Journal:

Article Title: Proinflammatory functions of vascular endothelial growth factor in alloimmunity

doi: 10.1172/JCI200317712

Figure Lengend Snippet: Effect of VEGF on endothelial cell chemokine expression. (a–d) Confluent cultures of human endothelial cells were treated for 4 hours (a and c) or as a time course (b and d) with VEGF alone or VEGF in combination with IFN-γ as indicated. Total RNA was harvested from endothelial cells and the expression of chemokines was analyzed by RNase protection assay. Concentration- (a) and time-dependent (b) effects of VEGF on chemokine expression. (c and d) Effect of IFN-γ alone or in combination with VEGF on chemokine expression. Note that treatment with IFN-γ alone resulted in IP-10 expression (c, and d lane 7), and a combination of IFN-γ with VEGF resulted in a synergistic induction of IP-10 (c, and d lanes 2–6). Bar graphs to the right of each blot represent the quantitative analysis of IP-10 mRNA expression in three representative RNase protection assays as illustrated in c and d. (e) The production of IP-10 by ELISA in culture supernatants of endothelial cells treated with IFN-γ (1,000 U/ml; black bars) or with VEGF (10 ng/ml) and IFN-γ (1,000 U/ml) (white bars) for different times as indicated. Representative of three similar experiments performed in triplicate (mean ± 1 SD).

Article Snippet: The recombinant human cytokines IFN-γ and human VEGF 165 were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Expressing, Rnase Protection Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Proinflammatory functions of vascular endothelial growth factor in alloimmunity

doi: 10.1172/JCI200317712

Figure Lengend Snippet: Function of VEGF in alloimmune T cell activation and allograft rejection. (a) Anti–human VEGF or anti–murine VEGF antiserum was added into the human or the mouse MLR, respectively. Proliferation was assessed by [3H]thymidine incorporation for the last 18 hours of coculture. (b) The production of IFN-γ and IL-2 was assessed by ELISA in coculture supernatants from a human MLR. As illustrated, blockade of VEGF had no effect on proliferation or cytokine production in the MLR. Bars indicate the mean ± 1 SD for triplicate wells. Data are representative of three experiments with similar results. S, stimulators alone; R, responders alone. (c) Frequency of IFN-γ-producing cells in murine recipients of cardiac transplants as assessed by ELISPOT. Illust production of IFN-γ from a syngeneic, an untreated, and an anti-VEGF-treated animal. Representative of three such experiments performed in triplicate.

Article Snippet: The recombinant human cytokines IFN-γ and human VEGF 165 were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal:

Article Title: Proinflammatory functions of vascular endothelial growth factor in alloimmunity

doi: 10.1172/JCI200317712

Figure Lengend Snippet: Function of VEGF-dependent regulation of IP-10 in allograft rejection. (a) Confluent cultures of murine myocardial endothelial cells were treated for 4 hours with recombinant murine VEGF or IFN-γ alone or VEGF in combination with IFN-γ as indicated. Total RNA was harvested from endothelial cells and the expression of IP-10 was analyzed by RNase protection assay. (b and c) The ability of VEGF to mediate IP-10-dependent trafficking and rejection was evaluated using anti-VEGF and anti–IP-10 in fully MHC-mismatched C57BL/6 (H-2b) or IP-10–/– (H-2b) donor hearts transplanted into BALB/c (H-2d) mice. Recipients of wild-type grafts were treated with anti-VEGF alone, or with anti-VEGF in combination with anti–IP-10. Recipients of IP-10–/– donor grafts were treated with anti-VEGF. Both anti–IP-10 and anti-VEGF were administered according to the schedule outlined in Methods. As illustrated in b, we found that addition of anti-VEGF with anti–IP-10 significantly prolonged allograft survival in wild-type combinations (P < 0.005); and in c, anti-VEGF prolonged survival in mice that received IP-10–/– donor hearts (P < 0.04). The survival of control untreated wild-type grafts are illustrated by the dotted line.

Article Snippet: The recombinant human cytokines IFN-γ and human VEGF 165 were purchased from R&D Systems (Minneapolis, Minnesota, USA).

Techniques: Recombinant, Expressing, Rnase Protection Assay

Journal:

Article Title: Calprotectin Expression by Gingival Epithelial Cells

doi: 10.1128/IAI.69.5.3248-3254.2001

Figure Lengend Snippet: Treatment of immortalized keratinocytes with IL-1β (0.1, 1, and 10 ng/ml) for 2 or 4 h. Immortalized keratinocytes were incubated with various concentrations of IL-1β (0.1, 1, and 10 ng/ml) for 2 h (black bars) or 4 h (crosshatched bars). Expression of mRNA for IL-8-, MRP8-, and MRP14-specific mRNA was determined by RNase protection assay. The signal intensity for the protected fragments was normalized to the abundance of the internal control GAPDH. The data shown are the mean ± the standard deviation (n = 3).

Article Snippet: Immortalized keratinocytes were stimulated by incubation with lipopolysaccharide from E. coli O55:B5 (Sigma, St. Louis, Mo.), phorbol 12-myristate 13-acetate (PMA; Sigma) or recombinant human IL-1β (R&D Systems, Inc., St. Paul, Minn.).

Techniques: Incubation, Expressing, Rnase Protection Assay, Standard Deviation

Journal: Physiological Reports

Article Title: Salivary vascular growth factor responses to prolonged and interrupted sitting in young, healthy adults

doi: 10.14814/phy2.70798

Figure Lengend Snippet: Changes in salivary growth factor concentrations during prolonged sitting (PS), low‐intensity (LI), and moderate‐intensity (MI) interruption sessions. Box‐and‐whisker plots display pre‐ (Pre, 0 h) and post‐session (Post, 4 h) concentrations for (a) VEGF, (b) EGF, and (c) angiogenin. Panel (d) shows pre‐post percent change for all three biomarkers. The line within each box represents the median; whiskers indicate minimum and maximum values. VEGF decreased significantly in LI and MI but not PS; EGF showed no significant changes; and angiogenin increased significantly in all conditions. No significant differences were observed between conditions in the magnitude of pre‐post change.

Article Snippet: Samples were then analyzed using sandwich Enzyme‐Linked Immunoabsorbent Assays (ELISA) to examine EGF (Human EGF DuoSet ELISA, R&D Systems Inc.), VEGF (Human VEGF DuoSet ELISA, R&D Systems Inc.), and angiogenin (Human Angiogenin DuoSet ELISA, R&D Systems Inc.) following the manufacturer's protocol with absorbance measured at 450 nm using a Cytation 5 microplate reader (Bio‐Tek Instrumentation, VT, USA).

Techniques: Whisker Assay