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Abstracts / Molecular Immunology 89 (2017) 200–206
Treating C3G murine models by mini-Factor H
Keenan 1,∗ ,
Zhang 1 ,
Markus J.
Harder 2 , Richard J.H. Smith 1,3 , Christoph Q.
Schmidt 2
Molecular Otolaryngology and Renal Research
Laboratories, University of Iowa, Iowa City, IA, USA
2 Institute of Pharmacology of Natural Products &
Clinical Pharmacology, Ulm University, Germany
3 Department of Pediatrics, Division of Nephrology,
Carver College of Medicine, University of Iowa, Iowa
City, IA, USA
Evaluation of risk of bacteria-mediated putative
“bystander” hemolysis of PNH red blood cells
in vitro: No evidence of significant
complement-mediated hemolysis induced by
Guangwei Yang 1,∗ , Xuan Yuan 2 , Manuel Galvan 1 ,
Jane A. Thanassi 1 , Yongsen Zhao 1 , Dharaben
Patel 1 , Joanne Fabrycki 1 , Amanda Luu 1 ,
Wengang Yang 1 , Jason Wiles 1 , Avinash Phadke 1 ,
Joel Barrish 1 , Mingjun Huang 1 , Robert Brodsky 2 ,
Steven D. Podos 1
Background: C3 glomerulopathy (C3G) is an ultra-rare
glomerular disease that progresses to end-stage renal failure within
a decade of diagnosis in 50% of patients. The underlying disease mechanism is uncontrolled C3 convertase activity of the
complement alternative pathway (AP) that causes predominant
glomerular C3 deposition in the absence or sparse presence of
immunoglobulins. Based on the location and shape of the glomerular deposits, two C3G pathological entities can be resolved by
electron microscopy – C3 glomerulonephritis (C3GN) and Dense
Deposit Disease (DDD). In C3GN, deposits are lighter and mostly
mesangial while in DDD, they are darker and intramembranous.
The central role of C3 convertase in disease pathogenesis makes
this molecular complex an excellent target for therapeutic intervention. Therapies that restore systemic control of C3 convertase
should be evaluated as potential treatment for this disease.
Materials and methods: We completed in vitro studies using
mini-Factor H (FH), an engineered short version of FH that consists
of only the complement regulatory domains and surface recognition domains, to determine whether this complement regulator is
able to control C3 convertase activity in sera from C3G patients. We
also completed in vivo studies in murine models of C3G using different infusion routes (intraperitoneal or subcutaneous) and following
renal pathology and function.
Results and conclusions: The in vitro study showed that miniFH out-performed FH in preventing complement-mediated lysis
of sheep erythrocytes. In vivo therapy (single or multiple doses)
with mini-FH in mice deficient in complement factor H (Cfh−/− )
and Cfh−/− mice transgenic for human CR1 (Cfh−/− tgCR1) stopped
uncontrolled activation of the AP, normalized murine serum C3
levels, and led to rapid reduction of complement debris in renal
glomeruli. Modulation of the bioavailability of mini-FH via subcutaneous application improved the effectiveness of the mini-FH
compound. These data suggest that mini-FH may offer a novel and
promising therapeutic option to C3G patients as a disease-specific
targeted therapy.
Achillion Pharmaceuticals, New Haven, CT, USA
Division of Hematology, Department of Medicine,
Johns Hopkins University School of Medicine,
Baltimore, MD, USA
Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a
clonal disease in which erythrocytes lacking the GPI-anchored complement regulators CD55 and CD59 are susceptible to C3 convertase
and terminal complex assembly on their membranes following the
normal slow activation of complement alternative pathway (AP)
in the fluid phase. The terminal complement inhibitor eculizumab
blocks intravascular hemolysis in PNH patients, yet breakthrough
hemolysis has been observed during inflammatory events such
as infection. The present study examines a possible mechanism
known as “bystander” hemolysis in which pathogen-dependent
complement activation could lead to transfer of activating fragments to PNH red blood cells (RBCs) through the fluid phase.
Materials and methods: Hemolysis of PNH RBCs (5 × 107 /mL)
was assessed in GVB++ buffer under approximately physiological serum conditions (80% final serum dilution), without or with
eight representative Gram-positive and Gram-negative bacteria
(5 × 106 /mL or 5 × 107 /mL) for 1 h at 37 ◦ C. Two N. meningitidis serotypes were assessed with matched sera possessing
AP-independent bactericidal activity. Complement activation by
bacteria was confirmed by loss of viability (susceptible strains) or
by C3 and C4 fragment deposition (non-susceptible strains).
Results and conclusions: PNH RBCs from four patients were
susceptible to hemolysis in 80% acidified serum, with moderate (patient A) or no (patients B, D, and E) significant hemolysis
observed at neutral pH. Although all complement pathways are
open to activation in GVB++ buffer, PNH RBC hemolysis in this assay
system was AP-dependent as shown by complete inhibition by the
small-molecule factor D inhibitor ACH-4471. PNH RBCs showed no
increase in hemolysis or C3 fragment deposition when assessed at
neutral pH with each of the eight bacterial strains, indicating no
“bystander” hemolysis under these assay conditions. The soluble
AP activator LPS also conferred no increase in hemolysis, further
establishing that fluid-phase activation does not mediate PNH RBC
hemolysis. These results suggest a limited risk to PNH patients
of breakthrough hemolysis by the “bystander” mechanism upon
infection with bacteria including the clinically important N. meningitidis.
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molimm, 236, 2017
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