/ml) for 12 h at 37 °C and compound levels were quantified. b BMDMs were stimulated with LPS (50 ng/ml) without or with UroA (blue line)/UAS03 (purple line) (0.1, 1, 10, 25, and 50 µM) for 6 h. IL-6 and TNF-α levels in supernatants were measured. c C57BL/6 mice (n = 3–4) were pretreated with UroA (20 mg/kg) and UAS03 (20 mg/kg). After 4 h, LPS (2 mg/kg) was injected intraperitoneally. Post 4 h of LPS administration, serum levels of IL-6 and TNF-α was measured. d – f HT29 or Caco2 cells were treated with vehicle (DMSO-0.01%) or UroA/UAS03 (50 μM) for 24 h. d The fold changes in mRNA levels of claudin 4 ( Cldn4 ), occludin ( Ocln ), and Zona occludens 1 ( ZO1 ) in HT29 cells were determined by RT PCR method. e UroA/UAS03 induced protein expression of Cldn4, Ocln, and ZO1 in HT29 cells were determined by immunoblots and quantified by Image J software. f Caco2 or HT29 cells were grown on coverslip bottom FluroDish and treated with Vehicle, UroA/UAS03 for 24 h. The cells were stained with anti-Cldn4 followed by secondary antibody tagged with Alexa-488. Nucleus was stained using DAPI. The confocal images were captured. The green intensity ( n = 15–20 cell membrane regions) was measured. Scale bars for Caco2 and HT29 cells indicate 50 and 25 μm respectively. g Monolayer HT29 or Caco2 cells on transmembranes were treated with vehicle or UroA/UAS03 (50 μM) for 24 h followed by treatment with LPS (50 ng/ml) for 2 h. FITC-dextran was added to these cells (top of the membrane) and incubated for 2 h and FITC-dextran levels in bottom chamber well was measured. Results are representative of three independent experiments with triplicates for each concentration. * p
Figure Legend Snippet: UAS03 is a potent anti-inflammatory structural analogue of UroA and induces tight junction proteins. a Chemical structures of UroA, UAS03. UroA/UAS03 stability was examined in the presence of gastric pH 2.0 and digestive enzymes. UroA and UAS03 (0.2 mg/ml) were incubated with digestive enzymes (esterases and proteases, 100 U/ml) for 12 h at 37 °C and compound levels were quantified. b BMDMs were stimulated with LPS (50 ng/ml) without or with UroA (blue line)/UAS03 (purple line) (0.1, 1, 10, 25, and 50 µM) for 6 h. IL-6 and TNF-α levels in supernatants were measured. c C57BL/6 mice (n = 3–4) were pretreated with UroA (20 mg/kg) and UAS03 (20 mg/kg). After 4 h, LPS (2 mg/kg) was injected intraperitoneally. Post 4 h of LPS administration, serum levels of IL-6 and TNF-α was measured. d – f HT29 or Caco2 cells were treated with vehicle (DMSO-0.01%) or UroA/UAS03 (50 μM) for 24 h. d The fold changes in mRNA levels of claudin 4 ( Cldn4 ), occludin ( Ocln ), and Zona occludens 1 ( ZO1 ) in HT29 cells were determined by RT PCR method. e UroA/UAS03 induced protein expression of Cldn4, Ocln, and ZO1 in HT29 cells were determined by immunoblots and quantified by Image J software. f Caco2 or HT29 cells were grown on coverslip bottom FluroDish and treated with Vehicle, UroA/UAS03 for 24 h. The cells were stained with anti-Cldn4 followed by secondary antibody tagged with Alexa-488. Nucleus was stained using DAPI. The confocal images were captured. The green intensity ( n = 15–20 cell membrane regions) was measured. Scale bars for Caco2 and HT29 cells indicate 50 and 25 μm respectively. g Monolayer HT29 or Caco2 cells on transmembranes were treated with vehicle or UroA/UAS03 (50 μM) for 24 h followed by treatment with LPS (50 ng/ml) for 2 h. FITC-dextran was added to these cells (top of the membrane) and incubated for 2 h and FITC-dextran levels in bottom chamber well was measured. Results are representative of three independent experiments with triplicates for each concentration. * p

Techniques Used: Incubation, Mouse Assay, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Software, Staining, Concentration Assay

UroA/UAS03 treatment attenuates TNBS-induced colitis in mice. Colitis was induced by intrarectal administration of TNBS (2.5 mg/mouse) in C57BL/6 (8 week age old, n = 5/group) mice. Mice were orally treated with vehicle or UroA (20 mg/kg) or UAS03 (20 mg/kg body weight) every 12 h post-TNBS instillation for 60 h and the experiment terminated at 72 h. Representative data from one of three independent experiments is shown. a Percent body weight loss (No TNBS- Solid black line; Veh + TNBS- Solid red line; UroA + TNBS- Solid blue line; UAS03 + TNBS- Solid purple line). b disease activity index, c intestinal permeability, d colon lengths were measured. e Gross morphological changes of colon, f ratio of colon weight/length, g colonic myeloperoxidase (MPO) levels, h serum IL-6, TNF-α, CXCL1, and IL-1β levels, i microphotographs of hematoxylin and eosin (H E) stained sections of colons and inflammation scores are shown. Scale bar indicates 300 μm. j Cldn4 expression in the colons of these mice ( n = 3) was measured by immunoblots and quantified. Statistical analysis was performed (unpaired t -test) using Graphpad Prism software. Error bars, ±SEM *** p
Figure Legend Snippet: UroA/UAS03 treatment attenuates TNBS-induced colitis in mice. Colitis was induced by intrarectal administration of TNBS (2.5 mg/mouse) in C57BL/6 (8 week age old, n = 5/group) mice. Mice were orally treated with vehicle or UroA (20 mg/kg) or UAS03 (20 mg/kg body weight) every 12 h post-TNBS instillation for 60 h and the experiment terminated at 72 h. Representative data from one of three independent experiments is shown. a Percent body weight loss (No TNBS- Solid black line; Veh + TNBS- Solid red line; UroA + TNBS- Solid blue line; UAS03 + TNBS- Solid purple line). b disease activity index, c intestinal permeability, d colon lengths were measured. e Gross morphological changes of colon, f ratio of colon weight/length, g colonic myeloperoxidase (MPO) levels, h serum IL-6, TNF-α, CXCL1, and IL-1β levels, i microphotographs of hematoxylin and eosin (H E) stained sections of colons and inflammation scores are shown. Scale bar indicates 300 μm. j Cldn4 expression in the colons of these mice ( n = 3) was measured by immunoblots and quantified. Statistical analysis was performed (unpaired t -test) using Graphpad Prism software. Error bars, ±SEM *** p

Techniques Used: Mouse Assay, Activity Assay, Permeability, Staining, Expressing, Western Blot, Software

17) Product Images from "The antifibrotic drug pirfenidone inhibits spondyloarthritis fibroblast-like synoviocytes and osteoblasts in vitro"

Article Title: The antifibrotic drug pirfenidone inhibits spondyloarthritis fibroblast-like synoviocytes and osteoblasts in vitro

Journal: BMC rheumatology

doi: 10.1186/s41927-018-0040-9

Effects of PFD on intracellular and membrane molecules characterizing differentiation and activity of SpA FLSs. a - b Images of membrane-based antibody array of SpA FLSs stimulated with TGFβ, TNFα, and IFNγ (TGFβ+TNFα+IFNγ) with or without pirfenidone (PFD). Black square marks MCP-1 and grey square marks YKL-40. c - d Column bar graph of MCP-1 and YKL-40 secretion by SpA FLSs untreated (UT) and stimulated with TGFβ, TNFα, IFNγ or all three cytokines (All) with or without PFD ( n = 4). Data were normalized to untreated cultures without PFD (ratio), log-transformed and analyzed with the paired t-test. Boxes and bars indicate median and IQR. * p
Figure Legend Snippet: Effects of PFD on intracellular and membrane molecules characterizing differentiation and activity of SpA FLSs. a - b Images of membrane-based antibody array of SpA FLSs stimulated with TGFβ, TNFα, and IFNγ (TGFβ+TNFα+IFNγ) with or without pirfenidone (PFD). Black square marks MCP-1 and grey square marks YKL-40. c - d Column bar graph of MCP-1 and YKL-40 secretion by SpA FLSs untreated (UT) and stimulated with TGFβ, TNFα, IFNγ or all three cytokines (All) with or without PFD ( n = 4). Data were normalized to untreated cultures without PFD (ratio), log-transformed and analyzed with the paired t-test. Boxes and bars indicate median and IQR. * p

Techniques Used: Activity Assay, Ab Array, Transformation Assay

18) Product Images from "Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function"

Article Title: Selective ORAI1 inhibition ameliorates autoimmune CNS inflammation by suppressing effector but not regulatory T cell function

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

doi: 10.4049/jimmunol.1501406

Progression and severity of EAE are attenuated by treatment of mice with CRAC channel inhibitor ( A ) Clinical EAE score in WT mice injected with MOG 35-55 in CFA. Mice were treated with 6 mg/kg of the CRAC channel inhibitor AMG1 or vehicle control for 10 days starting when EAE scores were ≥ 1. Graphs represent the average ± SEM of 8 mice per group. ( B ) Ca 2+ influx was measured in splenocytes isolated at the end of 10 day treatment with inhibitor or vehicle. Cells were loaded with Fura2-AM and stimulated with 1 μM thapsigargin (TG) in Ca 2+ free buffer, followed by readdition of 1 mM extracellular Ca 2+ to induce SOCE. ( C ) Absolute numbers of CD4 + and CD8 + T cells and CD11b + Gr-1 + polymorphonuclear (PMN) cells, CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - DC isolated from the CNS of mice at day 23 after EAE induction and analyzed by flow cytometry. ( D ) Frequencies of CD4 + Foxp3 + Treg cells in the CNS at day 23 after EAE induction. ( E ) Expression of IFN-γ, IL-17 and GM-CSF by CD4 + T cells isolated from the CNS at day 23 after EAE induction and stimulated with PMA and ionomycin for 6 h. Data represent the average ± SEM of 8 mice per group. Statistical analysis of EAE scores in A (days 13–23) was performed using a Mann-Whitney test; data in panels B–E were analyzed using an unpaired Student’s t test. * p
Figure Legend Snippet: Progression and severity of EAE are attenuated by treatment of mice with CRAC channel inhibitor ( A ) Clinical EAE score in WT mice injected with MOG 35-55 in CFA. Mice were treated with 6 mg/kg of the CRAC channel inhibitor AMG1 or vehicle control for 10 days starting when EAE scores were ≥ 1. Graphs represent the average ± SEM of 8 mice per group. ( B ) Ca 2+ influx was measured in splenocytes isolated at the end of 10 day treatment with inhibitor or vehicle. Cells were loaded with Fura2-AM and stimulated with 1 μM thapsigargin (TG) in Ca 2+ free buffer, followed by readdition of 1 mM extracellular Ca 2+ to induce SOCE. ( C ) Absolute numbers of CD4 + and CD8 + T cells and CD11b + Gr-1 + polymorphonuclear (PMN) cells, CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - DC isolated from the CNS of mice at day 23 after EAE induction and analyzed by flow cytometry. ( D ) Frequencies of CD4 + Foxp3 + Treg cells in the CNS at day 23 after EAE induction. ( E ) Expression of IFN-γ, IL-17 and GM-CSF by CD4 + T cells isolated from the CNS at day 23 after EAE induction and stimulated with PMA and ionomycin for 6 h. Data represent the average ± SEM of 8 mice per group. Statistical analysis of EAE scores in A (days 13–23) was performed using a Mann-Whitney test; data in panels B–E were analyzed using an unpaired Student’s t test. * p

Techniques Used: Mouse Assay, Injection, Isolation, Flow Cytometry, Cytometry, Expressing, MANN-WHITNEY

Inducible deletion of Orai1 gene in T cells during ongoing EAE ameliorates disease severity ( A,B ) In vitro deletion of Orai1 in CD4 + T cells isolated from Orai1 fl/fl Cre-ERT2 , WT and Orai1 fl/fl Cd4-Cre control mice. Cells were stimulated with anti-CD3/CD28 for 3 days in the presence of 1 μM tamoxifen (TAM) and analyzed for SOCE after stimulation with thapsigargin and addition of 1 mM Ca 2+ ( A ) and Orai1 mRNA expression by RT PCR ( B ). ( C–F ) For passive induction of EAE, Orai1 fl/fl Cre-ERT2 and WT mice were immunized with MOG peptide. T cells were isolated from spleen and LNs 12 days later and restimulated in vitro with MOG peptide in the presence of IL-23 for 3 days. 4 × 10 6 CD4 + T cells were transferred i.v. into sublethally irradiated CD45.1 recipient mice. After EAE symptoms developed, recipient mice were injected with tamoxifen or vehicle control from day 10–14 after T cell transfer. ( C ) Clinical EAE scores. ( D–F ) Absolute numbers of CD4 + and CD8 + T cells ( D ) as well as CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells ( E ) in the CNS 18 days after adoptive transfer. ( F ) Frequencies of CD4 + IFN-γ + and CD4 + IL-17 + T cells isolated from the CNS and restimulated in vitro with PMA and ionomycin for 6h. Data represent the average ± SEM of 3–4 mice per group. Statistical analysis of Orai1 expression in B was performed using a one-way ANOVA test. Statistical analysis of EAE scores (days 10–18) in C was performed using a Mann-Whitney test. Data in panels D–F were analyzed using an unpaired Student’s t test. * p
Figure Legend Snippet: Inducible deletion of Orai1 gene in T cells during ongoing EAE ameliorates disease severity ( A,B ) In vitro deletion of Orai1 in CD4 + T cells isolated from Orai1 fl/fl Cre-ERT2 , WT and Orai1 fl/fl Cd4-Cre control mice. Cells were stimulated with anti-CD3/CD28 for 3 days in the presence of 1 μM tamoxifen (TAM) and analyzed for SOCE after stimulation with thapsigargin and addition of 1 mM Ca 2+ ( A ) and Orai1 mRNA expression by RT PCR ( B ). ( C–F ) For passive induction of EAE, Orai1 fl/fl Cre-ERT2 and WT mice were immunized with MOG peptide. T cells were isolated from spleen and LNs 12 days later and restimulated in vitro with MOG peptide in the presence of IL-23 for 3 days. 4 × 10 6 CD4 + T cells were transferred i.v. into sublethally irradiated CD45.1 recipient mice. After EAE symptoms developed, recipient mice were injected with tamoxifen or vehicle control from day 10–14 after T cell transfer. ( C ) Clinical EAE scores. ( D–F ) Absolute numbers of CD4 + and CD8 + T cells ( D ) as well as CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells ( E ) in the CNS 18 days after adoptive transfer. ( F ) Frequencies of CD4 + IFN-γ + and CD4 + IL-17 + T cells isolated from the CNS and restimulated in vitro with PMA and ionomycin for 6h. Data represent the average ± SEM of 3–4 mice per group. Statistical analysis of Orai1 expression in B was performed using a one-way ANOVA test. Statistical analysis of EAE scores (days 10–18) in C was performed using a Mann-Whitney test. Data in panels D–F were analyzed using an unpaired Student’s t test. * p

Techniques Used: In Vitro, Isolation, Mouse Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Irradiation, Injection, Adoptive Transfer Assay, MANN-WHITNEY

Experimental autoimmune encephalomyelitis (EAE) is ameliorated in Orai1 fl/fl Cd4-Cre mice ( A ) RNA from wildtype and Orai1 fl/fl Cd4-Cre was isolated from CD4 + T cells and relative mRNA expression was measured by RT PCR. ( B ) SOCE was measured in Fura2-AM loaded freshly isolated CD4 + T cells from wildtype and Orai1 fl/fl Cd4-Cre mice after stimulation with thapsigargin (TG) followed by readdition of 1 mM Ca 2+ to the extracellular medium. ( C ) Clinical EAE scores in WT and Orai1 fl/fl Cd4-Cre mice injected with MOG 35-55 in CFA. ( D ) Summary of EAE with days to onset (±SEM), incidence of EAE and maximal EAE scores (±SEM). ( E ) H E and Luxol fast blue staining of spinal cords from WT and Orai1 fl/fl Cd4-Cre mice at 27 after EAE induction. Arrows indicate infiltrating cells and demyelination, respectively. Shown is one histological stain representative of 4 mice per group and 6 examined levels of spinal cord per mouse. ( F–I ) Cells were isolated from the spinal cord (CNS) of WT and Orai1 fl/fl Cd4-Cre mice at day 27 after EAE induction and analyzed by flow cytometry. ( F–H ) Absolute numbers of CD4 + and CD8 + T cells ( F ), frequencies of Foxp3 + CD4 + T cells ( G ) and absolute numbers of CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells in the CNS ( H ). ( I ) Cytokine production of T cells isolated from the CNS was analyzed by flow cytometry after stimulation with PMA and ionomycin in the presence of Brefeldin A for 6 h. Shown are absolute numbers of CD4 + T cells expressing IFN-γ, IL-17 or GM-CSF. Data in panels A and B represent the average ± SEM of 3 mice per group. Data in panels C–D and F–H represent the average ± SEM of 14–16 mice per group. Statistical analysis of EAE scores (starting at the first clinical signs of EAE) was performed using a Mann-Whitney test (panel C); data in panels B, F–H were analyzed using an unpaired Student’s t test. * p
Figure Legend Snippet: Experimental autoimmune encephalomyelitis (EAE) is ameliorated in Orai1 fl/fl Cd4-Cre mice ( A ) RNA from wildtype and Orai1 fl/fl Cd4-Cre was isolated from CD4 + T cells and relative mRNA expression was measured by RT PCR. ( B ) SOCE was measured in Fura2-AM loaded freshly isolated CD4 + T cells from wildtype and Orai1 fl/fl Cd4-Cre mice after stimulation with thapsigargin (TG) followed by readdition of 1 mM Ca 2+ to the extracellular medium. ( C ) Clinical EAE scores in WT and Orai1 fl/fl Cd4-Cre mice injected with MOG 35-55 in CFA. ( D ) Summary of EAE with days to onset (±SEM), incidence of EAE and maximal EAE scores (±SEM). ( E ) H E and Luxol fast blue staining of spinal cords from WT and Orai1 fl/fl Cd4-Cre mice at 27 after EAE induction. Arrows indicate infiltrating cells and demyelination, respectively. Shown is one histological stain representative of 4 mice per group and 6 examined levels of spinal cord per mouse. ( F–I ) Cells were isolated from the spinal cord (CNS) of WT and Orai1 fl/fl Cd4-Cre mice at day 27 after EAE induction and analyzed by flow cytometry. ( F–H ) Absolute numbers of CD4 + and CD8 + T cells ( F ), frequencies of Foxp3 + CD4 + T cells ( G ) and absolute numbers of CD11b + Gr-1 + polymorphonuclear cells (PMN), CD11b + CD11c - Gr-1 - macrophages and CD11c + Gr-1 - dendritic cells in the CNS ( H ). ( I ) Cytokine production of T cells isolated from the CNS was analyzed by flow cytometry after stimulation with PMA and ionomycin in the presence of Brefeldin A for 6 h. Shown are absolute numbers of CD4 + T cells expressing IFN-γ, IL-17 or GM-CSF. Data in panels A and B represent the average ± SEM of 3 mice per group. Data in panels C–D and F–H represent the average ± SEM of 14–16 mice per group. Statistical analysis of EAE scores (starting at the first clinical signs of EAE) was performed using a Mann-Whitney test (panel C); data in panels B, F–H were analyzed using an unpaired Student’s t test. * p

Techniques Used: Mouse Assay, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Injection, Staining, Flow Cytometry, Cytometry, MANN-WHITNEY

19) Product Images from "Reduced frequencies of polyfunctional CMV-specific T cell responses in infants with congenital CMV infection"

Article Title: Reduced frequencies of polyfunctional CMV-specific T cell responses in infants with congenital CMV infection

Journal: Journal of clinical immunology

doi: 10.1007/s10875-015-0139-3

Frequencies of detectable CMV pp65-specific T cell responses Box plots display the frequencies of CMV pp65-specific CD4 (left) or CD8 (right) T cells detected by individual measures of function (CD107, MIP1β, IFNγ, or IL2) for C infants, P adults, and CH adults. All detectable responses and timepoints are included. Horizontal lines represent the 25 th , 50 th (median), or 75 th percentiles. Diamonds indicate the mean frequencies, and the error bars indicate the minimum and maximum values. C infants showed significantly lower frequencies of pp65-specific CD8 T cells detectable by any function (i.e. gated on all responding cells, not shown; p =0.006 versus P adults; p =0.040 versus CH adults) or by MIP1β ( p =0.043 versus P adults; p =NS versus CH adults). Frequencies of responses measured by CD107, IFNγ, or IL2 were not significantly different between the groups
Figure Legend Snippet: Frequencies of detectable CMV pp65-specific T cell responses Box plots display the frequencies of CMV pp65-specific CD4 (left) or CD8 (right) T cells detected by individual measures of function (CD107, MIP1β, IFNγ, or IL2) for C infants, P adults, and CH adults. All detectable responses and timepoints are included. Horizontal lines represent the 25 th , 50 th (median), or 75 th percentiles. Diamonds indicate the mean frequencies, and the error bars indicate the minimum and maximum values. C infants showed significantly lower frequencies of pp65-specific CD8 T cells detectable by any function (i.e. gated on all responding cells, not shown; p =0.006 versus P adults; p =0.040 versus CH adults) or by MIP1β ( p =0.043 versus P adults; p =NS versus CH adults). Frequencies of responses measured by CD107, IFNγ, or IL2 were not significantly different between the groups

Techniques Used:

20) Product Images from "Cathelicidin Antimicrobial Peptide Expression Is Not Induced or Required for Bacterial Clearance during Salmonella enterica Infection of Human Monocyte-Derived Macrophages"

Article Title: Cathelicidin Antimicrobial Peptide Expression Is Not Induced or Required for Bacterial Clearance during Salmonella enterica Infection of Human Monocyte-Derived Macrophages

Journal: Infection and Immunity

doi: 10.1128/IAI.00672-12

Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and IL-6 (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P
Figure Legend Snippet: Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and IL-6 (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P

Techniques Used: Infection, Modification, Mutagenesis

21) Product Images from "A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice"

Article Title: A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice

Journal: Molecular Therapy

doi: 10.1016/j.ymthe.2017.06.023

Binding of Aptamers to Murine TIM-3 . (A) Six aptamers that bind to recombinant TIM-3-Fc fusion protein. (B) b0 aptamer binding to TIM-3-expressing CHO cells. (C) b0 aptamer binding to activated BALB/c lymphocytes. (A–C) Black, b0 aptamer incubated to naked beads, parental CHO cells, or non-activated lymphocytes. Blue, scrambled aptamer binding to TIM-3 targets. Red, TIM-3-specific aptamers. (D–F) TIM-3 antibody binding to TIM-3-Fc fusion-coated beads (D), TIM-3 expressing CHO cells (E) and activated lymphocytes (F). Blue, binding of isotype control antibody. Red, TIM-3-specific antibody.
Figure Legend Snippet: Binding of Aptamers to Murine TIM-3 . (A) Six aptamers that bind to recombinant TIM-3-Fc fusion protein. (B) b0 aptamer binding to TIM-3-expressing CHO cells. (C) b0 aptamer binding to activated BALB/c lymphocytes. (A–C) Black, b0 aptamer incubated to naked beads, parental CHO cells, or non-activated lymphocytes. Blue, scrambled aptamer binding to TIM-3 targets. Red, TIM-3-specific aptamers. (D–F) TIM-3 antibody binding to TIM-3-Fc fusion-coated beads (D), TIM-3 expressing CHO cells (E) and activated lymphocytes (F). Blue, binding of isotype control antibody. Red, TIM-3-specific antibody.

Techniques Used: Binding Assay, Recombinant, Expressing, Incubation

22) Product Images from "Inflammatory Th1 and Th17 cells in small intestine are each driven by functionally-specialized dendritic cells with distinct requirements for MyD88"

Article Title: Inflammatory Th1 and Th17 cells in small intestine are each driven by functionally-specialized dendritic cells with distinct requirements for MyD88

Journal: Cell reports

doi: 10.1016/j.celrep.2016.09.091

In small intestine inflammation CD103 + CD11b + DCs and CD103 − CD11b + DCs expand inflammatory Th17 cells ( a ) Representative flow plot of SI-LP DCs in co-housed, littermate A20 cko -DTA − mice and A20 cko -DTA + mice at 9-12 weeks of age. ( b ) Absolute number of each SI-LP DC subset in A20 wt or A20 cko mice, either DTA − or DTA + . ( c-f ) Absolute number of SI-LP CD4 T cells, either IL-17 + ( c ), IFNγ + ( d ), IL-17 + IFNγ + ( e ) or Foxp3 + CD4 T cells ( f ) in co-housed A20 wt or A20 cko mice, additionally either DTA − or DTA + . Results were combined from 3 independent experiments including at least one mouse of each genotype. Each dot represents a mouse. Error bars represent mean ± SEM. *, P
Figure Legend Snippet: In small intestine inflammation CD103 + CD11b + DCs and CD103 − CD11b + DCs expand inflammatory Th17 cells ( a ) Representative flow plot of SI-LP DCs in co-housed, littermate A20 cko -DTA − mice and A20 cko -DTA + mice at 9-12 weeks of age. ( b ) Absolute number of each SI-LP DC subset in A20 wt or A20 cko mice, either DTA − or DTA + . ( c-f ) Absolute number of SI-LP CD4 T cells, either IL-17 + ( c ), IFNγ + ( d ), IL-17 + IFNγ + ( e ) or Foxp3 + CD4 T cells ( f ) in co-housed A20 wt or A20 cko mice, additionally either DTA − or DTA + . Results were combined from 3 independent experiments including at least one mouse of each genotype. Each dot represents a mouse. Error bars represent mean ± SEM. *, P

Techniques Used: Flow Cytometry, Mouse Assay

In A20 cko and A20/Myd88 cko mice, intestinal DCs are phenotypically mature and expand pathological mucosal T cells that cause small intestine inflammation ( a ) Representative flow cytometry gating strategy in A20 wt mice to identify the three bona fide DC populations of SI-LP. ( b ) Expression of A20 mRNA by the indicated SI-LP DC subset or macrophages from wild-type mice, relative to hprt . ( c-f ) Cell surface expression of maturation markers CD80 ( c ), CD86 ( d ) CD40 ( e ) and total cell number ( f ) of SI-LP DCs and macrophages. Cellular expression of maturation markers is represented as mean fluorescence intensity (MFI) relative to that of the same population in wild-type (WT) mice. ( g ) Cell number of SI-LP CD4 + T cells, IFNγ + , IL-17 + , IFNγ + IL-17 + and Foxp3 + CD4 + T cells from mice of the indicated genotype. Data in ( c-g ) was combined from at least 3 independent experiments with mice between 10-14 weeks of age, including at least one mouse of each genotype per experiment. Each dot represents one mouse. ( h ) Organ weights of small intestine from Rag1 -/- mice of the indicated genotypes (n = 9-11 mice of each genotype, aged 12-14 weeks). Error bars represent mean ± SEM, *, P
Figure Legend Snippet: In A20 cko and A20/Myd88 cko mice, intestinal DCs are phenotypically mature and expand pathological mucosal T cells that cause small intestine inflammation ( a ) Representative flow cytometry gating strategy in A20 wt mice to identify the three bona fide DC populations of SI-LP. ( b ) Expression of A20 mRNA by the indicated SI-LP DC subset or macrophages from wild-type mice, relative to hprt . ( c-f ) Cell surface expression of maturation markers CD80 ( c ), CD86 ( d ) CD40 ( e ) and total cell number ( f ) of SI-LP DCs and macrophages. Cellular expression of maturation markers is represented as mean fluorescence intensity (MFI) relative to that of the same population in wild-type (WT) mice. ( g ) Cell number of SI-LP CD4 + T cells, IFNγ + , IL-17 + , IFNγ + IL-17 + and Foxp3 + CD4 + T cells from mice of the indicated genotype. Data in ( c-g ) was combined from at least 3 independent experiments with mice between 10-14 weeks of age, including at least one mouse of each genotype per experiment. Each dot represents one mouse. ( h ) Organ weights of small intestine from Rag1 -/- mice of the indicated genotypes (n = 9-11 mice of each genotype, aged 12-14 weeks). Error bars represent mean ± SEM, *, P

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

Microbiota are required for small intestinal inflammation and expansion of pathological mucosal T cells in A20 cko and A20/Myd88 cko mice ( a ) Organ weights of small intestine from mice of the indicated genotypes, treated with or without broad-spectrum antibiotics (Abx) for 9-10 weeks. Mice from these treatment groups were analyzed for cell number of SI-LP IL-17 + ( b ), IFNγ + ( c ) and IFNγ + IL-17 + CD4 T cells ( d ). Each dot represents one mouse. Results are combined from 4 independent experiments. Error bars show mean ± SEM, **, P
Figure Legend Snippet: Microbiota are required for small intestinal inflammation and expansion of pathological mucosal T cells in A20 cko and A20/Myd88 cko mice ( a ) Organ weights of small intestine from mice of the indicated genotypes, treated with or without broad-spectrum antibiotics (Abx) for 9-10 weeks. Mice from these treatment groups were analyzed for cell number of SI-LP IL-17 + ( b ), IFNγ + ( c ) and IFNγ + IL-17 + CD4 T cells ( d ). Each dot represents one mouse. Results are combined from 4 independent experiments. Error bars show mean ± SEM, **, P

Techniques Used: Mouse Assay

Each SI-LP DC subset possesses unique APC functions, and differential requirements for MyD88 signals SI-LP CD103 + CD11b − DCs were isolated from mice of the indicated genotype and co-cultured 1:1 with naïve OT-II T cells for 4 days. ( a ) Representative flow plots of IFNγ + and IL-17 + T cells in each CD103 + CD11b − DC–T cell co-culture. The percentages of IFNγ + or IL-17 + CD4 T cells ( b ) and ELISA quantification of IFNγ and IL-17 protein ( c ) in co-cultures of CD103 + CD11b − DCs from A20 wt , A20 cko and A20/Myd88 cko mice. OT-II co-cultures with CD103 + CD11b + DCs ( d-f ) or CD103 − CD11b + DCs ( g-i ) were assayed as above. Data are combined from 3 independent experiments. Each dot represents one experiment, or replicates within an experiment, using DCs pooled from at least 3 mice of each genotype, and including all 3 genotypes for each experiment. ( j-k ) CD103 − CD11b + DCs isolated from co-housed Myd88 wt and Myd88 cko mice were co-cultured with naïve OT-II T cells, as above. The percentages ( j ) and total cell number ( k ) of IFNγ + or IL-17 + T cells, or IFNγ or IL-17A protein in culture supernatant ( l ) assayed from two independent experiments, with at least 5 mice per genotype, and including both genotypes for each experiment. Each dot represents replicates within an experiment. Error bars represent mean ± SEM, *, P
Figure Legend Snippet: Each SI-LP DC subset possesses unique APC functions, and differential requirements for MyD88 signals SI-LP CD103 + CD11b − DCs were isolated from mice of the indicated genotype and co-cultured 1:1 with naïve OT-II T cells for 4 days. ( a ) Representative flow plots of IFNγ + and IL-17 + T cells in each CD103 + CD11b − DC–T cell co-culture. The percentages of IFNγ + or IL-17 + CD4 T cells ( b ) and ELISA quantification of IFNγ and IL-17 protein ( c ) in co-cultures of CD103 + CD11b − DCs from A20 wt , A20 cko and A20/Myd88 cko mice. OT-II co-cultures with CD103 + CD11b + DCs ( d-f ) or CD103 − CD11b + DCs ( g-i ) were assayed as above. Data are combined from 3 independent experiments. Each dot represents one experiment, or replicates within an experiment, using DCs pooled from at least 3 mice of each genotype, and including all 3 genotypes for each experiment. ( j-k ) CD103 − CD11b + DCs isolated from co-housed Myd88 wt and Myd88 cko mice were co-cultured with naïve OT-II T cells, as above. The percentages ( j ) and total cell number ( k ) of IFNγ + or IL-17 + T cells, or IFNγ or IL-17A protein in culture supernatant ( l ) assayed from two independent experiments, with at least 5 mice per genotype, and including both genotypes for each experiment. Each dot represents replicates within an experiment. Error bars represent mean ± SEM, *, P

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

23) Product Images from "MCAM-expressing CD4+ T cells in Peripheral Blood Secrete IL-17A and are Significantly Elevated in Inflammatory Autoimmune Diseases"

Article Title: MCAM-expressing CD4+ T cells in Peripheral Blood Secrete IL-17A and are Significantly Elevated in Inflammatory Autoimmune Diseases

Journal: Journal of autoimmunity

doi: 10.1016/j.jaut.2011.09.003

Cytokine secretion of CD4 + CD45RO + CD146 + T cells and CD4 + CD45RO + CD146 − T cells Single-cell suspensions from healthy individuals (n=7) were stained and sorted into 2 populations CD4 + CD45RO + CD146 + and CD4 + CD45RO + CD146 − . These subsets were stimulated with CD3/CD28 as described in the material and methods. Secretion of IL-17A (A), IFN-γ (B), IL-8 (C) and IL-6 (D) were measured in culture supernatant using multiplex bead array assays in CD4 + CD146 − or CD4 + CD146 + subsets.
Figure Legend Snippet: Cytokine secretion of CD4 + CD45RO + CD146 + T cells and CD4 + CD45RO + CD146 − T cells Single-cell suspensions from healthy individuals (n=7) were stained and sorted into 2 populations CD4 + CD45RO + CD146 + and CD4 + CD45RO + CD146 − . These subsets were stimulated with CD3/CD28 as described in the material and methods. Secretion of IL-17A (A), IFN-γ (B), IL-8 (C) and IL-6 (D) were measured in culture supernatant using multiplex bead array assays in CD4 + CD146 − or CD4 + CD146 + subsets.

Techniques Used: Staining, Multiplex Assay

Intracellular cytokines in freshly isolated CD4 + CD45RO + CD146 + and CD4 + CD45RO + CD146 − T lymphocytes PBMCs from healthy individuals (n=7) and patients with various inflammatory diseases (n=4) were stimulated using PMA/ionomycin and stained for intracellular cytokines IL-17A and IFN-γ. Data shown are the percentages of IFN-γ (left panel) and IL-17A (right panel) positive cells in CD4 + CD45RO + CD146 + and CD4 + CD45RO + CD146 − subsets of healthy donors (H) and patients (D).
Figure Legend Snippet: Intracellular cytokines in freshly isolated CD4 + CD45RO + CD146 + and CD4 + CD45RO + CD146 − T lymphocytes PBMCs from healthy individuals (n=7) and patients with various inflammatory diseases (n=4) were stimulated using PMA/ionomycin and stained for intracellular cytokines IL-17A and IFN-γ. Data shown are the percentages of IFN-γ (left panel) and IL-17A (right panel) positive cells in CD4 + CD45RO + CD146 + and CD4 + CD45RO + CD146 − subsets of healthy donors (H) and patients (D).

Techniques Used: Isolation, Staining

Intracellular cytokine expression of CD4 + CD45RO + CD146 + T cells and CD4 + CD45RO + CD146 − T cells in presence of Th17 polarizing conditions Single-cell suspensions from healthy individuals (n≥3) were stained and sorted into 2 populations: CD4 + CD45RO + CD146 + and CD4 + CD45RO + CD146 − . These sorted populations were subjected to different in vitro stimulations and the expression of IL-17A (x axis) and IFN-γ (y axis) was measured. CD4 + CD45RO + CD146 − T cells are shown in the left panel and CD4 + CD45RO + CD146 + T cells are shown in the center panel, and summary data are shown in the right panel. The condition of stimulations were: (A) plate bound CD3 and CD28 (B) plate bound CD3 and CD28 and IL-1β, (C) plate bound CD3 and CD28 and IL-23, (D) plate bound CD3 and CD28 and IL-1β+IL-23 and (E) plate bound CD3 and CD28 and IL-1β+IL-23+TGF-β+IL-6.
Figure Legend Snippet: Intracellular cytokine expression of CD4 + CD45RO + CD146 + T cells and CD4 + CD45RO + CD146 − T cells in presence of Th17 polarizing conditions Single-cell suspensions from healthy individuals (n≥3) were stained and sorted into 2 populations: CD4 + CD45RO + CD146 + and CD4 + CD45RO + CD146 − . These sorted populations were subjected to different in vitro stimulations and the expression of IL-17A (x axis) and IFN-γ (y axis) was measured. CD4 + CD45RO + CD146 − T cells are shown in the left panel and CD4 + CD45RO + CD146 + T cells are shown in the center panel, and summary data are shown in the right panel. The condition of stimulations were: (A) plate bound CD3 and CD28 (B) plate bound CD3 and CD28 and IL-1β, (C) plate bound CD3 and CD28 and IL-23, (D) plate bound CD3 and CD28 and IL-1β+IL-23 and (E) plate bound CD3 and CD28 and IL-1β+IL-23+TGF-β+IL-6.

Techniques Used: Expressing, Staining, In Vitro

24) Product Images from "Endocytosis of Albumin by Podocytes Elicits an Inflammatory Response and Induces Apoptotic Cell Death"

Article Title: Endocytosis of Albumin by Podocytes Elicits an Inflammatory Response and Induces Apoptotic Cell Death

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054817

Albumin exposure modulates pro-inflammatory cytokine expression and release in human podocytes. A , Time course of IL-1β RNA levels in podocytes treated with 5 mg/ml recombinant low endotoxin human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.0002 compared to dextran controls. B , Time course of TNF RNA expression in podocytes treated with 5 mg/ml human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.01 compared to dextran control. C , IL-6 RNA levels in podocytes treated with 5 mg/ml human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.008 compared to dextran control. D, Amount of IL-1β normalized to total cellular protein released into the medium after treatment of podocytes with 5 mg/ml recombinant human albumin (closed bars) or 5 mg/ml dextran (open bars) for varying amounts of time. * denotes P = 0.005 compared to dextran treated controls. E , Amount of TNF normalized to total cellular protein released into the medium by podocytes after treatment with albumin (closed bars) or dextran (open bars) for varying amounts of time. * denotes P = 0.003 compared to dextran treated control cells. F , Levels of IL-6 normalized to total cellular protein released into the medium by podocytes treated with albumin (closed bars) or dextran (open bars) for varying amounts of time. * denotes P
Figure Legend Snippet: Albumin exposure modulates pro-inflammatory cytokine expression and release in human podocytes. A , Time course of IL-1β RNA levels in podocytes treated with 5 mg/ml recombinant low endotoxin human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.0002 compared to dextran controls. B , Time course of TNF RNA expression in podocytes treated with 5 mg/ml human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.01 compared to dextran control. C , IL-6 RNA levels in podocytes treated with 5 mg/ml human albumin (closed bars) or 5 mg/ml dextran (open bars). * denotes P = 0.008 compared to dextran control. D, Amount of IL-1β normalized to total cellular protein released into the medium after treatment of podocytes with 5 mg/ml recombinant human albumin (closed bars) or 5 mg/ml dextran (open bars) for varying amounts of time. * denotes P = 0.005 compared to dextran treated controls. E , Amount of TNF normalized to total cellular protein released into the medium by podocytes after treatment with albumin (closed bars) or dextran (open bars) for varying amounts of time. * denotes P = 0.003 compared to dextran treated control cells. F , Levels of IL-6 normalized to total cellular protein released into the medium by podocytes treated with albumin (closed bars) or dextran (open bars) for varying amounts of time. * denotes P

Techniques Used: Expressing, Recombinant, RNA Expression

Albumin overload induces proteinuria and upregulates pro-inflammatory cytokine expression in isolated mouse glomeruli. A , Urinary protein normalized to urinary creatinine in mice injected with saline (open bar) or low endotoxin bovine serum albumin (BSA; closed bar; n = 4 animals per group). * denotes P = 0.004 compared to saline injected controls. B , IL-1β expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group). * denotes P = 0.02 compared to controls. C , TNF expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group). * denotes P = 0.02 compared to controls. D , IL-6 expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group).
Figure Legend Snippet: Albumin overload induces proteinuria and upregulates pro-inflammatory cytokine expression in isolated mouse glomeruli. A , Urinary protein normalized to urinary creatinine in mice injected with saline (open bar) or low endotoxin bovine serum albumin (BSA; closed bar; n = 4 animals per group). * denotes P = 0.004 compared to saline injected controls. B , IL-1β expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group). * denotes P = 0.02 compared to controls. C , TNF expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group). * denotes P = 0.02 compared to controls. D , IL-6 expression in isolated glomeruli from mice injected with saline (open bar) or BSA (closed bar; n = 4 animals per group).

Techniques Used: Expressing, Isolation, Mouse Assay, Injection

25) Product Images from "Lithium Controls Central Nervous System Autoimmunity through Modulation of IFN-? Signaling"

Article Title: Lithium Controls Central Nervous System Autoimmunity through Modulation of IFN-? Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0052658

Lithium inhibits Th1-induced, but not Th17-induced EAE. (A) Th1 cells (4–6×10 6 ), (B) Th17 cells (4–6×10 6 ), or (C and D) IL-17F-Thy1.1 cells (3–6×10 5 ) were adoptively transferred i.v. into naïve untreated or lithium–treated recipient mice to induce EAE (mean ± SEM, n = 8–11 mice/group, * p
Figure Legend Snippet: Lithium inhibits Th1-induced, but not Th17-induced EAE. (A) Th1 cells (4–6×10 6 ), (B) Th17 cells (4–6×10 6 ), or (C and D) IL-17F-Thy1.1 cells (3–6×10 5 ) were adoptively transferred i.v. into naïve untreated or lithium–treated recipient mice to induce EAE (mean ± SEM, n = 8–11 mice/group, * p

Techniques Used: Mouse Assay

26) Product Images from "Enhanced Insight into the Autoimmune Component of Glaucoma: IgG Autoantibody Accumulation and Pro-Inflammatory Conditions in Human Glaucomatous Retina"

Article Title: Enhanced Insight into the Autoimmune Component of Glaucoma: IgG Autoantibody Accumulation and Pro-Inflammatory Conditions in Human Glaucomatous Retina

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057557

Levels of pro-inflammatory cytokines in glaucomatous retinal tissue. Figure 1 depicts the altered concentrations of the pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, IL-8 and IFN-γ in detail. The arrowheads indicate the relative intensity (U) on the y-axis of each individual subject as sample means and the black bar summarizes the group means. All investigated pro-inflammatory cytokines were up-regulated in the glaucomatous group (glaucoma) compared to the healthy subjects (ctrl). (In some box-plots, the arrowheads of sample means overlaid.).
Figure Legend Snippet: Levels of pro-inflammatory cytokines in glaucomatous retinal tissue. Figure 1 depicts the altered concentrations of the pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, IL-8 and IFN-γ in detail. The arrowheads indicate the relative intensity (U) on the y-axis of each individual subject as sample means and the black bar summarizes the group means. All investigated pro-inflammatory cytokines were up-regulated in the glaucomatous group (glaucoma) compared to the healthy subjects (ctrl). (In some box-plots, the arrowheads of sample means overlaid.).

Techniques Used:

27) Product Images from "Involvement of DNA-PKcs in the IL-6 and IL-12 Response to CpG-ODN Is Mediated by Its Interaction with TRAF6 in Dendritic Cells"

Article Title: Involvement of DNA-PKcs in the IL-6 and IL-12 Response to CpG-ODN Is Mediated by Its Interaction with TRAF6 in Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0058072

DNA-PKcs is involved in the IL-6 and IL-12 response to CpG-ODN. Bone marrow-derived dendritic cells (DCs) from WT, DNA-PKcs −/− , TLR9 −/− and DNA-PKcs −/−/ TLR9 −/− mice were harvested based on their natural status: adhesion and suspension. ( A ). Combined DCs (adhesion and suspension) at day 7.5 were subjected to flow cytometry. The levels of CD11b, CD11c and MHC-II expression on DCs were determined. ( B–M ). Adhesion or suspension DCs at day 6.5 were seeded in 96-well plates at 1×10 5 /well in triplicate, and then treated with CpG-ODN (CpG, 0.2, 0.73, 2.2 or 7.3 µg/ml), LPS (0.1 µg/ml), R848 (0.125 µg/ml), or left untreated for indicated time durations. The levels of IL-6 ( B–G ) and IL-12p40 ( H–M ) were determined by ELISA using IL-6 or IL-12p40 ELISA kits. Similar results were obtained in at least 3 independent experiments. Note: bars represent the average of triplicates ± SD. * p
Figure Legend Snippet: DNA-PKcs is involved in the IL-6 and IL-12 response to CpG-ODN. Bone marrow-derived dendritic cells (DCs) from WT, DNA-PKcs −/− , TLR9 −/− and DNA-PKcs −/−/ TLR9 −/− mice were harvested based on their natural status: adhesion and suspension. ( A ). Combined DCs (adhesion and suspension) at day 7.5 were subjected to flow cytometry. The levels of CD11b, CD11c and MHC-II expression on DCs were determined. ( B–M ). Adhesion or suspension DCs at day 6.5 were seeded in 96-well plates at 1×10 5 /well in triplicate, and then treated with CpG-ODN (CpG, 0.2, 0.73, 2.2 or 7.3 µg/ml), LPS (0.1 µg/ml), R848 (0.125 µg/ml), or left untreated for indicated time durations. The levels of IL-6 ( B–G ) and IL-12p40 ( H–M ) were determined by ELISA using IL-6 or IL-12p40 ELISA kits. Similar results were obtained in at least 3 independent experiments. Note: bars represent the average of triplicates ± SD. * p

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

28) Product Images from "Ethyl Pyruvate Ameliorates Hepatic Ischemia-Reperfusion Injury by Inhibiting Intrinsic Pathway of Apoptosis and Autophagy"

Article Title: Ethyl Pyruvate Ameliorates Hepatic Ischemia-Reperfusion Injury by Inhibiting Intrinsic Pathway of Apoptosis and Autophagy

Journal: Mediators of Inflammation

doi: 10.1155/2013/461536

In the condition of hepatic ischemia-reperfusion injury, HMGB1 is predominantly released by stressed hepatocytes, which actively secrete HMGB1 into the circulation. After release, HMGB1, as a proinflammatory cytokine, combined with TLR4 was expressed on the surface of hepatocytes or other nonparenchymal cells. NF- κ B signal pathway is further activated to promote cytokine release, such as TNF- α and IL-6. Under the stimulation of TNF- α and IL-6, intrinsic pathway of apoptosis is initiated, exhibited as the upregulation of Bax and downregulation of Bcl-2. On the other hand, with the translocation of HMGB1 into cytoplasm from nuclei, HMGB1 competitively combined with Beclin-1 to promote the level of autophagy through representing the site of Bcl-2, which can maintain the inactive status of autophagy. Ethyl pyruvate successfully inhibits the expression and translocation of HMGB1 in stressed cells under the condition of hepatic I/R injury and further downregulates intrinsic pathway of apoptosis and autophagy to ameliorate hepatic I/R injury.
Figure Legend Snippet: In the condition of hepatic ischemia-reperfusion injury, HMGB1 is predominantly released by stressed hepatocytes, which actively secrete HMGB1 into the circulation. After release, HMGB1, as a proinflammatory cytokine, combined with TLR4 was expressed on the surface of hepatocytes or other nonparenchymal cells. NF- κ B signal pathway is further activated to promote cytokine release, such as TNF- α and IL-6. Under the stimulation of TNF- α and IL-6, intrinsic pathway of apoptosis is initiated, exhibited as the upregulation of Bax and downregulation of Bcl-2. On the other hand, with the translocation of HMGB1 into cytoplasm from nuclei, HMGB1 competitively combined with Beclin-1 to promote the level of autophagy through representing the site of Bcl-2, which can maintain the inactive status of autophagy. Ethyl pyruvate successfully inhibits the expression and translocation of HMGB1 in stressed cells under the condition of hepatic I/R injury and further downregulates intrinsic pathway of apoptosis and autophagy to ameliorate hepatic I/R injury.

Techniques Used: Translocation Assay, Expressing

29) Product Images from "Expression of the ARPC4 Subunit of Human Arp2/3 Severely Affects Mycobacterium tuberculosis Growth and Suppresses Immunogenic Response in Murine Macrophages"

Article Title: Expression of the ARPC4 Subunit of Human Arp2/3 Severely Affects Mycobacterium tuberculosis Growth and Suppresses Immunogenic Response in Murine Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069949

Activation of infected macrophages and inter-compartmental localization of mycobacteria within A. The plots depict reduced activation of H37RV/ARPC4 infected macrophages. a) Percentage of cells expressing PD1 among CD11B+ cells is shown in the dot plots with mean±STDEV. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B and PD1 antibodies and samples were acquired by flow cytometry. Data shown here are representative of three independent experiments. b) Cell death in H37Rv and H37Rv/ARPC4 Mtb infected macrophages. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains in 1∶10 ratio, were surface-stained with anti-CD11B, CD11C antibodies followed by PI staining for 20 minutes prior to acquisition by flow cytometry to assess cell death in infected macrophages. The percentage of cells expressing PI among CD11B±cells is shown with mean±STDEV. Data shown here are representative of three independent experiments. c) Expression of macrophage activation markers. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B, CD11C, MHCII, CD14, CD54, CD80, CD86 and CD69 antibodies and samples were acquired by flow cytometry. CD11B+ cells were gated for expression of MHCII, CD14, CD54, CD80 and CD86 and the percentage of cells expressing these markers are shown in the histogram overlay plots with mean±STDEV. Data shown here are representative of three independent experiments. B. Confocal Microscopy images showing that H37Rv/ARPC4 mycobacterial strain is less infectious and persistent in macrophage. FITC-labelled mycobacterial cells (green) were used for infecting macrophages (red) at an MOI of 5. At specific time-points, the cells were fixed, permeabilized and stained with anti-LAMP1 antibodies, followed by Alexa Fluor 594 goat anti-rat antibodies. (a)- (d) shows the infected macrophage cells at 0, 24, 48 and 72 hours' time-points post-infection, respectively. (e) and (f) are the graphical representation of the percentage of macrophage cells infected with FITC-labeled mycobacteria at different time-points and the percentage of bacteria localized in lysosomes in the infected cells, respectively. Experiments were repeated thrice and similar results were obtained.
Figure Legend Snippet: Activation of infected macrophages and inter-compartmental localization of mycobacteria within A. The plots depict reduced activation of H37RV/ARPC4 infected macrophages. a) Percentage of cells expressing PD1 among CD11B+ cells is shown in the dot plots with mean±STDEV. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B and PD1 antibodies and samples were acquired by flow cytometry. Data shown here are representative of three independent experiments. b) Cell death in H37Rv and H37Rv/ARPC4 Mtb infected macrophages. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains in 1∶10 ratio, were surface-stained with anti-CD11B, CD11C antibodies followed by PI staining for 20 minutes prior to acquisition by flow cytometry to assess cell death in infected macrophages. The percentage of cells expressing PI among CD11B±cells is shown with mean±STDEV. Data shown here are representative of three independent experiments. c) Expression of macrophage activation markers. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B, CD11C, MHCII, CD14, CD54, CD80, CD86 and CD69 antibodies and samples were acquired by flow cytometry. CD11B+ cells were gated for expression of MHCII, CD14, CD54, CD80 and CD86 and the percentage of cells expressing these markers are shown in the histogram overlay plots with mean±STDEV. Data shown here are representative of three independent experiments. B. Confocal Microscopy images showing that H37Rv/ARPC4 mycobacterial strain is less infectious and persistent in macrophage. FITC-labelled mycobacterial cells (green) were used for infecting macrophages (red) at an MOI of 5. At specific time-points, the cells were fixed, permeabilized and stained with anti-LAMP1 antibodies, followed by Alexa Fluor 594 goat anti-rat antibodies. (a)- (d) shows the infected macrophage cells at 0, 24, 48 and 72 hours' time-points post-infection, respectively. (e) and (f) are the graphical representation of the percentage of macrophage cells infected with FITC-labeled mycobacteria at different time-points and the percentage of bacteria localized in lysosomes in the infected cells, respectively. Experiments were repeated thrice and similar results were obtained.

Techniques Used: Activation Assay, Infection, Expressing, Isolation, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Confocal Microscopy, Labeling

30) Product Images from "Identification of Potential Cytokine Pathways for Therapeutic Intervention in Murine Primary Biliary Cirrhosis"

Article Title: Identification of Potential Cytokine Pathways for Therapeutic Intervention in Murine Primary Biliary Cirrhosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074225

Inflammatory cytokine production in mice immunized with 2OA-BSA. (A) Serum levels of IL-17A, IFN-γ, TNF-α and IL-6 in WT, IL-17A −/− , IL-17F −/− and IL-22 −/− mice, respectively, at 2 weeks following 2OA-BSA immunization. (B) Production of IFN-γ in supernatant fluids of cultured splenic and hepatic MNCs (n = 4) with anti-CD3/CD28 mAbs at 3 days. (C) The level of inflammatory cytokines in extracted liver protein from WT, IL-17A −/− , IL-17F −/− and IL-22 −/− mice, respectively. Each group n = 8. *p
Figure Legend Snippet: Inflammatory cytokine production in mice immunized with 2OA-BSA. (A) Serum levels of IL-17A, IFN-γ, TNF-α and IL-6 in WT, IL-17A −/− , IL-17F −/− and IL-22 −/− mice, respectively, at 2 weeks following 2OA-BSA immunization. (B) Production of IFN-γ in supernatant fluids of cultured splenic and hepatic MNCs (n = 4) with anti-CD3/CD28 mAbs at 3 days. (C) The level of inflammatory cytokines in extracted liver protein from WT, IL-17A −/− , IL-17F −/− and IL-22 −/− mice, respectively. Each group n = 8. *p

Techniques Used: Mouse Assay, Cell Culture

Pathological changes in liver of mice immunized with 2OA-BSA. (A) Representative H E staining profiles of liver from IL-17A −/− , IL-17F −/− and IL-22 −/− mice 8 weeks after immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed. IL-17F −/− demonstrating more epithelioid granulomas (red arrowhead) in liver compared to IL-17A −/− and IL-22 −/− mice. (B) Scoring of portal inflammation and bile duct damage in liver from WT (n = 25), IL-17A −/− (n = 19), IL-17F −/− (n = 13) and IL-22 −/− (n = 14) mice.
Figure Legend Snippet: Pathological changes in liver of mice immunized with 2OA-BSA. (A) Representative H E staining profiles of liver from IL-17A −/− , IL-17F −/− and IL-22 −/− mice 8 weeks after immunization. Portal inflammatory changes with interlobular bile duct damage (red arrow) were observed. IL-17F −/− demonstrating more epithelioid granulomas (red arrowhead) in liver compared to IL-17A −/− and IL-22 −/− mice. (B) Scoring of portal inflammation and bile duct damage in liver from WT (n = 25), IL-17A −/− (n = 19), IL-17F −/− (n = 13) and IL-22 −/− (n = 14) mice.

Techniques Used: Mouse Assay, Staining

31) Product Images from "BHBA Suppresses LPS-Induced Inflammation in BV-2 Cells by Inhibiting NF-κB Activation"

Article Title: BHBA Suppresses LPS-Induced Inflammation in BV-2 Cells by Inhibiting NF-κB Activation

Journal: Mediators of Inflammation

doi: 10.1155/2014/983401

Effects of BHBA on LPS-induced the gene expression and protein secretion of proinflammatory cytokines (TNF- α , IL-1 β , and IL-6) in BV-2 cells. Cells were pretreated with BHBA (0.5, 1.0, and 1.5 mM) 1 h prior to incubation of LPS (1 μ g/mL) for 4 h (mRNA) or 24 h (protein). Proteins and mRNA of TNF- α ((a), (b)), IL-1 β ((c), (d)), and IL-6 ((e), (f)) were determined by ELISA and quantitative real-time PCR as described in Section 2 . The relative mRNA level was normalized to β -actin mRNA. Results are expressed as mean ± SD for each group from three independent experiments. # Significantly different when compared with control alone, P
Figure Legend Snippet: Effects of BHBA on LPS-induced the gene expression and protein secretion of proinflammatory cytokines (TNF- α , IL-1 β , and IL-6) in BV-2 cells. Cells were pretreated with BHBA (0.5, 1.0, and 1.5 mM) 1 h prior to incubation of LPS (1 μ g/mL) for 4 h (mRNA) or 24 h (protein). Proteins and mRNA of TNF- α ((a), (b)), IL-1 β ((c), (d)), and IL-6 ((e), (f)) were determined by ELISA and quantitative real-time PCR as described in Section 2 . The relative mRNA level was normalized to β -actin mRNA. Results are expressed as mean ± SD for each group from three independent experiments. # Significantly different when compared with control alone, P

Techniques Used: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Effects of BHBA mediated by GPR109A. BV-2 cells were pretreated with vehicle or PTX for 1 h. Medium from each condition was removed and replaced with vehicle, BHBA (1.5 mM), PTX (100 ng/mL), LPS (1 μ g/mL), BHBA + LPS, or BHBA + LPS + PTX. BV-2 cells were sampled at 4 h. The mRNA of proinflammatory enzymes and proinflammatory cytokines were determined by quantitative real-time PCR. Attenuation by BHBA of induced mRNA of iNOS (a), COX-2 (b), TNF- α (c), IL-1 β (d), and IL-6 (e) from BV-2 cells; this effect is abolished with pretreatment with PTX (** P
Figure Legend Snippet: Effects of BHBA mediated by GPR109A. BV-2 cells were pretreated with vehicle or PTX for 1 h. Medium from each condition was removed and replaced with vehicle, BHBA (1.5 mM), PTX (100 ng/mL), LPS (1 μ g/mL), BHBA + LPS, or BHBA + LPS + PTX. BV-2 cells were sampled at 4 h. The mRNA of proinflammatory enzymes and proinflammatory cytokines were determined by quantitative real-time PCR. Attenuation by BHBA of induced mRNA of iNOS (a), COX-2 (b), TNF- α (c), IL-1 β (d), and IL-6 (e) from BV-2 cells; this effect is abolished with pretreatment with PTX (** P

Techniques Used: Real-time Polymerase Chain Reaction

32) Product Images from "Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M"

Article Title: Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

Journal: Respiratory Research

doi: 10.1186/s12931-014-0164-4

OSM stimulation with IL-17A, IL-4 or IL-13 induces MCP-1/CCL2 and IL-6. HASMC cultures were prepared and stimulated as in Figure 1 with the indicated cytokines (in quadruplicates) and concentrations of: (A) IL-17A up to 10 ng/ml and in combination with 0, 0.01, 0.1, 1, and 10 ng/ml OSM, (B) IL-4 at 0 to 10 ng/ml and in combination with 0 or 0.5 ng/ml OSM, (C) IL-13 at 0 to 10 ng/ml and in combination with 0 or 0.5 ng/ml OSM, (D) IFNγ at 0 to 10 ng/ml and in combination with 0 or 0.5 ng/ml OSM. Data shown are from one of two individual cell lines that showed identical trends. 24-hour supernatants were collected and cytokine concentrations were quantified by ELISA for MCP-1/CCL-2 (left panels) or IL-6 (right panels). * p
Figure Legend Snippet: OSM stimulation with IL-17A, IL-4 or IL-13 induces MCP-1/CCL2 and IL-6. HASMC cultures were prepared and stimulated as in Figure 1 with the indicated cytokines (in quadruplicates) and concentrations of: (A) IL-17A up to 10 ng/ml and in combination with 0, 0.01, 0.1, 1, and 10 ng/ml OSM, (B) IL-4 at 0 to 10 ng/ml and in combination with 0 or 0.5 ng/ml OSM, (C) IL-13 at 0 to 10 ng/ml and in combination with 0 or 0.5 ng/ml OSM, (D) IFNγ at 0 to 10 ng/ml and in combination with 0 or 0.5 ng/ml OSM. Data shown are from one of two individual cell lines that showed identical trends. 24-hour supernatants were collected and cytokine concentrations were quantified by ELISA for MCP-1/CCL-2 (left panels) or IL-6 (right panels). * p

Techniques Used: Enzyme-linked Immunosorbent Assay

Effects of STAT3 pharmacologic inhibition on HASMC responses. HASMC cultures were prepared and stimulated, as previous, with the indicated cytokines IL-4, IL-13, or IL-17A at 5 ng/ml in the absence or presence of 1 ng/ml OSM. Parallel cultures were stimulated in the presence of either (A) 2.5 uM Stattic (black bars), (B) 10 uM SB203580 (dashed lines) or (C) 5 uM Akt X (white bars). Cell lines were stimulated for 18 hours and cell culture supernatants were collected and assessed by ELISA for levels of MCP-1/CCL-2 (left panel) and IL-6 (right panels). * p
Figure Legend Snippet: Effects of STAT3 pharmacologic inhibition on HASMC responses. HASMC cultures were prepared and stimulated, as previous, with the indicated cytokines IL-4, IL-13, or IL-17A at 5 ng/ml in the absence or presence of 1 ng/ml OSM. Parallel cultures were stimulated in the presence of either (A) 2.5 uM Stattic (black bars), (B) 10 uM SB203580 (dashed lines) or (C) 5 uM Akt X (white bars). Cell lines were stimulated for 18 hours and cell culture supernatants were collected and assessed by ELISA for levels of MCP-1/CCL-2 (left panel) and IL-6 (right panels). * p

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

OSM and other gp130 cytokine stimulation and HASMC responses. (A-C) HASMC cultures were prepared and stimulated as in Figure 1 with the indicated cytokines and concentrations of 10 ng/ml for each. Data shown are from one of two individual cell lines that showed identical trends. 24-hour culture supernatants were assessed by ELISA for levels of MCP-1/CCL-2 (A), IL-6 (B) and eotaxin-1 (C) . *** p
Figure Legend Snippet: OSM and other gp130 cytokine stimulation and HASMC responses. (A-C) HASMC cultures were prepared and stimulated as in Figure 1 with the indicated cytokines and concentrations of 10 ng/ml for each. Data shown are from one of two individual cell lines that showed identical trends. 24-hour culture supernatants were assessed by ELISA for levels of MCP-1/CCL-2 (A), IL-6 (B) and eotaxin-1 (C) . *** p

Techniques Used: Enzyme-linked Immunosorbent Assay

HASMC responses to OSM and IL-17A. HASMC were cultured and prepared for stimulation as described in methods, where each treatment is completed in quadruplicates within each cell line. Cells were then stimulated in medium supplemented with 2% FBS and 0–50 ng/ml of the indicated gp130 cytokines. Protein concentrations in 24-hour supernatants were determined by ELISA for (A) MCP-1/CCL-2 (Left panel) and IL-6 (Right panel). *p
Figure Legend Snippet: HASMC responses to OSM and IL-17A. HASMC were cultured and prepared for stimulation as described in methods, where each treatment is completed in quadruplicates within each cell line. Cells were then stimulated in medium supplemented with 2% FBS and 0–50 ng/ml of the indicated gp130 cytokines. Protein concentrations in 24-hour supernatants were determined by ELISA for (A) MCP-1/CCL-2 (Left panel) and IL-6 (Right panel). *p

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

33) Product Images from "Zinc finger protein Zfp335 is required for the formation of the naïve T cell compartment"

Article Title: Zinc finger protein Zfp335 is required for the formation of the naïve T cell compartment

Journal: eLife

doi: 10.7554/eLife.03549

Impaired late-stage SP thymocyte development and early post-thymic peripheral T cell maturation in blt/blt mice. ( A ) Turnover of DN, DP, semi-mature, and mature CD4SP thymocytes assessed after 2–4 days of continuous in vivo BrdU labeling (mean ± s.d., n = 4). ( B ) Input-normalized fraction of donor CD45.2 + CD4 + CD62L hi CD44 lo naïve T cells (closed symbols) or CD4SP thymocytes (open symbols) that were blt/blt , recovered from spleen at indicated time points after transfer of blt/blt and control peripheral lymphocytes or thymocytes to lymphoreplete CD45.1 + hosts (mean ± s.d., n = 2–4). ( C ) (left) GFP signal in CD62L hi CD44 lo CD4 + and CD8 + T cells from spleen of Rag1-GFP transgenic blt /+ vs. blt / blt mice. (right) Percentage of GFP hi naïve T cells, gated as shown in histograms (mean ± s.d., n = 6–8). ( D ) Percentage of total naïve T cells, GFP hi RTEs and GFP lo mature naïve (MN) T cells from spleens of blt/blt mice relative to matched blt /+ littermate controls. Data obtained from mice analyzed between 7 to 10 weeks of age (mean ± s.d., n = 10). DOI: http://dx.doi.org/10.7554/eLife.03549.011
Figure Legend Snippet: Impaired late-stage SP thymocyte development and early post-thymic peripheral T cell maturation in blt/blt mice. ( A ) Turnover of DN, DP, semi-mature, and mature CD4SP thymocytes assessed after 2–4 days of continuous in vivo BrdU labeling (mean ± s.d., n = 4). ( B ) Input-normalized fraction of donor CD45.2 + CD4 + CD62L hi CD44 lo naïve T cells (closed symbols) or CD4SP thymocytes (open symbols) that were blt/blt , recovered from spleen at indicated time points after transfer of blt/blt and control peripheral lymphocytes or thymocytes to lymphoreplete CD45.1 + hosts (mean ± s.d., n = 2–4). ( C ) (left) GFP signal in CD62L hi CD44 lo CD4 + and CD8 + T cells from spleen of Rag1-GFP transgenic blt /+ vs. blt / blt mice. (right) Percentage of GFP hi naïve T cells, gated as shown in histograms (mean ± s.d., n = 6–8). ( D ) Percentage of total naïve T cells, GFP hi RTEs and GFP lo mature naïve (MN) T cells from spleens of blt/blt mice relative to matched blt /+ littermate controls. Data obtained from mice analyzed between 7 to 10 weeks of age (mean ± s.d., n = 10). DOI: http://dx.doi.org/10.7554/eLife.03549.011

Techniques Used: Mouse Assay, In Vivo, Labeling, Transgenic Assay

Normal expression of IL-7 receptor and Bcl2 family members. ( A ) Normalized expression levels of indicated Bcl2 family genes from Affymetrix array analysis of mRNA from sorted WT and blt / blt mature CD4SP thymocytes (mean ± s.d., n = 3). ( B ) Quantitative RT-PCR analysis of Il7r transcript in FACS-purified mature SP thymocytes and naïve T cells (mean ± s.d., n = 3). ( C ) Surface expression of IL-7Rα on WT (CD45.1 + CD45.2 + ) and blt / blt (CD45.2 + ) mature SP thymocytes and naïve T cells from mixed chimeras. Data are representative of eight mice. DOI: http://dx.doi.org/10.7554/eLife.03549.014
Figure Legend Snippet: Normal expression of IL-7 receptor and Bcl2 family members. ( A ) Normalized expression levels of indicated Bcl2 family genes from Affymetrix array analysis of mRNA from sorted WT and blt / blt mature CD4SP thymocytes (mean ± s.d., n = 3). ( B ) Quantitative RT-PCR analysis of Il7r transcript in FACS-purified mature SP thymocytes and naïve T cells (mean ± s.d., n = 3). ( C ) Surface expression of IL-7Rα on WT (CD45.1 + CD45.2 + ) and blt / blt (CD45.2 + ) mature SP thymocytes and naïve T cells from mixed chimeras. Data are representative of eight mice. DOI: http://dx.doi.org/10.7554/eLife.03549.014

Techniques Used: Expressing, Quantitative RT-PCR, FACS, Purification, Mouse Assay

Mice heterozygous for the bloto mutation do not exhibit a T cell phenotype. ( A ) Number of CD4 + and CD8 + naïve T cells from spleens of WT ( n = 6) and blt /+ ( n = 4) mice. ( B ) Ratio of blt /+ (CD45.2 + ) vs WT (CD45.1 + CD45.2 + ) cells in splenic NK cells and indicated thymocyte and T cell populations from irradiation chimeras reconstituted with a mix of blt /+ and WT bone marrow as in Figure 1F . DOI: http://dx.doi.org/10.7554/eLife.03549.005
Figure Legend Snippet: Mice heterozygous for the bloto mutation do not exhibit a T cell phenotype. ( A ) Number of CD4 + and CD8 + naïve T cells from spleens of WT ( n = 6) and blt /+ ( n = 4) mice. ( B ) Ratio of blt /+ (CD45.2 + ) vs WT (CD45.1 + CD45.2 + ) cells in splenic NK cells and indicated thymocyte and T cell populations from irradiation chimeras reconstituted with a mix of blt /+ and WT bone marrow as in Figure 1F . DOI: http://dx.doi.org/10.7554/eLife.03549.005

Techniques Used: Mouse Assay, Mutagenesis, Irradiation

34) Product Images from "Interferon-β Suppresses Murine Th1 Cell Function in the Absence of Antigen-Presenting Cells"

Article Title: Interferon-β Suppresses Murine Th1 Cell Function in the Absence of Antigen-Presenting Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124802

IFN-β suppresses the encephalitogenic potential of Th1 cells. CD4 + CD62L hi T cells were isolated from female 2D2 mouse spleens and lymph nodes, and were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions, in the presence or absence of 100 U mL -1 of IFN-β, for 5 days. They were then transferred to female, 6-week old, Rag1-/- mice (5x10 6 cells/mouse). Recipient mice were monitored for clinical signs of EAE. n = 5 Th1, n = 4 Th1+IFN-β. Right graph, linear regression curves of the disease courses. The slopes are significantly different between the disease courses (p
Figure Legend Snippet: IFN-β suppresses the encephalitogenic potential of Th1 cells. CD4 + CD62L hi T cells were isolated from female 2D2 mouse spleens and lymph nodes, and were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions, in the presence or absence of 100 U mL -1 of IFN-β, for 5 days. They were then transferred to female, 6-week old, Rag1-/- mice (5x10 6 cells/mouse). Recipient mice were monitored for clinical signs of EAE. n = 5 Th1, n = 4 Th1+IFN-β. Right graph, linear regression curves of the disease courses. The slopes are significantly different between the disease courses (p

Techniques Used: Isolation, Mouse Assay

IFN-β suppresses cytokine secretion from Th1 cells. A. CD4 + CD62L hi T cells were sorted from the spleens and lymph nodes of C57BL6/J mice and were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions, with the indicated concentrations of IFN-β, for 5 days. Cells were restimulated for 48 hours under Th1 conditions with the same concentration of IFN-β as at the initial stimulation. A. Generation of IFN-γ and IL-2 was assessed by intracellular cytokine staining and flow cytometry. Representative of three experiments. B. Expression of Tim-3 and PD-1 were assessed by flow cytometry. Representative of three experiments. C. Expression of T-bet was assessed by flow cytometry. Representative data from one of three mice assessed individually. Solid line with open histogram, Th1; dashed line with open histogram, Th1 + 10 U mL -1 IFN-β; dotted line with open histogram, Th1 + 100 U mL -1 IFN-β; shaded histogram, FMO control. Data in all panels gated on CD4 + cells.
Figure Legend Snippet: IFN-β suppresses cytokine secretion from Th1 cells. A. CD4 + CD62L hi T cells were sorted from the spleens and lymph nodes of C57BL6/J mice and were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions, with the indicated concentrations of IFN-β, for 5 days. Cells were restimulated for 48 hours under Th1 conditions with the same concentration of IFN-β as at the initial stimulation. A. Generation of IFN-γ and IL-2 was assessed by intracellular cytokine staining and flow cytometry. Representative of three experiments. B. Expression of Tim-3 and PD-1 were assessed by flow cytometry. Representative of three experiments. C. Expression of T-bet was assessed by flow cytometry. Representative data from one of three mice assessed individually. Solid line with open histogram, Th1; dashed line with open histogram, Th1 + 10 U mL -1 IFN-β; dotted line with open histogram, Th1 + 100 U mL -1 IFN-β; shaded histogram, FMO control. Data in all panels gated on CD4 + cells.

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

IFN-β regulates Stat1 and Stat4 expression on Th cells in the absence of APCs. CD4 + CD62L hi CD25 - T cells were sorted from the spleens and lymph nodes of C57BL6/J mice. They were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 or Th17 conditions, with the indicated concentrations of IFN-β, for 48 hours, and cell lysates were generated. A. Expression of Stat1 and pStat1(Y701) were assessed by Western blot. Representative of two experiments. B. Expression of Stat4 and pStat4(Y693) were assessed by Western blot. Representative of three experiments. GAPDH, loading control.
Figure Legend Snippet: IFN-β regulates Stat1 and Stat4 expression on Th cells in the absence of APCs. CD4 + CD62L hi CD25 - T cells were sorted from the spleens and lymph nodes of C57BL6/J mice. They were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 or Th17 conditions, with the indicated concentrations of IFN-β, for 48 hours, and cell lysates were generated. A. Expression of Stat1 and pStat1(Y701) were assessed by Western blot. Representative of two experiments. B. Expression of Stat4 and pStat4(Y693) were assessed by Western blot. Representative of three experiments. GAPDH, loading control.

Techniques Used: Expressing, Mouse Assay, Generated, Western Blot

IFN-β suppresses Th1 cell proliferation. A. WT CD4 + CD62L hi T cells were labeled with CFSE and stimulated for 5 days with either soluble anti-CD3 plus irradiated splenocytes (APCs), or with plate-bound anti-CD3+anti-CD28, under Th1 or Th17 conditions, with the indicated concentrations of IFN-β. CFSE dilution was assessed by flow cytometry. Data representative of three experiments. B. WT or IFNAR1-/- CD4 + CD62L hi T cells were labeled with CFSE and stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions with the indicated concentrations of IFN-β for 5 days. CFSE dilution was assessed by flow cytometry. Data representative of two experiments. Gates represent the percentage of cells that underwent at least one division.
Figure Legend Snippet: IFN-β suppresses Th1 cell proliferation. A. WT CD4 + CD62L hi T cells were labeled with CFSE and stimulated for 5 days with either soluble anti-CD3 plus irradiated splenocytes (APCs), or with plate-bound anti-CD3+anti-CD28, under Th1 or Th17 conditions, with the indicated concentrations of IFN-β. CFSE dilution was assessed by flow cytometry. Data representative of three experiments. B. WT or IFNAR1-/- CD4 + CD62L hi T cells were labeled with CFSE and stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions with the indicated concentrations of IFN-β for 5 days. CFSE dilution was assessed by flow cytometry. Data representative of two experiments. Gates represent the percentage of cells that underwent at least one division.

Techniques Used: Labeling, Irradiation, Flow Cytometry, Cytometry

35) Product Images from "Matrix Metalloprotease 9 Mediates Neutrophil Migration into the Airways in Response to Influenza Virus-Induced Toll-Like Receptor Signaling"

Article Title: Matrix Metalloprotease 9 Mediates Neutrophil Migration into the Airways in Response to Influenza Virus-Induced Toll-Like Receptor Signaling

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002641

Morbidity, inflammation, and neutrophil number are increased after infection with high dose influenza virus. (A) Kinetics of weight loss as a percentage of starting weight in C57BL/6 mice infected i.n. with 125 ( light-grey squares ), 1250 ( dark-grey triangles ), or 12500 ( black circles ) EID 50 PR8 virus. Mean ± SEM (n = 5, representative of four independent experiments). (B) Cytokine and chemokine levels in the BAL of uninfected mice (‘control’, clear bars ) or mice infected 6 days earlier with 125 ( light-grey bars ), 1250 ( grey bars ), or 12500 ( dark-grey bars ) EID 50 PR8 virus. Mean ± SEM (n = 3–4, representative of two independent experiments). (C) Histological examination of uninfected lungs (control, left ) or lungs 6 days after infection with 125 ( middle ) or 12500 ( right ) EID 50 PR8 virus. Perfused lungs were fixed in formalin and stained for hematoxylin and eosin (scalebar = 100 µm). Images are representative of multiple mice. b. bronchiole, a. arteriole, av. alveole, and arrowhead. venule. (D) Neutrophil numbers in the BAL and lung of uninfected mice (‘control’, clear bars ) or mice infected 6 days earlier with 125 ( light-grey bars ) or 12500 ( dark-grey bars ) EID 50 PR8 virus. Mean ± SEM (n = 3, representative of two independent experiments). (E) The percentage of blood neutrophils from LysM-GFP mice infected with 125 ( light-grey squares ) or 12500 ( black circles ) EID 50 PR8 virus compared to control (PBS, open squares ) or allantoic fluid at the same dilution as the highest viral dose ( open triangles ). Mean ± SEM (n = 4–5).
Figure Legend Snippet: Morbidity, inflammation, and neutrophil number are increased after infection with high dose influenza virus. (A) Kinetics of weight loss as a percentage of starting weight in C57BL/6 mice infected i.n. with 125 ( light-grey squares ), 1250 ( dark-grey triangles ), or 12500 ( black circles ) EID 50 PR8 virus. Mean ± SEM (n = 5, representative of four independent experiments). (B) Cytokine and chemokine levels in the BAL of uninfected mice (‘control’, clear bars ) or mice infected 6 days earlier with 125 ( light-grey bars ), 1250 ( grey bars ), or 12500 ( dark-grey bars ) EID 50 PR8 virus. Mean ± SEM (n = 3–4, representative of two independent experiments). (C) Histological examination of uninfected lungs (control, left ) or lungs 6 days after infection with 125 ( middle ) or 12500 ( right ) EID 50 PR8 virus. Perfused lungs were fixed in formalin and stained for hematoxylin and eosin (scalebar = 100 µm). Images are representative of multiple mice. b. bronchiole, a. arteriole, av. alveole, and arrowhead. venule. (D) Neutrophil numbers in the BAL and lung of uninfected mice (‘control’, clear bars ) or mice infected 6 days earlier with 125 ( light-grey bars ) or 12500 ( dark-grey bars ) EID 50 PR8 virus. Mean ± SEM (n = 3, representative of two independent experiments). (E) The percentage of blood neutrophils from LysM-GFP mice infected with 125 ( light-grey squares ) or 12500 ( black circles ) EID 50 PR8 virus compared to control (PBS, open squares ) or allantoic fluid at the same dilution as the highest viral dose ( open triangles ). Mean ± SEM (n = 4–5).

Techniques Used: Infection, Mouse Assay, Staining

Depletion of neutrophils abrogates MMP9 secretion after influenza virus infection. Neutrophils were depleted by injecting C57BL/6 mice with 400 µg anti-Ly6G antibody (αLy6G) or isotype control (IgG) one day before infection and every other day thereafter. (A) Depletion of Ly6G+ cells in αLy6G-treated C57BL/6 mice ( bottom panel ) compared to IgG ( top panel ) was verified by flow cytometric analysis 6 days after infection. (B) Kinetics of weight loss as a percentage of starting weight in uninfected (control, open symbols ) and infected ( closed symbols ) mice treated with the αLy6G ( squares ) or IgG isotype ( circles ). (C) MMP9 secretion by cells from BAL and lung from infected mice that were treated with αLy6G ( clear bars ) or IgG isotype ( grey bars ) was measured by ELISPOT. (D) Inflammatory cytokine release in airways after neutrophil depletion. BALs were collected 6 days after infection and supernatants assayed by bead array. (B–D) Mean ± SEM (n = 3, representative of two independent experiments). (B–C) Considered significant at * P
Figure Legend Snippet: Depletion of neutrophils abrogates MMP9 secretion after influenza virus infection. Neutrophils were depleted by injecting C57BL/6 mice with 400 µg anti-Ly6G antibody (αLy6G) or isotype control (IgG) one day before infection and every other day thereafter. (A) Depletion of Ly6G+ cells in αLy6G-treated C57BL/6 mice ( bottom panel ) compared to IgG ( top panel ) was verified by flow cytometric analysis 6 days after infection. (B) Kinetics of weight loss as a percentage of starting weight in uninfected (control, open symbols ) and infected ( closed symbols ) mice treated with the αLy6G ( squares ) or IgG isotype ( circles ). (C) MMP9 secretion by cells from BAL and lung from infected mice that were treated with αLy6G ( clear bars ) or IgG isotype ( grey bars ) was measured by ELISPOT. (D) Inflammatory cytokine release in airways after neutrophil depletion. BALs were collected 6 days after infection and supernatants assayed by bead array. (B–D) Mean ± SEM (n = 3, representative of two independent experiments). (B–C) Considered significant at * P

Techniques Used: Infection, Mouse Assay, Flow Cytometry, Enzyme-linked Immunospot

36) Product Images from "IL-33-Mediated Protection against Experimental Cerebral Malaria Is Linked to Induction of Type 2 Innate Lymphoid Cells, M2 Macrophages and Regulatory T Cells"

Article Title: IL-33-Mediated Protection against Experimental Cerebral Malaria Is Linked to Induction of Type 2 Innate Lymphoid Cells, M2 Macrophages and Regulatory T Cells

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004607

IL-33 expands ILC2 which adoptively protect mice against ECM. C57BL/6 mice were not infected (NI) or infected with PbA and treated daily with PBS or IL-33 from day 0. Splenic ILC2 were analysed by FACS on day 3. (A) Representative FACS showing % of ICOS + ST2 + cells gated on Lin − CD45 + cells. Cumulative percentage (B) and number (C) of ILC2 per spleen are shown. (D) Percentage of Ki67 + ILC2. Data are mean ± SEM (n = 5 per group) and representative of 2 independent experiments. *P
Figure Legend Snippet: IL-33 expands ILC2 which adoptively protect mice against ECM. C57BL/6 mice were not infected (NI) or infected with PbA and treated daily with PBS or IL-33 from day 0. Splenic ILC2 were analysed by FACS on day 3. (A) Representative FACS showing % of ICOS + ST2 + cells gated on Lin − CD45 + cells. Cumulative percentage (B) and number (C) of ILC2 per spleen are shown. (D) Percentage of Ki67 + ILC2. Data are mean ± SEM (n = 5 per group) and representative of 2 independent experiments. *P

Techniques Used: Mouse Assay, Infection, FACS

IL-33, ILC2 and M2 induce Tregs. (A-C) C57BL/6 mice were infected i.v. with PbA and treated daily with PBS or IL-33 from day 0. (A) Relative expression (% of Hprt1 ) of Foxp3 + mRNA in purified CD4 + cells from the spleen. (B) percentage of Foxp3 + cells gated on CD4 + cells and number of Foxp3 + CD4 + cells in the spleen. Data are mean ± SEM (n = 5 per group), representative of at least 2 independent experiments. *P
Figure Legend Snippet: IL-33, ILC2 and M2 induce Tregs. (A-C) C57BL/6 mice were infected i.v. with PbA and treated daily with PBS or IL-33 from day 0. (A) Relative expression (% of Hprt1 ) of Foxp3 + mRNA in purified CD4 + cells from the spleen. (B) percentage of Foxp3 + cells gated on CD4 + cells and number of Foxp3 + CD4 + cells in the spleen. Data are mean ± SEM (n = 5 per group), representative of at least 2 independent experiments. *P

Techniques Used: Mouse Assay, Infection, Expressing, Purification

ILC2 promote M2 macrophage polarization. (A) BMDM from C57BL/6 mice were cultured in the lower chamber of a 24-transwell plate in complete medium alone (M0) or supplemented with IL-4 (M2). In some experiments ILC2, sorted from naïve WT mice pre-treated with IL-33, were added to the upper chamber. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to Hprt1 ). (B) ST2-deficient BMDM were co-cultured in transwell plates as above with WT ILC2 in the presence of IL-33 alone or in combination with IL-7. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to Hprt1 ). Type 2 cytokines in the supernatants of ILC2 cultured in the presence of IL-33 or IL-33 + IL-7 were determined by ELISA (C), or by FACS (D, E). Data are mean ± SEM (n = 3 per group), representative of two independent experiments, *P
Figure Legend Snippet: ILC2 promote M2 macrophage polarization. (A) BMDM from C57BL/6 mice were cultured in the lower chamber of a 24-transwell plate in complete medium alone (M0) or supplemented with IL-4 (M2). In some experiments ILC2, sorted from naïve WT mice pre-treated with IL-33, were added to the upper chamber. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to Hprt1 ). (B) ST2-deficient BMDM were co-cultured in transwell plates as above with WT ILC2 in the presence of IL-33 alone or in combination with IL-7. After 48 h, BMDM were collected and assayed for the expression of M2 markers by qPCR (relative to Hprt1 ). Type 2 cytokines in the supernatants of ILC2 cultured in the presence of IL-33 or IL-33 + IL-7 were determined by ELISA (C), or by FACS (D, E). Data are mean ± SEM (n = 3 per group), representative of two independent experiments, *P

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

37) Product Images from "Bridelia ferruginea Produces Antineuroinflammatory Activity through Inhibition of Nuclear Factor-kappa B and p38 MAPK Signalling"

Article Title: Bridelia ferruginea Produces Antineuroinflammatory Activity through Inhibition of Nuclear Factor-kappa B and p38 MAPK Signalling

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2012/546873

(a) BFE inhibited COX-2 protein expression in LPS-stimulated primary microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of incubation period, COX-2 protein levels were measured using western blot using specific antibodies for each protein. (b) Quantitative densitometric analysis of COX-2 protein expression normalized to actin loading control. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
Figure Legend Snippet: (a) BFE inhibited COX-2 protein expression in LPS-stimulated primary microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of incubation period, COX-2 protein levels were measured using western blot using specific antibodies for each protein. (b) Quantitative densitometric analysis of COX-2 protein expression normalized to actin loading control. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

Techniques Used: Expressing, Incubation, Western Blot

(a) BFE inhibited iNOS protein expression in LPS-stimulated primary microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of incubation period, iNOS protein levels were measured using western blot using specific antibodies for each protein. (b) Quantitative densitometric analysis of iNOS protein expression normalized to actin loading control. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
Figure Legend Snippet: (a) BFE inhibited iNOS protein expression in LPS-stimulated primary microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of incubation period, iNOS protein levels were measured using western blot using specific antibodies for each protein. (b) Quantitative densitometric analysis of iNOS protein expression normalized to actin loading control. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

Techniques Used: Expressing, Incubation, Western Blot

Reduction of nitrite production in LPS-stimulated microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for nitrite measurement with the Griess assay. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
Figure Legend Snippet: Reduction of nitrite production in LPS-stimulated microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for nitrite measurement with the Griess assay. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

Techniques Used: Incubation, Griess Assay

BFE inhibited p38, but not p42/44 or JNK MAPK in LPS-stimulated BV-2 microglia. Lysates from LPS-stimulated microglia were analysed using ELISA for phospho-p38, phospho-42/44, and phospho-JNK. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
Figure Legend Snippet: BFE inhibited p38, but not p42/44 or JNK MAPK in LPS-stimulated BV-2 microglia. Lysates from LPS-stimulated microglia were analysed using ELISA for phospho-p38, phospho-42/44, and phospho-JNK. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

Techniques Used: Enzyme-linked Immunosorbent Assay

Reduction of TNF α ((a) and (b)) and IL-6 ((a) and (c)) production in LPS-stimulated BV-2 microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for TNF α and IL-6 measurement according to the manufacturer's instructions. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
Figure Legend Snippet: Reduction of TNF α ((a) and (b)) and IL-6 ((a) and (c)) production in LPS-stimulated BV-2 microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for TNF α and IL-6 measurement according to the manufacturer's instructions. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

Techniques Used: Incubation

Reduction of PGE 2 production in LPS-stimulated microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for PGE 2 measurement. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P
Figure Legend Snippet: Reduction of PGE 2 production in LPS-stimulated microglia. Cells were stimulated with LPS (100 ng/mL) in the presence or absence of BFE (25–200 μ g/mL) for 24 h. At the end of the incubation period, supernatants were collected for PGE 2 measurement. All values are expressed as mean ± SEM for 3 independent experiments. Data were analysed using one-way ANOVA for multiple comparison with post-hoc Student Newman-Keuls test. * P

Techniques Used: Incubation

38) Product Images from "RAGE deficiency predisposes mice to virus-induced paucigranulocytic asthma"

Article Title: RAGE deficiency predisposes mice to virus-induced paucigranulocytic asthma

Journal: eLife

doi: 10.7554/eLife.21199

Neonatal RAGE deficient mice do not display a pronounced T H 2 response to an early life infection with PVM. RAGE deficient and WT (RAGE + ) mice were infected with 10 PFU of PVM or mock at seven days of age and the inflammatory profile was assessed seven days post-infection ( A ) Inflammatory cells in the bronchoalveolar lavage fluid were enumerated by differential counting following Geimsa staining. ( B ) Cytokine levels in the bronchoalveolar lavage fluid of neonatal mice seven days post-PVM or mock infection (i.e. at 14-days of age). ( C ) Cytokine levels in the lungs of neonatal mice seven days post-PVM or mock infection (i.e. at 14-days of age). Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparisons test ( A ) or a two-way ANOVA ( B and C ). Statistical significance is denoted by asterisks (*p
Figure Legend Snippet: Neonatal RAGE deficient mice do not display a pronounced T H 2 response to an early life infection with PVM. RAGE deficient and WT (RAGE + ) mice were infected with 10 PFU of PVM or mock at seven days of age and the inflammatory profile was assessed seven days post-infection ( A ) Inflammatory cells in the bronchoalveolar lavage fluid were enumerated by differential counting following Geimsa staining. ( B ) Cytokine levels in the bronchoalveolar lavage fluid of neonatal mice seven days post-PVM or mock infection (i.e. at 14-days of age). ( C ) Cytokine levels in the lungs of neonatal mice seven days post-PVM or mock infection (i.e. at 14-days of age). Statistical significance was determined by a one-way ANOVA with Tukey’s multiple comparisons test ( A ) or a two-way ANOVA ( B and C ). Statistical significance is denoted by asterisks (*p

Techniques Used: Mouse Assay, Infection, Staining

RAGE deficient mice do not display a pronounced T H 2 response upon re-infection with PVM. Neonatal WT (RAGE + ), TLR7 deficient and RAGE deficient mice were infected with PVM (10 PFU) or mock at seven days of age and later re-infected with PVM (100 PFU) or mock 42 days after the primary infection. ( A ) Inflammatory cells in the bronchoalveolar lavage fluid were enumerated by differential counting following Geimsa staining seven days post-reinfection (i.e. at 56-days of age). ( B ) Cytokine levels in the bronchoalveolar lavage fluid of neonatal mice seven days post-re infection (i.e. at 56-days of age). ( C ) Cytokine levels in the lungs of neonatal mice seven days post- reinfection (i.e. at 56-days of age). Periostin levels were determined by immunohistochemistry and quantified as a percentage of the epithelial basement membrane length. Statistical significance was determined by a Student’s t-test ( A ) and a two-way ANOVA with Tukey’s multiple comparisons test ( B and D ). Statistical significance is denoted by asterisks (*p
Figure Legend Snippet: RAGE deficient mice do not display a pronounced T H 2 response upon re-infection with PVM. Neonatal WT (RAGE + ), TLR7 deficient and RAGE deficient mice were infected with PVM (10 PFU) or mock at seven days of age and later re-infected with PVM (100 PFU) or mock 42 days after the primary infection. ( A ) Inflammatory cells in the bronchoalveolar lavage fluid were enumerated by differential counting following Geimsa staining seven days post-reinfection (i.e. at 56-days of age). ( B ) Cytokine levels in the bronchoalveolar lavage fluid of neonatal mice seven days post-re infection (i.e. at 56-days of age). ( C ) Cytokine levels in the lungs of neonatal mice seven days post- reinfection (i.e. at 56-days of age). Periostin levels were determined by immunohistochemistry and quantified as a percentage of the epithelial basement membrane length. Statistical significance was determined by a Student’s t-test ( A ) and a two-way ANOVA with Tukey’s multiple comparisons test ( B and D ). Statistical significance is denoted by asterisks (*p

Techniques Used: Mouse Assay, Infection, Staining, Immunohistochemistry

39) Product Images from "12/15-lipoxygenase–mediated enzymatic lipid oxidation regulates DC maturation and function"

Article Title: 12/15-lipoxygenase–mediated enzymatic lipid oxidation regulates DC maturation and function

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI78490

DC-derived 12/15-LO regulates Th17 T cell differentiation. ( A ) Real-time PCR-based quantification of mRNA expression levels of the indicated cytokines in Alox15 +/+ and Alox15 –/– CD11c + BM-DCs after LPS-induced maturation (100 ng LPS /ml for 16 hours). ( B ) ELISA-based analysis of the cytokine production by BM-DCs from Alox15 +/+ and Alox15 –/– mice. Cells were pulse stimulated with LPS (100 ng/ml) for 6 hours, and the subsequent cytokine secretion was determined at the indicated time points. ( C ) Flow cytometric analysis of the frequency of the indicated Th cell subsets after cocultivation of CD4 + T cells together with Alox15 +/+ or Alox15 –/– CD11c + BM-DCs for 4 days under Th-skewing conditions. ( D ) Effect of oxPC (10 μg/ml) on the differentiation of Th17 T cells during cocultivation of CD4 + T cells together with Alox15 +/+ or Alox15 –/– CD11c + BM-DCs under Th17-skewing conditions. ( E ) Effect of oxPC (10 μg/ml) on the differentiation of Th17 T cells under Th0- or Th17-skewing conditions in the absence of BM-DCs. Data shown are representative of at least 3 independent experiments ( n = 3). Error bars represent SEM. * P
Figure Legend Snippet: DC-derived 12/15-LO regulates Th17 T cell differentiation. ( A ) Real-time PCR-based quantification of mRNA expression levels of the indicated cytokines in Alox15 +/+ and Alox15 –/– CD11c + BM-DCs after LPS-induced maturation (100 ng LPS /ml for 16 hours). ( B ) ELISA-based analysis of the cytokine production by BM-DCs from Alox15 +/+ and Alox15 –/– mice. Cells were pulse stimulated with LPS (100 ng/ml) for 6 hours, and the subsequent cytokine secretion was determined at the indicated time points. ( C ) Flow cytometric analysis of the frequency of the indicated Th cell subsets after cocultivation of CD4 + T cells together with Alox15 +/+ or Alox15 –/– CD11c + BM-DCs for 4 days under Th-skewing conditions. ( D ) Effect of oxPC (10 μg/ml) on the differentiation of Th17 T cells during cocultivation of CD4 + T cells together with Alox15 +/+ or Alox15 –/– CD11c + BM-DCs under Th17-skewing conditions. ( E ) Effect of oxPC (10 μg/ml) on the differentiation of Th17 T cells under Th0- or Th17-skewing conditions in the absence of BM-DCs. Data shown are representative of at least 3 independent experiments ( n = 3). Error bars represent SEM. * P

Techniques Used: Derivative Assay, Cell Differentiation, Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Mouse Assay

Deletion of 12/15-LO results in increased DC maturation and Th17 T cell differentiation as well as an aggravated form of EAE. ( A ) Flow cytometry–based measurement of the 12/15-LO expression in indicated splenic DC subsets during steady state and ( B ) follow-up of 12/15-LO expression in moDCs after induction of EAE. ( C ) Flow cytometric analysis of MHC class II molecule expression on moDC, lymphoid DC, and myeloid DCs, and ( D ) quantification of the mRNA expression profile of indicated cytokines, including Alox15 and Rorgt , in draining lymph nodes of WT and Alox15 –/– mice 3 days after induction of immunized (EAE) mice compared with nonimmunized mice (control). ( E ) Flow cytometric analysis of the frequency of IL-17–expressing Th17 T cells, FOXP3-expressing regulatory T cells, IFN-γ–expressing Th1 T cells, and IL-4–expressing Th2 T cells in the spleens of WT or Alox15 –/– mice 10 days after induction of EAE. Data shown are representative of at least 3 independent experiments. Error bars represent SEM. * P
Figure Legend Snippet: Deletion of 12/15-LO results in increased DC maturation and Th17 T cell differentiation as well as an aggravated form of EAE. ( A ) Flow cytometry–based measurement of the 12/15-LO expression in indicated splenic DC subsets during steady state and ( B ) follow-up of 12/15-LO expression in moDCs after induction of EAE. ( C ) Flow cytometric analysis of MHC class II molecule expression on moDC, lymphoid DC, and myeloid DCs, and ( D ) quantification of the mRNA expression profile of indicated cytokines, including Alox15 and Rorgt , in draining lymph nodes of WT and Alox15 –/– mice 3 days after induction of immunized (EAE) mice compared with nonimmunized mice (control). ( E ) Flow cytometric analysis of the frequency of IL-17–expressing Th17 T cells, FOXP3-expressing regulatory T cells, IFN-γ–expressing Th1 T cells, and IL-4–expressing Th2 T cells in the spleens of WT or Alox15 –/– mice 10 days after induction of EAE. Data shown are representative of at least 3 independent experiments. Error bars represent SEM. * P

Techniques Used: Cell Differentiation, Flow Cytometry, Expressing, Mouse Assay

40) Product Images from "Selective B cell depletion upon infusion of replication-incompetent anti-CD19 CAR lentivirus"

Article Title: Selective B cell depletion upon infusion of replication-incompetent anti-CD19 CAR lentivirus

Journal: bioRxiv

doi: 10.1101/2020.05.15.098335

Mouse weights (grams) pre- and post treatment. Figure 5A : 1D3-CD19CAR-GFP lentivirus, Figure 5B : FMC63-CD19CAR-GFP lentivirus, and Figure 5C : GFP-only lentivirus or PBS. In Figure 5A and 5B , the mice that were treated with the 1D3-CD19CAR-GFP lentivirus or the FMC63-CD19CAR-GFP showed a 5.5 +/− 2.97% (mean +/− SD) and 3.6 +/− 1.78% reduction in weight, respectively, within 5-7 days after receiving treatment. Once the mice weights recovered, they exhibited no other visible adverse effects.
Figure Legend Snippet: Mouse weights (grams) pre- and post treatment. Figure 5A : 1D3-CD19CAR-GFP lentivirus, Figure 5B : FMC63-CD19CAR-GFP lentivirus, and Figure 5C : GFP-only lentivirus or PBS. In Figure 5A and 5B , the mice that were treated with the 1D3-CD19CAR-GFP lentivirus or the FMC63-CD19CAR-GFP showed a 5.5 +/− 2.97% (mean +/− SD) and 3.6 +/− 1.78% reduction in weight, respectively, within 5-7 days after receiving treatment. Once the mice weights recovered, they exhibited no other visible adverse effects.

Techniques Used: Mouse Assay

In vitro 1D3- or FMC63-CD19CAR-GFP T cell cytolytic activity using C57BL/6 splenocytes. Debris, cell doublets, and clumps were eliminated by FCS-A vs SSC-A gating followed by FSC-A vs. FSC-H and SSC-A vs. SSC-H gating. T and B cells were then identified by CD3 vs. CD20 gating. CD20 positive cells were selected for and gated on FVS-780 to obtain the percentage of live vs. dead target cells. The flow collected data was analysed using Flow Jo v10, for windows and GraphPad Prism software version 8.0.0 for windows (GraphPad Software, California, USA) [ Figure 2 ]
Figure Legend Snippet: In vitro 1D3- or FMC63-CD19CAR-GFP T cell cytolytic activity using C57BL/6 splenocytes. Debris, cell doublets, and clumps were eliminated by FCS-A vs SSC-A gating followed by FSC-A vs. FSC-H and SSC-A vs. SSC-H gating. T and B cells were then identified by CD3 vs. CD20 gating. CD20 positive cells were selected for and gated on FVS-780 to obtain the percentage of live vs. dead target cells. The flow collected data was analysed using Flow Jo v10, for windows and GraphPad Prism software version 8.0.0 for windows (GraphPad Software, California, USA) [ Figure 2 ]

Techniques Used: In Vitro, Activity Assay, Software

1D3-CD19CAR-GFP, FMC63-CD19CAR-GFP, and GFP lentivirus transduction efficiency. Figure S4A : Titration of 1D3-CD19CAR-GFP and FMC63-CD19CAR-GFP viral supernatant titration over EL4 (green and purple, respectively) and K562 (red and blue, respectively) cell lines. While the transduction efficiency appears significantly better in the human K562s (p
Figure Legend Snippet: 1D3-CD19CAR-GFP, FMC63-CD19CAR-GFP, and GFP lentivirus transduction efficiency. Figure S4A : Titration of 1D3-CD19CAR-GFP and FMC63-CD19CAR-GFP viral supernatant titration over EL4 (green and purple, respectively) and K562 (red and blue, respectively) cell lines. While the transduction efficiency appears significantly better in the human K562s (p

Techniques Used: Transduction, Titration

Analysis of C57BL/6 whole blood pre- and post-treatment with 1D3-CD19CAR-GFP CAR, FMC63-CD19 CAR-GFP or control (GFP-only) lentiviruses. Figure 4A : Total (grey bars) and GFP + (white bars) CD3 + , CD90.2 + T cells, Figure 4B : Total (grey bars) and GFP + (white bars) CD20 + , CD45R/B220 + B cells, Figure 4C : Total (grey bars) and GFP + (white bars) CD11b + macrophage and other non-T cells, and Figure 4D : Total (grey bars) and GFP + (white bars) CD335 + NK and NKT cells.
Figure Legend Snippet: Analysis of C57BL/6 whole blood pre- and post-treatment with 1D3-CD19CAR-GFP CAR, FMC63-CD19 CAR-GFP or control (GFP-only) lentiviruses. Figure 4A : Total (grey bars) and GFP + (white bars) CD3 + , CD90.2 + T cells, Figure 4B : Total (grey bars) and GFP + (white bars) CD20 + , CD45R/B220 + B cells, Figure 4C : Total (grey bars) and GFP + (white bars) CD11b + macrophage and other non-T cells, and Figure 4D : Total (grey bars) and GFP + (white bars) CD335 + NK and NKT cells.

Techniques Used:

Analysis of Total CD20 + , CD45R/B220 + B cells and GFP + , CD3 + , CD90.2 + T cells isolated from C57BL/6 whole blood pre- and posttreatment with 1D3-CD19CAR-GFP CAR, FMC63-CD19 CAR-GFP or control (GFP-only) lentiviruses. Figure 3A : C57BL/6 wildtype mice were treated with a single IV injection lentivirus (1.8-2 x10 7 IU in 200μl PBS), to deliver either the 1D3-CD19CAR-GFP CAR, FMC63-CD19 CAR, or control (GFP-only). Mice treated with the ID3-CD19CAR lentivirus had a maximum 1D3-CD19CART cell population of 42.5 +/− 1.07% (mean +/− SD) within their peripheral blood by week 5 (white bars) which was accompanied by B cell aplasia (grey bars) by week 5 which was maintained until the end of the experiment (week 8). Mice treated with the FMC63-CD19CAR lentivirus had a maximum FMC63-CD19CAR T cell population of 26.1 +/− 1.01% within their peripheral blood by week 5 (white bars) which was accompanied by B cell aplasia (grey bars) by week 5 which was maintained until the end of the experiment (week 8). Figure 3B : Representative flowgrams from Figure 3A
Figure Legend Snippet: Analysis of Total CD20 + , CD45R/B220 + B cells and GFP + , CD3 + , CD90.2 + T cells isolated from C57BL/6 whole blood pre- and posttreatment with 1D3-CD19CAR-GFP CAR, FMC63-CD19 CAR-GFP or control (GFP-only) lentiviruses. Figure 3A : C57BL/6 wildtype mice were treated with a single IV injection lentivirus (1.8-2 x10 7 IU in 200μl PBS), to deliver either the 1D3-CD19CAR-GFP CAR, FMC63-CD19 CAR, or control (GFP-only). Mice treated with the ID3-CD19CAR lentivirus had a maximum 1D3-CD19CART cell population of 42.5 +/− 1.07% (mean +/− SD) within their peripheral blood by week 5 (white bars) which was accompanied by B cell aplasia (grey bars) by week 5 which was maintained until the end of the experiment (week 8). Mice treated with the FMC63-CD19CAR lentivirus had a maximum FMC63-CD19CAR T cell population of 26.1 +/− 1.01% within their peripheral blood by week 5 (white bars) which was accompanied by B cell aplasia (grey bars) by week 5 which was maintained until the end of the experiment (week 8). Figure 3B : Representative flowgrams from Figure 3A

Techniques Used: Isolation, Mouse Assay, IV Injection

Proliferation and cytolytic properties of CD3+ T cells from C57BL/6 splenocytestransduced with 1D3-CD19CAR-GFP, FMC63-CD19CAR-GFP, or GFP only transgenes. Figure 2A : 2.5×10 5 C57BL/6 splenocytes were transduced with 1D3-CD19CAR-GFP or GFP-only lentivirus at an MOI of 5. The transduced splenocytes were then co-cultured with an excess of irradiated C57BL/6 splenocytes which provided a source of B cells expressing the CD19 antigen. The 1D3-CD19CAR transduced splenocytes showed a 12.2+/− 0.09 (mean +/− SD) fold greater expansion (red, square), compared to GFP transduced (green, triangle) (p
Figure Legend Snippet: Proliferation and cytolytic properties of CD3+ T cells from C57BL/6 splenocytestransduced with 1D3-CD19CAR-GFP, FMC63-CD19CAR-GFP, or GFP only transgenes. Figure 2A : 2.5×10 5 C57BL/6 splenocytes were transduced with 1D3-CD19CAR-GFP or GFP-only lentivirus at an MOI of 5. The transduced splenocytes were then co-cultured with an excess of irradiated C57BL/6 splenocytes which provided a source of B cells expressing the CD19 antigen. The 1D3-CD19CAR transduced splenocytes showed a 12.2+/− 0.09 (mean +/− SD) fold greater expansion (red, square), compared to GFP transduced (green, triangle) (p

Techniques Used: Transduction, Cell Culture, Irradiation, Expressing

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Enzyme-linked Immunosorbent Assay:

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Article Snippet: .. The levels of TNF-α, IL-6 and IL-1β in mammary glands were determined using the mouse ELISA kits (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions. .. Cell Culture The mMECs were purchased from the American Type Culture Collection (ATCC, ATCC® CRL-3063™, Rockville, MD, USA), and these cells were cultured in DMEM medium (Gibco, Grand Island, NY 14072, USA) containing 10% FBS (Clark Bioscience, Richmond, VA, USA) at 37 °C in a humidified incubator with 5% CO2 .

Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells
Article Snippet: .. Cytokine Quantitation The levels of the following cytokines were evaluated in MDM supernatants using commercially available ELISA sets: IL-8, IL-6, IL-1β, TNFα, IL-10 (ELISA MAX Deluxe kits, BioLegend) and sICAM (DouSet ELISA, R & D Systems). .. MIF plasma levels were evaluated using an in-house ELISA constructed with an anti-human MIF antibody pair and an MIF standard obtained from BioLegend.

Incubation:

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis
Article Snippet: .. PBMCs were incubated with CD19 (HIB19), CD20 (2H7), CD24 (ML5), CD10 (HI10a), IgM (MHM-88), CD3 (UCHT1), CD8 (RPA-T8), CD45RO (UCHL1), CD45RA (HI100), CD138 (MI15), IL-21R (17A12), from Biolegend; CD38 (HIT2), CD27 (L128), CD4 (SK3), CD27 (L128), Blimp-1 (5E7), γC (TUGh4), STAT3 (M59-50), from BD, SP1 (D4C3) from CST and IL-6 (MQ2-13AS) from eBioscience. .. IL-6 and IgM levels were determined after brefeldinA (Biolegend)/monensin (Biolegend) stimulation for 4 h, fixed with cytofix (BD), permeabilized with cytoperm (BD) followed by intracellular staining.

Recombinase Polymerase Amplification:

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis
Article Snippet: .. PBMCs were incubated with CD19 (HIB19), CD20 (2H7), CD24 (ML5), CD10 (HI10a), IgM (MHM-88), CD3 (UCHT1), CD8 (RPA-T8), CD45RO (UCHL1), CD45RA (HI100), CD138 (MI15), IL-21R (17A12), from Biolegend; CD38 (HIT2), CD27 (L128), CD4 (SK3), CD27 (L128), Blimp-1 (5E7), γC (TUGh4), STAT3 (M59-50), from BD, SP1 (D4C3) from CST and IL-6 (MQ2-13AS) from eBioscience. .. IL-6 and IgM levels were determined after brefeldinA (Biolegend)/monensin (Biolegend) stimulation for 4 h, fixed with cytofix (BD), permeabilized with cytoperm (BD) followed by intracellular staining.

other:

Article Title: Protective Cancer Vaccine Using Genetically Modified Hematopoietic Stem Cells
Article Snippet: PE, PE/Cy7, PerCP, PerCP/Cy5.5, or APC conjugated Thy1.2 (Clone 53-2.1), TCRVβ5 (MR9-4), TCRVα2 TCR (B20.1), CD44 (Clone IM7), IFN-γ (Clone XMG1.2) and IL-2 (JES6-5H4) were purchased from Biolegend (San Diego, CA, USA).

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis
Article Snippet: We defined RA-IL-21Rhigh as subjects for whom the gMFI of IL-21R is two standard deviations (SD) above the mean of the control group.

Article Title: Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis
Article Snippet: In contrast, there was no correlation between IL-21R and IL-6 production or intracellular IL-6 levels following stimulation with PMA/ionomycin (Figure , Figure ), suggesting that the differences in IL-6 production are dependent on IL-21.

Quantitation Assay:

Article Title: Interaction Between Macrophage Migration Inhibitory Factor and CD74 in Human Immunodeficiency Virus Type I Infected Primary Monocyte-Derived Macrophages Triggers the Production of Proinflammatory Mediators and Enhances Infection of Unactivated CD4+ T Cells
Article Snippet: .. Cytokine Quantitation The levels of the following cytokines were evaluated in MDM supernatants using commercially available ELISA sets: IL-8, IL-6, IL-1β, TNFα, IL-10 (ELISA MAX Deluxe kits, BioLegend) and sICAM (DouSet ELISA, R & D Systems). .. MIF plasma levels were evaluated using an in-house ELISA constructed with an anti-human MIF antibody pair and an MIF standard obtained from BioLegend.

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    BioLegend interleukin 6
    Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and <t>IL-6</t> (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P
    Interleukin 6, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioLegend plasma il 6 levels
    NK cell depletion reduces lung viral burden and lung IFN-γ in vaccinated, influenza-challenged mice. Four days post infection in the model described in Fig. 2A , (A) Lung RNA was analyzed by qPCR for influenza viral burden (plotted against a dose curve of IFA with known HAU, giving HAU equivalents per 5ug RNA tested). (B-C) Transcript levels of inflammatory cytokine genes (B) Il6 and Ifnγ and (C) neutrophil-related chemokines Cxcl1 and Cxcl2 , along with neutrophil lipocalin protein (Lcn2) RNA induction normalized to housekeeping gene ß-actin and displayed as induction over mock-treated control mice. (D) Plasma levels of <t>IL-6</t> (pg/mL). (E) IL-6 levels in lung supernatants after whole lung enzymatic digestion for single cell isolation and viral burden quantification. (A-E) A pool of two independent experiments (n=5-9/group; (A-D) were female {F} and (E) were male {E} mice used for single cell isolation). Dots represent individual mice with bars showing mean. Significance determined by Mann-Whitney U test, ns = not significant. Spearman correlation coefficient shown.
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    BioLegend mouse il 6 elisa kit
    Direct effect of acetate on B cells. Ex vivo splenic B cells were purified from specific pathogen–free NOD mice gavaged with acetate or water for 10 to 12 weeks. The purified B cells were stimulated in vitro in the presence of 10 Mm acetate with 20 μg/mL anti-CD40 mAb and 10 μg/mL LPS for 5 days. ( A ) IgA concentration in the culture supernatant of stimulated B cells was measured by <t>ELISA</t> ( n = 8–9). ( B ) Representative flow cytometric plots of intracellular <t>IL-6</t> expression of B cells after acetate stimulation. ( C ) Summary of IL-6-expressing B cells. ( D ) Secreted IL-6, determined by ELISA, from the culture supernatant of B cells stimulated with acetate ( n = 6–7). ( E – H ) Gene expression of B cells, after acetate stimulation, was assessed by qPCR: Pst α ( E ), Pst2 β ( F ), Stat5b ( G ), and Irf4 ( H ). The expression levels were determined using the 2 −ΔΔCt method by normalizing the housekeeping gene Gapdh . Data combined from 2 independent experiments are presented as mean ± SEM and were analyzed using a 2-tailed Student’s t test ( A and C – H ).
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    BioLegend mouse il 6
    Proinflammatory cytokine and chemokine expression is decreased by hMSC administration . Twenty-four hours after LPS exposure, IL-1β, <t>IL-6,</t> IL-17, IP-10, MCP-1, MIP-1α and RANTES levels were measured by multiplex immunoassay in BAL fluid. Data are expressed as mean ± SEM (n = 6 per group). * P
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    Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and IL-6 (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P

    Journal: Infection and Immunity

    Article Title: Cathelicidin Antimicrobial Peptide Expression Is Not Induced or Required for Bacterial Clearance during Salmonella enterica Infection of Human Monocyte-Derived Macrophages

    doi: 10.1128/IAI.00672-12

    Figure Lengend Snippet: Levels of proinflammatory cytokines released from S . Typhi- or S . Typhimurium-infected MDMs. Shown are levels of TNF-α (A) and IL-6 (B) released from MDMs after 2.5 and 24 h of S . Typhi infection and TNF-α (C) and IL-6 (D) released from MDMs after 2.5 and 24 h of S . Typhimurium infection. Statistical significance was determined by Student's t test comparing cytokine levels between LPS modification mutant-infected samples and the WT-infected sample collected at that time point. *, P

    Article Snippet: The proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) were detected in cell culture supernatant by enzyme-linked immunosorbent assay (ELISA) (Biolegend, San Diego, CA, and R & D Systems, Minneapolis, MN, respectively).

    Techniques: Infection, Modification, Mutagenesis

    NK cell depletion reduces lung viral burden and lung IFN-γ in vaccinated, influenza-challenged mice. Four days post infection in the model described in Fig. 2A , (A) Lung RNA was analyzed by qPCR for influenza viral burden (plotted against a dose curve of IFA with known HAU, giving HAU equivalents per 5ug RNA tested). (B-C) Transcript levels of inflammatory cytokine genes (B) Il6 and Ifnγ and (C) neutrophil-related chemokines Cxcl1 and Cxcl2 , along with neutrophil lipocalin protein (Lcn2) RNA induction normalized to housekeeping gene ß-actin and displayed as induction over mock-treated control mice. (D) Plasma levels of IL-6 (pg/mL). (E) IL-6 levels in lung supernatants after whole lung enzymatic digestion for single cell isolation and viral burden quantification. (A-E) A pool of two independent experiments (n=5-9/group; (A-D) were female {F} and (E) were male {E} mice used for single cell isolation). Dots represent individual mice with bars showing mean. Significance determined by Mann-Whitney U test, ns = not significant. Spearman correlation coefficient shown.

    Journal: bioRxiv

    Article Title: Natural Killer cells dampen the pathogenic features of recall responses to influenza infection

    doi: 10.1101/846626

    Figure Lengend Snippet: NK cell depletion reduces lung viral burden and lung IFN-γ in vaccinated, influenza-challenged mice. Four days post infection in the model described in Fig. 2A , (A) Lung RNA was analyzed by qPCR for influenza viral burden (plotted against a dose curve of IFA with known HAU, giving HAU equivalents per 5ug RNA tested). (B-C) Transcript levels of inflammatory cytokine genes (B) Il6 and Ifnγ and (C) neutrophil-related chemokines Cxcl1 and Cxcl2 , along with neutrophil lipocalin protein (Lcn2) RNA induction normalized to housekeeping gene ß-actin and displayed as induction over mock-treated control mice. (D) Plasma levels of IL-6 (pg/mL). (E) IL-6 levels in lung supernatants after whole lung enzymatic digestion for single cell isolation and viral burden quantification. (A-E) A pool of two independent experiments (n=5-9/group; (A-D) were female {F} and (E) were male {E} mice used for single cell isolation). Dots represent individual mice with bars showing mean. Significance determined by Mann-Whitney U test, ns = not significant. Spearman correlation coefficient shown.

    Article Snippet: Plasma IL-6 levels were determined by sandwich enzyme-linked immunosorbent assay (ELISA) (Biolegend ELISA MAX Deluxe, London UK).

    Techniques: Mouse Assay, Infection, Real-time Polymerase Chain Reaction, Immunofluorescence, Single-cell Isolation, MANN-WHITNEY

    Depletion of NK cells after vaccination, and subsequent repopulation, does not alter lung viral burden, disease severity or systemic inflammation after challenge. (A) Transgenic C57BL/6 mice with NKp46 driven expression of diphtheria toxin (DT) receptor were vaccinated 42 days (d) prior to intranasal influenza (Flu) challenge and treated with DT (NK-depleted) 21 days prior to challenge with necropsy (nx) at 4 days post influenza challenge. (B) At 3 and 21 days post DT treatment, lungs were excised and single cells isolated for flow cytometry for the proportion (%) of NK1.1+, NKp46+ NK cells. (C) Weight loss at 4 days post influenza challenge. (D) Lung cell-free supernatants were analyzed by qPCR for influenza viral burden (plotted against a dose curve of Flu with known HAU, giving HAU equivalents). (E) Plasma levels of IL-6 (pg/mL). (B) NK cell depletion data a pool of two independent experiments, (n=2/group of male {M} mice) and (n=5-6/group of female {F} mice). (C-D) Data a pool of two independent experiment (n=4-10/group); all male {M} mice. Dots represent individual mice with bars showing mean. Significance determined by Mann-Whitney test, ns = not significant.

    Journal: bioRxiv

    Article Title: Natural Killer cells dampen the pathogenic features of recall responses to influenza infection

    doi: 10.1101/846626

    Figure Lengend Snippet: Depletion of NK cells after vaccination, and subsequent repopulation, does not alter lung viral burden, disease severity or systemic inflammation after challenge. (A) Transgenic C57BL/6 mice with NKp46 driven expression of diphtheria toxin (DT) receptor were vaccinated 42 days (d) prior to intranasal influenza (Flu) challenge and treated with DT (NK-depleted) 21 days prior to challenge with necropsy (nx) at 4 days post influenza challenge. (B) At 3 and 21 days post DT treatment, lungs were excised and single cells isolated for flow cytometry for the proportion (%) of NK1.1+, NKp46+ NK cells. (C) Weight loss at 4 days post influenza challenge. (D) Lung cell-free supernatants were analyzed by qPCR for influenza viral burden (plotted against a dose curve of Flu with known HAU, giving HAU equivalents). (E) Plasma levels of IL-6 (pg/mL). (B) NK cell depletion data a pool of two independent experiments, (n=2/group of male {M} mice) and (n=5-6/group of female {F} mice). (C-D) Data a pool of two independent experiment (n=4-10/group); all male {M} mice. Dots represent individual mice with bars showing mean. Significance determined by Mann-Whitney test, ns = not significant.

    Article Snippet: Plasma IL-6 levels were determined by sandwich enzyme-linked immunosorbent assay (ELISA) (Biolegend ELISA MAX Deluxe, London UK).

    Techniques: Transgenic Assay, Mouse Assay, Expressing, Isolation, Flow Cytometry, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    Direct effect of acetate on B cells. Ex vivo splenic B cells were purified from specific pathogen–free NOD mice gavaged with acetate or water for 10 to 12 weeks. The purified B cells were stimulated in vitro in the presence of 10 Mm acetate with 20 μg/mL anti-CD40 mAb and 10 μg/mL LPS for 5 days. ( A ) IgA concentration in the culture supernatant of stimulated B cells was measured by ELISA ( n = 8–9). ( B ) Representative flow cytometric plots of intracellular IL-6 expression of B cells after acetate stimulation. ( C ) Summary of IL-6-expressing B cells. ( D ) Secreted IL-6, determined by ELISA, from the culture supernatant of B cells stimulated with acetate ( n = 6–7). ( E – H ) Gene expression of B cells, after acetate stimulation, was assessed by qPCR: Pst α ( E ), Pst2 β ( F ), Stat5b ( G ), and Irf4 ( H ). The expression levels were determined using the 2 −ΔΔCt method by normalizing the housekeeping gene Gapdh . Data combined from 2 independent experiments are presented as mean ± SEM and were analyzed using a 2-tailed Student’s t test ( A and C – H ).

    Journal: JCI Insight

    Article Title: Gut microbial metabolites alter IgA immunity in type 1 diabetes

    doi: 10.1172/jci.insight.135718

    Figure Lengend Snippet: Direct effect of acetate on B cells. Ex vivo splenic B cells were purified from specific pathogen–free NOD mice gavaged with acetate or water for 10 to 12 weeks. The purified B cells were stimulated in vitro in the presence of 10 Mm acetate with 20 μg/mL anti-CD40 mAb and 10 μg/mL LPS for 5 days. ( A ) IgA concentration in the culture supernatant of stimulated B cells was measured by ELISA ( n = 8–9). ( B ) Representative flow cytometric plots of intracellular IL-6 expression of B cells after acetate stimulation. ( C ) Summary of IL-6-expressing B cells. ( D ) Secreted IL-6, determined by ELISA, from the culture supernatant of B cells stimulated with acetate ( n = 6–7). ( E – H ) Gene expression of B cells, after acetate stimulation, was assessed by qPCR: Pst α ( E ), Pst2 β ( F ), Stat5b ( G ), and Irf4 ( H ). The expression levels were determined using the 2 −ΔΔCt method by normalizing the housekeeping gene Gapdh . Data combined from 2 independent experiments are presented as mean ± SEM and were analyzed using a 2-tailed Student’s t test ( A and C – H ).

    Article Snippet: Cytokine ELISA.Murine IL-6 was measured using the Mouse IL-6 ELISA kit following the manufacturer’s instructions (BioLegend).

    Techniques: Ex Vivo, Purification, Mouse Assay, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction

    Proinflammatory cytokine and chemokine expression is decreased by hMSC administration . Twenty-four hours after LPS exposure, IL-1β, IL-6, IL-17, IP-10, MCP-1, MIP-1α and RANTES levels were measured by multiplex immunoassay in BAL fluid. Data are expressed as mean ± SEM (n = 6 per group). * P

    Journal: Stem Cell Research & Therapy

    Article Title: Human multipotent stromal cells attenuate lipopolysaccharide-induced acute lung injury in mice via secretion of tumor necrosis factor-?-induced protein 6

    doi: 10.1186/scrt68

    Figure Lengend Snippet: Proinflammatory cytokine and chemokine expression is decreased by hMSC administration . Twenty-four hours after LPS exposure, IL-1β, IL-6, IL-17, IP-10, MCP-1, MIP-1α and RANTES levels were measured by multiplex immunoassay in BAL fluid. Data are expressed as mean ± SEM (n = 6 per group). * P

    Article Snippet: The quantification of mouse IL-6 was performed with mouse IL-6 ELISA MAX™ Deluxe Set from BioLegend (San Diego, CA, USA) according to the manufacturer's instruction.

    Techniques: Expressing, Multiplex Assay