Journal: PLoS ONE

Article Title: Local Th17/IgA immunity correlate with protection against intranasal infection with Streptococcus pyogenes

doi: 10.1371/journal.pone.0175707

Figure Lengend Snippet: Female CB6F1 mice (n = 8) were vaccinated 3 x s.c. with two weeks interval. Vaccines consisted of heat killed GAS (HGAS) either alone or formulated in CAF01 Two weeks after the last immunization, three animals were sacrificed and PBMCs were stimulated with HGAS for 72 h before measuring levels of (A) IFNγ and (B) IL-17 in culture supernatants by ELISA. An identical follow-up experiment (n = 10) was performed with the CAF01 adjuvant using multiplex cytokine analysis (C) . Bars indicate means ± SEM. Statistical significance was evaluated by an ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism version 6.05. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. IgG responses in pooled serum samples were measured by antigen-specific ELISA (D) . Six weeks after the last immunization, the remaining mice (n = 5) were challenged with a lethal dose of MGAS5005 bacteria (1–1.5 10 7 CFU/mouse) by i.p. injection. Mice were monitored over a period of 24 hours according to a validated clinical scoring system from 0–4 ( E ). When reaching a score of 4, mice were euthanized and data was plotted in Kaplan-Meier survival curves ( F ) (**p = 0.008 with a Chi-square test between the non-vaccinated animals and the CAF01 group). Bacterial numbers were measured in the spleen when mice were euthanized or at the end of the experiment 24 h post infection ( G ).

Article Snippet: Female CB6F1 hybrid mice (offspring of female BALB/c and male C57BL/6 mice) at 6–8 weeks of age were purchased at Envigo Laboratories (The Netherlands) and randomly assigned to cages at the animal facility at Statens Serum Institut upon arrival.

Techniques: Vaccines, Enzyme-linked Immunosorbent Assay, Adjuvant, Multiplex Assay, Comparison, Bacteria, Injection, Infection

Journal: PLoS ONE

Article Title: Local Th17/IgA immunity correlate with protection against intranasal infection with Streptococcus pyogenes

doi: 10.1371/journal.pone.0175707

Figure Lengend Snippet: A . Vaccination and infection sch edule (‘Vac’:Vaccine, ‘Inf.’:Infection, ‘CFU’:Colony forming unit). Groups of female CB6F1 mice (n = 8) were vaccinated with HGAS formulated in CAF01 either via the s.c. or i.n. route. Mice were then rested before receiving repeated i.n. infections of 10 6 MGAS5005 bacteria at week 16, 19, 21 and 23. After each infection bacterial numbers were determined in throat swabs. Bacterial numbers were very similar for the four infections and are therefore combined into the same plots (for individual experiments see ). The percentage of throat culture positive animals are shown at day 1 (B) and at day 4 (C) post infection. The bacterial numbers in throat culture positive animals are shown in (D) at day 1 and (E) at day 4 post infection. Statistical significance was evaluated by an ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism version 6.05. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: Female CB6F1 hybrid mice (offspring of female BALB/c and male C57BL/6 mice) at 6–8 weeks of age were purchased at Envigo Laboratories (The Netherlands) and randomly assigned to cages at the animal facility at Statens Serum Institut upon arrival.

Techniques: Infection, Bacteria, Comparison

Journal: PLoS ONE

Article Title: Local Th17/IgA immunity correlate with protection against intranasal infection with Streptococcus pyogenes

doi: 10.1371/journal.pone.0175707

Figure Lengend Snippet: Groups of female CB6F1 mice (n = 7/8) were vaccinated with HGAS/CAF01 either via the s.c. or i.n. route. Mice were rested for 26 weeks before an i.n. infection with 5 x 10 7 MGAS5005 bacteria. Four mice were sacrificed right before infection while the remaining mice were sacrificed 7 days post infection. Lungs were harvested and cytokine expression in CD4 T cells was evaluated by ICS for IL-17 (A) and IFNγ (B) . A and B show two pooled experiments. All of the cytokine producing CD4 T cells were of a CD44 high phenotype. For complete gating strategy see C. Two examples of CD4 + CD44 + cells from lung of the the i.n. vaccinated mice. The plots show the IL-17/IFNγ expression. C and D . Antibody responses measured by antigen-specific ELISA from the same experiment in lungs and blood. Lungs were perfused, homogenized and IgG and IgA responses were analyzed in the supernatants the day before infection (pre-inf, only shown for lungs)) and 7 days post infection. Statistical significance was evaluated by an ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism version 6.05. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: Female CB6F1 hybrid mice (offspring of female BALB/c and male C57BL/6 mice) at 6–8 weeks of age were purchased at Envigo Laboratories (The Netherlands) and randomly assigned to cages at the animal facility at Statens Serum Institut upon arrival.

Techniques: Infection, Bacteria, Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: PLoS ONE

Article Title: Local Th17/IgA immunity correlate with protection against intranasal infection with Streptococcus pyogenes

doi: 10.1371/journal.pone.0175707

Figure Lengend Snippet: Groups of six female CB6F1 mice were vaccinated with HGAS formulated in CAF01 either via the s.c. or i.n. route. Lungs were harvested at day 3 post infection and cytokine expression in CD45.2 - , CD4 + T cells was evaluated by ICS for IL-17 (A) and IFNγ (B) . 250μl of the aCD45.2 solution was injected i.v. into each mouse via the tail vein, three minutes before euthanization of the mice. All of the cytokine producing CD4 T cells were of a CD44 high phenotype. C. Lungs were perfused, homogenized and IgA responses were analyzed in the supernatants 3 days post infection. Statistical significance was evaluated by an ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism version 6.05. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: Female CB6F1 hybrid mice (offspring of female BALB/c and male C57BL/6 mice) at 6–8 weeks of age were purchased at Envigo Laboratories (The Netherlands) and randomly assigned to cages at the animal facility at Statens Serum Institut upon arrival.

Techniques: Infection, Expressing, Injection, Comparison

Journal: PLoS ONE

Article Title: Local Th17/IgA immunity correlate with protection against intranasal infection with Streptococcus pyogenes

doi: 10.1371/journal.pone.0175707

Figure Lengend Snippet: Groups of female CB6F1 mice (n = 6) were vaccinated with HGAS/CAF01 formulated in CAF01 either via s.c.+i.n. route. Mice were then subjected to an i.n. infection with 5 x 10 7 MGAS5005 bacteria. At day 7 post infection lungs were harvested and IL-17 and IFNγ cytokine expression in CD44 + CD4 + T cells was evaluated by ICS and IgA antibody responses in the lungs was measured by antigen-specific ELISA (A). IgG levels (endpoint titers were determined in serum and PBMCs were stimulated with HGAS for 72 h before measuring levels of IFNγ and IL-17 in culture supernatants by ELISA (B). The sc+in group was part of the experiment shown in and the non-vaccinated control group are therefore shared. Statistical significance was evaluated by an ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism version 6.05. *p<0.05.

Article Snippet: Female CB6F1 hybrid mice (offspring of female BALB/c and male C57BL/6 mice) at 6–8 weeks of age were purchased at Envigo Laboratories (The Netherlands) and randomly assigned to cages at the animal facility at Statens Serum Institut upon arrival.

Techniques: Infection, Bacteria, Expressing, Enzyme-linked Immunosorbent Assay, Control, Comparison

Journal: Cell reports

Article Title: Impact of WIN site inhibitor on the WDR5 interactome

doi: 10.1016/j.celrep.2020.108636

Figure Lengend Snippet: (A) Endogenous PDPK1 was recovered from lysates of the indicated cell lines and probed for co-precipitating WDR5 by IB. Inputs for PDPK1 are 10%–20%. Inputs for WDR5 are 1%–5%. n = 3 biological replicates. (B) Proximity ligation assay with FLAG and WDR5 antibodies in U2OS cells stably expressing FLAG-tagged PDPK1. Cells were treated overnight (30 μM C6/C6nc) before analysis; scale bar, 20 μm. n = 3 biological replicates. (C) HEK293 cells were treated overnight with 30 μM C6 or 5 μM GSK470, lysates prepared, and a PDPK1 IP performed. IB was then performed for the indicated proteins. Inputs are 5% for PDPK1 and 1% for all others. n = 3 biological replicates. (D) HEK293 cells were fractionated into cytosolic (S2), soluble nuclear (S3), and chromatin-associated (P3) fractions. Equal amounts of each fraction were analyzed by IB with the antibodies against the indicated proteins. H3 (nuclear) and α-tubulin (cytosolic) are controls for fractionation. n = 3 biological replicates. (E) Cytosolic and nuclear lysates from HEK293 cells were subject to IP with PDPK1 antibody or an IgG control and immunoblotted with antibodies against the indicated proteins. A short and long exposure of the WDR5 IB are shown. n = 3 biological replicates. (F) PDPK1 possesses two WIN-like motifs centered on R3 and R238. (G) FLAG-tagged PDPK1 (WT and the R3A and R238A mutants) were transiently expressed in HEK293 cells; lysates were prepared and subject to IP with anti-FLAG beads. Immune complexes were probed for PDPK1 or endogenous WDR5 by IB. n = 3 biological replicates. (H) FLAG-tagged PDPK1 (WT and the R3A) was transiently expressed in HEK293 cells, recovered by FLAG-IP, resolved by SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were then incubated with recombinant WDR5 followed by anti-WDR5 antibody. n = 3 biological replicates. (I) In vitro -transcribed and -translated PDPK1-FLAG variants were incubated with recombinant 6xHis-SUMO-WDR5 proteins, recovered with Ni-NTA agarose, and analyzed by IB. n = 2 biological replicates. PH, pleckstrin homology domain. See also .

Article Snippet: PolyScreen PVDF Hybridization Transfer Membrane , PerkinElmer , Cat# NEF1002.

Techniques: Proximity Ligation Assay, Stable Transfection, Expressing, Fractionation, Control, SDS Page, Membrane, Incubation, Recombinant, In Vitro