Journal: Biomedicines

Article Title: Autoantibodies Against Factor B and Factor H Without Pathogenic Effects in a Patient with Immune Complex-Mediated Membranoproliferative Glomerulonephritis

doi: 10.3390/biomedicines13030648

Figure Lengend Snippet: Schematic view of complement activation. ( A ) Activation of the complement system via the classical, lectin and alternative pathways, all joining at the central C3 component and leading to the common terminal pathway activation. Complement can be activated via the classical pathway (CP) by immune complexes, the lectin pathway (LP) by microbial polysaccharides, or the alternative pathway (AP) that is constantly active due to the so-called “tick-over” mechanism. Any activator stimulus leads to the formation of a convertase enzyme complex (C4b2b in the CP and LP and C3bBb in the AP) that cleaves the central C3 molecule and propagates the formation of C5 convertases. These, in turn, cleave C5 into C5a and C5b and thus bring forth the cascade to the common terminal pathway, resulting in the formation of the C5b-9 membrane attack complex. The generated anaphylatoxins C3a and C5a are important inflammatory mediators. ( B ) A more detailed delineation of alternative pathway activation depicting potential intervention points of the analyzed FB- and FH-autoantibodies. At a low rate, the reactive thioester group of C3 hydrolyses and the generated C3(H 2 O) binds factor B (FB), which, after cleavage by factor D (FD), forms the initial fluid phase C3 convertase of the AP (C3(H 2 O)Bb). It cleaves C3 into C3a and C3b, the latter being able to bind to all kinds of surfaces. If not inactivated by regulators, e.g., cell surface regulators (not depicted) or factor H (FH) and factor I (FI), C3b serves as a binding partner for FB, and after cleavage by FD, they form the solid phase AP C3 convertase C3bBb, which can be stabilized by properdin (P). The C3bBb convertase cleaves further C3 molecules that initiate new C3 convertases by binding FB, thereby amplifying AP activity (amplification loop) or transforming the C3 convertase into C5 convertase (C3bBbC3b). FH, as a main regulator, can prevent convertase formation by inhibiting FB binding to C3b, force the decay of existing convertases by displacing Bb, and assist as a cofactor in the inactivation of C3b by FI. Autoantibodies against the convertases (nephritic factors, not depicted) can stabilize the convertases and thus prolong complement activation and increase anaphylatoxin generation. Autoantibodies against FB and FH can enhance or impair the above-mentioned functions, thereby shifting the balance of complement activation and regulation.

Article Snippet: The activity of the convertase was analyzed by adding 10 µg/mL C3 for 1 h at 37 °C and measuring the amount of the cleavage product C3a from the supernatant by ELISA (Quidel)Ortho, San Diego, CA, USA.

Techniques: Activation Assay, Membrane, Generated, Binding Assay, Activity Assay, Amplification

Journal: Biomedicines

Article Title: Autoantibodies Against Factor B and Factor H Without Pathogenic Effects in a Patient with Immune Complex-Mediated Membranoproliferative Glomerulonephritis

doi: 10.3390/biomedicines13030648

Figure Lengend Snippet: Complex-free autoantibodies reduced the activity of the solid phase C3 convertase (C3bBbP). ( A ) Patient IgG (GN IgG, shown in red) had no effect on the assembly of the solid phase C3 convertase when added together with the components (FB, FD, and properdin) of the convertase during assembly. Data are mean ± SD from three independent experiments. ( B ) Complex-free autoantibodies do not affect the FH-mediated decay when added to the convertase together with FH. Data are mean ± SD from three independent experiments. The results of two healthy controls have already been published . ( C ) Assembled convertase was preincubated with IgG from the patient or healthy donors, then C3 was added and incubated at 37 °C. The generated C3a was measured in the supernatant with a specific C3a ELISA. Autoantibodies of the patient reduced the activity of the convertase. Data were normalized to the values obtained without the addition of IgG. Data are mean ± SD from four independent experiments. One-Way ANOVA, * p = 0.0222.

Article Snippet: The activity of the convertase was analyzed by adding 10 µg/mL C3 for 1 h at 37 °C and measuring the amount of the cleavage product C3a from the supernatant by ELISA (Quidel)Ortho, San Diego, CA, USA.

Techniques: Activity Assay, Incubation, Generated, Enzyme-linked Immunosorbent Assay

Journal: Biomedicines

Article Title: Autoantibodies Against Factor B and Factor H Without Pathogenic Effects in a Patient with Immune Complex-Mediated Membranoproliferative Glomerulonephritis

doi: 10.3390/biomedicines13030648

Figure Lengend Snippet: Patient IgG does not impair fluid-phase complement activation. The complex-free IgG of the patient (GN IgG, shown in red) or healthy donors was incubated with normal human serum in microtubes and then added to LPS-coated microplate wells; as a positive control, serum was preincubated with RRBCs to activate complement. The remaining complement activity was measured as C3 ( A ) or C5b-9 ( B ) deposition. The patient’s IgG had no effect on the complement activation measured as C3 ( A ) or C5b-9 ( B ) deposition compared to healthy control IgGs. Data are mean ± SD from two independent experiments. RRBC: rabbit red blood cell.

Article Snippet: The activity of the convertase was analyzed by adding 10 µg/mL C3 for 1 h at 37 °C and measuring the amount of the cleavage product C3a from the supernatant by ELISA (Quidel)Ortho, San Diego, CA, USA.

Techniques: Activation Assay, Incubation, Positive Control, Activity Assay, Control