Journal: British Journal of Pharmacology

Article Title: Endocannabinoids modulate human blood–brain barrier permeability in vitro

doi: 10.1111/bph.13106

Figure Lengend Snippet: The effects of increasing concentrations of AEA on BBB permeability measured by TEER (A) with corresponding AUC (B) ( n = 9 inserts from three separate experiments). The effects of capsazepine (Cpz) (C) ( n = 7 inserts from three separate experiments) or AM630 (D) ( n = 7–8 inserts from three separate experiments) or CGRP (E) ( n = 5–6 inserts from three separate experiments) on the effect of AEA (10 μM). The effects of dexamethasone ( n = 6) and the CB 2 agonist HU308 on BBB permeability over time (F) and expressed as AUC (G). Data are given as mean ± SEM. * * * P < 0.001, * * P < 0.01, * P < 0.05; AEA compared with vehicle-treated inserts; †† P < 0.01; AEA and antagonist compared with AEA alone; # P < 0.05, ## P < 0.01; HU308 compared with vehicle; one-way anova with Dunnett's (B) or Bonferroni's test (C,D,E).

Article Snippet: AM251, AM630, GW6471, GW9662 (all 100 nM), capsazepine, O-1918 (both 1 μM) (all dissolved in dimethyl sulfoxide) and CGRP 8–37 (2 μM, dissolved in distilled water) were all purchased from Tocris and URB597 (1 μM, dissolved in dimethyl sulfoxide) was purchased from Sigma (Dorset, UK).

Techniques: Permeability

Journal: British Journal of Pharmacology

Article Title: Endocannabinoids modulate human blood–brain barrier permeability in vitro

doi: 10.1111/bph.13106

Figure Lengend Snippet: Expression profiling of potential target sites of action in HA and HBMEC cells. Shown are the ethidium bromide-stained gels of the products obtained by RT-PCR using primers specific for PPARα, PPARγ, CB 1 receptors, CB 2 receptors, TRPV1 channels, CGRP receptors and the control gene HPRT. cDNAs generated in the presence (+) or absence (−) of reverse transcriptase on total RNA from HA or HBMEC cells were used as template for the PCRs. The 100 bp DNA ladder was used in all gels except for PPARα and PPARγ where a 10 bp ladder was used. Sizes are in base pairs.

Article Snippet: AM251, AM630, GW6471, GW9662 (all 100 nM), capsazepine, O-1918 (both 1 μM) (all dissolved in dimethyl sulfoxide) and CGRP 8–37 (2 μM, dissolved in distilled water) were all purchased from Tocris and URB597 (1 μM, dissolved in dimethyl sulfoxide) was purchased from Sigma (Dorset, UK).

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Control, Generated, Reverse Transcription

Journal: British Journal of Pharmacology

Article Title: Endocannabinoids modulate human blood–brain barrier permeability in vitro

doi: 10.1111/bph.13106

Figure Lengend Snippet:

Article Snippet: AM251, AM630, GW6471, GW9662 (all 100 nM), capsazepine, O-1918 (both 1 μM) (all dissolved in dimethyl sulfoxide) and CGRP 8–37 (2 μM, dissolved in distilled water) were all purchased from Tocris and URB597 (1 μM, dissolved in dimethyl sulfoxide) was purchased from Sigma (Dorset, UK).

Techniques:

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A) Representative Fura-2 ratiometric fields (left) and calcium traces (center) of DRG neurons responding to filtered supernatant from S. pyogenes M1 (5×109 cfu/mL), capsaicin (1 µm), and KCl (40 mM). Proportions (Right) of capsaicin non-responsive (Cap−) and capsaicin responsive (Cap+) neurons that responded to M1 supernatant (n=3–4 fields/condition). (B–D) Representative Fura-2 ratiometric fields (B) and calcium traces (C) of DRG neurons stimulated with filtered supernatant from S. pyogenes M1 (wt) or isogenic mutants lacking SLS (ΔsagA), both SLO and SLS (ΔsloΔsagA), or double mutant bacteria in which sagA expression was restored (ΔsloΔsagA+pDL:sagA). (D) Proportions of responding DRG neurons to bacterial supernatant from S. pyogenes M1 (wt) or isogenic mutant strains (n=3 fields/condition). (E) DRG neurons stimulated for 30 min with supernatant from S. pyogenes M1 (wt), isogenic mutants, or medium, analyzed for in vitro release of CGRP (n=5 samples/group). Statistical analysis: (A) Two-way ANOVA, Bonferroni post-tests. (D,E) One-way ANOVA, Tukey post-tests. *p<0.05 ***p<0.001 ****p<0.0001. ns=not significant. Scale bars, 50 µm. Mean±SEM. See Figure S2 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Mutagenesis, Bacteria, Expressing, In Vitro

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A) Histopathology of flank biopsies from vehicle or RTX-treated mice 3 days after injection of S. pyogenes M1 (5×106 cfu). Scale bars, 50 µm. (B) Bacterial load recovery (log10 cfu) from flank lesions and spleens in RTX or vehicle-treated mice after S. pyogenes M1 injection (5×106 cfu, n=4/group). (C–E) Flow cytometry of leukocyte recruitment in necrotizing lesions 1 day after S. pyogenes M1 injection (5×106 cfu): (C) Representative FACS plots showing neutrophils (CD11b+Ly6G+ gates) in lesion samples. (D–E) Quantification of immune cell populations by flow cytometry in flank biopsies from infected Trpv1-Cre/Dta mice or control littermates (n=4/group), or from uninfected mice, infected vehicle-treated mice, or infected RTX-treated mice (n=4–5/group). (F–H) Measurement of CGRP release ex vivo from flank skin punch biopsies. (G) CGRP release from uninfected skin (0 h), 7 h, or 24 h after S. pyogenes M1 injection (5×106 cfu) (n=3/group). (H) CGRP release from uninfected skin or 7 h after S. pyogenes M1 (5×106 cfu) injection of Trpv1-Cre/Dta mice or control littermates, or Vehicle or RTX-treated mice (n=3/group). Statistical analysis: (B,D,E,H) Two-way ANOVA, Bonferroni post-tests. (G) One-way ANOVA, Tukey post-tests. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. ns=not significant. nd=none detected. Mean±SEM. See Figure S5 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Histopathology, Injection, Flow Cytometry, Infection, Control, Ex Vivo

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A–D) Subcutaneous administration of BoNT/A (25 pg/100 µL) or vehicle 6 days prior to S. pyogenes M1 injection in flank skin (5×106 cfu). (B) Representative images of lesions (day 8), (C) Dermonecrosis size measurements, and (D) Weight loss over time after injection of S. pyogenes (n=5–10/group). (E–H) Intrathecal administration of BoNT/A or vehicle 1 day prior to S. pyogenes M1 injection in flank skin (5×106 cfu). (F) Representative images of lesions (day 8), (G) Dermonecrosis size measurements, and (H) Weight loss over time after injection of S. pyogenes (n=6/group). (I) DRG neurons exposed to BoNT/A (25 pg/200 µL) or medium for 24 h were stimulated with S. pyogenes supernatant (5×109 cfu/mL) for 30 min, and CGRP was measured in neuronal supernatant (n=5/group). (J) CGRP release from skin punch biopsies of mice treated intrathecally or locally with BoNT/A, 7 h after S. pyogenes M1 (5×106 cfu) injection (n=3/group). Statistical analysis: (C,D,G,H) Two-way ANOVA, Bonferroni post-tests. (I,J) One-way ANOVA, Tukey post-tests. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. ns=not significant. Mean±SEM. See Figure S6 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Injection

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A) DRG neurons were pretreated with BoNT/A for 24 h, or with CGRP antagonists (CGRP8–37 or BIBN4096) immediately before co-incubation with mouse neutrophils and S. pyogenes M1 for 1 h. Bacterial survival was measured as the multiplication factor of surviving colonies/starting inoculum (n=3–4 replicates/group). (B) Mouse neutrophils were incubated with S. pyogenes M1 in presence of CGRP or vehicle for 1 h, and bacterial survival measured (n=4/group). (C) Human whole blood was incubated with S. pyogenes M1 in presence of CGRP or vehicle for 3 h, and bacterial survival measured (n=3/group). (D) Representative images of lesions at day 8 (left) and dermonecrosis size (right) of mice treated 2 h after S. pyogenes M1 injection (5×106 cfu) with vehicle, BoNT/A, or BIBN4096 (n=6–7/group). (E–G) Mice were treated subcutaneously with BoNT/A or vehicle at day 2 and day 9 following flank injection of S. pyogenes M1 (5×106 cfu). Representative images show lesions before and after treatment (E). Dermonecrotic lesions (F) and abscess sizes (G) were measured over time (n=10/group). Blue dots show injection sites at day 2 and day 9. Arrows show BoNT/A treatments. Statistical analysis: (A–C) One-way ANOVA, Tukey post-tests. (D–G) Two-way ANOVA, Bonferroni post-tests. (A–C,F–G) *p<0.05 **p<0.01 ***p<0.01 ****p<0.0001. (D) BIBN4096 vs veh: *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001, BoNT/A vs veh: †p<0.05 ††p<0.01 †††p<0.001 ††††p<0.0001. ns=not significant. Mean±SEM. See Figure S7 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Incubation, Injection