Journal: Pathogens

Article Title: Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin

doi: 10.3390/pathogens14040354

Figure Lengend Snippet: Inhibition of C3b and C4b deposition by r Em CRT and its functional binding regions measured by ELISA. ( A ) Pre-incubation of 100 μL C1qD (1:50) as source of natural MBL with different amounts of r Em CRT, r Em CRT-S, or r Em CRT-NP (0, 2, 4 μM) before adding into mannan-coated plates (50 μg/mL). After being washed, C1qD (1:150) was added as source of complement components to initiate lectin pathway of complement activation. Deposition of C3b ( a ) and C4b ( b ) was detected with anti-C3 or C4 monoclonal antibodies. ( B ) The similar ELISA procedure was performed using NHS as source of physiological MBL instead of C1qD serum to detect the generation of C3b (a) and C4b (b). Data are expressed as mean of OD 450 ± SDs of three independent experiments (* p < 0.005, ** p < 0.001, *** p < 0.0005, ns, no significant difference compared to plates without added r Em CRT).

Article Snippet: After washing with 1 × TBST containing 5 mM CaCl 2 , the C1qD diluted at 1:150 in 1 × Veronal Buffer (VB, Lonza, Basel, Switzerland) containing 0.1% gelatin, 0.05% Tween-20 was added (100 μL) as supplement of other complement components (without C1q) into each well of the plates and incubated at 37 °C for 1 h. The lectin pathway-activated C3b/C4b deposition was detected with rabbit anti-C3b monoclonal antibody (1:1000, BOSTER Biological Technology, Wuhan, China) and goat anti-C4b polyclonal antibody (1:3000, Abcam, Cambridge, UK).

Techniques: Inhibition, Functional Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay, Bioprocessing

Journal: Journal of Clinical Investigation

Article Title: Endothelial C3a receptor mediates vascular inflammation and blood-brain barrier permeability during aging

doi: 10.1172/jci140966

Figure Lengend Snippet: Figure 1. C3a/C3aR signaling regulates age-associated endothelial VCAM1 expression. (A) ELISA measurement of C3 levels in WT mouse brain lysates at 2, 12, and 20 months (n = 8/group). (B) Immunofluorescence staining using anti-GFAP and anti-C3 antibodies demonstrated localization of C3 to astrocytes. (C) Quantification confirming increased C3 staining within GFAP + astrocytes in the hippocampus with age (n = 5/age). (D) Triple immunostaining of isolated vessels from WT and C3ar1 –/– brains using anti-GFAP, anti–VE-cadherin, and anti-C3aR antibodies showing positive C3aR staining along endothelial cell surface that was not present in C3ar1–/– vessels. (E) Triple immunostaining of brain tissue with anti-Glut1, anti-C3aR, and anti-CD31 or anti–PDGFR-β, anti-C3aR, and anti–Col IV, demonstrating expression of C3aR on brain endothelial cells but not pericytes. (F and G) Immunofluorescence staining and quantification using anti-CD31 and anti-VCAM1 antibodies of WT or C3ar1–/– mouse cortices at 2, 12, and 20 months demonstrated an increase in VCAM1 with age in WT mice, but VCAM1 was rescued in the absence of C3aR. All data represent the mean ± SEM. Significance was calculated using 1-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Scale bars: 20 μm (B), 10 μm (D), 15 μm (E), and 50 μm (F).

Article Snippet: Confluent cells were treated with IL-1β (10 ng/mL, R&D Systems, 201-LB-005), C3a (500 nM, R&D Systems, 3677-C3-025), C5a (250 nM, R&D Systems, 2037-C5-025), ionomycin (10 μM, Cayman Chemical, 10004974), or in combination with one of the inhibitors SB290157 (5 μM, Calbiochem, 559410), W7 (50 μM, Tocris, 0369) or BAPTA-AM (1 μM, Tocris, 2787).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Triple Immunostaining, Isolation

Journal: Theranostics

Article Title: C5aR1 is a master regulator in Colorectal Tumorigenesis via Immune modulation

doi: 10.7150/thno.45058

Figure Lengend Snippet: The strong correlation between complement activation and CRC. ( A-D ) The associations between the mRNA levels of C3 (A), C5 (B), C5AR1 (C), and C5AR2 (D) and the overall survival of CRC patients (n=364). ( E, F ) The effect of C3 , C5 , C5ar1 or C5ar2 deficiency on AOM/DSS-induced colorectal tumorigenesis compared to WT control. Images of the colorectum, where CRC often developed at distal (rectum) sites (E), and quantitative analysis of tumor number and mass (F). The experiment was duplicated. Scale bar in (E), 1 cm. ( G ) Pathological analysis of control or tumor tissues, including H&E, Ki-67 and C3d staining. Scale bar, 100 µm for H&E and 50 µm for IHC. ( H ) C5a concentration in colon tissue homogenates (CTHs) measured by ELISA. n≥ 7 in each group of WT, C3 -KO, C5 -KO, C5ar1 -KO, or C5ar2 -KO mice. Data are represented as mean ± SEM; ns, not significant; * P <0.05; and *** P <0.001. NC, negative control.

Article Snippet: Then, mouse colon sections were incubated with an affinity purified pAb specific for mouse complement component C3d (1:80; R&D Systems, Minneapolis, MN), rabbit anti-Ki67 antibody (1 μg/ml; Abcam, Cambridge, MA) or rabbit anti-CD8 alpha antibody (1:1500; Abcam, Cambridge, MA) at 4°C overnight.

Techniques: Activation Assay, Control, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: PLoS ONE

Article Title: Inhibition of C3 Convertase Activity by Hepatitis C Virus as an Additional Lesion in the Regulation of Complement Components

doi: 10.1371/journal.pone.0101422

Figure Lengend Snippet: (A) Comparison of serum C3 convertase level in randomly chosen HCV infected patient sera (n = 12) and normal human sera (NHS) from healthy donors (n = 12) using a commercially available ELISA kit. (B) HCV infected patient sera (n = 12) and NHS (n = 12) were subjected to analysis for comparison of C3b deposition on E. coli surface in the presence or absence of purified C3 protein. Asterisks denote: *p<0.05; **p<0.01; ***p<0.001 when compared to control. (C) Complement mediated lysis was inhibited in HCV infected patient sera. Normal sera (n = 6) and HCV infected patient sera (n = 6) were incubated with sheep RBC after treatment with EDTA. Sera from each test group were studied, with lysis of erythrocytes completed using EDTA treated guinea pig serum. (D) Sheep RBCs were incubated with sera in the presence or absence of anti-factor B antibody to separate out alternate pathway mediated lysis from classical antibody mediated lysis, and lysis was carried out as described above.

Article Snippet: Mouse monoclonal antibody to human C3 (Abcam, MA), goat anti-mouse secondary antibody (Sigma, MO), purified human complement component C3 protein (Quidel, CA) were purchased.

Techniques: Comparison, Infection, Enzyme-linked Immunosorbent Assay, Purification, Control, Lysis, Incubation