Limit Complement Activation Research Articles

CL-11 circulates in serum as functionally distinct isoforms.

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway capable of interacting with collectin-10 (CL-10) and the MASPs to activate the complement cascade. Alternative splicing of the COLEC11 gene gives rise to two different isoforms found in serum (A and D). These isoforms vary in the length of their collagen-like region, which is involved in the stabilization of the trimeric subunit and the interaction with the MASPs. Here we aim at elucidating the biological differences of naturally occurring CL-11 isoforms A and D. We produced recombinant CL-11 as independent isoforms (CL-11A and CL-11D) and together with CL-10 (CL-10/11A, CL-10/11D). Both CL-11 isoforms associated with CL-10, but CL-11D did so to a lesser extent. CL-10/11 heterocomplexes were composed of trimeric subunits of CL-10 and CL-11, as opposed to CL-10 and CL-11 homotrimers. Heterocomplexes were more stable and migrated with higher apparent molecular weights. Immunoprecipitation of serum CL-11 and subsequent mass spectrometry analysis confirmed that native CL-11 circulates in the form of CL-10/11 heterocomplexes that associate with MASP-1, and MASP-3, but not necessarily MASP-2. Despite a shorter collagen region, CL-11D was capable to bind to the MASPs, suggesting that the missing exon 4 is not required for MASP association CL-11D had a reduced ligand binding compared to full-length CL-11A. Based on its reduced ability to oligomerize, form CL-10/11 heterocomplexes, and bind to ligands, we hypothesize that CL-11D may have a limited complement activation potential compared to full-length CL-11A.

Decreased Systemic Complement Activation Product C3a is Associated with a Reduction in Pancreatic β Cell Area in Islets of Female Rat Offspring following Chronic Placental Ischemia‐induced Hypertension

Preeclampsia is a pregnancy specific condition characterized by new-onset maternal hypertension and increased systemic inflammation, including increased complement activation. Our previous studies demonstrated that inhibition of complement activation attenuated placental ischemia-induced hypertension in the Reduced Uteroplacental Perfusion Pressure (RUPP) model of gestational hypertension in rat. Hypertensive pregnancy disorders can also result in increased risk of later-life cardiovascular and metabolic disease for the offspring. In fact, recent studies have demonstrated that gestational hypertension is associated with increased risk for Type 2 diabetes in offspring. Clearly, pancreatic β cell mass, established early in life, can influence whether an individual develops Type 2 diabetes. β cell area has also been linked to complement component C3 and activation product C3a, with increased C3a generation associated with prevention of human islet cell atrophy. Our previous studies demonstrated a significant reduction in β cell area in postnatal day (PD)13 female, but not male, offspring of rats subjected to chronic placental ischemia-induced hypertension in the RUPP model. Thus, we hypothesized that decreased C3a is associated with reductions in β cell area in the female offspring following placental insufficiency. The RUPP model was used to induce placental insufficiency by placing silver clips on the abdominal aorta and uterine arteries on gestation day 14 of a 21 day gestation. Serum was collected via the abdominal aorta from PD13 pups born from RUPP and Sham pregnancies. Pancreatic islets of PD13 male and female offspring of RUPP and Sham dams were isolated by collagenase perfusion and handpicking. We previously demonstrated that the transcript message for membrane-bound complement regulator CD55 (negative regulator of C3 activation) was significantly increased in islets isolated from female, but not male, RUPP vs Sham offspring, with no change in acinar tissue. In addition, no significant change was observed in message for the parent molecule C3. The relative amount of circulating C3a in serum of PD13 offspring was determined by Western blot and expressed as C3a units/ul based on signal intensity of 1 ul of a standard pool of adult rat serum activated by yeast. Circulating C3a in female RUPP offspring (1.35 ± 0.37 units/ul) was significantly lower (p<0.05) than C3a in female Sham offspring (2.54 ± 0.44 units/ul). No difference was noted comparing C3a in serum of male offspring, RUPP vs Sham, where no change in β cell area has been observed. Thus, these data suggest an increase in CD55 may limit complement activation and C3a production in female offspring. This dysregulation of complement activation may contribute to reduction of β cell area and long-term susceptibility for Type 2 diabetes. Future studies are needed to determine if local or circulating C3a is instrumental in controlling pancreatic β cell area in offspring of hypertensive pregnancies.

A porcine model of hemodialyzer reactions: roles of complement activation and rinsing back of extracorporeal blood

Hemodialysis reactions (HDRs) resemble complement-activation-related pseudoallergy (CARPA) to certain i.v. drugs, for which pigs provide a sensitive model. On this basis, to better understand the mechanism of human HDRs, we subjected pigs to hemodialysis using polysulfone (FX CorDiax 40, Fresenius) or cellulose triacetate (SureFlux-15UX, Nipro) dialyzers, or Dialysis exchange-set without membranes, as control. Experimental endpoints included typical biomarkers of porcine CARPA; pulmonary arterial pressure (PAP), blood cell counts, plasma sC5b-9 and thromboxane-B2 levels. Hemodialysis (60 min) was followed by reinfusion of extracorporeal blood into the circulation, and finally, an intravenous bolus injection of the complement activator zymosan. The data indicated low-extent steady rise of sC5b-9 along with transient leukopenia, secondary leukocytosis and thrombocytopenia in the two dialyzer groups, consistent with moderate complement activation. Surprisingly, small changes in baseline PAP and plasma thromboxane-B2 levels during hemodialysis switched into 30%–70% sharp rises in all three groups resulting in synchronous spikes within minutes after blood reinfusion. These observations suggest limited complement activation by dialyzer membranes, on which a membrane-independent second immune stimulus was superimposed, and caused pathophysiological changes also characteristic of HDRs. Thus, the porcine CARPA model raises the hypothesis that a second “hit” on anaphylatoxin-sensitized immune cells may be a key contributor to HDRs.

Abstract 2: Increased Complement Regulator CD55 Is Associated With A Reduction In Pancreatic Beta Cell Area In Islets Of Rat Offspring Following Chronic Placental Ischemia-induced Hypertension

Placental insufficiency can result in gestational hypertension and/or intrauterine growth restriction (IUGR) in the offspring. Gestational hypertension with or without low birth weight is associated with increased risk for Type 2 diabetes in the offspring. Pancreatic β cell mass, set very early in life, can influence whether an individual develops Type 2 diabetes. Our previous studies demonstrated a significant reduction in β cell area in embryonic day 19 and postnatal day (PD)13 female offspring of rats subjected to chronic placental ischemia-induced hypertension. Previous studies in human islets demonstrated important roles for complement C3 and activation product C3a in determining β cell area, with increased C3a generation associated with prevention of islet cell atrophy. Thus, we hypothesized that increases in self-proteins that limit complement system activation, i.e. complement regulators, are associated with reductions in β cell area in the offspring following placental insufficiency. The Reduced Uterine Perfusion Pressure (RUPP) model was used to induce placental insufficiency in the dam by placing silver clips on the abdominal aorta and uterine arteries on gestation day 14 of a 21 day gestation to mechanically reduce placental perfusion. This results in hypertension in the dam and IUGR in the offspring. Islets from the pancreas of (PD)13 female offspring of RUPP and Sham dams were isolated by collagenase perfusion and hand picking. The transcript message for complement regulators CD55 and Crry (regulators of C3 activation) and CD59 (terminal pathway regulator) was determined using qRT-PCR. Complement regulator CD55 was significantly increased in female RUPP offspring (2.01 + 0.29 fold) compared to female Sham offspring (1.00 + 0.13 fold change; p &lt; 0.05; 6-13 pups from 4-7 dams; mixed model ANOVA including litter effect), whereas the Crry increase of 1.38 + 0.20 fold in female RUPP vs Sham offspring was not significant (p = 0.22). No significant change was observed in message for CD59 or C3. Thus, these data suggest increased islet complement regulator CD55 may limit complement activation in islets of PD13 offspring following placental ischemia and thereby contribute to reduction of β cell area and long term susceptibility for Type 2 diabetes.

C5aR1 Activation Drives Early IFN-γ Production to Control Experimental Toxoplasma gondii Infection

Toxoplasma gondii (T. gondii) is a parasite infecting animals and humans. In intermediate hosts, such as humans or rodents, rapidly replicating tachyzoites drive vigorous innate and adaptive immune responses resulting in bradyzoites that survive within tissue cysts. Activation of the innate immune system is critical during the early phase of infection to limit pathogen growth and to instruct parasite-specific adaptive immunity. In rodents, dendritic cells (DCs) sense T. gondii through TLR11/12, leading to IL-12 production, which activates NK cells to produce IFN-γ as an essential mechanism for early parasite control. Further, C3 can bind to T. gondii resulting in limited complement activation. Here, we determined the role of C5a/C5aR1 axis activation for the early innate immune response in a mouse model of peritoneal T. gondii infection. We found that C5ar1−/− animals suffered from significantly higher weight loss, disease severity, mortality, and parasite burden in the brain than wild type control animals. Severe infection in C5ar1−/− mice was associated with diminished serum concentrations of IL-12, IL-27, and IFN-γ. Importantly, the serum levels of pro-inflammatory cytokines, including IL-1α, IL-6, and TNF-α, as well as several CXC and CC chemokines, were decreased in comparison to wt animals, whereas anti-inflammatory IL-10 was elevated. The defect in IFN-γ production was associated with diminished Ifng mRNA expression in the spleen and the brain, reduced frequency of IFN-γ+ NK cells in the spleen, and decreased Nos2 expression in the brain of C5ar1−/− mice. Mechanistically, DCs from the spleen of C5ar1−/− mice produced significantly less IL-12 in response to soluble tachyzoite antigen (STAg) stimulation in vivo and in vitro. Our findings suggest a model in which the C5a/C5aR1 axis promotes IL-12 induction in splenic DCs that is critical for IFN-γ production from NK cells and subsequent iNOS expression in the brain as a critical mechanism to control acute T. gondii infection.

Complement Receptor 1 (CR1/CD35)-expressing retinal pigment epithelial cells as a potential therapy for age-related macular degeneration

The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential “enhanced” cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.

The Human Platelet as an Innate Immune Cell: Interactions Between Activated Platelets and the Complement System.

Platelets play an essential role in maintaining homeostasis in the circulatory system after an injury by forming a platelet thrombus, but they also occupy a central node in the intravascular innate immune system. This concept is supported by their extensive interactions with immune cells and the cascade systems of the blood. In this review we discuss the close relationship between platelets and the complement system and the role of these interactions during thromboinflammation. Platelets are protected from complement-mediated damage by soluble and membrane-expressed complement regulators, but they bind several complement components on their surfaces and trigger complement activation in the fluid phase. Furthermore, localized complement activation may enhance the procoagulant responses of platelets through the generation of procoagulant microparticles by insertion of sublytic amounts of C5b9 into the platelet membrane. We also highlight the role of post-translational protein modifications in regulating the complement system and the critical role of platelets in driving these reactions. In particular, modification of disulfide bonds by thiol isomerases and protein phosphorylation by extracellular kinases have emerged as important mechanisms to fine-tune complement activity in the platelet microenvironment. Lastly, we describe disorders with perturbed complement activation where part of the clinical presentation includes uncontrolled platelet activation that results in thrombocytopenia, and illustrate how complement-targeting drugs are alleviating the prothrombotic phenotype in these patients. Based on these clinical observations, we discuss the role of limited complement activation in enhancing platelet activation and consider how these drugs may provide opportunities for further dissecting the complex interactions between complement and platelets.

Intravascular hemolysis activates complement via cell-free heme and heme-loaded microvesicles.

In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme triggered rapid P selectin, C3aR, and C5aR expression and downregulated CD46 on endothelial cells. Importantly, complement deposition was attenuated in vivo and in vitro by heme scavenger hemopexin. In conclusion, we demonstrate that intravascular hemolysis triggers complement activation in vivo, encouraging further studies on its role in SCD nephropathy. Conversely, heme inhibition using hemopexin may provide a novel therapeutic opportunity to limit complement activation in hemolytic diseases.

IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis.

Interleukin 17A (IL-17A) and complement (C') activation have each been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have reported that IL-17A induces epithelial injury via TGF-β in murine bronchiolitis obliterans; that TGF-β and the C' cascade present signaling interactions in mediating epithelial injury; and that the blockade of C' receptors mitigates lung fibrosis. In the present study, we investigated the role of IL-17A in regulating C' in lung fibrosis. Microarray analyses of mRNA isolated from primary normal human small airway epithelial cells indicated that IL-17A (100 ng/ml; 24 h; n = 5 donor lungs) induces C' components (C' factor B, C3, and GPCR kinase isoform 5), cytokines (IL8, -6, and -1B), and cytokine ligands (CXCL1, -2, -3, -5, -6, and -16). IL-17A induces protein and mRNA regulation of C' components and the synthesis of active C' 3a (C3a) in normal primary human alveolar type II epithelial cells (AECs). Wild-type mice subjected to IL-17A neutralization and IL-17A knockout (il17a-/- ) mice were protected against bleomycin (BLEO)-induced fibrosis and collagen deposition. Further, BLEO-injured il17a-/- mice had diminished levels of circulating Krebs Von Den Lungen 6 (alveolar epithelial injury marker), local caspase-3/7, and local endoplasmic reticular stress-related genes. BLEO-induced local C' activation [C3a, C5a, and terminal C' complex (C5b-9)] was attenuated in il17a-/- mice, and IL-17A neutralization prevented the loss of epithelial C' inhibitors (C' receptor-1 related isoform Y and decay accelerating factor), and an increase in local TUNEL levels. RNAi-mediated gene silencing of il17a in fibrotic mice arrested the progression of lung fibrosis, attenuated cellular apoptosis (caspase-3/7) and lung deposition of collagen and C' (C5b-9). Compared to normals, plasma from IPF patients showed significantly higher hemolytic activity. Our findings demonstrate that limiting complement activation by neutralizing IL-17A is a potential mechanism in ameliorating lung fibrosis.-Cipolla, E., Fisher, A. J., Gu, H., Mickler, E. A., Agarwal, M., Wilke, C. A., Kim, K. K., Moore, B. B., Vittal, R. IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis.

Rivaroxaban limits complement activation compared with warfarin in antiphospholipid syndrome patients with venous thromboembolism

Background Complement activation may play a major role in the pathogenesis of thrombotic antiphospholipid syndrome (APS). Coagulation proteases such as factor Xa can activate complement proteins. Aims To establish whether rivaroxaban, a direct factor Xa inhibitor, limits complement activation compared with warfarin in APS patients with previous venous thromboembolism (VTE). Methods A total of 111 APS patients with previous VTE, on warfarin target INR 2.5, had blood samples taken at baseline and at day 42 after randomization in the RAPS (Rivaroxaban in Antiphospholipid Syndrome) trial. Fifty-six patients remained on warfarin and 55 switched to rivaroxaban. Fifty-five normal controls (NC) were also studied. Markers of complement activation (C3a, C5a, terminal complement complex [SC5b-9] and Bb fragment) were assessed. Results APS patients had significantly higher complement activation markers compared with NC at both time-points irrespective of the anticoagulant. There were no differences between the two patient groups at baseline, or patients remaining on warfarin at day 42. In 55 patients randomized to rivaroxaban, C3a, C5a and SC5b-9 were lower at day 42 (median (ng mL-1 ) [confidence interval] 64 [29-125] vs. 83 [35-147], 9 [2-15] vs. 12 [4-18] and 171 [56-245] vs. 201 [66-350], respectively) but levels of Bb fragment were unchanged. There were no correlations between rivaroxaban levels and complement activation markers. Conclusions APS patients with previous VTE on warfarin exhibit increased complement activation, which is likely to occur via the classical pathway and is decreased by rivaroxaban administration. Rivaroxaban may therefore potentially provide an additional benefit to its anticoagulant effect in this patient group by limiting complement activation.

AHUS in children: A severe disease with increasingly better diagnostic and treatment options

Atypical haemolytic uraemic syndrome (aHUS) accounts for around 10% of children presenting with haemolytic uremic syndrome, a triad of thrombocytopenia, haemolytic anaemia and renal failure. As opposed to diarrhoeal causes usually associated with E. coli infection producing the toxin STEC, aHUS generally presents without diarrhoea and is frequently associated with defects in the complement pathway leading to over-activation of complement.The major cause of paediatric aHUS is due to defects in complement factor H, a regulatory protein that limits complement activation. Mutations in CFH that are both dominant and recessive have been described. There are now over 10 other genes that have been described in aHUS with around 40% of sporadic cases of aHUS without an identified genetic cause.Clinically patients often present acutely with an infectious prodrome, and may require supportive haemodialysis and transfusion. Children occasionally have extra renal manifestations involving GI and CNS. Therapy involves plasma exchange to replace complement factors and more recently the use of C5 inhibitors that have been highly effective.In those who progress to end-stage renal failure, transplantation carries a high risk of disease recurrence and this is highest with CFH mutations.Exclusion of infectious HUS and of TTP, a similar adult disease due to defects in ADAMTS13, is important. Genetic diagnosis has been significantly aided by massively parallel sequencing that can sequence the 10 known genes in one run. Further analysis using MLPA to identify rearrangements and deletions is also increasingly of use. Genetic identification assists in risk stratification and in identifying those children who will benefit from C5 inhibitors. Atypical haemolytic uraemic syndrome (aHUS) accounts for around 10% of children presenting with haemolytic uremic syndrome, a triad of thrombocytopenia, haemolytic anaemia and renal failure. As opposed to diarrhoeal causes usually associated with E. coli infection producing the toxin STEC, aHUS generally presents without diarrhoea and is frequently associated with defects in the complement pathway leading to over-activation of complement. The major cause of paediatric aHUS is due to defects in complement factor H, a regulatory protein that limits complement activation. Mutations in CFH that are both dominant and recessive have been described. There are now over 10 other genes that have been described in aHUS with around 40% of sporadic cases of aHUS without an identified genetic cause. Clinically patients often present acutely with an infectious prodrome, and may require supportive haemodialysis and transfusion. Children occasionally have extra renal manifestations involving GI and CNS. Therapy involves plasma exchange to replace complement factors and more recently the use of C5 inhibitors that have been highly effective. In those who progress to end-stage renal failure, transplantation carries a high risk of disease recurrence and this is highest with CFH mutations. Exclusion of infectious HUS and of TTP, a similar adult disease due to defects in ADAMTS13, is important. Genetic diagnosis has been significantly aided by massively parallel sequencing that can sequence the 10 known genes in one run. Further analysis using MLPA to identify rearrangements and deletions is also increasingly of use. Genetic identification assists in risk stratification and in identifying those children who will benefit from C5 inhibitors.

Rivaroxaban Limits Complement Activation Compared to Warfarin in Antiphospholipid Syndrome Patients with Venous Thromboembolism

BackgroundComplement activation may play a role in the pathogenesis of thrombosis and other pathological processes in the antiphospholipid syndrome (APS). Since coagulation proteases, such as factor Xa, can cleave complement proteins, we investigated complement activation in thrombotic APS patients receiving rivaroxaban, a direct factor Xa inhibitor.AimsTo assess markers of complement activation (C3a, C5a, terminal complement complex (SC5b-9) and Bb fragment) in patients with thrombotic APS treated with rivaroxaban or warfarin in a prospective randomised controlled trial.Methods116 APS patients with previous venous thromboembolism, including 22 with systemic lupus erythematosus (SLE), on long-term warfarin (target INR 2.5) were studied. 59 patients remained on warfarin and 57 (11 with SLE in each group) switched to rivaroxaban (20mg daily). EDTA samples were collected at baseline (all patients on warfarin) and on day 42 (2-4 hours after the last dose of rivaroxaban in patients on rivaroxaban). 5/116 patients were excluded (samples from four patients were haemolysed and one patient withdrew from the trial after randomisation), leaving 111 (55 rivaroxaban and 56 warfarin) patients for analysis at both baseline and day 42. Samples were also collected from 55 normal controls (NC). C3a, C5a SC5b-9 and Bb fragment were assessed using ELISA assay kits (QUIDEL Corp).ResultsMedian (95% CI) C3a, C5a, SC5b-9 and Bb fragment were 48.9 (30.1-100.2) ng/mL, 6.8 (2.2-11.8 ng/mL, 113.9 (50.5-170) ng/mL and 1.1 (0.64-1.86) µg/mL in NC, respectively. APS patients had significantly higher complement activation markers compared to NC at both time points irrespective of the anticoagulant (p<0.0001 for C3a, C5a, SC5b-9 and Bb). There were no differences in the markers between the two patient groups at baseline, or in patients remaining on warfarin at day 42 [median (95% CI) for C3a, C5a, SC5b-9 and Bb fragment levels in patient on warfarin on day 0 vs day 42 were: C3a (ng/mL) 77.2 (33.4-180.1) vs 73.6 (34.7-156), C5a (ng/mL) 10.8 (3.2-19.4) vs 10.3 (3.7-19.8), SC5b-9 (ng/mL) 203.5 (70.5-440.3) vs 214.4 (78.3-470.4) and Bb fragment (µg/mL) 1.3 (0.6-2.8) vs 1.4 (0.7-2.4)].In 55 patients randomised to rivaroxaban, C3a, C5a and SC5b-9 decreased significantly compared with baseline values on warfarin [day 0 versus day 42: C3a (ng/mL): 82.8 (34.6-146.6) vs 64.0 (29.2-125.1), (p=0.004); C5a (ng/mL):12.0 (4.1-17.9) vs 9.0 (2.4-14.8), p=0.01; SC5b-9 (ng/mL): 201.0 (65.6-350.2) vs 171.5 (55.6-245.5), (p=0.001)]. However, Bb fragment levels were unchanged.ConclusionsComplement activation occurs in APS despite anticoagulation with warfarin. Rivaroxaban decreased complement activation compared to warfarin, although levels of the markers did not normalise in the majority of patients. This action of rivaroxaban appears to occur via the classical pathway, since Bb fragment (a marker of alternative pathway activation) was unchanged. The observations in rivaroxaban-treated patients may reflect inhibition of factor Xa cleavage of complement proteins, or inhibition of its pro-inflammatory effects (and consequent complement activation). These data suggest that rivaroxaban may have an additional therapeutic modality in thrombotic APS patients by limiting complement activation. DisclosuresMackie:Volution Immuno Pharmaceuticals (Uk) Ltd: Research Funding. Cohen:Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Honoraria diverted to local charity, Research Funding, Speakers Bureau.

Complement Is a Central Mediator of Radiotherapy-Induced Tumor-Specific Immunity and Clinical Response

Radiotherapy induces DNA damage and cell death, but recent data suggest that concomitant immune stimulation is an integral part of the therapeutic action of ionizing radiation. It is poorly understood how radiotherapy supports tumor-specific immunity. Here we report that radiotherapy induced tumor cell death and transiently activated complement both in murine and human tumors. The local production of pro-inflammatory anaphylatoxins C3a and C5a was crucial to the tumor response to radiotherapy and concomitant stimulation of tumor-specific immunity. Dexamethasone, a drug frequently given during radiotherapy, limited complement activation and the anti-tumor effects of the immune system. Overall, our findings indicate that anaphylatoxins are key players in radiotherapy-induced tumor-specific immunity and the ensuing clinical responses.

An effective treatment of atypical hemolytic uremic syndrome with plasma exchange and eculizumab: A case report

Atypical hemolytic uremic syndrome is a rare thrombotic microangiopathy caused by chronic defective regulation of the complement activation. This activation results in systemic endothelial damage leading to renal failure. Eculizumab, an anti-C5 antibody, is effective in limiting complement activation in patients with aHUS and has recently came out as a therapeutic option for aHUS. Here we present a case showing that first-line eculizumab treatment successfully prevents the induction of the terminal complement cascade and blocked the progression of thrombotic microangiopathy in aHUS.

Heparin molecularly imprinted surfaces for the attenuation of complement activation in blood.

Heparin-imprinted synthetic polymer surfaces with the ability to attenuate activation of both the complement and the coagulation system in whole blood were successfully produced. Imprinting was achieved using a template coated with heparin, a highly sulfated glycosaminoglycan known for its anticoagulant properties. The N,N'-diacryloylpiperazine-methacrylic acid copolymers were characterized using goniometry, AFM and XPS. The influence of the molecular imprinting process on morphology and template rebinding was demonstrated by radioligand binding assays. Surface hemocompatibility was evaluated using human whole blood without anticoagulants followed by measurement of complement activation markers C3a and sC5b-9 and platelet consumption as a surrogate coagulation activation marker. The observed low thrombogenicity of this copolymer combined with the attenuation of complement activation induced by the molecular imprint offer potential for the development of self-regulating surfaces with important potential clinical applications. We propose a mechanism for the observed phenomena based upon the recruitment of endogenous sulfated glycosaminoglycans with heparin-like activities.

Self-assembled 3D biocompatible and bioactive layer at the macro-interface via graphene-based supermolecules

Research on the interface self-assembly of functional layers is fuelled by diverse biomedical needs, like drug encapsulation and release, stem cell proliferation and differentiation, cell and tissue cultures, as well as artificial organs. In this study, a novel and biocompatible 3D composite layer is fabricated on a membrane substrate by the layer-by-layer (LBL) self-assembly of graphene-based 2D supermolecules. The graphene-based 2D supermolecules are prepared by grafting poly(styrenesulfonate) (PSS, polyanion) and poly(acrylamide) (PAM, polycation) onto graphene oxide (GO) through free radical polymerization. Then, the prepared graphene-based supermolecules are taken to construct a 3D porous thin film layer through a LBL process. Polyethersulfone (PES) membrane is chosen as the model substrate. The chemical composition, surface morphology, water contact angle, surface zeta-potential, blood compatibility and the cell compatibility are systematically investigated. The results indicate that the 2D graphene-based supermolecules are successfully assembled into a 3D porous thin film layer structure on the membrane surface. The assembled layer shows increased hydrophilicity, suppressed platelet adhesion, and a limited hemolysis ratio and complement activation compared to the pristine substrate. The cell morphology observation and cytotoxicity assays demonstrate that the 3D graphene layer shows superior performance both in endothelial and hepatocyte cells proliferation and morphology differentiation. In addition, the excellent anticoagulant bioactivities indicate that the 2D graphene-based supermolecules have heparin-mimicking biological activity. In general, the protocol of interface thin film layer self-assembly confers the modified substrates with integrated blood compatibility, cell proliferation and biological activity; which may forward the fabrication of multi-functional biomedical devices by using the 2D graphene-based supermolecules.

Islet Amyloid Polypeptide Triggers Limited Complement Activation and Binds Complement Inhibitor C4b-binding Protein, Which Enhances Fibril Formation

Islet amyloid polypeptide (IAPP) is synthesized in pancreatic β-cells and co-secreted with insulin. Aggregation and formation of IAPP-amyloid play a critical role in β-cell death in type 2 diabetic patients. Because Aβ-fibrils in Alzheimer disease activate the complement system, we have here investigated specific interactions between IAPP and complement factors. IAPP fibrils triggered limited activation of complement in vitro, involving both the classical and the alternative pathways. Direct binding assays confirmed that IAPP fibrils interact with globular head domains of complement initiator C1q. Furthermore, IAPP also bound complement inhibitors factor H and C4b-binding protein (C4BP). Recombinant C4BP mutants were used to show that complement control protein (CCP) domains 8 and 2 of the α-chain were responsible for the strong, hydrophobic binding of C4BP to IAPP. Immunostaining of pancreatic sections from type 2 diabetic patients revealed the presence of complement factors in the islets and varying degree of co-localization between IAPP fibrils and C1q, C3d, as well as C4BP and factor H but not membrane attack complex. Furthermore, C4BP enhanced formation of IAPP fibrils in vitro. We conclude that C4BP binds to IAPP thereby limiting complement activation and may be enhancing formation of IAPP fibrils from cytotoxic oligomers.

The salivary scavenger and agglutinin binds MBL and regulates the lectin pathway of complement in solution and on surfaces

The salivary scavenger and agglutinin (SALSA), also known as gp340, salivary agglutinin and deleted in malignant brain tumor 1, is a 340-kDa glycoprotein expressed on mucosal surfaces and secreted into several body fluids. SALSA binds to a broad variety of microbes and endogenous ligands, such as complement factor C1q, surfactant proteins D and A, and IgA. Our search for novel ligands of SALSA by direct protein-interaction studies led to the identification of mannan-binding lectin (MBL) as a new binding partner. We observed that surface-associated SALSA activates complement via binding of MBL. On the other hand, soluble SALSA was found to inhibit Candida albicans-induced complement activation. Thus, SALSA has a dual complement activation modifying function. It activates the lectin pathway when bound to a surface and inhibits it when free in the fluid phase. These activities are mediated via a direct interaction with MBL. This suggests that SALSA could target the innate immune responses to certain microorganisms and simultaneously limit complement activation in the fluid phase.

Islet amyloid polypeptide triggers limited complement activation and binds complement inhibitor C4b-binding protein, which enhances fibril formation

Islet amyloid polypeptide (IAPP) is synthesized in pancreatic β-cells and co-secreted with insulin. Aggregation and formation of IAPP-amyloid play a critical role in β-cell death in type 2 diabetic patients. Because Aβ-fibrils in Alzheimer disease activate the complement system, we have here investigated specific interactions between IAPP and complement factors. IAPP fibrils triggered limited activation of complement in vitro, involving both the classical and the alternative pathways. Direct binding assays confirmed that IAPP fibrils interact with globular head domains of complement initiator C1q. Furthermore, IAPP also bound complement inhibitors factor H and C4b-binding protein (C4BP). Recombinant C4BP mutants were used to show that complement control protein (CCP) domains 8 and 2 of the α-chain were responsible for the strong, hydrophobic binding of C4BP to IAPP. Immunostaining of pancreatic sections from type 2 diabetic patients revealed the presence of complement factors in the islets and varying degree of co-localization between IAPP fibrils and C1q, C3d, as well as C4BP and factor H but not membrane attack complex. Furthermore, C4BP enhanced formation of IAPP fibrils in vitro. We conclude that C4BP binds to IAPP thereby limiting complement activation and may be enhancing formation of IAPP fibrils from cytotoxic oligomers.

Binding of Flavivirus Nonstructural Protein NS1 to C4b Binding Protein Modulates Complement Activation

The complement system plays a pivotal protective role in the innate immune response to many pathogens including flaviviruses. Flavivirus nonstructural protein 1 (NS1) is a secreted nonstructural glycoprotein that accumulates in plasma to high levels and is displayed on the surface of infected cells but absent from viral particles. Previous work has defined an immune evasion role of flavivirus NS1 in limiting complement activation by forming a complex with C1s and C4 to promote cleavage of C4 to C4b. In this study, we demonstrate a second mechanism, also involving C4 and its active fragment C4b, by which NS1 antagonizes complement activation. Dengue, West Nile, or yellow fever virus NS1 directly associated with C4b binding protein (C4BP), a complement regulatory plasma protein that attenuates the classical and lectin pathways. Soluble NS1 recruited C4BP to inactivate C4b in solution and on the plasma membrane. Mapping studies revealed that the interaction sites of NS1 on C4BP partially overlap with the C4b binding sites. Together, these studies further define the immune evasion potential of NS1 in reducing the functional capacity of C4 in complement activation and control of flavivirus infection.