Initiate Complement Activation Research Articles

Complement in immune thrombocytopenia (ITP): The role of complement in refractory ITP.

Immune thrombocytopenia (ITP) is a disorder characterized by low platelets due to increased clearance and decreased platelet production. While ITP has been characterized as an acquired disorder of the adaptive immune system, the resulting platelet autoantibodies provide ancillary links to the innate immune system via antibody interaction with the complement system. Most autoantibodies in patients with ITP are of the IgG1 subclass, which can be potent activators of the classical complement pathway. Antibody-coated platelets can initiate complement activation via the classical pathway leading to both direct platelet destruction and enhanced clearance of C3b-coated platelets by complement receptors. Similar autoantibody interactions with bone marrow megakaryocytes can also result in complement injury and ineffective thrombopoiesis. The development of novel therapeutic complement inhibitors has revived interest in the role of complement in autoantibody-mediated disorders, such as ITP. A recent early-phase clinical trial of a classical complement pathway inhibitor has demonstrated efficacy in a subset of ITP patients refractory to conventional immune modulation. In this review, we will analyse the role of complement in refractory ITP.

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity.

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

Terminal complement pathway activation drives synaptic loss in Alzheimer’s disease models

Complement is involved in developmental synaptic pruning and pathological synapse loss in Alzheimer’s disease. It is posited that C1 binding initiates complement activation on synapses; C3 fragments then tag them for microglial phagocytosis. However, the precise mechanisms of complement-mediated synaptic loss remain unclear, and the role of the lytic membrane attack complex (MAC) is unexplored. We here address several knowledge gaps: (i) is complement activated through to MAC at the synapse? (ii) does MAC contribute to synaptic loss? (iii) can MAC inhibition prevent synaptic loss? Novel methods were developed and optimised to quantify C1q, C3 fragments and MAC in total and regional brain homogenates and synaptoneurosomes from WT and AppNL−G−F Alzheimer’s disease model mouse brains at 3, 6, 9 and 12 months of age. The impact on synapse loss of systemic treatment with a MAC blocking antibody and gene knockout of a MAC component was assessed in Alzheimer’s disease model mice. A significant increase in C1q, C3 fragments and MAC was observed in AppNL−G−F mice compared to controls, increasing with age and severity. Administration of anti-C7 antibody to AppNL−G−F mice modulated synapse loss, reflected by the density of dendritic spines in the vicinity of plaques. Constitutive knockout of C6 significantly reduced synapse loss in 3xTg-AD mice. We demonstrate that complement dysregulation occurs in Alzheimer’s disease mice involving the activation (C1q; C3b/iC3b) and terminal (MAC) pathways in brain areas associated with pathology. Inhibition or ablation of MAC formation reduced synapse loss in two Alzheimer’s disease mouse models, demonstrating that MAC formation is a driver of synapse loss. We suggest that MAC directly damages synapses, analogous to neuromuscular junction destruction in myasthenia gravis.

P1530: PERITRANFUSIONAL C1-INHIBITOR IN PATIENTS WITH SEVERE COMPLEMENT-MEDIATED AUTOIMMUNE HEMOLYTIC ANEMIA: AN OPEN LABEL PHASE 2 TRIAL.

Background: Complement-mediated autoimmune hemolytic anemia (AIHA) is characterized by destruction of red blood cells (RBCs) due to autoantibodies that initiate complement activation via the classical pathway. This may result in fulminant intra- and extravascular hemolysis with need for RBC transfusions, of which the efficacy can be compromised by the antibodies reacting with donor RBCs. Hemolysis induces a pro-inflammatory response that can contribute to hypercoagulability, as seen in patients with other complement-mediated hemolytic disorders such as paroxysmal nocturnal hemoglobinuria. C1-inhibitor (C1-INH; Cinryze) is an endogenous plasma regulator of the classical and lectin complement pathway approved for patients with hereditary angioedema. In a single case report and in vitro, we observed that C1-INH administration attenuated complement deposition on RBCs and subsequent hemolysis. Therefore, we hypothesized that peritransfusional administration of C1-INH in complement AIHA would reduce complement activation and hemolysis and thus improve transfusion outcome. Aims: To assess the efficacy and safety of intravenous C1-INH in patients with transfusion-dependent complement-mediated AIHA on parameters for hemolytic activity, complement activation and inflammation. Methods: We conducted a prospective, single center, phase 2 open label trial. Patients with AIHA and confirmed C3c/d-positive direct antiglobulin test, baseline hemoglobin (Hb) of ≤8 g/dL and an indication for ≥ two RBC transfusion units were included after informed consent. Treatment consisted of four peritransfusional doses of C1-INH intravenously (6000, 3000, 2000 and 1000 U) with 12 hour (hr) intervals. Blood samples at 0, 12, 24, 36, 48, 72 hrs, 7 and 14 days were analyzed for hemolytic parameters, RBC opsonization and markers of complement activation and systemic inflammation (nucleosomes and human neutrophil elastase-α1-antitrypsin complexes (HNE-α1-ATc)). Safety was monitored and scored (CTCAE version 4.0). Results: Ten patients were included in the study. At baseline, we found increased complement C3d, IgG and IgM deposition on RBCs compared to healthy controls (p=0.002, 0.005 and 0.002). Circulating C3b/c levels, nucleosomes and HNE-α1-ATc were increased compared to healthy controls (p<0.001, p=0.046 and p=0.001). C1-INH administration resulted in an increase of C1-INH antigen and -activity, peaking at 48 hrs (Fig. A). At 48 hrs after first administration, treatment significantly reduced C3d deposition on RBCs (-21%, p=0.016), but not systemic complement activation as shown by C3b/c levels (-14.96 nM, p=0.874; Fig. B). With declining C1-INH levels, C3 deposition increased again. Shortly after transfusion, Hb increased (1.59 g/dL at 12 h; p=0.008), but at 48 hrs, hemolytic and inflammation parameters did not improve (LDH -169.5 U/L, p=0.572; bilirubin total -4.306 µmol/L, p=0.987; Hb -1.217 g/dL; p=0.056; nucleosomes -17%, p=0.938; HNE-α1-ATc +21%;, p=0.297; Fig. C, D). No serious adverse events were reported. Image:Summary/Conclusion: Peritransfusional C1-INH in AIHA temporarily reduced local complement activation as evidenced by C3d deposition on RBCs, but did not affect hemolysis or systemic complement activation. Increased Hb levels at later timepoints were observed, but this effect might be confounded by additional treatments and transfusions. In conclusion, we found no (clinically) significant effect of C1-INH on hemolysis and systemic complement activation. This may be explained by the dosing of C1-INH, which possibly was too low to counter the severe complement-mediated AIHA and additional transfusion effects.

Novel Monoclonal Antibodies Against Mouse C1q: Characterisation and Development of a Quantitative ELISA for Mouse C1q

Recent studies have identified roles for complement in synaptic pruning, both physiological during development and pathological in Alzheimer’s disease (AD). These reports suggest that C1q initiates complement activation on synapses and C3 fragments then tag them for removal by microglia. There is an urgent need to characterise these processes in rodent AD models; this requires the development of reagents and methods for detection and quantification of rodent C1q in fluids and pathological tissues. These will enable better evaluation of the role of C1q in disease and its value as disease biomarker. We describe the generation in C1q-deficient mice of novel monoclonal antibodies against mouse and rat C1q that enabled development of a sensitive, specific, and quantitative ELISA for mouse and rat C1q capable of measuring C1q in biological fluids and tissue extracts. Serum C1q levels were measured in wild-type (WT), C1q knockout (KO), C3 KO, C7 KO, Crry KO, and 3xTg and APPNL-G-F AD model mice through ageing. C1q levels significantly decreased in WT, APPNL-G-F, and C7 KO mice with ageing. C1q levels were reduced in APPNL-G-F compared to WT at all ages and in 3xTg at 12 months; C3 KO and C7 KO, but not Crry KO mice, also demonstrated significantly lower C1q levels compared to matched WT. In brain homogenates, C1q levels increased with age in both WT and APPNL-G-F mice. This robust and adaptable assay for quantification of mouse and rat C1q provides a vital tool for investigating the expression of C1q in rodent models of AD and other complement-driven pathologies.

In vitro Measurement of Membrane Attack Complex in RPE Cells.

Initiation of the complement system results in the formation of a multiprotein pore termed the membrane attack complex (MAC, C5b-C9). MAC pores accumulate on a cell surface and can result in cell lysis. The retinal pigment epithelium (RPE) is a single monolayer of pigmented epithelial cells located at the posterior poll of the eye that forms the outer blood retinal barrier. RPE cells are highly polarized with apical microvilli and basolateral contact with Bruch's membrane. In order to obtain biologically relevant polarized RPE cultures in vitro, RPE cells are seeded onto the apical side of a transwell filter and cultured for 4 weeks in low serum media. MAC formation on RPE cells has been reported to be sub-lytic. MAC formation can be achieved in vitro by introduction of normal human serum (NHS) to media following serum starvation for 24 h. NHS contains all serum complement proteins required to initiate complement activation and MAC formation. We combined in vitro RPE polarization and complement activation to visualize MAC formation in vitro utilizing confocal microscopy allowing for high resolution MAC imaging.

Eculizumab impairs Neisseria meningitidis serogroup B killing in whole blood despite 4CMenB vaccination of PNH patients

AbstractComplement C5 inhibitor eculizumab has a great impact on the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH). However, this treatment success has a major drawback: a substantially increased susceptibility for life-threatening Neisseria meningitidis infections. Therefore, N meningitidis vaccination is strongly advised before initiating complement C5–blocking therapy. In this study, we show that the multicomponent N meningitidis serogroup B (4CMenB) vaccination of PNH patients treated with eculizumab results in a significant increase in anti–N meningitidis serogroup B (MenB) plasma immunoglobulin G (IgG) levels. Anti-MenB IgG was able to bind to the bacterial surface and initiate complement activation; however, inhibition of the membrane attack complex formation completely blocked whole blood–mediated killing of MenB. This would suggest that, despite 4CMenB vaccination, PNH patients taking C5 inhibitors are not sufficiently protected against MenB infection, which is in line with the fact that vaccinated PNH patients still experience meningococcal infections.

Associations of Ficolins With Hematological Malignancies in Patients Receiving High-Dose Chemotherapy and Autologous Hematopoietic Stem Cell Transplantations.

A prospective study of 312 patients [194 with multiple myeloma (MM) and 118 with lymphomas (LYMPH)] receiving high-dose chemotherapy and autologous hematopoietic stem cell transplantation (auto-HSCT) was conducted. Ficolins are innate immune defense factors, able to distinguish between “self” “abnormal self,” and “non-self” and contribute to the elimination of the last two by direct opsonization and/or initiation of complement activation via the lectin pathway. Concentrations of ficolin-1, ficolin-2, and ficolin-3 in serially taken serum samples were determined as were the polymorphisms of the corresponding (FCN1, FCN2, and FCN3) genes. Serum samples were collected before conditioning chemotherapy, before HSCT, and once weekly post-HSCT (four to five samples in total); some patients were also sampled at 1 and/or 3 months post-transplantation. The control group (C) consisted of 267 healthy unrelated individuals. Median ficolin-1 and ficolin-2 (but not ficolin-3) levels in MM patients' sera taken before chemotherapy were lower (and correspondingly frequencies of the lowest concentrations were higher) compared with controls. That appeared to be associated with the malignant disease itself rather than with post-HSCT complications (febrile neutropenia, infections accompanied, or not with bacteremia). Higher frequencies of the FCN1 genotype G/A-C/C-G/G (corresponding to polymorphisms at positions −542, −144, and +6658, respectively) and FCN2 gene heterozygosity for the −857 C>A polymorphism were found among patients diagnosed with MM compared with the C group. Furthermore, FCN2 G/G homozygosity (−557 A>G) was found more frequently and heterozygosity G/T at +6424 less frequently among LYMPH patients than among the healthy subjects. Heterozygosity for +1637delC mutation of the FCN3 gene was more common among patients diagnosed with lymphomas who experienced hospital infections. Although no evidence for an association of low ficolin-1 or ficolin-2 with infections during neutropenia following chemotherapy before HSCT was found, we observed a possible protective effect of ficolins during follow-up.

L-Fucose prevention of renal ischaemia/reperfusion injury in Mice.

In a recent study, we identified a fucosylated damage‐associated ligand exposed by ischemia on renal tubule epithelial cells, which after recognition by collectin‐11 (CL‐11 or collectin kidney 1 (CL‐K1)), initiates complement activation and acute kidney injury. We exploited the ability to increase the local tissue concentration of free l‐fucose following systemic administration, in order to block ligand binding by local CL‐11 and prevent complement activation. We achieved a thirty‐five‐fold increase in the intrarenal concentration of l‐fucose following an IP bolus given before the ischemia induction procedure ‐ a concentration found to significantly block in vitro binding of CL‐11 on hypoxia‐stressed renal tubule cells. At this l‐fucose dose, complement activation and acute post‐ischemic kidney injury are prevented, with additional protection achieved by a second bolus after the induction procedure. CL‐11−/− mice gained no additional protection from l‐fucose administration, indicating that the mechanism of l‐fucose therapy was largely CL‐11‐dependent. The hypothesis is that a high dose of l‐fucose delivered to the kidney obstructs the carbohydrate recognition site on CL‐11 thereby reducing complement‐mediated damage following ischemic insult. Further work will examine the utility in preventing post‐ischemic injury during renal transplantation, where acute kidney injury is known to correlate with poor graft survival.

Recombinant C1q variants modulate macrophage responses but do not activate the classical complement pathway

Complement protein C1q plays a dual role in a number of inflammatory diseases such as atherosclerosis. While in later stages classical complement pathway activation by C1q exacerbates disease progression, C1q also plays a beneficial role in early disease. Independent of its role in complement activation, we and others have identified a number of potentially beneficial interactions of C1q with phagocytes in vitro, including triggering phagocytosis of cellular and molecular debris and polarizing macrophages toward an anti-inflammatory phenotype. These interactions may also be important in preventing autoimmunity. Here, we characterize variants of recombinant human C1q (rC1q) which no longer initiate complement activation, through mutation of the C1r2C1s2 interaction site. For insight into the structural location of the site of C1q that is important for interaction with phagocytes, we investigated the effect of these mutations on phagocytosis and macrophage inflammatory polarization, as compared to wild-type C1q. Phagocytosis of antibody coated sheep erythrocytes and oxidized LDL was measured in human monocytes and monocyte-derived macrophages (HMDM) respectively that had interacted with rC1q wild-type or variants. Secreted levels of cytokines were also measured in C1q stimulated HMDM. All variants of C1q increased phagocytosis in HMDM compared to controls, similar to native or wild-type rC1q. In addition, levels of certain pro-inflammatory cytokines and chemokines secreted by HMDM were modulated in cells that interacted with C1q variants, similar to wild-type rC1q and native C1q. This includes downregulation of IL-1α, IL-1β, TNFα, MIP-1α, and IL-12p40 by native and rC1q in both resting and M1-polarized HMDM. This suggests that the site responsible for C1q interaction with phagocytes is independent of the C1r2C1s2 interaction site. Further studies with these classical pathway-null variants of C1q should provide greater understanding of the complement-independent role of C1q, and allow for potential therapeutic exploitation.

Mannose-binding lectin (MBL) in adult patients with inflammatory bowel disease.

Inflammatory bowel disease (IBD) refers to disorders associated with progressive inflammatory processes in the gastrointestinal system. IBD consists of two major forms, Crohn's disease (CD), and ulcerative colitis (UC). IBD became a global disease in the 21st century. Its pathogenesis is still not fully understood. Mannose-binding lectin (MBL) is a pattern-recognising molecule, involved in anti-microbial and anti-cancer immunity. It is able to opsonize microorganisms and abnormal host cells, and to initiate complement activation. The aim of this study was to investigate possible involvement of MBL in inflammatory bowel disease in adults. Forty persons diagnosed with CD and 28 with ulcerative colitis were recruited. The control group consisted of 136 healthy persons. Single nucleotide polymorphisms of the MBL2 gene (localised to both promoter and exon 1) were determined as were serum MBL concentrations. The exon 1 variant alleles and MBL deficiency-associated genotypes were more frequent among patients compared with controls, although this difference was not statistically significant. No differences of MBL levels were found between the major groups. However in MBL2 A/A homozygous IBD patients, the median was significantly higher than in corresponding healthy subjects. That was particularly evident in the case of active Crohn's disease (1493 ng/ml vs. 800 ng/ml, p = 0.021). It may suggest that MBL and MBL-dependent complement activation contributes to excessive inflammation and its adverse effects in the course of CD. It cannot also be excluded that high MBL activity constitutes in some cases part of a multifactorial network conducing to development of CD.

Complement C1r serine protease contributes to kidney fibrosis.

We have previously reported that complement activation precedes the development of kidney fibrosis; however, little is known about the cellular mechanisms involved in this transition. We hypothesized that increased expression of C1 complex protease C1r, the initiator of complement activation, contributes to tubulointerstitial fibrosis and tested this idea in mice with global deletion of C1r. Although expression of C1r in untreated wild-type (WT) mice was higher in the liver compared with kidney tissue, administration of folic acid (FA) led to upregulation of C1r mRNA and protein levels only in kidney tissue. Immunohistochemistry and in situ hybridization experiments localized increased expression of C1r and C1s proteases to renal tubular epithelial cells. C1r-null mice had reduced acute tubular injury and inflammation measured 2 days after FA administration compared with WT mice. C1r deletion reduced expression of C1s, C3 fragment formation, and organ fibrosis measured 14 days after FA administration. Differential gene expression performed in kidney tissue demonstrated that C1r-null mice had reduced expression of genes associated with the acute phase response, complement, proliferation of connective tissue cells (e.g., platelet-derived growth factor receptor-β), and reduced expression of genes associated with inflammation compared with FA-treated WT mice. In vitro experiments in renal epithelial cells demonstrated that C1s expression is dependent on increased C1r expression and that interferon-γ induces the expression of these two proteases. We conclude that increased expression of C1 complex proteases is associated with increased tissue inflammation and complement C3 formation and represents an important pathogenic mechanism leading to FA-mediated tubulointerstitial fibrosis.

The Interplay Between Blood Proteins, Complement, and Macrophages on Nanomedicine Performance and Responses.

In the blood, depending on their physicochemical characteristics, nanoparticles attract a wide range of plasma biomolecules. The majority of blood biomolecules bind nonspecifically to nanoparticles. On the other hand, biomolecules such as pattern-recognition complement-sensing proteins may recognize some structural determinants of the pristine surface, causing complement activation. Adsorption of nonspecific blood proteins could also recruit natural antibodies and initiate complement activation, and this seems to be a global process with many preclinical and clinical nanomedicines. We discuss these issues, since complement activation has ramifications in nanomedicine stability and pharmacokinetics, as well as in inflammation and disease progression. Some studies have also predicted a role for complement systems in infusion-related reactions, whereas others show a direct role for macrophages and other immune cells independent of complement activation. We comment on these discrepancies and suggest directions for exploring the underlying mechanisms.

A Novel Injury Site-Targeted Complement Inhibitor Which Protects against Lung Transplant Ischemia Reperfusion Injury

Purpose Self-reactive natural Abs initiate complement activation and injury following ischemia and reperfusion of certain tissues, but their role in post-ischemic lung injury is unknown. Here, we investigated the expression of two previously identified post-ischemic neoepitopes, namely a modified annexin IV epitope recognized by B4 IgM mAb, and an epitope consisting of a subset of phospholipids recognized by C2 IgM mAb, and additionally determined their role in lung transplant ischemia reperfusion injury (IRI). Methods Lungs from C57Bl/6 mice were orthotopically transplanted into Rag1-/- (antibody-deficient) mice reconstituted with either B4 and C2 mAbs. Results Rag1-/- mice were protected from lung IRI, but reconstitution with either B4 or C2 natural IgM mAbs restored lung IRI. The C2 mAb was more effective than the B4 mAb at restoring injury, as determined by histological and micro-CT analysis. Based on this information, we developed and characterized a therapeutic strategy that exploited the post-ischemia recognition system of natural antibodies. We constructed a single-chain antibody (scFv) derived from C2 mAb and linked it to Crry, an inhibitor of C3 activation (C2scFv-Crry). In models of syngeneic (B6-B6) and allogeneic (Balb/c-B6) lung transplant models in which recipients contain a full natural antibody repertoire, a single post-transplant injection of C2scFv-Crry blocked the binding of endogenous IgM to the graft, inhibited complement activation, and significantly reduced graft inflammation and injury as determined by histology, μCT, and immunhistochemical analysis. The severity of IRI is associated with the onset of acute rejection. Conclusion We identified pathophysiologically important epitopes expressed within the lung after lung ischemia and reperfusion post transplantation. We further describe a novel translatable strategy for complement inhibition that is specifically targeted to the transplanted lung, and that has several advantages over currently available approaches that rely on systemic complement inhibition. Significantly, the C2 epitope has been identified in both mouse and man, increasing the translational relevance of the approach.

Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway

AbstractThe mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder “phenotype” (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.

Eculizumab in cold agglutinin disease (DECADE): an open-label, prospective, bicentric, nonrandomized phase 2 trial

AbstractCold agglutinin disease (CAD) is a complement-dependent disorder, with extravascular and intravascular hemolysis resulting from initial or terminal complement activation, respectively. We tested the efficacy and safety of eculizumab, an inhibitor of the terminal complement pathway. Treatment-requiring patients received 600 mg eculizumab weekly for 4 weeks, followed 1 week later by 900 mg every other week through week 26. The primary end point was the difference in the lactate dehydrogenase level between the first and the last day of therapy. Twelve patients with chronic CAD and 1 patient with an acute cold agglutinin syndrome were included. The median lactate dehydrogenase level decreased from 572 U/L (interquartile range [IQR], 534-685) to 334 U/L (IQR, 243-567; P = .0215), paralleled by an increase in hemoglobin from 9.35 g/dL (IQR, 8.80-10.80) to 10.15 g/dL (IQR, 9.00-11.35; P = .0391; Wilcoxon signed-rank test). Three patients maintained and 8 patients acquired transfusion independence, and 1 patient each showed a reduced or increased transfusion requirement, respectively (P = .0215; exact McNemar's test). Patients with cold agglutinins with a thermal amplitude of 37°C tended to have less pronounced lactate dehydrogenase responses than patients with cold agglutinins with narrower thermal amplitudes. In the latter, responses were observed at lower serum levels of eculizumab than they were in the former. In contrast to hemolysis, cold-induced circulatory symptoms remained unaffected. In conclusion, eculizumab significantly reduced hemolysis and transfusion requirement in patients with CAD. Suppression of hemolysis caused by cold agglutinins with a wide thermal amplitude may require higher eculizumab doses than used here. The trial is registered with EudraCT (#2009-016966-97) and www.clinicaltrials.gov (#NCT01303952).

Immunomodulatory Role of Complement Proteins in the Neuropathology Associated with Opiate Abuse and HIV-1 Co-Morbidity

ABSTRACTThe complement system which is a critical mediator of innate immunity plays diverse roles in the neuropathogenesis of HIV-1 infection such as clearing HIV-1 and promoting productive HIV-1 replication. In the development of HIV-1 associated neurological disorders (HAND), there may be an imbalance between complement activation and regulation, which may contribute to the neuronal damage as a consequence of HIV-1 infection. It is well recognized that opiate abuse exacerbates HIV-1 neuropathology, however, little is known about the role of complement proteins in opiate induced neuromodulation, specifically in the presence of co-morbidity such as HIV-1 infection. Complement levels are significantly increased in the HIV-1-infected brain, thus HIV-induced complement synthesis may represent an important mechanism for the pathogenesis of AIDS in the brain, but remains underexplored. Anti-HIV-1 antibodies are able to initiate complement activation in HIV-1 infected CNS cells such as microglia and astrocytes during the course of disease progression; however, this complement activation fails to clear and eradicate HIV-1 from infected cells. In addition, the antiretroviral agents used for HIV therapy cause dysregulation of lipid metabolism, endothelial, and adipocyte cell function, and activation of pro-inflammatory cytokines. We speculate that both HIV-1 and opiates trigger a cytokine-mediated pro-inflammatory stimulus that modulates the complement cascade to exacerbate the virus-induced neurological damage. We examined the expression levels of C1q, SC5b-9, C5L2, C5aR, C3aR, and C9 key members of the complement cascade both in vivo in post mortem brain frontal cortex tissue from patients with HAND who used/did not use heroin, and in vitro using human microglial cultures treated with HIV tat and/or heroin. We observed significant expression of C1q and SC5b-9 by immunofluorescence staining in both the brain cortical and hippocampal region in HAND patients who abused heroin. Additionally, we observed increased gene expression of C5aR, C3aR, and C9 in the brain tissue of both HIV-1 infected patients with HAND who abused and did not abuse heroin, as compared to HIV negative controls. Our results show a significant increase in the expression of complement proteins C9, C5L2, C5aR, and C3aR in HIV transfected microglia and an additional increase in the levels of these complement proteins in heroin-treated HIV transfected microglia. This study highlights the a) potential roles of complement proteins in the pathogenesis of HIV-1-related neurodegenerative disorders; b) the combined effect of an opiate, like heroin, and HIV viral protein like HIV tat on complement proteins in normal human microglial cells and HIV transfected microglial cells. In the context of HAND, targeting selective steps in the complement cascade could help ameliorating the HIV burden in the CNS, thus investigations of complement-related therapeutic approaches for the treatment of HAND are warranted.

Age-related macular degeneration associated polymorphism rs10490924 in ARMS2 results in deficiency of a complement activator

BackgroundAge-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The polymorphism rs10490924 in the ARMS2 gene is highly associated with AMD and linked to an indel mutation (del443ins54), the latter inducing mRNA instability. At present, the function of the ARMS2 protein, the exact cellular sources in the retina and the biological consequences of the rs10490924 polymorphism are unclear.MethodsRecombinant ARMS2 was expressed in Pichia pastoris, and protein functions were studied regarding cell surface binding and complement activation in human serum using fluoresence-activated cell sorting (FACS) as well as laser scanning microscopy (LSM). Biolayer interferometry defined protein interactions. Furthermore, endogenous ARMS2 gene expression was studied in human blood derived monocytes and in human induced pluripotent stem cell-derived microglia (iPSdM) by PCR and LSM. The ARMS2 protein was localized in human genotyped retinal sections and in purified monocytes derived from AMD patients without the ARMS2 risk variant by LSM. ARMS2 expression in monocytes under oxidative stress was determined by Western blot analysis.ResultsHere, we demonstrate for the first time that ARMS2 functions as surface complement regulator. Recombinant ARMS2 binds to human apoptotic and necrotic cells and initiates complement activation by recruiting the complement activator properdin. ARMS2-properdin complexes augment C3b surface opsonization for phagocytosis. We also demonstrate for the first time expression of ARMS2 in human monocytes especially under oxidative stress and in microglia cells of the human retina. The ARMS2 protein is absent in monocytes and also in microglia cells, derived from patients homozygous for the ARMS2 AMD risk variant (rs10490924).ConclusionsARMS2 is likely involved in complement-mediated clearance of cellular debris. As AMD patients present with accumulated proteins and lipids on Bruch’s membrane, ARMS2 protein deficiency due to the genetic risk variant might be involved in drusen formation.

Vascular changes and perifascicular muscle fiber damage in dermatomyositis: another question of the chicken or the egg that is on our mind.

Dermatomyositis (DM) is a multisystem disease with symptoms spanning diverse body functions including the skin, the joints, the heart and the lungs. The skin manifestations involve a sun-sensitive rash on the face, neck and upper trunk, and erythema of knees, elbows and knuckles. In classical cases, where these typical skin manifestations combine with muscle weakness, diagnosis can be set on clinical grounds. In other cases, diagnosis is confirmed by assessing the level of serum muscle enzymes, electromyographic findings, and a muscle biopsy (1). In the skeletal muscle tissue of DM, the primary target of the immune response is the vascular endothelium of perimysial and perifascicular blood vessels, and inflammatory infiltrates often occur at perivascular sites. Complement deposition of the terminal C5b-9 membrane attack complex (MAC) leads to blood vessel necrosis (2). The trigger that initiates complement activation remains however unclear.

Platelet‐borne complement proteins and their role in platelet–bacteria interactions

Essentials Platelets play an important role in pathogen recognition.Platelets contain several complement factors and can interact with E. coli.Platelet's complement protein C3 differs from plasmatic C3 in its electrophoretic mobility.Upon contact with bacteria, platelets are activated and can enhance complement activation. SummaryBackgroundThe role of platelets in immune defense is increasingly being recognized. Platelets bind complement proteins from plasma, initiate complement activation, and interact with bacteria. However, the contribution of platelets to complement‐mediated defense against bacterial infections is not known in detail.ObjectivesTo assess platelet interactions with Escherichia coli strains, and evaluate the contributions of platelet complement proteins to host defense.MethodsWe studied the cell–cell interactions of a pathogenic and a non‐pathogenic E. coli strain with platelet concentrates, washed platelets and manually isolated platelets by flow cytometry and ELISA. The presence of complement proteins and complement RNA in megakaryocytes and platelets was analyzed by PCR, RT‐PCR, confocal microscopy, and western blotting.ResultsIncubation with E. coli leads to platelet activation, as indicated by the expression of CD62P and CD63 on the platelet surface. RNA and protein analyses show that megakaryocytes and platelets contain complement C3, and that platelet C3 migrates differently on polyacrylamide gels than plasmatic C3. Activation of platelets by bacteria leads to translocation of C3 to the cell surface. This translocation is not induced by thrombin receptor activating peptide or lipopolysaccharide. Interaction of platelets with E. coli occurs even in the absence of plasma proteins, and is independent of platelet toll‐like receptor 4 and α2bβ3 (glycoprotein IIbIIIa).ConclusionPlatelets contain a specific form of C3. Importantly, they can modulate immune defense against bacteria by enhancing plasmatic complement activation.