Journal:

Article Title: Brucella abortus Rough Mutants Induce Macrophage Oncosis That Requires Bacterial Protein Synthesis and Direct Interaction with the Macrophage

doi: 10.1128/IAI.74.5.2667-2675.2006

Figure Lengend Snippet: Roles of TNF-α and nitric oxide in macrophage cell death induced by CA180 infection. J774.A1 macrophages were infected with CA180 at an MOI of 100 and treated with L-NAME and goat anti-mouse TNF-α. The viability of the macrophages was determined by detecting LDH release at 24 h p.i. The results are from three independent experiments.

Article Snippet: Goat anti-murine tumor necrosis factor alpha (TNF-α) and goat immunoglobulin G (IgG) control were purchased from PeproTech Inc. (Rocky Hill, NJ).

Techniques: Infection

Journal: Life

Article Title: Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα

doi: 10.3390/life12101527

Figure Lengend Snippet: Antibodies and dyes used.

Article Snippet: Goat anti-human TNFα , Peprotech, Princeton, NJ, USA , 500-P31AG , 0.5 mg/mL , 1:50.

Techniques: Concentration Assay

Journal: Life

Article Title: Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα

doi: 10.3390/life12101527

Figure Lengend Snippet: ( A ) Native images of synoviocyte cell lines, K4IM and HSE (100 × magnification, scale bar = 100 µm). ( B ) Representative overlay images of live-dead staining of the synoviocytes after 24 h stimulation with 1 µg/mL NP, 10 ng/mL TNFα or the combination. Fluorescein diacetate (green: vital cells) and propidium iodide (red: dead cells) have been used for staining. ( C ) Comparison of the vitality of unstimulated K4IM and HSE after 24 h, analyzed using ImageJ. n = 4. ( D , E ) Comparison of the vitality of stimulated K4IM and HSE, respectively, after 24 h stimulation. Number of living cells in relation to total cells (100%) measured in live-dead staining images of the synoviocytes, with control normalized to 100 as reference. D (n = 3), E (n = 4). ( F ) Cytotoxicity was assessed (CellTiter 96® AQueous One Solution Cell Proliferation Assay) on the hEC after 24 h stimulation with 1 µg/mL NP and 10% DMSO as positive control. n = 3. Control has been normalized to 100. Mean and the standard deviation (SD) are depicted. One sample t -test with significance in relation to control (*). Repeated Measures one-way ANOVA using Tukey’s multiple comparisons (#). */# = p ≤ 0.05; ** = p ≤ 0.01.

Article Snippet: Goat anti-human TNFα , Peprotech, Princeton, NJ, USA , 500-P31AG , 0.5 mg/mL , 1:50.

Techniques: Staining, Comparison, Control, Proliferation Assay, Positive Control, Standard Deviation

Journal: Life

Article Title: Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα

doi: 10.3390/life12101527

Figure Lengend Snippet: Comparison of the relative cell proliferation in ( A ) unstimulated (n = 8), ( B – D ) stimulated K4IM and HSE cell lines (n = 4, 1 µg/mL NP, 10 ng/mL TNFα or the combination) as well as ( E , F ) stimulated human endothelial cells (hEC) assessed by CyQUANT® NF Cell Proliferation Assay. E (n = 4), F (n = 5). Mean with standard deviation (SD). Control has been normalized to 100. One sample t -test with significance in relation to control (*). Repeated Measures one-way ANOVA using Tukey’s multiple comparisons (#). * = p ≤ 0.05; **/## = p ≤ 0.01; *** = p ≤ 0.001, **** = p ≤ 0.0001.

Article Snippet: Goat anti-human TNFα , Peprotech, Princeton, NJ, USA , 500-P31AG , 0.5 mg/mL , 1:50.

Techniques: Comparison, CyQUANT Assay, Proliferation Assay, Standard Deviation, Control

Journal: Life

Article Title: Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα

doi: 10.3390/life12101527

Figure Lengend Snippet: ( A ) Representative overlay of immunofluorescence images of Ki67-stained unstimulated and stimulated K4IM and HSE fibroblasts (200× magnification, 1 µg/mL NP, 10 ng/mL TNFα or the combination). Red (Cy3) = Ki67, blue (DAPI) = cell nuclei. n = 3. ( B – D ) Graphic representation of the ratio between proliferation marker Ki67-positive cells and total cells (DAPI) in percentage, mean with standard deviation (SD). One sample t-test with significance in relation to control (*). Repeated Measures one-way ANOVA using Tukey’s multiple comparisons. * = p ≤ 0.05. Scale bar = 100 µm.

Article Snippet: Goat anti-human TNFα , Peprotech, Princeton, NJ, USA , 500-P31AG , 0.5 mg/mL , 1:50.

Techniques: Immunofluorescence, Staining, Marker, Standard Deviation, Control

Journal: Life

Article Title: Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα

doi: 10.3390/life12101527

Figure Lengend Snippet: Graphic representation of relative gene expression of complement factors (C5aR1, CD46, CD55 and CD59) and cytokines (TNFα and IL-6) in non-stimulated synoviocyte cell lines K4IM and HSE ( A ) as well as K4IM ( B ) or HSE ( C ) when stimulated separately for 24 h with 1 µg/mL NP, 10 ng/mL TNFα or the combination of both. n = 3–5. Mean with standard deviation (SD). Control has been normalized to 100. One sample t -test with significance in relation to control (*). Repeated Measures one-way ANOVA using Tukey’s multiple comparisons. * = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.001.

Article Snippet: Goat anti-human TNFα , Peprotech, Princeton, NJ, USA , 500-P31AG , 0.5 mg/mL , 1:50.

Techniques: Gene Expression, Standard Deviation, Control

Journal: Life

Article Title: Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα

doi: 10.3390/life12101527

Figure Lengend Snippet: ( A ) Representative images of K4IM cell line after 24 h stimulation (630× magnification, 1 µg/mL NP, 10 ng/mL TNFα or the combination), immunolabeled with C5aR1 and CD55 specific antibodies and negative control of the staining. Green (Alexa 488) = C5aR1, red (Cy3) = CD55, blue (DAPI) = cell nuclei. Scale bar = 25 µm. Graphic representation of relative ( B ) C5aR1 and ( C ) CD55 protein fluorescence intensity, n = 4, mean with standard deviation (SD). Control has been normalized to 100. One sample t-test with significance in relation to control. Repeated Measures one-way ANOVA using Tukey’s multiple comparisons.

Article Snippet: Goat anti-human TNFα , Peprotech, Princeton, NJ, USA , 500-P31AG , 0.5 mg/mL , 1:50.

Techniques: Immunolabeling, Negative Control, Staining, Fluorescence, Standard Deviation, Control

Journal: Life

Article Title: Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα

doi: 10.3390/life12101527

Figure Lengend Snippet: ( A ) Representative images of HSE cell line after 24 h stimulation (630× magnification, 1 µg/mL NP, 10 ng/mL TNFα or the combination), immunolabeled with C5aR1 and CD55 specific antibodies and negative control of the staining. Green (Alexa 488) = C5aR1, red (Cy3) = CD55, blue (DAPI) = cell nuclei. Scale bar = 25 µm. Graphic representation of relative ( B ) C5aR1 and ( C ) CD55 protein fluorescence intensity, n = 4, mean with standard deviation (SD). Control has been normalized to 100. One sample t-test with significance in relation to control. Repeated Measures one-way ANOVA using Tukey’s multiple comparisons.

Article Snippet: Goat anti-human TNFα , Peprotech, Princeton, NJ, USA , 500-P31AG , 0.5 mg/mL , 1:50.

Techniques: Immunolabeling, Negative Control, Staining, Fluorescence, Standard Deviation, Control

Journal: Life

Article Title: Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα

doi: 10.3390/life12101527

Figure Lengend Snippet: ( A ) Representative images of K4IM cell line after 24 h stimulation (630× magnification, 1 µg/mL NP, 10 ng/mL TNFα or the combination), immunolabeled with TNFα and IL-6 specific antibodies and negative control of the staining. Green (Alexa 488) = TNFα, red (Alexa 555) = IL-6, blue (DAPI) = cell nuclei. Scale bar = 25 µm. Graphic representation of relative ( B ) TNFα and ( C ) IL-6 protein fluorescence intensity, n = 3, mean with standard deviation (SD). Control has been normalized to 100. One sample t-test with significance in relation to control. Repeated Measures one-way ANOVA using Tukey’s multiple comparisons.

Article Snippet: Goat anti-human TNFα , Peprotech, Princeton, NJ, USA , 500-P31AG , 0.5 mg/mL , 1:50.

Techniques: Immunolabeling, Negative Control, Staining, Fluorescence, Standard Deviation, Control

Journal: Life

Article Title: Complement Regulation in Immortalized Fibroblast-like Synoviocytes and Primary Human Endothelial Cells in Response to SARS-CoV-2 Nucleocapsid Protein and Pro-Inflammatory Cytokine TNFα

doi: 10.3390/life12101527

Figure Lengend Snippet: ( A ) Representative images of HSE cell line after 24 h stimulation (630× magnification, 1 µg/mL NP, 10 ng/mL TNFα or the combination), immunolabeled with TNFα and IL-6 specific antibodies and negative control of the staining. Green (Alexa 488) = TNFα, red (Alexa 555) = IL-6, blue (DAPI) = cell nuclei. Scale bar = 25 µm. Graphic representation of relative ( B ) TNFα and ( C ) IL-6 protein fluorescence intensity, n = 3, mean with standard deviation (SD). Control has been normalized to 100. One sample t -test with significance in relation to control (**). Repeated Measures one-way ANOVA using Tukey’s multiple comparisons. ** = p ≤ 0.01.

Article Snippet: Goat anti-human TNFα , Peprotech, Princeton, NJ, USA , 500-P31AG , 0.5 mg/mL , 1:50.

Techniques: Immunolabeling, Negative Control, Staining, Fluorescence, Standard Deviation, Control

Journal: PLoS ONE

Article Title: The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0012127

Figure Lengend Snippet: HCP and HcpA induced release of IL-8 and TNF-α but not IL-2, IL-6, and IL-10 (A). * P <0.0001 and HcpA ** P <0.001. High levels of IL-8 and TNF-α released from HT-29, T84, and HeLa cells in the presence of HCP and HcpA (B) and (C). HCP and HcpA induced similar levels of IL-8 (1658.3 pg/ml from HT-29; 1558.33 pg/ml from T84; and 1423 pg/ml from HeLa cells) (C). Both proteins induced comparable levels of TNF-α release from HT-29, T84, and HeLa cells [HCP (1346.6, 1258.3, and 1205.3 pg/ml) and HcpA (1272.6, 1205.3, and 1223.3 pg/ml)], respectively (B). Polarized intestinal and non-intestinal cells were incubated alone with DMEM as a negative control.

Article Snippet: A sandwich ELISA using 96-well plates coated overnight at 4°C, and using a concentration of 10 µg/ml goat anti-human antibodies of IL-8 and TNF-α (BD Biosciences Pharmingen, San Diego, CA, USA) was performed as previously described .

Techniques: Incubation, Negative Control

Journal: PLoS ONE

Article Title: The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0012127

Figure Lengend Snippet: An ELISA-based assay demonstrating the induction of TNF-α in polarized HT-29 cells in response to different concentrations of purified HCP and HcpA (A). Western blot showing the kinetics of secreted proteins and cell lysates produced after the incubation of polarized HT-29 cells with HCP and HcpA (B). Both proteins stimulate the production of TNF-α in HT-29 cells in a dose-dependent manner. Purified HCP (100 pg/ml) and HcpA (100 pg/ml) were incubated with polarized HT-29 cells at different intervals (C). The supernatant recovered from the basolateral surface showed a significant time-dependent increase of TNF-α as shown by Western blotting (D). Detection of actin was used as a protein loading control.

Article Snippet: A sandwich ELISA using 96-well plates coated overnight at 4°C, and using a concentration of 10 µg/ml goat anti-human antibodies of IL-8 and TNF-α (BD Biosciences Pharmingen, San Diego, CA, USA) was performed as previously described .

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Western Blot, Produced, Incubation

Journal: PLoS ONE

Article Title: The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0012127

Figure Lengend Snippet: Purified HCP or HcpA protein (100 pg/ml) were pre-incubated with anti-HCP antibodies in DMEM low glucose medium before addition to the apical surface of HT-29 cells. A dose-dependent inhibition for IL-8 and TNF-α from HT-29 cells was obtained: between 95–98% at the 1∶200 dilution of the antibody (A and B). * P <0.0002.

Article Snippet: A sandwich ELISA using 96-well plates coated overnight at 4°C, and using a concentration of 10 µg/ml goat anti-human antibodies of IL-8 and TNF-α (BD Biosciences Pharmingen, San Diego, CA, USA) was performed as previously described .

Techniques: Purification, Incubation, Inhibition

Journal: PLoS ONE

Article Title: The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0012127

Figure Lengend Snippet: The highest level of IL-8 and TNF-α release was observed after growth of EDL933 in Minca (A and B) (* P <0.0001). The hcpA mutant was reduced for IL-8 and TNF-α induction compared to the wild-type strain (** P <0.0012). Comparison of IL-8 and TNF-α induction by EDL933, EDL933Δ hcpA , EDL933Δ fliC and EDL933Δ hcpA /Δ fliC previously cultured in Minca (C). Asterisks and ampersands indicate that a value is significantly different from the value for the mutant in hcpA , fliC and the double mutant hcpA / fliC cultured in Minca, ** P <0.0012, && P <0.005, *** P <0.001, and **** P <0.0001.

Article Snippet: A sandwich ELISA using 96-well plates coated overnight at 4°C, and using a concentration of 10 µg/ml goat anti-human antibodies of IL-8 and TNF-α (BD Biosciences Pharmingen, San Diego, CA, USA) was performed as previously described .

Techniques: Mutagenesis, Cell Culture

Journal:

Article Title: Afa/Dr Diffusely Adhering Escherichia coli Infection in T84 Cell Monolayers Induces Increased Neutrophil Transepithelial Migration, Which in Turn Promotes Cytokine-Dependent Upregulation of Decay-Accelerating Factor (CD55), the Receptor for Afa/Dr Adhesins

doi: 10.1128/IAI.71.4.1774-1783.2003

Figure Lengend Snippet: TNF-α and IL-1β production during the time-course of f-MLP-induced PMNL transepithelial migration (TM) in T84 cells. Cytokine production was measured by ELISAs. f-MLP-treated T84 cells or T84 cell monolayers infected for 1 or 3 h with strain C1845 served as negative controls. T84 cells treated with LPS or phorbol myristate acetate (PMA) served as positive controls (n = 4).

Article Snippet: Phosphatase-conjugated goat anti-TNF-α and anti-IL-1β polyclonal antibodies were from Sandoz Pharmaceutical (Rueil-Malmaison, France).

Techniques: Migration, Infection

Journal:

Article Title: Afa/Dr Diffusely Adhering Escherichia coli Infection in T84 Cell Monolayers Induces Increased Neutrophil Transepithelial Migration, Which in Turn Promotes Cytokine-Dependent Upregulation of Decay-Accelerating Factor (CD55), the Receptor for Afa/Dr Adhesins

doi: 10.1128/IAI.71.4.1774-1783.2003

Figure Lengend Snippet: DAF upregulation observed in T84 monolayers during f-MLP-induced PMNL transmigration is linked to TNF-α and IL-1β production. DAF expression in T84 cells was analyzed by Western blotting after incubation of cells with or without MAbs against TNF-α and/or IL-1β added to the lower reservoir and subsequent PMNL transepithelial migration for 6 h. DAF upregulation observed after f-MLP-induced PMNL transepithelial migration or incubation of epithelial cells with TNF-α and/or IL-1β was decreased after treatment of T84 monolayers with either the anti-IL-1β or the anti-TNF-α MAb alone and was further decreased when the two MAbs were combined. In control (Ctl) cells, exposure to PI-PLC, which breaks down the GPI anchor of DAF, caused DAF to disappear. Micrographs are representative of three to four experiments.

Article Snippet: Phosphatase-conjugated goat anti-TNF-α and anti-IL-1β polyclonal antibodies were from Sandoz Pharmaceutical (Rueil-Malmaison, France).

Techniques: Transmigration Assay, Expressing, Western Blot, Incubation, Migration, Control

Journal:

Article Title: The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood-brain barrier disruption: Implications for multiple sclerosis treatment

doi:

Figure Lengend Snippet: Comparison of the histology of spinal cords in vehicle- and SR 57746A-treated rats. Typical histopathological slices from MBP68–82-immunized rats treated with vehicle, showed several inflammatory infiltrates (A), localized around vessels (B) in the white matter in the lumbar part of spinal cords. In contrast, sections from 10 mg/kg SR 57746A-treated rats immunized with MBP68–82 were similar to those from normal rats. TNF-α (C) and IFN-γ (D) were produced only by perivascular leukocytes (arrows) from rats with EAE.

Article Snippet: In parallel, series of adjacent vibratome sections were incubated sequentially with goat anti-rat TNF-α and IFN-γ antibodies (Genzyme), horseradish peroxidase-conjugated anti-goat antibody (Dako), and diaminobenzidine chromogen.

Techniques: Produced

Journal:

Article Title: The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood-brain barrier disruption: Implications for multiple sclerosis treatment

doi:

Figure Lengend Snippet: SR 57746A inhibited the expression of the proinflammatory cytokines TNF-α and IFN-γ. Inflammatory cells were isolated on day 10 postimmunization from the spinal cord of 25 normal rats, 25 rats immunized with MBP68–82, and 25 rats immunized with MBP and treated daily with SR 57746A at a 10 mg/kg dose. Quantification of mRNA was performed by quantitative PCR. The level of mRNA in each sample was normalized for their β-2 microglobulin content and plotted as a percentage of β-2 microglobulin mRNA content. (Insets) Electrophoresed 30-cycle PCR products stained with ethidium bromide.

Article Snippet: In parallel, series of adjacent vibratome sections were incubated sequentially with goat anti-rat TNF-α and IFN-γ antibodies (Genzyme), horseradish peroxidase-conjugated anti-goat antibody (Dako), and diaminobenzidine chromogen.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Staining

Journal:

Article Title: The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood-brain barrier disruption: Implications for multiple sclerosis treatment

doi:

Figure Lengend Snippet: SR 57746A did not prevent ex vivo release of Th1 and Th2 cytokines by MBP68–82-specific PLNC. PLNC were harvested 21 days after EAE induction and cultured for 96 h with 111 μg/ml MBP68–82 or 1 μg/ml Con A. Supernatants were evaluated for the presence of IFN-γ and IL-10 by ELISA. The dotted lines indicate IFN-γ and IL-10 levels in the supernatant of PLNC cultured without stimulators.

Article Snippet: In parallel, series of adjacent vibratome sections were incubated sequentially with goat anti-rat TNF-α and IFN-γ antibodies (Genzyme), horseradish peroxidase-conjugated anti-goat antibody (Dako), and diaminobenzidine chromogen.

Techniques: Ex Vivo, Cell Culture, Enzyme-linked Immunosorbent Assay