mouse c5a receptor  (Hycult Biotech)

Journal: The Journal of General Virology

Article Title: MERS-CoV infection causes brain damage in human DPP4-transgenic mice through complement-mediated inflammation

doi: 10.1099/jgv.0.001667

Figure Lengend Snippet: Anti-C5aR1 antibody treatment decreased inflammation and viral spread in brains of hDPP4-transgenic mice. Representative images of immunohistochemical staining of cleaved caspase-3 ( a, b ), phosphorylated P38 ( c, d ), IBA-1 ( e, f ), and antiviral NP ( g, h ) in similar regions of cerebral cortex in brains of hDPP4 transgenic mice 7 days after infection with MERS-CoV and treatment with anti-C5aR1 monoclonal antibody. Fewer immunopositive cells were detected in the anti-C5aR1 antibody treatment group ( n =3 per group).

Article Snippet: Mice were treated intravenously (600 µg kg −1 ) with a monoclonal antibody (mAb) to the mouse C5a receptor (C5aR1, HM1076; Hycult Biotech, PB Uden, The Netherlands) for complement inhibition immediately after virus challenge or with the same volume of isotype antibody (HI4041, Hycult Biotech, PB Uden, The Netherlands) as a control.

Techniques: Transgenic Assay, Immunohistochemical staining, Staining, Infection

Journal: The Journal of General Virology

Article Title: MERS-CoV infection causes brain damage in human DPP4-transgenic mice through complement-mediated inflammation

doi: 10.1099/jgv.0.001667

Figure Lengend Snippet: Anti-C5aR1 antibody treatment decreased brain damage in hDPP4 transgenic mice. ( a, b ) Representative images of H&E staining of brain sections of hDPP4-transgenic mice 7 days after infection with MERS-CoV and treatment with anti-C5aR1 or sham control. The representative image of the brains in anti-C5aR1 treatment mice showed less oedema, fewer infiltrating inflammatory cells, especially around vessels in the cerebellum compared to those receiving sham treatment. ( c, d ) Evans blue staining of mice brain on day 7. The brain of a MERS-CoV-infected mouse appeared blue compared with that of a mouse treated with anti-C5aR1 antibody. ( e–h ) Representative images of immunohistochemical staining for neutrophil infiltration ( e, f ) and NF-κB localization ( g, h ). ( i, j ) Semiquantitative analysis of brain damage via H&E scores ( i ) and neutrophil infiltration ( j ). #, Undetectable; ** P <0.01 (Student’s t -test with Welch’s correction)

Article Snippet: Mice were treated intravenously (600 µg kg −1 ) with a monoclonal antibody (mAb) to the mouse C5a receptor (C5aR1, HM1076; Hycult Biotech, PB Uden, The Netherlands) for complement inhibition immediately after virus challenge or with the same volume of isotype antibody (HI4041, Hycult Biotech, PB Uden, The Netherlands) as a control.

Techniques: Transgenic Assay, Staining, Infection, Immunohistochemical staining

Journal: The Journal of General Virology

Article Title: MERS-CoV infection causes brain damage in human DPP4-transgenic mice through complement-mediated inflammation

doi: 10.1099/jgv.0.001667

Figure Lengend Snippet: Diagram illustrating damage to brain tissues in human DPP4-transgenic mice. Neurons infected by MERS-CoV secrete complement components which could activate microglia, which, in turn, could also secrete complement components in brain. Excessive complement activation could activate the endothelial cells of BBB, enhancing the infiltration of inflammatory cells, such as neutrophils and macrophages, into brain parenchyma. The infiltrated inflammatory cells secrete proinflammatory cytokines which could further enhance neuronal damage. However, the inhibition of C5a-C5aR1 interaction could inhibit BBB damage and decrease second damage owing to the excessive inflammatory response.

Article Snippet: Mice were treated intravenously (600 µg kg −1 ) with a monoclonal antibody (mAb) to the mouse C5a receptor (C5aR1, HM1076; Hycult Biotech, PB Uden, The Netherlands) for complement inhibition immediately after virus challenge or with the same volume of isotype antibody (HI4041, Hycult Biotech, PB Uden, The Netherlands) as a control.

Techniques: Transgenic Assay, Infection, Activation Assay, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Mechanisms Involved in the Promoting Activity of Fibroblasts in HTLV-1-Mediated Lymphomagenesis: Insights into the Plasticity of Lymphomatous Cells

doi: 10.3390/ijms221910562

Figure Lengend Snippet: Characterization of HFF and C91/PL cells after short-term transwell coculture. ( a ) Flow cytometry analysis of CD10 and GPR77 expression in HFF in normal culture conditions (upper panels) and after 24 h of coculture with C91/PL cells (lower panels). Fibroblasts were stained with anti-CD10 and anti-GPR77 monoclonal antibodies; isotype controls (left-hand side panels) were used to determine background staining. A representative example of one of the four independent experiments is shown. The percentage of indicated CD10 + GPR77 + HFF is enlarged. ( b ) Aldehyde dehydrogenase 1 (ALDH1) activity in C91/PL cells. Control C91/PL cells and HFF-cocultured C91/PL cells were collected at different time points and analysed for ALDH1 activity. Representative dot plots are shown for C91/PL cells alone (upper panels) or after 48 h of coculture with HFF (lower panels). N,N-diethylaminobenzaldehyde (DEAB) inhibitor was used to provide a negative control (left-hand side panels) for threshold set up. Flow cytometry data are shown as ALDH1 activity vs side scatter signal (SSC).

Article Snippet: HFF were analysed with the following anti-human monoclonal antibodies: allophycocyanin (APC)—conjugated mouse anti-human CD10 (clone eBioCB-CALLA, eBioscience, Thermo Fisher Scientific) in combination with phycoerythrin (PE)—conjugated mouse anti-human C5a receptor-like 2 (GPR77 or C5L2, clone 1D9-M12, BioLegend, San Diego, CA, USA) and, in parallel, with APC-IgG2b, k (eBioscience) and PE-IgG2a, k (BioLegend) isotype controls.

Techniques: Flow Cytometry, Expressing, Staining, Activity Assay, Control, Negative Control

Journal: Cancer research

Article Title: Complement activation via a C3a receptor pathway alters CD4+ T lymphocytes and mediates lung cancer progression

doi: 10.1158/0008-5472.CAN-17-0240

Figure Lengend Snippet: Age matched WT and C3−/− mice were injected with CMT-luc tumors, and the primary tumors, organs, and whole blood were collected at the terminal sacrifice 28 days post-injection. (A) ELISA specific for murine C3a was used to measure C3a in the plasma samples collected from naïve or tumor-bearing WT and C3−/− mice (Naïve WT n = 3; WT+CMT-luc n = 5; naïve C3−/− n = 4; C3−/−+CMT-luc n = 9). (B) Immunofluorescence co-staining for C3 (green) and IgM (red) or C3 (green) and C4 (red) in the primary tumor specimens from WT mice were evaluated by confocal microscopy. (C) Primary tumor volumes 10 or 28 days after CMT-luc implantation in WT or C3−/− mice are shown. (Day 10: WT n = 6 and C3−/− n = 6; Day 28: WT n = 10 and C3−/− n = 9) (D) Number of metastases to the secondary pulmonary space were counted from ex vivo images that captured the luciferase activities of CMT-luc metastases. (WT n = 11; C3−/− n = 9) (E) Flank tumor volumes 28 days after CMT-luc implantation in WT or C3−/− are shown (WT n = 31; Day 28 C3−/− n = 12) (F-G) WT mice are administered with (F) C3a receptor antagonist (C3aRA; SB290157) or (G) PMX-53 (C5a receptor antagonist, C5aRA), starting a day prior to tumor implantation into WT mice. Primary tumor volumes 28 days after tumor implantation in the treated groups and vehicle or control peptide group are shown (F, n = 4 and G, n = 10 each group). *p <0.05. Error bars represent mean ± SEM.

Article Snippet: C3a/C5a Receptor Antagonist Treatment Mice were intraperitoneally injected daily with PBS or C3aRA, SB290157, trifluoroacetate salt (1mg/kg, Cayman Chemical Company, 15783), starting a day prior to tumor implantation.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Confocal Microscopy, Ex Vivo, Luciferase, Tumor Implantation

Journal: Cancer research

Article Title: Complement activation via a C3a receptor pathway alters CD4+ T lymphocytes and mediates lung cancer progression

doi: 10.1158/0008-5472.CAN-17-0240

Figure Lengend Snippet: Age and gender-matched WT and C3−/− mice were injected with LLC-luc tumors, and primary tumors, organs, and whole blood were collected at the terminal sacrifice 21 days post-injection. (A) ELISA specific for murine C3a was used to measure C3a in the plasma samples collected from naïve or LLC-luc tumor-bearing WT and C3−/− mice (Naïve WT n = 3; WT+LLC-luc n = 21; naïve C3−/− n = 4; C3−/−+LLC-luc n = 4). Same data from naïve WT and C3−/− mice are used in this graph. (B) Immunofluorescence co-staining for C3 (green) and IgM (red) or C3 (green) and C4 (red) in tumor specimens induced by LLC-luc from C3−/− were evaluated by confocal microscopy. (C) Primary tumor volumes 21 days after LLC-luc implantation in WT or C3−/− mice are shown (WT n = 18; C3−/− n = 11). (D and E) Number of metastases to the secondary pulmonary space and liver were counted from ex vivo images that captured the luciferase activities of LLC-luc metastases (Pulmonary metastasis - WT n = 18; C3−/− n = 11 and Liver metastasis - WT n = 14; C3−/− n = 9). (F-G) WT mice are administered with (F) C3a receptor antagonist (C3aRA; SB290157) or (G) PMX-53 (C5a receptor antagonist, C5aRA), starting a day prior to tumor implantation into WT mice. Primary tumor volumes 21 days after tumor implantation in both treated groups and vehicle or control peptide group are shown (F, n = 5 and G, n = 5 each group). *p <0.05. Error bars represent mean ± SEM.

Article Snippet: C3a/C5a Receptor Antagonist Treatment Mice were intraperitoneally injected daily with PBS or C3aRA, SB290157, trifluoroacetate salt (1mg/kg, Cayman Chemical Company, 15783), starting a day prior to tumor implantation.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Confocal Microscopy, Ex Vivo, Luciferase, Tumor Implantation

Journal: The Journal of Immunology Author Choice

Article Title: Endoplasmic Reticulum Stress of Neutrophils Is Required for Ischemia/Reperfusion–Induced Acute Lung Injury

doi: 10.4049/jimmunol.1500073

Figure Lengend Snippet: C5a interacting with C5aR induces ER stress in neutrophils. (A) Neutrophils prepared from mouse peripheral blood or murine myeloid cell line 32Dcl3 were lysed for immunoblotting with C5aR Ab. Images shown are representative of three experiments. Band intensities were determined with ImageJ. (B) Murine myeloid cells (32Dcl3) were transiently transfected with siC5aR overnight and then lysed. Lysates were analyzed by immunoblotting with C5aR Ab. Images shown are representatives of three experiments. (C) Murine myeloid cells (32Dcl3) were transiently transfected with siC5aR overnight, followed by treatment with C5a and/or C5a neutralizing Ab for 4 h and then lysed. Lysates were analyzed by immunoblotting with indicated Abs. Images shown are representatives of three experiments. (D) Supernatants from cultured neutrophils described as in (C) were collected, and MPO was measured using ELISA. Data are expressed as mean ± SD of three independent experiments. *p < 0.05.

Article Snippet: The anti-mouse complement 5a (C5a) receptor (C5aR) Ab was obtained from OriGene (Rockville, MD).

Techniques: Western Blot, Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay