mouse monoclonal anti fgf8 antibody  (R&D Systems)

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: Kinetics of FGF8 production in polymicrobial sepsis. A , Male C57BL/6 mice (n = 5 per group) were subjected to sham or CLP using a 24-gauge needle. PLF, serum, lung, spleen and kidney were collected at the indicated times (6, 24, 48 hours) after CLP. FGF8 concentrations were measured by ELISA. B , Representative fluorescence images of FGF8 expression in kidney, spleen, and lung after CLP. Scale bar = 50 μm. Quantitative results are shown (n = 3 per group). C , FGF8 concentrations in serum and PLF of TLR2 −/− , TLR4 −/− , TLR2/4 −/− , and WT mice 24 hours (n = 3–4 per group) after CLP. D , Supernatant of heat-killed Pseudomonas aeruginosa (MOI = 1:100) challenged macrophages was collected at the indicated times (6, 12, 24 hours). FGF8 levels were measured by ELISA (n = 4 per group). A–D , Data are representative of 3 independent experiments; Kruskal-Wallis test followed by Dunn multiple comparisons posttest. * P < .05, ** P < .01, *** P < .001, **** P < .0001 compared within 2 groups. Abbreviations: CLP, cecal ligation and puncture; Ctrl, control; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; MOI, multiplicity of infection; ns, not significant; PLF, peritoneal lavage fluid; TLR, Toll-like receptor; WT, wild type.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Expressing, Ligation, Control, Infection

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: Inhibition of endogenous FGF8 exacerbated polymicrobial sepsis. A , Survival of septic mice (n = 20 per group) with or without FGF8 neutralization after CLP using a 24-gauge needle. B , Dilutions of PLF, blood, liver, lung, and spleen tissues were obtained from septic mice (n = 10–11 per group) treated with or without monoclonal anti-FGF8 antibody at 24 hours after CLP; samples were cultured on blood agar plates and the numbers of bacterial colonies were then determined. C , Representative examples of hematoxylin and eosin-stained tissues as indicated from mice (n = 5 per group) treated or not treated with monoclonal anti-FGF8 antibody at 24 hours after CLP. The pathology scores are shown on the right side of histological images. D , Serological markers of organ injury including ALT, AST, LDH, creatinine, and urea in septic mice (n = 11–12 per group) treated with or without monoclonal anti-FGF8 antibody at 24 hours after CLP. Each dot represents an individual mouse. B–D , Nonparametric Mann-Whitney U test; ( A ) Kaplan-Meier analysis followed by log-rank test. * P < .05, ** P < .01, *** P < .001 compared within 2 groups. Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; CLP, cecal ligation and puncture; FGF, fibroblast growth factor; IgG, immunoglobulin G; LDH, lactate dehydrogenase; ns, not significant; PLF, peritoneal lavage fluid.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Inhibition, Neutralization, Cell Culture, Staining, MANN-WHITNEY, Ligation

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGF8 treatment improves outcomes in a murine sepsis model. A , Different levels of rFGF8 (0, 5, 12.5 μg/kg) were injected intraperitoneally into mice (n = 20 per group) immediately after CLP and survival was monitored for 14 days. B , Dilutions of blood, lungs, PLF, and spleens obtained from septic mice (n = 9–10 per group) treated with or without rFGF8 (12.5 μg/kg) 48 hours after CLP. C , Representative examples of hematoxylin and eosin-stained lung, liver, spleen, and kidney tissues from CLP mice treated with or without rFGF8 (12.5 μg/kg) after CLP. Scale bars = 400 μm. Histological scores (n = 5 per group) are shown. D , Serological markers of organ injury in septic mice (n = 13 per group) treated with or without rFGF8 (12.5 μg/kg) at 48 hours after CLP. B–D , Data are representatives of 3 independent experiments; nonparametric Mann-Whitney U test; ( A ) Kaplan-Meier analysis followed by log-rank test. * P < .05, ** P < .01 compared within 2 groups. Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; CLP, cecal ligation and puncture; FGF, fibroblast growth factor; LDH, lactate dehydrogenase; ns, not significant; PBS, phosphate-buffered saline; PLF, peritoneal lavage fluid.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Injection, Staining, MANN-WHITNEY, Ligation, Saline

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGF8 directly enhances bacterial phagocytosis and killing by macrophages. A , Peritoneal macrophages (1 × 10 6 cells) were stimulated with or without rFGF8 (200 ng/mL) for 6 hours and then challenged with FITC-labeled Pseudomonas aeruginosa (MOI = 1:100) for 30 minutes at 37°C. Representative images from 3 independent experiments are shown. Dot plot depicts macrophage phagocytosis levels (n = 5 per group). B , Bacterial killing of P. aeruginosa in peritoneal macrophages (5 × 10 5 cells) treated with PBS or the indicated doses of rFGF8. Dot plot depicts macrophage killing (n = 4 per group). C , Mortality of rFGF8-treated (12.5 μg/kg) septic mice in the presence or absence of macrophage depletion after CLP (n = 10 per group). D , Bacterial loads in PLF and blood of rFGF8-treated (12.5 μg/kg) septic mice in the presence or absence of macrophage depletion after CLP (n = 5 per group). E , Survival after transfer of rFGF8- or PBS-treated peritoneal macrophages in mice (n = 12 per group) after CLP. A, B, and D , Data are representatives of 3 independent experiments; ( A ) nonparametric Mann-Whitney U test; ( B and D ) Kruskal-Wallis test followed by Dunn multiple comparisons posttest; ( C and E ) Kaplan-Meier analysis followed by log-rank test. * P < .05, ** P < .01, *** P < .001, **** P < .0001 compared within 2 groups. Abbreviations: CLP, cecal ligation and puncture; CFU, colony-forming unit; DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; MOI, multiplicity of infection; ns, not significant; PBS, phosphate-buffered saline; rFGF, recombinant fibroblast growth factor; TRITC, tetraethyl rhodamine isothiocyanate.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Labeling, MANN-WHITNEY, Ligation, Infection, Saline, Recombinant

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGFR1 plays a critical role in FGF8-induced protection against experimental sepsis. A , Representative confocal images of the colocalization of FGF8 (Cy3) and FGFR (FITC) in peritoneal macrophages treated with rFGF8 (200 ng/mL). Scale bar = 20 μm. B , Peritoneal macrophages were pretreated with or without rFGF8 (200 ng/mL) followed by incubation with or without heat-inactivated Pseudomonas aeruginosa. Representative fluorescence images of phospho-FGFR1 expression are shown. Scale bar = 25 μm. C , Peritoneal macrophages (n = 4 per group) were pretreated with or without the FGFR1 inhibitor PD173074 (100 nM) for 1 hour followed by incubation with or without rFGF8 (200 ng/mL). In vitro bacterial phagocytosis and killing of P. aeruginosa . D , Mortality after blocking FGFR1 with PD173074 (1 mg/kg) and subsequent treatment with rFGF8 (12.5 μg/kg) or PBS control after CLP (n = 15 per group). Except for survival ( D ), data are presented as means and are representative of 3 independent experiments; ( C ) Kruskal-Wallis test followed by Dunn multiple comparisons posttest; ( D ) Kaplan-Meier analysis followed by log-rank test. * P < .05, *** P < .001, **** P < .0001 compared within 2 groups. Abbreviations: CLP, cecal ligation and puncture; FGFR, FGF receptor; ns, not significant; rFGF, recombinant fibroblast growth factor; FITC, Fluorescein Isothiocyanate; Cy3, Cyanine 3.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Incubation, Fluorescence, Expressing, In Vitro, Blocking Assay, Control, Ligation, Recombinant

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGF8-enhanced antibacterial functions of macrophages are regulated by ERK1/2 signaling pathways. A , Peritoneal murine macrophages were treated with or without rFGF8 (200 ng/mL) for the indicated times and examined for the presence of phosphorylated ERK1/2, P38, STAT1, and Akt. Three independent experiments were performed with comparable results and representative blots are shown. B , Effects of rFGF8 on the activation of MAPK, STAT, PI3 K signaling pathways in murine macrophages. Peritoneal macrophages were treated with or without rFGF8 (200 ng/mL) and then incubated with heat-inactivated Pseudomonas aeruginosa . Total cellular proteins were extracted from murine macrophages for the detection of phosphorylated ERK1/2, P38, STAT1, and Akt with the indicated antibodies by western blot analysis. Experiments were performed in 3 independent experiments with consistent results and representative blots are shown. Peritoneal macrophages (n = 4 per group) were pretreated with the ERK1/2 inhibitor U0126 (20 μM) for 1 hour followed by incubation with or without rFGF8 (200 ng/mL). C , Analysis of in vitro bacterial phagocytosis and killing of P. aeruginosa . D , Mortality after blocking ERK1/2 signaling pathway with U0126 (10 mg/kg) and subsequent treatment with rFGF8 (12.5 μg/kg) or PBS control after CLP (n = 20 per group). Except for survival rate ( D ), data are presented as means and are representative of 3 independent experiments; ( C ) Kruskal-Wallis test followed by Dunn multiple comparisons posttest; ( D ) Kaplan-Meier analysis followed by log-rank test. * P < .05, ** P < .01, **** P < .0001 compared within 2 groups. Abbreviations: CFU, colony-forming unit; CLP, cecal ligation and puncture; DMSO, dimethyl sulfoxide; ns, not significant; PBS, phosphate-buffered saline; rFGF, recombinant fibroblast growth factor.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Protein-Protein interactions, Activation Assay, Incubation, Western Blot, In Vitro, Blocking Assay, Control, Ligation, Saline, Recombinant

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGF8 is a candidate biomarker for sepsis. A , Levels of FGF8 at admission measured by ELISA in serum samples collected from 73 adult patients with sepsis, 96 child patients with sepsis, and corresponding healthy controls. B , Receiver operating characteristic analysis of FGF8 for diagnosis of sepsis (AUC = 0.89 for adult and AUC = 0.81 for children). C , Comparison of serum FGF8 levels between male and female patients with sepsis and healthy controls. D , Comparison of serum FGF8 levels between adults and children in healthy controls and patients with sepsis. A , C , and D , Nonparametric Mann-Whitney U test. **** P < .0001 compared within 2 groups. Abbreviations: AUC, area under the curve; CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; ns, not significant.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Comparison, MANN-WHITNEY

Journal: Biology Open

Article Title: FGF8 morphogen gradients are differentially regulated by heparan sulphotransferases Hs2st and Hs6st1 in the developing brain

doi: 10.1242/bio.028605

Figure Lengend Snippet: Differential HS sulphation regulates [Fgf8] gradient formation with differential kinetics. (A) Fgf8 mRNA is located at the CSB angle (arrowhead). This image is also shown enlarged in Fig. 3 C. Sep, septum; Ctx, cortex; GE, ganglionic eminences. (B) Schematic of ex vivo explant culture with Fgf8-infused bead implanted at the CSB angle (arrowhead). (C) Fgf8 immunofluorescence (here shown as greyscale) illustrating concentric rings (yellow) used to quantify [Fgf8] at increasing distance (d) from the Fgf8-bead edge. (D-G) Representative images of Fgf8 immunofluorescence (red) in sections though cultured explants under different conditions and time-points indicated next to panels, asterisk marks bead centre. (H-K) [Fgf8] gradient up to 200 µm from the bead. Data for 1, 2, and 4 h time-points coloured black, red, and blue respectively with BSA control data coloured green, asterisks indicate significant difference between each particular condition with its corresponding WT (Kolmogorov–Smirnov test; P <0.05 following a Bonferroni correction for multiple pairwise comparisons). (L-O) Total Fgf8 level within 200 µm of the bead. Cross indicates significant differences between bracketed time-points within a culture condition indicating fluctuating amplitude through time, while asterisks indicate significant difference at a particular time-point between each particular condition and WT (two-way ANOVA followed by Holm-Sidak post hoc test; P <0.05). (P-S) Fitted curve of H-I in log 10 [Fgf8] vs. log 10 [d] . Each plot shows all data points and average line of best fit (solid line) with dotted line indicating extrapolation to log­ 10 [Fgf8] axis with numbers on indicating intercept and underlined numbers the slope. Number of explants analysed (N): WT , 8; Heparanase, 5; Hs2st −/− , 5; Hs6st1 −/− , 4. Scale bar in D applies to D-G: 100 µm. In H-S values are shown as mean±s.e.m.

Article Snippet: Primary antibodies: rabbit anti-phospho-MAPK1/2 (1/200) (D13.14.4E) (Cell Signalling Technology); mouse rabbit monoclonal antibody anti-Fgf receptor 1 (1/200) (D8E4) (Cell Signalling Technology); mouse anti-10E4 (1/200) (370255-1) (Amsbio); anti-Fgf8 mouse monoclonal antibody (1/2500) (MAB323) (R&D Systems).

Techniques: Ex Vivo, Immunofluorescence, Cell Culture, Control

Journal: Biology Open

Article Title: FGF8 morphogen gradients are differentially regulated by heparan sulphotransferases Hs2st and Hs6st1 in the developing brain

doi: 10.1242/bio.028605

Figure Lengend Snippet: Differential HS sulphation differentially regulate Erk response to Fgf8. (A-D) Representative images of pErk immunofluorescence in sections though cultured explants representing different conditions and time-points indicated on panels, asterisk marks bead centre. (E-H) [pErk] gradient formed at different time points up to 200 µm from the bead. Asterisks indicate significant difference between each particular condition with its corresponding WT [Kolmogorov–Smirnov test ( P <0.05) following a Bonferroni correction for multiple pairwise comparisons]. (I-L) [Fgf8]/[pErk] dose response curves. Number of explants analysed: WT , 8; Heparanase, 5; Hs2st −/− , 5; Hs6st1 −/− , 4. Data for 1, 2, and 4 h time-points coloured black, red, and blue respectively. Scale bar in D applies to A-D: 100 µm. In E-L values are shown as mean±s.e.m.

Article Snippet: Primary antibodies: rabbit anti-phospho-MAPK1/2 (1/200) (D13.14.4E) (Cell Signalling Technology); mouse rabbit monoclonal antibody anti-Fgf receptor 1 (1/200) (D8E4) (Cell Signalling Technology); mouse anti-10E4 (1/200) (370255-1) (Amsbio); anti-Fgf8 mouse monoclonal antibody (1/2500) (MAB323) (R&D Systems).

Techniques: Immunofluorescence, Cell Culture

Journal: Biology Open

Article Title: FGF8 morphogen gradients are differentially regulated by heparan sulphotransferases Hs2st and Hs6st1 in the developing brain

doi: 10.1242/bio.028605

Figure Lengend Snippet: Analysis of Fgf8/Erk signalling components in the CSB region of E14.5. WT , Hs2st −/− , and Hs6st1 −/− embryos show correlation to Hs2st and Hs6st1 action ex vivo . (A) Hs2st and (B) Hs6st1 expression visualized by LacZ staining of Hs2st LacZ/+ and Hs6st1 LacZ/+ sections, respectively. Cross marks an area with very low Hs6st1 LacZ expression, N =4. (C-E) Fgf8 mRNA expression. N =5. Note that the image in C is an enlargement of that shown in Fig. 1 A. (F-H) Fgf8 protein expression. N =3. (I-K) Higher magnification of VZ angle in F-H. (L) Quantification of Fgf8 fluorescent intensities in I-K. P values are as depicted on graph. Error bars indicate standard error of mean. (M) Dose response of WT , Hs2st −/− , Hs6st1 −/− tissue showing increased sensitivity to Fgf8 in Hs2st −/− while Hs6st1 −/− tissue have decreased sensitivity to Fgf8 when compared to WT tissue. (N,O) Expression of Fgfr1 protein. N =4 for WT , N =3 for Hs2st −/− and Hs6st1 −/− . (Q-S) pErk protein expression. N =3. (T-V) Higher magnification of ventricular zone in Q-S. In A, B, C-K and N-V arrowheads indicate apical surface of the ventricular zone at the CSB angle; arrows in H, P, S indicate Fgf8 High , Fgfr1 Low , pErk Low septal area in Hs6st1 −/− embryos. N =3. Ctx, cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; Sep, septum. Scale bars: A applies also to B: 200 µm; C applies to C-E: 100 µm; F applies to F-H: 100 µm; I applies to I-K: 100 µm; N applies to N-P: 100 µm; and Q applies to Q-S: 100 µm; T applies to T-V: 100 µm.

Article Snippet: Primary antibodies: rabbit anti-phospho-MAPK1/2 (1/200) (D13.14.4E) (Cell Signalling Technology); mouse rabbit monoclonal antibody anti-Fgf receptor 1 (1/200) (D8E4) (Cell Signalling Technology); mouse anti-10E4 (1/200) (370255-1) (Amsbio); anti-Fgf8 mouse monoclonal antibody (1/2500) (MAB323) (R&D Systems).

Techniques: Ex Vivo, Expressing, Staining