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