As a role for complement is identified in a growing number of diseases and multiple complement inhibitors become available in clinical practice, novel assays that can identify patients likely to respond to complement-directed therapies and predict responses to specific complement inhibitors are needed. Supplementary Material supplemental figuresClick here to view.(452K, pdf) Acknowledgments Funding This work was supported by the National Institutes of Health (NIH)/National Heart, CX3CL1 Lung, and Blood Institute (NHLBI) [R01HL133113 2016] and NIH [K08 HL138142]. Footnotes Appendix A. positive control, we measured cell killing in the mHam as an indicator of terminal complement activation using the following: (1) Sialidase (Sia), which cleaves sialic acid residues on the cell surface thereby diminishing the inhibitory effect of factor H on the alternative pathway; (2) Shiga toxin 1 (Stx1), which activates complement through all three complement pathways [20,21]; (3) cobra venom factor (CVF), which binds to factor B and upregulates the formation of C3b and C5b [22]; and (4) lipopolysaccharide (LPS), which is known to induce C3 convertase [23]. Addition of Sia and Stx1 led to a marked increase in cell killing in the mHam (Fig. 2A and ?andB).B). Sia added to cells at a dose of 50 units/ml increased complement-mediated cell killing by more than 3-fold compared to NHS alone (Fig. 2A). Stx1 at a concentration of 10 g/ml increased cell death by more than 4-fold compared to NHS alone (Fig. 2B). Increasing doses of CVF and LPS produced minimal cell death ( 20% cell killing) (Fig. 2C and ?andDD). Acetylcholine iodide Open in a separate window Fig. 2. Sialidase and Shiga toxin type 1 induce complement-mediated cell killing in a dose-dependent manner that is blocked by addition of ACH-145951 and anti-C5 antibody. Cell killing measured by the mHam with the addition of (A) Sialidase, (B) Shiga toxin 1, (C) Lipopolysaccharide and (D) Cobra venom factor at escalating doses. (E) Sia-induced cell killing in the mHam in the presence of ACH-145951 and anti-C5Ab. (F) Stx1-induced cell killing in the presence of ACH-145951 and anti-C5Ab. Data shown as mean SEM of triplicate wells and are Acetylcholine iodide representative of three independent experiments. Normal Human Serum (NHS), Sialidase (Sia), Shiga toxin 1 (Stx1), Lipopolysaccharide (LPS), Cobra venom factor (CVF), factor D inhibitor (ACH-145951), anti-C5 antibody (anti-C5Ab) * 0.05, ** p 0.01, ****p 0.0001. When NHS was incubated with increasing concentrations of anti-C5Ab or ACH-145951, the complement-mediated killing induced by Sia was inhibited in a dose-dependent manner (Fig. 2E). ACH-145951 at a concentration of 0.3 M reduced cell killing of Sia-treated cells to the level of NHS alone (Fig. 2E). This observation is consistent with the mechanism of Sia-induced complement activation through upregulation of the alternative pathway. In contrast, Stx1 induced cell killing was more effectively blocked by anti-C5Ab than ACH-145951 (Fig. 2F). Complement inhibition with ACH-145951 on Stx1-treated cells was concentration dependent (Fig. 2F). However, 0.3 M ACH-145951 only partially blocked Stx1-induced cell killing, whereas anti-C5Ab blocked this completely, suggesting additional complement activation by Stx1 occurs outside of the alternative pathway. 3.3. Sia promotes C5b-9 deposition predominantly via the alternative pathway We next evaluated C5b-9 and C3c deposition on TF1 0.001, *** p 0.001, **** p 0.0001. Table 1 Clinical characteristics of atypical hemolytic uremic syndrome and antiphospholipid antibody syndrome Acetylcholine iodide patients and aHUSpatient was completely blocked after the addition of 50 g/ml anti-C5 antibody but only 50% reduced with addition of 1 1.0 M ACH-145951 (Fig. 4B). The inhibitory effect of anti-C5 antibody and ACH-145951 on C5b-9 deposition reflected the pattern of cell killing in the mHam. Treating cells with serum from aHUSand aHUSin the alternative pathway-specific (AP) buffer induced levels of C5b-9 deposition similar to those observed in the all complement pathway buffer (Fig. 4C), whereas C5b-9 deposition triggered by aHUSserum was.