Journal: Virulence

Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

doi: 10.1080/21505594.2016.1216294

Figure Lengend Snippet: BilRI bound to various human inflammatory cytokines but not to fibrinogen or collagen. A) Recombinant BilRI containing an 8-histidine-long C-terminal tag bound to various recombinant human cytokines in a microplate assay. BSA served as a negative control and was used as a blocking agent in the assays. The bound BilRI was detected with HRP-labeled HisProbe™. The BilRI binding to IL-8 was high compared to the binding to the control protein BSA (**:p = 0.008, paired-samples T-test). B) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to fibrinogen-coated wells in a microplate assay when detected with europium-labeled antibody against the histidine tag. Although the positive control recombinant ClfA of S. aureus bound fibrinogen efficiently, the recombinant FgbA, which has been reported to bind fibrinogen, did not show positive binding in the assay. Only BilRI binding to IL-8 showed a statistically significant positive difference compared with the BSA control (p = 0.028, Mann-Whitney U-test) from the test experiments. C) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to collagen-coated wells in the microplate assays when detected with europium-labeled antibody against the histidine tag. The positive control recombinant YadA of Y. enterocolitica bound collagen efficiently. Only BilRI binding IL-8 showed a statistically significant positive difference compared with control BSA (p = 0.028, Mann-Whitney U-test) from the test experiments.

Article Snippet: The staining was performed with Dako TechMate™ 500 Plus Autostainer (Dako, Glostrup, Denmark) using 20 μg/mL of primary polyclonal rabbit IgG against IL-8 (NBP2-16958; Novus Biologicals, Cambridge, UK) and IL-6 (NBP2-16957; Novus Biologicals) and a Dako REAL™ Detection System, Peroxidase/DAB+, Rabbit/Mouse (Code K5001; Dako) as instructed by the producer.

Techniques: Recombinant, Negative Control, Blocking Assay, Labeling, Binding Assay, Positive Control, MANN-WHITNEY

Journal: Virulence

Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

doi: 10.1080/21505594.2016.1216294

Figure Lengend Snippet: Viable wild-type A. actinomycetemcomitans biofilm bound IL-8 and IL-6, and internalized IL-1β when co-cultured with organotypic gingival mucosa. A) A. actinomycetemcomitans wild-type biofilm bound both IL-8 and IL-6 when co-cultured with organotypic gingival mucosa in the absence of the antibiotics penicillin and streptomycin. In the presence of these antibiotics, the biofilm bound less IL-8 and IL-6 whereas the epithelium contained elevated amounts of IL-8 and IL-6. B) The amount of IL-8 produced was approximately 10 times the amount of IL-6 in the organotypic gingival mucosa tissue culture model. The co-culture system released slightly more IL-8 and IL-6 to the culture medium when stimulated with A. actinomycetemcomitans biofilm in the presence of antibiotics than in the absence of antibiotics. Due to the standard deviation between the samples, the difference was not statistically significant. N = 3. C) In the organotypic gingival tissue culture model, which produced approximately 200 pg of IL-1β to the culture medium during a 24-h incubation with viable A. actinomycetemcomitans wild-type biofilm, the uptake efficiency of IL-1β was estimated by counting the number of gold particles in anti-IL-1β-stained immuno-EM samples.

Article Snippet: The staining was performed with Dako TechMate™ 500 Plus Autostainer (Dako, Glostrup, Denmark) using 20 μg/mL of primary polyclonal rabbit IgG against IL-8 (NBP2-16958; Novus Biologicals, Cambridge, UK) and IL-6 (NBP2-16957; Novus Biologicals) and a Dako REAL™ Detection System, Peroxidase/DAB+, Rabbit/Mouse (Code K5001; Dako) as instructed by the producer.

Techniques: Cell Culture, Produced, Co-Culture Assay, Standard Deviation, Incubation, Staining

Journal: Virulence

Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

doi: 10.1080/21505594.2016.1216294

Figure Lengend Snippet: A. actinomycetemcomitans wild-type and bilRI − mutant strains internalized all tested inflammatory cytokines: IL-1β, IL-8 and IL-6. The outer membrane lipoprotein BilRI had a role in the uptake of only IL-1β in the test system. A) Both A. actinomycetemcomitans wild-type and bilRI − mutant biofilm cells internalized IL-1β, IL-8 and IL-6 when incubated for 24 h with human gingival keratinocyte monolayers. Cytokine uptake was studied with anti-cytokine IgG antibodies combined with protein A-gold labeling and transmission electron microscopy. B) Deletion of the bilRI gene decreased only IL-1β uptake (p = 0.007, Mann-Whitney U-test), while IL-8 and IL-6 uptake levels were not affected. The uptake efficiencies were estimated by counting the amounts of gold labeling in the positively stained cells.

Article Snippet: The staining was performed with Dako TechMate™ 500 Plus Autostainer (Dako, Glostrup, Denmark) using 20 μg/mL of primary polyclonal rabbit IgG against IL-8 (NBP2-16958; Novus Biologicals, Cambridge, UK) and IL-6 (NBP2-16957; Novus Biologicals) and a Dako REAL™ Detection System, Peroxidase/DAB+, Rabbit/Mouse (Code K5001; Dako) as instructed by the producer.

Techniques: Mutagenesis, Incubation, Labeling, Transmission Assay, Electron Microscopy, MANN-WHITNEY, Staining

Journal: Virulence

Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

doi: 10.1080/21505594.2016.1216294

Figure Lengend Snippet: The produced recombinant proteins YadA (23 kDa), ClfA (36 kDa), FgbA (12 kDa), IL-8 (9 kDa) and BilRI (18 kDa) were pure, as observed in a Coomassie-stained SDS-PAGE gel. A total amount of 1 μg of each protein was run in the gel.

Article Snippet: The staining was performed with Dako TechMate™ 500 Plus Autostainer (Dako, Glostrup, Denmark) using 20 μg/mL of primary polyclonal rabbit IgG against IL-8 (NBP2-16958; Novus Biologicals, Cambridge, UK) and IL-6 (NBP2-16957; Novus Biologicals) and a Dako REAL™ Detection System, Peroxidase/DAB+, Rabbit/Mouse (Code K5001; Dako) as instructed by the producer.

Techniques: Produced, Recombinant, Staining, SDS Page

Journal: Virulence

Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

doi: 10.1080/21505594.2016.1216294

Figure Lengend Snippet: BilRI bound to various human inflammatory cytokines but not to fibrinogen or collagen. A) Recombinant BilRI containing an 8-histidine-long C-terminal tag bound to various recombinant human cytokines in a microplate assay. BSA served as a negative control and was used as a blocking agent in the assays. The bound BilRI was detected with HRP-labeled HisProbe™. The BilRI binding to IL-8 was high compared to the binding to the control protein BSA (**:p = 0.008, paired-samples T-test). B) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to fibrinogen-coated wells in a microplate assay when detected with europium-labeled antibody against the histidine tag. Although the positive control recombinant ClfA of S. aureus bound fibrinogen efficiently, the recombinant FgbA, which has been reported to bind fibrinogen, did not show positive binding in the assay. Only BilRI binding to IL-8 showed a statistically significant positive difference compared with the BSA control (p = 0.028, Mann-Whitney U-test) from the test experiments. C) Recombinant BilRI containing an 8-histidine-long C-terminal tag did not bind to collagen-coated wells in the microplate assays when detected with europium-labeled antibody against the histidine tag. The positive control recombinant YadA of Y. enterocolitica bound collagen efficiently. Only BilRI binding IL-8 showed a statistically significant positive difference compared with control BSA (p = 0.028, Mann-Whitney U-test) from the test experiments.

Article Snippet: The primary antibodies, rabbit anti-IL-1β (NB600-633; Novus Biologicals), rabbit anti-IL-8 (NBP2-16958; Novus Biologicals), and rabbit anti-IL-6 (NBP2-16957), were diluted in 1% BSA-PBS 2 and incubated with the samples for 60 min. After washing with 1% BSA-PBS 2 , the bound antibodies were detected by incubating with protein A-gold complex (10 nm) diluted in 0.1% BSA-PBS 2 .

Techniques: Recombinant, Negative Control, Blocking Assay, Labeling, Binding Assay, Positive Control, MANN-WHITNEY

Journal: Virulence

Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

doi: 10.1080/21505594.2016.1216294

Figure Lengend Snippet: Viable wild-type A. actinomycetemcomitans biofilm bound IL-8 and IL-6, and internalized IL-1β when co-cultured with organotypic gingival mucosa. A) A. actinomycetemcomitans wild-type biofilm bound both IL-8 and IL-6 when co-cultured with organotypic gingival mucosa in the absence of the antibiotics penicillin and streptomycin. In the presence of these antibiotics, the biofilm bound less IL-8 and IL-6 whereas the epithelium contained elevated amounts of IL-8 and IL-6. B) The amount of IL-8 produced was approximately 10 times the amount of IL-6 in the organotypic gingival mucosa tissue culture model. The co-culture system released slightly more IL-8 and IL-6 to the culture medium when stimulated with A. actinomycetemcomitans biofilm in the presence of antibiotics than in the absence of antibiotics. Due to the standard deviation between the samples, the difference was not statistically significant. N = 3. C) In the organotypic gingival tissue culture model, which produced approximately 200 pg of IL-1β to the culture medium during a 24-h incubation with viable A. actinomycetemcomitans wild-type biofilm, the uptake efficiency of IL-1β was estimated by counting the number of gold particles in anti-IL-1β-stained immuno-EM samples.

Article Snippet: The primary antibodies, rabbit anti-IL-1β (NB600-633; Novus Biologicals), rabbit anti-IL-8 (NBP2-16958; Novus Biologicals), and rabbit anti-IL-6 (NBP2-16957), were diluted in 1% BSA-PBS 2 and incubated with the samples for 60 min. After washing with 1% BSA-PBS 2 , the bound antibodies were detected by incubating with protein A-gold complex (10 nm) diluted in 0.1% BSA-PBS 2 .

Techniques: Cell Culture, Produced, Co-Culture Assay, Standard Deviation, Incubation, Staining

Journal: Virulence

Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

doi: 10.1080/21505594.2016.1216294

Figure Lengend Snippet: A. actinomycetemcomitans wild-type and bilRI − mutant strains internalized all tested inflammatory cytokines: IL-1β, IL-8 and IL-6. The outer membrane lipoprotein BilRI had a role in the uptake of only IL-1β in the test system. A) Both A. actinomycetemcomitans wild-type and bilRI − mutant biofilm cells internalized IL-1β, IL-8 and IL-6 when incubated for 24 h with human gingival keratinocyte monolayers. Cytokine uptake was studied with anti-cytokine IgG antibodies combined with protein A-gold labeling and transmission electron microscopy. B) Deletion of the bilRI gene decreased only IL-1β uptake (p = 0.007, Mann-Whitney U-test), while IL-8 and IL-6 uptake levels were not affected. The uptake efficiencies were estimated by counting the amounts of gold labeling in the positively stained cells.

Article Snippet: The primary antibodies, rabbit anti-IL-1β (NB600-633; Novus Biologicals), rabbit anti-IL-8 (NBP2-16958; Novus Biologicals), and rabbit anti-IL-6 (NBP2-16957), were diluted in 1% BSA-PBS 2 and incubated with the samples for 60 min. After washing with 1% BSA-PBS 2 , the bound antibodies were detected by incubating with protein A-gold complex (10 nm) diluted in 0.1% BSA-PBS 2 .

Techniques: Mutagenesis, Incubation, Labeling, Transmission Assay, Electron Microscopy, MANN-WHITNEY, Staining

Journal: Virulence

Article Title: A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

doi: 10.1080/21505594.2016.1216294

Figure Lengend Snippet: The produced recombinant proteins YadA (23 kDa), ClfA (36 kDa), FgbA (12 kDa), IL-8 (9 kDa) and BilRI (18 kDa) were pure, as observed in a Coomassie-stained SDS-PAGE gel. A total amount of 1 μg of each protein was run in the gel.

Article Snippet: The primary antibodies, rabbit anti-IL-1β (NB600-633; Novus Biologicals), rabbit anti-IL-8 (NBP2-16958; Novus Biologicals), and rabbit anti-IL-6 (NBP2-16957), were diluted in 1% BSA-PBS 2 and incubated with the samples for 60 min. After washing with 1% BSA-PBS 2 , the bound antibodies were detected by incubating with protein A-gold complex (10 nm) diluted in 0.1% BSA-PBS 2 .

Techniques: Produced, Recombinant, Staining, SDS Page