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

Journal: Cell

Article Title: Cutaneous TRPV1 + Neurons Trigger Protective Innate Type-17 Anticipatory Immunity

doi: 10.1016/j.cell.2019.06.022

Figure Lengend Snippet: A) Il23a, Il6, Tnfα mRNA levels in flank skin isolated from photostimulated Ai32 (open circles) or TRPV1-Ai32 (red squares) mice at the indicated skin site 6 hours after initiation of photostimulation. (B) C. albicans and (C) S. aureus CFU is shown from the indicated skin region of epicutaneously infected Ai32 or TRPV1-Ai32 mice on day 3 post-infection with photostimulation on days 0, 1, and 2. (D) CGRP protein and (E) Il23a, Il6, Tnfα mRNA levels at the indicated site from photostimulated TRPV1-Ai32 mice pre-treated with 5mM bupivacaine (blue squares) or vehicle (red squares) at the Stim site. (F-G) As in (D-E) except TRPV1-Ai32 mice were pretreated at the stim site with 0.3U Botulinum toxin (BoNT/A) or vehicle. (H) C. albicans CFU is shown from the indicated skin region of TRPV1-Ai32 mice treated twice daily with vehicle (red squares) or 5mM Bupivacaine (blue squares) at the Stim site. Tissue harvested on day 3 post-infection with photostimulation on days 0, 1, and 2. (I) As in (H) except that 0.5ug CGRP8-37 (blue squares) or vehicle (red squares) was injected daily at the adjacent site. (J) As in (H) except mice were pretreated i.p. with neutralizing mAbs to IL-17A (blue squares) or isotype control (red squares). Each symbol represents an individual animal. Results in all panels are represented as mean ± SEM from 2-4 independent experiments. Significance was calculated using a Student’s t-test. *p<0.05, **p<.001, ***p<.0001. See also supplemental figure S5

Article Snippet: CGRP 8-37 , Fisher Scientific , 1169500U.

Techniques: Isolation, Infection, Injection, Control

Journal: Cell

Article Title: Cutaneous TRPV1 + Neurons Trigger Protective Innate Type-17 Anticipatory Immunity

doi: 10.1016/j.cell.2019.06.022

Figure Lengend Snippet: (A) Il23a, Il6, Tnfα mRNA expression in whole photostimulated (“+”) or sham treated (“−”) ears from TRPV1-Ai32 mice pre-treated with vehicle (red squares) or 0.3U Botulinum toxin (BoNT/A) (blue squares) isolated at 6 hours after initiation of photostimulation. (B) ELISA quantification of CGRPα in supernatants from cultured DRG isolated from Ai32 (black circles) or TRPV1-Ai32 (red squares) mice after 30 minutes of sham (−) or photostimulation(+). (C) ELISA quantification of CGRPα in supernatants from ex vivo skin explant organ cultures of Ai32 or TRPV1-Ai32 mice harvested 6 hours after initiation of in vivo photostimulation. (D) Il23a, Il6, Tnfα mRNA expression in whole photostimulated (“+”) or sham treated (“−”) ears from TRPV1-Ai32 mice pre-treated with vehicle (red squares) or 0.5ug CGRP8-37 (blue squares) isolated at 6 hours after initiation of photostimulation. (E) As in D except that ears were harvested at the indicated time point following initiation of photostimulation. (F) Summary data of IL-17A expression by PMA/Ionomycin stimulated TCRγδ and CD4 T cells isolated from ears of TRPV1-Ai32 and Ai32 mice treated daily with vehicle or CGRP8-37 during 4 days of photostimulation. Each symbol in A, C, D, and F represents an individual animal. Each symbol in B represents pooled DRG neurons from an individual mouse. Each symbol in E represents the mean in a group size of 4-7 mice. Results in all panels are represented as mean ± SEM from 2-3 independent experiments. Significance was calculated using a one way ANOVA, *p<0.05, **p<.001, ***p<.0001.

Article Snippet: CGRP 8-37 , Fisher Scientific , 1169500U.

Techniques: Expressing, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture, Ex Vivo, In Vivo

Journal: Cell

Article Title: Cutaneous TRPV1 + Neurons Trigger Protective Innate Type-17 Anticipatory Immunity

doi: 10.1016/j.cell.2019.06.022

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CGRP 8-37 , Fisher Scientific , 1169500U.

Techniques: Plasmid Preparation, Virus, Recombinant, Electron Microscopy, Protease Inhibitor, Lysis, Extraction, Reverse Transcription, SYBR Green Assay, Software, Microscopy, Flow Cytometry, Imaging

Journal:

Article Title: Influence of CGRP (8-37), but not adrenomedullin (22-52), on the haemodynamic responses to lipopolysaccharide in conscious rats

doi: 10.1038/sj.bjp.0702718

Figure Lengend Snippet: Cardiovascular responses to CGRP at 0.1 nmol kg−1 (n=8) or 1 nmol kg−1 (n=6) in the absence or presence of ADM (22-52) (500 nmol kg−1 h−1) in conscious Long Evans rats. The mesenteric vasoconstrictor effect of the higher dose of CGRP was enhanced in the presence of ADM (22-52), possibly due to the mesenteric vasodilator influence of the latter. Values are mean and vertical bars show s.e.mean.

Article Snippet: Adrenomedullin and human adrenomedullin (22-52) were obtained from the Peptide Research Institute (Osaka, Japan), CGRP (8-37) was a gift from Celltech (Slough, U.K.), losartan potassium was a gift from Dr R.D.

Techniques:

Journal:

Article Title: Influence of CGRP (8-37), but not adrenomedullin (22-52), on the haemodynamic responses to lipopolysaccharide in conscious rats

doi: 10.1038/sj.bjp.0702718

Figure Lengend Snippet: Cardiovascular responses to infusion of LPS (150 μg kg−1 h−1) in the presence of dexamethasone and SB 209670 in conscious Long Evans rats. At 6 h, animals were infused with CGRP (8-37) (6 μmol kg−1 h−1) (n=9) or saline (n=7; data as in Figure 3). CGRP (8-37) caused small, but significant inhibitions of the hypotension and renal, mesenteric and hindquarters vasodilatations. In animals receiving saline infusions (n=6), CGRP (8-37) at 6 h had no effects. Values are mean and vertical bars show s.e.mean. *P<0.05 versus the 6 h value (Friedman's test).

Article Snippet: Adrenomedullin and human adrenomedullin (22-52) were obtained from the Peptide Research Institute (Osaka, Japan), CGRP (8-37) was a gift from Celltech (Slough, U.K.), losartan potassium was a gift from Dr R.D.

Techniques: Saline

Journal:

Article Title: Influence of CGRP (8-37), but not adrenomedullin (22-52), on the haemodynamic responses to lipopolysaccharide in conscious rats

doi: 10.1038/sj.bjp.0702718

Figure Lengend Snippet: Cardiovascular responses to infusion of LPS (150 μg kg−1 h−1) in conscious Long Evans rats pretreated with SB 209670, and infused with CGRP (8-37) (6 μmol kg−1 h−1) at 6 h (n=6), or pretreated with losartan and SB 209670, and infused with CGRP (8-37) at 6 h (n=3), or pretreated with losartan and SB 209670 and infused with saline at 6 h (n=6). Values are mean and vertical bars show s.e.mean. *P<0.05 versus the 6 h value (Friedman's test).

Article Snippet: Adrenomedullin and human adrenomedullin (22-52) were obtained from the Peptide Research Institute (Osaka, Japan), CGRP (8-37) was a gift from Celltech (Slough, U.K.), losartan potassium was a gift from Dr R.D.

Techniques: Saline

Journal:

Article Title: Influence of CGRP (8-37), but not adrenomedullin (22-52), on the haemodynamic responses to lipopolysaccharide in conscious rats

doi: 10.1038/sj.bjp.0702718

Figure Lengend Snippet: Cardiovascular responses to infusion of LPS (150 μg kg−1 h−1) in conscious Long Evans rats (n=3) pretreated with losartan and SB 209670, and given CGRP (8-37) starting 40 min after the onset of LPS infusion. For comparison, the data from animals infused with LPS following pretreatment with losartan and SB 209670 (from Figure 5) are included. Values are mean and vertical bars show s.e.mean. CGRP (8-37) did not reverse the hypotension or vasodilatations associated with LPS infusion.

Article Snippet: Adrenomedullin and human adrenomedullin (22-52) were obtained from the Peptide Research Institute (Osaka, Japan), CGRP (8-37) was a gift from Celltech (Slough, U.K.), losartan potassium was a gift from Dr R.D.

Techniques: Comparison