Journal:

Article Title: Selective Inhibition of the C5a Chemotactic Cofactor Function of the Vitamin D Binding Protein by 1,25(OH) 2 Vitamin D 3

doi: 10.1016/j.molimm.2005.07.023

Figure Lengend Snippet: Effect of 1,25(OH)2D3 on chemotaxis to C5a, LTB4, formyl peptide and CXCL8. Purified DBP (100 nM) was treated for 5 min at 37°C with 10 nM 1,25(OH)2D3. Neutrophils (4 x 106/ml) were then preincubated for 40 min at 37°C with either DBP plus 1,25(OH)2D3 or buffer. Neutrophil movement then was assayed for 30 min at 37°C. Numbers represent mean ± SEM of 3 experiments using cells from different donors.

Article Snippet: Recombinant CXCL8 (IL-8) was obtained from R&D Systems (Minneapolis, MN).

Techniques: Chemotaxis Assay, Purification

Journal: Frontiers in Microbiology

Article Title: Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

doi: 10.3389/fmicb.2019.00879

Figure Lengend Snippet: Production of C. difficile spores, toxins, and host responses to infection. (A) Colony counts of spores recovered after heat treatment, and total cells in the cell-associated C. difficile fraction (infected cell lysates) in the 2D epithelial model. Data shown are the mean of three independent experiments and error bars indicate SD, * p < 0.05 as determined by two-way ANOVA. (B) ELISA for C. difficile toxins A and B shows increased toxin production after extended infection. Toxins were measured from medium obtained from the apical compartment containing uninfected cell layers incubated for 24 or 48 h (Control) or cells infected with C. difficile for 3, 6, 24, or 48 h. Data shown are the mean of three independent experiments and error bars indicate SD, * p < 0.05 as determined by two-way ANOVA. Gray line represents the sensitivity of the test at 0.5 ng/ml. (C) ELISA for human IL-8 indicates increased IL-8 production at 24 and 48 h p.i. IL-8 was measured in medium obtained from the basolateral compartment containing uninfected cell layers incubated for 3, 6, 24, or 48 h (Control) or cells infected with C. difficile for 3–48 h. Gray line represents the limit of detection at 32 pg/ml. Data shown are the mean of three independent experiments and error bars indicate SD, * p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001 as determined by one-way ANOVA with Tukey’s test for multiple comparison.

Article Snippet: IL-8 production was also determined by analysis of basolateral supernatants from the VDC using a human IL-8 ELISA kit (R&D systems, Minneapolis, MN, United States) following the manufacturer’s instruction.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation, Control, Comparison

Journal: Frontiers in Microbiology

Article Title: Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

doi: 10.3389/fmicb.2019.00879

Figure Lengend Snippet: C. difficile spores and toxin production, and host response to infection in the 3D-VDC model. (A) Colony counts of spores and total cells in the host cell-associated C. difficile fraction (infected cell lysates). Data shown are the mean of three independent experiments and error bars indicate SD, ns, not significant as determined by two-way ANOVA. (B) Toxin A and B levels from apical compartment supernatants as determined by ELISA in the 3D model. Data shown are the mean of three independent experiments and error bars indicate SD, ns, not significant as determined by two-way ANOVA. Gray line represents the sensitivity of the test at 0.5 ng/ml. (C) Human IL-8 levels in supernatants from the basolateral compartments with uninfected cells incubated for 24 h or with cells infected with C. difficile for 3 and 24 h, as determined by ELISA. Gray line represents the limit of detection at 32 pg/ml. Data shown are the mean of three independent experiments and error bars indicate SD, ** p < 0.01, as determined by the one-way ANOVA with Tukey’s test for multiple comparison.

Article Snippet: IL-8 production was also determined by analysis of basolateral supernatants from the VDC using a human IL-8 ELISA kit (R&D systems, Minneapolis, MN, United States) following the manufacturer’s instruction.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation, Comparison

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Chemokine induction in Hct-8 cells infected with LEE-negative and LEE-positive STEC strains. Hct-8 cells were infected with the indicated STEC strains (Table ​(Table1).1). (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, culture supernatants were collected and assayed for IL-8 protein by ELISA as described in Materials and Methods. Data are shown as the means ± standard errors of the means (SEM) from two experiments. The significance of differences between IL-8 secretion by infected versus uninfected cells is indicated as follows: **, P < 0.001; *, P < 0.05.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Stimulation of Hct-8 cells by strain EDL933 derivatives. Cells were infected with strains 98NK2, EDL933, EDL933 expressing wild-type or defective copies of the ler gene carried on a multicopy plasmid (designated EDL933 Ler and 95SF2 Ler, respectively), and EDL933 with a deletion mutation in eae (Δeae) (all described previously [23]). (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. *, significant difference relative to control cells (P < 0.001).

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Infection, Expressing, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells treated with purified Stx1 and Stx2. Hct-8 cells were treated with purified Stx1 or Stx2 at the indicated concentrations or with STEC strain 98NK2 or EDL933. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. Significant differences relative to untreated control cells are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Purification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells treated with H21 flagellin. Hct-8 cells were stimulated with H21 flagellin at the indicated concentrations or with STEC strains 98NK2 and EDL933. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. Significant differences relative to untreated control cells are indicated as follows: *, P < 0.001.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells infected with strain 98NK2 stx2 and fliC deletion mutants. Hct-8 cells were stimulated with strain 98NK2, 98NK2Δstx2, or 98NK2ΔfliC. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. *, significant difference relative to strain 98NK2-treated cells (P < 0.01).

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells treated with flagellin from strains 95HE4 and 95ZG1. Hct-8 cells were stimulated with strain 95HE4 (H7) or 95ZG1 flagellin at the indicated concentrations or with STEC strain 95HE4, 95ZG1, or 98NK2. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. Significant differences relative to untreated control cells are indicated as follows: *, P < 0.001.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay