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R&D Systems il 13
Impact of stimulation with 100 ng/mL of IL-4 or <t>IL-13</t> on HCT 116 cells—a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) control for IL-13 at 72 h; ( m ) stimulated with IL-13 for 0 h; ( n ) stimulated with IL-13 at 24 h; ( o ) stimulated with IL-13 at 48 h; ( p ) stimulated with IL-13 at 72 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The ratios in interleukin-stimulated cells significantly ( p
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1) Product Images from "Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility"

Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

Journal: Cancers

doi: 10.3390/cancers12061463

Impact of stimulation with 100 ng/mL of IL-4 or IL-13 on HCT 116 cells—a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) control for IL-13 at 72 h; ( m ) stimulated with IL-13 for 0 h; ( n ) stimulated with IL-13 at 24 h; ( o ) stimulated with IL-13 at 48 h; ( p ) stimulated with IL-13 at 72 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The ratios in interleukin-stimulated cells significantly ( p
Figure Legend Snippet: Impact of stimulation with 100 ng/mL of IL-4 or IL-13 on HCT 116 cells—a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) control for IL-13 at 72 h; ( m ) stimulated with IL-13 for 0 h; ( n ) stimulated with IL-13 at 24 h; ( o ) stimulated with IL-13 at 48 h; ( p ) stimulated with IL-13 at 72 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The ratios in interleukin-stimulated cells significantly ( p

Techniques Used: Wound Healing Assay

Effect of IL-4 and IL-13 stimulation on the expression of epithelial-mesenchymal transition markers and tight junction proteins: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p
Figure Legend Snippet: Effect of IL-4 and IL-13 stimulation on the expression of epithelial-mesenchymal transition markers and tight junction proteins: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

Techniques Used: Expressing

Effect of IL-4 and IL-13 stimulation on the expression of mediators of proliferation and apoptosis: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (control) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p
Figure Legend Snippet: Effect of IL-4 and IL-13 stimulation on the expression of mediators of proliferation and apoptosis: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (control) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

Techniques Used: Expressing

Effect of exogenous IL-13 (1–250 ng/mL) on cancer cell viability under normal (10% FBS) and nutritional-stress (0.5% FBS) conditions as determined with sulforhodamine B (SRB) assay: ( a ) HCT 116 cultured in 10% FBS; ( b ) HCT 116 cultured in 0.5% FBS; (c ) HT-29 cultured in 10% FBS; ( d ) HT-29 cultured in 0.5% FBS; ( e ) Caco-2 cultured in 10% FBS; ( f ) Caco-2 cultured in 0.5% FBS. Data presented as percentage (% control) of viability of unstimulated cells, marked at 100% as reference red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p
Figure Legend Snippet: Effect of exogenous IL-13 (1–250 ng/mL) on cancer cell viability under normal (10% FBS) and nutritional-stress (0.5% FBS) conditions as determined with sulforhodamine B (SRB) assay: ( a ) HCT 116 cultured in 10% FBS; ( b ) HCT 116 cultured in 0.5% FBS; (c ) HT-29 cultured in 10% FBS; ( d ) HT-29 cultured in 0.5% FBS; ( e ) Caco-2 cultured in 10% FBS; ( f ) Caco-2 cultured in 0.5% FBS. Data presented as percentage (% control) of viability of unstimulated cells, marked at 100% as reference red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

Techniques Used: Sulforhodamine B Assay, Cell Culture

Effect of IL-4 and IL-13 on their respective receptor expression: ( a ) IL13Ra1 and IL4 expression following 24-h stimulation with 250 ng/mL of IL-4; ( b ) IL13Ra1 expression following 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative expression of receptor transcripts in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p
Figure Legend Snippet: Effect of IL-4 and IL-13 on their respective receptor expression: ( a ) IL13Ra1 and IL4 expression following 24-h stimulation with 250 ng/mL of IL-4; ( b ) IL13Ra1 expression following 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative expression of receptor transcripts in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

Techniques Used: Expressing

Patients’-matched analysis of tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein in ESCC ( n = 18); ( b ) IL13 mRNA in ESCC ( n = 16); ( c ) IL13Ra1 mRNA in ESCC ( n = 16); ( d ) IL-13 protein in GC ( n = 14); ( e ) IL13 mRNA in GC ( n = 14); ( f ) IL13Ra1 mRNA in GC ( n = 14). Data were analyzed as logs using t -test for paired samples and presented as geometric means with 95% confidence interval ( CI ). Mean difference in protein concentration (MD) between groups is accompanied by 95% CI . Transcriptomic data are presented as normalized relative quantities (NRQ). CRC, colorectal cancer; ESCC, esophageal squamous cell carcinoma; GC, gastric adenocarcinoma.
Figure Legend Snippet: Patients’-matched analysis of tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein in ESCC ( n = 18); ( b ) IL13 mRNA in ESCC ( n = 16); ( c ) IL13Ra1 mRNA in ESCC ( n = 16); ( d ) IL-13 protein in GC ( n = 14); ( e ) IL13 mRNA in GC ( n = 14); ( f ) IL13Ra1 mRNA in GC ( n = 14). Data were analyzed as logs using t -test for paired samples and presented as geometric means with 95% confidence interval ( CI ). Mean difference in protein concentration (MD) between groups is accompanied by 95% CI . Transcriptomic data are presented as normalized relative quantities (NRQ). CRC, colorectal cancer; ESCC, esophageal squamous cell carcinoma; GC, gastric adenocarcinoma.

Techniques Used: Expressing, Protein Concentration

Effect of anatomical site on tumor-to-adjacent expression ratios of IL13 mRNA (IL13m), IL-13 protein (IL13p), and IL13Ra1 . Data analyzed as logs using one-way ANOVA and presented as geometric means with 95% confidence interval (whiskers). Red triangles represent mean values in colorectal cancers (denoted as C); blue squares represent mean values in esophageal squamous cell carcinoma (denoted as E); green circles represent mean values in gastric adenocarcinoma (denoted as G). p values for IL13 mRNA analysis are denoted as P m , for IL-13 protein analysis as P p , and for IL13Ra1 mRNA analysis as P R .
Figure Legend Snippet: Effect of anatomical site on tumor-to-adjacent expression ratios of IL13 mRNA (IL13m), IL-13 protein (IL13p), and IL13Ra1 . Data analyzed as logs using one-way ANOVA and presented as geometric means with 95% confidence interval (whiskers). Red triangles represent mean values in colorectal cancers (denoted as C); blue squares represent mean values in esophageal squamous cell carcinoma (denoted as E); green circles represent mean values in gastric adenocarcinoma (denoted as G). p values for IL13 mRNA analysis are denoted as P m , for IL-13 protein analysis as P p , and for IL13Ra1 mRNA analysis as P R .

Techniques Used: Expressing

Analysis of the scratch assay data. Effect of IL-4 and IL-13 stimulation (100 ng/mL) on: ( a ) Caco-2 cells; ( b ) HCT 116 cells. Results at each time point are expressed as a percentage of gap area considering the gap at the time 0 as 100%. Each bar represents mean ± SE of three (Caco-2) or four (HCT116) biological replicates. Statistically significant differences as compared to time 0, analyzed using t -test for one mean, are marked as follows: p
Figure Legend Snippet: Analysis of the scratch assay data. Effect of IL-4 and IL-13 stimulation (100 ng/mL) on: ( a ) Caco-2 cells; ( b ) HCT 116 cells. Results at each time point are expressed as a percentage of gap area considering the gap at the time 0 as 100%. Each bar represents mean ± SE of three (Caco-2) or four (HCT116) biological replicates. Statistically significant differences as compared to time 0, analyzed using t -test for one mean, are marked as follows: p

Techniques Used: Wound Healing Assay

Effect of IL-4 and IL-13 stimulation on expression of various proteins relevant for cancer growth: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by horizontal red line. Statistically significant differences ( p
Figure Legend Snippet: Effect of IL-4 and IL-13 stimulation on expression of various proteins relevant for cancer growth: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by horizontal red line. Statistically significant differences ( p

Techniques Used: Expressing

Effect of anatomical site on tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein; ( b ) IL13 mRNA; ( c ) IL13Ra1 mRNA. Data analyzed as logs using one-way ANOVA and presented as geometric means with 95% confidence interval (whiskers). Blue triangles represent mean values in colorectal cancers (denoted as C); red squares represent mean values in esophageal squamous cell carcinoma (denoted as E); green circles represent mean values in gastric adenocarcinoma (denoted as G). p values for the analysis in adjacent tissue are denoted as P a and for tumor tissue as P t . Statistically significant differences between groups are marked with symbols of the same type (*, #, etc.). Transcriptomic data presented as normalized relative quantities (NRQ).
Figure Legend Snippet: Effect of anatomical site on tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein; ( b ) IL13 mRNA; ( c ) IL13Ra1 mRNA. Data analyzed as logs using one-way ANOVA and presented as geometric means with 95% confidence interval (whiskers). Blue triangles represent mean values in colorectal cancers (denoted as C); red squares represent mean values in esophageal squamous cell carcinoma (denoted as E); green circles represent mean values in gastric adenocarcinoma (denoted as G). p values for the analysis in adjacent tissue are denoted as P a and for tumor tissue as P t . Statistically significant differences between groups are marked with symbols of the same type (*, #, etc.). Transcriptomic data presented as normalized relative quantities (NRQ).

Techniques Used: Expressing

Systemic concentration of interleukins in gastrointestinal tract cancers: ( a ) IL-4; ( b ) IL-13. Data presented as medians (red triangles) with 95% confidence interval (whiskers) and analyzed using Kruskal-Wallis H test. Statistically significant differences between groups are marked with symbols of the same type (*, #, etc.). CN, healthy controls; EC, patients with esophageal squamous cell carcinoma; CC, patients with adenocarcinoma of gastric cardia; GC, patients with gastric adenocarcinoma; CRC, patients with colorectal cancer. An outlying observation is marked by a closed square.
Figure Legend Snippet: Systemic concentration of interleukins in gastrointestinal tract cancers: ( a ) IL-4; ( b ) IL-13. Data presented as medians (red triangles) with 95% confidence interval (whiskers) and analyzed using Kruskal-Wallis H test. Statistically significant differences between groups are marked with symbols of the same type (*, #, etc.). CN, healthy controls; EC, patients with esophageal squamous cell carcinoma; CC, patients with adenocarcinoma of gastric cardia; GC, patients with gastric adenocarcinoma; CRC, patients with colorectal cancer. An outlying observation is marked by a closed square.

Techniques Used: Concentration Assay

Impact of stimulation with 100 ng/mL of IL-4 or IL-13 on Caco-2 cells–a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0 h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) stimulated with IL-13 at 0 h; ( m ) stimulated with IL-13 at 24 h; ( n ) stimulated with IL-13 at 48 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The rates in interleukin-stimulated cells significantly ( p
Figure Legend Snippet: Impact of stimulation with 100 ng/mL of IL-4 or IL-13 on Caco-2 cells–a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0 h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) stimulated with IL-13 at 0 h; ( m ) stimulated with IL-13 at 24 h; ( n ) stimulated with IL-13 at 48 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The rates in interleukin-stimulated cells significantly ( p

Techniques Used: Wound Healing Assay

Patients’-matched analysis of tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein in CRC ( n = 17); ( b ) IL13 mRNA in CRC ( n = 21); ( c ) IL13Ra1 mRNA in CRC ( n = 21). Data were analyzed as logs using t -test for paired samples and presented as geometric means with 95% confidence interval ( CI ). Mean difference in protein concentration (MD) between groups is accompanied by 95% CI . Transcriptomic data are presented as normalized relative quantities (NRQ). CRC, colorectal cancer; ESCC, esophageal squamous cell carcinoma; GC, gastric adenocarcinoma.
Figure Legend Snippet: Patients’-matched analysis of tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein in CRC ( n = 17); ( b ) IL13 mRNA in CRC ( n = 21); ( c ) IL13Ra1 mRNA in CRC ( n = 21). Data were analyzed as logs using t -test for paired samples and presented as geometric means with 95% confidence interval ( CI ). Mean difference in protein concentration (MD) between groups is accompanied by 95% CI . Transcriptomic data are presented as normalized relative quantities (NRQ). CRC, colorectal cancer; ESCC, esophageal squamous cell carcinoma; GC, gastric adenocarcinoma.

Techniques Used: Expressing, Protein Concentration

2) Product Images from "Pulmonary Immune Responses to Propionibacterium acnes in C57BL/6 and BALB/c Mice"

Article Title: Pulmonary Immune Responses to Propionibacterium acnes in C57BL/6 and BALB/c Mice

Journal:

doi: 10.1165/rcmb.2005-0285OC

Th2 cytokines in the lung after sensitization and challenge with PA. Cytokine levels were measured in lung homogenate protein extracts by ELISA on the indicated days after challenge. IL-4 ( A ) and IL-13 ( B ) levels in C57BL/6 mice sensitized/challenged
Figure Legend Snippet: Th2 cytokines in the lung after sensitization and challenge with PA. Cytokine levels were measured in lung homogenate protein extracts by ELISA on the indicated days after challenge. IL-4 ( A ) and IL-13 ( B ) levels in C57BL/6 mice sensitized/challenged

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay

3) Product Images from "Interleukin-13 and Interleukin-4 Induce Vascular Endothelial Growth Factor Release from Airway Smooth Muscle Cells"

Article Title: Interleukin-13 and Interleukin-4 Induce Vascular Endothelial Growth Factor Release from Airway Smooth Muscle Cells

Journal:

doi: 10.1165/rcmb.2005-0147OC

( A ) Real-time PCR measurements of VEGF mRNA in HASM cells that were left untreated or treated for 6 h with IL-4 (0.3–30 ng/ml) or IL-13 (30 ng/ml). Results are mean ± SE of data from five to nine RNA samples in each case and are normalized
Figure Legend Snippet: ( A ) Real-time PCR measurements of VEGF mRNA in HASM cells that were left untreated or treated for 6 h with IL-4 (0.3–30 ng/ml) or IL-13 (30 ng/ml). Results are mean ± SE of data from five to nine RNA samples in each case and are normalized

Techniques Used: Real-time Polymerase Chain Reaction

Effect of the −460/−152/+405 haplotype on VEGF release from untreated HASM cells (Ctrl) and from cells treated with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean value obtained
Figure Legend Snippet: Effect of the −460/−152/+405 haplotype on VEGF release from untreated HASM cells (Ctrl) and from cells treated with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean value obtained

Techniques Used:

Effect of +405 genotype ( A ) and of −460/−152 genotype ( B ) on VEGF release under basal conditions (Ctrl) and after treatment with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean
Figure Legend Snippet: Effect of +405 genotype ( A ) and of −460/−152 genotype ( B ) on VEGF release under basal conditions (Ctrl) and after treatment with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean

Techniques Used:

( A ) Effects of increasing doses of IL-4 or IL-13 administered for 24 h on VEGF release from HASM cells. Values represent mean ± SE from 4–13 donors. Each donor was studied in duplicate.* P
Figure Legend Snippet: ( A ) Effects of increasing doses of IL-4 or IL-13 administered for 24 h on VEGF release from HASM cells. Values represent mean ± SE from 4–13 donors. Each donor was studied in duplicate.* P

Techniques Used:

Effect of the presence of at least one +936T allele on VEGF release from untreated HASM cells (Ctrl) and from cells treated with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean value obtained for
Figure Legend Snippet: Effect of the presence of at least one +936T allele on VEGF release from untreated HASM cells (Ctrl) and from cells treated with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean value obtained for

Techniques Used:

4) Product Images from "HDAC Inhibitor, CG-745, Enhances the Anti-Cancer Effect of Anti-PD-1 Immune Checkpoint Inhibitor by Modulation of the Immune Microenvironment"

Article Title: HDAC Inhibitor, CG-745, Enhances the Anti-Cancer Effect of Anti-PD-1 Immune Checkpoint Inhibitor by Modulation of the Immune Microenvironment

Journal: Journal of Cancer

doi: 10.7150/jca.44622

CG-745 induces T cell activation and M2 macrophage polarization suppression: (A) Jurkat T cells were cultured with 1 μM of HDACis for 12 hours and harvested for total RNA extraction followed by PCR; (B) Jurkat T cells were cultured with CG-745 in a dose-dependent manner (0, 0.5, 1, 2.5, 5 μM) for 12 hours and harvested for total RNA extraction followed by PCR; (C) Jurkat T cells were stimulated with PMA (50 nM)/Ionomycin (500 nM) or PMA (50 nM)/Ionomycin (500 nM)/CG-745 (1 μM) and harvested for total RNA extraction in a time-dependent manner; (D) THP-1 macrophages were pre-treated with CG-745 (5 μM) for 1 h and polarized during 48 h with either LPS (10 pg/ml)/ IFN-γ (20 ng/ml) or IL-4 (20 ng/ml)/ IL-13 (20 ng/ml). The mRNA level of macrophage markers (M1 markers: CCR7, CD80, CXCL10 , and IL12RB2 ; M2 markers: CCL17, CD163, CD206, and MRC1 ) was measured by real-time RT-PCR and normalized by RPL13A . ns=not significant, * p
Figure Legend Snippet: CG-745 induces T cell activation and M2 macrophage polarization suppression: (A) Jurkat T cells were cultured with 1 μM of HDACis for 12 hours and harvested for total RNA extraction followed by PCR; (B) Jurkat T cells were cultured with CG-745 in a dose-dependent manner (0, 0.5, 1, 2.5, 5 μM) for 12 hours and harvested for total RNA extraction followed by PCR; (C) Jurkat T cells were stimulated with PMA (50 nM)/Ionomycin (500 nM) or PMA (50 nM)/Ionomycin (500 nM)/CG-745 (1 μM) and harvested for total RNA extraction in a time-dependent manner; (D) THP-1 macrophages were pre-treated with CG-745 (5 μM) for 1 h and polarized during 48 h with either LPS (10 pg/ml)/ IFN-γ (20 ng/ml) or IL-4 (20 ng/ml)/ IL-13 (20 ng/ml). The mRNA level of macrophage markers (M1 markers: CCR7, CD80, CXCL10 , and IL12RB2 ; M2 markers: CCL17, CD163, CD206, and MRC1 ) was measured by real-time RT-PCR and normalized by RPL13A . ns=not significant, * p

Techniques Used: Activation Assay, Cell Culture, RNA Extraction, Polymerase Chain Reaction, Quantitative RT-PCR

5) Product Images from "A Novel Bivalent Mannosylated Targeting Ligand Displayed on Nanoparticles Selectively Targets Anti-Inflammatory M2 Macrophages"

Article Title: A Novel Bivalent Mannosylated Targeting Ligand Displayed on Nanoparticles Selectively Targets Anti-Inflammatory M2 Macrophages

Journal: Pharmaceutics

doi: 10.3390/pharmaceutics12030243

( A ) Macrophage expression of the M1 marker iNOS. iNOS mRNA expression in rat peritoneal macrophages (MΦ) treated with IFN-γ or IL-4/IL-13 for 6–72 h was characterized using qRT-PCR ( n = 5). The 65-, 28-, 13- and 19-fold differences between IFN-γ and IL-4/IL-13-treated macrophages were observed at 6, 24, 48 and 72 h, respectively (*: t test, p
Figure Legend Snippet: ( A ) Macrophage expression of the M1 marker iNOS. iNOS mRNA expression in rat peritoneal macrophages (MΦ) treated with IFN-γ or IL-4/IL-13 for 6–72 h was characterized using qRT-PCR ( n = 5). The 65-, 28-, 13- and 19-fold differences between IFN-γ and IL-4/IL-13-treated macrophages were observed at 6, 24, 48 and 72 h, respectively (*: t test, p

Techniques Used: Expressing, Marker, Quantitative RT-PCR

Time course of protein expression levels of the M1 marker iNOS, the M2 markers Arg-1 and MR, and the housekeeping protein actin in activated MPs. Western blot was performed using cell lysates from activated MPs. IFN-γ-treated MPs expressed high levels of iNOS protein at 48 and 72 h, while untreated resting and IL-4/IL-13-treated MPs expressed no or low levels of iNOS protein at 48 and 72 h. Conversely, IL-4/IL-13-treated MPs expressed high MR and Arg-1 levels at 48 and 72 h, while the IFN-γ-treated MPs expressed no or low levels of MR and Arg-1 proteins. The result shown represents one of the independent experiments.
Figure Legend Snippet: Time course of protein expression levels of the M1 marker iNOS, the M2 markers Arg-1 and MR, and the housekeeping protein actin in activated MPs. Western blot was performed using cell lysates from activated MPs. IFN-γ-treated MPs expressed high levels of iNOS protein at 48 and 72 h, while untreated resting and IL-4/IL-13-treated MPs expressed no or low levels of iNOS protein at 48 and 72 h. Conversely, IL-4/IL-13-treated MPs expressed high MR and Arg-1 levels at 48 and 72 h, while the IFN-γ-treated MPs expressed no or low levels of MR and Arg-1 proteins. The result shown represents one of the independent experiments.

Techniques Used: Expressing, Marker, Western Blot

6) Product Images from "Alnus hirsuta (Spach) Rupr. Attenuates Airway Inflammation and Mucus Overproduction in a Murine Model of Ovalbumin-Challenged Asthma"

Article Title: Alnus hirsuta (Spach) Rupr. Attenuates Airway Inflammation and Mucus Overproduction in a Murine Model of Ovalbumin-Challenged Asthma

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2021.614442

AH reduces inflammatory cell number, inflammatory cytokines, eotaxin, MUC5AC, total IgE and OVA-specific IgE in bronchoalveolar lavage fluid or serum of mice 48 h after final OVA challenge. (A) The inflammatory cells were attached on slide and stained with Diff-Quik stain reagent. (B) The levels of IL-4, IL-5, IL-6, IL-13, eotaxin and MUC5AC in the BALF were determined by ELISA. (C) Total IgE and OVA-specific IgE were determined using ELISA. NC: normal control mice; OVA: OVA-challenged mice; DEX: dexamethasone (3 mg/kg) + OVA-challenged mice; AH: AH (50 or 100 mg/kg) + OVA-challenged mice. The values are expressed as the means ± SD (n = 7/group). ## p
Figure Legend Snippet: AH reduces inflammatory cell number, inflammatory cytokines, eotaxin, MUC5AC, total IgE and OVA-specific IgE in bronchoalveolar lavage fluid or serum of mice 48 h after final OVA challenge. (A) The inflammatory cells were attached on slide and stained with Diff-Quik stain reagent. (B) The levels of IL-4, IL-5, IL-6, IL-13, eotaxin and MUC5AC in the BALF were determined by ELISA. (C) Total IgE and OVA-specific IgE were determined using ELISA. NC: normal control mice; OVA: OVA-challenged mice; DEX: dexamethasone (3 mg/kg) + OVA-challenged mice; AH: AH (50 or 100 mg/kg) + OVA-challenged mice. The values are expressed as the means ± SD (n = 7/group). ## p

Techniques Used: Mouse Assay, Staining, Diff-Quik, Enzyme-linked Immunosorbent Assay

Effects of AH on cell viability, inflammatory cytokines (TNF-α, IL-6), Th2 cytokines (IL-4, IL-5 and IL-13) and MUC5AC in TNF-α-stimulated NCI-H292 cells. The (A) cell viability was measured using a WST-1 reagent, and AH was treated with 10, 20, 40 and 80 μg/ml for 24 h. The levels of (B) TNF-α and (C) IL-6 was determined by ELISA. The culture medium were changed RPMI1640 (0.1% FBS) and treated with AH (20, 40 and 80 μg/ml) for 1 h and incubated with TNF-α (30 ng/ml) for 24 min (TNF-α, IL-6). (D) The mRNA levels of IL-4, IL-5, IL-13 and MUC5AC were measured by real-time PCR in TNF-α-stimulated NCI-H292 cells. Cells were treated with AH (20, 40 and 80 μg/ml) for 1 h and incubated with TNF-α (30 ng/ml) for 18 h. NC: Normal control NCI-H292 cells; TNF-α: TNF-α-stimulated NCI-H292 cells; DEX: dexamethasone (20 μg/ml) + TNF-α-stimulated NCI-H292 cells; AH: AH (20, 40 and 80 mg/kg) + TNF-α-stimulated NCI-H292 cells. The values are expressed as the means ± SD (n = 3). ## p
Figure Legend Snippet: Effects of AH on cell viability, inflammatory cytokines (TNF-α, IL-6), Th2 cytokines (IL-4, IL-5 and IL-13) and MUC5AC in TNF-α-stimulated NCI-H292 cells. The (A) cell viability was measured using a WST-1 reagent, and AH was treated with 10, 20, 40 and 80 μg/ml for 24 h. The levels of (B) TNF-α and (C) IL-6 was determined by ELISA. The culture medium were changed RPMI1640 (0.1% FBS) and treated with AH (20, 40 and 80 μg/ml) for 1 h and incubated with TNF-α (30 ng/ml) for 24 min (TNF-α, IL-6). (D) The mRNA levels of IL-4, IL-5, IL-13 and MUC5AC were measured by real-time PCR in TNF-α-stimulated NCI-H292 cells. Cells were treated with AH (20, 40 and 80 μg/ml) for 1 h and incubated with TNF-α (30 ng/ml) for 18 h. NC: Normal control NCI-H292 cells; TNF-α: TNF-α-stimulated NCI-H292 cells; DEX: dexamethasone (20 μg/ml) + TNF-α-stimulated NCI-H292 cells; AH: AH (20, 40 and 80 mg/kg) + TNF-α-stimulated NCI-H292 cells. The values are expressed as the means ± SD (n = 3). ## p

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

7) Product Images from "HMGB1 amplifies ILC2-induced type-2 inflammation and airway smooth muscle remodelling"

Article Title: HMGB1 amplifies ILC2-induced type-2 inflammation and airway smooth muscle remodelling

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1008651

Anti-HMGB1 diminishes type-2 inflammation and ASM growth in neonatal IRF7 -/- mice. IRF7 -/- mice were inoculated with PVM as described in Fig 2 , and treated with anti-HMGB1 or anti-IL-33 as described in S3 Fig . (a) HMGB1 and (b) IL-33 protein expression in BALF. (c) ILC2 numbers in lung. (d) IL-13 protein expression in BALF. (e) ASM area. (f) TGF-β1 expression by ASM cells. Data are representative of n = 2 experiments with 4–8 mice in each group and are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers). Data were analysed by t-test; *, P
Figure Legend Snippet: Anti-HMGB1 diminishes type-2 inflammation and ASM growth in neonatal IRF7 -/- mice. IRF7 -/- mice were inoculated with PVM as described in Fig 2 , and treated with anti-HMGB1 or anti-IL-33 as described in S3 Fig . (a) HMGB1 and (b) IL-33 protein expression in BALF. (c) ILC2 numbers in lung. (d) IL-13 protein expression in BALF. (e) ASM area. (f) TGF-β1 expression by ASM cells. Data are representative of n = 2 experiments with 4–8 mice in each group and are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers). Data were analysed by t-test; *, P

Techniques Used: Mouse Assay, Expressing, Whisker Assay

Activated ILC2s induce ASM proliferation in vitro in an HMGB1-dependent manner. (a-b) Primary ASM cells were isolated from naïve WT neonatal mice and cultured with ILC2s (purified from the lungs of IRF7 -/- mice at 10 dpi) in the presence of various stimuli as indicated. (a) ASM proliferation (indicated by decreasing median fluorescence intensity (MFI)), IL-13, active TGFβ and HMGB1 concentration in the culture supernatant. * Compared to ASM cells cultured in the absence of ILC2s or cytokine stimulation. (b) ASM proliferation in response to IL-13 (3 ng/ml), disulphide HMGB1 (30 ng/ml), or a non-oxidisable mutant HMGB1 (30 ng/ml). * Compared to IL-13-stimulated ASM cells with disulphide-HMGB1-stimulated ASM cells or disulphide-HMGB1-stimulated ASM cells with non-oxidisable mutant HMGB1-stimulated ASM cells as indicated. (c) HMGB1 protein expression in the supernatant of ASM cells cultured in the presence of IL-33 or IL-13 as indicated. (d) Representative micrograph (x1000 magnification) of HMGB1 immunoreactivity (red) in the lung at 10 dpi, arrows indicate expression by ASM cells. Bar, 5 μm. Quantification of HMGB1 expression by ASM cells. For panel a-d, data are representative of one experiment, performed twice with similar results. For panel e, data are representative of n = 2 experiments with four to eight neonates in each group. Data are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a-d) or as mean ± SEM (e [right]). Data were analysed by One-way ANOVA with Dunnett post hoc test (a-d) or Two-way ANOVA with Tukey post hoc test (e ([right]; *, P
Figure Legend Snippet: Activated ILC2s induce ASM proliferation in vitro in an HMGB1-dependent manner. (a-b) Primary ASM cells were isolated from naïve WT neonatal mice and cultured with ILC2s (purified from the lungs of IRF7 -/- mice at 10 dpi) in the presence of various stimuli as indicated. (a) ASM proliferation (indicated by decreasing median fluorescence intensity (MFI)), IL-13, active TGFβ and HMGB1 concentration in the culture supernatant. * Compared to ASM cells cultured in the absence of ILC2s or cytokine stimulation. (b) ASM proliferation in response to IL-13 (3 ng/ml), disulphide HMGB1 (30 ng/ml), or a non-oxidisable mutant HMGB1 (30 ng/ml). * Compared to IL-13-stimulated ASM cells with disulphide-HMGB1-stimulated ASM cells or disulphide-HMGB1-stimulated ASM cells with non-oxidisable mutant HMGB1-stimulated ASM cells as indicated. (c) HMGB1 protein expression in the supernatant of ASM cells cultured in the presence of IL-33 or IL-13 as indicated. (d) Representative micrograph (x1000 magnification) of HMGB1 immunoreactivity (red) in the lung at 10 dpi, arrows indicate expression by ASM cells. Bar, 5 μm. Quantification of HMGB1 expression by ASM cells. For panel a-d, data are representative of one experiment, performed twice with similar results. For panel e, data are representative of n = 2 experiments with four to eight neonates in each group. Data are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a-d) or as mean ± SEM (e [right]). Data were analysed by One-way ANOVA with Dunnett post hoc test (a-d) or Two-way ANOVA with Tukey post hoc test (e ([right]; *, P

Techniques Used: In Vitro, Isolation, Mouse Assay, Cell Culture, Purification, Fluorescence, Concentration Assay, Mutagenesis, Expressing, Whisker Assay

IRF7 deficiency predisposes to Pneumovirus-induced type-2 inflammation and ASM growth in early-life. WT (IRF7 +/+ ) and IRF7 -/- mice were inoculated with PVM at postnatal day 7 and endpoints assessed at the times indicated. (a) IL-13 and IL-5 protein (10 dpi) expression in BALF. (b) ILC2 numbers in the lung (c) Expression of IL-13 (dsRed) and IL-4 (AmCyan) by ILC2 in the lungs of 4C13R IRF7 +/+ and 4C13R IRF7 -/- mice at 10 dpi. Numbers in quadrants show the percentages of gated cells. Percentage of IL-13-expressing ILC2 (right panel). (d) Numbers of IL-13-producing ILC2, CD4 + and CD8 + cells in left lung lobe at 10 dpi. (e) ASM area. (f) Active TGFβ protein expression in BALF. (g) Representative micrograph (x400 magnification) of TGF-β1 immunoreactivity (red) in the lung at 10 dpi. Bars, 20 μm (left panels). Quantification of TGF-β1 expression by ASM cells (right panel). Data are representative of n = 2 experiments with 4 to 8 mice in each group and are presented as mean ± SEM (a [left], b, e) or as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a [right], c [right], d, f). Data were analysed by Two-way ANOVA with Tukey post hoc test (a [left], b, e) or One-way ANOVA with Dunnett post hoc test (a [right], f [right[) or T-test (c [right], d); *, P
Figure Legend Snippet: IRF7 deficiency predisposes to Pneumovirus-induced type-2 inflammation and ASM growth in early-life. WT (IRF7 +/+ ) and IRF7 -/- mice were inoculated with PVM at postnatal day 7 and endpoints assessed at the times indicated. (a) IL-13 and IL-5 protein (10 dpi) expression in BALF. (b) ILC2 numbers in the lung (c) Expression of IL-13 (dsRed) and IL-4 (AmCyan) by ILC2 in the lungs of 4C13R IRF7 +/+ and 4C13R IRF7 -/- mice at 10 dpi. Numbers in quadrants show the percentages of gated cells. Percentage of IL-13-expressing ILC2 (right panel). (d) Numbers of IL-13-producing ILC2, CD4 + and CD8 + cells in left lung lobe at 10 dpi. (e) ASM area. (f) Active TGFβ protein expression in BALF. (g) Representative micrograph (x400 magnification) of TGF-β1 immunoreactivity (red) in the lung at 10 dpi. Bars, 20 μm (left panels). Quantification of TGF-β1 expression by ASM cells (right panel). Data are representative of n = 2 experiments with 4 to 8 mice in each group and are presented as mean ± SEM (a [left], b, e) or as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a [right], c [right], d, f). Data were analysed by Two-way ANOVA with Tukey post hoc test (a [left], b, e) or One-way ANOVA with Dunnett post hoc test (a [right], f [right[) or T-test (c [right], d); *, P

Techniques Used: Mouse Assay, Expressing, Whisker Assay

HMGB1/RAGE axis amplifies ILC2 type-2 cytokine production and contributes to ASM growth in vivo . (a [left]) Lung ILC2s were purified from IRF7 -/- mice at 10 dpi and cultured with IL-2 for 4 days. Representative micrograph (x1000 magnification) of HMGB1 expression (red) in IL-33 stimulated ILC2s. Bar, 5 μm. (a [right]) HMGB1 levels in the culture supernatant after a 4 day culture +/- IL-33 (3 ng/ml) and +/- sIL-13Rα2 (5 μg/ml) as indicated. (b) IL-13 and HMGB1 protein expression by ILC2s. (c) RAGE expression detected by flow cytometry in lung ILC2s. (d) IL-5, IL-9 and IL-13 protein expression by lung ILC2s purified from IRF7 -/- mice cultured in IL-2 +/- difulphide-HMGB1 (30 ng/ml) or non-oxidisable mutant HMGB1 (30 ng/ml) as indicated. * Compared to IL-2-stimulated ILC2s. (e) Lung ILC2s purified from 4C13R IRF7 -/- mice were cultured in IL-2 +/- IL-33, and treated with anti-HMGB1 (Clone 2G7, 5 μg/ml), TLR4 antagonist (LPS-RS 1 μg/ml) and RAGE antagonist (FPS-ZMI; 300 nM) as indicated. DsRed (IL-13) intensity was quantified by flow cytometry. * Compared to IL-2/IL-33-stimulated ILC2s. (f) IL-5 and IL-9 production by ILC2s, treated as described in panel e. (g-m) IRF7 -/- mice were inoculated with PVM as described in Fig 2 , and treated daily from 3–10 dpi with a RAGE antagonist (FPS-ZM1). (g) IL-33 and (h) HMGB1 protein expression in BALF. (i) Lung ILC2s, (j) IL-5 protein in BALF, (k) Eosinophils in BALF, (l) ASM area, and (m) Quantification of TGF-β1 expression by ASM cells. For panel a-f, data are representative of one experiment, performed twice with similar results. For panel g-m, data are representative of n = 2 experiments with 4–9 mice in each group and are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a [right], d, g-m) or as mean ± SEM (b) or as scatter plot (e [right]-f). Data were analysed by One-way ANOVA with Dunnett post hoc test (a, d, e [right]-f) or T-test (g-m); *, P
Figure Legend Snippet: HMGB1/RAGE axis amplifies ILC2 type-2 cytokine production and contributes to ASM growth in vivo . (a [left]) Lung ILC2s were purified from IRF7 -/- mice at 10 dpi and cultured with IL-2 for 4 days. Representative micrograph (x1000 magnification) of HMGB1 expression (red) in IL-33 stimulated ILC2s. Bar, 5 μm. (a [right]) HMGB1 levels in the culture supernatant after a 4 day culture +/- IL-33 (3 ng/ml) and +/- sIL-13Rα2 (5 μg/ml) as indicated. (b) IL-13 and HMGB1 protein expression by ILC2s. (c) RAGE expression detected by flow cytometry in lung ILC2s. (d) IL-5, IL-9 and IL-13 protein expression by lung ILC2s purified from IRF7 -/- mice cultured in IL-2 +/- difulphide-HMGB1 (30 ng/ml) or non-oxidisable mutant HMGB1 (30 ng/ml) as indicated. * Compared to IL-2-stimulated ILC2s. (e) Lung ILC2s purified from 4C13R IRF7 -/- mice were cultured in IL-2 +/- IL-33, and treated with anti-HMGB1 (Clone 2G7, 5 μg/ml), TLR4 antagonist (LPS-RS 1 μg/ml) and RAGE antagonist (FPS-ZMI; 300 nM) as indicated. DsRed (IL-13) intensity was quantified by flow cytometry. * Compared to IL-2/IL-33-stimulated ILC2s. (f) IL-5 and IL-9 production by ILC2s, treated as described in panel e. (g-m) IRF7 -/- mice were inoculated with PVM as described in Fig 2 , and treated daily from 3–10 dpi with a RAGE antagonist (FPS-ZM1). (g) IL-33 and (h) HMGB1 protein expression in BALF. (i) Lung ILC2s, (j) IL-5 protein in BALF, (k) Eosinophils in BALF, (l) ASM area, and (m) Quantification of TGF-β1 expression by ASM cells. For panel a-f, data are representative of one experiment, performed twice with similar results. For panel g-m, data are representative of n = 2 experiments with 4–9 mice in each group and are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a [right], d, g-m) or as mean ± SEM (b) or as scatter plot (e [right]-f). Data were analysed by One-way ANOVA with Dunnett post hoc test (a, d, e [right]-f) or T-test (g-m); *, P

Techniques Used: In Vivo, Purification, Mouse Assay, Cell Culture, Expressing, Flow Cytometry, Mutagenesis, Whisker Assay

Secondary Pneumovirus infection promotes an asthma-like pathology in IRF7-deficienct mice. WT (IRF7 +/+ ) and IRF7 -/- mice were inoculated with PVM at postnatal day 7 and re-infected 6 weeks later. Samples were collected prior to re-infection (t = 0) and at 3, 7 and 21 dpi. (a) Representative micrograph (x400 magnification) of α-smooth muscle actin immunoreactivity (red) in the lung at 7 dpi and ASM area quantification. Bars, 20 μm. (b) AHR at 30 mg/ml of methacholine. (c) IL-13 protein levels in BALF. (d) Total eosinophil numbers in BALF. (e) Mucus-producing airway epithelial cells (AECs). (f) IL-33 (left) and HMGB1 (right) protein expression in BALF at 3 dpi. Data are representative of n = 2 experiments with 4–9 mice in each group and are presented as mean ± SEM (a ([right], b—e) or as box-and-whisker plots showing quartiles (boxes) and range (whiskers; f). Data were analyzed by Two-way ANOVA with Tukey post hoc test (a ([right], b—e) or One-way ANOVA with Dunnett post hoc test (f); *, P
Figure Legend Snippet: Secondary Pneumovirus infection promotes an asthma-like pathology in IRF7-deficienct mice. WT (IRF7 +/+ ) and IRF7 -/- mice were inoculated with PVM at postnatal day 7 and re-infected 6 weeks later. Samples were collected prior to re-infection (t = 0) and at 3, 7 and 21 dpi. (a) Representative micrograph (x400 magnification) of α-smooth muscle actin immunoreactivity (red) in the lung at 7 dpi and ASM area quantification. Bars, 20 μm. (b) AHR at 30 mg/ml of methacholine. (c) IL-13 protein levels in BALF. (d) Total eosinophil numbers in BALF. (e) Mucus-producing airway epithelial cells (AECs). (f) IL-33 (left) and HMGB1 (right) protein expression in BALF at 3 dpi. Data are representative of n = 2 experiments with 4–9 mice in each group and are presented as mean ± SEM (a ([right], b—e) or as box-and-whisker plots showing quartiles (boxes) and range (whiskers; f). Data were analyzed by Two-way ANOVA with Tukey post hoc test (a ([right], b—e) or One-way ANOVA with Dunnett post hoc test (f); *, P

Techniques Used: Infection, Mouse Assay, Expressing, Whisker Assay

8) Product Images from "EP4 signalling is essential for controlling islet inflammation by causing a shift in macrophage polarization in obesity/type 2 diabetes"

Article Title: EP4 signalling is essential for controlling islet inflammation by causing a shift in macrophage polarization in obesity/type 2 diabetes

Journal: Diabetes & Vascular Disease Research

doi: 10.1177/1479164120945675

EP4 receptor-activation in M1 macrophages inhibits inflammatory activation and recovers impaired insulin production of pancreatic β-cells. (a) Peritoneal macrophages from wild type (WT) mice were incubated with 1 μg/mL of LPS or with 20 ng/mL of IL-4 and IL-13, as well as with 1 μM of EP4 agonist (ONO-AE1-329) or vehicle. After 8 h, the secretion of TNFα, IL-6 and IL-10 was measured. (b)–(d) MIN6 cells were co-cultured with M1-polarized peritoneal macrophages with or without EP4 agonist. After 14 h of incubation, macrophages were removed and MIN6 cells were incubated for another 24 h. The mRNA levels of (b) insulin, Pdx1 , (c) TNFα, TLR4 in MIN6 cells and the protein levels of (d) TNFα and IL-6 in culture supernatant were measured. All values are the mean scores ± SEM ( n = 6 for each condition). * p
Figure Legend Snippet: EP4 receptor-activation in M1 macrophages inhibits inflammatory activation and recovers impaired insulin production of pancreatic β-cells. (a) Peritoneal macrophages from wild type (WT) mice were incubated with 1 μg/mL of LPS or with 20 ng/mL of IL-4 and IL-13, as well as with 1 μM of EP4 agonist (ONO-AE1-329) or vehicle. After 8 h, the secretion of TNFα, IL-6 and IL-10 was measured. (b)–(d) MIN6 cells were co-cultured with M1-polarized peritoneal macrophages with or without EP4 agonist. After 14 h of incubation, macrophages were removed and MIN6 cells were incubated for another 24 h. The mRNA levels of (b) insulin, Pdx1 , (c) TNFα, TLR4 in MIN6 cells and the protein levels of (d) TNFα and IL-6 in culture supernatant were measured. All values are the mean scores ± SEM ( n = 6 for each condition). * p

Techniques Used: Activation Assay, Mouse Assay, Incubation, Cell Culture

9) Product Images from "Characterization of allergic inflammation in chronic uterine cervicitis"

Article Title: Characterization of allergic inflammation in chronic uterine cervicitis

Journal: Clinical and Experimental Immunology

doi: 10.1093/cei/uxab026

Th2 cell and cytokine in VLF. VLF was obtained from 80 NC subjects and 80 CC patients, and analyzed by ELISA and FACC. ( A ) boxplots show CD4 + T-cell-derived cytokine levels in VLF. ( B ) FSC/SSC plots. ( C ) CD3 + CD4 + T cells were gated. ( D ) Gated FACC plots show frequency of Th2 cells (IL-4 + , IL-5 + , and IL-13 + ) and Th1 cells (IFN-γ + ). ( E ) violin plots show summarized cell counts of Th2 and Th1 cells in VLF. The data of boxplots are presented as median (IQR). ∗∗∗ P
Figure Legend Snippet: Th2 cell and cytokine in VLF. VLF was obtained from 80 NC subjects and 80 CC patients, and analyzed by ELISA and FACC. ( A ) boxplots show CD4 + T-cell-derived cytokine levels in VLF. ( B ) FSC/SSC plots. ( C ) CD3 + CD4 + T cells were gated. ( D ) Gated FACC plots show frequency of Th2 cells (IL-4 + , IL-5 + , and IL-13 + ) and Th1 cells (IFN-γ + ). ( E ) violin plots show summarized cell counts of Th2 and Th1 cells in VLF. The data of boxplots are presented as median (IQR). ∗∗∗ P

Techniques Used: Enzyme-linked Immunosorbent Assay, Derivative Assay

Inducing VLF T cells by DME. CD4 + T cells were isolated from VLF samples collected from 10 CCp patients and 10 CCn patients, and respectively exposed to DME (saline or BSA were used as controls) in the culture (10 6 cells/ml) overnight in the presence of DCs. The supernatant was harvested, analyzed by ELISA. Boxplots show the levels of IL-4 ( A ), IL-5 ( B ), IL-13 ( C ), IL-25 ( D ), IFN-γ ( E ), and IL-17 ( F ) in supernatant. ( G ) cell counts after overnight culture. ∗∗∗ P
Figure Legend Snippet: Inducing VLF T cells by DME. CD4 + T cells were isolated from VLF samples collected from 10 CCp patients and 10 CCn patients, and respectively exposed to DME (saline or BSA were used as controls) in the culture (10 6 cells/ml) overnight in the presence of DCs. The supernatant was harvested, analyzed by ELISA. Boxplots show the levels of IL-4 ( A ), IL-5 ( B ), IL-13 ( C ), IL-25 ( D ), IFN-γ ( E ), and IL-17 ( F ) in supernatant. ( G ) cell counts after overnight culture. ∗∗∗ P

Techniques Used: Isolation, Enzyme-linked Immunosorbent Assay

10) Product Images from "Dual blockade of IL‐4 and IL‐13 with dupilumab, an IL‐4Rα antibody, is required to broadly inhibit type 2 inflammation, et al. Dual blockade of IL‐4 and IL‐13 with dupilumab, an IL‐4Rα antibody, is required to broadly inhibit type 2 inflammation"

Article Title: Dual blockade of IL‐4 and IL‐13 with dupilumab, an IL‐4Rα antibody, is required to broadly inhibit type 2 inflammation, et al. Dual blockade of IL‐4 and IL‐13 with dupilumab, an IL‐4Rα antibody, is required to broadly inhibit type 2 inflammation

Journal: Allergy

doi: 10.1111/all.14151

IL‐4 dominates IgE‐dependent human mast cell responses. A, In vitro differentiated human mast cells were treated for 2 d with increasing concentrations of IL‐4 or IL‐13, or serial dilutions of IL‐4Rα Ab or a control (ctrl) IgG in the presence of 50 pmol/L IL‐4 or 1 nmol/L IL‐13, and FcεRIα surface expression was assessed by flow cytometry. Error bars represent SD from samples run in triplicate. Black arrows indicate cytokine concentrations used for the corresponding blocking assays. IC50 is shown. Data are representative of at least three independent experiments using different donors. B, Heatmap of cytokine‐ and chemokine‐related genes upon IgE crosslinking (sensitization with two different Fel d 1‐specific IgE followed by activation with Fel d 1 antigen) of human mast cells. 51 cytokine/chemokine‐related genes were induced (FDR ≤ 0.05; |FC|≥2) by either IL‐4, IL‐13, Fel d 1‐specific IgE (Fel d 1 IgE)+Fel d 1, IL‐4 + Fel d 1 IgE + Fel d 1, or IL‐13 + Fel d 1 IgE + Fel d 1. The color gradient represents fold‐change values comparing each treatment to the control samples. C, mRNA expression levels measured by real‐time qPCR and expressed relative to GAPDH mRNA expression; *= P
Figure Legend Snippet: IL‐4 dominates IgE‐dependent human mast cell responses. A, In vitro differentiated human mast cells were treated for 2 d with increasing concentrations of IL‐4 or IL‐13, or serial dilutions of IL‐4Rα Ab or a control (ctrl) IgG in the presence of 50 pmol/L IL‐4 or 1 nmol/L IL‐13, and FcεRIα surface expression was assessed by flow cytometry. Error bars represent SD from samples run in triplicate. Black arrows indicate cytokine concentrations used for the corresponding blocking assays. IC50 is shown. Data are representative of at least three independent experiments using different donors. B, Heatmap of cytokine‐ and chemokine‐related genes upon IgE crosslinking (sensitization with two different Fel d 1‐specific IgE followed by activation with Fel d 1 antigen) of human mast cells. 51 cytokine/chemokine‐related genes were induced (FDR ≤ 0.05; |FC|≥2) by either IL‐4, IL‐13, Fel d 1‐specific IgE (Fel d 1 IgE)+Fel d 1, IL‐4 + Fel d 1 IgE + Fel d 1, or IL‐13 + Fel d 1 IgE + Fel d 1. The color gradient represents fold‐change values comparing each treatment to the control samples. C, mRNA expression levels measured by real‐time qPCR and expressed relative to GAPDH mRNA expression; *= P

Techniques Used: In Vitro, Expressing, Flow Cytometry, Blocking Assay, Activation Assay, Real-time Polymerase Chain Reaction

Dual IL‐4/IL‐13 blockade prevents HDM‐induced impairment of lung function. Il4ra hu/hu Il4 hu/hu mice were exposed to saline or HDM for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIg G or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. A, Representative PAS‐stained lung histology sections of a mouse not exposed to HDM, an untreated HDM‐exposed mouse, a HDM‐exposed mouse treated with ctrl hIgG, ctrl mIgG, IL‐4Rα Ab, IL‐4 Ab, or mIL‐13Rα2‐Fc. White arrows show examples of epithelial cell nuclei. Black arrows indicate examples of PAS‐positive goblet cells; bar, 25 µm. B, GCM quantification. PAS‐positive goblet cells and total epithelial cells were counted in a millimeter length of primary bronchus (approximately 100 epithelial cells), and GCM was expressed as the percentage of PAS‐positive epithelial cells. †= P
Figure Legend Snippet: Dual IL‐4/IL‐13 blockade prevents HDM‐induced impairment of lung function. Il4ra hu/hu Il4 hu/hu mice were exposed to saline or HDM for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIg G or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. A, Representative PAS‐stained lung histology sections of a mouse not exposed to HDM, an untreated HDM‐exposed mouse, a HDM‐exposed mouse treated with ctrl hIgG, ctrl mIgG, IL‐4Rα Ab, IL‐4 Ab, or mIL‐13Rα2‐Fc. White arrows show examples of epithelial cell nuclei. Black arrows indicate examples of PAS‐positive goblet cells; bar, 25 µm. B, GCM quantification. PAS‐positive goblet cells and total epithelial cells were counted in a millimeter length of primary bronchus (approximately 100 epithelial cells), and GCM was expressed as the percentage of PAS‐positive epithelial cells. †= P

Techniques Used: Mouse Assay, Staining

Dual IL‐4/IL‐13 blockade prevents infiltration of eosinophils into the lungs of HDM‐exposed mice and their activation. A, Il4ra hu/hu Il4 hu/hu mice were exposed to saline or HDM for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. On day 30, mice were injected with anti‐CD45 to label all circulating immune cells and killed after 5 min. Lungs were harvested and dissociated for flow cytometric analysis of circulating lung and lung tissue eosinophils. Representative flow cytometric plots are shown (left panels). Circulating lung (middle panel) and lung tissue (right panel) eosinophils are reported as a frequency of live cells in the suspension. Each symbol represents one mouse. n ≥ 5 mice per group. Saline (white boxes) or HDM (shaded boxes). †= P
Figure Legend Snippet: Dual IL‐4/IL‐13 blockade prevents infiltration of eosinophils into the lungs of HDM‐exposed mice and their activation. A, Il4ra hu/hu Il4 hu/hu mice were exposed to saline or HDM for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. On day 30, mice were injected with anti‐CD45 to label all circulating immune cells and killed after 5 min. Lungs were harvested and dissociated for flow cytometric analysis of circulating lung and lung tissue eosinophils. Representative flow cytometric plots are shown (left panels). Circulating lung (middle panel) and lung tissue (right panel) eosinophils are reported as a frequency of live cells in the suspension. Each symbol represents one mouse. n ≥ 5 mice per group. Saline (white boxes) or HDM (shaded boxes). †= P

Techniques Used: Mouse Assay, Activation Assay, Injection

Both IL‐4 and IL‐13 drive airway inflammation in mice. A, Il4ra hu/hu Il4 hu/hu mice were exposed to PBS, human IL‐4 or human IL‐13 for 12 d. B‐F, On day 12, mice were injected with anti‐CD45 to label all circulating immune cells and killed after 5 min. Lungs were harvested and dissociated for flow cytometric analysis of circulating lung and lung tissue CD45 + cells (B), CD4 T cells (C), alveolar macrophages (D), neutrophils (E), and eosinophils (F). Circulating lung and lung tissue cell populations are reported as a frequency of live cells in the suspension. Each symbol represents one mouse. n = 5 mice per group. PBS (white boxes), human IL‐4 (dark blue boxes), or human IL‐13 (light blue boxes). G, Cytokine and chemokine gene heatmap. Fold change relative to control mice of lung tissue mRNA expression levels was measured by real‐time qPCR and expressed relative to β‐actin (ACTB) mRNA expression. H, Left , representative PAS‐stained lung histology sections of a mouse not exposed to cytokines (left panel), an IL‐4‐exposed mouse (middle panel), or an IL‐13‐exposed mouse (right panel). White arrows show examples of epithelial cell nuclei. Black arrows indicate examples of PAS‐positive goblet cells; Bar, 25 µm. Right , GCM quantification. PAS‐positive goblet cells and total epithelial cells were counted in a millimeter length of primary bronchus (approximately 100 epithelial cells), and GCM was expressed as the percentage of PAS‐positive epithelial cells. †,*= P
Figure Legend Snippet: Both IL‐4 and IL‐13 drive airway inflammation in mice. A, Il4ra hu/hu Il4 hu/hu mice were exposed to PBS, human IL‐4 or human IL‐13 for 12 d. B‐F, On day 12, mice were injected with anti‐CD45 to label all circulating immune cells and killed after 5 min. Lungs were harvested and dissociated for flow cytometric analysis of circulating lung and lung tissue CD45 + cells (B), CD4 T cells (C), alveolar macrophages (D), neutrophils (E), and eosinophils (F). Circulating lung and lung tissue cell populations are reported as a frequency of live cells in the suspension. Each symbol represents one mouse. n = 5 mice per group. PBS (white boxes), human IL‐4 (dark blue boxes), or human IL‐13 (light blue boxes). G, Cytokine and chemokine gene heatmap. Fold change relative to control mice of lung tissue mRNA expression levels was measured by real‐time qPCR and expressed relative to β‐actin (ACTB) mRNA expression. H, Left , representative PAS‐stained lung histology sections of a mouse not exposed to cytokines (left panel), an IL‐4‐exposed mouse (middle panel), or an IL‐13‐exposed mouse (right panel). White arrows show examples of epithelial cell nuclei. Black arrows indicate examples of PAS‐positive goblet cells; Bar, 25 µm. Right , GCM quantification. PAS‐positive goblet cells and total epithelial cells were counted in a millimeter length of primary bronchus (approximately 100 epithelial cells), and GCM was expressed as the percentage of PAS‐positive epithelial cells. †,*= P

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

Dual IL‐4/IL‐13 blockade broadly inhibits chemokine and type 2 proinflammatory cytokine expression in the lungs of HDM‐exposed mice. A, Volcano plot of HDM signature. After 4 wk of HDM exposure, significant changes were detected in 873 transcripts that were differentially expressed in the lungs of HDM‐exposed mice compared with the lungs of mice not exposed to HDM (adjusted P ‐value ≤ .05; |fold change|≥2). B and D, Venn diagrams of HDM signature vs IL‐4Rα Ab (B), IL‐4 Ab (C), and mIL‐13Rα2‐Fc (D) signatures (adjusted P ‐value ≤ .05; |fold change|≥2). Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. Treatment signatures correspond to statistically significant changes in gene expression compared to mice receiving corresponding ctrl IgG. ↓↑, treatment‐responsive genes inversely differentially regulated in the HDM signature. E, Heatmap of cytokine/chemokine‐related genes. 29 cytokine/chemokine‐related genes from the HDM signature were inversely regulated by IL‐4Rα Ab, IL‐4 Ab, and/or mouse IL‐13Rα2‐Fc. The color gradient represents z‐score of the gene expression values in various treatment and control samples. F, Il4ra hu/hu Il4 hu/hu mice were exposed to saline (white boxes) or HDM (shaded boxes) for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. At the end of the study, lung tissue mRNA expression levels were measured by real‐time qPCR and are expressed relative to β‐actin ( ACTB ) mRNA expression. Each symbol represents one mouse. n ≥ 5 mice per group. †= P
Figure Legend Snippet: Dual IL‐4/IL‐13 blockade broadly inhibits chemokine and type 2 proinflammatory cytokine expression in the lungs of HDM‐exposed mice. A, Volcano plot of HDM signature. After 4 wk of HDM exposure, significant changes were detected in 873 transcripts that were differentially expressed in the lungs of HDM‐exposed mice compared with the lungs of mice not exposed to HDM (adjusted P ‐value ≤ .05; |fold change|≥2). B and D, Venn diagrams of HDM signature vs IL‐4Rα Ab (B), IL‐4 Ab (C), and mIL‐13Rα2‐Fc (D) signatures (adjusted P ‐value ≤ .05; |fold change|≥2). Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. Treatment signatures correspond to statistically significant changes in gene expression compared to mice receiving corresponding ctrl IgG. ↓↑, treatment‐responsive genes inversely differentially regulated in the HDM signature. E, Heatmap of cytokine/chemokine‐related genes. 29 cytokine/chemokine‐related genes from the HDM signature were inversely regulated by IL‐4Rα Ab, IL‐4 Ab, and/or mouse IL‐13Rα2‐Fc. The color gradient represents z‐score of the gene expression values in various treatment and control samples. F, Il4ra hu/hu Il4 hu/hu mice were exposed to saline (white boxes) or HDM (shaded boxes) for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. At the end of the study, lung tissue mRNA expression levels were measured by real‐time qPCR and are expressed relative to β‐actin ( ACTB ) mRNA expression. Each symbol represents one mouse. n ≥ 5 mice per group. †= P

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

The IL‐4Rα antibody dupilumab blocks human IL‐4 and IL‐13/IL‐13Rα1 complex binding to IL‐4Rα and prevents IL‐4 and IL‐13 signaling through Type I and Type II receptors. A, Type I signaling: Ramos.2G6.4C10/STAT3/Luciferase cells were treated with increasing concentrations of IL‐4, or serial dilutions of IL‐4Rα Ab, IL‐4 Ab, or an isotype control antibody (ctrl IgG) in the presence of 300 pmol/L IL‐4. B and C, Type II signaling: HEK293/STAT6/Luciferase cells were treated with increasing concentrations of IL‐4 (B), IL‐13 (C), or serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or ctrl IgG in the presence of 10 pmol/L IL‐4 (B) or 25 pmol/L IL‐13 (C). Inhibition of IL‐4‐ or IL‐13‐mediated signaling was quantified by measuring luminescence (RLU). Error bars represent SD from samples run in triplicate. Black arrows indicate cytokine concentrations used for the corresponding blocking assays. IC50 is shown. Data are representative of at least two independent experiments. D, Schematic diagram representing APC‐Th2 coculture cell interactions in the presence of SEB. E, Cocultures of B cells with Th2 cells in the presence of SEB were coincubated with serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or control antibody (ctrl IgG) for 2 d. Inhibition of CD23 induction at the surface of B cells was monitored by flow cytometry. F, Cocultures of moDCs with Th2 cells in the presence of SEB were coincubated with serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or ctrl IgG for 2 d. Inhibition of IL‐12p70 release from moDCs was determined by MSD. Error bars represent SD from samples run in duplicate. All data are representative of at least three independent experiments using different donors. G, To assess the ability of IL‐4Rα Ab to block IL‐4 and IL‐13/IL‐13Rα1 binding to IL‐4Rα, human IL‐4, human IL‐13, human IL‐13Rα1.hFc, or IL‐13/IL‐13Rα1.hFc complex was injected over human IL‐4Rα surfaces that were prebound with IL‐4Rα Ab or a control antibody (ctrl IgG). Graph presents surface‐bound proteins in resonance units (RU). H, Schematic diagram showing dupilumab (IL‐4Rα Ab) mechanism of action
Figure Legend Snippet: The IL‐4Rα antibody dupilumab blocks human IL‐4 and IL‐13/IL‐13Rα1 complex binding to IL‐4Rα and prevents IL‐4 and IL‐13 signaling through Type I and Type II receptors. A, Type I signaling: Ramos.2G6.4C10/STAT3/Luciferase cells were treated with increasing concentrations of IL‐4, or serial dilutions of IL‐4Rα Ab, IL‐4 Ab, or an isotype control antibody (ctrl IgG) in the presence of 300 pmol/L IL‐4. B and C, Type II signaling: HEK293/STAT6/Luciferase cells were treated with increasing concentrations of IL‐4 (B), IL‐13 (C), or serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or ctrl IgG in the presence of 10 pmol/L IL‐4 (B) or 25 pmol/L IL‐13 (C). Inhibition of IL‐4‐ or IL‐13‐mediated signaling was quantified by measuring luminescence (RLU). Error bars represent SD from samples run in triplicate. Black arrows indicate cytokine concentrations used for the corresponding blocking assays. IC50 is shown. Data are representative of at least two independent experiments. D, Schematic diagram representing APC‐Th2 coculture cell interactions in the presence of SEB. E, Cocultures of B cells with Th2 cells in the presence of SEB were coincubated with serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or control antibody (ctrl IgG) for 2 d. Inhibition of CD23 induction at the surface of B cells was monitored by flow cytometry. F, Cocultures of moDCs with Th2 cells in the presence of SEB were coincubated with serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or ctrl IgG for 2 d. Inhibition of IL‐12p70 release from moDCs was determined by MSD. Error bars represent SD from samples run in duplicate. All data are representative of at least three independent experiments using different donors. G, To assess the ability of IL‐4Rα Ab to block IL‐4 and IL‐13/IL‐13Rα1 binding to IL‐4Rα, human IL‐4, human IL‐13, human IL‐13Rα1.hFc, or IL‐13/IL‐13Rα1.hFc complex was injected over human IL‐4Rα surfaces that were prebound with IL‐4Rα Ab or a control antibody (ctrl IgG). Graph presents surface‐bound proteins in resonance units (RU). H, Schematic diagram showing dupilumab (IL‐4Rα Ab) mechanism of action

Techniques Used: Binding Assay, Luciferase, Inhibition, Blocking Assay, Flow Cytometry, Injection

11) Product Images from "Effect of chemically modified IL-13 short interfering RNA on development of airway hyperresponsiveness in mice"

Article Title: Effect of chemically modified IL-13 short interfering RNA on development of airway hyperresponsiveness in mice

Journal: The Journal of Allergy and Clinical Immunology

doi: 10.1016/j.jaci.2007.08.029

Reduction of IL-13 mRNA (A) and cytokine (B) by siRNA in BMMCs. BMMCs were electroporated with 100 nmol/L siRNA before stimulation with anti-OVA IgE and OVA. For A , levels of IL-13 and IL-4 mRNA measured by RT-PCR were normalized to GAPDH. For B , cell media was harvested 24 hours after activation, and IL-13 cytokine levels were measured by ELISA and normalized to control siRNA-treated samples. The results are from 3 independent experiments. ∗ P
Figure Legend Snippet: Reduction of IL-13 mRNA (A) and cytokine (B) by siRNA in BMMCs. BMMCs were electroporated with 100 nmol/L siRNA before stimulation with anti-OVA IgE and OVA. For A , levels of IL-13 and IL-4 mRNA measured by RT-PCR were normalized to GAPDH. For B , cell media was harvested 24 hours after activation, and IL-13 cytokine levels were measured by ELISA and normalized to control siRNA-treated samples. The results are from 3 independent experiments. ∗ P

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Activation Assay, Enzyme-linked Immunosorbent Assay

Lack of effect of siRNA on airway inflammation in sensitized and challenged mice. Tissue was obtained from mice that were challenged only (A) , sensitized and challenged with antigen (B) , or sensitized and challenged and exposed to either a control siRNA targeted to GFP (C) or an IL-13-specific siRNA (D) according to the protocol outlined in Fig 3 , A . Sections were stained with PAS with HE counterstain to identify mucus-containing cells and inflammatory cell infiltrate.
Figure Legend Snippet: Lack of effect of siRNA on airway inflammation in sensitized and challenged mice. Tissue was obtained from mice that were challenged only (A) , sensitized and challenged with antigen (B) , or sensitized and challenged and exposed to either a control siRNA targeted to GFP (C) or an IL-13-specific siRNA (D) according to the protocol outlined in Fig 3 , A . Sections were stained with PAS with HE counterstain to identify mucus-containing cells and inflammatory cell infiltrate.

Techniques Used: Mouse Assay, Staining

Chemically modified IL-13 siRNAs (A) and their potencies in vitro (B) . Anti-IL-13 siRNA activities were tested in vitro using the cell-based dual-luciferase reporter assay described in the text. Each siRNA was tested in triplicate. The ratio of renilla to firefly luciferase activity was normalized to that observed for each reporter plasmid after transfection with a control siRNA of the same stabilization motif.
Figure Legend Snippet: Chemically modified IL-13 siRNAs (A) and their potencies in vitro (B) . Anti-IL-13 siRNA activities were tested in vitro using the cell-based dual-luciferase reporter assay described in the text. Each siRNA was tested in triplicate. The ratio of renilla to firefly luciferase activity was normalized to that observed for each reporter plasmid after transfection with a control siRNA of the same stabilization motif.

Techniques Used: Modification, In Vitro, Luciferase, Reporter Assay, Activity Assay, Plasmid Preparation, Transfection

Reduction of airway resistance in sensitized and challenged mice after treatment with IL-13 siRNA. A, Schematic diagram of allergen sensitization and challenge and siRNA administration protocol. B, IL-13 siRNA decreased airway resistance in sensitized and challenged mice. With increasing MCh challenges, sensitized and challenged mice exhibited increases in airway resistance compared with challenged only mice. Treatment with IL-13 siRNA decreased airway resistance in the mice. n = 8, ∗ P
Figure Legend Snippet: Reduction of airway resistance in sensitized and challenged mice after treatment with IL-13 siRNA. A, Schematic diagram of allergen sensitization and challenge and siRNA administration protocol. B, IL-13 siRNA decreased airway resistance in sensitized and challenged mice. With increasing MCh challenges, sensitized and challenged mice exhibited increases in airway resistance compared with challenged only mice. Treatment with IL-13 siRNA decreased airway resistance in the mice. n = 8, ∗ P

Techniques Used: Mouse Assay

12) Product Images from "Schizophyllum commune induces IL-17-mediated neutrophilic airway inflammation in OVA-induced asthma model mice"

Article Title: Schizophyllum commune induces IL-17-mediated neutrophilic airway inflammation in OVA-induced asthma model mice

Journal: Scientific Reports

doi: 10.1038/s41598-019-55836-x

S. commune induces Th17-related cytokine production in OVA-induced asthma model mice. IFN-γ, IL-4, IL-13, IL-17A, and IL-17F levels in BAL fluid were measured by ELISA. All results are expressed as mean ± SEM (n = 7 mice/group). Each symbol represents an individual sample. *P
Figure Legend Snippet: S. commune induces Th17-related cytokine production in OVA-induced asthma model mice. IFN-γ, IL-4, IL-13, IL-17A, and IL-17F levels in BAL fluid were measured by ELISA. All results are expressed as mean ± SEM (n = 7 mice/group). Each symbol represents an individual sample. *P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

13) Product Images from "Th2 Allergic Immune Response to Inhaled Fungal Antigens is Modulated By TLR-4-Independent Bacterial Products"

Article Title: Th2 Allergic Immune Response to Inhaled Fungal Antigens is Modulated By TLR-4-Independent Bacterial Products

Journal:

doi: 10.1002/eji.200838932

C. albicans (Ca) induces Th2 cytokine production while P. aeruginosa (Pa) induces Th1 and Th17 cytokine profile. (A) IL-4, (B) IL-5, (C) IL-13, (D) IFNγ and (E) IL-17 production by ex vivo restimulated splenic CD4 T cells after in vivo exposure
Figure Legend Snippet: C. albicans (Ca) induces Th2 cytokine production while P. aeruginosa (Pa) induces Th1 and Th17 cytokine profile. (A) IL-4, (B) IL-5, (C) IL-13, (D) IFNγ and (E) IL-17 production by ex vivo restimulated splenic CD4 T cells after in vivo exposure

Techniques Used: Ex Vivo, In Vivo

14) Product Images from "Inflammatory M1-like macrophages polarized by NK-4 undergo enhanced phenotypic switching to an anti-inflammatory M2-like phenotype upon co-culture with apoptotic cells"

Article Title: Inflammatory M1-like macrophages polarized by NK-4 undergo enhanced phenotypic switching to an anti-inflammatory M2-like phenotype upon co-culture with apoptotic cells

Journal: Journal of Inflammation (London, England)

doi: 10.1186/s12950-020-00267-z

NK-4 polarizes THP-1 macrophages toward an M1-like phenotype based on morphology and cytokine production. THP-1 macrophages were cultured with various concentrations of NK-4 and/or with 20 ng/ml IL-4/IL-13 for 2–3 days. Morphological features were assessed by light microscopy ( a ). Levels of TNF-α at day 3 were measured in culture supernatants by ELISA ( b ). Cell numbers at day 3 were determined by cell counting kit-8 ( c ). 3 days post-NK-4 treatment, cells were stimulated with 1 μg/ml LPS for 2 days. Levels of TNF-α (d) and IL-10 ( e ) were measured in the culture supernatants by ELISA. THP-1 macrophages were stimulated with various concentrations of LPS (f), Pam3CSK4 ( g ), or poly(I:C) ( h ) in the presence or absence of 4 μM NK-4 for 1–3 days. Levels of TNF-α in the culture supernatants on day 1 ( f, g ) and day 3 ( h ) were determined by ELISA. Graphs show the mean ± S.D. of triplicate cultures and are representative of three independent experiments with similar results. * p
Figure Legend Snippet: NK-4 polarizes THP-1 macrophages toward an M1-like phenotype based on morphology and cytokine production. THP-1 macrophages were cultured with various concentrations of NK-4 and/or with 20 ng/ml IL-4/IL-13 for 2–3 days. Morphological features were assessed by light microscopy ( a ). Levels of TNF-α at day 3 were measured in culture supernatants by ELISA ( b ). Cell numbers at day 3 were determined by cell counting kit-8 ( c ). 3 days post-NK-4 treatment, cells were stimulated with 1 μg/ml LPS for 2 days. Levels of TNF-α (d) and IL-10 ( e ) were measured in the culture supernatants by ELISA. THP-1 macrophages were stimulated with various concentrations of LPS (f), Pam3CSK4 ( g ), or poly(I:C) ( h ) in the presence or absence of 4 μM NK-4 for 1–3 days. Levels of TNF-α in the culture supernatants on day 1 ( f, g ) and day 3 ( h ) were determined by ELISA. Graphs show the mean ± S.D. of triplicate cultures and are representative of three independent experiments with similar results. * p

Techniques Used: Cell Culture, Light Microscopy, Enzyme-linked Immunosorbent Assay, Cell Counting

15) Product Images from "M2 Macrophages Promote HCC Cells Invasion and Migration via miR-149-5p/MMP9 Signaling"

Article Title: M2 Macrophages Promote HCC Cells Invasion and Migration via miR-149-5p/MMP9 Signaling

Journal: Journal of Cancer

doi: 10.7150/jca.35444

M2 macrophages can increase the invasion and migration capacity of HCC cells. (A) qRT-PCR was applied in macrophages using primers for M2 markers (CD206, CD163, Arginase-1,TGFβ, IL-10) after induced by IL-4 and IL-13. (B) Pattern diagram for co-culture, invasion and wound healing migration system. (C-D) Invasion assay was used to measure invasion capacity of HCC SK-HEP-1 and HA22T cells after co-cultured with M2 macrophages. (E-F) Migration capacity was measured in SK-HEP-1 and HA22T cells after co-cultured with M2 macrophages. All quantifications are mean ± SD, *p
Figure Legend Snippet: M2 macrophages can increase the invasion and migration capacity of HCC cells. (A) qRT-PCR was applied in macrophages using primers for M2 markers (CD206, CD163, Arginase-1,TGFβ, IL-10) after induced by IL-4 and IL-13. (B) Pattern diagram for co-culture, invasion and wound healing migration system. (C-D) Invasion assay was used to measure invasion capacity of HCC SK-HEP-1 and HA22T cells after co-cultured with M2 macrophages. (E-F) Migration capacity was measured in SK-HEP-1 and HA22T cells after co-cultured with M2 macrophages. All quantifications are mean ± SD, *p

Techniques Used: Migration, Quantitative RT-PCR, Co-Culture Assay, Invasion Assay, Cell Culture

16) Product Images from "Lindera obtusiloba Attenuates Oxidative Stress and Airway Inflammation in a Murine Model of Ovalbumin-Challenged Asthma"

Article Title: Lindera obtusiloba Attenuates Oxidative Stress and Airway Inflammation in a Murine Model of Ovalbumin-Challenged Asthma

Journal: Antioxidants

doi: 10.3390/antiox9070563

Effects of LOL on cell viability, pro-inflammatory cytokines and Th2 cytokines, including IL-4, IL-5 and IL-13, in TNF-α-stimulated NCI-H292 cells. The ( A ) cell viability was measured using a WST-1 reagent, and LOL was treated with 10, 25, 50, and 100 μg/mL for 24 h. The levels of ( B ) TNF-α and ( C ) IL-6 were determined by ELISA. The culture medium were changed RPMI1640 (0.1% FBS) and treated with LOL (25, 50 and 100 μg/mL) and DEX (20 μg/mL) for 1 h and incubated with TNF-α (30 ng/mL) for 30 min (TNF-α) or 24 h (IL-6). The mRNA levels of ( D ) IL-4, ( E ) IL-5, and ( F ) IL-13 were measured by real-time PCR in TNF-α-stimulated NCI-H292 cells. Cells were treated with LOL (25, 50 and 100 μg/mL) and DEX (20 μg/mL) for 1 h and incubated with TNF-α (30 ng/mL) for 18 h. The values are expressed as the means ± SD ( n = 3). # p
Figure Legend Snippet: Effects of LOL on cell viability, pro-inflammatory cytokines and Th2 cytokines, including IL-4, IL-5 and IL-13, in TNF-α-stimulated NCI-H292 cells. The ( A ) cell viability was measured using a WST-1 reagent, and LOL was treated with 10, 25, 50, and 100 μg/mL for 24 h. The levels of ( B ) TNF-α and ( C ) IL-6 were determined by ELISA. The culture medium were changed RPMI1640 (0.1% FBS) and treated with LOL (25, 50 and 100 μg/mL) and DEX (20 μg/mL) for 1 h and incubated with TNF-α (30 ng/mL) for 30 min (TNF-α) or 24 h (IL-6). The mRNA levels of ( D ) IL-4, ( E ) IL-5, and ( F ) IL-13 were measured by real-time PCR in TNF-α-stimulated NCI-H292 cells. Cells were treated with LOL (25, 50 and 100 μg/mL) and DEX (20 μg/mL) for 1 h and incubated with TNF-α (30 ng/mL) for 18 h. The values are expressed as the means ± SD ( n = 3). # p

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

Effects of LOL on inflammatory cell number, Th2 cytokines, eotaxin, MUC5AC, total IgE and OVA-specific IgE in OVA-challenged asthma model. ( A ) The inflammatory cells were attached on slide and stained with Diff-Quik stain reagent. ( B ) The levels of IL-4, IL-5, IL-13, eotaxin, and MUC5AC in the BALF were determined by ELISA. ( C ) The total IgE and OVA-specific IgE in the serum were determined using ELISA. NC: normal control mice; OVA: OVA-challenged mice; DEX: dexamethasone (3 mg/kg) + OVA-challenged mice; LOL: LOL (50 or 100 mg/kg) + OVA-challenged mice. The values are expressed as the means ± SD ( n = 7/group). # p
Figure Legend Snippet: Effects of LOL on inflammatory cell number, Th2 cytokines, eotaxin, MUC5AC, total IgE and OVA-specific IgE in OVA-challenged asthma model. ( A ) The inflammatory cells were attached on slide and stained with Diff-Quik stain reagent. ( B ) The levels of IL-4, IL-5, IL-13, eotaxin, and MUC5AC in the BALF were determined by ELISA. ( C ) The total IgE and OVA-specific IgE in the serum were determined using ELISA. NC: normal control mice; OVA: OVA-challenged mice; DEX: dexamethasone (3 mg/kg) + OVA-challenged mice; LOL: LOL (50 or 100 mg/kg) + OVA-challenged mice. The values are expressed as the means ± SD ( n = 7/group). # p

Techniques Used: Staining, Diff-Quik, Enzyme-linked Immunosorbent Assay, Mouse Assay

17) Product Images from "Paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid pH"

Article Title: Paracellular bicarbonate flux across human cystic fibrosis airway epithelia tempers changes in airway surface liquid pH

Journal: The Journal of Physiology

doi: 10.1113/JP280120

Proinflammatory cytokines increased airway surface liquid pH and induce HCO 3 − absorption or minimal HCO 3 − flux 1 of 6 cystic fibrosis (CF) donors was excluded from the IL‐13 analysis because of a high IL‐13‐induced paracellular HCO 3 − conductance of 0.868 mS cm −2 , Grubb's test α ≤ 0.01. Red circles represent HCO 3 − data and black circles represent Cl − data, each for a single donor. A , paracellular P HCO3 reported in Table 2 and Table 3 ; two‐tailed paired Student's t test, P values shown. B , G HCO3 calculated using Eqn 6 , paracellular G HCO3 increased due to increased apical [HCO 3 − ]; two‐tailed paired Student's t test, P values shown. C , paracellular HCO 3 − flux calculated using Eqn 9 and Faraday's constant. Minimal HCO 3 − secretion was reduced or absorptive with cytokine treatments; two‐tailed paired Student's t test, P values shown.
Figure Legend Snippet: Proinflammatory cytokines increased airway surface liquid pH and induce HCO 3 − absorption or minimal HCO 3 − flux 1 of 6 cystic fibrosis (CF) donors was excluded from the IL‐13 analysis because of a high IL‐13‐induced paracellular HCO 3 − conductance of 0.868 mS cm −2 , Grubb's test α ≤ 0.01. Red circles represent HCO 3 − data and black circles represent Cl − data, each for a single donor. A , paracellular P HCO3 reported in Table 2 and Table 3 ; two‐tailed paired Student's t test, P values shown. B , G HCO3 calculated using Eqn 6 , paracellular G HCO3 increased due to increased apical [HCO 3 − ]; two‐tailed paired Student's t test, P values shown. C , paracellular HCO 3 − flux calculated using Eqn 9 and Faraday's constant. Minimal HCO 3 − secretion was reduced or absorptive with cytokine treatments; two‐tailed paired Student's t test, P values shown.

Techniques Used: Two Tailed Test

18) Product Images from "Obesity-Associated Extracellular Matrix Remodeling Promotes a Macrophage Phenotype Similar to Tumor-Associated Macrophages"

Article Title: Obesity-Associated Extracellular Matrix Remodeling Promotes a Macrophage Phenotype Similar to Tumor-Associated Macrophages

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2019.06.005

Obesity-associated extracellular matrix (ECM) regulates macrophage morphology and proliferation in vitro . A: Representative photomicrographs illustrating the distribution of human adipose tissue interstitial ECM in between adipocytes via hematoxylin and eosin (H E; top panel ) and visualizing fibronectin (Fn) and collagen, two key components of interstitial fibrosis, by immunohistochemistry (IHC; middle panel ) and second harmonic generation (SHG) imaging ( bottom panel ), respectively. B: Schematic illustrating protocol for adipose stromal cell (ASC) isolation from the inguinal fat pad of lean wild-type and obese ( ob/ob ) age-matched mice and their use for preparation of decellularized ECMs. C: Representative confocal microscopy images of decellularized ECM assembled by lean and obese ASCs after immunostaining for Fn. D – F: Bone marrow–derived macrophages (BMDMs) cultured on lean and obese decellularized matrices ( dashed lines highlight the long axis of the macrophages; D ) are more aligned with the matrix (0 on x axis represents perfect alignment; E ) and exhibit increased elongation ( F ), as determined by confocal microscopy and image analysis (top 25% of cells plotted) after stimulation with IL-4 and IL-13. F: U -test was performed. G: BMDMs cultured on obese decellularized ECM are more proliferative, as determined by bromodeoxyuridine (BrdU) incorporation with immunofluorescence image analysis. A t -test analysis was performed. Data are expressed as median and interquartile range ( F ) or means ± SD ( G ). n = 3 independent experiments with 10 images per condition per experiment ( D and E ); n = 3 independent experiments with 30 representative images per condition per experiment ( G ). *** P
Figure Legend Snippet: Obesity-associated extracellular matrix (ECM) regulates macrophage morphology and proliferation in vitro . A: Representative photomicrographs illustrating the distribution of human adipose tissue interstitial ECM in between adipocytes via hematoxylin and eosin (H E; top panel ) and visualizing fibronectin (Fn) and collagen, two key components of interstitial fibrosis, by immunohistochemistry (IHC; middle panel ) and second harmonic generation (SHG) imaging ( bottom panel ), respectively. B: Schematic illustrating protocol for adipose stromal cell (ASC) isolation from the inguinal fat pad of lean wild-type and obese ( ob/ob ) age-matched mice and their use for preparation of decellularized ECMs. C: Representative confocal microscopy images of decellularized ECM assembled by lean and obese ASCs after immunostaining for Fn. D – F: Bone marrow–derived macrophages (BMDMs) cultured on lean and obese decellularized matrices ( dashed lines highlight the long axis of the macrophages; D ) are more aligned with the matrix (0 on x axis represents perfect alignment; E ) and exhibit increased elongation ( F ), as determined by confocal microscopy and image analysis (top 25% of cells plotted) after stimulation with IL-4 and IL-13. F: U -test was performed. G: BMDMs cultured on obese decellularized ECM are more proliferative, as determined by bromodeoxyuridine (BrdU) incorporation with immunofluorescence image analysis. A t -test analysis was performed. Data are expressed as median and interquartile range ( F ) or means ± SD ( G ). n = 3 independent experiments with 10 images per condition per experiment ( D and E ); n = 3 independent experiments with 30 representative images per condition per experiment ( G ). *** P

Techniques Used: In Vitro, Immunohistochemistry, Imaging, Isolation, Mouse Assay, Confocal Microscopy, Immunostaining, Derivative Assay, Cell Culture, BrdU Incorporation Assay, Immunofluorescence

Interactions with obese extracellular matrix (ECM) promote macrophage proangiogenic function. A: CD206 + macrophages and CD31 + vascular profiles are positively correlated on the basis of immunohistochemistry image analysis of clinical samples. Red dotted line illustrates the best fit line, as determined by linear regression. B: Endothelial cell transwell migration toward bone marrow–derived macrophages (BMDMs) is significantly increased when IL-4– and IL-13–stimulated BMDMs are in contact with obese versus lean or fibronectin (Fn) control ECMs; Kruskal-Wallis analysis was performed. C: Endothelial cell tubulogenesis is increased in the presence of conditioned medium collected from unstimulated BMDMs cultured on obese versus lean ECM or Fn control substrates. Kruskal-Wallis analysis was performed. Data are expressed as median and interquartile range ( B and C ). n = 43 ( A ); n = 3 independent experiments with 3 transwells per condition per experiment and 4 images per transwell ( B ); n = 3 independent experiments with 3 wells per condition and 1 representative image per well ( C ). * P
Figure Legend Snippet: Interactions with obese extracellular matrix (ECM) promote macrophage proangiogenic function. A: CD206 + macrophages and CD31 + vascular profiles are positively correlated on the basis of immunohistochemistry image analysis of clinical samples. Red dotted line illustrates the best fit line, as determined by linear regression. B: Endothelial cell transwell migration toward bone marrow–derived macrophages (BMDMs) is significantly increased when IL-4– and IL-13–stimulated BMDMs are in contact with obese versus lean or fibronectin (Fn) control ECMs; Kruskal-Wallis analysis was performed. C: Endothelial cell tubulogenesis is increased in the presence of conditioned medium collected from unstimulated BMDMs cultured on obese versus lean ECM or Fn control substrates. Kruskal-Wallis analysis was performed. Data are expressed as median and interquartile range ( B and C ). n = 43 ( A ); n = 3 independent experiments with 3 transwells per condition per experiment and 4 images per transwell ( B ); n = 3 independent experiments with 3 wells per condition and 1 representative image per well ( C ). * P

Techniques Used: Immunohistochemistry, Migration, Derivative Assay, Cell Culture

Obesity-associated extracellular matrix (ECM) impacts macrophage polarization in vitro . A and B: A greater proportion of bone marrow–derived macrophages cultured on obese decellularized ECM expresses the anti-inflammatory macrophage markers CD206 ( A ) and arginase-1 ( B ), when exposed to IL-4 and IL-13, as determined by immunofluorescence confocal image analysis. A t -test analysis was used. Data are expressed as means ± SD ( A and B ). n = 3 independent experiments, with 15 images per condition per experiment ( A and B ). * P
Figure Legend Snippet: Obesity-associated extracellular matrix (ECM) impacts macrophage polarization in vitro . A and B: A greater proportion of bone marrow–derived macrophages cultured on obese decellularized ECM expresses the anti-inflammatory macrophage markers CD206 ( A ) and arginase-1 ( B ), when exposed to IL-4 and IL-13, as determined by immunofluorescence confocal image analysis. A t -test analysis was used. Data are expressed as means ± SD ( A and B ). n = 3 independent experiments, with 15 images per condition per experiment ( A and B ). * P

Techniques Used: In Vitro, Derivative Assay, Cell Culture, Immunofluorescence

19) Product Images from "Vinpocetine alleviates lung inflammation via macrophage inflammatory protein-1β inhibition in an ovalbumin-induced allergic asthma model"

Article Title: Vinpocetine alleviates lung inflammation via macrophage inflammatory protein-1β inhibition in an ovalbumin-induced allergic asthma model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0251012

Effects of IBMX and Vinp on OVA-induced increase in allergic inflammatory mediators. Plasma samples obtained from mice were analyzed by ELISA to investigate systemic allergic inflammation. ( A ) Anti-OVA IgE in serum and ( B ) the release of IL-13 in BALF were measured by ELISA. The mRNA expression levels of ( C ) IL-13 in lung tissues were measured by RT-qPCR. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA (Data were considered significant at ** P
Figure Legend Snippet: Effects of IBMX and Vinp on OVA-induced increase in allergic inflammatory mediators. Plasma samples obtained from mice were analyzed by ELISA to investigate systemic allergic inflammation. ( A ) Anti-OVA IgE in serum and ( B ) the release of IL-13 in BALF were measured by ELISA. The mRNA expression levels of ( C ) IL-13 in lung tissues were measured by RT-qPCR. Data are expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA (Data were considered significant at ** P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

20) Product Images from "The conformational structural change of β-lactoglobulin via acrolein treatment reduced the allergenicity"

Article Title: The conformational structural change of β-lactoglobulin via acrolein treatment reduced the allergenicity

Journal: Food Chemistry: X

doi: 10.1016/j.fochx.2021.100120

Effects of acrolein (1 mmol/L) treated BLG in IgE-mediated allergic responses in RBL-2H3 cells sensitized with mouse sera IgE. A, The release of β-hexosaminidase; B, The release of histamine; C, The release of cysteinyl leukotriene; D, The release of prostaglandin D2; E, The release of IL-4; F, The release of IL-13. *p
Figure Legend Snippet: Effects of acrolein (1 mmol/L) treated BLG in IgE-mediated allergic responses in RBL-2H3 cells sensitized with mouse sera IgE. A, The release of β-hexosaminidase; B, The release of histamine; C, The release of cysteinyl leukotriene; D, The release of prostaglandin D2; E, The release of IL-4; F, The release of IL-13. *p

Techniques Used:

21) Product Images from "Development and characterization of a human Th17‐driven ex vivo skin inflammation model, et al. Development and characterization of a human Th17‐driven ex vivo skin inflammation model"

Article Title: Development and characterization of a human Th17‐driven ex vivo skin inflammation model, et al. Development and characterization of a human Th17‐driven ex vivo skin inflammation model

Journal: Experimental Dermatology

doi: 10.1111/exd.14160

Characterization of cytokine release consecutive to Th17/Th1 activation. NativeSkin® models were either cultured under basal conditions or in the presence of the Th17 polarization cocktail (negative control), or injected with rhIL‐2, anti‐CD3/CD28 antibodies (in situ activation) in the absence (negative control) or presence of the Th17 polarization cocktail (InflammaSkin®) for 7 days. Protein levels of IL‐17A (A), IL‐17AF (B), IL‐17F (C), IL‐22 (D), IFN‐γ (E), TNF‐α (F), IL‐15 (G), IL‐10 (H), IL‐4 (I) and IL‐13 (J) were measured by ELISA (IL‐22) and MSD (all the other cytokines) in supernatant samples harvested after the last 24 h of 7 d of culture. Lowest limit of detection (LLOD) for each cytokine is indicated by a dotted line. Data are expressed as mean values ± SEM of pg/mL of secreted cytokines of models obtained from three different skin donors (n = 3). Two‐way ANOVA statistical analysis followed by Tukey's multiple comparison tests was used to compare protein levels. **** P
Figure Legend Snippet: Characterization of cytokine release consecutive to Th17/Th1 activation. NativeSkin® models were either cultured under basal conditions or in the presence of the Th17 polarization cocktail (negative control), or injected with rhIL‐2, anti‐CD3/CD28 antibodies (in situ activation) in the absence (negative control) or presence of the Th17 polarization cocktail (InflammaSkin®) for 7 days. Protein levels of IL‐17A (A), IL‐17AF (B), IL‐17F (C), IL‐22 (D), IFN‐γ (E), TNF‐α (F), IL‐15 (G), IL‐10 (H), IL‐4 (I) and IL‐13 (J) were measured by ELISA (IL‐22) and MSD (all the other cytokines) in supernatant samples harvested after the last 24 h of 7 d of culture. Lowest limit of detection (LLOD) for each cytokine is indicated by a dotted line. Data are expressed as mean values ± SEM of pg/mL of secreted cytokines of models obtained from three different skin donors (n = 3). Two‐way ANOVA statistical analysis followed by Tukey's multiple comparison tests was used to compare protein levels. **** P

Techniques Used: Activation Assay, Cell Culture, Negative Control, Injection, In Situ, Enzyme-linked Immunosorbent Assay

22) Product Images from "Inhibition of the Inflammasome Activity of NLRP3 Attenuates HDM-Induced Allergic Asthma"

Article Title: Inhibition of the Inflammasome Activity of NLRP3 Attenuates HDM-Induced Allergic Asthma

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2021.718779

NLRP3 deficiency attenuates airway inflammation HDM-induced asthma. (A–E) WT and Nlrp3 -/- mice were sensitized and challenged with 10μg HDM i.n. on day 0 and days 7-11. The mice in control group were sensitized and challenged by inhaling saline. On day 14, all mice were euthanized for analysis. (A) counts of total BALF cells, eosinophils, neutrophils and lymphocytes. (B) ELISA analysis of IgE and HDM-specific IgE in serum. (C) Histological analysis of lung sections stained with H E and PAS (Scale bar = 200 μm); n = 3 biologically independent experiments. ELISA analysis of IL-4, IL-5, IL-13, IL-6 and IFNγ in BALF (D) and supernatants of MLN cells obtained from control or challenged mice after or without restimulation with HDM (E) . n = 3-6 biologically independent mice (A, B, D, E) . Statistical significances were analyzed by unpaired Student’s t -test: * P
Figure Legend Snippet: NLRP3 deficiency attenuates airway inflammation HDM-induced asthma. (A–E) WT and Nlrp3 -/- mice were sensitized and challenged with 10μg HDM i.n. on day 0 and days 7-11. The mice in control group were sensitized and challenged by inhaling saline. On day 14, all mice were euthanized for analysis. (A) counts of total BALF cells, eosinophils, neutrophils and lymphocytes. (B) ELISA analysis of IgE and HDM-specific IgE in serum. (C) Histological analysis of lung sections stained with H E and PAS (Scale bar = 200 μm); n = 3 biologically independent experiments. ELISA analysis of IL-4, IL-5, IL-13, IL-6 and IFNγ in BALF (D) and supernatants of MLN cells obtained from control or challenged mice after or without restimulation with HDM (E) . n = 3-6 biologically independent mice (A, B, D, E) . Statistical significances were analyzed by unpaired Student’s t -test: * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining

RRx-001 treatment has therapeutic effects on HDM-induced allergic asthma. (A) Mice were induced allergic asthma with 10μg HDM i.n. on day 0 and days 7-11, and given 10mg/kg RRx-001 or vehicle i.p. on days 7, 9, and 11. The mice in control group were challenged by inhaling saline, and given 10mg/kg RRx-001 or vehicle i.p. on days 7, 9, and 11. On day 14, all mice were euthanized for analysis. (B–F) WT mice were induced allergic asthma with HDM and treated with RRx-001 or vehicle. (B) Counts of total BALF cells, eosinophils, neutrophils and lymphocytes. (C) ELISA analysis of IgE and HDM-specific IgE in serum. (D) Histological analysis of lung sections stained with H E and PAS (Scale bar = 200 μm); n = 3 biologically independent experiments. ELISA analysis of IL-4, IL-5, IL-13, IL-6 and IFNγ in BALF (E) and supernatants of MLN cells obtained from control or challenged mice after or without restimulation with HDM (F) . n = 3-5 biologically independent mice (B, C, E, F) . Statistical significances were analyzed by unpaired Student’s t -test: * P
Figure Legend Snippet: RRx-001 treatment has therapeutic effects on HDM-induced allergic asthma. (A) Mice were induced allergic asthma with 10μg HDM i.n. on day 0 and days 7-11, and given 10mg/kg RRx-001 or vehicle i.p. on days 7, 9, and 11. The mice in control group were challenged by inhaling saline, and given 10mg/kg RRx-001 or vehicle i.p. on days 7, 9, and 11. On day 14, all mice were euthanized for analysis. (B–F) WT mice were induced allergic asthma with HDM and treated with RRx-001 or vehicle. (B) Counts of total BALF cells, eosinophils, neutrophils and lymphocytes. (C) ELISA analysis of IgE and HDM-specific IgE in serum. (D) Histological analysis of lung sections stained with H E and PAS (Scale bar = 200 μm); n = 3 biologically independent experiments. ELISA analysis of IL-4, IL-5, IL-13, IL-6 and IFNγ in BALF (E) and supernatants of MLN cells obtained from control or challenged mice after or without restimulation with HDM (F) . n = 3-5 biologically independent mice (B, C, E, F) . Statistical significances were analyzed by unpaired Student’s t -test: * P

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining

23) Product Images from "Effect of Obstructive Sleep Apnea on Immunity in Cases of Chronic Rhinosinusitis With Nasal Polyps"

Article Title: Effect of Obstructive Sleep Apnea on Immunity in Cases of Chronic Rhinosinusitis With Nasal Polyps

Journal: Clinical and Experimental Otorhinolaryngology

doi: 10.21053/ceo.2020.02250

Correlations of upregulated inflammatory mediators and serum markers in eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP) patients who had moderate-to-severe obstructive sleep apnea (OSA). (A) Associations of CRP with IL-6, CXCL-1, IL-17A. (B) Associations of serum eosinophil count with IL-4, IL-13, CCL-11, and CCL-24. CRP, C-reactive protein; IL, interleukin; CXCL, chemokine [C-X-C motif] ligand; CCL, C-C motif chemokine.
Figure Legend Snippet: Correlations of upregulated inflammatory mediators and serum markers in eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP) patients who had moderate-to-severe obstructive sleep apnea (OSA). (A) Associations of CRP with IL-6, CXCL-1, IL-17A. (B) Associations of serum eosinophil count with IL-4, IL-13, CCL-11, and CCL-24. CRP, C-reactive protein; IL, interleukin; CXCL, chemokine [C-X-C motif] ligand; CCL, C-C motif chemokine.

Techniques Used:

24) Product Images from "Dual blockade of IL‐4 and IL‐13 with dupilumab, an IL‐4Rα antibody, is required to broadly inhibit type 2 inflammation, et al. Dual blockade of IL‐4 and IL‐13 with dupilumab, an IL‐4Rα antibody, is required to broadly inhibit type 2 inflammation"

Article Title: Dual blockade of IL‐4 and IL‐13 with dupilumab, an IL‐4Rα antibody, is required to broadly inhibit type 2 inflammation, et al. Dual blockade of IL‐4 and IL‐13 with dupilumab, an IL‐4Rα antibody, is required to broadly inhibit type 2 inflammation

Journal: Allergy

doi: 10.1111/all.14151

IL‐4 dominates IgE‐dependent human mast cell responses. A, In vitro differentiated human mast cells were treated for 2 d with increasing concentrations of IL‐4 or IL‐13, or serial dilutions of IL‐4Rα Ab or a control (ctrl) IgG in the presence of 50 pmol/L IL‐4 or 1 nmol/L IL‐13, and FcεRIα surface expression was assessed by flow cytometry. Error bars represent SD from samples run in triplicate. Black arrows indicate cytokine concentrations used for the corresponding blocking assays. IC50 is shown. Data are representative of at least three independent experiments using different donors. B, Heatmap of cytokine‐ and chemokine‐related genes upon IgE crosslinking (sensitization with two different Fel d 1‐specific IgE followed by activation with Fel d 1 antigen) of human mast cells. 51 cytokine/chemokine‐related genes were induced (FDR ≤ 0.05; |FC|≥2) by either IL‐4, IL‐13, Fel d 1‐specific IgE (Fel d 1 IgE)+Fel d 1, IL‐4 + Fel d 1 IgE + Fel d 1, or IL‐13 + Fel d 1 IgE + Fel d 1. The color gradient represents fold‐change values comparing each treatment to the control samples. C, mRNA expression levels measured by real‐time qPCR and expressed relative to GAPDH mRNA expression; *= P
Figure Legend Snippet: IL‐4 dominates IgE‐dependent human mast cell responses. A, In vitro differentiated human mast cells were treated for 2 d with increasing concentrations of IL‐4 or IL‐13, or serial dilutions of IL‐4Rα Ab or a control (ctrl) IgG in the presence of 50 pmol/L IL‐4 or 1 nmol/L IL‐13, and FcεRIα surface expression was assessed by flow cytometry. Error bars represent SD from samples run in triplicate. Black arrows indicate cytokine concentrations used for the corresponding blocking assays. IC50 is shown. Data are representative of at least three independent experiments using different donors. B, Heatmap of cytokine‐ and chemokine‐related genes upon IgE crosslinking (sensitization with two different Fel d 1‐specific IgE followed by activation with Fel d 1 antigen) of human mast cells. 51 cytokine/chemokine‐related genes were induced (FDR ≤ 0.05; |FC|≥2) by either IL‐4, IL‐13, Fel d 1‐specific IgE (Fel d 1 IgE)+Fel d 1, IL‐4 + Fel d 1 IgE + Fel d 1, or IL‐13 + Fel d 1 IgE + Fel d 1. The color gradient represents fold‐change values comparing each treatment to the control samples. C, mRNA expression levels measured by real‐time qPCR and expressed relative to GAPDH mRNA expression; *= P

Techniques Used: In Vitro, Expressing, Flow Cytometry, Blocking Assay, Activation Assay, Real-time Polymerase Chain Reaction

Dual IL‐4/IL‐13 blockade prevents HDM‐induced impairment of lung function. Il4ra hu/hu Il4 hu/hu mice were exposed to saline or HDM for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIg G or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. A, Representative PAS‐stained lung histology sections of a mouse not exposed to HDM, an untreated HDM‐exposed mouse, a HDM‐exposed mouse treated with ctrl hIgG, ctrl mIgG, IL‐4Rα Ab, IL‐4 Ab, or mIL‐13Rα2‐Fc. White arrows show examples of epithelial cell nuclei. Black arrows indicate examples of PAS‐positive goblet cells; bar, 25 µm. B, GCM quantification. PAS‐positive goblet cells and total epithelial cells were counted in a millimeter length of primary bronchus (approximately 100 epithelial cells), and GCM was expressed as the percentage of PAS‐positive epithelial cells. †= P
Figure Legend Snippet: Dual IL‐4/IL‐13 blockade prevents HDM‐induced impairment of lung function. Il4ra hu/hu Il4 hu/hu mice were exposed to saline or HDM for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIg G or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. A, Representative PAS‐stained lung histology sections of a mouse not exposed to HDM, an untreated HDM‐exposed mouse, a HDM‐exposed mouse treated with ctrl hIgG, ctrl mIgG, IL‐4Rα Ab, IL‐4 Ab, or mIL‐13Rα2‐Fc. White arrows show examples of epithelial cell nuclei. Black arrows indicate examples of PAS‐positive goblet cells; bar, 25 µm. B, GCM quantification. PAS‐positive goblet cells and total epithelial cells were counted in a millimeter length of primary bronchus (approximately 100 epithelial cells), and GCM was expressed as the percentage of PAS‐positive epithelial cells. †= P

Techniques Used: Mouse Assay, Staining

Dual IL‐4/IL‐13 blockade prevents infiltration of eosinophils into the lungs of HDM‐exposed mice and their activation. A, Il4ra hu/hu Il4 hu/hu mice were exposed to saline or HDM for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. On day 30, mice were injected with anti‐CD45 to label all circulating immune cells and killed after 5 min. Lungs were harvested and dissociated for flow cytometric analysis of circulating lung and lung tissue eosinophils. Representative flow cytometric plots are shown (left panels). Circulating lung (middle panel) and lung tissue (right panel) eosinophils are reported as a frequency of live cells in the suspension. Each symbol represents one mouse. n ≥ 5 mice per group. Saline (white boxes) or HDM (shaded boxes). †= P
Figure Legend Snippet: Dual IL‐4/IL‐13 blockade prevents infiltration of eosinophils into the lungs of HDM‐exposed mice and their activation. A, Il4ra hu/hu Il4 hu/hu mice were exposed to saline or HDM for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. On day 30, mice were injected with anti‐CD45 to label all circulating immune cells and killed after 5 min. Lungs were harvested and dissociated for flow cytometric analysis of circulating lung and lung tissue eosinophils. Representative flow cytometric plots are shown (left panels). Circulating lung (middle panel) and lung tissue (right panel) eosinophils are reported as a frequency of live cells in the suspension. Each symbol represents one mouse. n ≥ 5 mice per group. Saline (white boxes) or HDM (shaded boxes). †= P

Techniques Used: Mouse Assay, Activation Assay, Injection

Both IL‐4 and IL‐13 drive airway inflammation in mice. A, Il4ra hu/hu Il4 hu/hu mice were exposed to PBS, human IL‐4 or human IL‐13 for 12 d. B‐F, On day 12, mice were injected with anti‐CD45 to label all circulating immune cells and killed after 5 min. Lungs were harvested and dissociated for flow cytometric analysis of circulating lung and lung tissue CD45 + cells (B), CD4 T cells (C), alveolar macrophages (D), neutrophils (E), and eosinophils (F). Circulating lung and lung tissue cell populations are reported as a frequency of live cells in the suspension. Each symbol represents one mouse. n = 5 mice per group. PBS (white boxes), human IL‐4 (dark blue boxes), or human IL‐13 (light blue boxes). G, Cytokine and chemokine gene heatmap. Fold change relative to control mice of lung tissue mRNA expression levels was measured by real‐time qPCR and expressed relative to β‐actin (ACTB) mRNA expression. H, Left , representative PAS‐stained lung histology sections of a mouse not exposed to cytokines (left panel), an IL‐4‐exposed mouse (middle panel), or an IL‐13‐exposed mouse (right panel). White arrows show examples of epithelial cell nuclei. Black arrows indicate examples of PAS‐positive goblet cells; Bar, 25 µm. Right , GCM quantification. PAS‐positive goblet cells and total epithelial cells were counted in a millimeter length of primary bronchus (approximately 100 epithelial cells), and GCM was expressed as the percentage of PAS‐positive epithelial cells. †,*= P
Figure Legend Snippet: Both IL‐4 and IL‐13 drive airway inflammation in mice. A, Il4ra hu/hu Il4 hu/hu mice were exposed to PBS, human IL‐4 or human IL‐13 for 12 d. B‐F, On day 12, mice were injected with anti‐CD45 to label all circulating immune cells and killed after 5 min. Lungs were harvested and dissociated for flow cytometric analysis of circulating lung and lung tissue CD45 + cells (B), CD4 T cells (C), alveolar macrophages (D), neutrophils (E), and eosinophils (F). Circulating lung and lung tissue cell populations are reported as a frequency of live cells in the suspension. Each symbol represents one mouse. n = 5 mice per group. PBS (white boxes), human IL‐4 (dark blue boxes), or human IL‐13 (light blue boxes). G, Cytokine and chemokine gene heatmap. Fold change relative to control mice of lung tissue mRNA expression levels was measured by real‐time qPCR and expressed relative to β‐actin (ACTB) mRNA expression. H, Left , representative PAS‐stained lung histology sections of a mouse not exposed to cytokines (left panel), an IL‐4‐exposed mouse (middle panel), or an IL‐13‐exposed mouse (right panel). White arrows show examples of epithelial cell nuclei. Black arrows indicate examples of PAS‐positive goblet cells; Bar, 25 µm. Right , GCM quantification. PAS‐positive goblet cells and total epithelial cells were counted in a millimeter length of primary bronchus (approximately 100 epithelial cells), and GCM was expressed as the percentage of PAS‐positive epithelial cells. †,*= P

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

Dual IL‐4/IL‐13 blockade broadly inhibits chemokine and type 2 proinflammatory cytokine expression in the lungs of HDM‐exposed mice. A, Volcano plot of HDM signature. After 4 wk of HDM exposure, significant changes were detected in 873 transcripts that were differentially expressed in the lungs of HDM‐exposed mice compared with the lungs of mice not exposed to HDM (adjusted P ‐value ≤ .05; |fold change|≥2). B and D, Venn diagrams of HDM signature vs IL‐4Rα Ab (B), IL‐4 Ab (C), and mIL‐13Rα2‐Fc (D) signatures (adjusted P ‐value ≤ .05; |fold change|≥2). Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. Treatment signatures correspond to statistically significant changes in gene expression compared to mice receiving corresponding ctrl IgG. ↓↑, treatment‐responsive genes inversely differentially regulated in the HDM signature. E, Heatmap of cytokine/chemokine‐related genes. 29 cytokine/chemokine‐related genes from the HDM signature were inversely regulated by IL‐4Rα Ab, IL‐4 Ab, and/or mouse IL‐13Rα2‐Fc. The color gradient represents z‐score of the gene expression values in various treatment and control samples. F, Il4ra hu/hu Il4 hu/hu mice were exposed to saline (white boxes) or HDM (shaded boxes) for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. At the end of the study, lung tissue mRNA expression levels were measured by real‐time qPCR and are expressed relative to β‐actin ( ACTB ) mRNA expression. Each symbol represents one mouse. n ≥ 5 mice per group. †= P
Figure Legend Snippet: Dual IL‐4/IL‐13 blockade broadly inhibits chemokine and type 2 proinflammatory cytokine expression in the lungs of HDM‐exposed mice. A, Volcano plot of HDM signature. After 4 wk of HDM exposure, significant changes were detected in 873 transcripts that were differentially expressed in the lungs of HDM‐exposed mice compared with the lungs of mice not exposed to HDM (adjusted P ‐value ≤ .05; |fold change|≥2). B and D, Venn diagrams of HDM signature vs IL‐4Rα Ab (B), IL‐4 Ab (C), and mIL‐13Rα2‐Fc (D) signatures (adjusted P ‐value ≤ .05; |fold change|≥2). Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. Treatment signatures correspond to statistically significant changes in gene expression compared to mice receiving corresponding ctrl IgG. ↓↑, treatment‐responsive genes inversely differentially regulated in the HDM signature. E, Heatmap of cytokine/chemokine‐related genes. 29 cytokine/chemokine‐related genes from the HDM signature were inversely regulated by IL‐4Rα Ab, IL‐4 Ab, and/or mouse IL‐13Rα2‐Fc. The color gradient represents z‐score of the gene expression values in various treatment and control samples. F, Il4ra hu/hu Il4 hu/hu mice were exposed to saline (white boxes) or HDM (shaded boxes) for 4 wk. Five groups of HDM‐exposed mice received injections of an isotype control antibody (ctrl hIgG or ctrl mIgG), IL‐4Rα Ab, IL‐4 Ab, or mouse IL‐13Rα2‐Fc. At the end of the study, lung tissue mRNA expression levels were measured by real‐time qPCR and are expressed relative to β‐actin ( ACTB ) mRNA expression. Each symbol represents one mouse. n ≥ 5 mice per group. †= P

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

The IL‐4Rα antibody dupilumab blocks human IL‐4 and IL‐13/IL‐13Rα1 complex binding to IL‐4Rα and prevents IL‐4 and IL‐13 signaling through Type I and Type II receptors. A, Type I signaling: Ramos.2G6.4C10/STAT3/Luciferase cells were treated with increasing concentrations of IL‐4, or serial dilutions of IL‐4Rα Ab, IL‐4 Ab, or an isotype control antibody (ctrl IgG) in the presence of 300 pmol/L IL‐4. B and C, Type II signaling: HEK293/STAT6/Luciferase cells were treated with increasing concentrations of IL‐4 (B), IL‐13 (C), or serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or ctrl IgG in the presence of 10 pmol/L IL‐4 (B) or 25 pmol/L IL‐13 (C). Inhibition of IL‐4‐ or IL‐13‐mediated signaling was quantified by measuring luminescence (RLU). Error bars represent SD from samples run in triplicate. Black arrows indicate cytokine concentrations used for the corresponding blocking assays. IC50 is shown. Data are representative of at least two independent experiments. D, Schematic diagram representing APC‐Th2 coculture cell interactions in the presence of SEB. E, Cocultures of B cells with Th2 cells in the presence of SEB were coincubated with serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or control antibody (ctrl IgG) for 2 d. Inhibition of CD23 induction at the surface of B cells was monitored by flow cytometry. F, Cocultures of moDCs with Th2 cells in the presence of SEB were coincubated with serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or ctrl IgG for 2 d. Inhibition of IL‐12p70 release from moDCs was determined by MSD. Error bars represent SD from samples run in duplicate. All data are representative of at least three independent experiments using different donors. G, To assess the ability of IL‐4Rα Ab to block IL‐4 and IL‐13/IL‐13Rα1 binding to IL‐4Rα, human IL‐4, human IL‐13, human IL‐13Rα1.hFc, or IL‐13/IL‐13Rα1.hFc complex was injected over human IL‐4Rα surfaces that were prebound with IL‐4Rα Ab or a control antibody (ctrl IgG). Graph presents surface‐bound proteins in resonance units (RU). H, Schematic diagram showing dupilumab (IL‐4Rα Ab) mechanism of action
Figure Legend Snippet: The IL‐4Rα antibody dupilumab blocks human IL‐4 and IL‐13/IL‐13Rα1 complex binding to IL‐4Rα and prevents IL‐4 and IL‐13 signaling through Type I and Type II receptors. A, Type I signaling: Ramos.2G6.4C10/STAT3/Luciferase cells were treated with increasing concentrations of IL‐4, or serial dilutions of IL‐4Rα Ab, IL‐4 Ab, or an isotype control antibody (ctrl IgG) in the presence of 300 pmol/L IL‐4. B and C, Type II signaling: HEK293/STAT6/Luciferase cells were treated with increasing concentrations of IL‐4 (B), IL‐13 (C), or serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or ctrl IgG in the presence of 10 pmol/L IL‐4 (B) or 25 pmol/L IL‐13 (C). Inhibition of IL‐4‐ or IL‐13‐mediated signaling was quantified by measuring luminescence (RLU). Error bars represent SD from samples run in triplicate. Black arrows indicate cytokine concentrations used for the corresponding blocking assays. IC50 is shown. Data are representative of at least two independent experiments. D, Schematic diagram representing APC‐Th2 coculture cell interactions in the presence of SEB. E, Cocultures of B cells with Th2 cells in the presence of SEB were coincubated with serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or control antibody (ctrl IgG) for 2 d. Inhibition of CD23 induction at the surface of B cells was monitored by flow cytometry. F, Cocultures of moDCs with Th2 cells in the presence of SEB were coincubated with serial dilutions of IL‐4Rα Ab, IL‐4 Ab, IL‐13 Ab, or ctrl IgG for 2 d. Inhibition of IL‐12p70 release from moDCs was determined by MSD. Error bars represent SD from samples run in duplicate. All data are representative of at least three independent experiments using different donors. G, To assess the ability of IL‐4Rα Ab to block IL‐4 and IL‐13/IL‐13Rα1 binding to IL‐4Rα, human IL‐4, human IL‐13, human IL‐13Rα1.hFc, or IL‐13/IL‐13Rα1.hFc complex was injected over human IL‐4Rα surfaces that were prebound with IL‐4Rα Ab or a control antibody (ctrl IgG). Graph presents surface‐bound proteins in resonance units (RU). H, Schematic diagram showing dupilumab (IL‐4Rα Ab) mechanism of action

Techniques Used: Binding Assay, Luciferase, Inhibition, Blocking Assay, Flow Cytometry, Injection

25) Product Images from "Anti-inflammatory roles of interleukin-35 in the pathogenesis of Japanese cedar pollinosis"

Article Title: Anti-inflammatory roles of interleukin-35 in the pathogenesis of Japanese cedar pollinosis

Journal: Asia Pacific Allergy

doi: 10.5415/apallergy.2021.11.e34

Effects of interleukin (IL)-35 on cell proliferation and mRNA expression of IL-5, IL-13, and GATA3 in group 2 innate lymphoid cells (ILC2s). (A) Sorted ILC2s were incubated with IL-2 (50 ng/mL) in the presence or absence of IL-35 (100 ng/mL) for 2 weeks. (B) The numbers of viable ILC2s were counted using a cell counting kit-8, and are shown as optical density. (C) Proliferated ILC2s (1× 10 4 cells/ mL) were stimulated with IL-33 (100 ng/mL) in the presence or absence of IL-35 (100 ng/mL) for 6 hours, and mRNA expression of IL-5, IL-13, and GATA3 were measured by reverse transcription-polymerase chain reaction. * p
Figure Legend Snippet: Effects of interleukin (IL)-35 on cell proliferation and mRNA expression of IL-5, IL-13, and GATA3 in group 2 innate lymphoid cells (ILC2s). (A) Sorted ILC2s were incubated with IL-2 (50 ng/mL) in the presence or absence of IL-35 (100 ng/mL) for 2 weeks. (B) The numbers of viable ILC2s were counted using a cell counting kit-8, and are shown as optical density. (C) Proliferated ILC2s (1× 10 4 cells/ mL) were stimulated with IL-33 (100 ng/mL) in the presence or absence of IL-35 (100 ng/mL) for 6 hours, and mRNA expression of IL-5, IL-13, and GATA3 were measured by reverse transcription-polymerase chain reaction. * p

Techniques Used: Expressing, Incubation, Cell Counting, Reverse Transcription Polymerase Chain Reaction

(A, B) Effects of interleukin (IL)-35 on IL-33-induced cell differentiation and mRNA expression of IL5, IL-13, and GATA3 in Th2 cells. Isolated CD4 + T cells were cultured with IL-33 (100 ng/mL) in the presence or absence of IL-35 (10–1,000 ng/mL) for 5 days and the ratio of Th2 (CD4 + ST2 + ) cells in total CD4 + T cells was examined using flow cytometry. Sorted Th2 cells (C) were cultured with recombinant human IL-33 (100 ng/mL) in the presence or absence of recombinant human IL-35 (10–1,000 ng/mL) for 6 hours, and mRNA expression of IL-5, IL-13, and GATA3 was measured by reverse transcription-polymerase chain reaction (D). * p
Figure Legend Snippet: (A, B) Effects of interleukin (IL)-35 on IL-33-induced cell differentiation and mRNA expression of IL5, IL-13, and GATA3 in Th2 cells. Isolated CD4 + T cells were cultured with IL-33 (100 ng/mL) in the presence or absence of IL-35 (10–1,000 ng/mL) for 5 days and the ratio of Th2 (CD4 + ST2 + ) cells in total CD4 + T cells was examined using flow cytometry. Sorted Th2 cells (C) were cultured with recombinant human IL-33 (100 ng/mL) in the presence or absence of recombinant human IL-35 (10–1,000 ng/mL) for 6 hours, and mRNA expression of IL-5, IL-13, and GATA3 was measured by reverse transcription-polymerase chain reaction (D). * p

Techniques Used: Cell Differentiation, Expressing, Isolation, Cell Culture, Flow Cytometry, Recombinant, Reverse Transcription Polymerase Chain Reaction

Cry j 1-induced production of interleukin (IL)-5, IL-13, and IL-35 from peripheral blood mononuclears cells (PBMCs). PBMCs were incubated with Cry j 1, a major allergen of Japanese cedar pollen for 5 days. Concentrations of IL-5, IL-13, and IL-35 in cell-free supernatants were measured by enzyme-linked immunosorbent assay. * p
Figure Legend Snippet: Cry j 1-induced production of interleukin (IL)-5, IL-13, and IL-35 from peripheral blood mononuclears cells (PBMCs). PBMCs were incubated with Cry j 1, a major allergen of Japanese cedar pollen for 5 days. Concentrations of IL-5, IL-13, and IL-35 in cell-free supernatants were measured by enzyme-linked immunosorbent assay. * p

Techniques Used: Incubation, Enzyme-linked Immunosorbent Assay

26) Product Images from "Monocyte chemotactic protein-inducing protein 1 negatively regulating asthmatic airway inflammation and mucus hypersecretion involving γ-aminobutyric acid type A receptor signaling pathway in vivo and in vitro"

Article Title: Monocyte chemotactic protein-inducing protein 1 negatively regulating asthmatic airway inflammation and mucus hypersecretion involving γ-aminobutyric acid type A receptor signaling pathway in vivo and in vitro

Journal: Chinese Medical Journal

doi: 10.1097/CM9.0000000000001154

Plasmid-MCPIP1 abrogated IL-13-induced MCPIP1, MCP1, TSLP, and MUC5AC in BEAS-2B cells. Plasmid-MCPIP1-transfected and non-transfected BEAS-2B cells were stimulated with IL-13 (10 ng/mL) for 24 h. (A, C, E, and G) Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of MCPIP1, MCP1, TSLP, and MUC5AC; (B, D, F, and H) western blot was used to measure the protein expression of MCPIP1, MCP1, TSLP, and MUC5AC. Quantitative data were presented as mean ± standard error ( n = 5). ∗ P
Figure Legend Snippet: Plasmid-MCPIP1 abrogated IL-13-induced MCPIP1, MCP1, TSLP, and MUC5AC in BEAS-2B cells. Plasmid-MCPIP1-transfected and non-transfected BEAS-2B cells were stimulated with IL-13 (10 ng/mL) for 24 h. (A, C, E, and G) Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of MCPIP1, MCP1, TSLP, and MUC5AC; (B, D, F, and H) western blot was used to measure the protein expression of MCPIP1, MCP1, TSLP, and MUC5AC. Quantitative data were presented as mean ± standard error ( n = 5). ∗ P

Techniques Used: Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Plasmid-MCPIP1 attenuated IL-13-induced GABAARβ2 expression in BEAS-2B cells. Plasmid-MCPIP1-transfected and non-transfected BEAS-2B cells were stimulated with IL-13 (10 ng/mL) for 24 h. (A) Immunofluorescence was used to observe the expression of GABAARβ2 in BEAS-2B cells. The figure demonstrates a representative view (original magnification, ×400) from each group; (B) Quantitative real-time polymerase chain reaction was performed to measure the mRNA expression of GABAARβ2. (C) Western blot was used to measure the protein expression of GABAARβ2. Quantitative data were presented as mean ± standard error ( n = 5). ∗ P
Figure Legend Snippet: Plasmid-MCPIP1 attenuated IL-13-induced GABAARβ2 expression in BEAS-2B cells. Plasmid-MCPIP1-transfected and non-transfected BEAS-2B cells were stimulated with IL-13 (10 ng/mL) for 24 h. (A) Immunofluorescence was used to observe the expression of GABAARβ2 in BEAS-2B cells. The figure demonstrates a representative view (original magnification, ×400) from each group; (B) Quantitative real-time polymerase chain reaction was performed to measure the mRNA expression of GABAARβ2. (C) Western blot was used to measure the protein expression of GABAARβ2. Quantitative data were presented as mean ± standard error ( n = 5). ∗ P

Techniques Used: Plasmid Preparation, Expressing, Transfection, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot

27) Product Images from "Distinct roles for IL-13 and IL-4 via IL-13 receptor ?1 and the type II IL-4 receptor in asthma pathogenesis"

Article Title: Distinct roles for IL-13 and IL-4 via IL-13 receptor ?1 and the type II IL-4 receptor in asthma pathogenesis

Journal:

doi: 10.1073/pnas.0802465105

Assessment of IL-13Rα1-mediated responses in a model of IL-13-induced airway inflammation. Forty-eight hours after the final IL-13 challenge, the mice were assessed for BALF chemokine ( A–D ) and active TGF-β ( E ) levels, mucus production
Figure Legend Snippet: Assessment of IL-13Rα1-mediated responses in a model of IL-13-induced airway inflammation. Forty-eight hours after the final IL-13 challenge, the mice were assessed for BALF chemokine ( A–D ) and active TGF-β ( E ) levels, mucus production

Techniques Used: Mouse Assay

Assessment of baseline serum IL-13Rα2 and IgE. ( A and B ) Assessment of total sIL-13Rα2 (free and bound to IL-13) and IL-13/sIL-13Rα2 complexes. The y axes in are shown in pg/ml where each dot represents a different mouse ( n = 10
Figure Legend Snippet: Assessment of baseline serum IL-13Rα2 and IgE. ( A and B ) Assessment of total sIL-13Rα2 (free and bound to IL-13) and IL-13/sIL-13Rα2 complexes. The y axes in are shown in pg/ml where each dot represents a different mouse ( n = 10

Techniques Used:

28) Product Images from "Interferon-?1 (interleukin-29) preferentially down-regulates interleukin-13 over other T helper type 2 cytokine responses in vitro"

Article Title: Interferon-?1 (interleukin-29) preferentially down-regulates interleukin-13 over other T helper type 2 cytokine responses in vitro

Journal:

doi: 10.1111/j.1365-2567.2008.02862.x

Interferon (IFN)-λ1 inhibits the production and secretion of interleukin (IL)-13. (a) Cell culture supernatants were harvested and subjected to enzyme-linked immunosorbent assays (ELISAs) as described. Data for each donor are shown, with and without
Figure Legend Snippet: Interferon (IFN)-λ1 inhibits the production and secretion of interleukin (IL)-13. (a) Cell culture supernatants were harvested and subjected to enzyme-linked immunosorbent assays (ELISAs) as described. Data for each donor are shown, with and without

Techniques Used: Cell Culture

Interferon (IFN)-λ1 inhibits interleukin (IL)-13 secretion under T helper type 2 (Th2)-promoting conditions. (a) Peripheral blood mononuclear cells (PBMC) were stimulated with Concanavalin-A (ConA) under the following conditions: A, ConA alone;
Figure Legend Snippet: Interferon (IFN)-λ1 inhibits interleukin (IL)-13 secretion under T helper type 2 (Th2)-promoting conditions. (a) Peripheral blood mononuclear cells (PBMC) were stimulated with Concanavalin-A (ConA) under the following conditions: A, ConA alone;

Techniques Used:

Interferon (IFN)-λ1 lowers the numbers of interleukin (IL)-4, IL-5 and IL-13-positive CD4 T cells. Cells were harvested and prepared for flow cytometric analysis as described. (a, b) typical fluorescence-activated cell sorting (FACS) analysis
Figure Legend Snippet: Interferon (IFN)-λ1 lowers the numbers of interleukin (IL)-4, IL-5 and IL-13-positive CD4 T cells. Cells were harvested and prepared for flow cytometric analysis as described. (a, b) typical fluorescence-activated cell sorting (FACS) analysis

Techniques Used: Flow Cytometry, Fluorescence, FACS

29) Product Images from "Antiviral Activity of Nrf2 in a Murine Model of Respiratory Syncytial Virus Disease"

Article Title: Antiviral Activity of Nrf2 in a Murine Model of Respiratory Syncytial Virus Disease

Journal:

doi: 10.1164/rccm.200804-535OC

Differential increase of pulmonary cytokines in Nrf2 +/+ and Nrf2 −/− mice after respiratory syncytial virus (RSV) infection. Bronchoalveolar lavage (BAL) fluid levels of IL-6, IL-10, IL-13, IL-18, and IFN-γ in Nrf2
Figure Legend Snippet: Differential increase of pulmonary cytokines in Nrf2 +/+ and Nrf2 −/− mice after respiratory syncytial virus (RSV) infection. Bronchoalveolar lavage (BAL) fluid levels of IL-6, IL-10, IL-13, IL-18, and IFN-γ in Nrf2

Techniques Used: Mouse Assay, Infection

30) Product Images from "Monocyte chemotactic protein-inducing protein 1 negatively regulating asthmatic airway inflammation and mucus hypersecretion involving γ-aminobutyric acid type A receptor signaling pathway in vivo and in vitro"

Article Title: Monocyte chemotactic protein-inducing protein 1 negatively regulating asthmatic airway inflammation and mucus hypersecretion involving γ-aminobutyric acid type A receptor signaling pathway in vivo and in vitro

Journal: Chinese Medical Journal

doi: 10.1097/CM9.0000000000001154

Plasmid-MCPIP1 abrogated IL-13-induced MCPIP1, MCP1, TSLP, and MUC5AC in BEAS-2B cells. Plasmid-MCPIP1-transfected and non-transfected BEAS-2B cells were stimulated with IL-13 (10 ng/mL) for 24 h. (A, C, E, and G) Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of MCPIP1, MCP1, TSLP, and MUC5AC; (B, D, F, and H) western blot was used to measure the protein expression of MCPIP1, MCP1, TSLP, and MUC5AC. Quantitative data were presented as mean ± standard error ( n = 5). ∗ P
Figure Legend Snippet: Plasmid-MCPIP1 abrogated IL-13-induced MCPIP1, MCP1, TSLP, and MUC5AC in BEAS-2B cells. Plasmid-MCPIP1-transfected and non-transfected BEAS-2B cells were stimulated with IL-13 (10 ng/mL) for 24 h. (A, C, E, and G) Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of MCPIP1, MCP1, TSLP, and MUC5AC; (B, D, F, and H) western blot was used to measure the protein expression of MCPIP1, MCP1, TSLP, and MUC5AC. Quantitative data were presented as mean ± standard error ( n = 5). ∗ P

Techniques Used: Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Plasmid-MCPIP1 attenuated IL-13-induced GABAARβ2 expression in BEAS-2B cells. Plasmid-MCPIP1-transfected and non-transfected BEAS-2B cells were stimulated with IL-13 (10 ng/mL) for 24 h. (A) Immunofluorescence was used to observe the expression of GABAARβ2 in BEAS-2B cells. The figure demonstrates a representative view (original magnification, ×400) from each group; (B) Quantitative real-time polymerase chain reaction was performed to measure the mRNA expression of GABAARβ2. (C) Western blot was used to measure the protein expression of GABAARβ2. Quantitative data were presented as mean ± standard error ( n = 5). ∗ P
Figure Legend Snippet: Plasmid-MCPIP1 attenuated IL-13-induced GABAARβ2 expression in BEAS-2B cells. Plasmid-MCPIP1-transfected and non-transfected BEAS-2B cells were stimulated with IL-13 (10 ng/mL) for 24 h. (A) Immunofluorescence was used to observe the expression of GABAARβ2 in BEAS-2B cells. The figure demonstrates a representative view (original magnification, ×400) from each group; (B) Quantitative real-time polymerase chain reaction was performed to measure the mRNA expression of GABAARβ2. (C) Western blot was used to measure the protein expression of GABAARβ2. Quantitative data were presented as mean ± standard error ( n = 5). ∗ P

Techniques Used: Plasmid Preparation, Expressing, Transfection, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot

31) Product Images from "Interleukin-13 and Interleukin-4 Induce Vascular Endothelial Growth Factor Release from Airway Smooth Muscle Cells"

Article Title: Interleukin-13 and Interleukin-4 Induce Vascular Endothelial Growth Factor Release from Airway Smooth Muscle Cells

Journal:

doi: 10.1165/rcmb.2005-0147OC

( A ) Real-time PCR measurements of VEGF mRNA in HASM cells that were left untreated or treated for 6 h with IL-4 (0.3–30 ng/ml) or IL-13 (30 ng/ml). Results are mean ± SE of data from five to nine RNA samples in each case and are normalized
Figure Legend Snippet: ( A ) Real-time PCR measurements of VEGF mRNA in HASM cells that were left untreated or treated for 6 h with IL-4 (0.3–30 ng/ml) or IL-13 (30 ng/ml). Results are mean ± SE of data from five to nine RNA samples in each case and are normalized

Techniques Used: Real-time Polymerase Chain Reaction

Effect of the −460/−152/+405 haplotype on VEGF release from untreated HASM cells (Ctrl) and from cells treated with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean value obtained
Figure Legend Snippet: Effect of the −460/−152/+405 haplotype on VEGF release from untreated HASM cells (Ctrl) and from cells treated with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean value obtained

Techniques Used:

Effect of +405 genotype ( A ) and of −460/−152 genotype ( B ) on VEGF release under basal conditions (Ctrl) and after treatment with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean
Figure Legend Snippet: Effect of +405 genotype ( A ) and of −460/−152 genotype ( B ) on VEGF release under basal conditions (Ctrl) and after treatment with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean

Techniques Used:

( A ) Effects of increasing doses of IL-4 or IL-13 administered for 24 h on VEGF release from HASM cells. Values represent mean ± SE from 4–13 donors. Each donor was studied in duplicate.* P
Figure Legend Snippet: ( A ) Effects of increasing doses of IL-4 or IL-13 administered for 24 h on VEGF release from HASM cells. Values represent mean ± SE from 4–13 donors. Each donor was studied in duplicate.* P

Techniques Used:

Effect of the presence of at least one +936T allele on VEGF release from untreated HASM cells (Ctrl) and from cells treated with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean value obtained for
Figure Legend Snippet: Effect of the presence of at least one +936T allele on VEGF release from untreated HASM cells (Ctrl) and from cells treated with IL-13 (3 ng/ml), IL-4 (1 ng/ml), or TNF-α (10 ng/ml). Each data point represents the mean value obtained for

Techniques Used:

32) Product Images from "Syk Activation in Dendritic Cells Is Essential for Airway Hyperresponsiveness and Inflammation"

Article Title: Syk Activation in Dendritic Cells Is Essential for Airway Hyperresponsiveness and Inflammation

Journal:

doi: 10.1165/rcmb.2005-0298OC

Decreased IL-13 release from OVA-sensitized spleen cells cultured with Syk inhibitor-treated BMDCs. BMDCs were preincubated with 0.3–3 μM of R406 for 1 h. The cells were pulsed with anti-OVA IgG/OVA for 1 h, thoroughly washed, and co-cultured
Figure Legend Snippet: Decreased IL-13 release from OVA-sensitized spleen cells cultured with Syk inhibitor-treated BMDCs. BMDCs were preincubated with 0.3–3 μM of R406 for 1 h. The cells were pulsed with anti-OVA IgG/OVA for 1 h, thoroughly washed, and co-cultured

Techniques Used: Cell Culture

33) Product Images from "HMGB1 amplifies ILC2-induced type-2 inflammation and airway smooth muscle remodelling"

Article Title: HMGB1 amplifies ILC2-induced type-2 inflammation and airway smooth muscle remodelling

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1008651

Anti-HMGB1 diminishes type-2 inflammation and ASM growth in neonatal IRF7 -/- mice. IRF7 -/- mice were inoculated with PVM as described in Fig 2 , and treated with anti-HMGB1 or anti-IL-33 as described in S3 Fig . (a) HMGB1 and (b) IL-33 protein expression in BALF. (c) ILC2 numbers in lung. (d) IL-13 protein expression in BALF. (e) ASM area. (f) TGF-β1 expression by ASM cells. Data are representative of n = 2 experiments with 4–8 mice in each group and are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers). Data were analysed by t-test; *, P
Figure Legend Snippet: Anti-HMGB1 diminishes type-2 inflammation and ASM growth in neonatal IRF7 -/- mice. IRF7 -/- mice were inoculated with PVM as described in Fig 2 , and treated with anti-HMGB1 or anti-IL-33 as described in S3 Fig . (a) HMGB1 and (b) IL-33 protein expression in BALF. (c) ILC2 numbers in lung. (d) IL-13 protein expression in BALF. (e) ASM area. (f) TGF-β1 expression by ASM cells. Data are representative of n = 2 experiments with 4–8 mice in each group and are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers). Data were analysed by t-test; *, P

Techniques Used: Mouse Assay, Expressing, Whisker Assay

Activated ILC2s induce ASM proliferation in vitro in an HMGB1-dependent manner. (a-b) Primary ASM cells were isolated from naïve WT neonatal mice and cultured with ILC2s (purified from the lungs of IRF7 -/- mice at 10 dpi) in the presence of various stimuli as indicated. (a) ASM proliferation (indicated by decreasing median fluorescence intensity (MFI)), IL-13, active TGFβ and HMGB1 concentration in the culture supernatant. * Compared to ASM cells cultured in the absence of ILC2s or cytokine stimulation. (b) ASM proliferation in response to IL-13 (3 ng/ml), disulphide HMGB1 (30 ng/ml), or a non-oxidisable mutant HMGB1 (30 ng/ml). * Compared to IL-13-stimulated ASM cells with disulphide-HMGB1-stimulated ASM cells or disulphide-HMGB1-stimulated ASM cells with non-oxidisable mutant HMGB1-stimulated ASM cells as indicated. (c) HMGB1 protein expression in the supernatant of ASM cells cultured in the presence of IL-33 or IL-13 as indicated. (d) Representative micrograph (x1000 magnification) of HMGB1 immunoreactivity (red) in the lung at 10 dpi, arrows indicate expression by ASM cells. Bar, 5 μm. Quantification of HMGB1 expression by ASM cells. For panel a-d, data are representative of one experiment, performed twice with similar results. For panel e, data are representative of n = 2 experiments with four to eight neonates in each group. Data are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a-d) or as mean ± SEM (e [right]). Data were analysed by One-way ANOVA with Dunnett post hoc test (a-d) or Two-way ANOVA with Tukey post hoc test (e ([right]; *, P
Figure Legend Snippet: Activated ILC2s induce ASM proliferation in vitro in an HMGB1-dependent manner. (a-b) Primary ASM cells were isolated from naïve WT neonatal mice and cultured with ILC2s (purified from the lungs of IRF7 -/- mice at 10 dpi) in the presence of various stimuli as indicated. (a) ASM proliferation (indicated by decreasing median fluorescence intensity (MFI)), IL-13, active TGFβ and HMGB1 concentration in the culture supernatant. * Compared to ASM cells cultured in the absence of ILC2s or cytokine stimulation. (b) ASM proliferation in response to IL-13 (3 ng/ml), disulphide HMGB1 (30 ng/ml), or a non-oxidisable mutant HMGB1 (30 ng/ml). * Compared to IL-13-stimulated ASM cells with disulphide-HMGB1-stimulated ASM cells or disulphide-HMGB1-stimulated ASM cells with non-oxidisable mutant HMGB1-stimulated ASM cells as indicated. (c) HMGB1 protein expression in the supernatant of ASM cells cultured in the presence of IL-33 or IL-13 as indicated. (d) Representative micrograph (x1000 magnification) of HMGB1 immunoreactivity (red) in the lung at 10 dpi, arrows indicate expression by ASM cells. Bar, 5 μm. Quantification of HMGB1 expression by ASM cells. For panel a-d, data are representative of one experiment, performed twice with similar results. For panel e, data are representative of n = 2 experiments with four to eight neonates in each group. Data are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a-d) or as mean ± SEM (e [right]). Data were analysed by One-way ANOVA with Dunnett post hoc test (a-d) or Two-way ANOVA with Tukey post hoc test (e ([right]; *, P

Techniques Used: In Vitro, Isolation, Mouse Assay, Cell Culture, Purification, Fluorescence, Concentration Assay, Mutagenesis, Expressing, Whisker Assay

IRF7 deficiency predisposes to Pneumovirus-induced type-2 inflammation and ASM growth in early-life. WT (IRF7 +/+ ) and IRF7 -/- mice were inoculated with PVM at postnatal day 7 and endpoints assessed at the times indicated. (a) IL-13 and IL-5 protein (10 dpi) expression in BALF. (b) ILC2 numbers in the lung (c) Expression of IL-13 (dsRed) and IL-4 (AmCyan) by ILC2 in the lungs of 4C13R IRF7 +/+ and 4C13R IRF7 -/- mice at 10 dpi. Numbers in quadrants show the percentages of gated cells. Percentage of IL-13-expressing ILC2 (right panel). (d) Numbers of IL-13-producing ILC2, CD4 + and CD8 + cells in left lung lobe at 10 dpi. (e) ASM area. (f) Active TGFβ protein expression in BALF. (g) Representative micrograph (x400 magnification) of TGF-β1 immunoreactivity (red) in the lung at 10 dpi. Bars, 20 μm (left panels). Quantification of TGF-β1 expression by ASM cells (right panel). Data are representative of n = 2 experiments with 4 to 8 mice in each group and are presented as mean ± SEM (a [left], b, e) or as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a [right], c [right], d, f). Data were analysed by Two-way ANOVA with Tukey post hoc test (a [left], b, e) or One-way ANOVA with Dunnett post hoc test (a [right], f [right[) or T-test (c [right], d); *, P
Figure Legend Snippet: IRF7 deficiency predisposes to Pneumovirus-induced type-2 inflammation and ASM growth in early-life. WT (IRF7 +/+ ) and IRF7 -/- mice were inoculated with PVM at postnatal day 7 and endpoints assessed at the times indicated. (a) IL-13 and IL-5 protein (10 dpi) expression in BALF. (b) ILC2 numbers in the lung (c) Expression of IL-13 (dsRed) and IL-4 (AmCyan) by ILC2 in the lungs of 4C13R IRF7 +/+ and 4C13R IRF7 -/- mice at 10 dpi. Numbers in quadrants show the percentages of gated cells. Percentage of IL-13-expressing ILC2 (right panel). (d) Numbers of IL-13-producing ILC2, CD4 + and CD8 + cells in left lung lobe at 10 dpi. (e) ASM area. (f) Active TGFβ protein expression in BALF. (g) Representative micrograph (x400 magnification) of TGF-β1 immunoreactivity (red) in the lung at 10 dpi. Bars, 20 μm (left panels). Quantification of TGF-β1 expression by ASM cells (right panel). Data are representative of n = 2 experiments with 4 to 8 mice in each group and are presented as mean ± SEM (a [left], b, e) or as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a [right], c [right], d, f). Data were analysed by Two-way ANOVA with Tukey post hoc test (a [left], b, e) or One-way ANOVA with Dunnett post hoc test (a [right], f [right[) or T-test (c [right], d); *, P

Techniques Used: Mouse Assay, Expressing, Whisker Assay

HMGB1/RAGE axis amplifies ILC2 type-2 cytokine production and contributes to ASM growth in vivo . (a [left]) Lung ILC2s were purified from IRF7 -/- mice at 10 dpi and cultured with IL-2 for 4 days. Representative micrograph (x1000 magnification) of HMGB1 expression (red) in IL-33 stimulated ILC2s. Bar, 5 μm. (a [right]) HMGB1 levels in the culture supernatant after a 4 day culture +/- IL-33 (3 ng/ml) and +/- sIL-13Rα2 (5 μg/ml) as indicated. (b) IL-13 and HMGB1 protein expression by ILC2s. (c) RAGE expression detected by flow cytometry in lung ILC2s. (d) IL-5, IL-9 and IL-13 protein expression by lung ILC2s purified from IRF7 -/- mice cultured in IL-2 +/- difulphide-HMGB1 (30 ng/ml) or non-oxidisable mutant HMGB1 (30 ng/ml) as indicated. * Compared to IL-2-stimulated ILC2s. (e) Lung ILC2s purified from 4C13R IRF7 -/- mice were cultured in IL-2 +/- IL-33, and treated with anti-HMGB1 (Clone 2G7, 5 μg/ml), TLR4 antagonist (LPS-RS 1 μg/ml) and RAGE antagonist (FPS-ZMI; 300 nM) as indicated. DsRed (IL-13) intensity was quantified by flow cytometry. * Compared to IL-2/IL-33-stimulated ILC2s. (f) IL-5 and IL-9 production by ILC2s, treated as described in panel e. (g-m) IRF7 -/- mice were inoculated with PVM as described in Fig 2 , and treated daily from 3–10 dpi with a RAGE antagonist (FPS-ZM1). (g) IL-33 and (h) HMGB1 protein expression in BALF. (i) Lung ILC2s, (j) IL-5 protein in BALF, (k) Eosinophils in BALF, (l) ASM area, and (m) Quantification of TGF-β1 expression by ASM cells. For panel a-f, data are representative of one experiment, performed twice with similar results. For panel g-m, data are representative of n = 2 experiments with 4–9 mice in each group and are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a [right], d, g-m) or as mean ± SEM (b) or as scatter plot (e [right]-f). Data were analysed by One-way ANOVA with Dunnett post hoc test (a, d, e [right]-f) or T-test (g-m); *, P
Figure Legend Snippet: HMGB1/RAGE axis amplifies ILC2 type-2 cytokine production and contributes to ASM growth in vivo . (a [left]) Lung ILC2s were purified from IRF7 -/- mice at 10 dpi and cultured with IL-2 for 4 days. Representative micrograph (x1000 magnification) of HMGB1 expression (red) in IL-33 stimulated ILC2s. Bar, 5 μm. (a [right]) HMGB1 levels in the culture supernatant after a 4 day culture +/- IL-33 (3 ng/ml) and +/- sIL-13Rα2 (5 μg/ml) as indicated. (b) IL-13 and HMGB1 protein expression by ILC2s. (c) RAGE expression detected by flow cytometry in lung ILC2s. (d) IL-5, IL-9 and IL-13 protein expression by lung ILC2s purified from IRF7 -/- mice cultured in IL-2 +/- difulphide-HMGB1 (30 ng/ml) or non-oxidisable mutant HMGB1 (30 ng/ml) as indicated. * Compared to IL-2-stimulated ILC2s. (e) Lung ILC2s purified from 4C13R IRF7 -/- mice were cultured in IL-2 +/- IL-33, and treated with anti-HMGB1 (Clone 2G7, 5 μg/ml), TLR4 antagonist (LPS-RS 1 μg/ml) and RAGE antagonist (FPS-ZMI; 300 nM) as indicated. DsRed (IL-13) intensity was quantified by flow cytometry. * Compared to IL-2/IL-33-stimulated ILC2s. (f) IL-5 and IL-9 production by ILC2s, treated as described in panel e. (g-m) IRF7 -/- mice were inoculated with PVM as described in Fig 2 , and treated daily from 3–10 dpi with a RAGE antagonist (FPS-ZM1). (g) IL-33 and (h) HMGB1 protein expression in BALF. (i) Lung ILC2s, (j) IL-5 protein in BALF, (k) Eosinophils in BALF, (l) ASM area, and (m) Quantification of TGF-β1 expression by ASM cells. For panel a-f, data are representative of one experiment, performed twice with similar results. For panel g-m, data are representative of n = 2 experiments with 4–9 mice in each group and are presented as box-and-whisker plots showing quartiles (boxes) and range (whiskers; a [right], d, g-m) or as mean ± SEM (b) or as scatter plot (e [right]-f). Data were analysed by One-way ANOVA with Dunnett post hoc test (a, d, e [right]-f) or T-test (g-m); *, P

Techniques Used: In Vivo, Purification, Mouse Assay, Cell Culture, Expressing, Flow Cytometry, Mutagenesis, Whisker Assay

Secondary Pneumovirus infection promotes an asthma-like pathology in IRF7-deficienct mice. WT (IRF7 +/+ ) and IRF7 -/- mice were inoculated with PVM at postnatal day 7 and re-infected 6 weeks later. Samples were collected prior to re-infection (t = 0) and at 3, 7 and 21 dpi. (a) Representative micrograph (x400 magnification) of α-smooth muscle actin immunoreactivity (red) in the lung at 7 dpi and ASM area quantification. Bars, 20 μm. (b) AHR at 30 mg/ml of methacholine. (c) IL-13 protein levels in BALF. (d) Total eosinophil numbers in BALF. (e) Mucus-producing airway epithelial cells (AECs). (f) IL-33 (left) and HMGB1 (right) protein expression in BALF at 3 dpi. Data are representative of n = 2 experiments with 4–9 mice in each group and are presented as mean ± SEM (a ([right], b—e) or as box-and-whisker plots showing quartiles (boxes) and range (whiskers; f). Data were analyzed by Two-way ANOVA with Tukey post hoc test (a ([right], b—e) or One-way ANOVA with Dunnett post hoc test (f); *, P
Figure Legend Snippet: Secondary Pneumovirus infection promotes an asthma-like pathology in IRF7-deficienct mice. WT (IRF7 +/+ ) and IRF7 -/- mice were inoculated with PVM at postnatal day 7 and re-infected 6 weeks later. Samples were collected prior to re-infection (t = 0) and at 3, 7 and 21 dpi. (a) Representative micrograph (x400 magnification) of α-smooth muscle actin immunoreactivity (red) in the lung at 7 dpi and ASM area quantification. Bars, 20 μm. (b) AHR at 30 mg/ml of methacholine. (c) IL-13 protein levels in BALF. (d) Total eosinophil numbers in BALF. (e) Mucus-producing airway epithelial cells (AECs). (f) IL-33 (left) and HMGB1 (right) protein expression in BALF at 3 dpi. Data are representative of n = 2 experiments with 4–9 mice in each group and are presented as mean ± SEM (a ([right], b—e) or as box-and-whisker plots showing quartiles (boxes) and range (whiskers; f). Data were analyzed by Two-way ANOVA with Tukey post hoc test (a ([right], b—e) or One-way ANOVA with Dunnett post hoc test (f); *, P

Techniques Used: Infection, Mouse Assay, Expressing, Whisker Assay

34) Product Images from "Altered Frequencies and Functions of Innate Lymphoid Cells in Melanoma Patients Are Modulated by Immune Checkpoints Inhibitors"

Article Title: Altered Frequencies and Functions of Innate Lymphoid Cells in Melanoma Patients Are Modulated by Immune Checkpoints Inhibitors

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.811131

Oscillations in ILC subpopulations induced by Nivolumab treatment in stage IV melanoma patients. Statistical analysis of the frequency of the indicated ILC subsets in stage IV melanoma patients before (squares) and after (triangles) two months of Nivolumab therapy. For total ILCs and CD117 - ILC2s, the analysis was performed cumulating data from 24 patients. For IL-13 and TNFα, the analysis was performed cumulating data from 14 patients. Analyses were performed by paired Student’s t-test for normally distributed samples or Wilcoxon matched-pairs signed rank test for not-normally distributed samples. Cumulative data are shown as mean ± SD. *p-value
Figure Legend Snippet: Oscillations in ILC subpopulations induced by Nivolumab treatment in stage IV melanoma patients. Statistical analysis of the frequency of the indicated ILC subsets in stage IV melanoma patients before (squares) and after (triangles) two months of Nivolumab therapy. For total ILCs and CD117 - ILC2s, the analysis was performed cumulating data from 24 patients. For IL-13 and TNFα, the analysis was performed cumulating data from 14 patients. Analyses were performed by paired Student’s t-test for normally distributed samples or Wilcoxon matched-pairs signed rank test for not-normally distributed samples. Cumulative data are shown as mean ± SD. *p-value

Techniques Used:

Cross-talk between ILC subsets and melanoma cells in vitro . (A) Gating strategy used to identify ILCs within co-cultures. (B, C) Representative plots of TNFα up-regulation in all the three ILC subsets (B) and IL-13 up-regulation in ILC2s (C) co-cultured with melanoma cells at 5:1 ratio for 3 days. (D, E) Percentage of positive cells (upper lines) and expression (lower lines) of TNFα in all the three ILC subsets (D) and of IL-13 in ILC2s (E) . Cumulative data derived from eight independent experiments performed using two different melanoma cell lines are shown as mean ± SD. For each cell line, at least three independent experiments were carried out. Analysis was performed by paired Student t test. **p-value
Figure Legend Snippet: Cross-talk between ILC subsets and melanoma cells in vitro . (A) Gating strategy used to identify ILCs within co-cultures. (B, C) Representative plots of TNFα up-regulation in all the three ILC subsets (B) and IL-13 up-regulation in ILC2s (C) co-cultured with melanoma cells at 5:1 ratio for 3 days. (D, E) Percentage of positive cells (upper lines) and expression (lower lines) of TNFα in all the three ILC subsets (D) and of IL-13 in ILC2s (E) . Cumulative data derived from eight independent experiments performed using two different melanoma cell lines are shown as mean ± SD. For each cell line, at least three independent experiments were carried out. Analysis was performed by paired Student t test. **p-value

Techniques Used: In Vitro, Cell Culture, Expressing, Derivative Assay

Nivolumab effects on ILCs subsets in stage IV melanoma. The inhibitory receptor PD-1 regulates the secretory activity of both ILC2s and ILC3s as well as CD117 + ILC precursors. In stage IV melanoma patients, PD-1 targeting through Nivolumab enhances the production of TNFα and IL-13 from circulating CD117 + ILCs and CD117 + ILC2s, respectively. Furthermore, Nivolumab induces the expansion of blood CD117 - ILC2s. These effects are likely due to Nivolumab-mediated disruption of the interaction between PD-1, expressed on ILCs, and its ligand PD-L1, expressed on melanoma cells and/or other immune cells such as macrophages, MDSCs and Tregs.
Figure Legend Snippet: Nivolumab effects on ILCs subsets in stage IV melanoma. The inhibitory receptor PD-1 regulates the secretory activity of both ILC2s and ILC3s as well as CD117 + ILC precursors. In stage IV melanoma patients, PD-1 targeting through Nivolumab enhances the production of TNFα and IL-13 from circulating CD117 + ILCs and CD117 + ILC2s, respectively. Furthermore, Nivolumab induces the expansion of blood CD117 - ILC2s. These effects are likely due to Nivolumab-mediated disruption of the interaction between PD-1, expressed on ILCs, and its ligand PD-L1, expressed on melanoma cells and/or other immune cells such as macrophages, MDSCs and Tregs.

Techniques Used: Activity Assay

35) Product Images from "Altered Frequencies and Functions of Innate Lymphoid Cells in Melanoma Patients Are Modulated by Immune Checkpoints Inhibitors"

Article Title: Altered Frequencies and Functions of Innate Lymphoid Cells in Melanoma Patients Are Modulated by Immune Checkpoints Inhibitors

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.811131

Oscillations in ILC subpopulations induced by Nivolumab treatment in stage IV melanoma patients. Statistical analysis of the frequency of the indicated ILC subsets in stage IV melanoma patients before (squares) and after (triangles) two months of Nivolumab therapy. For total ILCs and CD117 - ILC2s, the analysis was performed cumulating data from 24 patients. For IL-13 and TNFα, the analysis was performed cumulating data from 14 patients. Analyses were performed by paired Student’s t-test for normally distributed samples or Wilcoxon matched-pairs signed rank test for not-normally distributed samples. Cumulative data are shown as mean ± SD. *p-value
Figure Legend Snippet: Oscillations in ILC subpopulations induced by Nivolumab treatment in stage IV melanoma patients. Statistical analysis of the frequency of the indicated ILC subsets in stage IV melanoma patients before (squares) and after (triangles) two months of Nivolumab therapy. For total ILCs and CD117 - ILC2s, the analysis was performed cumulating data from 24 patients. For IL-13 and TNFα, the analysis was performed cumulating data from 14 patients. Analyses were performed by paired Student’s t-test for normally distributed samples or Wilcoxon matched-pairs signed rank test for not-normally distributed samples. Cumulative data are shown as mean ± SD. *p-value

Techniques Used:

Cross-talk between ILC subsets and melanoma cells in vitro . (A) Gating strategy used to identify ILCs within co-cultures. (B, C) Representative plots of TNFα up-regulation in all the three ILC subsets (B) and IL-13 up-regulation in ILC2s (C) co-cultured with melanoma cells at 5:1 ratio for 3 days. (D, E) Percentage of positive cells (upper lines) and expression (lower lines) of TNFα in all the three ILC subsets (D) and of IL-13 in ILC2s (E) . Cumulative data derived from eight independent experiments performed using two different melanoma cell lines are shown as mean ± SD. For each cell line, at least three independent experiments were carried out. Analysis was performed by paired Student t test. **p-value
Figure Legend Snippet: Cross-talk between ILC subsets and melanoma cells in vitro . (A) Gating strategy used to identify ILCs within co-cultures. (B, C) Representative plots of TNFα up-regulation in all the three ILC subsets (B) and IL-13 up-regulation in ILC2s (C) co-cultured with melanoma cells at 5:1 ratio for 3 days. (D, E) Percentage of positive cells (upper lines) and expression (lower lines) of TNFα in all the three ILC subsets (D) and of IL-13 in ILC2s (E) . Cumulative data derived from eight independent experiments performed using two different melanoma cell lines are shown as mean ± SD. For each cell line, at least three independent experiments were carried out. Analysis was performed by paired Student t test. **p-value

Techniques Used: In Vitro, Cell Culture, Expressing, Derivative Assay

Nivolumab effects on ILCs subsets in stage IV melanoma. The inhibitory receptor PD-1 regulates the secretory activity of both ILC2s and ILC3s as well as CD117 + ILC precursors. In stage IV melanoma patients, PD-1 targeting through Nivolumab enhances the production of TNFα and IL-13 from circulating CD117 + ILCs and CD117 + ILC2s, respectively. Furthermore, Nivolumab induces the expansion of blood CD117 - ILC2s. These effects are likely due to Nivolumab-mediated disruption of the interaction between PD-1, expressed on ILCs, and its ligand PD-L1, expressed on melanoma cells and/or other immune cells such as macrophages, MDSCs and Tregs.
Figure Legend Snippet: Nivolumab effects on ILCs subsets in stage IV melanoma. The inhibitory receptor PD-1 regulates the secretory activity of both ILC2s and ILC3s as well as CD117 + ILC precursors. In stage IV melanoma patients, PD-1 targeting through Nivolumab enhances the production of TNFα and IL-13 from circulating CD117 + ILCs and CD117 + ILC2s, respectively. Furthermore, Nivolumab induces the expansion of blood CD117 - ILC2s. These effects are likely due to Nivolumab-mediated disruption of the interaction between PD-1, expressed on ILCs, and its ligand PD-L1, expressed on melanoma cells and/or other immune cells such as macrophages, MDSCs and Tregs.

Techniques Used: Activity Assay

36) Product Images from "Chinese herbal component, Praeruptorin E, enhances anti-asthma efficacy and prevents toxicity of aminophylline by targeting the NF-κB/PXR/CYP3A4 pathway"

Article Title: Chinese herbal component, Praeruptorin E, enhances anti-asthma efficacy and prevents toxicity of aminophylline by targeting the NF-κB/PXR/CYP3A4 pathway

Journal: Annals of Translational Medicine

doi: 10.21037/atm-22-386

PE facilitates aminophylline inhibition of inflammation factors in asthmatic mice. (A-C) Concentrations of IL-4, IL-5, and IL-13 in BALF. (D,E) Concentrations of IgE and NF-κB p65 in serum. (F,G) Expression levels of NF-κB p65 in the lungs and bronchi detected by immunofluorescence assay. Magnification ×400, green: NF-κB p65, blue: nucleus. OVA + Ami group, OVA + Ami + PE 2.5 mg/kg group, OVA + Ami + PE 10 mg/kg group and OVA + Ami + PE 40 mg/kg group compared with the OVA group, *P
Figure Legend Snippet: PE facilitates aminophylline inhibition of inflammation factors in asthmatic mice. (A-C) Concentrations of IL-4, IL-5, and IL-13 in BALF. (D,E) Concentrations of IgE and NF-κB p65 in serum. (F,G) Expression levels of NF-κB p65 in the lungs and bronchi detected by immunofluorescence assay. Magnification ×400, green: NF-κB p65, blue: nucleus. OVA + Ami group, OVA + Ami + PE 2.5 mg/kg group, OVA + Ami + PE 10 mg/kg group and OVA + Ami + PE 40 mg/kg group compared with the OVA group, *P

Techniques Used: Inhibition, Mouse Assay, Expressing, Immunofluorescence

37) Product Images from "BCG vaccination reduces the mortality of Mycobacterium tuberculosis–infected type 2 diabetes mellitus mice"

Article Title: BCG vaccination reduces the mortality of Mycobacterium tuberculosis–infected type 2 diabetes mellitus mice

Journal: JCI Insight

doi: 10.1172/jci.insight.133788

IL-13 produced by Tregs is involved in the expansion of antiinflammatory M2 macrophages. ( A ) At four months p.i., the formalin fixed and paraffin embedded lungs sections from Mtb-infected BCG-vaccinated and BCG-vaccinated T2DM were analyzed by confocal microscopy for Foxp3 + IL13 + cells. Foxp3 (red), IL-13 (green), and DAPI (blue) expression is shown. Scale bar: 20 μm. BCG-vaccinated T2DM mouse splenocytes were isolated and cultured at 1 × 10 6 cells/well in RPMI-1640 containing penicillin (Invitrogen) and 10% heat-inactivated FCS, with or without γ-irradiated Mtb H37Rv (10 µg/mL) at 37°C and 5% CO 2 . In some wells, Treg-depleted splenocytes were stimulated with γ-irradiated Mtb (10 µg/mL). After 72 hours, the cells were collected and phenotypically characterized for M1/M2 macrophages, and the supernatants were used to measure levels of the cytokine IL-13. ( B ) Mean fluorescence intensity (MFI) values showing the CD45 + F4/80 + iNos + and CD45 + F4/80 + Arg1 + cells, and IL-13 levels in culture supernatants, were measured by ELISA. ( C ) BCG-vaccinated nondiabetic and BCG-vaccinated T2DM mouse splenocytes were stimulated with γ-irradiated Mtb H37Rv (10 µg/mL) and treated with anti–IL-13R (10 µg/mL) for 72 hours. After 72 hours, flow cytometry was performed to determine the M2 macrophage phenotype (CD45 + F4/80 + Arg1 + cells). ( D ) MFI values and histogram plot showing IL-13R expression gated on CD45 + F4/80 + Arg1 + cells. ( E ) Macrophages isolated from BCG-vaccinated mice were cocultured with autologous Tregs or Tregs isolated from BCG-vaccinated T2DM mice and stimulated with γ-irradiated Mtb (10 µg/mL). After 72-hour incubation, the macrophages were analyzed for M1 and M2 phenotype by flow cytometry. MFI values showing the CD45 + F4/80 + iNos + cells and CD45 + F4/80 + Arg1 + cells. ( F ) For neutralization studies, BCG-vaccinated T2DM mouse macrophages were stimulated with γ-irradiated Mtb (10 µg/mL) with or without autologous Tregs, and in some wells, IgG control or anti–IL-13R (10 µg/mL) antibodies were added. After 72 hours of incubation, the M1 and M2 phenotypes of cultured macrophages were determined by flow cytometry, and TNF-α and IL-6 levels were measured by ELISA in the culture supernatants. rIL-13 (10 ng/mL) was added to the macrophages from BCG-vaccinated T2DM mice and stimulated with γ-irradiated Mtb (10 µg/mL). (G) After 72 hours of incubation, the M1 and M2 phenotypes of cultured macrophages were determined by flow cytometry, and MFI values for CD45 + F4/80 + iNos + cells and CD45 + F4/80 + Arg1 + cells are shown. Experiments were performed 2 times, and each time, 2–3 mice per group were used. The data are shown as mean ± SDs of n = 5 mice per group. The statistical analysis was performed by 1-way ANOVA, followed by Tukey’s multiple comparisons test. * P
Figure Legend Snippet: IL-13 produced by Tregs is involved in the expansion of antiinflammatory M2 macrophages. ( A ) At four months p.i., the formalin fixed and paraffin embedded lungs sections from Mtb-infected BCG-vaccinated and BCG-vaccinated T2DM were analyzed by confocal microscopy for Foxp3 + IL13 + cells. Foxp3 (red), IL-13 (green), and DAPI (blue) expression is shown. Scale bar: 20 μm. BCG-vaccinated T2DM mouse splenocytes were isolated and cultured at 1 × 10 6 cells/well in RPMI-1640 containing penicillin (Invitrogen) and 10% heat-inactivated FCS, with or without γ-irradiated Mtb H37Rv (10 µg/mL) at 37°C and 5% CO 2 . In some wells, Treg-depleted splenocytes were stimulated with γ-irradiated Mtb (10 µg/mL). After 72 hours, the cells were collected and phenotypically characterized for M1/M2 macrophages, and the supernatants were used to measure levels of the cytokine IL-13. ( B ) Mean fluorescence intensity (MFI) values showing the CD45 + F4/80 + iNos + and CD45 + F4/80 + Arg1 + cells, and IL-13 levels in culture supernatants, were measured by ELISA. ( C ) BCG-vaccinated nondiabetic and BCG-vaccinated T2DM mouse splenocytes were stimulated with γ-irradiated Mtb H37Rv (10 µg/mL) and treated with anti–IL-13R (10 µg/mL) for 72 hours. After 72 hours, flow cytometry was performed to determine the M2 macrophage phenotype (CD45 + F4/80 + Arg1 + cells). ( D ) MFI values and histogram plot showing IL-13R expression gated on CD45 + F4/80 + Arg1 + cells. ( E ) Macrophages isolated from BCG-vaccinated mice were cocultured with autologous Tregs or Tregs isolated from BCG-vaccinated T2DM mice and stimulated with γ-irradiated Mtb (10 µg/mL). After 72-hour incubation, the macrophages were analyzed for M1 and M2 phenotype by flow cytometry. MFI values showing the CD45 + F4/80 + iNos + cells and CD45 + F4/80 + Arg1 + cells. ( F ) For neutralization studies, BCG-vaccinated T2DM mouse macrophages were stimulated with γ-irradiated Mtb (10 µg/mL) with or without autologous Tregs, and in some wells, IgG control or anti–IL-13R (10 µg/mL) antibodies were added. After 72 hours of incubation, the M1 and M2 phenotypes of cultured macrophages were determined by flow cytometry, and TNF-α and IL-6 levels were measured by ELISA in the culture supernatants. rIL-13 (10 ng/mL) was added to the macrophages from BCG-vaccinated T2DM mice and stimulated with γ-irradiated Mtb (10 µg/mL). (G) After 72 hours of incubation, the M1 and M2 phenotypes of cultured macrophages were determined by flow cytometry, and MFI values for CD45 + F4/80 + iNos + cells and CD45 + F4/80 + Arg1 + cells are shown. Experiments were performed 2 times, and each time, 2–3 mice per group were used. The data are shown as mean ± SDs of n = 5 mice per group. The statistical analysis was performed by 1-way ANOVA, followed by Tukey’s multiple comparisons test. * P

Techniques Used: Produced, Infection, Confocal Microscopy, Expressing, Isolation, Cell Culture, Irradiation, Fluorescence, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Mouse Assay, Incubation, Neutralization

BCG vaccination alters cytokine and chemokine levels in T2DM mice infected with Mtb . PBS control, BCG-vaccinated, T2DM, or BCG-vaccinated T2DM mice were infected with ~100 CFU of aerosolized Mtb H37Rv. ( A ) At 1 and 4 months p.i., lung homogenates from PBS control, BCG-vaccinated control, T2DM, BCG-vaccinated T2DM, infected control, infected T2DM, infected BCG-vaccinated control, and infected BCG-vaccinated T2DM mice were collected, and cytokine and chemokine levels were measured in a multiplex ELISA. ( A–I ) Quantitative results of the cytokines and chemokines IL-1β ( A ), IFN-γ ( B ), IL-17 ( C ), IL-6 ( D ), IL-13 ( E ), IL-15 ( F ), IL-21 ( G ), IL-22 ( H ), and CXCL10/IP-10 ( I ) are shown. Experiments were performed 2 times, and each time, 2–3 mice per group were used. The data are shown as mean ± SDs of n = 5 mice per group. The statistical analysis was performed by 2-way ANOVA, followed by Tukey’s multiple comparisons test. * P
Figure Legend Snippet: BCG vaccination alters cytokine and chemokine levels in T2DM mice infected with Mtb . PBS control, BCG-vaccinated, T2DM, or BCG-vaccinated T2DM mice were infected with ~100 CFU of aerosolized Mtb H37Rv. ( A ) At 1 and 4 months p.i., lung homogenates from PBS control, BCG-vaccinated control, T2DM, BCG-vaccinated T2DM, infected control, infected T2DM, infected BCG-vaccinated control, and infected BCG-vaccinated T2DM mice were collected, and cytokine and chemokine levels were measured in a multiplex ELISA. ( A–I ) Quantitative results of the cytokines and chemokines IL-1β ( A ), IFN-γ ( B ), IL-17 ( C ), IL-6 ( D ), IL-13 ( E ), IL-15 ( F ), IL-21 ( G ), IL-22 ( H ), and CXCL10/IP-10 ( I ) are shown. Experiments were performed 2 times, and each time, 2–3 mice per group were used. The data are shown as mean ± SDs of n = 5 mice per group. The statistical analysis was performed by 2-way ANOVA, followed by Tukey’s multiple comparisons test. * P

Techniques Used: Mouse Assay, Infection, Multiplex Assay, Enzyme-linked Immunosorbent Assay

38) Product Images from "Linking the effects of helminth infection, diet and the gut microbiota with human whole-blood signatures"

Article Title: Linking the effects of helminth infection, diet and the gut microbiota with human whole-blood signatures

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1008066

Helminth dependent covariates of microbial and blood profiles. (A) Blood covariates that responded significantly to deworming, with changes in values that resembled values seen in urban participants, as demonstrated using the multi-group boxplots (left). Values for boxplots are based on 18 urban participants and 46 Orang Asli participants with pre-deworming serum chemistry values, as well as 34 Orang Asli who received deworming treatment and had serum iron and serum globulin levels measured post-deworming and 33 Orang Asli who received deworming treatment and had serum zinc levels measured post-deworming. (Right) Connected line plots for paired comparisons of 30 pairs of pre- and post-deworming samples for serum zinc levels, as well as 31 pairs of pre- and post-deworming samples for serum iron and globulin levels. P-values for two-group comparisons (pre-deworming Orang Asli vs. urban participants) are based on t-tests (when data is normally distributed) and Mann-Whitney tests (when data is not normally distributed). Normality of data distribution is determined using the Shapiro-Wilk test of normality. Statistical significance of deworming changes are based on paired t-test of pre- and post-deworming paired values. Microbial OTUs associated with changes in (B) serum zinc levels and (C) serum iron levels post-deworming treatment are shown; values are coefficients from SPLS-regression models. Selected microbial OTUs are labeled with taxonomic assignments of the finest possible resolution and color coded by taxonomic order. Repeated taxa names represent two distinct OTUs with the same taxonomic assignment. (D) A PCA plot based on the 1835 most variable genes (85 th percentile) shows separation between transcriptional profiles of macrophages that were treated (teal) or untreated (pink) with zinc sulfate. Some samples were treated with IL-4 (squares) and IL-13 (triangles) as well. (E) Heatmap of the 41 immune-related genes (GO:0002376) that are significantly differentially expressed in zinc-stimulated versus unstimulated experiments (cutoff p = 0.01, |log2 fold-change| > 1).
Figure Legend Snippet: Helminth dependent covariates of microbial and blood profiles. (A) Blood covariates that responded significantly to deworming, with changes in values that resembled values seen in urban participants, as demonstrated using the multi-group boxplots (left). Values for boxplots are based on 18 urban participants and 46 Orang Asli participants with pre-deworming serum chemistry values, as well as 34 Orang Asli who received deworming treatment and had serum iron and serum globulin levels measured post-deworming and 33 Orang Asli who received deworming treatment and had serum zinc levels measured post-deworming. (Right) Connected line plots for paired comparisons of 30 pairs of pre- and post-deworming samples for serum zinc levels, as well as 31 pairs of pre- and post-deworming samples for serum iron and globulin levels. P-values for two-group comparisons (pre-deworming Orang Asli vs. urban participants) are based on t-tests (when data is normally distributed) and Mann-Whitney tests (when data is not normally distributed). Normality of data distribution is determined using the Shapiro-Wilk test of normality. Statistical significance of deworming changes are based on paired t-test of pre- and post-deworming paired values. Microbial OTUs associated with changes in (B) serum zinc levels and (C) serum iron levels post-deworming treatment are shown; values are coefficients from SPLS-regression models. Selected microbial OTUs are labeled with taxonomic assignments of the finest possible resolution and color coded by taxonomic order. Repeated taxa names represent two distinct OTUs with the same taxonomic assignment. (D) A PCA plot based on the 1835 most variable genes (85 th percentile) shows separation between transcriptional profiles of macrophages that were treated (teal) or untreated (pink) with zinc sulfate. Some samples were treated with IL-4 (squares) and IL-13 (triangles) as well. (E) Heatmap of the 41 immune-related genes (GO:0002376) that are significantly differentially expressed in zinc-stimulated versus unstimulated experiments (cutoff p = 0.01, |log2 fold-change| > 1).

Techniques Used: MANN-WHITNEY, Labeling

39) Product Images from "Restoration of Tumor Immune Surveillance via Targeting of IL-13Receptor-?2"

Article Title: Restoration of Tumor Immune Surveillance via Targeting of IL-13Receptor-?2

Journal:

doi: 10.1158/0008-5472.CAN-07-5301

IL-13 receptor expression and TGF-β 1 production after CT-26 injection. ( A ) IL-13Rα 1 and IL-13Rα 2 expression during the CT-26 lung tumor model in BALB/c mice. IL-13Rα 1 mRNA expression was determined by RT-PCR of RNA extracted
Figure Legend Snippet: IL-13 receptor expression and TGF-β 1 production after CT-26 injection. ( A ) IL-13Rα 1 and IL-13Rα 2 expression during the CT-26 lung tumor model in BALB/c mice. IL-13Rα 1 mRNA expression was determined by RT-PCR of RNA extracted

Techniques Used: Expressing, Injection, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

40) Product Images from "Interferon-?1 (interleukin-29) preferentially down-regulates interleukin-13 over other T helper type 2 cytokine responses in vitro"

Article Title: Interferon-?1 (interleukin-29) preferentially down-regulates interleukin-13 over other T helper type 2 cytokine responses in vitro

Journal:

doi: 10.1111/j.1365-2567.2008.02862.x

Interferon (IFN)-λ1 inhibits the production and secretion of interleukin (IL)-13. (a) Cell culture supernatants were harvested and subjected to enzyme-linked immunosorbent assays (ELISAs) as described. Data for each donor are shown, with and without
Figure Legend Snippet: Interferon (IFN)-λ1 inhibits the production and secretion of interleukin (IL)-13. (a) Cell culture supernatants were harvested and subjected to enzyme-linked immunosorbent assays (ELISAs) as described. Data for each donor are shown, with and without

Techniques Used: Cell Culture

Interferon (IFN)-λ1 inhibits interleukin (IL)-13 secretion under T helper type 2 (Th2)-promoting conditions. (a) Peripheral blood mononuclear cells (PBMC) were stimulated with Concanavalin-A (ConA) under the following conditions: A, ConA alone;
Figure Legend Snippet: Interferon (IFN)-λ1 inhibits interleukin (IL)-13 secretion under T helper type 2 (Th2)-promoting conditions. (a) Peripheral blood mononuclear cells (PBMC) were stimulated with Concanavalin-A (ConA) under the following conditions: A, ConA alone;

Techniques Used:

Interferon (IFN)-λ1 lowers the numbers of interleukin (IL)-4, IL-5 and IL-13-positive CD4 T cells. Cells were harvested and prepared for flow cytometric analysis as described. (a, b) typical fluorescence-activated cell sorting (FACS) analysis
Figure Legend Snippet: Interferon (IFN)-λ1 lowers the numbers of interleukin (IL)-4, IL-5 and IL-13-positive CD4 T cells. Cells were harvested and prepared for flow cytometric analysis as described. (a, b) typical fluorescence-activated cell sorting (FACS) analysis

Techniques Used: Flow Cytometry, Fluorescence, FACS

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    R&D Systems interleukin 13
    Schematic outline of the irradiation procedure for macrophages. THP-1 monocytes were differentiated into macrophages (M0) with 150 nM PMA in cloning cylinder, placed at the center of the irradiation chamber. Macrophages were polarized in M1 phenotype with 10 pg/ml LPS and 20 ng/ml IFN-γ during 24 h incubation or were polarized in M2 phenotype with 20 ng/ml IL-4 and <t>IL-13</t> during 48 h incubation. THP-1 monocytes were differentiated on the appropriate day, in order to obtain the three phenotypes on day 4. Following the experiment that was performed, the biological material was collected 8, 12, 16, or 24 h after proton or X-ray irradiation
    Interleukin 13, supplied by R&D Systems, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems rabbit mabs
    Synergistic and antagonistic effects of multiple <t>NiV-G</t> N-glycans in combination on cell-cell fusion. (A) Relative levels of 293T cell surface expression (HA), binding to <t>MAbs</t> (MAb26, MAb45, and MAb213), binding to soluble ephrinB2 receptor, and cell-cell
    Rabbit Mabs, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems recombinant human il13rα1
    IL13Rα2-CAR T cells release cytokines after stimulation with recombinant IL13Rα2 protein or IL13Rα2-positive cells . IL13Rα2-CAR or nontransduced (NT) T cells were stimulated with recombinant <t>IL13Rα1,</t> IL13Rα2,
    Recombinant Human Il13rα1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    R&D Systems il 13
    Impact of stimulation with 100 ng/mL of IL-4 or <t>IL-13</t> on HCT 116 cells—a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) control for IL-13 at 72 h; ( m ) stimulated with IL-13 for 0 h; ( n ) stimulated with IL-13 at 24 h; ( o ) stimulated with IL-13 at 48 h; ( p ) stimulated with IL-13 at 72 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The ratios in interleukin-stimulated cells significantly ( p
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    Schematic outline of the irradiation procedure for macrophages. THP-1 monocytes were differentiated into macrophages (M0) with 150 nM PMA in cloning cylinder, placed at the center of the irradiation chamber. Macrophages were polarized in M1 phenotype with 10 pg/ml LPS and 20 ng/ml IFN-γ during 24 h incubation or were polarized in M2 phenotype with 20 ng/ml IL-4 and IL-13 during 48 h incubation. THP-1 monocytes were differentiated on the appropriate day, in order to obtain the three phenotypes on day 4. Following the experiment that was performed, the biological material was collected 8, 12, 16, or 24 h after proton or X-ray irradiation

    Journal: Cell Death & Disease

    Article Title: Proton irradiation orchestrates macrophage reprogramming through NFκB signaling

    doi: 10.1038/s41419-018-0757-9

    Figure Lengend Snippet: Schematic outline of the irradiation procedure for macrophages. THP-1 monocytes were differentiated into macrophages (M0) with 150 nM PMA in cloning cylinder, placed at the center of the irradiation chamber. Macrophages were polarized in M1 phenotype with 10 pg/ml LPS and 20 ng/ml IFN-γ during 24 h incubation or were polarized in M2 phenotype with 20 ng/ml IL-4 and IL-13 during 48 h incubation. THP-1 monocytes were differentiated on the appropriate day, in order to obtain the three phenotypes on day 4. Following the experiment that was performed, the biological material was collected 8, 12, 16, or 24 h after proton or X-ray irradiation

    Article Snippet: Macrophages were polarized in M1 phenotype with 10 pg/ml lipopolysaccharides (LPS, Sigma; #8630) and 20 ng/ml interferon γ (IFN-γ, R & D Systems, #285-IF) during 24 h incubation or were polarized in M2 phenotype with 20 ng/ml interleukin 4 (IL-4, R & D Systems, #204-IL) and 20 ng/ml interleukin 13 (IL-13, R & D Systems, #213-ILB) during 48 h incubation as described in ref. .

    Techniques: Irradiation, Clone Assay, Incubation

    Synergistic and antagonistic effects of multiple NiV-G N-glycans in combination on cell-cell fusion. (A) Relative levels of 293T cell surface expression (HA), binding to MAbs (MAb26, MAb45, and MAb213), binding to soluble ephrinB2 receptor, and cell-cell

    Journal: Journal of Virology

    Article Title: N-Glycans on the Nipah Virus Attachment Glycoprotein Modulate Fusion and Viral Entry as They Protect against Antibody Neutralization

    doi: 10.1128/JVI.01304-12

    Figure Lengend Snippet: Synergistic and antagonistic effects of multiple NiV-G N-glycans in combination on cell-cell fusion. (A) Relative levels of 293T cell surface expression (HA), binding to MAbs (MAb26, MAb45, and MAb213), binding to soluble ephrinB2 receptor, and cell-cell

    Article Snippet: The binding of anti-NiV-F antiserum 834 to surface NiV-F and the binding of the anti-NiV-G specific rabbit MAbs (MAb26, MAb45, or MAb213) or mouse anti-hemagglutinin (anti-HA) MAb or soluble ephinB2 fused to human Fc (B2-hFc; R & D Systems, Minneapolis, MN) to cell surface NiV-G were measured by flow cytometry on NiV-F/G-transfected cells.

    Techniques: Expressing, Binding Assay

    IL13Rα2-CAR T cells release cytokines after stimulation with recombinant IL13Rα2 protein or IL13Rα2-positive cells . IL13Rα2-CAR or nontransduced (NT) T cells were stimulated with recombinant IL13Rα1, IL13Rα2,

    Journal: Molecular Therapy

    Article Title: Characterization and Functional Analysis of scFv-based Chimeric Antigen Receptors to Redirect T Cells to IL13Rα2-positive Glioma

    doi: 10.1038/mt.2015.199

    Figure Lengend Snippet: IL13Rα2-CAR T cells release cytokines after stimulation with recombinant IL13Rα2 protein or IL13Rα2-positive cells . IL13Rα2-CAR or nontransduced (NT) T cells were stimulated with recombinant IL13Rα1, IL13Rα2,

    Article Snippet: Nontissue culture 24-well plates were precoated with recombinant human IL13Rα1, IL13Rα2, or IL4Rα proteins, (R & D Systems) at a final concentration of 500 ng/well.

    Techniques: Recombinant

    Impact of stimulation with 100 ng/mL of IL-4 or IL-13 on HCT 116 cells—a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) control for IL-13 at 72 h; ( m ) stimulated with IL-13 for 0 h; ( n ) stimulated with IL-13 at 24 h; ( o ) stimulated with IL-13 at 48 h; ( p ) stimulated with IL-13 at 72 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The ratios in interleukin-stimulated cells significantly ( p

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Impact of stimulation with 100 ng/mL of IL-4 or IL-13 on HCT 116 cells—a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) control for IL-13 at 72 h; ( m ) stimulated with IL-13 for 0 h; ( n ) stimulated with IL-13 at 24 h; ( o ) stimulated with IL-13 at 48 h; ( p ) stimulated with IL-13 at 72 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The ratios in interleukin-stimulated cells significantly ( p

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Wound Healing Assay

    Effect of IL-4 and IL-13 stimulation on the expression of epithelial-mesenchymal transition markers and tight junction proteins: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Effect of IL-4 and IL-13 stimulation on the expression of epithelial-mesenchymal transition markers and tight junction proteins: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Expressing

    Effect of IL-4 and IL-13 stimulation on the expression of mediators of proliferation and apoptosis: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (control) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Effect of IL-4 and IL-13 stimulation on the expression of mediators of proliferation and apoptosis: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (control) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Expressing

    Effect of exogenous IL-13 (1–250 ng/mL) on cancer cell viability under normal (10% FBS) and nutritional-stress (0.5% FBS) conditions as determined with sulforhodamine B (SRB) assay: ( a ) HCT 116 cultured in 10% FBS; ( b ) HCT 116 cultured in 0.5% FBS; (c ) HT-29 cultured in 10% FBS; ( d ) HT-29 cultured in 0.5% FBS; ( e ) Caco-2 cultured in 10% FBS; ( f ) Caco-2 cultured in 0.5% FBS. Data presented as percentage (% control) of viability of unstimulated cells, marked at 100% as reference red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Effect of exogenous IL-13 (1–250 ng/mL) on cancer cell viability under normal (10% FBS) and nutritional-stress (0.5% FBS) conditions as determined with sulforhodamine B (SRB) assay: ( a ) HCT 116 cultured in 10% FBS; ( b ) HCT 116 cultured in 0.5% FBS; (c ) HT-29 cultured in 10% FBS; ( d ) HT-29 cultured in 0.5% FBS; ( e ) Caco-2 cultured in 10% FBS; ( f ) Caco-2 cultured in 0.5% FBS. Data presented as percentage (% control) of viability of unstimulated cells, marked at 100% as reference red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Sulforhodamine B Assay, Cell Culture

    Effect of IL-4 and IL-13 on their respective receptor expression: ( a ) IL13Ra1 and IL4 expression following 24-h stimulation with 250 ng/mL of IL-4; ( b ) IL13Ra1 expression following 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative expression of receptor transcripts in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Effect of IL-4 and IL-13 on their respective receptor expression: ( a ) IL13Ra1 and IL4 expression following 24-h stimulation with 250 ng/mL of IL-4; ( b ) IL13Ra1 expression following 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative expression of receptor transcripts in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by red horizontal line. Statistically significant differences in treated cells as compared to their respective controls, analyzed using t -test for one mean, are marked as follows: p

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Expressing

    Patients’-matched analysis of tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein in ESCC ( n = 18); ( b ) IL13 mRNA in ESCC ( n = 16); ( c ) IL13Ra1 mRNA in ESCC ( n = 16); ( d ) IL-13 protein in GC ( n = 14); ( e ) IL13 mRNA in GC ( n = 14); ( f ) IL13Ra1 mRNA in GC ( n = 14). Data were analyzed as logs using t -test for paired samples and presented as geometric means with 95% confidence interval ( CI ). Mean difference in protein concentration (MD) between groups is accompanied by 95% CI . Transcriptomic data are presented as normalized relative quantities (NRQ). CRC, colorectal cancer; ESCC, esophageal squamous cell carcinoma; GC, gastric adenocarcinoma.

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Patients’-matched analysis of tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein in ESCC ( n = 18); ( b ) IL13 mRNA in ESCC ( n = 16); ( c ) IL13Ra1 mRNA in ESCC ( n = 16); ( d ) IL-13 protein in GC ( n = 14); ( e ) IL13 mRNA in GC ( n = 14); ( f ) IL13Ra1 mRNA in GC ( n = 14). Data were analyzed as logs using t -test for paired samples and presented as geometric means with 95% confidence interval ( CI ). Mean difference in protein concentration (MD) between groups is accompanied by 95% CI . Transcriptomic data are presented as normalized relative quantities (NRQ). CRC, colorectal cancer; ESCC, esophageal squamous cell carcinoma; GC, gastric adenocarcinoma.

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Expressing, Protein Concentration

    Effect of anatomical site on tumor-to-adjacent expression ratios of IL13 mRNA (IL13m), IL-13 protein (IL13p), and IL13Ra1 . Data analyzed as logs using one-way ANOVA and presented as geometric means with 95% confidence interval (whiskers). Red triangles represent mean values in colorectal cancers (denoted as C); blue squares represent mean values in esophageal squamous cell carcinoma (denoted as E); green circles represent mean values in gastric adenocarcinoma (denoted as G). p values for IL13 mRNA analysis are denoted as P m , for IL-13 protein analysis as P p , and for IL13Ra1 mRNA analysis as P R .

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Effect of anatomical site on tumor-to-adjacent expression ratios of IL13 mRNA (IL13m), IL-13 protein (IL13p), and IL13Ra1 . Data analyzed as logs using one-way ANOVA and presented as geometric means with 95% confidence interval (whiskers). Red triangles represent mean values in colorectal cancers (denoted as C); blue squares represent mean values in esophageal squamous cell carcinoma (denoted as E); green circles represent mean values in gastric adenocarcinoma (denoted as G). p values for IL13 mRNA analysis are denoted as P m , for IL-13 protein analysis as P p , and for IL13Ra1 mRNA analysis as P R .

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Expressing

    Analysis of the scratch assay data. Effect of IL-4 and IL-13 stimulation (100 ng/mL) on: ( a ) Caco-2 cells; ( b ) HCT 116 cells. Results at each time point are expressed as a percentage of gap area considering the gap at the time 0 as 100%. Each bar represents mean ± SE of three (Caco-2) or four (HCT116) biological replicates. Statistically significant differences as compared to time 0, analyzed using t -test for one mean, are marked as follows: p

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Analysis of the scratch assay data. Effect of IL-4 and IL-13 stimulation (100 ng/mL) on: ( a ) Caco-2 cells; ( b ) HCT 116 cells. Results at each time point are expressed as a percentage of gap area considering the gap at the time 0 as 100%. Each bar represents mean ± SE of three (Caco-2) or four (HCT116) biological replicates. Statistically significant differences as compared to time 0, analyzed using t -test for one mean, are marked as follows: p

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Wound Healing Assay

    Effect of IL-4 and IL-13 stimulation on expression of various proteins relevant for cancer growth: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by horizontal red line. Statistically significant differences ( p

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Effect of IL-4 and IL-13 stimulation on expression of various proteins relevant for cancer growth: ( a ) 24-h stimulation with 250 ng/mL of IL-4; ( b ) 24-h stimulation with 250 ng/mL of IL-13. Bars represent mean ± SE ( n = 4) of relative gene expression in stimulated as compared to unstimulated (controls) cells expressed as a percentage. Reference expression level of untreated cells (100%) is represented by horizontal red line. Statistically significant differences ( p

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Expressing

    Effect of anatomical site on tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein; ( b ) IL13 mRNA; ( c ) IL13Ra1 mRNA. Data analyzed as logs using one-way ANOVA and presented as geometric means with 95% confidence interval (whiskers). Blue triangles represent mean values in colorectal cancers (denoted as C); red squares represent mean values in esophageal squamous cell carcinoma (denoted as E); green circles represent mean values in gastric adenocarcinoma (denoted as G). p values for the analysis in adjacent tissue are denoted as P a and for tumor tissue as P t . Statistically significant differences between groups are marked with symbols of the same type (*, #, etc.). Transcriptomic data presented as normalized relative quantities (NRQ).

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Effect of anatomical site on tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein; ( b ) IL13 mRNA; ( c ) IL13Ra1 mRNA. Data analyzed as logs using one-way ANOVA and presented as geometric means with 95% confidence interval (whiskers). Blue triangles represent mean values in colorectal cancers (denoted as C); red squares represent mean values in esophageal squamous cell carcinoma (denoted as E); green circles represent mean values in gastric adenocarcinoma (denoted as G). p values for the analysis in adjacent tissue are denoted as P a and for tumor tissue as P t . Statistically significant differences between groups are marked with symbols of the same type (*, #, etc.). Transcriptomic data presented as normalized relative quantities (NRQ).

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Expressing

    Systemic concentration of interleukins in gastrointestinal tract cancers: ( a ) IL-4; ( b ) IL-13. Data presented as medians (red triangles) with 95% confidence interval (whiskers) and analyzed using Kruskal-Wallis H test. Statistically significant differences between groups are marked with symbols of the same type (*, #, etc.). CN, healthy controls; EC, patients with esophageal squamous cell carcinoma; CC, patients with adenocarcinoma of gastric cardia; GC, patients with gastric adenocarcinoma; CRC, patients with colorectal cancer. An outlying observation is marked by a closed square.

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Systemic concentration of interleukins in gastrointestinal tract cancers: ( a ) IL-4; ( b ) IL-13. Data presented as medians (red triangles) with 95% confidence interval (whiskers) and analyzed using Kruskal-Wallis H test. Statistically significant differences between groups are marked with symbols of the same type (*, #, etc.). CN, healthy controls; EC, patients with esophageal squamous cell carcinoma; CC, patients with adenocarcinoma of gastric cardia; GC, patients with gastric adenocarcinoma; CRC, patients with colorectal cancer. An outlying observation is marked by a closed square.

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Concentration Assay

    Impact of stimulation with 100 ng/mL of IL-4 or IL-13 on Caco-2 cells–a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0 h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) stimulated with IL-13 at 0 h; ( m ) stimulated with IL-13 at 24 h; ( n ) stimulated with IL-13 at 48 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The rates in interleukin-stimulated cells significantly ( p

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Impact of stimulation with 100 ng/mL of IL-4 or IL-13 on Caco-2 cells–a scratch assay (representative data): ( a ) control for IL-4 at 0 h; ( b ) control for IL-4 at 24 h; ( c ) control for IL-4 at 48 h; ( d ) control for IL-4 at 72 h; ( e ) stimulated with IL-4 at 0 h; ( f ) stimulated with IL-4 at 24 h; ( g ) stimulated with IL-4 at 48 h; ( h ) stimulated with IL-4 at 72 h; ( i ) control for IL-13 at 0 h; ( j ) control for IL-13 at 24 h; ( k ) control for IL-13 at 48 h; ( l ) stimulated with IL-13 at 0 h; ( m ) stimulated with IL-13 at 24 h; ( n ) stimulated with IL-13 at 48 h. Scale bars (200 µm) are indicated in lower right corner of each photograph. Numeric data in the upper right corner of each photograph indicate a ratio of gap (scratch) area at given time point to gap area at time 0. The rates in interleukin-stimulated cells significantly ( p

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Wound Healing Assay

    Patients’-matched analysis of tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein in CRC ( n = 17); ( b ) IL13 mRNA in CRC ( n = 21); ( c ) IL13Ra1 mRNA in CRC ( n = 21). Data were analyzed as logs using t -test for paired samples and presented as geometric means with 95% confidence interval ( CI ). Mean difference in protein concentration (MD) between groups is accompanied by 95% CI . Transcriptomic data are presented as normalized relative quantities (NRQ). CRC, colorectal cancer; ESCC, esophageal squamous cell carcinoma; GC, gastric adenocarcinoma.

    Journal: Cancers

    Article Title: Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility

    doi: 10.3390/cancers12061463

    Figure Lengend Snippet: Patients’-matched analysis of tumor and tumor-adjacent tissue expression of: ( a ) IL-13 protein in CRC ( n = 17); ( b ) IL13 mRNA in CRC ( n = 21); ( c ) IL13Ra1 mRNA in CRC ( n = 21). Data were analyzed as logs using t -test for paired samples and presented as geometric means with 95% confidence interval ( CI ). Mean difference in protein concentration (MD) between groups is accompanied by 95% CI . Transcriptomic data are presented as normalized relative quantities (NRQ). CRC, colorectal cancer; ESCC, esophageal squamous cell carcinoma; GC, gastric adenocarcinoma.

    Article Snippet: IL-4 and IL-13 concentrations in clarified tissue homogenates were measured immunoenzymatically using respective Quantikine high-sensitive (HS) ELISA assays for human IL-4 and IL-13, purchased from RnD Systems (Minneapolis, MN, USA).

    Techniques: Expressing, Protein Concentration