Q2. What is the significance of the mutations in aHUS?
Mutations identified in complement genes in aHUS patients indicate that complement dysregulation is involved in disease development.
Q3. What is the effect of mAbs on autoantibody binding?
(B) Autoantibody binding to FH was blocked with mAbs (25 µg/ml) specific to the C-terminal domains SCR19-20 of FH, i.e. C02, C14 and C18, whereas mAbs N11, M12, M13 and M15, which bind in the N-terminal and middle regions of FH, did not inhibit autoantibody binding.
Q4. What was the significance of the ELISA assay?
FH-autoantibodies of aHUS patients bind to the C-terminus of FH Plasma samples from 60 patients with hemolytic uremic syndrome (51 with aHUS and 9 with D+ HUS) were screened for the presence of FH-autoantibodies.
Q5. How many sheep erythrocytes were analyzed for aHUS?
4Hemolysis assays were performed in 100 µl buffer (20 mM HEPES, 7 mM MgCl2, 10 mM EGTA, 144 mM NaCl, 1% BSA, pH 7.4) containing 5×106 sheep erythrocytes (SRBC,BioTrend Chemikalien GmbH, Cologne, Germany) and 10-40% plasma.
Q6. What is the common cause of HUS?
HUS is most often caused by bacterial infection and is associated with diarrhea (D+ HUS), or arises rarely as atypical HUS (aHUS).
Q7. What is the effect of the autoantibody on FH?
FH-autoantibody inhibits C-terminus mediated function of FH Reduced C3b binding of FH and impaired C3b processing on the cell surface, which results in enhanced complement activation and cell damage, is caused by the mAbs C18 and C14,11 which share binding epitopes with the autoantibodies of all five analyzed aHUS patients (Figure 1C).
Q8. What is the OD value of the plasma samples?
(B) Dose-dependent binding of the autoantibody positive plasma samples of five aHUS patients to immobilized FH, indicating different autoantibody titers.
Q9. What is the effect of the autoantibody on the aHUS patients?
the hemolytic activity was reversed in a dose-dependent manner by addition of excess FH to the patient’s plasma, and thus increasing the concentration of autoantibody-free FH (Figure 2C).
Q10. What was the effect of the autoantibody on FH?
C3b binding was strongly reduced when FH was preincubated with patient derived IgG, which contained FH-autoantibodies, whereas control IgG had no effect (Figure 2A).
Q11. What was the OD value of the plasma samples?
(A) Plasma samples of aHUS (n = 51) and D+ HUS (n = 9) patients as well as healthy donors (n = 30) were analyzed for binding on immobilized purified FH in an ELISA assay.
Q12. What is the risk of a kidney transplant?
for the autoantibody positive patients kidney transplant is likely at high risk, as the autoreactive antibodies will remain in plasma.
Q13. What is the role of factor H in the alternative complement pathway?
In addition, factor H (FH)-autoantibodies have been reported for three aHUS-patients.3FH is a major regulator of the alternative complement pathway.
Q14. What is the effect of mAbs on the binding of FH to the C-?
6,11 In all five cases, binding of the autoantibody to FH was reduced by mAbs which bind to the FH C-terminus, namely mAbs C02 (binding in SCR19), C14 and C18 (both binding within SCR20).
Q15. What is the effect of the autoantibody on SRBC?
This effect of the autoantibody is in line with the observation, that the C-terminally binding mAbs C18 and C14 cause enhanced SRBC lysis when added to normal human plasma (data not shown).