Journal: Cellular microbiology

Article Title: Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo.

doi: 10.1111/j.1462-5822.2005.00673.x

Figure Lengend Snippet: Fig. 4. TLR5 immunostaining, increased chemokine expression and mucosal neutrophil influx in H7 flagellin stimulated human colon xenografts. A. Sections of unstimulated human colon xenograft were stained with anti-TLR5 (red) (panel A), an isotype control antibody (panel D), or anti- TLR5 antibody preabsorbed with TLR5 peptide (panel G). Panels B, E and H are the same sections stained with Alexa 488-phalloidin for F-actin (green). Panels C, F and I are merged images of panels A and B, D and E and G and H respectively. Original magnification 400 ×. B. EHEC H7 flagellin or PBS was instilled in the lumen of human colon xenografts. Xenografts were harvested 6 h later, epithelial cells were isolated and CXCL8 mRNA expression levels were determined by real-time PCR. Data are mean + SEM from three xenografts. *P < 0.01 compared with PBS control. C. Human intestinal xenografts were injected with EHEC H7 flagellin (panel A) or PBS (panel C), harvested 6 h later and immunostained for CCL20. No immunostaining was seen with an isotype control (not shown). Panels B and D are nuclear counterstaining of panels A and C, respectively, with Hoechst 33258 dye. Original magnification 200 ×. D. Human intestinal xenografts injected with EHEC H7 flagellin (top panel) or PBS (bottom panel), were harvested after 6 h and stained with hematoxylin and eosin. Top panel shows a small cell infiltrate consisting mostly of neutrophils (arrows); original magnification 400 ×.

Article Snippet: Mouse antihuman IL-8 (CXCL8) monoclonal antibody (MAb), biotinylated goat anti-human IL-8 (CXCL8) antibody and mouse anti-human MIP3α (CCL20) MAb were from R and D systems (Minneapolis, MN).

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

Journal: Cellular microbiology

Article Title: Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo.

doi: 10.1111/j.1462-5822.2005.00673.x

Figure Lengend Snippet: Fig. 5. Induction of CXCL8 by H7 flagellin, Stx2 and isogenic mutants of EHEC. A. Caco-2 cells were stimulated with titrated doses of H7 flagellin alone or in combination with titrated concentrations of Stx2 for 6 h after which CXCL8 protein in culture media was assayed by ELISA. Data are mean + SEM of three repeated experiments. B. Caco-2 cells were infected with wild-type EHEC 86–24 (stx+/fliC+), or isogenic mutants of EHEC 86–24 that lack fliC (stx+/fliC–), Stx (stx–/fliC+) or both (stx–/fliC–) at an moi of 100 for 3 h after which cells were washed and cultures were incubated in media containing gen- tamicin for an additional 6 h. CXCL8 protein in culture supernatants was measured by ELISA. Data are mean + SEM of three repeated experiments. *P < 0.05 compared with wild-type EHEC. Motility in soft agar is shown for each mutant.

Article Snippet: Mouse antihuman IL-8 (CXCL8) monoclonal antibody (MAb), biotinylated goat anti-human IL-8 (CXCL8) antibody and mouse anti-human MIP3α (CCL20) MAb were from R and D systems (Minneapolis, MN).

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

Journal: Cellular microbiology

Article Title: Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo.

doi: 10.1111/j.1462-5822.2005.00673.x

Figure Lengend Snippet: Fig. 6. TLR5 distribution and flagellin stimula- tion of polarized Caco-2 cells. A. Polarized Caco-2 cells were immunostained for TLR5 (red) and F-actin (green) and analy- sed by confocal microscopy. Top panels show merged apical, middle and basal sections. Bot- tom panel is a merged X-Z reconstruction. Orig- inal magnification 600 ×. B. Titrated concentrations of EHEC H7 flagellin were added to either the apical or basolateral chambers of polarized Caco-2 cells. Superna- tants from the basolateral chamber were removed after 6 h and CXCL8 was assayed by ELISA. Transepithelial resistance of the mono- layers remained unchanged during the time course of the experiments. Values are mean + SEM of three repeated experiments.

Article Snippet: Mouse antihuman IL-8 (CXCL8) monoclonal antibody (MAb), biotinylated goat anti-human IL-8 (CXCL8) antibody and mouse anti-human MIP3α (CCL20) MAb were from R and D systems (Minneapolis, MN).

Techniques: Confocal Microscopy, Enzyme-linked Immunosorbent Assay