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Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. AMD is a multifactorial disorder but complement-mediated inflammation at the level of the retina plays a pivotal role. Oral zinc supplementation can reduce the progression of AMD but the precise mechanism of this protective effect is as yet unclear. We investigated whether zinc supplementation directly affects the degree of complement activation in AMD and whether there is a relation between serum complement catabolism during zinc administration and the complement factor H (CFH) gene or the Age-Related Maculopathy susceptibility 2 (ARMS2) genotype. In this open-label clinical study, 72 randomly selected AMD patients in various stages of AMD received a daily supplement of 50 mg zinc sulphate and 1 mg cupric sulphate for three months. Serum complement catabolism-defined as the C3d/C3 ratio-was measured at baseline, throughout the three months of supplementation and after discontinuation of zinc administration. Additionally, downstream inhibition of complement catabolism was evaluated by measurement of anaphylatoxin C5a. Furthermore, we investigated the effect of zinc on complement activation in vitro. AMD patients with high levels of complement catabolism at baseline exhibited a steeper decline in serum complement activation (p<0.001) during the three month zinc supplementation period compared to patients with low complement levels. There was no significant association of change in complement catabolism and CFH and ARMS2 genotype. In vitro zinc sulphate directly inhibits complement catabolism in hemolytic assays and membrane attack complex (MAC) deposition on RPE cells. This study provides evidence that daily administration of 50 mg zinc sulphate can inhibit complement catabolism in AMD patients with increased complement activation. This could explain part of the mechanism by which zinc slows AMD progression. The Netherlands National Trial Register NTR2605.

Lutein is selectively taken up by the primate retina and plays an important role as a filter for harmful blue light and as an antioxidant. Recent studies have shown that lutein has systemic anti-inflammatory properties. Dietary lutein has been associated with reduced circulating levels of inflammatory biomarkers such as CRP and sICAM. Whether lutein also affects activation of the complement system has not yet been addressed and was the purpose of the study described here. Seventy-two subjects with signs of early macular degeneration were randomly assigned to receive either a 10 mg lutein supplement or a placebo during one year. EDTA blood samples were collected at 0, 4, 8 and 12 months. Complement factor D (CFD), a rate limiting component of the alternative pathway of complement activation and the complement activation products C5a and C3d were determined in the plasma samples by ELISA. A significant 0.11 µg/ml monthly decrease in plasma CFD concentration was observed in the lutein group (p<0.001), resulting in a 51% decrease from 2.3 µg/ml at baseline to 1.0 µg/ml at 12 months. The C5a concentration showed a significant 0.063ng/ml monthly decrease in the lutein group (p<0.001) resulting in a 36% decrease from 2.2ng/ml at baseline to 1.6ng/ml at 12 months. The C3d concentration showed a significant 0.19µg/ml monthly decrease in the lutein group (p=0.004) that gave rise to a 9% decrease from 15.4µg/ml at baseline to 14.4µg/ml at 12 months. In the placebo group we found a significant 0.04 µg/ml monthly decrease in plasma CFD concentration, whereas no changes were observed for C5a and C3d. Lutein supplementation markedly decreases circulating levels of the complement factors CFD, C5a and C3d levels, which might allow a simple method to control this inflammatory pathway of the innate immune system.

The alternative pathway of complement is important in innate immunity, attacking not only microbes but all unprotected biological surfaces through powerful amplification. It is unresolved how host and nonhost surfaces are distinguished at the molecular level, but key components are domains 19-20 of the complement regulator factor H (FH), which interact with host (i.e., nonactivator surface glycosaminoglycans or sialic acids) and the C3d part of C3b. Our structure of the FH19-20:C3d complex at 2.3-Å resolution shows that FH19-20 has two distinct binding sites, FH19 and FH20, for C3b. We show simultaneous binding of FH19 to C3b and FH20 to nonactivator surface glycosaminoglycans, and we show that both of these interactions are necessary for full binding of FH to C3b on nonactivator surfaces (i.e., for target discrimination). We also show that C3d could replace glycosaminoglycan binding to FH20, thus providing a feedback control for preventing excess C3b deposition and complement amplification. This explains the molecular basis of atypical hemolytic uremic syndrome, where mutations on the binding interfaces between FH19-20 and C3d or between FH20 and glycosaminoglycans lead to complement attack against host surfaces.

Atypical hemolytic uremic syndrome (aHUS) is a rare and severe thrombotic microangiopathy caused by genetic or acquired abnormalities leading to activation of the complement alternative pathway on cell surfaces. This process leads to endothelial dysfunction and microvascular thrombosis. The introduction of anti-C5 antibodies has dramatically improved aHUS prognosis; however, these treatments require regular intravenous infusions and block systemic complement activity, exposing the patient to risk of infections. Recently complement inhibitors have been developed to selectively bind injury-associated target molecules, thereby concentrating the drug at specific cellular or tissue sites while preserving systemic complement function. This study evaluated the local complement inhibitory activity of new molecules that exploit the natural localization of C3d at complement activation sites on cells: namely the anti-C3d monoclonal antibody 3d8b conjugated with the first 10 or 17 short consensus repeats (SCRs) of complement receptor 1 (CR1 1–10 and CR1 1-17 , respectively) or the first 5 SCRs of complement factor H (FH 1-5 ). To this purpose we tested their capability to block C3 deposition and C5b-9 formation on microvascular endothelial cells (HMEC-1) exposed to serum from patients with aHUS. We also assessed their ability to prevent loss of anti-thrombogenic properties in HMEC-1 pre-exposed to aHUS serum and then perfused with control blood. We demonstrate that anti-C3d-antibody conjugated with CR1 1-10 , or CR1 1-17 , or FH 1–5 effectively prevented aHUS serum-induced complement activation on HMEC-1, outperforming their non-targeted soluble counterparts. The efficacy of C3 convertase inhibition varied depending on the complement inhibitory component (CR1 1-17 > CR1 1-10 > FH 1-5 ). However, all the inhibitors successfully blocked C5 convertase activity and eliminated the pro-thrombogenic effects of aHUS patients’ serum. These findings support the potential of tissue-targeted complement inhibition as a novel, non-systemic therapeutic strategy for aHUS and other diseases characterized by localized complement dysregulation.

Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation.

Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues 1 and in plasma samples 2 from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.

Factor H exists as a 155,000 dalton, extended protein composed of twenty small domains which is flexible enough that it folds back on itself. Factor H regulates complement activation through its interactions with C3b and polyanions. Three binding sites for C3b and multiple polyanion binding sites have been identified on Factor H. In intact Factor H these sites appear to act synergistically making their individual contributions difficult to distinguish. Recombinantly expressed fragments of human Factor H were examined using surface plasmon resonance (SPR) for interactions with C3, C3b, iC3b, C3c, and C3d. Eleven recombinant proteins of lengths from one to twenty domains were used to show that the three C3b-binding sites exhibit 100-fold different affinities for C3b. The N-terminal site [complement control protein (CCP) domains 1-6] bound C3b with a of 0.08 μM and this interaction was not influenced by the presence or absence of domains 7 and 8. Full length Factor H similarly exhibited a for C3b of 0.1 μM. Unexpectedly, the N-terminal site (CCP 1-6) bound native C3 with a of 0.4 μM. The C-terminal domains (CCP 19-20) exhibited a of 1.7 μM for C3b. We localized a weak third C3b binding site in the CCP 13-15 region with a estimated to be ~15 μM. The C-terminal site (CCP 19-20) bound C3b, iC3b, and C3d equally well with a of 1 to 2 μM. In order to identify and compare regions of Factor H that interact with polyanions a family of 18 overlapping three domain recombinant proteins spanning the entire length of Factor H were expressed and purified. Immobilized heparin was used as a model polyanion and SPR confirmed the presence of heparin binding sites in CCP 6-8 ( 1.2 μM) and in CCP 19-20 (4.9 μM) and suggested the existence of a weak third polyanion binding site in the center of Factor H (CCP 11-13). Our results unveil the relative contributions of different regions of Factor H to its regulation of complement, and may contribute to the understanding of how defects in certain Factor H domains lead to disease.

The interaction of complement receptor 2 (CR2)—which is present on B cells and follicular dendritic cells—with its antigen-bound ligand C3d results in an enhanced antibody response, thus providing an important link between the innate and adaptive immune systems. Although a cocrystal structure of a complex between C3d and the ligand-binding domains of CR2 has been published, several aspects of this structure, including the position in C3d of the binding interface, remained controversial because of disagreement with biochemical data. We now report a cocrystal structure of a CR2(SCR1-2):C3d complex at 3.2 angstrom resolution in which the interaction interfaces differ markedly from the previously published structure and are consistent with the biochemical data. It is likely that, in the previous structure, the interaction was influenced by the presence of zinc acetate additive in the crystallization buffer, leading to a nonphysiological complex. Detailed knowledge of the binding interface now at hand gives the potential to exploit the interaction in vaccine design or in therapeutics directed against autoreactive B cells.

Background Degos disease is a frequently fatal and incurable occlusive vasculopathy most commonly affecting the skin, gastrointestinal tract and brain. Vascular C5b-9 deposition and a type I interferon (IFN) rich microenvironment are held to be pathogenetically important in the evolution of the vascular changes. We recently discovered the use of eculizumab as a salvage drug in the treatment of near fatal Malignant atrophic papulosis (MAP). The effects of eculizumab on the pathology of MAP are explored. Methods Archival skin and gastrointestinal biopsy material was procured over a 2.5-year period before and after eculizumab therapy in our index case. Routine light microscopy and immunohistochemical assessment for C3d, C4d, C5b-9, MxA and caspase 3 were examined. Direct immunofluorescent studies were also conducted on select biopsy material. Results The patient had received eculizumab as a emergent life saving measure and following rapid improvement he continued with biweekly infusions for 4 years. Although improved he continues to have signs and symptoms of persistent abdominal disease. Pre-Eculizumab biopsies showed an active thrombotic microangiopathy associated with a high type I interferon signature and extensive vascular deposits of C5b-9 in skin and gastrointestinal biopsies. Endothelial cell apoptosis as revealed by Caspase 3 expression was noted. Inflammation comprising lymphocytes and macrophages along with mesenchymal mucin was observed as well. Post-eculizumab biopsies did not show active luminal thrombosis but only chronic sequelae of prior episodes of vascular injury. There was no discernible caspase 3 expression. After 12 months of therapy, C5b-9 was no longer detectable in tissue. The high type I IFN signature and inflammation along with mucin deposition was not altered by the drug. In addition, there was little effect of the drug on the occlusive fibrointimal arteriopathy which appears to be one characterized by extensive myofibroblastic expansion of the intima potentially as revealed by staining for smooth muscle actin without immunoreactivity for desmin and myogenin. Conclusions Complement activation and enhanced endothelial cell apoptosis play an important role in the thrombotic complications of MAP. However, the larger vessel proliferative intimal changes appear to be independent of complement activation and may be on the basis of other upstream mechanisms. Monitoring C5b-9 deposition in tissue is likely not of great value in assessing treatment response to eculizumab given the persistence of C5b-9 in tissue for several months despite clinically effective C5 blocking therapy. A more integrated approach addressing upstream and downstream pathways in addition to those attributable to complement activation are critical for the successful treatment of MAP. Eculizumab may be used as salvage therapy in critically ill patients with thrombotic microangiopathy.

Inflammatory rheumatic diseases (IRD) are associated with accelerated coronary artery disease (CAD), which may result from both systemic and vascular wall inflammation. There are indications that complement may be involved in the pathogenesis of CAD in Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA). This study aimed to evaluate the associations between circulating complement and complement activation products with mononuclear cell infiltrates (MCI, surrogate marker of vascular inflammation) in the aortic media and adventitia in IRDCAD and non-IRDCAD patients undergoing coronary artery bypass grafting (CABG). Furthermore, we compared complement activation product deposition patterns in rare aorta adventitial and medial biopsies from SLE, RA and non-IRD patients. We examined plasma C3 (p-C3) and terminal complement complexes (p-TCC) in 28 IRDCAD (SLE = 3; RA = 25), 52 non-IRDCAD patients, and 32 IRDNo CAD (RA = 32) from the Feiring Heart Biopsy Study. Aortic biopsies taken from the CAD only patients during CABG were previously evaluated for adventitial MCIs. The rare aortic biopsies from 3 SLE, 3 RA and 3 non-IRDCAD were assessed for the presence of C3 and C3d using immunohistochemistry. IRDCAD patients had higher p-TCC than non-IRDCAD or IRDNo CAD patients (p<0.0001), but a similar p-C3 level (p = 0.42). Circulating C3 was associated with IRD duration (ρ, p-value: 0.46, 0.03). In multiple logistic regression analysis, IRD remained significantly related to the presence and size of MCI (p<0.05). C3 was present in all tissue samples. C3d was detected in the media of all patients and only in the adventitia of IRD patients (diffuse in all SLE and focal in one RA). The independent association of IRD status with MCI and the observed C3d deposition supports the unique relationship between rheumatic disease, and, in particular, SLE with the complement system. Exaggerated systemic and vascular complement activation may accelerate CVD, serve as a CVD biomarker, and represent a target for new therapies.