Journal: Clinical & Translational Immunology
Article Title: The contribution of the alternative pathway in complement activation on cell surfaces depends on the strength of classical pathway initiation
doi: 10.1002/cti2.1436
Figure Lengend Snippet: Complement activation on Neisseria meningitidis bypasses the amplification loop in post‐vaccination serum. Neisseria meningitidis was incubated with 10% FD‐deficient patient serum before (▲) and after (●) MenB‐4C vaccination. (a, b) show C3 and C5b‐9 binding, respectively, as determined by flow cytometry on whole bacteria. Both are statistically significant increased by the addition of FD to the deficient serum before vaccination, but this effect was lost postvaccination. The addition of anti‐C1q resulted in complete inhibition of C3 deposition and strong reduction of C5b‐9 deposition in all circumstances. (c) Bacterial survival upon serum incubation as determined by CFU, demonstrating that bacterially killing was increased by addition of FD in the prevaccination serum, but not the postvaccination serum. When anti‐C1q was added, bacterial survival was around 100%, indicating that CP activation was required for bacterial killing. (d) Complement‐mediated E. coli killing was inhibited by addition of anti‐C1q alone to a similar extent as addition of anti‐C1q, anti‐FB and anti‐FD combined. Anti‐FB and anti‐FD alone were unable to prevent complement‐mediated bacterial killing. (a–c) Mean and individual data points from four independent experiments, (d) mean + SD from n = 3 independent experiments. Statistical significance in a–c was tested using the paired t ‐test, * P < 0.05, ** P < 0.01.
Article Snippet: Surface‐bound complement C3 and complement complex C5b‐9 were detected with 1:500‐diluted FITC‐labelled polyclonal goat anti‐human C3 (MP biomedicals) and 1:100‐diluted monoclonal mouse antihuman C5b‐9 (Clone aE11, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by 1:500‐diluted Alexa647‐labelled donkey antimouse IgG (Invitrogen).
Techniques: Activation Assay, Amplification, Incubation, Binding Assay, Flow Cytometry, Inhibition
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