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0

unctional characterization of mutations in complement C3hat predispose to aHUS

lizabeth C. Miller a, Lubka Roumenina b, Veronique Fremeaux-acchi b, John P. Atkinson a

Department of Medicine, Division of Rheumatology, Washington Uni-ersity, St. Louis, MO 63110, United StatesCentre de recherché des Cordeliers, Paris, France

Atypical haemolytic uraemic syndrome (aHUS) is a throm-oangiopathy predominantly of the kidney microvasculatureharacterised by haemolytic anaemia, thrombocytopenia and acuteenal failure. Mutations in the complement regulatory proteins,actor H (FH), Membrane Cofactor Protein (MCP; CD46), and Factor(FI) lead to a decreased ability to regulate the alternative pathwayAP). Mutations in Factor B (FB) and C3 also predispose to aHUSnd are primary or secondary gain of function mutations. In all ofhese cases, a hyperactive AP is the functional hallmark. Previously,e analyzed C3 mutations in nine aHUS patients initially selected

or analysis due to low serum C3 levels. In the majority, we identi-ed defects in their interactions with negative regulators. We haveow characterised five additional C3 mutations in aHUS patientsresenting with normal C3. In these studies, we transiently expresshe mutant proteins and then perform functional analyses includ-ng MCP, FH, and CR1 binding ELISAs and fluid phase cofactor assayss well as FB and properdin binding assays. The five mutations are43N, K133Q, R139W, I1073S and P1092L. All of these proteinsere expressed normally and migrated on SDS-PAGE similarly toT. K43N, in MG1, exhibited decreased binding to MCP but normal

H binding. K133Q, in MG2, had normal interactions with MCP andH. Interestingly, P1092L, in the TED, bound to MCP comparably toT C3 but had decreased binding to FH. R139W, in MG2, and twoutations in MG6 from our initial study, R570Q/W, demonstrated

ncreased affinity for properdin. Of note, R570Q/W also had a defectn regulation by MCP and FH in our initial studies. Thus, relative tohe AP, enhanced activity secondary to either decreased regulationy inhibitors (FH, MCP or FI) or increased activity of an enhancerproperdin) or a combination thereof leads to aHUS. These data alsoend new insight as to where properdin may bind C3(H20)/C3b.

oi:10.1016/j.molimm.2010.05.273

1

actor H autoantibodies are associated with MPGN

sabel Y. Pappworth a, Mark Denton b, David Kavanagh a, Iainoore a, Lisa Strain c, Paul N. Barlow d, Andrew P. Herbert d,

hristoph Q. Schmidt c

Institutes of Cellular Medicine and Human Genetics, Newcastle Uni-ersity, Newcastle-upon-Tyne, United KingdomDepartment of Renal Medicine, Derriford Hospital, Plymouth Hospi-

als NHS Trust, Plymouth, United KingdomNorthern Molecular Genetics Service, Newcastle upon Tyne HospitalsHS Foundation Trust, Newcastle-upon-Tyne, United KingdomSchool of Chemistry, University of Edinburgh, Edinburgh, Unitedingdom

Factor H autoantibodies are found in ∼10% of atypicalaemolytic uraemic syndrome (aHUS) patients. The majority aressociated with complete deficiency of factor H related proteins

/3 and bind to the C terminal recognition domain. Membranopro-

iferative glomerulonephritis (MPGN), like aHUS, is characterisedy complement activation. Therefore, we examined the hypoth-sis that factor H autoantibodies are associated with MPGN. We

gy 47 (2010) 2198–2294 2291

screened sera from 14 MPGN patients and 100 normal controlsfor factor H autoantibodies using enzyme-linked immunosorbentassay (ELISA). We detected strongly positive IgG factor H autoan-tibodies in two patients, which were confirmed by titration andwestern blotting. Further serum samples collected from both con-firmed these findings and in one showed an increasing antibodytitre with time. One patient (male aged 24 years) had type II (densedeposit disease; DDD) MPGN and one patient (female aged 26years) had type I MPGN and both were C3 NeF negative. We identi-fied the binding site of the autoantibodies using small SCR domainfragments in the ELISA and showed that the autoantibodies in bothpatients bound predominately to the N terminal complement reg-ulatory domain of factor H. We measured CFHR1/3 copy numberin paired DNA samples using multiplex ligation-dependent probeamplification (MLPA) and established both patients had two copiesof CFHR1/3. Finally, functionality of the detected factor H autoan-tibodies was examined using purified patient IgG in haemolyticassays. We observed increased haemolysis when purified Ig fromboth patients (but not from control) was added to normal humansera prior to incubation with rabbit red blood cells. Thus, in a smallcohort of MPGN patients, we have found a high titre of function-ally significant factor H autoantibodies in two patients. Antibodydepleting therapy may have a role in such patients and we suggestthat screening for factor H autoantibodies should be undertaken inall patients with MPGN (particularly DDD).

doi:10.1016/j.molimm.2010.05.274

92

Factor I autoantibodies are associated with atypical haemolyticuraemic syndrome

David Kavanagh a, Isabel Y. Pappworth a, Pietro Roversi d, JohnS. Tapson b, Iain Moore a, Lisa Strain c, Susan Lea d, Timothy H.J.Goodship a, Kevin J. Marchbank a

a Institutes of Cellular Medicine and Human Genetics, Newcastle Uni-versity, Newcastle-upon-Tyne, UKb Renal Services Centre, Newcastle upon Tyne Hospitals NHS Founda-tion Trust, Newcastle-upon-Tyne, UKc Northern Molecular Genetics Service, Newcastle upon Tyne HospitalsNHS Foundation Trust, Newcastle-upon-Tyne, UKd Sir William Dunn School of Pathology, University of Oxford, SouthParks Road, UK

Background: Atypical haemolytic uraemic syndrome (aHUS) isa disease of complement over activation. Mutations in the genesencoding both regulators (factor H, membrane cofactor protein andfactor I) and activators (factor B and C3) are associated with aHUS.Factor H autoantibodies have also been demonstrated in around∼10% of aHUS patients. These autoantibodies bind to the C-terminalrecognition domain of factor H and impair complement regulationat the cell surface. In this study we have examined the hypothesisthat factor I (fI) autoantibodies are associated with aHUS.

Methods: We screened sera from 142 aHUS patients and 100controls for fI autoantibodies using ELISA. We confirmed posi-tive results by titration and western blotting. We screened CFH,CFI, MCP, CFB and C3 for mutations using direct fluorescentsequencing. We screened for genomic disorders using muliplexligation-dependent probe amplification and assessed the func-tional significance of co-existing mutations in co-factor assaysusing recombinant proteins.

Results: We detected IgG fI autoantibodies in the sera of threeaHUS patients. One was strongly positive and two were moder-ately positive. We confirmed these results with western blotting,which also showed that the autoantibodies bind predominantly to

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292 Abstracts / Molecular Imm

he heavy chain of fI. In one individual, archived serum samplesllowed serial measurements of autoantibody titre demonstratingn increased titre at the time of recurrence of aHUS in a renal trans-lant. In the same patient, we also detected functionally significantutations in CFH (c.3468dupA) and CFI (−c.1657 C > T− Pro553Ser).Conclusions: We have found fI autoantibodies in three aHUS

atients. In one patient we have found additional mutations in CFHnd CFI and in this individual we observed an increased titre at theime of transplant recurrence. This observation provides furthervidence that multiple concurrent risk factors may be necessary inndividual patients for disease manifestation.

oi:10.1016/j.molimm.2010.05.275

3

he monoclonal antibody, 3E7, is a multi-functional inhibitorf the alternative pathway that blocks NeF-triggered activation

anielle Paixao-Cavalcante a, Margaret A. Lindorfer b, B. Paulorgan a, Ronald P. Taylor b, Claire L. Harris a

Department of Infection, Immunity and Biochemistry, School ofedicine, Cardiff University, Cardiff, UKDepartment of Biochemistry and Molecular Genetics, University ofirginia Health Science Center, Charlottesville, VA, USA

Uncontrolled activation of the alternative pathway (AP) drivesathology in various diseases, including membranoproliferativelomerulonephritis (MPGN) and atypical hemolytic uremic syn-rome. The monoclonal antibody 3E7 has previously been showno bind C3b and iC3b and selectively inhibit the AP, leaving thelassical pathway free to function. Here we investigate further theechanism by which 3E7 inhibits activation of the AP and influ-

nces regulation of the convertase by factor H (fH). The action ofE7 was dissected using C3 convertase ELISAs, haemolytic assaynd Biacore analyses. We show that 3E7 binds C3b and preventsinding of factor B (fB) and assembly of the C3 convertase; 3E7 alsoinds pre-formed C3 convertase, suggesting that it shares a bind-

ng site with the Ba domain of fB. Binding of 3E7 to the preformedonvertase induced weak accelerated decay of C3bBb; however, itrevented binding of the decay accelerator, fH. Although fH bindingas prevented, 3E7 inhibited cleavage of the convertase substrate,3, thus acting as a powerful inhibitor of the AP amplification loop.

mportantly, 3E7 prevented C3 convertase formation even in theresence of type I C3 nephritic factor (NeF), a pathogenic autoan-ibody which stabilises and prevents decay of the C3 convertase.

hen bound to the preformed NeF-stabilised enzyme, 3E7 blockedurther AP amplification and formation of downstream activationroducts such as C5b and MAC. These data suggest that 3E7 may be

n effective therapy in disorders mediated by dysregulation of theP C3 convertase and particularly in NeF-associated diseases suchs MPGN.

oi:10.1016/j.molimm.2010.05.276

gy 47 (2010) 2198–2294

94

Non-invasive assessment of disease activity in a model of lupusnephritis using complement receptor-2 conjugated to super-paramagnetic iron oxide nanoparticles

Kendra M. Hasebroock a,b, Natalie J. Serkova a,b, S. AnnaSargsyan a,b, Brandon Renner a,b, Brian Larsen a,b, ConradStoldt a,b, V. Michael Holers a,b, Joshua M. Thurman a,b

a University of Colorado Denver, Aurora, CO, United Statesb University of Colorado Boulder, Boulder, CO, United States

Lupus nephritis is characterized by immune-complex deposi-tion as well as complement C3 activation within the glomeruli.Currently, percutaneous renal biopsy is the gold standard formonitoring disease activity in patients with lupus nephritis. Wehave developed a non-invasive method for detecting tissue-boundiC3b/C3d using superparamagnetic particles of iron oxide (SPIO)linked to the iC3b/C3d binding region of complement receptortype 2 (CR2). SPIO cause a reduction in the spin–spin T2-relaxationtime which leads to negative enhancement (i.e. darkening) of T2-weighted magnetic resonance images (MRI). We hypothesized thatsystemically administered CR2-targeted SPIO, through their bind-ing to iC3b/C3d deposition site, could be used to monitor diseaseactivity in a murine model of lupus nephritis based on decreasedT2-relaxation times. SPIO were synthesized by a solvothermalmethod yielding ∼10 nm magnetite nanoparticles that were thenencapsulated using amine-functionalized phospholipids as ∼75 nmaggregates. The particles were conjugated to a recombinant proteinthat contains the SCR1-2 iC3b/C3d binding region of CR2, gener-ating CR2-targeted SPIO. MRL/lpr mice spontaneously develop aprogressive complement-dependent lupus-like glomerulonephri-tis as they age. MRL/lpr mice and control MRL/MpJ mice wereinjected with CR2-targeted SPIO at 12, 16, 20 and 24 weeks of age.T2-weighted MR images were acquired prior to and at 48 h post-injection. We found that CR2-targeted SPIO caused a reduction inthe T2-relaxation time in the cortex and outer medullas of the kid-neys of MRL/lpr mice, but did not affect the T2-relaxation time in thekidneys of control mice. Negative enhancement of the kidneys afterinjection with CR2-SPIO was strongest in the 20-week-old MRL/lprmice. Furthermore, histological examination for C3d deposition inthe diseased kidneys showed that the reduction in T2-relaxationtime positively correlated with the degree of iC3b/C3d depositionin the glomeruli and tubulointerstium of diseased animals. Molecu-lar imaging of the kidneys by MRI after injection with CR2-targetedSPIO may provide a quantitative non-invasive alternative for mon-itoring disease activity in patients with lupus nephritis.

doi:10.1016/j.molimm.2010.05.277

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The complement inhibitor Crry is critical for the prevention ofcomplement activation on murine renal tubular epithelial cells

Brandon Renner a,b, Kathrin E. Coleman a,b, Claudia Amura a,b,Hector Molina a,b, V. Michael Holers a,b, Joshua M. Thurman a,b

a Department of Medicine, University of Colorado School of Medicine,Denver, CO, United Statesb Department of Medicine, Washington University School of Medicine,St. Louis, MO, United States

We have previously demonstrated that renal ischemia reduces

the expression of the membrane complement regulatory proteinCrry by renal tubular epithelial cells (TECs). The current studieswere undertaken to test the hypothesis that reduced expression ofCrry on TECs is sufficient to cause complement mediated injury of