Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: Cytotoxic necrotizing factor 1 (CNF1), but not CNF1-C866S, induces phenotypic maturation of immature monocyte-derived dendritic cells (moDCs). ( A – C ) Immature moDCs were cultured for 24 and 48 h without toxin (Ctrl) or with different concentrations of CNF1 or CNF1-C866S before the expression of phenotypic maturation markers was measured by flow cytometry. ( A ) Representative pseudocolor dot plot showing surface expression of CD83 and CD86. ( B ) Graphs summarizing the mean percentages of CD83+CD86+ cells from 3 independent experiments, each using moDCs derived from different donors ( n = 3). Error bars represent SEM and * denotes a significant difference ( p < 0.05) compared to immature moDCs cultured without toxin using a two-way ANOVA with Sidak’s multiple comparisons test. ( C ) Histogram plots showing the surface expression of HLA-DR on moDCs cultured without toxin (green), with CNF1 (red) or with CNF1-C866S (blue). Arrows indicate increasing concentrations (0.004, 0.02 and 0.1 nM) of CNF1 and CNF1-C866S. The plots are representative of 3 independent experiments each using moDCs derived from different donors ( n = 3).

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Derivative Assay, Cell Culture, Expressing, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: CNF1-induced moDC maturation is not driven by endotoxin or other putative contaminants in the toxin preparation. ( A ) Culture media alone (Ctrl) or culture media containing 0.1 nM CNF1, 50 ng/mL lipopolysaccharide (LPS) or 50 ng/mL tumor necrosis factor alpha (TNFα) was pre-incubated for 30 min at room temperature with 5 µg/mL Polymyxin B (PMB) or vehicle (endotoxin-free water). Then, immature moDCs were added to each media sample and cultured for 24 h before the expression of CD83 and CD86 was determined by flow cytometry. Shown is the mean percentage of CD83+CD86+ moDCs from 4 different donors ( n = 4) analyzed in 3 independent experiments. Error bars represent SEM and * denotes a significant difference ( p < 0.05) between vehicle- and PMB-treated cells using a paired Student’s t -test. ( B ) CNF1 diluted in moDC medium was incubated with (2) uncoated Protein A agarose beads or (3) Protein A agarose beads that had been pre-coated with a monoclonal anti-CNF1 (αCNF1) antibody or (4) an isotype control (IC) antibody. As a negative control, (1) moDC medium without CNF1 was in parallel incubated with uncoated Protein A agarose beads. After removal of the beads, the level of CNF1 in the supernatants was analyzed by western blotting. ( C ) Immature moDCs were cultured for 24 h with the supernatants (1–4) obtained in ( B ) and the expression of CD83 and CD86 analyzed by flow cytometry.

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Incubation, Cell Culture, Expressing, Flow Cytometry, Negative Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: CNF1 does not induce apoptosis or cell death in moDCs. ( A , B ) Immature moDCs were cultured for 24 and 48 h without toxin (Ctrl) or with 0.1 nM CNF1 before the percentages of Annexin V- and propidium iodide (PI)-positive cells were determined by flow cytometry. moDCs cultured in medium without fetal bovine serum and cytokines (Starved) were included in the analysis as a positive control for induction of apoptotic cell death. ( A ) Histogram summarizing the mean percentages of Annexin V+ and/or PI+ cells from 3 independent experiments, each using moDCs derived from different donors ( n = 3). Error bars represent SEM and * denotes a significant difference ( p < 0.05) compared to Ctrl moDCs at the same timepoint using a two-tailed paired Student’s t -test. ( B ) A representative pseudocolor dot plot of an Annexin V and PI staining after 48 h of culture.

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Cell Culture, Flow Cytometry, Positive Control, Derivative Assay, Two Tailed Test, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Bacterial Toxin CNF1 Induces Activation and Maturation of Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms19051408

Figure Lengend Snippet: CNF1 induces functional maturation of moDCs. ( A , B ) Immature moDCs were cultured for 24 h without toxin (Ctrl), with 0.1 nM CNF1 or with 0.1 nM CNF1-C866S before the concentrations of ( A ) interleukin-6 (IL-6) and ( B ) TNFα in the cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). ( A , B ) Shown is the mean cytokine concentration + SEM in cell culture supernatants from 3 independent experiments each using moDCs derived from different donors ( n = 3). ( C ) Immature moDCs were cultured for 48 h with 0.1 nM CNF1, 0.1 nM CNF1-C866S, 50 ng/mL LPS or without toxin (Ctrl). The moDCs were subsequently washed and different numbers cultured with allogenic naïve CD4+ T cells for 4 days. Then [ 3 H]-thymidine was added, and the cells cultured for 24 h further, before determination of [ 3 H]-thymidine incorporation. Shown are the mean counts per minute (CPM) from 3 independent experiments. Each independent experiment was performed using moDCs and allogenic naïve CD4+ T cells derived from different donors (total of 6 donors). Error bars represent SEM and * denotes a significant difference ( p < 0.05) compared to CD4+ T cells cultured with the same number of Ctrl moDCs using a two-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Protein A-agarose macrobeads (Sigma-Aldrich) were coated overnight at 4 °C with an anti-CNF1 antibody (Clone NG8, Hycult Biotech, Plymouth Meeting, PA, USA), an IgG2a isotype control antibody (R&D systems, Minneapolis, MN, USA) or PBS as control.

Techniques: Functional Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Derivative Assay

Journal:

Article Title: A Functional Virulence Complex Composed of Gingipains, Adhesins, and Lipopolysaccharide Shows High Affinity to Host Cells and Matrix Proteins and Escapes Recognition by Host Immune Systems

doi: 10.1128/IAI.73.2.883-893.2005

Figure Lengend Snippet: Modification of the cell-associated gingipain complex by attachment of LPS. (A) The purified complex was applied to native polyacrylamide gels. The proteins separated on the gel were then transferred to nitrocellulose membranes and immunostained with the monoclonal antibody to lipid A of LPS. (B) The complex was subjected to SDS-PAGE and stained with Sudan III. (C) The complex was subjected to two-dimensional PAGE followed by immunoblot (IB) analyses. Upper panel, anti-lipid A antibody; middle panel, antibodies recognizing the catalytic domains of both Kgp and Rgp; bottom panel, a combination of anti-Hgp44 antibodies and anti-Hgp27 antibodies, respectively. Immunoreacting spots to anti-lipid A antibody are coincident with the catalytic domains of Rgp (43 kDa) (closed arrowheads), Kgp (51 kDa) (open arrowheads), Hgp44, (asterisks), and Hgp39 (arrow).

Article Snippet: A monoclonal antibody that recognizes lipid A of LPS was from HyCult Biotechnology B.V. (Uden, The Netherlands).

Techniques: Modification, Purification, SDS Page, Staining, Western Blot