Journal: The FASEB Journal

Article Title: Inhibition of biomaterial-induced complement activation attenuates the inflammatory host response to implantation

doi: 10.1096/fj.12-225888

Figure Lengend Snippet: C5aR signaling is involved in shaping the host immune response after deep-tissue implantation of PP mesh. A) Image of a PP mesh on the day of implantation into the peritoneal space (D0) and on the day of its retrieval (D5). B, C) Levels of granulocytes (B) and macrophages (C) isolated from the tissue surrounding the PP mesh, 5 d after implantation in the peritoneal space of C5aR−/− and C5aR+/+ mice, as assessed by flow cytometry (n=7/strain). D, E) Effect of C5aR antagonism on granulocyte (D) and macrophage (E) accumulation around the implanted mesh in WT mice treated with either C5aRa or an inactive analog (control C5aRa) (n=4/treatment). F, G) Histological analysis of the meshes retrieved from WT mice treated with either C5aRa or inactive control compound. Cell density in the stroma (F) and mesh interface (G) was evaluated in H&E-stained sections (n=4/treatment); cell density is expressed as described for Fig. 3B, C. H) Immunofluorescent staining for C3 cleavage products in tissue sections retrieved from WT mice 5 d after implantation. Bright green staining indicates the presence of deposited C3c fragments at the interface. Incubation of slides with PBS, instead of anti-C3c Ab, was used as a negative control. *P < 0.05; unpaired t test.

Article Snippet: For the complement inhibition studies, blood samples were preincubated with 20 μM of the compstatin analog Cp20 (AcI[CVW(Me)QDWSarAHRC]mI-NH 2 ), linear inactive peptide (IAVVQDWGHHRAT-NH 2 ; ref. 21 , 22 ), 10 μM of the C5a receptor antagonist PMX53 (AcF-[OPdChaWR]; synthesized by GL Biochem, Shanghai, China; ref. 23 ), or its inactive control [AcdChaPWFRO-NH 2 ; synthesized in house], for 5 min prior to biomaterial addition.

Techniques: Isolation, Flow Cytometry, Control, Staining, Incubation, Negative Control

Journal: Oncotarget

Article Title: Overexpression of complement component C5a accelerates the development of atherosclerosis in ApoE-knockout mice

doi: 10.18632/oncotarget.11180

Figure Lengend Snippet: A. , Fluorescence images of HEK293 cells after transfection with phosphate buffered saline (PBS) or different multiplicities of infection (MOI) of adenovirus C5a (Ad-C5a) for 24 hr. B. , Migration assay of chemotaxis of recombinant C5a to macrophages. HEK293 cells were transfected with Ad-GFP and different MOI (1:1, 10:1 and 100:1) of Ad-C5a. At 24 hr, the supernatants underwent trans-well assay. C. , Quantitative analysis of trans-well assay. Data are mean ± SEM from 5 separate fields in each sample from 3 independent experiments. ** P < 0.01. D. , Detection of recombinant mouse C5a protein in ApoE −/− mouse plasma ( n = 5). * P < 0.05 and ** P < 0.01 vs day 0.

Article Snippet: The C5a receptor antagonist AcF [OPdChaWR] was synthesized by GL Biochem (Shanghai).

Techniques: Fluorescence, Transfection, Saline, Infection, Migration, Chemotaxis Assay, Recombinant, Clinical Proteomics

Journal: Oncotarget

Article Title: Overexpression of complement component C5a accelerates the development of atherosclerosis in ApoE-knockout mice

doi: 10.18632/oncotarget.11180

Figure Lengend Snippet: A. , Quantitative analysis of atherosclerotic lesions by en face staining after C5a receptor antagonist treatment. B. and C. , Quantitative analysis of atherosclerotic lesions by en face staining (B) and in aortic roots (C) after a high fat diet for 8 weeks. D. and E. , Representative photomicrographs showing atherosclerotic lesions on the surface of aorta ( en face staining, D) and aortic roots (E). F. and G. Quantitative analysis of atherosclerotic lesions by en face staining (G) and in aortic roots (F). Data are mean±SEM in 6 sections for each mouse sample. N = 12 per group. *P < 0.05, ** P < 0.01.

Article Snippet: The C5a receptor antagonist AcF [OPdChaWR] was synthesized by GL Biochem (Shanghai).

Techniques: Staining

Journal: Oncotarget

Article Title: Overexpression of complement component C5a accelerates the development of atherosclerosis in ApoE-knockout mice

doi: 10.18632/oncotarget.11180

Figure Lengend Snippet: A. , Representative photomicrographs of atherosclerotic plaque stained for macrophages in the aortic root of ApoE −/− mice. B. , Morphometric analysis of stained areas for macrophages. Data are mean±SEM percentage positive area to total lesion area in 6 sections for each mouse. N = 8 per group. * P < 0.05. C. , Migration assay of chemotaxis of 200 nM C5a or cell culture supernatant to macrophages. HEK293 cells were treated with 100:1 MOI Ad-C5a for 24 hr. The supernatant, 200 nM C5a and different concentrations of C5a receptor antagonist were used in the trans-well assay. D. , Quantitative analysis of trans-well assay. Data are mean ± SEM from 5 separate fields in each sample from 3 independent experiments. ** P < 0.01.

Article Snippet: The C5a receptor antagonist AcF [OPdChaWR] was synthesized by GL Biochem (Shanghai).

Techniques: Staining, Migration, Chemotaxis Assay, Cell Culture

Journal: Oncotarget

Article Title: Overexpression of complement component C5a accelerates the development of atherosclerosis in ApoE-knockout mice

doi: 10.18632/oncotarget.11180

Figure Lengend Snippet: Body weight and serum cholesterol level of ApoE −/− mice

Article Snippet: The C5a receptor antagonist AcF [OPdChaWR] was synthesized by GL Biochem (Shanghai).

Techniques:

Journal: Oncotarget

Article Title: Overexpression of complement component C5a accelerates the development of atherosclerosis in ApoE-knockout mice

doi: 10.18632/oncotarget.11180

Figure Lengend Snippet: Serum from blood samples was taken 14 days after Ad-C5a injection. ELISA of the serum level of interferon γ (IFN-γ) A. , interleukin 6 (IL-6) B. and tumor necrosis factor α (TNF-α) C. N = 5 mice per treatment group. Data are means ± SEM. ** P < 0.01.

Article Snippet: The C5a receptor antagonist AcF [OPdChaWR] was synthesized by GL Biochem (Shanghai).

Techniques: Injection, Enzyme-linked Immunosorbent Assay