anti-interleukin il Search Results


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  • 93
    Millipore anti il 6
    Stimulation of rRNA transcription by <t>IL-6</t> downregulates p53 expression and activity. ( a ) Real-time–PCR evaluation of the TP53 mRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment. ( b ) Representative western blot and densitometric analysis of p53 expression in NCM460, HepG2, SW1990 and LS174T cells treated with IL-6 for 24 h. ( c ) Representative western blot and time-course analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. The values relative to p53 expression at 0.5, 1 and 2 h of CHX treatment are significantly higher in control than in IL-6-exposed cells ( P
    Anti Il 6, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam interleukin 6
    ATR down-regulation enhances the paracrine pro-carcinogenic effects of breast stromal fibroblasts in an <t>SDF-1/IL-6-dependent</t> manner (A) SFCM collected from the indicated cells were added separately to MCF-7 cells previously seeded into 96 wells, and cell proliferation was assessed by the real-time cell electronic sensing system. (B) MCF-7 cells were seeded onto the upper compartment of the migration and invasion plates, and then were incubated for 24 h in the presence of SFCM from the indicated cells. Error bars represent mean ± S.D (n=3). * P
    Interleukin 6, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 383 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti interleukin 2 il 2 apc
    Percentages of cytokine-positive CD8 + T lymphocyte responses in three vaccine groups. PBL from each vaccinated monkey were evaluated for secretion of IFN-γ, TNF-α, and <t>IL-2</t> in an intracellular cytokine assay following stimulation with
    Anti Interleukin 2 Il 2 Apc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti il6 antibody
    The expression of inflammatory cytokines and fibrotic factors are reduced in STZ-induced diabetic rats. ( A ) The kidney tissues were fixed in formalin and then subjected to immunofluorescence detection of TNF-α (arrow heads pointing to dark-brown dots indicating TNF-α expression). n = 5 per group, ( B ) <t>IL6</t> protein level ( C ) MCP1 protein level ( D ) TGF-β mRNA level, ( E ) TGF-β protein level with a representative blot, ( F ) Fibronectin (FN) mRNA level in kidney tissues. Data are shown as the means ± SEM *p
    Anti Il6 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti interleukin 2
    The ratio of <t>IL-2/IFN-γ</t> production by CMV-specific CD4 T cells is decreased in elderly donors. Whole blood stimulation and cytokine flow cytometry were used to determine IFN-γ and IL-2 production in response to CMV viral lysate. PBMC were stimulated with CMV lysate, and the percentage of cells which produced IL-2 or IFN-γ was determined and expressed as a ratio (IL-2/IFN-γ). Data from each donor are expressed as a single data point, with the median indicated by a horizontal line. The median value was 0.63 in the young donors and 0.37 in elderly donors ( P = 0.003).
    Anti Interleukin 2, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore anti interleukin 1beta antibody
    The ratio of <t>IL-2/IFN-γ</t> production by CMV-specific CD4 T cells is decreased in elderly donors. Whole blood stimulation and cytokine flow cytometry were used to determine IFN-γ and IL-2 production in response to CMV viral lysate. PBMC were stimulated with CMV lysate, and the percentage of cells which produced IL-2 or IFN-γ was determined and expressed as a ratio (IL-2/IFN-γ). Data from each donor are expressed as a single data point, with the median indicated by a horizontal line. The median value was 0.63 in the young donors and 0.37 in elderly donors ( P = 0.003).
    Anti Interleukin 1beta Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc interleukin il 1β
    The ratio of <t>IL-2/IFN-γ</t> production by CMV-specific CD4 T cells is decreased in elderly donors. Whole blood stimulation and cytokine flow cytometry were used to determine IFN-γ and IL-2 production in response to CMV viral lysate. PBMC were stimulated with CMV lysate, and the percentage of cells which produced IL-2 or IFN-γ was determined and expressed as a ratio (IL-2/IFN-γ). Data from each donor are expressed as a single data point, with the median indicated by a horizontal line. The median value was 0.63 in the young donors and 0.37 in elderly donors ( P = 0.003).
    Interleukin Il 1β, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad anti bovine il 10
    Bovine <t>IL-10</t> dose-dependently inhibits DC surface marker expression and cytokine production. A: Typical example of raw data of flow cytometric analysis. Data shown is CD80 expression during LPS induced DC maturation of one donor. The solid line indicates CD80 staining and the dashed line the isotype control. On top of the graphs is indicated whether medium, LPS or LPS plus different doses of IL-10 (ng/ml) were used. B: Bovine IL-10 dose-dependently modulates DC surface marker expression (CD83, p = 0.002; CD40, p = 0.030; CD80, p = 0.018) during LPS induced maturation. Relative values are shown from three different donors. Mean fluorescent intensities were divided by the isotype control and expressed relative to the positive control (LPS, without IL-10), which was set at 100%. C: Recombinant bovine IL-10 dose dependently modulates the production of cytokines by human DC's during LPS induced maturation (IL-12, p =
    Anti Bovine Il 10, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti il 10 antibody
    Over-expression of c-rel restores IL-12 p40 induction in <t>IL-10-treated</t> macrophages. The RAW 264.7 macrophages were transfected with either the c-rel plasmid construct or with both c-rel and p65 NF-κB plasmids using lipofectin. Cells were incubated
    Anti Il 10 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti il1 beta antibody
    Over-expression of c-rel restores IL-12 p40 induction in <t>IL-10-treated</t> macrophages. The RAW 264.7 macrophages were transfected with either the c-rel plasmid construct or with both c-rel and p65 NF-κB plasmids using lipofectin. Cells were incubated
    Anti Il1 Beta Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 313 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Novartis anti interleukin 2 receptor antibodies
    Over-expression of c-rel restores IL-12 p40 induction in <t>IL-10-treated</t> macrophages. The RAW 264.7 macrophages were transfected with either the c-rel plasmid construct or with both c-rel and p65 NF-κB plasmids using lipofectin. Cells were incubated
    Anti Interleukin 2 Receptor Antibodies, supplied by Novartis, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore anti il 4
    LCL growth inhibition assay with a CD4 + T-cell line from a representative seropositive donor. (a) Photograph of a U-bottomed microtitre plate showing the inhibition of LCL growth after 4 weeks co-culture with an autologous CD4 + T-cell line. Triplicate wells (columns 1–3) were seeded with doubling dilutions of LCL at 10 4 cells per well in row A down to 80 cells per well in row H. Growth of LCL cells can be seen as a large central pellet of cells in rows A–H. Triplicate wells (columns 4–6) were seeded with identical numbers of LCL cells and, in addition, 10 4 autologous CD4 + T cells were added to each well. After an initial period of growth, as indicated by a small central spot in each well, there was subsequent arrest and no further outgrowth of the LCL in any of the wells. The dot plots below the plate show that of 22% of CD4 + T cells from this donor produced <t>IL-4</t> and 5% IFNγ when stimulated with PMA and ionomycin. (b) Photograph of a second plate set up in an identical manner to (a). In this case, the growth of LCL was inhibited in all but the top two rows. CD4 + T cells from this donor produced 7% IL-4 and 53% IFNγ. (c) Photograph of a third plate set up in an identical manner to (a). The larger pellets of growing cells in columns 4–6 show that the growth of LCL was only poorly inhibited by this CD4 + T-cell line. Microscopic examination confirmed that complete inhibition of growth occurred at T Cell:LCL ratios of 1:32 or more. The T cells in this particular line had a strong Th1 bias, with only 2% of cells producing IL-4 but 95% producing IFNγ. Flow cytometry confirmed that the live cells present after 4 weeks culture were all LCL cells, and no T cells were detected in any wells examined at this time.
    Anti Il 4, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti il6
    The protein expression of <t>IL6,</t> p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3 and STAT3 in different transfected groups. Error bars indicate means ± SD. *P
    Anti Il6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore anti il 2
    <t>IL-2</t> enhances the differentiation of memory precursors via PI3K in vitro . (A,B) Blockade of IL-2 reduces the frequency of CD49b + CXCR3 + subpopulation in in vitro -activated CD4 T cells. Naive CD4 T cells from C57BL/6 mice were stimulated with anti-CD3/CD28 for 48 h in the presence of anti-IL-2 or isotype control and then incubated for 48 h. The frequencies of CD49b + CXCR3 + in activated CD4 T cells (A) and Ly-6C hi cells in CXCR3 + activated CD4 T cells (B) are shown. (C,D) Inhibition of PI3K reduces the frequency of CD49b + CXCR3 + subpopulation in in vitro -activated CD4 T cells. Naive CD4 T cells from C57BL/6 mice were stimulated with anti-CD3/CD28 for 48 h in the presence of Wortmannin, STAT5 inhibitor or DMSO and then incubated for 48 h. The frequencies of CD49b + CXCR3 + in activated CD4 T cells (C) and Ly-6C hi cells in CXCR3 + activated CD4 T cells (D) are shown n = 6. These data are representative of two independent experiments. Data are shown as mean ± SD. *** p
    Anti Il 2, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam anti interleukin il 1β
    <t>Interleukin-1β</t> expression decreased in group B and group C compared to that in group A.
    Anti Interleukin Il 1β, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti interleukin il 1β
    Hypoxia/ischemia induces microglial activation and the release of pro-inflammatory cytokines and neurotoxic factors. ( A - C ) BV-2 cells were exposed to oxygen-glucose deprivation (OGD), and the mRNA levels of the pro-inflammatory cytokines ( A )tumor necrosis factor (TNF)-α, ( B ) <t>interleukin</t> <t>(IL)-1β</t> and ( C )inducible nitric oxide synthase (iNOS) were evaluated using real-time PCR at 3 hoursafter OGD treatment. ( D - E ) The release of( D ) TNF-α ( E ) and IL-1β into the medium of BV-2 cells was measured by ELISA at 48 hours after OGD. ( F ) The release of NO into the medium of BV-2 cells was measured by the Griess assay. Results are presented as the mean ± SE from three independent experiments. ( G ) Brain homogenates were obtained from the hippocampus 3 daysafter transient global cerebral ischemia/reperfusion (I/R) injury. The supernatant concentrations of IL-1β and TNF-α were tested by ELISA. ( H ) Sections from rat brain taken 3 daysafter I/R injury were incubated with primary antibodies against CD11b and iNOS. Representative immunoreactivities in the hippocampal CA1 region are shown. Photomicrographs are shown at ×400 magnification. ‘Ctrl’ (control) represents the microglial cells that were not subjected to OGD treatment. * P
    Anti Interleukin Il 1β, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti interleukin
    Hypoxia/ischemia induces microglial activation and the release of pro-inflammatory cytokines and neurotoxic factors. ( A - C ) BV-2 cells were exposed to oxygen-glucose deprivation (OGD), and the mRNA levels of the pro-inflammatory cytokines ( A )tumor necrosis factor (TNF)-α, ( B ) <t>interleukin</t> <t>(IL)-1β</t> and ( C )inducible nitric oxide synthase (iNOS) were evaluated using real-time PCR at 3 hoursafter OGD treatment. ( D - E ) The release of( D ) TNF-α ( E ) and IL-1β into the medium of BV-2 cells was measured by ELISA at 48 hours after OGD. ( F ) The release of NO into the medium of BV-2 cells was measured by the Griess assay. Results are presented as the mean ± SE from three independent experiments. ( G ) Brain homogenates were obtained from the hippocampus 3 daysafter transient global cerebral ischemia/reperfusion (I/R) injury. The supernatant concentrations of IL-1β and TNF-α were tested by ELISA. ( H ) Sections from rat brain taken 3 daysafter I/R injury were incubated with primary antibodies against CD11b and iNOS. Representative immunoreactivities in the hippocampal CA1 region are shown. Photomicrographs are shown at ×400 magnification. ‘Ctrl’ (control) represents the microglial cells that were not subjected to OGD treatment. * P
    Anti Interleukin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti il 1β
    Immunohistochemical localization of IL-1α, <t>IL-1β,</t> TNF-α and IL-6 in the median nerves from normal control rats (C) or from reach limbs of rats that performed a HRNF task for 5 weeks. No immunostaining for these cytokines is visible
    Anti Il 1β, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad rabbit anti sheep polyclonal antibodies
    Immunohistochemical localization of IL-1α, <t>IL-1β,</t> TNF-α and IL-6 in the median nerves from normal control rats (C) or from reach limbs of rats that performed a HRNF task for 5 weeks. No immunostaining for these cytokines is visible
    Rabbit Anti Sheep Polyclonal Antibodies, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Stimulation of rRNA transcription by IL-6 downregulates p53 expression and activity. ( a ) Real-time–PCR evaluation of the TP53 mRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment. ( b ) Representative western blot and densitometric analysis of p53 expression in NCM460, HepG2, SW1990 and LS174T cells treated with IL-6 for 24 h. ( c ) Representative western blot and time-course analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. The values relative to p53 expression at 0.5, 1 and 2 h of CHX treatment are significantly higher in control than in IL-6-exposed cells ( P

    Journal: Oncogene

    Article Title: Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer

    doi: 10.1038/onc.2014.1

    Figure Lengend Snippet: Stimulation of rRNA transcription by IL-6 downregulates p53 expression and activity. ( a ) Real-time–PCR evaluation of the TP53 mRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment. ( b ) Representative western blot and densitometric analysis of p53 expression in NCM460, HepG2, SW1990 and LS174T cells treated with IL-6 for 24 h. ( c ) Representative western blot and time-course analysis of p53 protein expression in control and 24 h IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. The values relative to p53 expression at 0.5, 1 and 2 h of CHX treatment are significantly higher in control than in IL-6-exposed cells ( P

    Article Snippet: The sections were subsequently incubated with primary anti-E-cadherin (clone 32A8, Cell Signaling Technology, Beverly, MA, USA), anti-IL-6 (Sigma-Aldrich) and anti-p53 (Novocastra) antibodies diluted in 1% bovine serum albumin in Tris buffer saline overnight at 4 °C using the appropriate dilutions.

    Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Concentration Assay

    Suggested pathway linking chronic inflammation to cancer. In inflamed tissues, such as colon mucosa with UC, a high amount of IL-6 is produced. IL-6 upregulates c-myc protein expression, which, in turn, enhances ribosome biogenesis. The enhanced ribosome biogenesis is responsible for an increased MDM2-mediated p53 degradation. This, on one hand, may favor the EMT of the epithelial cells, and, on the other hand, may reduce the cell response to genotoxic DNA damages. Both consequences can greatly favor cancer onset.

    Journal: Oncogene

    Article Title: Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer

    doi: 10.1038/onc.2014.1

    Figure Lengend Snippet: Suggested pathway linking chronic inflammation to cancer. In inflamed tissues, such as colon mucosa with UC, a high amount of IL-6 is produced. IL-6 upregulates c-myc protein expression, which, in turn, enhances ribosome biogenesis. The enhanced ribosome biogenesis is responsible for an increased MDM2-mediated p53 degradation. This, on one hand, may favor the EMT of the epithelial cells, and, on the other hand, may reduce the cell response to genotoxic DNA damages. Both consequences can greatly favor cancer onset.

    Article Snippet: The sections were subsequently incubated with primary anti-E-cadherin (clone 32A8, Cell Signaling Technology, Beverly, MA, USA), anti-IL-6 (Sigma-Aldrich) and anti-p53 (Novocastra) antibodies diluted in 1% bovine serum albumin in Tris buffer saline overnight at 4 °C using the appropriate dilutions.

    Techniques: Produced, Expressing

    IL-6 induces EMT in a p53-dependent manner. ( a ) Representative western blot analysis of E-cadherin and SLUG expression in NCM460, HepG2 and HCT116 p53 −/− cells exposed to IL-6 for 24 h. NCM460 were either (p53 − ) or not (SCR) silenced for TP53 expression. ( b ) Visualization of SLUG and E-cadherin distribution in control and IL-6-treated NCM460 cells. Cells were labeled with monoclonal antibodies versus SLUG or E-cadherin; the antibodies were revealed by FITC-conjugated secondary antibodies. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar=40 μm. ( c ) Invasion assay of control (SCR) and TP53-silenced (p53 − ) NCM460 and HepG2 cells. The cells were exposed, 48 h after the end of the silencing procedure, to IL-6 for 24 h. ( d ) Real-time–PCR evaluation of 45S rRNA and western blot analysis of p53 expression in NCM460 and HepG2 cells transfected with control sequences (SCR) and in POLR1A-silenced cells (Pol1 − ). At 48 h after the end of the silencing procedure cells were exposed to IL-6 for 24 h. ( e ) Western blot and densitometric analysis of E-cadherin expression in control (SCR) and POLR1A-silenced (Pol1 − ) HepG2 cells. At 48 h after the end of the silencing procedure the cells were exposed to IL-6 for 24 h. ( f ) Invasion assay of control (SCR) and POLR1A-silenced NCM460 and HepG2 cells. At 48 h after the end of the silencing procedure, the cells were exposed to IL-6 for 24 h. Histograms show the values (mean±s.d.) of three experiments. * P

    Journal: Oncogene

    Article Title: Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer

    doi: 10.1038/onc.2014.1

    Figure Lengend Snippet: IL-6 induces EMT in a p53-dependent manner. ( a ) Representative western blot analysis of E-cadherin and SLUG expression in NCM460, HepG2 and HCT116 p53 −/− cells exposed to IL-6 for 24 h. NCM460 were either (p53 − ) or not (SCR) silenced for TP53 expression. ( b ) Visualization of SLUG and E-cadherin distribution in control and IL-6-treated NCM460 cells. Cells were labeled with monoclonal antibodies versus SLUG or E-cadherin; the antibodies were revealed by FITC-conjugated secondary antibodies. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Scale bar=40 μm. ( c ) Invasion assay of control (SCR) and TP53-silenced (p53 − ) NCM460 and HepG2 cells. The cells were exposed, 48 h after the end of the silencing procedure, to IL-6 for 24 h. ( d ) Real-time–PCR evaluation of 45S rRNA and western blot analysis of p53 expression in NCM460 and HepG2 cells transfected with control sequences (SCR) and in POLR1A-silenced cells (Pol1 − ). At 48 h after the end of the silencing procedure cells were exposed to IL-6 for 24 h. ( e ) Western blot and densitometric analysis of E-cadherin expression in control (SCR) and POLR1A-silenced (Pol1 − ) HepG2 cells. At 48 h after the end of the silencing procedure the cells were exposed to IL-6 for 24 h. ( f ) Invasion assay of control (SCR) and POLR1A-silenced NCM460 and HepG2 cells. At 48 h after the end of the silencing procedure, the cells were exposed to IL-6 for 24 h. Histograms show the values (mean±s.d.) of three experiments. * P

    Article Snippet: The sections were subsequently incubated with primary anti-E-cadherin (clone 32A8, Cell Signaling Technology, Beverly, MA, USA), anti-IL-6 (Sigma-Aldrich) and anti-p53 (Novocastra) antibodies diluted in 1% bovine serum albumin in Tris buffer saline overnight at 4 °C using the appropriate dilutions.

    Techniques: Western Blot, Expressing, Labeling, Staining, Invasion Assay, Real-time Polymerase Chain Reaction, Transfection

    IL-6 treatment stimulates rRNA transcription by activation of c-myc protein in NCM460 and HepG2 cell lines. ( a ) Real-time–PCR analysis of the 45S rRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment performed with a dose of 50 ng/ml. ( b ) Visualization of rRNA synthesis in control and IL-6-treated NCM460 and HepG2 cells. Cells were labeled with 5-FU for 15 min, and 5-FU revealed by specific FITC-conjugated monoclonal antibody. DAPI counterstaining. Scale bar=20 μm. ( c ) Representative western blot and densitometric analysis of c-myc expression in NCM460 and HepG2 cells treated with IL-6 for 1 and 3 h. ( d ) Real-time–PCR analysis of the c-MYC mRNA levels in NCM460 and HepG2 after 1 and 3 h of IL-6 treatment. ( e ) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in control and 4 h IL-6-treated NCM460 and HepG2 cells after 8 h transfection with a bicistronic mRNA transcribed either from pRF (top) or from pR-c-MYC-IRES-F (bottom). ( f ) Time-course analysis of c-myc protein expression in control and 24 h-IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. ( g ) Real-time–PCR and western blot analysis of c-MYC expression in scrambled control siRNA (SCR) and in 24 h c-MYC-silenced (MYC−) HepG2 cells exposed to IL-6 for 24 h. The right panel shows the 45S rRNA expression in the same experimental condition. Histograms show the values (mean±s.d.) of three experiments. * P

    Journal: Oncogene

    Article Title: Interleukin 6 downregulates p53 expression and activity by stimulating ribosome biogenesis: a new pathway connecting inflammation to cancer

    doi: 10.1038/onc.2014.1

    Figure Lengend Snippet: IL-6 treatment stimulates rRNA transcription by activation of c-myc protein in NCM460 and HepG2 cell lines. ( a ) Real-time–PCR analysis of the 45S rRNA expression in NCM460, HepG2, SW1990 and LS174T cells after 24 h of IL-6 treatment performed with a dose of 50 ng/ml. ( b ) Visualization of rRNA synthesis in control and IL-6-treated NCM460 and HepG2 cells. Cells were labeled with 5-FU for 15 min, and 5-FU revealed by specific FITC-conjugated monoclonal antibody. DAPI counterstaining. Scale bar=20 μm. ( c ) Representative western blot and densitometric analysis of c-myc expression in NCM460 and HepG2 cells treated with IL-6 for 1 and 3 h. ( d ) Real-time–PCR analysis of the c-MYC mRNA levels in NCM460 and HepG2 after 1 and 3 h of IL-6 treatment. ( e ) IRES-mediated translation assessed by measuring the FLuc and RLuc activity in control and 4 h IL-6-treated NCM460 and HepG2 cells after 8 h transfection with a bicistronic mRNA transcribed either from pRF (top) or from pR-c-MYC-IRES-F (bottom). ( f ) Time-course analysis of c-myc protein expression in control and 24 h-IL-6-stimulated HepG2 cells, exposed to cycloheximide (CHX) at a concentration of 20 μg/ml. ( g ) Real-time–PCR and western blot analysis of c-MYC expression in scrambled control siRNA (SCR) and in 24 h c-MYC-silenced (MYC−) HepG2 cells exposed to IL-6 for 24 h. The right panel shows the 45S rRNA expression in the same experimental condition. Histograms show the values (mean±s.d.) of three experiments. * P

    Article Snippet: The sections were subsequently incubated with primary anti-E-cadherin (clone 32A8, Cell Signaling Technology, Beverly, MA, USA), anti-IL-6 (Sigma-Aldrich) and anti-p53 (Novocastra) antibodies diluted in 1% bovine serum albumin in Tris buffer saline overnight at 4 °C using the appropriate dilutions.

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Expressing, Labeling, Western Blot, Activity Assay, Transfection, Concentration Assay

    ATR down-regulation enhances the paracrine pro-carcinogenic effects of breast stromal fibroblasts in an SDF-1/IL-6-dependent manner (A) SFCM collected from the indicated cells were added separately to MCF-7 cells previously seeded into 96 wells, and cell proliferation was assessed by the real-time cell electronic sensing system. (B) MCF-7 cells were seeded onto the upper compartment of the migration and invasion plates, and then were incubated for 24 h in the presence of SFCM from the indicated cells. Error bars represent mean ± S.D (n=3). * P

    Journal: Oncotarget

    Article Title: ATR suppresses the pro-tumorigenic functions of breast stromal fibroblasts

    doi: 10.18632/oncotarget.26159

    Figure Lengend Snippet: ATR down-regulation enhances the paracrine pro-carcinogenic effects of breast stromal fibroblasts in an SDF-1/IL-6-dependent manner (A) SFCM collected from the indicated cells were added separately to MCF-7 cells previously seeded into 96 wells, and cell proliferation was assessed by the real-time cell electronic sensing system. (B) MCF-7 cells were seeded onto the upper compartment of the migration and invasion plates, and then were incubated for 24 h in the presence of SFCM from the indicated cells. Error bars represent mean ± S.D (n=3). * P

    Article Snippet: Antibodies directed against alpha smooth muscle actin (α-SMA), Ki-67, transforming growth factor beta 1 (TGF-β1), Stromal-derived factor-1 (SDF-1), Twist-1, Vimentin (RV202), MMP-9, (ab38898), ATR (ab54793), N-cadherin and interleukin-6 (IL-6) were purchased from Abcam (Cambridge, MA); STAT3, pSTAT3-Tyr705 (D3A7), Snail (C15D3), E-cadherin (24E10), EpCAM (UV1D9), JAK-2 (D2E12) and phospho-JAK-2 (TYR1007/1008), CyclinD1 (2922), Akt, phospho-Akt (193H12), MMP-2 (4022), ERK1/2 (137F5) and phospho-ERK1/2 from Cell Signaling (Danvers, MA); ZEB-1 (4C4) from Abnova (Taipei, Taiwan); p21 (F-5), p53 (DO-1) and Glyceraldehydes-3-phosphate dehydrogenase (GAPDH, FL-335) were purchased from Santa Cruz (Santa Cruz, CA).

    Techniques: Migration, Incubation

    SsnB administration attenuates GW chemical-induced expression of pro-inflammatory cytokines and damage-associated molecular pattern high mobility group box 1 (HMGB1). Representative immunohistochemistry images of small intestine showing immunoreactivity (reactivity was majorly observed in the villi marked with black arrows) for HMGB1 ( Figure 2 A), IL-1β ( Figure 2 B), and IL-6 ( Figure 2 C) in CONT ( n = 3) (wild-type mice which were treated with vehicle), GW ( n = 3) (GW chemical exposed group), GW + SsnB ( n = 3) (group exposed with GW chemicals and SsnB), and SsnB ( n = 3) (group treated with SsnB only). Images were taken in 20× magnification. Bar graphs depicting morphometric analysis of HMGB1 ( Figure 2 D), IL-1β ( Figure 2 E), and IL-6 ( Figure 2 F) immunoreactivity are represented as mean ± SD of the %ROI (mean value calculated from three separate fields of a section of the small intestine). Significance was analyzed by unpaired T-test where * p

    Journal: Brain Sciences

    Article Title: TLR Antagonism by Sparstolonin B Alters Microbial Signature and Modulates Gastrointestinal and Neuronal Inflammation in Gulf War Illness Preclinical Model

    doi: 10.3390/brainsci10080532

    Figure Lengend Snippet: SsnB administration attenuates GW chemical-induced expression of pro-inflammatory cytokines and damage-associated molecular pattern high mobility group box 1 (HMGB1). Representative immunohistochemistry images of small intestine showing immunoreactivity (reactivity was majorly observed in the villi marked with black arrows) for HMGB1 ( Figure 2 A), IL-1β ( Figure 2 B), and IL-6 ( Figure 2 C) in CONT ( n = 3) (wild-type mice which were treated with vehicle), GW ( n = 3) (GW chemical exposed group), GW + SsnB ( n = 3) (group exposed with GW chemicals and SsnB), and SsnB ( n = 3) (group treated with SsnB only). Images were taken in 20× magnification. Bar graphs depicting morphometric analysis of HMGB1 ( Figure 2 D), IL-1β ( Figure 2 E), and IL-6 ( Figure 2 F) immunoreactivity are represented as mean ± SD of the %ROI (mean value calculated from three separate fields of a section of the small intestine). Significance was analyzed by unpaired T-test where * p

    Article Snippet: Anti-Claudin 2, anti-Occludin, anti-interleukin-6 (IL-6), anti-nod-like receptor protein 3 (NLRP3), anti-Caspase 1, and anti-β-actin were purchased from Abcam (Cambridge, MA USA).

    Techniques: Expressing, Immunohistochemistry, Mouse Assay

    Intravenous injection of allogeneic UC-MSCs decreased inflammatory factors in the infarct area and peri-infarct area of the LV myocardium at week 8 after AMI. Protein expression of tumor necrosis factor (TNF) alpha and interleukin-6 in the infarct area ( a – c ), border area ( d – f ), and remote area ( g – i ). Data are presented as the mean ± SD. Phosphate-buffered saline (PBS) group, n = 3; low-dose group, n = 4; and high-dose group, n = 4. * P

    Journal: Stem Cell Research & Therapy

    Article Title: Intravenous injection of allogeneic umbilical cord-derived multipotent mesenchymal stromal cells reduces the infarct area and ameliorates cardiac function in a porcine model of acute myocardial infarction

    doi: 10.1186/s13287-018-0888-z

    Figure Lengend Snippet: Intravenous injection of allogeneic UC-MSCs decreased inflammatory factors in the infarct area and peri-infarct area of the LV myocardium at week 8 after AMI. Protein expression of tumor necrosis factor (TNF) alpha and interleukin-6 in the infarct area ( a – c ), border area ( d – f ), and remote area ( g – i ). Data are presented as the mean ± SD. Phosphate-buffered saline (PBS) group, n = 3; low-dose group, n = 4; and high-dose group, n = 4. * P

    Article Snippet: The transferred membranes were blocked using 5% bovine serum albumin (BSA) in TBST and incubated with the primary antibodies troponin I (1:200, Santa Cruz), Connexin 43 (Cx43; 1:200, Santa Cruz), tumor necrosis factor (TNF) alpha (1:1000, Abcam), and interleukin (IL)-6 (1:1000, Abcam) overnight and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody β-actin (1:1000, Cell Signaling) for 2 h. Bands were visualized using enhanced chemiluminescent (ECL; Merck Millipore, USA) detection reagents and scanned images were quantified using Image J software (NIH).

    Techniques: Injection, Expressing

    Percentages of cytokine-positive CD8 + T lymphocyte responses in three vaccine groups. PBL from each vaccinated monkey were evaluated for secretion of IFN-γ, TNF-α, and IL-2 in an intracellular cytokine assay following stimulation with

    Journal: Journal of Virology

    Article Title: Comparison of Immunogenicity in Rhesus Macaques of Transmitted-Founder, HIV-1 Group M Consensus, and Trivalent Mosaic Envelope Vaccines Formulated as a DNA Prime, NYVAC, and Envelope Protein Boost

    doi: 10.1128/JVI.00383-15

    Figure Lengend Snippet: Percentages of cytokine-positive CD8 + T lymphocyte responses in three vaccine groups. PBL from each vaccinated monkey were evaluated for secretion of IFN-γ, TNF-α, and IL-2 in an intracellular cytokine assay following stimulation with

    Article Snippet: Following fixing and permeabilization with Cytofix/Cytoperm solution (BD Biosciences), cells were stained with anti-CD69-ECD (TP1.55.3; Beckman Coulter), anti-IFN-γ phycoerythrin (PE)-Cy7 (B27; BD Biosciences), anti-tumor necrosis factor alpha (TNF-α)-fluorescein isothiocyanate (FITC) (monoclonal antibody 11 [MAb11]; BD Biosciences), and anti-interleukin 2 (IL-2) APC (MQ1-17H12; BD Biosciences) and fixed with 1% formaldehyde.

    Techniques: Cytokine Assay

    The expression of inflammatory cytokines and fibrotic factors are reduced in STZ-induced diabetic rats. ( A ) The kidney tissues were fixed in formalin and then subjected to immunofluorescence detection of TNF-α (arrow heads pointing to dark-brown dots indicating TNF-α expression). n = 5 per group, ( B ) IL6 protein level ( C ) MCP1 protein level ( D ) TGF-β mRNA level, ( E ) TGF-β protein level with a representative blot, ( F ) Fibronectin (FN) mRNA level in kidney tissues. Data are shown as the means ± SEM *p

    Journal: Aging (Albany NY)

    Article Title: Attenuation of diabetic kidney injury in DPP4-deficient rats; role of GLP-1 on the suppression of AGE formation by inducing glyoxalase 1

    doi: 10.18632/aging.102643

    Figure Lengend Snippet: The expression of inflammatory cytokines and fibrotic factors are reduced in STZ-induced diabetic rats. ( A ) The kidney tissues were fixed in formalin and then subjected to immunofluorescence detection of TNF-α (arrow heads pointing to dark-brown dots indicating TNF-α expression). n = 5 per group, ( B ) IL6 protein level ( C ) MCP1 protein level ( D ) TGF-β mRNA level, ( E ) TGF-β protein level with a representative blot, ( F ) Fibronectin (FN) mRNA level in kidney tissues. Data are shown as the means ± SEM *p

    Article Snippet: The antibodies used were as follows: anti-actin (#8457, Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (#MAB374, Millipore), anti-RAGE (sc-365154, Santa Cruz Biotechnology, Dallas, TX, USA), anti-DPP4/CD26 (5E8) (sc-8422, Santa Cruz), anti-glyoxalase-1 (sc-101537, Santa Cruz), anti-transforming growth factor (TGF)-β (#3711, Cell Signaling Technology), anti-Lamin A/C (#4777, Cell Signaling Technology, Danvers, MA, USA), anti-nuclear factor-erythroid 2 p45 subunit-related factor-2 (Nrf-2) (ab137550, Abcam), anti-monocyte chemoattractant protein 1 (MCP-1) (ab25124, Abcam), and anti-interleukin 6 (IL-6) (ab9324, Abcam).

    Techniques: Expressing, Immunofluorescence

    Ex-4 treatment reduces MGO-induced inflammatory cytokine expression in rat mesangial cells. Rat mesangial cells were treated either with 1 mM MGO, 10 nM Ex-4, or both for 10 h after synchronization with 1% fetal bovine serum for 13-16 h. ( A ) TNF-α mRNA level, ( B ) MCP-1 mRNA level, ( C ) IL6 mRNA level in rat mesangial cells. Data are shown as the means ± SEM. * p

    Journal: Aging (Albany NY)

    Article Title: Attenuation of diabetic kidney injury in DPP4-deficient rats; role of GLP-1 on the suppression of AGE formation by inducing glyoxalase 1

    doi: 10.18632/aging.102643

    Figure Lengend Snippet: Ex-4 treatment reduces MGO-induced inflammatory cytokine expression in rat mesangial cells. Rat mesangial cells were treated either with 1 mM MGO, 10 nM Ex-4, or both for 10 h after synchronization with 1% fetal bovine serum for 13-16 h. ( A ) TNF-α mRNA level, ( B ) MCP-1 mRNA level, ( C ) IL6 mRNA level in rat mesangial cells. Data are shown as the means ± SEM. * p

    Article Snippet: The antibodies used were as follows: anti-actin (#8457, Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH (#MAB374, Millipore), anti-RAGE (sc-365154, Santa Cruz Biotechnology, Dallas, TX, USA), anti-DPP4/CD26 (5E8) (sc-8422, Santa Cruz), anti-glyoxalase-1 (sc-101537, Santa Cruz), anti-transforming growth factor (TGF)-β (#3711, Cell Signaling Technology), anti-Lamin A/C (#4777, Cell Signaling Technology, Danvers, MA, USA), anti-nuclear factor-erythroid 2 p45 subunit-related factor-2 (Nrf-2) (ab137550, Abcam), anti-monocyte chemoattractant protein 1 (MCP-1) (ab25124, Abcam), and anti-interleukin 6 (IL-6) (ab9324, Abcam).

    Techniques: Expressing

    The ratio of IL-2/IFN-γ production by CMV-specific CD4 T cells is decreased in elderly donors. Whole blood stimulation and cytokine flow cytometry were used to determine IFN-γ and IL-2 production in response to CMV viral lysate. PBMC were stimulated with CMV lysate, and the percentage of cells which produced IL-2 or IFN-γ was determined and expressed as a ratio (IL-2/IFN-γ). Data from each donor are expressed as a single data point, with the median indicated by a horizontal line. The median value was 0.63 in the young donors and 0.37 in elderly donors ( P = 0.003).

    Journal: Journal of Virology

    Article Title: The Cytomegalovirus-Specific CD4+ T-Cell Response Expands with Age and Markedly Alters the CD4+ T-Cell Repertoire ▿

    doi: 10.1128/JVI.01262-06

    Figure Lengend Snippet: The ratio of IL-2/IFN-γ production by CMV-specific CD4 T cells is decreased in elderly donors. Whole blood stimulation and cytokine flow cytometry were used to determine IFN-γ and IL-2 production in response to CMV viral lysate. PBMC were stimulated with CMV lysate, and the percentage of cells which produced IL-2 or IFN-γ was determined and expressed as a ratio (IL-2/IFN-γ). Data from each donor are expressed as a single data point, with the median indicated by a horizontal line. The median value was 0.63 in the young donors and 0.37 in elderly donors ( P = 0.003).

    Article Snippet: The following monoclonal antibodies were used in this study: anti-gamma interferon (anti-IFN-γ; fluorescein isothiocyanate [FITC] and phycoerythrin [PE]), anti-interleukin-2 (anti-IL-2; FITC and PE), anti-tumor necrosis factor alpha (anti-TNF-α; FITC and PE), mouse immunoglobulin G2a (IgG2a; FITC and PE), mouse IgG1 (FITC and PE), and anti-CD49d and anti-CD28 were obtained from Becton-Dickinson Immunocytometry Systems.

    Techniques: Flow Cytometry, Cytometry, Produced

    The frequency of CMV-specific CD4 T cells is increased in elderly donors. Whole blood from CMV-seropositive donors was incubated with CMV viral lysate at 37°C for 6 h or with mock lysate as a control. The blood was then lysed, fixed, permeabilized, and stained with monoclonal antibodies. The frequency of the cells responsive to CMV was determined by the fraction of CD4 T cells which upregulated CD69 and expressed detectable amounts of intracellular TNF-α, IFN-γ, and IL-2. A. Representative flow cytometric profiles from a single elderly donor for the isotype control (i) and IFN-γ (ii) and IL-2 (iii) cytokine responses to CMV lysate. B. The frequency of CMV-specific CD4 T cells in individual donors is expressed as a percentage of the total CD4 T-cell pool and shown as a single data point. The mean value within the group is indicated as a horizontal line. (i) The mean percentage of CMV-specific CD4 T cell producing IFN-γ was 2% in young donors (range, 0.4 to 5.78%) and 4.3% in elderly donors (0.3 to 32%) ( P = 0.03). (ii) The mean percentage of CMV-specific CD4 T cells producing TNF-α was 2.2% in young donors (range, 0.51 to 4.7%) and 4.7% in elderly donors (0.3 to 16.7%) ( P = 0.08). (iii) The mean percentage of CMV-specific CD4 T cells producing IL-2 was 1.1% in young donors (range, 0.1 to 4.8%) and 1.3% in elderly donors (0.26 to 4.9%) ( P = 0.9).

    Journal: Journal of Virology

    Article Title: The Cytomegalovirus-Specific CD4+ T-Cell Response Expands with Age and Markedly Alters the CD4+ T-Cell Repertoire ▿

    doi: 10.1128/JVI.01262-06

    Figure Lengend Snippet: The frequency of CMV-specific CD4 T cells is increased in elderly donors. Whole blood from CMV-seropositive donors was incubated with CMV viral lysate at 37°C for 6 h or with mock lysate as a control. The blood was then lysed, fixed, permeabilized, and stained with monoclonal antibodies. The frequency of the cells responsive to CMV was determined by the fraction of CD4 T cells which upregulated CD69 and expressed detectable amounts of intracellular TNF-α, IFN-γ, and IL-2. A. Representative flow cytometric profiles from a single elderly donor for the isotype control (i) and IFN-γ (ii) and IL-2 (iii) cytokine responses to CMV lysate. B. The frequency of CMV-specific CD4 T cells in individual donors is expressed as a percentage of the total CD4 T-cell pool and shown as a single data point. The mean value within the group is indicated as a horizontal line. (i) The mean percentage of CMV-specific CD4 T cell producing IFN-γ was 2% in young donors (range, 0.4 to 5.78%) and 4.3% in elderly donors (0.3 to 32%) ( P = 0.03). (ii) The mean percentage of CMV-specific CD4 T cells producing TNF-α was 2.2% in young donors (range, 0.51 to 4.7%) and 4.7% in elderly donors (0.3 to 16.7%) ( P = 0.08). (iii) The mean percentage of CMV-specific CD4 T cells producing IL-2 was 1.1% in young donors (range, 0.1 to 4.8%) and 1.3% in elderly donors (0.26 to 4.9%) ( P = 0.9).

    Article Snippet: The following monoclonal antibodies were used in this study: anti-gamma interferon (anti-IFN-γ; fluorescein isothiocyanate [FITC] and phycoerythrin [PE]), anti-interleukin-2 (anti-IL-2; FITC and PE), anti-tumor necrosis factor alpha (anti-TNF-α; FITC and PE), mouse immunoglobulin G2a (IgG2a; FITC and PE), mouse IgG1 (FITC and PE), and anti-CD49d and anti-CD28 were obtained from Becton-Dickinson Immunocytometry Systems.

    Techniques: Incubation, Staining, Flow Cytometry

    T cell responses in the spleen after infection with Pb ANKA in the presence or absence of M. tuberculosis . C57BL/6 mice were infected via the aerosol route with M. tuberculosis H37Rv and 30 days later with 1 × 10 5 pRBCs i.p. Spleens were collected 4 or 6 days after Pb ANKA infection, and single-cell suspensions were analyzed for the presence and activation status of CD4 + and CD8 + T cells by flow cytometry. (A) Splenocytes were gated on CD90.2 and analyzed for the total numbers of CD4 + and CD8 + T cells, for the numbers of effector memory T cells (CD62L − CD44 + ), and for the expression of CXCR3. Data are presented as box and whisker plots with medians. (B) Spleen cells were restimulated ex vivo with anti-CD3 and anti-CD28 (5 μg/ml, respectively) and analyzed by flow cytometry for the presence of IFN-γ-, TNF-α-, IL-10-, or IL-2-producing CD44 + CD4 + and CD44 + CD8 + T cells gated for CD90.2 (presented as means ± SD). Data from two (d4) or 3 (d6) independent experiments are shown ( n = 8 to 15, Kruskal-Wallis test with Dunn′s posttest). *, P

    Journal: Infection and Immunity

    Article Title: Mycobacterium tuberculosis Coinfection Has No Impact on Plasmodium berghei ANKA-Induced Experimental Cerebral Malaria in C57BL/6 Mice

    doi: 10.1128/IAI.01290-15

    Figure Lengend Snippet: T cell responses in the spleen after infection with Pb ANKA in the presence or absence of M. tuberculosis . C57BL/6 mice were infected via the aerosol route with M. tuberculosis H37Rv and 30 days later with 1 × 10 5 pRBCs i.p. Spleens were collected 4 or 6 days after Pb ANKA infection, and single-cell suspensions were analyzed for the presence and activation status of CD4 + and CD8 + T cells by flow cytometry. (A) Splenocytes were gated on CD90.2 and analyzed for the total numbers of CD4 + and CD8 + T cells, for the numbers of effector memory T cells (CD62L − CD44 + ), and for the expression of CXCR3. Data are presented as box and whisker plots with medians. (B) Spleen cells were restimulated ex vivo with anti-CD3 and anti-CD28 (5 μg/ml, respectively) and analyzed by flow cytometry for the presence of IFN-γ-, TNF-α-, IL-10-, or IL-2-producing CD44 + CD4 + and CD44 + CD8 + T cells gated for CD90.2 (presented as means ± SD). Data from two (d4) or 3 (d6) independent experiments are shown ( n = 8 to 15, Kruskal-Wallis test with Dunn′s posttest). *, P

    Article Snippet: For flow cytometric analysis of surface markers and intracellular cytokines, single-cell suspensions of brains and spleens were stained with optimal concentrations of the following specific antibodies (Abs): CD45-V450, CD4-V500, CD8a fluorescein isothiocyanate (FITC), CD44-peridinin chlorophyll protein (PerCP)-Cy5.5, CD62L-allophycocyanin (APC), CXC chemokine receptor 3 (CXCR3)-phycoerythrin (PE), CD3e-APC-Cy7, IFN-γ–APC, and interleukin-2 (IL-2)–PE–Cy7 from BD Biosciences; CD8a-Pacific Blue, CD80-AF488, CD11c-PE, CD11b-PerCP-Cy5.5, I-A/I-E–PE-Cy7, CD86–APC, Ly6G-APC-Cy7, CD19-PE, TNF-α–Pacific Blue, and IL-10–PE from BioLegend; CD90.2-eFluor780 from eBioscience; and major histocompatibility complex class I (MHC-I) Ova Pentamer from ProImmune (Oxford, United Kingdom).

    Techniques: Infection, Mouse Assay, Activation Assay, Flow Cytometry, Cytometry, Expressing, Whisker Assay, Ex Vivo

    Bovine IL-10 dose-dependently inhibits DC surface marker expression and cytokine production. A: Typical example of raw data of flow cytometric analysis. Data shown is CD80 expression during LPS induced DC maturation of one donor. The solid line indicates CD80 staining and the dashed line the isotype control. On top of the graphs is indicated whether medium, LPS or LPS plus different doses of IL-10 (ng/ml) were used. B: Bovine IL-10 dose-dependently modulates DC surface marker expression (CD83, p = 0.002; CD40, p = 0.030; CD80, p = 0.018) during LPS induced maturation. Relative values are shown from three different donors. Mean fluorescent intensities were divided by the isotype control and expressed relative to the positive control (LPS, without IL-10), which was set at 100%. C: Recombinant bovine IL-10 dose dependently modulates the production of cytokines by human DC's during LPS induced maturation (IL-12, p =

    Journal: PLoS ONE

    Article Title: Modulation of Human Immune Responses by Bovine Interleukin-10

    doi: 10.1371/journal.pone.0018188

    Figure Lengend Snippet: Bovine IL-10 dose-dependently inhibits DC surface marker expression and cytokine production. A: Typical example of raw data of flow cytometric analysis. Data shown is CD80 expression during LPS induced DC maturation of one donor. The solid line indicates CD80 staining and the dashed line the isotype control. On top of the graphs is indicated whether medium, LPS or LPS plus different doses of IL-10 (ng/ml) were used. B: Bovine IL-10 dose-dependently modulates DC surface marker expression (CD83, p = 0.002; CD40, p = 0.030; CD80, p = 0.018) during LPS induced maturation. Relative values are shown from three different donors. Mean fluorescent intensities were divided by the isotype control and expressed relative to the positive control (LPS, without IL-10), which was set at 100%. C: Recombinant bovine IL-10 dose dependently modulates the production of cytokines by human DC's during LPS induced maturation (IL-12, p =

    Article Snippet: Bovine IL-10 ELISA A bovine IL-10 specific capture ELISA was developed in house using an anti-bovine IL-10 specific monoclonal (MCA2110, AbD Serotec, Oxford, UK) as a capture antibody and biotinylated anti-bovine IL-10 IgY as a detecting antibody.

    Techniques: Marker, Expressing, Flow Cytometry, Staining, Positive Control, Recombinant

    Dose-dependent inhibition during LPS-induced DC maturation is comparable for human and bovine IL-10. Data shown are from 3 different donors tested, error bars indicate standard error. A: Dose dependent inhibition of CD83 (p = 0.753) and CD86 (p = 0.936) by human and bovine IL-10. Data were divided by the isotype control and expressed relative to the positive control of only LPS, which was set at 100%. B: Dose dependent inhibition of TNF-α (p = 0.916) and IL-12p70 (p = 0.962) production by human and bovine IL-10, shown in pg/ml.

    Journal: PLoS ONE

    Article Title: Modulation of Human Immune Responses by Bovine Interleukin-10

    doi: 10.1371/journal.pone.0018188

    Figure Lengend Snippet: Dose-dependent inhibition during LPS-induced DC maturation is comparable for human and bovine IL-10. Data shown are from 3 different donors tested, error bars indicate standard error. A: Dose dependent inhibition of CD83 (p = 0.753) and CD86 (p = 0.936) by human and bovine IL-10. Data were divided by the isotype control and expressed relative to the positive control of only LPS, which was set at 100%. B: Dose dependent inhibition of TNF-α (p = 0.916) and IL-12p70 (p = 0.962) production by human and bovine IL-10, shown in pg/ml.

    Article Snippet: Bovine IL-10 ELISA A bovine IL-10 specific capture ELISA was developed in house using an anti-bovine IL-10 specific monoclonal (MCA2110, AbD Serotec, Oxford, UK) as a capture antibody and biotinylated anti-bovine IL-10 IgY as a detecting antibody.

    Techniques: Inhibition, Positive Control

    Comparison of human and bovine IL-10 in the human IL-10R. A: ClustalW sequence alignment of IL-10 from different species. IL-10 sequences were retrieved from the NCBI and Uniprot databases and analyzed for signal peptides (SignalP 3.0) which were removed from the sequences before performing the alignment (18 amino acids for human and bovine IL-10). At the top left the overall sequence identity is shown. In grey (Ib) and black (Ia) background shading the IL-10 receptor 1 binding sites are indicated as published by Josephson [8] . The underlined residues indicate > 5 Å 2 surface area in IL-10RA site I. At the bottom of each row the consensus (*) sequence is shown; “.” and “:” indicated homologous amino acids. B: Bovine IL-10 was modeled using human IL-10 in the human IL-10/IL-10R complex as template. The human IL-10R is shown in green and amino acid substitution between human and bovine IL-10 are depicted in cyan. Cysteine residues are colored yellow, the amino acid colored red (indicated with an arrow for one of the two IL-10 chains) is His 44, which is and amino acid substitution in close contact with the human Il-10R. C: The same model as in B, but displayed using spheres.

    Journal: PLoS ONE

    Article Title: Modulation of Human Immune Responses by Bovine Interleukin-10

    doi: 10.1371/journal.pone.0018188

    Figure Lengend Snippet: Comparison of human and bovine IL-10 in the human IL-10R. A: ClustalW sequence alignment of IL-10 from different species. IL-10 sequences were retrieved from the NCBI and Uniprot databases and analyzed for signal peptides (SignalP 3.0) which were removed from the sequences before performing the alignment (18 amino acids for human and bovine IL-10). At the top left the overall sequence identity is shown. In grey (Ib) and black (Ia) background shading the IL-10 receptor 1 binding sites are indicated as published by Josephson [8] . The underlined residues indicate > 5 Å 2 surface area in IL-10RA site I. At the bottom of each row the consensus (*) sequence is shown; “.” and “:” indicated homologous amino acids. B: Bovine IL-10 was modeled using human IL-10 in the human IL-10/IL-10R complex as template. The human IL-10R is shown in green and amino acid substitution between human and bovine IL-10 are depicted in cyan. Cysteine residues are colored yellow, the amino acid colored red (indicated with an arrow for one of the two IL-10 chains) is His 44, which is and amino acid substitution in close contact with the human Il-10R. C: The same model as in B, but displayed using spheres.

    Article Snippet: Bovine IL-10 ELISA A bovine IL-10 specific capture ELISA was developed in house using an anti-bovine IL-10 specific monoclonal (MCA2110, AbD Serotec, Oxford, UK) as a capture antibody and biotinylated anti-bovine IL-10 IgY as a detecting antibody.

    Techniques: Sequencing, IA, Binding Assay

    Bovine IL-10 can regulate TLR ligand induced cytokine production by monocytes by binding the IL-10R. Figures 2B-2D are box plots, showing the median (black horizontal bar), 50% data range (box) and 99% data range (error bars). On the x-axes is the addition indicated (+) of TLR stimuli (panel A), or 10 ng/ml LPS (B–D), 10 ng/ml IL-10 (A-D), bovine IL-10 or human IL-10R blocking antibodies (anti bIL-10 or IL-10R) or isotype control (C–D). A : Freshly isolated monocytes were stimulated for 24 hours with different ligands (lipopolysaccharide (LPS); Flagellin (Flag); peptidoglycan (PGN)) with or without recombinant bovine IL-10. IL-1β (p

    Journal: PLoS ONE

    Article Title: Modulation of Human Immune Responses by Bovine Interleukin-10

    doi: 10.1371/journal.pone.0018188

    Figure Lengend Snippet: Bovine IL-10 can regulate TLR ligand induced cytokine production by monocytes by binding the IL-10R. Figures 2B-2D are box plots, showing the median (black horizontal bar), 50% data range (box) and 99% data range (error bars). On the x-axes is the addition indicated (+) of TLR stimuli (panel A), or 10 ng/ml LPS (B–D), 10 ng/ml IL-10 (A-D), bovine IL-10 or human IL-10R blocking antibodies (anti bIL-10 or IL-10R) or isotype control (C–D). A : Freshly isolated monocytes were stimulated for 24 hours with different ligands (lipopolysaccharide (LPS); Flagellin (Flag); peptidoglycan (PGN)) with or without recombinant bovine IL-10. IL-1β (p

    Article Snippet: Bovine IL-10 ELISA A bovine IL-10 specific capture ELISA was developed in house using an anti-bovine IL-10 specific monoclonal (MCA2110, AbD Serotec, Oxford, UK) as a capture antibody and biotinylated anti-bovine IL-10 IgY as a detecting antibody.

    Techniques: Binding Assay, Blocking Assay, Isolation, Recombinant

    Over-expression of c-rel restores IL-12 p40 induction in IL-10-treated macrophages. The RAW 264.7 macrophages were transfected with either the c-rel plasmid construct or with both c-rel and p65 NF-κB plasmids using lipofectin. Cells were incubated

    Journal: Immunology

    Article Title: Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor

    doi: 10.1111/j.1365-2567.2005.02107.x

    Figure Lengend Snippet: Over-expression of c-rel restores IL-12 p40 induction in IL-10-treated macrophages. The RAW 264.7 macrophages were transfected with either the c-rel plasmid construct or with both c-rel and p65 NF-κB plasmids using lipofectin. Cells were incubated

    Article Snippet: The RAW 264.7 cells were either left untreated or were pretreated for 1 hr with rIL-10 or anti-IL-10 antibody or leptomycin B or with both leptomycin B and anti-IL-10 antibody and stimulated further with LPS + IFN-γ for 4 hr followed by another 4 hr incubation with 20 μg/ml brefeldin A (Sigma-Aldrich) that retained the cytokine within the cell.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Construct, Incubation

    Inhibition of endogenous IL-10 by treating macrophages with anti-IL-10 antibody (anti-IL-10 Ab) significantly increases nuclear c -rel level in addition to p65 NF-κB and causes up-regulation of IL-12 production. The RAW 264.7 macrophages were pretreated

    Journal: Immunology

    Article Title: Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor

    doi: 10.1111/j.1365-2567.2005.02107.x

    Figure Lengend Snippet: Inhibition of endogenous IL-10 by treating macrophages with anti-IL-10 antibody (anti-IL-10 Ab) significantly increases nuclear c -rel level in addition to p65 NF-κB and causes up-regulation of IL-12 production. The RAW 264.7 macrophages were pretreated

    Article Snippet: The RAW 264.7 cells were either left untreated or were pretreated for 1 hr with rIL-10 or anti-IL-10 antibody or leptomycin B or with both leptomycin B and anti-IL-10 antibody and stimulated further with LPS + IFN-γ for 4 hr followed by another 4 hr incubation with 20 μg/ml brefeldin A (Sigma-Aldrich) that retained the cytokine within the cell.

    Techniques: Inhibition

    Inhibition of c-rel import by leptomycin B prevents IL-12 up-regulation by anti-IL-10 Ab. RAW macrophages were pretreated with 10 n m of lepotmycin B for 1 hr and further stimulated with LPS + IFN-γ either for 1 hr for assaying nuclear accumulation

    Journal: Immunology

    Article Title: Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor

    doi: 10.1111/j.1365-2567.2005.02107.x

    Figure Lengend Snippet: Inhibition of c-rel import by leptomycin B prevents IL-12 up-regulation by anti-IL-10 Ab. RAW macrophages were pretreated with 10 n m of lepotmycin B for 1 hr and further stimulated with LPS + IFN-γ either for 1 hr for assaying nuclear accumulation

    Article Snippet: The RAW 264.7 cells were either left untreated or were pretreated for 1 hr with rIL-10 or anti-IL-10 antibody or leptomycin B or with both leptomycin B and anti-IL-10 antibody and stimulated further with LPS + IFN-γ for 4 hr followed by another 4 hr incubation with 20 μg/ml brefeldin A (Sigma-Aldrich) that retained the cytokine within the cell.

    Techniques: Inhibition

    In vitro transient expression of p65 NF-κB does not rescue the inhibitory effect of IL-10 on IL-12. The plasmid constructs were transfected into RAW 264.7 macrophage cells using the cationic lipid suspension lipofectin and incubation for 18 hr.

    Journal: Immunology

    Article Title: Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor

    doi: 10.1111/j.1365-2567.2005.02107.x

    Figure Lengend Snippet: In vitro transient expression of p65 NF-κB does not rescue the inhibitory effect of IL-10 on IL-12. The plasmid constructs were transfected into RAW 264.7 macrophage cells using the cationic lipid suspension lipofectin and incubation for 18 hr.

    Article Snippet: The RAW 264.7 cells were either left untreated or were pretreated for 1 hr with rIL-10 or anti-IL-10 antibody or leptomycin B or with both leptomycin B and anti-IL-10 antibody and stimulated further with LPS + IFN-γ for 4 hr followed by another 4 hr incubation with 20 μg/ml brefeldin A (Sigma-Aldrich) that retained the cytokine within the cell.

    Techniques: In Vitro, Expressing, Plasmid Preparation, Construct, Transfection, Incubation

    Exogenous IL-10 inhibits nuclear c-rel levels and also IL-12 production. The RAW 264.7 macrophages were cultured with LPS + IFN-γ in the absence or presence of 5 ng/ml of rIL-10. The nuclear accumulation of c-rel was examined by immunoblot analysis

    Journal: Immunology

    Article Title: Interleukin-10 (IL-10) mediated suppression of IL-12 production in RAW 264.7 cells also involves c-rel transcription factor

    doi: 10.1111/j.1365-2567.2005.02107.x

    Figure Lengend Snippet: Exogenous IL-10 inhibits nuclear c-rel levels and also IL-12 production. The RAW 264.7 macrophages were cultured with LPS + IFN-γ in the absence or presence of 5 ng/ml of rIL-10. The nuclear accumulation of c-rel was examined by immunoblot analysis

    Article Snippet: The RAW 264.7 cells were either left untreated or were pretreated for 1 hr with rIL-10 or anti-IL-10 antibody or leptomycin B or with both leptomycin B and anti-IL-10 antibody and stimulated further with LPS + IFN-γ for 4 hr followed by another 4 hr incubation with 20 μg/ml brefeldin A (Sigma-Aldrich) that retained the cytokine within the cell.

    Techniques: Cell Culture

    Protective Effect of Intermittent Expression of NT-3 or IL-10 from the Vectors on the Development of Paclitaxel-Induced Peripheral Neuropathy Animals were subcutaneously inoculated with either vector vL2rtNT-3 or vL2rtIL-10 or control vector vL2rtGFP and injected i.p. with paclitaxel (16 mg/kg) once a week for 5 weeks to mimic paclitaxel-induced peripheral neuropathy. Naive animals and animals receiving paclitaxel only served as the symptom-negative and -positive controls. Sensorimotor coordination and sensory nerve electrophysiological function (amplitude and conduction velocity) were evaluated 5 weeks after the initiation of paclitaxel treatment. (A) Schematic of the treatment protocol. (B) Effect of intermittent expression of NT-3 (top, sensory nerve amplitude; center, sensory nerve conduction velocity; bottom, sensorimotor coordination). (C) Effect of intermittent expression of IL-10 (top, sensory nerve amplitude; center, sensory nerve conduction velocity; bottom, sensorimotor coordination). *p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy

    doi: 10.1016/j.omtm.2017.06.004

    Figure Lengend Snippet: Protective Effect of Intermittent Expression of NT-3 or IL-10 from the Vectors on the Development of Paclitaxel-Induced Peripheral Neuropathy Animals were subcutaneously inoculated with either vector vL2rtNT-3 or vL2rtIL-10 or control vector vL2rtGFP and injected i.p. with paclitaxel (16 mg/kg) once a week for 5 weeks to mimic paclitaxel-induced peripheral neuropathy. Naive animals and animals receiving paclitaxel only served as the symptom-negative and -positive controls. Sensorimotor coordination and sensory nerve electrophysiological function (amplitude and conduction velocity) were evaluated 5 weeks after the initiation of paclitaxel treatment. (A) Schematic of the treatment protocol. (B) Effect of intermittent expression of NT-3 (top, sensory nerve amplitude; center, sensory nerve conduction velocity; bottom, sensorimotor coordination). (C) Effect of intermittent expression of IL-10 (top, sensory nerve amplitude; center, sensory nerve conduction velocity; bottom, sensorimotor coordination). *p

    Article Snippet: Immunoblots were incubated with the primary antibodies anti-NT-3 (1:1,000, Abcam), anti-IL-10 (1:1,000, Sigma), or anti-β-actin (1:1,000, Sigma) for 2 hr at RT followed by incubation with an HRP-conjugated secondary antibody (1;2000, Santa Cruz Biotechnology, Dallas, Texas) 1 hr at room temperature (RT) after being washed by 0.05% Tween 20-containing PBS (PBST) three times.

    Techniques: Expressing, Plasmid Preparation, Injection

    Regulated Expression of NT-3 and IL-10 from the Vectors by DOX In Vitro Complementing 7b cells were infected by vL2rtNT-3 or vL2rtIL-10 with an MOI of 0.5 and exposed to DOX at different concentrations 1 hr after infection. (A and D) NT-3 or IL-10 concentration in the medium (A, NT-3; D, IL-10) was measured by NT-3 or IL-10 ELISA kit after 2 days of DOX treatment. (B, C, E, and F). To test the kinetics of the turn-on and turn-off of NT-3 or IL-10 expression from the vectors, 7b cells were infected with either vL2rtNT-3 or vL2rtIL-10 at an MOI of 0.01 and cultured either in 1 μg/mL DOX-containing medium for 2 days and subsequently in normal medium for 4 days (B, NT-3; E, IL-10) or cultured in normal medium for the first 2 days after vector infection, followed by 4-day culturing with 1 μg/ml /mL DOX-containing medium (C, NT-3; F, IL-10). Under each culture condition, 50 μL of medium was collected every 2 days, and NT-3 or IL-10 concentration in the medium was measured by ELISA.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy

    doi: 10.1016/j.omtm.2017.06.004

    Figure Lengend Snippet: Regulated Expression of NT-3 and IL-10 from the Vectors by DOX In Vitro Complementing 7b cells were infected by vL2rtNT-3 or vL2rtIL-10 with an MOI of 0.5 and exposed to DOX at different concentrations 1 hr after infection. (A and D) NT-3 or IL-10 concentration in the medium (A, NT-3; D, IL-10) was measured by NT-3 or IL-10 ELISA kit after 2 days of DOX treatment. (B, C, E, and F). To test the kinetics of the turn-on and turn-off of NT-3 or IL-10 expression from the vectors, 7b cells were infected with either vL2rtNT-3 or vL2rtIL-10 at an MOI of 0.01 and cultured either in 1 μg/mL DOX-containing medium for 2 days and subsequently in normal medium for 4 days (B, NT-3; E, IL-10) or cultured in normal medium for the first 2 days after vector infection, followed by 4-day culturing with 1 μg/ml /mL DOX-containing medium (C, NT-3; F, IL-10). Under each culture condition, 50 μL of medium was collected every 2 days, and NT-3 or IL-10 concentration in the medium was measured by ELISA.

    Article Snippet: Immunoblots were incubated with the primary antibodies anti-NT-3 (1:1,000, Abcam), anti-IL-10 (1:1,000, Sigma), or anti-β-actin (1:1,000, Sigma) for 2 hr at RT followed by incubation with an HRP-conjugated secondary antibody (1;2000, Santa Cruz Biotechnology, Dallas, Texas) 1 hr at room temperature (RT) after being washed by 0.05% Tween 20-containing PBS (PBST) three times.

    Techniques: Expressing, In Vitro, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Plasmid Preparation

    Induction of NT-3 and IL-10 Expression from the Vector by DOX in Animals Animals were inoculated subcutaneously into the skin of both hindfeet with either vL2rtNT-3 or vL2rtIL-10 and fed DOX-containing chow for 1, 3, and 7 days to examine the induced expression of the transgenes from the vectors, or animals receiving the vectors were fed DOX-containing chow for 3 days, followed by normal food for 4 days, to test the shut-off of the expression of the transgenes from the vectors in the absence of DOX. After each DOX schedule was completed, L4-6 DRGs of both sides were dissected, and NT-3 or IL-10 mRNA and proteins in the DRGs were analyzed by semiquantitative PCR and western blot. β-Actin served as a loading control. (A) Semiquantitative NT-3 PCR and NT-3 mRNA relative amount. 3/4 off, 3 days with DOX treatment/4 days without DOX treatment. (B) NT-3 western blot and NT-3 protein relative amount. (C) Semiquantitative IL-10 PCR and IL-10 mRNA relative amount. (D) IL-10 western blot and IL-10 protein relative amount. *p

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy

    doi: 10.1016/j.omtm.2017.06.004

    Figure Lengend Snippet: Induction of NT-3 and IL-10 Expression from the Vector by DOX in Animals Animals were inoculated subcutaneously into the skin of both hindfeet with either vL2rtNT-3 or vL2rtIL-10 and fed DOX-containing chow for 1, 3, and 7 days to examine the induced expression of the transgenes from the vectors, or animals receiving the vectors were fed DOX-containing chow for 3 days, followed by normal food for 4 days, to test the shut-off of the expression of the transgenes from the vectors in the absence of DOX. After each DOX schedule was completed, L4-6 DRGs of both sides were dissected, and NT-3 or IL-10 mRNA and proteins in the DRGs were analyzed by semiquantitative PCR and western blot. β-Actin served as a loading control. (A) Semiquantitative NT-3 PCR and NT-3 mRNA relative amount. 3/4 off, 3 days with DOX treatment/4 days without DOX treatment. (B) NT-3 western blot and NT-3 protein relative amount. (C) Semiquantitative IL-10 PCR and IL-10 mRNA relative amount. (D) IL-10 western blot and IL-10 protein relative amount. *p

    Article Snippet: Immunoblots were incubated with the primary antibodies anti-NT-3 (1:1,000, Abcam), anti-IL-10 (1:1,000, Sigma), or anti-β-actin (1:1,000, Sigma) for 2 hr at RT followed by incubation with an HRP-conjugated secondary antibody (1;2000, Santa Cruz Biotechnology, Dallas, Texas) 1 hr at room temperature (RT) after being washed by 0.05% Tween 20-containing PBS (PBST) three times.

    Techniques: Expressing, Plasmid Preparation, Polymerase Chain Reaction, Western Blot

    Prolonged Regulated HSV Vector Prolonged regulated gene expression was achieved using a modified tet-on system we previously developed in which the expression of the transactivator was under the control of HSV latency-associated promoter 2 (LAP2). (A) The transactivator in the tet-on system is constitutively expressed and binds to the tetracycline response element (TRE)-minimal human cytomegalovirus immediate early promoter (HCMV IEp) of the inducible transgene expression element in the presence of DOX, thus resulting in the expression of the transgene. (B) In the vectors, two copies of the regulatable transgene expression units were inserted into the ICP4 loci of the replication-deficient parental HSV virus. DOX, doxycycline; NT-3, neurotrophin 3; IL-10, interleukin 10; rtTA, reverse tet-controlled transactivator; K, kozak sequence; GOI, gene of interest.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy

    doi: 10.1016/j.omtm.2017.06.004

    Figure Lengend Snippet: Prolonged Regulated HSV Vector Prolonged regulated gene expression was achieved using a modified tet-on system we previously developed in which the expression of the transactivator was under the control of HSV latency-associated promoter 2 (LAP2). (A) The transactivator in the tet-on system is constitutively expressed and binds to the tetracycline response element (TRE)-minimal human cytomegalovirus immediate early promoter (HCMV IEp) of the inducible transgene expression element in the presence of DOX, thus resulting in the expression of the transgene. (B) In the vectors, two copies of the regulatable transgene expression units were inserted into the ICP4 loci of the replication-deficient parental HSV virus. DOX, doxycycline; NT-3, neurotrophin 3; IL-10, interleukin 10; rtTA, reverse tet-controlled transactivator; K, kozak sequence; GOI, gene of interest.

    Article Snippet: Immunoblots were incubated with the primary antibodies anti-NT-3 (1:1,000, Abcam), anti-IL-10 (1:1,000, Sigma), or anti-β-actin (1:1,000, Sigma) for 2 hr at RT followed by incubation with an HRP-conjugated secondary antibody (1;2000, Santa Cruz Biotechnology, Dallas, Texas) 1 hr at room temperature (RT) after being washed by 0.05% Tween 20-containing PBS (PBST) three times.

    Techniques: Plasmid Preparation, Expressing, Modification, Sequencing

    No Effect of Repeated Paclitaxel Treatments on Induced Expression of the Transgenes from the Vectors To analyze the effect of repeated paclitaxel treatments on the induced expression of the transgenes from the vectors, naive animals and animals receiving paclitaxel alone were fed normal chow for 4 more days, whereas vector-infected animals were fed DOX-containing chow for 4 additional days 1 week after the fifth injection in the repeated paclitaxel treatment schedule. L4-6 DRGs of both hindfeet were dissected for determination of NT-3 or IL-10 mRNA and protein, measured by semiquantitative PCR and western blot. (A) Semiquantitative NT-3 PCR and NT-3 mRNA relative amount. (B) NT-3 western blot and NT-3 protein relative amount. (C) Semiquantitative IL-10 PCR and IL-10 mRNA relative amount. (D) IL-10 western blot and IL-10 protein relative amount. A, naive animals fed normal chow; B, paclitaxel-treated animals fed normal chow; C, vector-inoculated, paclitaxel-treated animals previously fed DOX-containing chow 4 days per week fed DOX-containing chow for 4 additional days; D, vector-inoculated, paclitaxel-treated animals previously fed DOX-containing chow continuously fed DOX-containing chow for 4 additional days.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy

    doi: 10.1016/j.omtm.2017.06.004

    Figure Lengend Snippet: No Effect of Repeated Paclitaxel Treatments on Induced Expression of the Transgenes from the Vectors To analyze the effect of repeated paclitaxel treatments on the induced expression of the transgenes from the vectors, naive animals and animals receiving paclitaxel alone were fed normal chow for 4 more days, whereas vector-infected animals were fed DOX-containing chow for 4 additional days 1 week after the fifth injection in the repeated paclitaxel treatment schedule. L4-6 DRGs of both hindfeet were dissected for determination of NT-3 or IL-10 mRNA and protein, measured by semiquantitative PCR and western blot. (A) Semiquantitative NT-3 PCR and NT-3 mRNA relative amount. (B) NT-3 western blot and NT-3 protein relative amount. (C) Semiquantitative IL-10 PCR and IL-10 mRNA relative amount. (D) IL-10 western blot and IL-10 protein relative amount. A, naive animals fed normal chow; B, paclitaxel-treated animals fed normal chow; C, vector-inoculated, paclitaxel-treated animals previously fed DOX-containing chow 4 days per week fed DOX-containing chow for 4 additional days; D, vector-inoculated, paclitaxel-treated animals previously fed DOX-containing chow continuously fed DOX-containing chow for 4 additional days.

    Article Snippet: Immunoblots were incubated with the primary antibodies anti-NT-3 (1:1,000, Abcam), anti-IL-10 (1:1,000, Sigma), or anti-β-actin (1:1,000, Sigma) for 2 hr at RT followed by incubation with an HRP-conjugated secondary antibody (1;2000, Santa Cruz Biotechnology, Dallas, Texas) 1 hr at room temperature (RT) after being washed by 0.05% Tween 20-containing PBS (PBST) three times.

    Techniques: Expressing, Plasmid Preparation, Infection, Injection, Polymerase Chain Reaction, Western Blot

    LCL growth inhibition assay with a CD4 + T-cell line from a representative seropositive donor. (a) Photograph of a U-bottomed microtitre plate showing the inhibition of LCL growth after 4 weeks co-culture with an autologous CD4 + T-cell line. Triplicate wells (columns 1–3) were seeded with doubling dilutions of LCL at 10 4 cells per well in row A down to 80 cells per well in row H. Growth of LCL cells can be seen as a large central pellet of cells in rows A–H. Triplicate wells (columns 4–6) were seeded with identical numbers of LCL cells and, in addition, 10 4 autologous CD4 + T cells were added to each well. After an initial period of growth, as indicated by a small central spot in each well, there was subsequent arrest and no further outgrowth of the LCL in any of the wells. The dot plots below the plate show that of 22% of CD4 + T cells from this donor produced IL-4 and 5% IFNγ when stimulated with PMA and ionomycin. (b) Photograph of a second plate set up in an identical manner to (a). In this case, the growth of LCL was inhibited in all but the top two rows. CD4 + T cells from this donor produced 7% IL-4 and 53% IFNγ. (c) Photograph of a third plate set up in an identical manner to (a). The larger pellets of growing cells in columns 4–6 show that the growth of LCL was only poorly inhibited by this CD4 + T-cell line. Microscopic examination confirmed that complete inhibition of growth occurred at T Cell:LCL ratios of 1:32 or more. The T cells in this particular line had a strong Th1 bias, with only 2% of cells producing IL-4 but 95% producing IFNγ. Flow cytometry confirmed that the live cells present after 4 weeks culture were all LCL cells, and no T cells were detected in any wells examined at this time.

    Journal: Clinical and Experimental Immunology

    Article Title: In vitro cytokine production and growth inhibition of lymphoblastoid cell lines by CD4+ T cells from Epstein-Barr virus (EBV) seropositive donors

    doi: 10.1046/j.1365-2249.2001.01641.x

    Figure Lengend Snippet: LCL growth inhibition assay with a CD4 + T-cell line from a representative seropositive donor. (a) Photograph of a U-bottomed microtitre plate showing the inhibition of LCL growth after 4 weeks co-culture with an autologous CD4 + T-cell line. Triplicate wells (columns 1–3) were seeded with doubling dilutions of LCL at 10 4 cells per well in row A down to 80 cells per well in row H. Growth of LCL cells can be seen as a large central pellet of cells in rows A–H. Triplicate wells (columns 4–6) were seeded with identical numbers of LCL cells and, in addition, 10 4 autologous CD4 + T cells were added to each well. After an initial period of growth, as indicated by a small central spot in each well, there was subsequent arrest and no further outgrowth of the LCL in any of the wells. The dot plots below the plate show that of 22% of CD4 + T cells from this donor produced IL-4 and 5% IFNγ when stimulated with PMA and ionomycin. (b) Photograph of a second plate set up in an identical manner to (a). In this case, the growth of LCL was inhibited in all but the top two rows. CD4 + T cells from this donor produced 7% IL-4 and 53% IFNγ. (c) Photograph of a third plate set up in an identical manner to (a). The larger pellets of growing cells in columns 4–6 show that the growth of LCL was only poorly inhibited by this CD4 + T-cell line. Microscopic examination confirmed that complete inhibition of growth occurred at T Cell:LCL ratios of 1:32 or more. The T cells in this particular line had a strong Th1 bias, with only 2% of cells producing IL-4 but 95% producing IFNγ. Flow cytometry confirmed that the live cells present after 4 weeks culture were all LCL cells, and no T cells were detected in any wells examined at this time.

    Article Snippet: The antibodies used were anti-IFNγ clone D9D10 (Serotec, Oxford, UK), anti-IL-2 clone 5334·2, anti-IL-4 clone 3007·11, anti-IL-6 clone 1927·311 (Sigma), anti-TNFα clone Mab 11, anti-TNFβ clone 359-81-11, anti-IL-10 clone JES3–9D7 and anti, IL-13 clone JES10-5a2 (Pharmingen, San Diego, CA, USA).

    Techniques: Growth Inhibition Assay, Inhibition, Co-Culture Assay, Produced, Flow Cytometry, Cytometry

    Dual colour intracellular cytokine staining of a CD4 + T-cell line reactivated with PMA/ionomycin. (a) CD4 + T cells stained with isotype-matched FITC and PE control antibody conjugates. (b) CD4 + T cells stained with anti-IL-4 PE conjugate. (c) CD4 + T cells stained with anti-IFNγ FITC conjugate. (d) Dual staining of CD4 + T-cell lines with anti-IFNγ FITC and anti-IL-4 PE, showing that many of the CD4 + T cells produce both cytokines simultaneously. (e) CD4 + T cells stained with anti-IL-13 PE conjugate. (f) Dual staining of CD4 + T-cell lines with anti-IFNγ FITC and anti-IL-13 PE, showing that many of the CD4 + T cells also produce both these cytokines simultaneously.

    Journal: Clinical and Experimental Immunology

    Article Title: In vitro cytokine production and growth inhibition of lymphoblastoid cell lines by CD4+ T cells from Epstein-Barr virus (EBV) seropositive donors

    doi: 10.1046/j.1365-2249.2001.01641.x

    Figure Lengend Snippet: Dual colour intracellular cytokine staining of a CD4 + T-cell line reactivated with PMA/ionomycin. (a) CD4 + T cells stained with isotype-matched FITC and PE control antibody conjugates. (b) CD4 + T cells stained with anti-IL-4 PE conjugate. (c) CD4 + T cells stained with anti-IFNγ FITC conjugate. (d) Dual staining of CD4 + T-cell lines with anti-IFNγ FITC and anti-IL-4 PE, showing that many of the CD4 + T cells produce both cytokines simultaneously. (e) CD4 + T cells stained with anti-IL-13 PE conjugate. (f) Dual staining of CD4 + T-cell lines with anti-IFNγ FITC and anti-IL-13 PE, showing that many of the CD4 + T cells also produce both these cytokines simultaneously.

    Article Snippet: The antibodies used were anti-IFNγ clone D9D10 (Serotec, Oxford, UK), anti-IL-2 clone 5334·2, anti-IL-4 clone 3007·11, anti-IL-6 clone 1927·311 (Sigma), anti-TNFα clone Mab 11, anti-TNFβ clone 359-81-11, anti-IL-10 clone JES3–9D7 and anti, IL-13 clone JES10-5a2 (Pharmingen, San Diego, CA, USA).

    Techniques: Staining

    Intracellular staining for cytokine production by a CD4 + T-cell line following re-stimulation with PMA and ionomycin. The results are typical of EBV-reactive CD4 + T-cell lines and show a mixture of Th1 and Th2 type cytokines, but with very high levels of IFNγ compared with IL-4.

    Journal: Clinical and Experimental Immunology

    Article Title: In vitro cytokine production and growth inhibition of lymphoblastoid cell lines by CD4+ T cells from Epstein-Barr virus (EBV) seropositive donors

    doi: 10.1046/j.1365-2249.2001.01641.x

    Figure Lengend Snippet: Intracellular staining for cytokine production by a CD4 + T-cell line following re-stimulation with PMA and ionomycin. The results are typical of EBV-reactive CD4 + T-cell lines and show a mixture of Th1 and Th2 type cytokines, but with very high levels of IFNγ compared with IL-4.

    Article Snippet: The antibodies used were anti-IFNγ clone D9D10 (Serotec, Oxford, UK), anti-IL-2 clone 5334·2, anti-IL-4 clone 3007·11, anti-IL-6 clone 1927·311 (Sigma), anti-TNFα clone Mab 11, anti-TNFβ clone 359-81-11, anti-IL-10 clone JES3–9D7 and anti, IL-13 clone JES10-5a2 (Pharmingen, San Diego, CA, USA).

    Techniques: Staining

    The protein expression of IL6, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3 and STAT3 in different transfected groups. Error bars indicate means ± SD. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Overexpression of miR-574-3p suppresses proliferation and induces apoptosis of chronic myeloid leukemia cells via targeting IL6/JAK/STAT3 pathway

    doi: 10.3892/etm.2018.6700

    Figure Lengend Snippet: The protein expression of IL6, p-JAK1, JAK1, p-JAK2, JAK2, p-STAT3 and STAT3 in different transfected groups. Error bars indicate means ± SD. *P

    Article Snippet: After blocked with 5% blocking agent, the membranes were immunoblotted with primary antibodies, including anti-IL6, anti-JAK1, anti-p-JAK1, anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3 and anti-β-actin (1:1,000, cell signaling, MA, USA) overnight at 4°C in shaker. β-actin was used as the loading control.

    Techniques: Expressing, Transfection

    IL6 was the direct target of miR-574-3p. (A) Luciferase report assay showed that miR-574-3p directly targeted the IL6 3′UTR-wt. (B) The mRNA expression of IL6 in different transfection groups determined by qRT-PCR. (C) The protein expression of IL6 in different transfection groups determined by western blot. (D) The mRNA expression of IL6 in peripheral blood obtained from CML patients and healthy controls determined by qRT-PCR. (E) The concentration of IL6 in peripheral blood obtained from CML patients and healthy controls determined by ELISA. Error bars indicate means ± SD. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Overexpression of miR-574-3p suppresses proliferation and induces apoptosis of chronic myeloid leukemia cells via targeting IL6/JAK/STAT3 pathway

    doi: 10.3892/etm.2018.6700

    Figure Lengend Snippet: IL6 was the direct target of miR-574-3p. (A) Luciferase report assay showed that miR-574-3p directly targeted the IL6 3′UTR-wt. (B) The mRNA expression of IL6 in different transfection groups determined by qRT-PCR. (C) The protein expression of IL6 in different transfection groups determined by western blot. (D) The mRNA expression of IL6 in peripheral blood obtained from CML patients and healthy controls determined by qRT-PCR. (E) The concentration of IL6 in peripheral blood obtained from CML patients and healthy controls determined by ELISA. Error bars indicate means ± SD. *P

    Article Snippet: After blocked with 5% blocking agent, the membranes were immunoblotted with primary antibodies, including anti-IL6, anti-JAK1, anti-p-JAK1, anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3 and anti-β-actin (1:1,000, cell signaling, MA, USA) overnight at 4°C in shaker. β-actin was used as the loading control.

    Techniques: Luciferase, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

    The effects of IL6 on the proliferation and apoptosis of CML K562 cells. K562 cells were transfected with pcDNA-IL6, blank vector, si-IL6 and si-control. (A) MTT assay showed the cell viability in different transfected groups after different times of transfection. The maximum absorption value is 490. (B) The expression of IL6 in different transfected groups after 2 days of transfection. (C) Colony formation assay showed the colony number in different transfected groups after 2 days of transfection. (D) Flow cytometry showed the apoptosis in different transfected groups after 2 days of transfection. Error bars indicate means ± SD. *P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Overexpression of miR-574-3p suppresses proliferation and induces apoptosis of chronic myeloid leukemia cells via targeting IL6/JAK/STAT3 pathway

    doi: 10.3892/etm.2018.6700

    Figure Lengend Snippet: The effects of IL6 on the proliferation and apoptosis of CML K562 cells. K562 cells were transfected with pcDNA-IL6, blank vector, si-IL6 and si-control. (A) MTT assay showed the cell viability in different transfected groups after different times of transfection. The maximum absorption value is 490. (B) The expression of IL6 in different transfected groups after 2 days of transfection. (C) Colony formation assay showed the colony number in different transfected groups after 2 days of transfection. (D) Flow cytometry showed the apoptosis in different transfected groups after 2 days of transfection. Error bars indicate means ± SD. *P

    Article Snippet: After blocked with 5% blocking agent, the membranes were immunoblotted with primary antibodies, including anti-IL6, anti-JAK1, anti-p-JAK1, anti-JAK2, anti-p-JAK2, anti-STAT3, anti-p-STAT3 and anti-β-actin (1:1,000, cell signaling, MA, USA) overnight at 4°C in shaker. β-actin was used as the loading control.

    Techniques: Transfection, Plasmid Preparation, MTT Assay, Expressing, Colony Assay, Flow Cytometry, Cytometry

    IL-2 enhances the differentiation of memory precursors via PI3K in vitro . (A,B) Blockade of IL-2 reduces the frequency of CD49b + CXCR3 + subpopulation in in vitro -activated CD4 T cells. Naive CD4 T cells from C57BL/6 mice were stimulated with anti-CD3/CD28 for 48 h in the presence of anti-IL-2 or isotype control and then incubated for 48 h. The frequencies of CD49b + CXCR3 + in activated CD4 T cells (A) and Ly-6C hi cells in CXCR3 + activated CD4 T cells (B) are shown. (C,D) Inhibition of PI3K reduces the frequency of CD49b + CXCR3 + subpopulation in in vitro -activated CD4 T cells. Naive CD4 T cells from C57BL/6 mice were stimulated with anti-CD3/CD28 for 48 h in the presence of Wortmannin, STAT5 inhibitor or DMSO and then incubated for 48 h. The frequencies of CD49b + CXCR3 + in activated CD4 T cells (C) and Ly-6C hi cells in CXCR3 + activated CD4 T cells (D) are shown n = 6. These data are representative of two independent experiments. Data are shown as mean ± SD. *** p

    Journal: Frontiers in Immunology

    Article Title: Enhanced Cell Division Is Required for the Generation of Memory CD4 T Cells to Migrate Into Their Proper Location

    doi: 10.3389/fimmu.2019.03113

    Figure Lengend Snippet: IL-2 enhances the differentiation of memory precursors via PI3K in vitro . (A,B) Blockade of IL-2 reduces the frequency of CD49b + CXCR3 + subpopulation in in vitro -activated CD4 T cells. Naive CD4 T cells from C57BL/6 mice were stimulated with anti-CD3/CD28 for 48 h in the presence of anti-IL-2 or isotype control and then incubated for 48 h. The frequencies of CD49b + CXCR3 + in activated CD4 T cells (A) and Ly-6C hi cells in CXCR3 + activated CD4 T cells (B) are shown. (C,D) Inhibition of PI3K reduces the frequency of CD49b + CXCR3 + subpopulation in in vitro -activated CD4 T cells. Naive CD4 T cells from C57BL/6 mice were stimulated with anti-CD3/CD28 for 48 h in the presence of Wortmannin, STAT5 inhibitor or DMSO and then incubated for 48 h. The frequencies of CD49b + CXCR3 + in activated CD4 T cells (C) and Ly-6C hi cells in CXCR3 + activated CD4 T cells (D) are shown n = 6. These data are representative of two independent experiments. Data are shown as mean ± SD. *** p

    Article Snippet: In vitro Stimulation of Naive CD4 T Cells Naive CD4 T cells from C57BL/6 mice were isolated as described above and stimulated with plate-coated anti-CD3 (145-2C11, 1 μg/ml) and anti-CD28 (37–51, 10 μg/ml) in the presence of anti-IL-2 (S4B6, 20 μg/ml) or isotype control, in case of inhibitors, in the presence of Wortmannin (0.8 μM, Sigma), STAT5 inhibitor (20 μM, Tocris) or DMSO for 48 h at 37°C.

    Techniques: In Vitro, Mouse Assay, Incubation, Inhibition

    Loss of IL-2 signal inhibits the differentiation of BM memory precursors. (A) Knock-down of IL-2Rβ reduces numbers of CD49b + CXCR3 + memory precursors. C57BL/6 mice were transferred with preactivated and IL-2Rβ shRNA- or scrambled (Scr.) shRNA-transduced Thy1.1 + LCMV GP-specific CD4 T cells, immunized with LCMV-gp61 and LPS and analyzed on day 6 after immunization by flow cytometry. The histogram shows the efficiency of IL-2Rβ shRNA-mediated knock-down (filled histogram, isotype control; dashed line, Scr. shRNA; solid line, IL-2Rβ shRNA) and the dot plots show the expression of CD49b and CXCR3 in Thy1.1 + CD44 hi CD4 + B220 − NK1.1 − PI − cells. The bar charts display the numbers of total Thy1.1 + LCMV GP-specific CD4 T cells (left) and the frequency of CD49b + CXCR3 + subpopulation (right) n = 6. (B–D) Injection of anti-IL-2 reduces numbers of CD49b + CXCR3 + memory precursors. As described in Figure 1 , C57BL/6 mice were transferred, immunized and analyzed by flow cytometry on day 6 after immunization, receiving 1 mg of anti-IL-2 or isotype control on days 0, 2, and 4. The dot plots show the expression of CD49b and CXCR3 in Thy1.1 + CD44 hi CD4 T cells. The bar charts display the numbers of total Thy1.1 + CD4 T cells ( B , left) and the frequency of CD49b + CXCR3 + subpopulation ( B , right), the dilution of CFSE in each subpopulation in the spleen (C) and the BM (D) n = 4. These data are representative of two independent experiments. Data are shown as mean ± SD. * p

    Journal: Frontiers in Immunology

    Article Title: Enhanced Cell Division Is Required for the Generation of Memory CD4 T Cells to Migrate Into Their Proper Location

    doi: 10.3389/fimmu.2019.03113

    Figure Lengend Snippet: Loss of IL-2 signal inhibits the differentiation of BM memory precursors. (A) Knock-down of IL-2Rβ reduces numbers of CD49b + CXCR3 + memory precursors. C57BL/6 mice were transferred with preactivated and IL-2Rβ shRNA- or scrambled (Scr.) shRNA-transduced Thy1.1 + LCMV GP-specific CD4 T cells, immunized with LCMV-gp61 and LPS and analyzed on day 6 after immunization by flow cytometry. The histogram shows the efficiency of IL-2Rβ shRNA-mediated knock-down (filled histogram, isotype control; dashed line, Scr. shRNA; solid line, IL-2Rβ shRNA) and the dot plots show the expression of CD49b and CXCR3 in Thy1.1 + CD44 hi CD4 + B220 − NK1.1 − PI − cells. The bar charts display the numbers of total Thy1.1 + LCMV GP-specific CD4 T cells (left) and the frequency of CD49b + CXCR3 + subpopulation (right) n = 6. (B–D) Injection of anti-IL-2 reduces numbers of CD49b + CXCR3 + memory precursors. As described in Figure 1 , C57BL/6 mice were transferred, immunized and analyzed by flow cytometry on day 6 after immunization, receiving 1 mg of anti-IL-2 or isotype control on days 0, 2, and 4. The dot plots show the expression of CD49b and CXCR3 in Thy1.1 + CD44 hi CD4 T cells. The bar charts display the numbers of total Thy1.1 + CD4 T cells ( B , left) and the frequency of CD49b + CXCR3 + subpopulation ( B , right), the dilution of CFSE in each subpopulation in the spleen (C) and the BM (D) n = 4. These data are representative of two independent experiments. Data are shown as mean ± SD. * p

    Article Snippet: In vitro Stimulation of Naive CD4 T Cells Naive CD4 T cells from C57BL/6 mice were isolated as described above and stimulated with plate-coated anti-CD3 (145-2C11, 1 μg/ml) and anti-CD28 (37–51, 10 μg/ml) in the presence of anti-IL-2 (S4B6, 20 μg/ml) or isotype control, in case of inhibitors, in the presence of Wortmannin (0.8 μM, Sigma), STAT5 inhibitor (20 μM, Tocris) or DMSO for 48 h at 37°C.

    Techniques: Mouse Assay, shRNA, Flow Cytometry, Expressing, Injection

    Interleukin-1β expression decreased in group B and group C compared to that in group A.

    Journal: Annals of Dermatology

    Article Title: Efficacy of Red or Infrared Light-Emitting Diodes in a Mouse Model of Propionibacterium acnes-Induced Inflammation

    doi: 10.5021/ad.2016.28.2.186

    Figure Lengend Snippet: Interleukin-1β expression decreased in group B and group C compared to that in group A.

    Article Snippet: The primary antibodies were as follows: integrin α6 (diluted 1:150; Santa Cruz Biotechnology Inc., Dallas, TX, USA), neutrophils (diluted 1:80; Abcam, Cambridge, UK), interleukin (IL)-1β (diluted 1:150; Abcam), matrix metalloproteinase (MMP)-2 (diluted 1:300; Abcam), and MMP-9 (diluted 1:250; Abcam).

    Techniques: Expressing

    Hypoxia/ischemia induces microglial activation and the release of pro-inflammatory cytokines and neurotoxic factors. ( A - C ) BV-2 cells were exposed to oxygen-glucose deprivation (OGD), and the mRNA levels of the pro-inflammatory cytokines ( A )tumor necrosis factor (TNF)-α, ( B ) interleukin (IL)-1β and ( C )inducible nitric oxide synthase (iNOS) were evaluated using real-time PCR at 3 hoursafter OGD treatment. ( D - E ) The release of( D ) TNF-α ( E ) and IL-1β into the medium of BV-2 cells was measured by ELISA at 48 hours after OGD. ( F ) The release of NO into the medium of BV-2 cells was measured by the Griess assay. Results are presented as the mean ± SE from three independent experiments. ( G ) Brain homogenates were obtained from the hippocampus 3 daysafter transient global cerebral ischemia/reperfusion (I/R) injury. The supernatant concentrations of IL-1β and TNF-α were tested by ELISA. ( H ) Sections from rat brain taken 3 daysafter I/R injury were incubated with primary antibodies against CD11b and iNOS. Representative immunoreactivities in the hippocampal CA1 region are shown. Photomicrographs are shown at ×400 magnification. ‘Ctrl’ (control) represents the microglial cells that were not subjected to OGD treatment. * P

    Journal: Journal of Neuroinflammation

    Article Title: The microRNA miR-181c controls microglia-mediated neuronal apoptosis by suppressing tumor necrosis factor

    doi: 10.1186/1742-2094-9-211

    Figure Lengend Snippet: Hypoxia/ischemia induces microglial activation and the release of pro-inflammatory cytokines and neurotoxic factors. ( A - C ) BV-2 cells were exposed to oxygen-glucose deprivation (OGD), and the mRNA levels of the pro-inflammatory cytokines ( A )tumor necrosis factor (TNF)-α, ( B ) interleukin (IL)-1β and ( C )inducible nitric oxide synthase (iNOS) were evaluated using real-time PCR at 3 hoursafter OGD treatment. ( D - E ) The release of( D ) TNF-α ( E ) and IL-1β into the medium of BV-2 cells was measured by ELISA at 48 hours after OGD. ( F ) The release of NO into the medium of BV-2 cells was measured by the Griess assay. Results are presented as the mean ± SE from three independent experiments. ( G ) Brain homogenates were obtained from the hippocampus 3 daysafter transient global cerebral ischemia/reperfusion (I/R) injury. The supernatant concentrations of IL-1β and TNF-α were tested by ELISA. ( H ) Sections from rat brain taken 3 daysafter I/R injury were incubated with primary antibodies against CD11b and iNOS. Representative immunoreactivities in the hippocampal CA1 region are shown. Photomicrographs are shown at ×400 magnification. ‘Ctrl’ (control) represents the microglial cells that were not subjected to OGD treatment. * P

    Article Snippet: The following antibodies were used: anti-CD11b (Millipore, Bedford, MA, USA), anti-TNF-α, anti-inducible nitric oxide synthase (iNOS; Abcam, Cambridge, MA, USA), anti-interleukin (IL)-1β (Santa Cruz Biotechnology, CA, USA), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology, Beverly, MA, USA).

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

    Immunohistochemical localization of IL-1α, IL-1β, TNF-α and IL-6 in the median nerves from normal control rats (C) or from reach limbs of rats that performed a HRNF task for 5 weeks. No immunostaining for these cytokines is visible

    Journal:

    Article Title: Increase in inflammatory cytokines in median nerves in a rat model of repetitive motion injury

    doi: 10.1016/j.jneuroim.2005.06.013

    Figure Lengend Snippet: Immunohistochemical localization of IL-1α, IL-1β, TNF-α and IL-6 in the median nerves from normal control rats (C) or from reach limbs of rats that performed a HRNF task for 5 weeks. No immunostaining for these cytokines is visible

    Article Snippet: Sections were incubated over-night at room temperature with one of the following primary antibodies from Chemicon diluted with 4% goat serum in phosphate buffered saline (PBS): anti-IL-1α (Chemicon, #AB1415, 1:250), anti-IL-1β (Chemicon, #AB1832P, 1:250), anti-TNF-α (Chemicon, #AB1837P, 1:300), anti-IL-6 (Chemicon, #AB1833P, 1:250), and anti-IL-10 (Chemicon, #AB1492, 1:250).

    Techniques: Immunohistochemistry, Immunostaining