Journal: JCI Insight

Article Title: Pravastatin therapy during preeclampsia prevents long-term adverse health effects in mice

doi: 10.1172/jci.insight.120147

Figure Lengend Snippet: Protective effects of pravastatin. Glomerular lesions were evaluated by immunohistochemistry. The glomerular lesions observed during and after preeclampsia (PE), characterized by increased tissue factor, fibrin, and complement C3b/iC3b/C3d expression/deposition are focal, and within the affected glomeruli, the pathological markers are global. Collagen deposition and α-smooth muscle actin (ASMA) expression in the glomeruli from mothers after PE are also focal and global. Representative images of the affected glomeruli are shown. (A) Immunohistochemical (IHC) detection of tissue factor, fibrin, and complement activation product C3b/iC3b/C3d in glomeruli from maternal kidneys harvested before, during, and after pregnancy in control mice, PE mice, and PE+PRAV mice. Scale bar: 20 μm. Five to six images per experimental group (n = 6–7 mice/group) and ten glomeruli per slide were analyzed. (B) IHC detection of collagen I (red fluorescence) and ASMA (green fluorescence) staining in maternal kidneys after normal pregnancies (control) and preeclamptic pregnancies (untreated [PE] and treated with pravastatin [PE+PRAV]). Scale bar: 50 μm. Five to six images per experimental group (n = 6–7 mice/group) and ten glomeruli per slide were analyzed. (C) Quantification of IHC staining of tissue factor, fibrin, and C3b/iC3b/C3d and collagen I and ASMA using ImageJ. Five to six images per experimental group (n = 6–7 mice/group) and ten glomeruli per slide were analyzed. Red squares represent the mean. Comparisons between groups were performed by 1-way ANOVA with Bonferroni’s post hoc test.*P < 0.01, statistically different from control at same time.

Article Snippet: Kidney cryostatic sections (5 μm) were then incubated with primary antibodies directed to mouse fibrinogen/fibrin (Abcam, ab34269), tissue factor (Abcam, ab151748), and complement C3b/iC3b/C3d (Hycult Biotech, mAb 3/26) followed by incubation with specific secondary IgG antibodies conjugated with FITC (Abcam).

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining, Activation Assay, Fluorescence, Staining

Journal: Kidney international

Article Title: Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis.

doi: 10.1038/sj.ki.5002407

Figure Lengend Snippet: Figure 4 | Complement activation in the kidney of mice hyperimmunized with 28-kDa SPE B mutant protein C192S. BALB/c mice were immunized four times with (a) BSA or (b) C192S. Complement C3 deposits were detected using FITC-conjugated anti-C3d antibody with fluorescence microscopy (n ¼ 10 per group). (c) The mean integral fluorescent intensity of glomeruli was analyzed in 10 fields per mouse (n ¼ 10 mice). Original magnification 200.

Article Snippet: Complement C3 was detected using sheep anti-human C3d (Serotec Ltd), which cross-reacts with mouse C3d, and then using FITC-conjugated goat anti-sheep IgG antibody (Jackson ImmunoResearch Laboratories Inc.).

Techniques: Activation Assay, Mutagenesis, Fluorescence, Microscopy

Journal: Kidney international

Article Title: Streptococcal pyrogenic exotoxin B antibodies in a mouse model of glomerulonephritis.

doi: 10.1038/sj.ki.5002407

Figure Lengend Snippet: Figure 8 | Immunoglobulin deposition and complement activation in mice after passive immunization with monoclonal anti-SPE B antibody 10G. BALB/c mice were intravenously immunized each day with 500 mg of monoclonal 9C or 10G IgG for 3 days and, after 7 days, the kidney sections were analyzed using (a, c) FITC-conjugated anti-mouse IgG or (b, d) anti-C3d antibody (n ¼ 3 per group). Original magnification 200.

Article Snippet: Complement C3 was detected using sheep anti-human C3d (Serotec Ltd), which cross-reacts with mouse C3d, and then using FITC-conjugated goat anti-sheep IgG antibody (Jackson ImmunoResearch Laboratories Inc.).

Techniques: Activation Assay

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