Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Summary of the genetic findings of our 8 patients

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques:

Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Mutation of Col4a2 promotes inflammation and astrocyte activation in CTX-TNA cells. CTX-TNA cells were stimulated with LPS and transfected with the Col4a2 Wt -AdV vector or the Col4a2 Mut -AdV vector. (A) The fluorescence intensity of GFAP (red) was measured in 5 different groups, with 50 CTX-TNA cells per group from 3 repeated experiments. The scale bar represents 25 μm. (B) Quantitative graphs displaying the mean fluorescence intensity of the GFAP-positive cells in (A). (C-E) Enzyme-linked immunosorbent assays were used to quantify IL-1β, IL-6, and TNF-α levels in the supernatant of CTX-TNA cells from 3 repeated experiments. The figure includes results from one-way ANOVA with calculated p values. (F) Western blot analysis was performed for GFAP, IL-1β, IL-6, TNF-α, JAK2, p-JAK2, STAT3, and p-STAT3. The proteins for the western blot analysis were extracted from cell lysates. **: vs. con, p<0.01; ++: vs. LPS+NC, p<0.01; ##: vs. LPS+ Col4a2 Wt , p <0.01.

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Col4a2 mutation promotes inflammation and astrocyte activation in primary astrocytes. Primary astrocytes were stimulated with LPS and transfected with either the Col4a2 Wt -AdV vector or the Col4a2 Mut -AdV vector. (A) The fluorescence intensity of GFAP (red) was measured in 5 different primary astrocyte groups, with 50 cells per group from 3 repeated experiments. The scale bar indicates 25 μm. (B) Quantitative graphs displaying the mean fluorescence intensity of the GFAP-positive cells in (A). (C-E) Enzyme-linked immunosorbent assays were used to measure IL-1β, IL-6, and TNF-α levels in primary astrocyte supernatants from 3 repeated experiments. The figure includes one-way ANOVA results with corresponding p values. (F) Western blot analysis was performed for GFAP, IL-1β, IL-6, TNF-α, JAK2, p-JAK2, STAT3, and p-STAT3. The proteins for the western blot analysis were extracted from cell lysates. **: vs. control, p<0.01; ++: vs. LPS+NC, p<0.01; ##: vs. LPS+ Col4a2 Wt , p <0.01.

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Western Blot, Control

Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Col4a2 mutation promotes inflammation and astrocyte activation by stimulating JAK/STAT signaling in CTX-TNA cells. CTX-TNA cells were stimulated with LPS and transfected with the Col4a2 Wt -AdV vector or the Col4a2 Mut -AdV vector or treated with WP1066 (a JAK2/STAT inhibitor). (A) The fluorescence intensity of GFAP was observed in 6 different groups (50 CTX-TNA cells per group) from 3 repeated experiments. The scale bar represents 25 μm. (B) Quantitative graphs displaying the mean fluorescence intensity of the GFAP-positive cells in (A). The figure includes the results of one-way ANOVA with calculated p values. (C) Western blot analysis of GFAP, IL-1β, IL-6, TNF-α, JAK2, p-JAK2, STAT3, and p-STAT3 was performed. The proteins for the western blot analysis were extracted from cell lysates. **: vs. con, p<0.01; ++: vs. LPS+NC, p<0.01; ##: vs. LPS+ Col4a2 Wt , p <0.01; ^^: vs. LPS+ Col4a2 Mut , p<0.01.

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Western Blot

Journal: International Journal of Medical Sciences

Article Title: Col4a2 Mutations Contribute to Infantile Epileptic Spasm Syndrome and Neuroinflammation

doi: 10.7150/ijms.97164

Figure Lengend Snippet: Col4a2 mutation promotes inflammation and astrocyte activation by stimulating JAK/STAT signaling in primary astrocytes. Primary astrocytes were stimulated with LPS and transfected with the Col4a2 Wt -AdV vector or the Col4a2 Mut -AdV vector or treated with WP1066 (a JAK2/STAT inhibitor). (A) The fluorescence intensity of GFAP in six different primary astrocyte groups (50 cells per group) from 3 repeated experiments is shown. The scale bar represents 25 μm. (B) Quantitative graphs displaying the mean fluorescence intensity of the GFAP-positive cells in (A). One-way ANOVA with calculated p values is presented in the figure. (C) Western blot analysis was conducted for GFAP, IL-1β, IL-6, TNF-α, JAK2, p-JAK2, STAT3, and p-STAT3. The proteins for the western blot analysis were extracted from cell lysates. **: vs. control, p<0.01; ++: vs. LPS+NC, p<0.01; ##: vs. LPS+ Col4a2 Wt , p <0.01; ^^: vs. LPS+ Col4a2 Mut , p<0.01.

Article Snippet: The experimental groups included the control group (no drug treatment), LPS group (cells stimulated with 5 μg/mL LPS for 48 hours to induce neuroinflammation), LPS+NC group (cells stimulated with LPS and transfected with an empty adenoviral vector), LPS+ Col4a2 Wt group (cells stimulated with LPS and transfected with the Col4a2 Wt -AdV vector), LPS+Col4a2 Mut group (cells stimulated with LPS and transfected with the Col4a2 Mut -AdV vector carrying the c.1838G>T, p.G613V mutation), and LPS+ Col4a2 Mut +WP1066 group (cells treated with the JAK2/STAT inhibitor WP1066 (5 μM, HY-15312; MCE, China) after stimulation with LPS and transfection with the Col4a2 Mut -AdV vector).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Fluorescence, Western Blot, Control

Journal: Immunology

Article Title: Distinct contribution of protein kinase C δ and protein kinase C ε in the lifespan and immune response of human blood monocyte subpopulations

doi: 10.1111/imm.12412

Figure Lengend Snippet: Protein kinase Cε (PKCε) regulates tumour necrosis factor-α (TNF-α) production in both monocyte subsets. (a) TNF-α production determined by ELISA in CD16− and CD16+ monocytes stimulated with 10 ng/ml LPS for 2, 4 and 8 hr. (b) Release of TNF-α in CD16− and CD16+ monocytes transfected with PKCε-small interfering RNA (siPKCε), PKCδ-siRNA (siPKCδ) or siRNA-Control (siCtrl) and subsequently stimulated with 10 ng/ml LPS for 8 hr. Data represent mean ± SEM (n = 3, *P < 0·05, **P < 0·01, ***P < 0·001).

Article Snippet: LPS stimulation and ELISA analysis Freshly isolated monocytes (1 × 10 6 cells/ml) were stimulated for 2, 4 and 8 hr with 10 ng/ml LPS ( Escherichia coli 0127:B8, BD Biosciences).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Small Interfering RNA

Journal: Blood Advances

Article Title: Runx1 negatively regulates inflammatory cytokine production by neutrophils in response to Toll-like receptor signaling

doi: 10.1182/bloodadvances.2019000785

Figure Lengend Snippet: Increased secretion of inflammatory cytokines, chemokines, and growth factors by Runx1 KO BM. (A-E) Absolute quantification by CBA of inflammatory factor levels in the supernatant of whole BM cells stimulated for 8 hours with vehicle or 100 ng/mL LPS. Bar graphs include independent data points. Error bars represent mean ± standard deviation (SD). Five replicates from 4 experiments were performed for each condition with all results above the limit of detection (blue arrowhead) plotted. For all factors except KC (C), a 2-tailed unpaired Student t test was performed comparing the factor concentration between the control and Runx1 KO LPS-treated samples. Because of limited detection of KC in the control LPS-treated sample (C), a 1-sample Student t test was performed comparing the mean of the Runx1 KO LPS-treated sample to a hypothetical mean = 0. (F-G) Representative FACS plots gated on live singlets and corresponding quantification of the frequencies of monocytes, neutrophils, and eosinophils in the BM (n = 3 from 3 experiments, mean ± SD, 2-tailed unpaired Student t test). Eos, eosinophils; FSC-A, forward scatter area; Mo, monocytes; PE, phycoerythrin. *P ≤ .05; **P ≤ .01.

Article Snippet: Cells were stimulated at 37°C with LPS (100 ng/mL) ( Escherichia coli O111:B4; Imgen Technologies) in Hanks media.

Techniques: Standard Deviation, Concentration Assay

Journal: Blood Advances

Article Title: Runx1 negatively regulates inflammatory cytokine production by neutrophils in response to Toll-like receptor signaling

doi: 10.1182/bloodadvances.2019000785

Figure Lengend Snippet: Increased TNF-α production by Runx1 KO neutrophils in response to TLR4 stimulation. (A) Representative FACS plots of intracellular TNF-α production by monocytes (CD11b+Ly6G−) after stimulation of whole BM with vehicle or 100 ng/mL LPS for 4 hours. (B) Quantification of the frequency of TNF-α+ monocytes and relative MFI of TNF-α in the TNF-α+ monocytes normalized to control monocytes run in the same experiment (n = 6 from 4 experiments). (C) Representative FACS plots of intracellular TNF-α production by neutrophils (CD11b+Ly6G+) after stimulation of whole BM with vehicle or 100 ng/mL LPS for 4 hours. (D) Quantification of the frequency of TNF-α+ neutrophils and relative MFI of TNF-α in the TNF-α+ neutrophils normalized to control neutrophils run in the same experiment (n = 6 from 4 experiments). (B,D) Bar graphs depict independent data points with the mean ± SD; 2-tailed unpaired Student t tests. (E) Quantification of the frequency of TNF-α+ neutrophils (CD11b+Ly6G+) after stimulation of whole BM for 4 hours with TLR agonists (n = 4 to 6, as indicated from 6 experiments). Bar graphs depict independent data points with the mean ± SD. Statistics represent the results of a 1-way analysis of variance followed by Sidak’s multiple comparison test to compare the means of the control and Runx1 KO samples for each TLR agonist. (F-H) Absolute quantification by CBA of inflammatory factor levels in the supernatant of 200 000 FACS-purified neutrophils (CD11b+SiglecF−F4/80−Ly6G+) stimulated for 8 hours with vehicle or 100 ng/mL LPS. (I) Quantification by CBA of TNF in the supernatant of 200 000 purified monocytes stimulated for 8 hours with vehicle or 100 ng/mL LPS. (F-I) Bar graphs depict independent data points with the mean ± SD. Five replicates from 3 experiments were performed for each condition with all results above the limit of detection (blue arrowhead) plotted. Statistics represent 2-tailed unpaired Student t tests. **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.

Article Snippet: Cells were stimulated at 37°C with LPS (100 ng/mL) ( Escherichia coli O111:B4; Imgen Technologies) in Hanks media.

Techniques: Purification

Journal: Blood Advances

Article Title: Runx1 negatively regulates inflammatory cytokine production by neutrophils in response to Toll-like receptor signaling

doi: 10.1182/bloodadvances.2019000785

Figure Lengend Snippet: Dysregulated expression of inflammatory pathways in Runx1 KO neutrophils. (A-D) RNA-seq data collected from FACS-purified neutrophils (CD11b+SiglecF−F4/80−Ly6G+) stimulated with vehicle or 100 ng/mL LPS for 2 hours (n = 3 from 3 experiments). Expression values are normalized to total cell number with a spike in control. (A) GO analysis of differentially expressed genes upregulated in Runx1 KO neutrophils as compared with control neutrophils with vehicle treatment. GO analysis of differentially expressed genes downregulated in vehicle-treated Runx1 KO neutrophils as compared with controls and up- or downregulated in LPS-treated Runx1 KO neutrophils as compared with controls is shown in supplemental Figure 6A. (B) Heat maps of TLR4 pathway genes in vehicle-treated Runx1 KO (RV1, RV2, RV3) compared with control neutrophils (CV1, CV2, CV3) ordered by fold-change in expression (high to low). Statistically significantly upregulated genes are marked with an asterisk and green text (false discovery rate <0.05). (C) Normalized expression of TLR4 pathway genes in vehicle-treated neutrophils (P = 1.45e-5, Student t test) (D) Schematic of TLR4 pathway genes with all statistically significantly upregulated genes in vehicle-treated Runx1 KO neutrophils denoted in green. Schematic adapted from O’Neill et al.45 (E) Cell surface TLR4 on control and Runx1 KO neutrophils (CD11b+Ly6G+) as compared with a representative isotype control (normalized to mode). Bar graphs depict absolute TLR4 MFI of individual samples (n = 3, mean ± SD, 2-tailed unpaired Student t test). Data are representative of 3 experiments. **P ≤ .001.

Article Snippet: Cells were stimulated at 37°C with LPS (100 ng/mL) ( Escherichia coli O111:B4; Imgen Technologies) in Hanks media.

Techniques: Expressing, RNA Sequencing Assay, Purification

Journal: Immunology

Article Title: A comparison between isolated blood dendritic cells and monocyte-derived dendritic cells in pigs

doi: 10.1111/j.1365-2567.2009.03192.x

Figure Lengend Snippet: The effect of lipopolysaccharide (LPS) stimulation on CD80/86 cell surface expression in monocyte-derived dendritic cells (MoDCs) (n = 4 animals) and blood dendritic cells (BDCs) (n = 6 animals). MoDCs at day 6 (a) and BDCs (b) were isolated from blood mononuclear cells and rested overnight before being cultured with LPS (100 ng/ml) for 24-hr. The expression of CD80/86 was determined by flow cytometry to examine DCs stimulated with LPS compared with DCs in medium. Results are expressed as the median of the percentages of positive cells. aP< 0·05 versus the control.

Article Snippet: Cell stimulation For stimulation with LPS, day 6 MoDCs and day 1 BDCs were cultured at 1 × 10 6 cells/ml and stimulated with 100 ng/ml of LPS ( Escherichia coli O55:B5; Cambrex Bioscience, Walkersville, MD) for 6-hr for gene expression studies or for 24-hr for ELISA and flow cytometry.

Techniques: Expressing, Derivative Assay, Isolation, Cell Culture, Flow Cytometry, Control

Journal: Immunology

Article Title: A comparison between isolated blood dendritic cells and monocyte-derived dendritic cells in pigs

doi: 10.1111/j.1365-2567.2009.03192.x

Figure Lengend Snippet: The effect of lipopolysaccharide (LPS) stimulation on CCR7 expression in monocyte-derived dendritic cells (MoDCs) (n = 4 animals) and blood dendritic cells (BDCs) (n = 4 animals). MoDCs at day 6 and BDCs at day 1 were cultured with LPS (100 ng/ml) for 6-hr. Samples were assessed for changes in gene expression of CCR7 by quantitative real-time polymerase chain reaction using ribosomal protein L19 as the reference gene. Results are shown as the median of the fold changes relative to the control.

Article Snippet: Cell stimulation For stimulation with LPS, day 6 MoDCs and day 1 BDCs were cultured at 1 × 10 6 cells/ml and stimulated with 100 ng/ml of LPS ( Escherichia coli O55:B5; Cambrex Bioscience, Walkersville, MD) for 6-hr for gene expression studies or for 24-hr for ELISA and flow cytometry.

Techniques: Expressing, Derivative Assay, Cell Culture, Gene Expression, Real-time Polymerase Chain Reaction, Control

Journal: Immunology

Article Title: A comparison between isolated blood dendritic cells and monocyte-derived dendritic cells in pigs

doi: 10.1111/j.1365-2567.2009.03192.x

Figure Lengend Snippet: Changes in gene expression of (a) CCL-2, CCL-4, CCL-20 and CXCL2 and (b) interleukin-6 (IL-6), IL-8 and tumour necrosis factor-α (TNF-α) in monocyte-derived dendritic cells (MoDCs) and IL-12 in blood dendritic cells (BDCs) following a 6-hr stimulation with lipopolysaccharide (LPS). MoDCs at day 6 and BDCs at day 1 were cultured with LPS (100 ng/ml). Samples were assessed for changes in gene expression by quantitative real-time polymerase chain reaction using ribosomal protein L19 as the reference gene. Results are shown as the median fold change relative to the control (n = 4 animals). a,b,c,dP < 0·05 MoDC versus BDC.

Article Snippet: Cell stimulation For stimulation with LPS, day 6 MoDCs and day 1 BDCs were cultured at 1 × 10 6 cells/ml and stimulated with 100 ng/ml of LPS ( Escherichia coli O55:B5; Cambrex Bioscience, Walkersville, MD) for 6-hr for gene expression studies or for 24-hr for ELISA and flow cytometry.

Techniques: Gene Expression, Derivative Assay, Cell Culture, Real-time Polymerase Chain Reaction, Control

Journal: Immunology

Article Title: A comparison between isolated blood dendritic cells and monocyte-derived dendritic cells in pigs

doi: 10.1111/j.1365-2567.2009.03192.x

Figure Lengend Snippet: Changes in interleukin-6 (IL-6), IL-8, and IL-12 concentrations following 24-hr lipopolysaccharide (LPS) stimulation and tumour necrosis factor-α (TNF-α) following an 8-hr stimulation

Article Snippet: Cell stimulation For stimulation with LPS, day 6 MoDCs and day 1 BDCs were cultured at 1 × 10 6 cells/ml and stimulated with 100 ng/ml of LPS ( Escherichia coli O55:B5; Cambrex Bioscience, Walkersville, MD) for 6-hr for gene expression studies or for 24-hr for ELISA and flow cytometry.

Techniques: Control