Figure 2
Complement activation on glomerular endothelial cells, incubated with sera from R139W-aHUS patients and their healthy relatives. (A) C3 deposition on resting or TNFα/IFNγ activated GEnCs in the presence of sera from 50 individual normal donors, FHdpl (4 different lots), R139W-aHUS patients (P2, P5, and P14), healthy relatives of patient P5 bearing the mutation (5.F indicates father; and 5.S1 and 5.S2, sisters) or mutation-free relatives of patient P5, indicated as family (5.M indicates mother; and 5.B, brother). C3 depositions (RFI) obtained with each patient or healthy donor were normalized by the C3 deposition from one normal human serum on resting cells, considered as a standard and run in each experiment to obtain the fold increase. Each point is a mean of 3-5 independent experiments. Statistical significance (***P < .001) was calculated by ANOVA. (B) Levels of C3a, C5a, and soluble C5b-9, released after incubation of the TNFα/IFNγ activated GEnCs with serum from normal donors (n = 6) or R139W sera (n = 3), were measured by ELISA. The level of C3a, C5a, or sC5b-9 in the supernatant (one-third diluted serum) from resting cells was subtracted from the corresponding levels on activated cells, to obtain the specific amount of C5a and sC5b-9 released because of complement activation. Results are expressed as fold increase, compared with a standard normal serum as in panel A. The statistical analysis was a Mann-Whitney test. (C) Tissue-factor expression on TNFα/IFNγ activated or resting GEnCs after overnight incubation with sera from normal donors (n = 6) or R139W-positive sera (n = 4). The percentage of TF-positive cells was measured by flow cytometry. The statistical analysis was the Mann-Whitney test.

Complement activation on glomerular endothelial cells, incubated with sera from R139W-aHUS patients and their healthy relatives. (A) C3 deposition on resting or TNFα/IFNγ activated GEnCs in the presence of sera from 50 individual normal donors, FHdpl (4 different lots), R139W-aHUS patients (P2, P5, and P14), healthy relatives of patient P5 bearing the mutation (5.F indicates father; and 5.S1 and 5.S2, sisters) or mutation-free relatives of patient P5, indicated as family (5.M indicates mother; and 5.B, brother). C3 depositions (RFI) obtained with each patient or healthy donor were normalized by the C3 deposition from one normal human serum on resting cells, considered as a standard and run in each experiment to obtain the fold increase. Each point is a mean of 3-5 independent experiments. Statistical significance (***P < .001) was calculated by ANOVA. (B) Levels of C3a, C5a, and soluble C5b-9, released after incubation of the TNFα/IFNγ activated GEnCs with serum from normal donors (n = 6) or R139W sera (n = 3), were measured by ELISA. The level of C3a, C5a, or sC5b-9 in the supernatant (one-third diluted serum) from resting cells was subtracted from the corresponding levels on activated cells, to obtain the specific amount of C5a and sC5b-9 released because of complement activation. Results are expressed as fold increase, compared with a standard normal serum as in panel A. The statistical analysis was a Mann-Whitney test. (C) Tissue-factor expression on TNFα/IFNγ activated or resting GEnCs after overnight incubation with sera from normal donors (n = 6) or R139W-positive sera (n = 4). The percentage of TF-positive cells was measured by flow cytometry. The statistical analysis was the Mann-Whitney test.

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