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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.

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 binding activity of donor-specific HLA antibodies (DSA) has been suggested as a new tool to stratify immunologic risk in kidney transplantation (KT). The objective of this study was to evaluate the clinical implication of C1q/C3d binding activity of de novo DSA (dnDSA) in KT recipients. A total of 161 pretransplant DSA-negative recipients were monitored for dnDSA at the time of biopsy. C1q/C3d binding activities of dnDSA were assessed using C1qScreen assay (One lambda, USA) and Lifecodes C3d detection assay (Immucor, USA), respectively. Clinical outcomes including biopsy-proven antibody mediated rejection (AMR), C4d detection and post-biopsy graft survival were investigated. De-novo DSAs were detected in fifty-four (33.5%) patients (HLA class I only, n = 19; class II only, n = 29; both class I and II, n = 6). Of them, complement binding activities were detected in 26 (48.1%) patients, including 17 C1q+ and 24 C3d+ patients. Both C1q and C3d positivity were associated with increased mean fluorescence intensity values of dnDSA. Complement binding activity of dnDSA enhanced the incidence of AMR (25.0% in C1q-C3d-, 36.4% in C1q+/C3d- or C1q-/C3d+, and 60.0% in C1q+/C3d+ patients) (P <0.001). The incidence of AMR was not different between patients with C1q+ and those with C3d+ dnDSA (64.7%, 11/17 versus 45.8%, 11/24, P = 0.238). In comparison between C1q and C3d assay according to HLA specificity, C1q+ HLA class I ± II dnDSA was the best predictor for AMR (odds ratio: 27.2). C1q+/C3d+ dnDSA was associated with more C4d deposition in allograft tissue and inferior post-biopsy graft survival. Clinical outcomes were not significantly different between C1q+ and C3d+ dnDSA-positive patients. Detection of complement binding activity using both C1q and C3d assays can be a further prognostic marker for predicting AMR and allograft outcome in dnDSA+ kidney transplant patients.

Antibody incompatible transplantation (AIT) may be an only option for highly sensitized patients. Severe form of early antibody mediated rejection (AMR) adversely affects graft survival after AIT. The aim of this study was to identify individuals at risk of AMR. We analyzed 213 living donor AITs performed at our center. Among 120 ABOi, 58 HLAi and 35 DSA + FCXM-negative cases, the rates of early AMR were 6%, 31%, and 9%, respectively ( p < 0.001). On multivariate analysis for graft loss, early AMR had a HR of 3.28 ( p < 0.001). The HLAi group had worse death-censored graft survival ( p = 0.003). In the HLAi group, Patients with aggressive variant AMR (AAMR) had greater percentage of C3d complement fixing DSA, higher baseline class I and total DSA MFI levels and B-cell FCXM RMF. C1q and C3d complement fixing DSA and strong positivity of baseline B- or T-cell FXCM as predictors of AAMR had 100% sensitivity. Early AMR is of significant clinical concern in AIT as it results in poor graft survival and is not well described in literature. An aggressive variant is characterized by massive rise in DSA levels at rejection. Baseline DSA, C1q, and C3d and baseline FCXM values can be used to risk-stratify candidates for AIT.

During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation-associated tissue inflammation.

Cleavage of C3 to C3a and C3b plays a central role in the generation of complement-mediated defences. Although the thioester-mediated surface deposition of C3b has been well-studied, fluid phase dimers of C3 fragments remain largely unexplored. Here we show C3 cleavage results in the spontaneous formation of C3b dimers and present the first X-ray crystal structure of a disulphide-linked human C3d dimer. Binding studies reveal these dimers are capable of crosslinking complement receptor 2 and preliminary cell-based analyses suggest they could modulate B cell activation to influence tolerogenic pathways. Altogether, insights into the physiologically-relevant functions of C3d(g) dimers gained from our findings will pave the way to enhancing our understanding surrounding the importance of complement in the fluid phase and could inform the design of novel therapies for immune system disorders in the future.

Age-related macular degeneration (AMD) is a common condition that leads to severe vision loss and dysregulation of the complement system is thought to be associated with the disease. To investigate associations of polymorphisms in AMD susceptibility genes with systemic complement activation, 2655 individuals were genotyped for 32 single nucleotide polymorphisms (SNPs) in or near 23 AMD associated risk genes. Component 3 (C3) and its catabolic fragment C3d were measured in serum and AMD staging was performed using multimodal imaging. The C3d/C3 ratio was calculated and associations with environmental factors, SNPs and various haplotypes of complement factor H (CFH) genes and complement factor B (CFB) genes were analyzed. Linear models were built to measure the influence of genetic variants on the C3d/C3 ratio. The study cohort included 1387 patients with AMD and 1268 controls. Higher C3d/C3 ratios were found for current smoker (p = 0.002), higher age (p = 1.56 × 10(-7)), AMD phenotype (p = 1.15 × 10(-11)) and the two SNPs in the C3 gene rs6795735 (p = 0.04) and rs2230199 (p = 0.04). Lower C3d/C3 ratios were found for diabetes (p = 2.87 × 10(-6)), higher body mass index (p = 1.00 × 10(-13)), the SNPs rs1410996 (p = 0.0001), rs800292 (p = 0.003), rs12144939 (p = 4.60 × 10(-6)) in CFH, rs4151667 (p = 1.01 × 10(-5)) in CFB and individual haplotypes in CFH and CFB. The linear model revealed a corrected R-square of 0.063 including age, smoking status, gender, and genetic polymorphisms explaining 6.3% of the C3d/C3 ratio. After adding the AMD status the corrected R-square was 0.067. In conclusion, none of the evaluated genetic polymorphisms showed an association with increased systemic complement activation apart from two SNPs in the C3 gene. Major genetic and non-genetic factors for AMD were not associated with systemic complement activation.

Previous studies indicate a pivotal role for complement in mediating both local and remote injury following ischemia and reperfusion of the intestine. Here, we report on the use of a mouse model of intestinal ischemia/reperfusion injury to investigate the strategy of targeting complement inhibition to sites of complement activation by linking an iC3b/C3dg-binding fragment of mouse complement receptor 2 (CR2) to a mouse complement-inhibitory protein, Crry. We show that the novel CR2-Crry fusion protein targets sites of local and remote (lung) complement activation following intestinal ischemia and reperfusion injury and that CR2-Crry requires a 10-fold lower dose than its systemic counterpart, Crry-Ig, to provide equivalent protection from both local and remote injury. CR2-Crry has a significantly shorter serum half-life than Crry-Ig and, unlike Crry-Ig, had no significant effect on serum complement activity at minimum effective therapeutic doses. Furthermore, the minimum effective dose of Crry-Ig significantly enhanced susceptibility to infection in a mouse model of acute septic peritonitis, whereas the effect of CR2-Crry on susceptibility to infection was indistinguishable from that of PBS control. Thus, compared with systemic inhibition, CR2-mediated targeting of a complement inhibitor of activation improved bioavailability, significantly enhanced efficacy, and maintained host resistance to infection.

Leprosy is a treatable infectious disease caused by Mycobacterium leprae. However, there is additional morbidity from leprosy-associated pathologic immune reactions, reversal reaction (RR) and erythema nodosum leprosum (ENL), which occur in 1 in 3 people with leprosy, even with effective treatment of M. leprae. There is currently no predictive marker in use to indicate which people with leprosy will develop these debilitating immune reactions. Our peripheral blood mononuclear cell (PBMC) transcriptome analysis revealed that activation of the classical complement pathway is common to both RR and ENL. Additionally, differential expression of immunoglobulin receptors and B cell receptors during RR and ENL support a role for the antibody-mediated immune response during both RR and ENL. In this study, we investigated B-cell immunophenotypes, total and M. leprae-specific antibodies, and complement levels in leprosy patients with and without RR or ENL. The objective was to determine the role of these immune mediators in pathogenesis and assess their potential as biomarkers of risk for immune reactions in people with leprosy. We followed newly diagnosed leprosy cases (n = 96) for two years for development of RR or ENL. They were compared with active RR (n = 35), active ENL (n = 29), and healthy household contacts (n = 14). People with leprosy who subsequently developed ENL had increased IgM, IgG1, and C3d-associated immune complexes with decreased complement 4 (C4) at leprosy diagnosis. People who developed RR also had decreased C4 at leprosy diagnosis. Additionally, elevated anti-M. leprae antibody levels were associated with subsequent RR or ENL. Differential co-receptor expression and immunoglobulin levels before and during immune reactions intimate a central role for humoral immunity in RR and ENL. Decreased C4 and elevated anti-M. leprae antibodies in people with new diagnosis of leprosy may be risk factors for subsequent development of leprosy immune reactions.