Role Of Complement Activation Research Articles

The role of complement in antineutrophil cytoplasmic antibody-associated vasculitis.

To provide a comprehensive overview of the current insight into the role of complement activation in antineutrophil cytoplasmic antibody-associated vasculitis (AAV). In addition, the therapeutic options targeting the complement system in AAV are discussed. It has become increasingly clear that complement, and more specifically signalling through the C5a receptor, contributes to the immunopathology of AAV. This has led to the design of clinical trials with a C5a receptor blocker. The first results show a reduction in tissue damage and a favourable safety profile, as other parts of the complement defence system are left intact. Although AAV was initially regarded as a pauci-immune disease, it is now well established that, in addition to autoantibodies, complement plays an essential role in the disease process. Animal models delivered the first insight, but the effective therapeutic interventions using complement inhibitors provided the proof that indeed complement activation contributes to disease activity and tissue damage in human AAV.

Use of Eculizumab for the Treatment of Hyperhaemolysis in Pregnancy in Sickle Cell Disease: A Case Report

Hyperhaemolysis is a rare but well recognized complication of transfusion in sickle cell disease, associated with increased mortality. The pathogenesis is not well understood and is likely to be multifactorial. There is no gold standard management of hyperhaemolysis; immunosuppression with steroids and intravenous immuoglobulins has been the mainstay of treatment. More recently Rituximab and Eculizumab are increasingly used. Hyperhaemolysis in pregnancy is an even more complex problem with compounded risk to the mother and foetus.This case reports a 29 year old female with HbSS who presented with hyperhaemolysis in pregnancy at 25 weeks gestation, 7 days after a red cell transfusion for a vaso-occlusive crisis. The patient received 1 g methylprednisolone and 2g/kg immunoglobulins in divided doses. Despite this, she required further blood transfusion as the haemoglobin was declining further and was symptomatic for her anaemia. Her haemoglobin reached a nadir level of 39 g/l. Therefore she was treated with Eculizumab 900 mg iv pre transfusion to prevent exacerbation of the haemolysis triggered by the transfusion. Her haemoglobin stabilised and the haemolysis resolved within 48 hours. She delivered safely at 34 weeks gestation.The DAT remained negative throughout and no new red cell antibodies were detected. Urine HPLC confirmed the presence of HbA and HbS in the urine, confirming the diagnosis. The urine HPLC 48 hr post Eculizumab and 2 further red cell units was clear, demonstrating no further haemolysis with the additional red cells given.This is the first case report detailing the use of Eculizumab for the management of hyperhaemolysis in pregnancy in sickle cell disease. The patient tolerated the treatment which resulted in resolution of the haemolysis, with safe delivery at 34 weeks gestation. The possible role of complement activation in hyperhaemolysis is discussed. The use of Eculizumab in other case reports of hyperhaemolysis is reviewed. The ideal dosing schedule remains uncertain. This report strongly suggests that Eculizumab should be accessible and considered in severe cases of hyperhaemolysis which are refractory to standard treatment. It also suggests safe usage in pregnancy in sickle cell patients. DisclosuresScully:Novartis: Honoraria, Other: Member of Advisory Board, Speakers Bureau. Porter:Cerus: Honoraria; Novartis: Consultancy; Agios: Honoraria.

Complements are involved in alcoholic fatty liver disease, hepatitis and fibrosis.

The complement system is a key component of the body’s immune system. When abnormally activated, this system can induce inflammation and damage to normal tissues and participate in the development and progression of a variety of diseases. In the past, many scholars believed that alcoholic liver disease (ALD) is induced by the stress of ethanol on liver cells, including oxidative stress and dysfunction of mitochondria and protease bodies, causing hepatocyte injury and apoptosis. Recent studies have shown that complement activation is also involved in the genesis and development of ALD. This review focuses on the roles of complement activation in ALD and of therapeutic intervention in complement-activation pathways. We intend to provide new ideas on the diagnosis and treatment of ALD.

Characterizing a pH‐switch anti‐C5 antibody as a tool for human and mouse complement C5 purification and cross‐species inhibition of classical and reactive lysis

SummaryC5 plays a major role in complement activation; C5 convertase cleaves C5 into the pro‐inflammatory C5a, and C5b, the nidus for the formation of the lytic membrane attack complex. C5 is a major target for anti‐complement drugs, necessitating better methods for the study of C5 function. Purification of C5 is complicated; classical methods involve precipitation or pH shifts that result in functional loss and low yield. We here present a method for C5 purification using a novel anti‐C5 monoclonal antibody (mAb); RO7112689 (C5i mAb, SKY59), pH‐switch engineered to induce antibody–antigen dissociation in the acidic endosome (~ pH 5·5). RO7112689 was bound on an affinity column; applied serum was completely depleted of C5. Elution at pH 5 produced fully active C5 at 98% yield. The mAb also bound C5b in the C5b6 complex, preventing C5b6 binding to target membranes and enabling purification of C5b6 from activated serum. RO7112689 inhibited C5 in mouse serum and efficiently purified mouse C5. Used as capture, RO7112689 produced sensitive and specific assays for human and mouse C5. This novel antibody enables efficient production of intact, fully active, pure human and mouse C5, and quantification of C5 in these species. The demonstration that RO7112689 binds C5b6 adds an additional mechanism of membrane attack complex inhibition by this mAb.

Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models.

Complement is essential for the protection against infections; however, dysregulation of complement activation can cause onset and progression of numerous inflammatory diseases. Convertase enzymes play a central role in complement activation and produce the key mediators of complement: C3 convertases cleave C3 to generate chemoattractant C3a and label target cells with C3b, which promotes phagocytosis; C5 convertases cleave C5 into chemoattractant C5a, and C5b, which drives formation of the membrane attack complex. Since convertases mediate nearly all complement effector functions, they are ideal targets for therapeutic complement inhibition. A unique feature of convertases is their covalent attachment to target cells, which effectively confines complement activation to the cell surface. However, surface localization precludes detailed analysis of convertase activation and inhibition. In our previous work, we developed a model system to form purified alternative pathway (AP) C5 convertases on C3b-coated beads and quantify C5 conversion via functional analysis of released C5a. Here, we developed a C3aR cell reporter system that enables functional discrimination between C3 and C5 convertases. By regulating the C3b density on the bead surface, we observe that high C3b densities are important for conversion of C5, but not C3, by AP convertases. Screening of well-characterized complement-binding molecules revealed that differential inhibition of AP C3 convertases (C3bBb) and C5 convertases [C3bBb(C3b)n] is possible. Although both convertases contain C3b, the C3b-binding molecules Efb-C/Ecb and FHR5 specifically inhibit C5 conversion. Furthermore, using a new classical pathway convertase model, we show that these C3b-binding proteins not only block AP C3/C5 convertases but also inhibit formation of a functional classical pathway C5 convertase under well-defined conditions. Our models enable functional characterization of purified convertase enzymes and provide a platform for the identification and development of specific convertase inhibitors for treatment of complement-mediated disorders.

Therapy of scleroderma renal crisis: State of the art

Scleroderma renal crisis (SRC) is an uncommon but still life-threatening manifestation of systemic sclerosis (SSc). The incidence of SRC has decreased in the last few decades, probably due to a widespread use of vasodilators in SSc patients. It is well-recognized that exposure to different drugs can trigger SRC (corticosteroids, cyclosporine) or might prevent its occurrence (iloprost, calcium channel blockers). The prognosis of this life-threatening manifestation has not substantially improved since 1980s, when ACE-inhibitors were introduced in its treatment. ACE-inhibitors remain the mainstay in the therapy of SRC due to their efficacy in controlling malignant hypertension; indeed, the prognosis largely depends on the rapid improvement of the ongoing renal ischemia. Calcium-channel blockers and in third line diuretics and alpha-blockers should be used as additional therapy if blood pressure control remains suboptimal despite maximum tolerated doses of ACE-inhibitors. Given the growing evidence on the role of complement activation and endothelin-1 in the pathogenesis of SRC, recent case-series and case reports have suggested the use of C5-inhibitors and endothelin receptor antagonists in the therapy of SRC, mainly in the refractory cases. Plasma-exchange seems to give some benefits in patients with SRC and microangiopathy or intolerant to ACE-inhibitors. Renal transplantation is the last treatment option and its outcome is similar to that reported in other connective tissue disorders, with a 5-year patient survival rate of about 82%. In this review we summarize the current knowledge in the treatment of SRC.

Complement Activation in Liver Transplantation: Role of Donor Macrosteatosis and Implications in Delayed Graft Function.

The complement system anchors the innate inflammatory response by triggering both cell-mediated and antibody-mediated immune responses against pathogens. The complement system also plays a critical role in sterile tissue injury by responding to damage-associated molecular patterns. The degree and duration of complement activation may be a critical variable controlling the balance between regenerative and destructive inflammation following sterile injury. Recent studies in kidney transplantation suggest that aberrant complement activation may play a significant role in delayed graft function following transplantation, confirming results obtained from rodent models of renal ischemia/reperfusion (I/R) injury. Deactivating the complement cascade through targeting anaphylatoxins (C3a/C5a) might be an effective clinical strategy to dampen reperfusion injury and reduce delayed graft function in liver transplantation. Targeting the complement cascade may be critical in donor livers with mild to moderate steatosis, where elevated lipid burden amplifies stress responses and increases hepatocyte turnover. Steatosis-driven complement activation in the donor liver may also have implications in rejection and thrombolytic complications following transplantation. This review focuses on the roles of complement activation in liver I/R injury, strategies to target complement activation in liver I/R, and potential opportunities to translate these strategies to transplanting donor livers with mild to moderate steatosis.

Infusion Reactions Associated with the Medical Application of Monoclonal Antibodies: The Role of Complement Activation and Possibility of Inhibition by Factor H.

Human application of monoclonal antibodies (mAbs), enzymes, as well as contrast media and many other particulate drugs and agents referred to as “nanomedicines”, can initiate pseudoallergic hypersensitivity reactions, also known as infusion reactions. These may in part be mediated by the activation of the complement system, a major humoral defense system of innate immunity. In this review, we provide a brief outline of complement activation-related pseudoallergy (CARPA) in general, and then focus on the reactions caused by mAb therapy. Because the alternative pathway of complement activation may amplify such adverse reactions, we highlight the potential use of complement factor H as an inhibitor of CARPA.

The role of complement activation in rhabdomyolysis-induced acute kidney injury.

Rhabdomyolysis (RM) may cause kidney damage and results primarily in acute kidney injury (AKI). Complement is implicated in the pathogenesis of renal diseases and ischemia-reperfusion injury (IRI), but the role of complement, especially its activation pathway(s) and its effect in RM-induced AKI, is not clear. This study established a rat model of AKI induced by RM via intramuscular treatment with glycerol. Cobra venom factor (CVF) was administered via tail vein injection to deplete complement 12 h prior to intramuscular injection of glycerol. We found that the complement components, including complement 3 (C3), C1q, MBL-A, factor B(fB), C5a, C5b-9, and CD59, were significantly increased in rat kidneys after intramuscular glycerol administration. However, the levels of serum BUN and Cr, renal tubular injury scores, and the number of TUNEL-positive cells decreased significantly in the CVF+AKI group. These results suggest that complement plays an important role in RM-induced AKI and that complement depletion may improve renal function and decrease renal tissue damage by reducing the inflammatory response and apoptosis.

Modulation of the Alternative Pathway of Complement by Murine Factor H-Related Proteins.

Factor H (FH) is a key alternative pathway regulator that controls complement activation both in the fluid phase and on specific cell surfaces, thus allowing the innate immune response to discriminate between self and foreign pathogens. However, the interrelationships between FH and a group of closely related molecules, designated the FH-related (FHR) proteins, are currently not well understood. Whereas some studies have suggested that human FHR proteins possess complement regulatory abilities, recent studies have shown that FHR proteins are potent deregulators. Furthermore, the roles of the FHR proteins have not been explored in any in vivo models of inflammatory disease. In this study, we report the cloning and expression of recombinant mouse FH and three FHR proteins (FHR proteins A-C). Results from functional assays show that FHR-A and FHR-B proteins antagonize the protective function of FH in sheep erythrocyte hemolytic assays and increase cell-surface C3b deposition on a mouse kidney proximal tubular cell line (TEC) and a human retinal pigment epithelial cell line (ARPE-19). We also report apparent KD values for the binding interaction of mouse C3d with mouse FH (3.85 μM), FHR-A (136 nM), FHR-B (546 nM), and FHR-C (1.04 μM), which directly correlate with results from functional assays. Collectively, our work suggests that similar to their human counterparts, a subset of mouse FHR proteins have an important modulatory role in complement activation. Further work is warranted to define the in vivo context-dependent roles of these proteins and determine whether FHR proteins are suitable therapeutic targets for the treatment of complement-driven diseases.

Shiga toxin triggers endothelial and podocyte injury: the role of complement activation.

Shiga toxin (Stx)-producing Escherichia coli (STEC) is the offending agent in post-diarrhea-associated hemolytic uremic syndrome (HUS), a disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia, and acute kidney failure, with thrombi occluding the renal microvasculature. Endothelial dysfunction has been recognized as the trigger event in the development of microangiopathic processes. Glomerular endothelial cells are susceptible to the toxic effects of Stxs that, via nuclear factor kappa B (NF-κB) activation, induce the expression of genes encoding for adhesion molecules and chemokines, culminating in leukocyte adhesion and platelet thrombus formation on the activated endothelium. Complement activation via the alternative pathway has been seen in patients during the acute phase of STEC-associated HUS. Experimental evidence has highlighted the role of complement proteins in driving glomerular endothelium toward a thrombogenic phenotype. At the glomerular level, podocytes are also an important target of Stx-induced complement activation. Glomerular injury as a consequence of podocyte dysfunction and loss is thus a mechanism that might affect long-term renal outcomes in the disease. New approaches to targeting the complement system may be useful therapeutic options for patients with STEC-HUS.

Circulating complement component 4d (C4d) correlates with tumor volume, chemotherapeutic response and survival in patients with malignant pleural mesothelioma

Only limited information is available on the role of complement activation in malignant pleural mesothelioma (MPM). Thus, we investigated the circulating and tissue levels of the complement component 4d (C4d) in MPM. Plasma samples from 55 MPM patients, 21 healthy volunteers (HV) and 14 patients with non-malignant pleural diseases (NMPD) were measured by ELISA for C4d levels. Tissue specimens from 32 patients were analyzed by C4d immunohistochemistry. Tumor volumetry was measured in 20 patients. We found no C4d labeling on tumor cells, but on ectopic lymphoid structures within the tumor stroma. Plasma C4d levels did not significantly differ between MPM, HV or NMPD. Late-stage MPM patients had higher plasma C4d levels compared to early-stage (p = 0.079). High circulating C4d was associated with a higher tumor volume (p = 0.047). Plasma C4d levels following induction chemotherapy were significantly higher in patients with stable/progressive disease compared to those with partial/major response (p = 0.005). Strikingly, patients with low C4d levels at diagnosis had a significantly better overall survival, confirmed in a multivariate cox regression model (hazard ratio 0.263, p = 0.01). Our findings suggest that circulating plasma C4d is a promising new prognostic biomarker in patients with MPM and, moreover, helps to select patients for surgery following induction chemotherapy.

Complement Activation in Patients With Diabetic Nephropathy

IntroductionComplement activation plays a role in various organs in patients with diabetes. However, in diabetic nephropathy (DN), the role of complement activation is poorly understood. We examined the prevalence and clinical significance of complement deposits in the renal tissue of cases with type 1 and type 2 diabetes with and without DN.MethodsWe measured the prevalence of glomerular C4d, C1q, mannose-binding lectin (MBL), and C5b-9 deposits in 101 autopsied diabetic cases with DN, 59 autopsied diabetic cases without DN, and 41 autopsied cases without diabetes or kidney disease. The presence of complement deposits was scored by researchers who were blinded with respect to the clinical and histological data.ResultsC4d deposits were more prevalent in cases with DN than in cases without DN in both the glomeruli (46% vs. 26%) and the arterioles (28% vs. 12%). C1q deposits were also increased in the glomerular hili (77% vs. 55%) and arterioles (33% vs.14%), and were correlated with DN (P < 0.01). MBL deposits were only rarely observed. C5b-9 deposits were more prevalent in the cases with diabetes mellitus (DM) than in the cases without DM (69% vs. 32%; P < 0.001). Finally, glomerular C4d and C5b-9 deposits were correlated with the severity of DN (ρ = 0.341 and 0.259, respectively; P < 0.001).ConclusionComplement activation is correlated with both the presence and severity of DN, suggesting that the complement system is involved in the development of renal pathology in patients with diabetes and is a promising target for inhibiting and/or preventing DN in these patients.

Construction of human MASP-2-CCP1/2SP, CCP2SP, SP plasmid DNA nanolipoplexes and the effects on tuberculosis in BCG-infected mice.

The lectin pathway, one of the complement cascade systems, provides the primary line of defense against invading pathogens. The serine protease of MASP-2 plays an essential role in complement activation of the lectin pathway. The C-terminal segment of MASP-2 is comprised of the CCP1-CCP2-SP domains, and is the crucial catalytic segment. However, what is the effect of CCP1-CCP2-SP domains in controlling chronic infection is unknown. In order to evaluate the potential impact of CCP1-CCP2-SP domains on tuberculosis, we constructed the human MASP-2 CCP1/2SP, CCP2SP and SP recombinant plasmids, and delivered these plasmids by DNA-DOTAP:cholesterol cationic nanolipoplexes to BCG-infected mice. After 21 days post DNA-DOTAP:chol nanolipoplexes application, we analyzed bacteria loads of pulmonary, pathology of granuloma, lymphocyte subpopulations. The C3a, C4a and MASP-2 levels in serum were measured with enzyme-linked immunosorbent assays. Compared to the control group that received GFP DNA-DOTAP:chol nanolipoplexes, MASP-2 CCP1/2SP DNA-DOTAP:chol nanolipoplexes treated group showed significantly enlarged pulmonary granulomas lesion (P<0.05) and did not reduce bacteria loads in the lung tissue (P<0.05). Furthermore, the levels of C3a in serum were decreased (P<0.05), the number and percentage of PD1+ and Tim3+ cells subgroups were increased in BCG-infected mice after treated with MASP-2 CCP1/2SP DNA-DOTAP:chol nanolipoplexes (P<0.05). But, there was no statistical difference in the serum C4a and MASP-2 level among DNA nanolipoplexes treated groups (P>0.05). These findings provided experimental evidence that MASP-2 CCP1/2SP DNA nanolipoplexes shown the negative efficacy in controlling Mycobacterium tuberculosis infection, and displayed a potential role of down-regulating T-cell-mediated immunity in tuberculosis.

In Situ complement activation and T-cell immunity in leprosy spectrum: An immunohistological study on leprosy lesional skin.

Mycobacterium leprae (M. leprae) infection causes nerve damage and the condition worsens often during and long after treatment. Clearance of bacterial antigens including lipoarabinomannan (LAM) during and after treatment in leprosy patients is slow. We previously demonstrated that M. leprae LAM damages peripheral nerves by in situ generation of the membrane attack complex (MAC). Investigating the role of complement activation in skin lesions of leprosy patients might provide insight into the dynamics of in situ immune reactivity and the destructive pathology of M. leprae. In this study, we analyzed in skin lesions of leprosy patients, whether M. leprae antigen LAM deposition correlates with the deposition of complement activation products MAC and C3d on nerves and cells in the surrounding tissue. Skin biopsies of paucibacillary (n = 7), multibacillary leprosy patients (n = 7), and patients with erythema nodosum leprosum (ENL) (n = 6) or reversal reaction (RR) (n = 4) and controls (n = 5) were analyzed. The percentage of C3d, MAC and LAM deposition was significantly higher in the skin biopsies of multibacillary compared to paucibacillary patients (p = <0.05, p = <0.001 and p = <0.001 respectively), with a significant association between LAM and C3d or MAC in the skin biopsies of leprosy patients (r = 0.9578, p< 0.0001 and r = 0.8585, p<0.0001 respectively). In skin lesions of multibacillary patients, MAC deposition was found on axons and co-localizing with LAM. In skin lesions of paucibacillary patients, we found C3d positive T-cells in and surrounding granulomas, but hardly any MAC deposition. In addition, MAC immunoreactivity was increased in both ENL and RR skin lesions compared to non-reactional leprosy patients (p = <0.01 and p = <0.01 respectively). The present findings demonstrate that complement is deposited in skin lesions of leprosy patients, suggesting that inflammation driven by complement activation might contribute to nerve damage in the lesions of these patients. This should be regarded as an important factor in M. leprae nerve damage pathology.

Complement activation turnover on surfaces of nanoparticles

The complement system is an important component of the innate immune system, which contributes to non-specific host defence. Particulate matters, such as invading pathogens and nanomedicines, in the blood may activate the complement system through classical, lectin and alternative pathways. Complement activation can aid recognition and clearance of particulate matters by immune cells, but uncontrolled complement activation can inflict damage and be life threatening. Plasma proteins on adsorption to surfaces of nanoparticles also play a significant role in complement activation and particularly through the alternative pathway. This process is continuous and changeable in vivo; protein-complement complexes are formed on the nanoparticle surface and then released and the cycle repeats on further plasma protein deposition. This complement activation turnover poses a challenge for design of immune-safe nanomedicines.

Correlation of complement depletion and the severity of sepsis in children

Objective To analyze the relationship between complement key component C3, C4 and the severity of sepsis in children, in order to explore the role of complement activation in the progression of sepsis and provide a reference for diagnosis and treatment of severe sepsis. Methods Four hundred and twenty-four children diagnosed as sepsis from December 2012 to December 2015 in Children′s Hospital of Nanjing Medical University were enrolled in this study, among whom 347 children with sepsis were eligible for the following research including 169 cases of common sepsis and 178 cases of severe sepsis. Blood specimens were collected in 24 hours after their admission into pediatric intensive care unit(PICU) for the analysis of lymphocyte subsets, humoral immunity, blood routine analysis, coagulation, liver and renal function analysis. General information was collected by consulting their medical records, laboratory analysis and clinical treatment. The relationship between complement C3, C4 and the severity of sepsis was analyzed, and the correlation between C3 and coagulation, liver, renal, myocardium damage was also studied. Logistic regression was used to analyze the relationship between C3 and the progression to severe sepsis and multiple organ dysfunction syndrome(MODS), while Cox regression was used for survival analysis. Results Natural killer(NK) cell percentage was lower in severe sepsis group than that in common sepsis group[6.6%(3.7%, 10.7%) vs. 8.5%(4.7%, 13.3%), Z=2.635, P=0.008], while C3 decreased in severe sepsis group compared with common sepsis group [0.653(0.462, 0.985) g/L vs. 0.991(0.678, 1.265) g/L, Z=5.684, P<0.001], and C4 decreased in severe sepsis group compared with common sepsis group [0.160(0.102, 0.244) g/L vs. 0.190(0.121, 0.265) g/L, Z=2.513, P=0.012]. The proportion of severe pneumonia was higher in severe sepsis group than that in co-mmon sepsis group (34.3% vs. 19.5%, χ2=9.540, P=0.002), and liver function damage proportion was increased in severe sepsis group than that in common sepsis group (48.3% vs. 16.0%, χ2=41.28, P<0.001), and the duration of PICU treatment was longer in severe sepsis group than that in common sepsis group[10.7(6.5, 17.4) d vs. 7.5(4.0, 12.4) d, Z=-4.039, P<0.001]. C3 was significantly decreased in children with single organ dysfunction, multiple organ dysfunction and death group compared with common sepsis group (K=33.04, P=0.001), and the median of each group decreased with the severity of sepsis, but C4 had no difference among 4 groups(K=7.36, P=0.061). C3 was positively correlated with coagulation marker platelet (ρ=0.31, P<0.001) and fibrinogen (ρ=0.53, P<0.001), but negatively correlated with international normalized ratio (INR) (ρ=-0.39, P<0.001) and activated partial thromboplastin time (ρ=-0.34, P<0.001). C3 was also negatively correlated with liver damage marker alanine transaminase (ρ=-0.30, P<0.001) and total bilirubin (ρ=-0.28, P<0.001), and had a negative correlation with renal function marker creatinine (ρ=-0.24, P<0.001) and myocardial damage marker creatine kinase-MB (ρ=-0.27, P<0.001). The depletion of C3 was a risk factor of severe sepsis(OR=3.45, P<0.001) and MODS(OR=3.03, P=0.005) after being adjusted for confounding factors by using Logistic regression. In stratification analysis, C3 depletion was still a risk factor of severe sepsis(OR=2.78, P=0.019) and MODS(OR=3.57, P=0.015) among children less than 1 year old, and was also a risk factor of severe sepsis(OR=4.76, P=0.008) among children more than 1 year old as well. In children without liver function damage, C3 depletion was still a risk factor of severe sepsis(OR=4.17, P=0.002) and MODS(OR=9.09, P=0.002). Cox regression showed that C3 depletion was a hazard in 28-day mortality(HR=3.57, P=0.026) in children with sepsis. Conclusion The decrease of C3 is correlated with coagulation dysfunction and organ damage marker, while C3 depletion was a risk factor of severe sepsis, MODS and 28-day mortality, and could be a potential prognostic marker of children with sepsis. Key words: Sepsis; Severe sepsis; Complement C3; Complement C4

Of mice and women: Statins as a potential treatment to prevent preeclampsia in antiphospholipid syndrome.

Pregnancy complications in antiphospholipid syndrome APS have been associated with placental insufficiency. While in vitro and ex vivo studies suggest that the placenta is the main target organ in obstetric APS, in vivo data was missing. Using 111In labelled antiphospholipid antibodies (aPL) and single photon emission computed tomography we identified the placenta as the main organ target in a new mouse model of obstetric APS that closely resembles the clinical scenario. The cause of placental insufficiency in APS was attributed to the procoagulant effects of aPL antibodies. However, we demonstrated that APS is a proinflammatory syndrome. Based on our previous findings on the crucial role of complement activation in placental mal perfusion in APS, we developed an MRI-based method in which anti-complement C3-targeted ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles were used for the noninvasive detection of complement activation in placenta in utero. C3 deposition was detected in the placenta using this novel method, confirming the role of inflammation in obstetric APS. Management of obstetric APS is based on attenuating the procoagulant state. In many cases, however, there is no evidence of decidual thrombosis and instead inflammatory signs are present. Current treatment fails in a significant number of pregnancies- raising the need to explore other therapies to improve obstetrical outcome. Based on our preclinical studies in which pravastatin prevented adverse pregnancy outcomes in mouse models of preeclampsia and APS we performed translational studies in women. Our aim was to investigate the clinical use of pravastatin in APS women refractory to classical treatment LDA+LMWH that developed PE and/or severe IUGR. Eleven pregnant women with APS that developed PE or severe IUGR despite conventional antithrombotic therapy were treated with pravastatin (20mg/day) when signs of placental insufficiency (PE, IUGR) were observed. After pravastatin addition, placental blood flow and maternal symptoms of PE improved significantly leading to live birth in 100% of the patients. The beneficial effects of pravastatin were observed as early as 10 days, with a mean response time of 14.08 ± 3.25. Pregnancies survived 14 weeks [IQR 12–15] in patients cotreated with pravastatin. In this group. all babies were born close to term and alive and are now healthy while the neonates in the group that did not receive pravastatin experienced still births, preterm birth, were admitted at NICU and some of tem showed developmental abnormalities. Our study indicates that the addition of pravastatin at the time of onset of PE or severe IUGR to conventional treatment is worthy of further assessment in the management of women with APS and preeclampsia. RCT should be organized to confirm these observations.

Classical complement pathway activation in the nasal tissue of patients with chronic rhinosinusitis

Complement plays a major role in inflammatory diseases, but its involvement and mechanisms of activation in patients with chronic rhinosinusitis (CRS) are not known. After earlier studies discovering autoantibodies in patients with CRS, we sought to investigate the nature, extent, and location of complement activation in nasal tissue of patients with CRS. Specifically, we were interested in whether antibody-mediated activation through the classical pathway was a major mechanism for complement activation in patients with CRS. Nasal tissue was obtained from patients with CRS and control subjects. Tissue homogenates were analyzed for complement activation products (ELISA-C5b-9, C4d, activated C1, and C5a) and major complement-fixing antibodies (Luminex). Tissue sections were stained for C5b-9, C4d, and laminin. Antibodies were purified with protein A/G columns from nasal polyps (NP), matching patient serum, and control serum and assayed for basement membrane binding by means of ELISA. C5b-9 levels were significantly increased in NP tissue compared with uncinate tissue (UT) of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and those with chronic rhinosinusitis without nasal polyps (CRSsNP; P<.01). Similarly, C4d levels were increased in NPs compared with UT of patients with CRSwNP, patients with CRSsNP, and control subjects (P<.05). Activated C1 levels were also increased in NP tissue compared with UT of patients with CRSsNP and control subjects (P<.05) and correlated with levels of C5a (P<.01), local immunoglobulins (especially IgM, P<.0001), and anti-double-stranded DNA IgG (P<.05). Immunofluorescence showed that C5b-9 and C4d deposition occurred linearly along the epithelial basement membrane. NP tissue extracts had significantly more anti-basement membrane antibodies than sera from patients with CRSwNP and control subjects (P<.0001). Levels of C5b-9, C4d, and activated C1 were significantly increased locally in NP tissue. C5b-9 and C4d were almost universally deposited linearly along the basement membrane of NP tissue. Furthermore, activated C1 levels were best correlated with local immunoglobulin and C5a levels. Together, these data suggest that the classical pathway plays a major role in complement activation in patients with CRS.

Antiphospholipid Syndrome (APS) Patient Plasmas Consistently Activate Complement: A Novel Approach to Detecting a Pathogenic Mechanism for APS

INTRODUCTION: The antiphospholipid syndrome (APS) has remained an enigmatic condition for over 3 decades. Pathogenic mechanism(s) have not been established, and the disorder has therefore continued to be defined by non-mechanistic assays such as the lupus anticoagulant and antiphospholipid immunoassays. Although evidence from animal models has indicated a role for complement activation in the disease process, it is has been remarkable that activation markers are not consistently detected in human APS plasmas. We hypothesized that this might be due to rapid clearance of activation products and that we might be better able to detect a role for complement in APS through a 2 stage system - i.e. by first allowing APS immune complexes to form on phospholipid vesicles and then measuring whether those vesicles might activate complement in normal serum.METHODS: Complement activation markers - specifically C5a and the C5b-9 membrane attack complex - were measured in plasmas of 5 groups of plasmas (n= 10 for each group): normal healthy controls, APS with clinical histories for thrombosis (8 with venous thromboembolism (VTE), 1 venous stroke, and 1 arterial stroke), cancer patients without thrombosis, systemic lupus erythematosus (SLE) without APS, and VTE without APS. These were compared to measurements of complement activation induced by phospholipid vesicles that were pre-incubated with the same plasmas. Additional experiments were then done to: 1) characterize whether the effects could be reproduced with purified APS vs control IgGs and β2-glycoprotein I (β2GPI), 2) define the complement activation pathways involved with factor B and C2 deficient sera and with serum in the presence of EGTA and MgCl2, and 3) determine whether the activation could be inhibited by the monoclonal anti-C5 antibody, eculizimab.RESULTS: When the plasmas themselves were assayed, we found no significant differences in the plasmas levels of C5a or C5b-9 between APS and controls or among the 5 groups (Fig 1 A, B). In contrast, preincubation of phospholipid vesicles with plasmas resulted in significant elevations of both C5a (Fig 1C) and C5b-9 (Fig 1D) in APS compared to controls. Also, C5a levels in cancer, SLE and VTE were significantly lower than in APS, but above the controls. C5b-9 levels were elevated in APS and the other disease groups compared to controls, with the APS levels that were significantly higher than only the cancer group. The results were confirmed using purified IgGs and β2GPI in place of the whole plasmas. In addition, we found that APS-treated vesicles triggered complement activation via both the classical/lectin pathway and the alternative pathway. Eculizimab completely abrogated the complement activation.CONCLUSIONS: To our knowledge, this is the first demonstration that APS patient plasmas clearly and consistently activate complement. This was achieved through the novel approach of first incubating the plasmas with phospholipid vesicles which serve as platforms for the assembly of APS plasma-derived protein complexes that, in turn, activate complement. In addition, these findings may provide the basis for a mechanistic biomarker for disease activity in APS, by detecting complement activation at the site of injury.* P<0.05 vs Controls. # P<0.05 vs APS [Display omitted] DisclosuresNo relevant conflicts of interest to declare.