Journal:

Article Title: Cooperative Interactions between Flagellin and SopE2 in the Epithelial Interleukin-8 Response to Salmonella enterica Serovar Typhimurium Infection

doi: 10.1128/IAI.72.9.5052-5062.2004

Figure Lengend Snippet: (A) Salmonella-induced IL-8 secretion by T84 cells is regulated by SopE2. Polarized T84 monolayers were left uninfected (control) or infected apically for 1 h with wild-type (F98), SopE2-deficient (SE2.1), or SopE2-transcomplemented (SE2.1/pSE2) strains of Salmonella. IL-8 in the basolateral culture medium was assayed by ELISA 5 h later. The results represent the means and standard deviations from eight separate experiments, with each experiment consisting of triplicate infections. P values are indicated above the corresponding bars. The Western blot in the upper part of the figure indicates the expression of SopE2 in lysates of the various bacterial strains. The amount of SE2.1/pSE2 lysate loaded was approximately 1/10 that of the F98 lysate. The number to the left of the blot indicates the position of the 29-kDa molecular mass standard. (B) IL-8 production by T84 cells in response to Salmonella infection in nonpolarized T84 cells. Nonpolarized T84 monolayers were left uninfected (control) or infected for 1 h with wild-type (F98), SopE2-deficient (SE2.1), or SopE2-transcomplemented (SE2.1/pSE2) strains of Salmonella. IL-8 in the supernatant was assayed by ELISA 5 h later. The results indicate the means and standard deviations of triplicate wells and are representative of two separate experiments. P values are indicated above the corresponding bars. (C) The effect of SopE2 on IL-8 secretion requires GEF activity. Polarized T84 monolayers were left uninfected (control) or infected apically for 1 h with Salmonella strains F98 (wild-type), SE2.1 (SopE2-deficient), SE2.1/pSE2 (SE2.1 expressing wild-type SopE2), or SE2.1/pSE2* (SE2.1 expressing mutant SopE2). IL-8 in the basolateral culture medium was assayed by ELISA 5 h later. The results represent the means and standard deviations of triplicate wells and are representative of two separate experiments. The P value for the difference between SE2.1/pSE2- and SE2.1/pSE2*-infected cells is indicated. The Western blot in the upper part of the figure indicates the expression of SopE2 in bacterial supernatants of SE2.1, SE2.1/pSE2, and SE2.1/pSE2*. The number to the left of the blot indicates the position of the 29-kDa molecular mass standard.

Article Snippet: IL-8 in the basolateral medium was measured by ELISA, using anti-human IL-8 antibodies from Pharmingen (San Diego, Calif.).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Activity Assay, Mutagenesis

Journal:

Article Title: Cooperative Interactions between Flagellin and SopE2 in the Epithelial Interleukin-8 Response to Salmonella enterica Serovar Typhimurium Infection

doi: 10.1128/IAI.72.9.5052-5062.2004

Figure Lengend Snippet: (A) Invasiveness of the Salmonella strains for T84 cells is regulated by SopE2. Polarized T84 monolayers were infected apically for 1 h with the wild-type (F98) or SopE2-deficient (SE2.1), SopE2-transcomplemented (SE2.1/pSE2), or SopE2 (SE2.1/pSE2*) mutant strains. The number of internalized bacteria was determined by gentamicin protection assay and expressed as a percentage of the number of infecting bacteria. The results indicate the means and standard deviations from triplicate wells and are representative of two separate experiments. (B) Effect of cytochalasin D on cell invasion and IL-8 induction by Salmonella. Left histogram, polarized T84 monolayers were infected apically for 1 h with the SopE2-transcomplemented strain SE2.1/pSE2 in the presence (+) or absence (−) of 15 μg of cytochalasin D/ml. The number of internalized bacteria was determined by gentamicin protection assay and expressed as a percentage of the number of infecting bacteria. The results indicate the means and standard deviations for triplicate wells and are representative of two separate experiments. Right histogram, polarized T84 monolayers were left uninfected or were infected apically for 1 h with the SopE2-transcomplemented strain SE2.1/pSE2 in the presence (+) or absence (−) of 15 μg of cytochalasin D/ml. IL-8 in the basolateral culture medium was assayed by ELISA 5 h later. The results indicate the means and standard deviations for triplicate wells and are representative of two separate experiments.

Article Snippet: IL-8 in the basolateral medium was measured by ELISA, using anti-human IL-8 antibodies from Pharmingen (San Diego, Calif.).

Techniques: Infection, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Cooperative Interactions between Flagellin and SopE2 in the Epithelial Interleukin-8 Response to Salmonella enterica Serovar Typhimurium Infection

doi: 10.1128/IAI.72.9.5052-5062.2004

Figure Lengend Snippet: (A) The effect of SopE2 on cell invasion in flagellin-deficient and wild-type strains. T84 cells were infected with SopE2-overexpressing or parental strains of SL3201 and SL3201 ΔfljB ΔfliC (indicated as BC−), with brief centrifugation (500 × g for 10 min) applied where noted. The number of internalized bacteria was determined by gentamicin protection assay and expressed as a percentage of the number of infecting bacteria. The results indicate the means and standard deviations for triplicate wells and are representative of two separate experiments. P values are indicated above the corresponding bars. The Western blot in the upper part of the figure indicates the expression of SopE2 in bacterial lysates of the various strains. The number to the left of the blot indicates the position of the 29-kDa molecular mass standard. (B) The role of SopE2 and flagellin in IL-8 secretion by nonpolarized T84 cells. Cells were left uninfected (control) or infected with SopE2-overexpressing or parental strains of SL3201 and SL3201 ΔfljB ΔfliC (indicated as BC−), with brief centrifugation (500 × g for 10 min) applied where noted. IL-8 in the supernatant medium was assayed by ELISA 5 h later. The results shown indicate the means and standard deviations from two separate experiments, each consisting of triplicate infections. P values are indicated above the corresponding bars.

Article Snippet: IL-8 in the basolateral medium was measured by ELISA, using anti-human IL-8 antibodies from Pharmingen (San Diego, Calif.).

Techniques: Infection, Centrifugation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Cooperative Interactions between Flagellin and SopE2 in the Epithelial Interleukin-8 Response to Salmonella enterica Serovar Typhimurium Infection

doi: 10.1128/IAI.72.9.5052-5062.2004

Figure Lengend Snippet: A model of cooperative interactions between SopE2 and flagellin in the induction of IL-8. Flagellin is required for the optimal translocation of SopE2. Signals activated by SopE2 and flagellin act together to increase production of IL-8.

Article Snippet: IL-8 in the basolateral medium was measured by ELISA, using anti-human IL-8 antibodies from Pharmingen (San Diego, Calif.).

Techniques: Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Immunomodulatory Activity of Dietary Fiber: Arabinoxylan and Mixed-Linked Beta-Glucan Isolated from Barley Show Modest Activities in Vitro

doi: 10.3390/ijms12010570

Figure Lengend Snippet: IL-8 secretion from Caco-2 ( A ) and HT-29 ( B ) cells in response to treatment with fiber fractions extracted from the barley variety Tyra, zymosan and PMII (all 1 mg/mL). Cells were incubated with fiber of the respective fiber fractions in cell culture medium for 24 hours before IL-8 secretion was measured. Each bar represents the average ± SD of one representative experiment from a total of three independent experiments. * p < 0.05.

Article Snippet: Monoclonal mouse anti-human IL-8 antibody (BD Bioscience Pharmingen, San Diego, CA) suspended in coating buffer (0.1 M Carbonate/Bicarbonate buffer pH 9.6) was added to MaxiSorp TM ELISA plates (Nunc, Roskilde, Denmark) and incubated over night at 4 °C.

Techniques: Incubation, Cell Culture