Plasma C3a Research Articles

Inflammatory and Immune Proteins in Umbilical Cord Blood: Association with Hearing Screening Test Failure in Preterm Neonates.

Objective We aimed to determine whether elevated levels of various inflammatory and immune proteins in umbilical cord blood are associated with an increased risk of newborn hearing screening (NHS) test failure in preterm neonates. Methods This retrospective cohort study included 127 premature singleton infants who were born at ≤33.6 weeks. Umbilical cord plasma at birth was assayed for interleukin (IL)-6, complement C3a and C5a, matrix metalloproteinase (MMP)-9, macrophage colony-stimulating factor (M-CSF), and endostatin levels using ELISA kits. Neonatal blood C-reactive protein (CRP) levels were measured within 2 hours of birth. The primary outcome measure was a uni- or bilateral refer result on an NHS test. Univariate and multivariate analyses were applied. Results Fifteen (11.8%) infants failed the NHS test. In the univariate analyses, high IL-6 and low C3a levels in umbilical cord plasma, funisitis, and an elevated CRP level (>5 mg/L) in the immediate postnatal period were significantly associated with NHS test failure. However, the levels of umbilical cord plasma MMP-9, C5a, M-CSF, and endostatin were not significantly different between infants who passed and those who failed the NHS test. Multiple logistic regression analyses indicated that elevated umbilical cord plasma C3a levels were independently associated with a reduced risk of NHS test failure, whereas elevated levels of umbilical cord plasma IL-6 and high CRP levels in the immediate postnatal period were significantly associated with NHS test failure. Conclusions Our data demonstrated that in preterm neonates, a systemic fetal inflammatory response reflected by umbilical cord plasma IL-6 and immediate postnatal CRP levels may contribute to the risk for NHS test failure, whereas the changes in complement activation fragments initiated in utero may have protective effect of hearing screen failure.

Elevated C5a Plasma Concentrations in Mongolian Patients with Atherosclerotic Vascular Diseases

Objectives: Complement activation product C5a is increased in acute cardiovascular events and infl ammation. However, investigation of C5a in patients with atherosclerotic vascular changes among Mongolian populations is still lacking. Methods: A total of 43 patients were selected using special criteria for this study. Plasma C5a and lipid levels were measured by high-sensitive enzyme-linked immune sorbent assay and colorimetric enzyme assay at the Mongolian National University of Medical Sciences, Core Laboratory and Second General Hospital. All data were analyzed for normality and then parametric or nonparametric tests using GraphPad Prism 6.0 software. The Pearson r correlation analysis was performed using Microsoft Excel. Results: Patients were divided into a healthy group ((n=23, carotid intima-media thickness (cIMT) <0.7 mm)) and a group with atherosclerosis (n=20 with cIMT >1.0 mm by Doppler-ultrasound imaging). The suffi cient number of patients within groups was approved by using column statistical analysis. A signifi cantly elevated plasma C5a protein concentration was found in patients with atherosclerosis compared to healthy controls (276.28±24.82 vs. 61.56±4.35 pg/mL, P<0.0001), which was directly correlated with increased total plasma cholesterol levels (r=0.68, P<0.01). Conclusion: Our study supported the notion that complement anaphylatoxin C5a may be involved in the pathogenesis of atherosclerotic vascular diseases.

Plasma inflammatory and immune proteins as predictors of intra-amniotic infection and spontaneous preterm delivery in women with preterm labor: a retrospective study

BackgroundWe investigated whether various inflammatory and immune proteins in plasma predict intra-amniotic infection and imminent preterm delivery in women with preterm labor and compared their predictive ability with that of amniotic fluid (AF) interleukin (IL)-6 and serum C-reactive protein (CRP).MethodsThis retrospective cohort study included 173 consecutive women with preterm labor who underwent amniocentesis for diagnosis of infection and/or inflammation in the AF. The AF was cultured, and assayed for IL-6. CRP levels and cervical length by transvaginal ultrasound were measured at the time of amniocentesis. The stored maternal plasma was assayed for IL-6, matrix metalloproteinase (MMP)-9, and complements C3a and C5a using ELISA kits. The primary and secondary outcome criteria were positive AF cultures and spontaneous preterm delivery (SPTD) within 48 h, respectively. Univariate, multivariate, and receiver operating characteristic analysis were used for the statistical analysis.ResultsIn bivariate analyses, elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery, whereas elevated plasma levels of MMP-9, C3a, and C5a were not associated with these two outcomes. On multivariate analyses, an elevated plasma IL-6 level was significantly associated with intra-amniotic infection and imminent preterm delivery after adjusting for confounders, including high serum CRP levels and short cervical length. In predicting intra-amniotic infection, the area under the curve (AUC) was significantly lower for plasma IL-6 than for AF IL-6 but was similar to that for serum CRP. Differences in the AUCs between plasma IL-6, AF IL-6, and serum CRP were not statistically significant in predicting imminent preterm delivery.ConclusionsMaternal plasma IL-6 independently predicts intra-amniotic infection in women with preterm labor; however, it has worse diagnostic performance than that of AF IL-6 and similar performance to that of serum CRP. To predict imminent preterm delivery, plasma IL-6 had an overall diagnostic performance similar to that of AF IL-6 and serum CRP. Plasma MMP-9, C3a, and C5a levels could not predict intra-amniotic infection or imminent preterm delivery.

In vitro characteristics and in vivo outcome of α1,3-galactosyltransferase gene-knockout miniature pig cornea in full-thickness corneal xenotransplantation using nonhuman primate

Abstract We investigated the biofunctional profiles of α1, 3-galactosyltransferase gene knockout (GTKO) miniature pig cornea and its outcome in pig-to-rhesus full thickness corneal xenotransplantation (FTCX). Ten eyes of GTKO miniature pigs (n = 5) were evaluated for biofunctional characteristics. Five Chinese rhesus macaques underwent FTCX (mean donor age: 11.3 mo). Systemic tacrolimus, basiliximab, and IVIG were administered as programmed schedules. T cell subsets, antibodies (Ab) in blood, and C3a in aqueous humor (AH) were evaluated. The results were compared with those of the wild type miniature pig corneal grafted group (n = 4, all grafts survived > 24 weeks, using anti-CD154 Ab) as controls. Mean central corneal thickness was 763 μm. Na+/K+ ATPase was well expressed 7 days after preservation. Mean endothelial cell density was 4729/mm2, and was maintained >2800/mm2 through 7 days. Mean doubling time was 30.4 hour which was comparable to that in SNU miniature pigs (30.7 hour, p = .413). Median graft survival was 72 days. Regulatory T cells at rejection decreased from baseline (p = .043) and were lower than those of controls at postoperative (postop) 24 weeks (p = .014), however the other T cell subsets did not change. At rejection, anti-non-αGal Ab increased from baseline (p = .043), however Anti-αGal Ab did not. C3a in AH at postop 4 weeks and at rejection increased from baseline (all p = .043). At postop 4 weeks, AH and plasma C3a were higher than those of controls (p = .016 and .036, respectively). The biofunctional characteristics of GTKO miniature pig cornea are feasible for FTCX. Rejection of GTKO pig-to-rhesus FTCX is related with non-αGal Ab, complement and decrease of regulatory T cells. Addition of B cell suppressing regimen should be considered.

Complement system activation in obstructive sleep apnea.

The complement system may play a role in the systemic inflammation characterising obstructive sleep apnea; however, this has not been investigated before. We aimed to study the involvement of effector complement elements in obstructive sleep apnea, namely C3a, C5a and SC5b-9. Venous blood was collected in 50 patients with obstructive sleep apnea and 26 control subjects in the evening and the following morning. Plasma complement proteins were analysed with ELISA. Complement factor levels were compared between the two groups and correlated with clinical variables. Plasma C3a concentration was elevated in obstructive sleep apnea both in the evening (84.1 [0-338.5] ng ml-1 ) and in the morning (85.5 [0-247.8] ng ml-1 ) compared with controls (30.3 [0-176.8] ng ml-1 and 36.3 [0-167.1] ng ml-1 , evening and morning, respectively, both p < 0.05). On the contrary, C5a and SC5b-9 levels were comparable between patients and controls at each time point (p > 0.05). There was no change in complement factors from evening to morning in either group (p > 0.05), except for C5a that decreased from evening to morning in obstructive sleep apnea (from 11.6 [1.6-47.4] ng ml-1 to 9.3 [0-46.4] ng ml-1 , p = 0.01). Elevated C3a levels were directly related to obstructive sleep apnea severity, and were significantly associated with male gender, weight, body mass index, hypertension, high C-reactive protein and low high-density lipoprotein cholesterol (p < 0.05). The complement system is activated in obstructive sleep apnea, which is correlated with disease severity. Our findings highlight the potential role of complement system in the pathophysiology of obstructive sleep apnea, thus facilitating further research.

The complement system as a biomarker of disease activity and response to treatment in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system. The complement system has an established role in the pathogenesis of MS, and evidence suggests that its components can be used as biomarkers of disease-state activity and response to treatment in MS. Plasma C4a levels have been found to be significantly elevated in patients with active relapsing-remitting MS (RRMS), as compared to both controls and patients with stable RRMS. C3 levels are also significantly elevated in the cerebrospinal fluid (CSF) of patients with RRMS, and C3 levels are correlated with clinical disability. Furthermore, increased levels of factor H can predict the transition from relapsing to progressive disease, since factor H levels have been found to increase progressively with disease progression over a 2-year period in patients transitioning from RRMS to secondary progressive (SP) MS. In addition, elevations in C3 are seen in primary progressive (PP) MS. Complement components can also differentiate RRMS from neuromyelitis optica. Response gene to complement (RGC)-32, a novel molecule induced by complement activation, is a possible biomarker of relapse and response to glatiramer acetate (GA) therapy, since RGC-32 mRNA expression is significantly decreased during relapse and increased in responders to GA treatment. The predictive accuracy of RGC-32 as a potential biomarker (by ROC analysis) is 90% for detecting relapses and 85% for detecting a response to GA treatment. Thus, complement components can serve as biomarkers of disease activity to differentiate MS subtypes and to measure response to therapy.

A novel 2-stage approach that detects complement activation in patients with antiphospholipid antibody syndrome

IntroductionThe antiphospholipid syndrome (APS) is marked by autoantibodies that recognize anionic phospholipids in a cofactor-dependent manner. A role for complement has been implicated in the pathophysiology, however, elevations of complement activation markers have not been consistently demonstrated in clinical studies. We therefore designed a proof-of-principle study to determine whether complement activation might be detectable in APS by first exposing plasmas to phospholipid vesicles. MethodsWe examined complement activation markers in patients with APS, non-APS thrombosis, systemic lupus erythematosus, cancer, patients with antiphospholipid antibodies without thrombosis (APL) and healthy controls. Direct measurements of plasma C5a and sC5b-9 levels were compared to levels that were generated in normal serum by phospholipid vesicles that had been pre-incubated with the same plasmas. We then determined the effects of the C5 inhibitor, eculizumab, examined the complement pathways involved, and determined whether the effects could be reproduced with purified IgGs and β2-glycoprotein I (β2GPI). ResultsPlasma levels of C5a and sC5b-9 were higher, but not significantly increased in APS patients compared to healthy controls. In contrast, phospholipid vesicles pre-incubated with APS plasmas generated significantly higher levels than healthy controls and the other groups, except for APL patients. Complement activation was abrogated by addition of eculizumab. The results with substrate sera indicated that the alternative and classical/lectin pathways were involved. The results were reproducible with purified IgGs and β2GPI. ConclusionThis proof-of-principle study confirms a role for complement in APS and opens the possibility of monitoring complement activation by including phospholipid vesicles in assay systems.

Upregulation of Early and Downregulation of Terminal Pathway Complement Genes in Subcutaneous Adipose Tissue and Adipocytes in Acquired Obesity.

Inflammation is an important mediator of obesity-related complications such as the metabolic syndrome but its causes and mechanisms are unknown. As the complement system is a key mediator of inflammation, we studied whether it is activated in acquired obesity in subcutaneous adipose tissue (AT) and isolated adipocytes. We used a special study design of genetically matched controls of lean and heavy groups, rare monozygotic twin pairs discordant for body mass index (BMI) [n = 26, within-pair difference (Δ) in body mass index, BMI >3 kg/m2] with as much as 18 kg mean Δweight. Additionally, 14 BMI-concordant (BMI <3 kg/m2) served as a reference group. The detailed measurements included body composition (DEXA), fat distribution (MRI), glucose, insulin, adipokines, C3a and SC5b-9 levels, and the expression of complement and insulin signaling pathway-related genes in AT and adipocytes. In both AT and isolated adipocytes, the classical and alternative pathway genes were upregulated, and the terminal pathway genes downregulated in the heavier co-twins of the BMI-discordant pairs. The upregulated genes included C1q, C1s, C2, ficolin-1, factor H, receptors for C3a and C5a (C5aR1), and the iC3b receptor (CR3). While the terminal pathway components C5 and C6 were downregulated, its inhibitor clusterin was upregulated. Complement gene upregulation in AT and adipocytes correlated positively with adiposity and hyperinsulinemia and negatively with the expression of insulin signaling-related genes. Plasma C3a, but not SC5b-9, levels were elevated in the heavier co-twins. There were no differences between the co-twins in BMI-concordant pairs. Obesity is associated with increased expression of the early, but not late, complement pathway components and of key receptors. The twins with acquired obesity have therefore an inflated inflammatory activity in the AT. The results suggest that complement is likely involved in orchestrating clearance of apoptotic debris and inflammation in the AT.

Proteomic Profiling Analysis Reveals a Link between Experimental Autoimmune Uveitis and Complement Activation in Rats

Uveitis is an autoimmune disease that usually damages the vision function, leading to poor visual quality in patients. As an autoimmune ocular inflammatory disease, the pathogenesis of uveitis is associated with abnormal expression of some proteins and aberrant regulation of multiple signalling pathways. Nevertheless, the detailed mechanism remains unclear. In this study, we induced an experimental autoimmune uveitis (EAU) model in rats. We determined the levels of C3a and membrane attack complex C5b-9 (soluble C5b-9, sC5b-9) in both plasma and aqueous humour, identified the differentially expressed proteins in plasma by liquid chromatography-tandem mass spectrometry and employed bioinformatics algorithms to analyse differentially expressed proteins in EAU rat plasma. The results demonstrate that there were 168 differentially expressed plasma proteins in EAU rats versus control subjects. The levels of sC5b-9 and C3a were elevated in the plasmas and aqueous humours of EAU rats. Gene ontology enrichment analysis showed that the differentially expressed proteins in EAU rat plasma were mainly involved in metabolic and immune processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway annotation, database for annotation, visualization and integrated discovery (DAVID) and protein-protein interaction analyses revealed that the differentially expressed proteins in EAU rat plasmas were closely associated with complement and coagulation cascades, metabolic pathways, NF-kappa B, PI3K-Akt, Toll-like receptors and autophagy. Overall, the differentially expressed proteins in EAU rat plasmas are mainly involved in the complement and coagulation cascades. The pathogenesis of uveitis closely correlates with complement activation.

Effect of halloysite nanotubes on the structure and function of important multiple blood components

Many researchers have investigated the application of halloysite nanotubes (HNTs) in biomedicine, because of their special nanoscale hollow tubular structure. Although the cytocompatibility of HNTs has been studied, their blood compatibility has not been systematically investigated. In this work, the effect of HNTs on the structure and function of different blood components has been studied, including the morphology and hemolysis of red blood cells (RBCs). Based on scanning electron microscopy (SEM) observations, optical density test and flow cytometry analysis, we found that HNTs can affect the morphology and membrane integrity of RBCs in phosphate buffered saline (PBS) in a content-dependent way. In particular, based on UV–vis absorption spectra, fluorescence spectra and circular dichroism (CD) spectra, HNTs can alter the secondary structure and conformation of human fibrinogen and γ-globulins. In addition, the detection of biomarker molecules C3a and C5a in plasma suggests that HNTs can trigger complement activation. In the blood clotting assay, HNTs were found to significantly prolong the activated partial thromboplastin time (APTT), shorten the prothrombin time (PT) of platelet-poor plasma (PPP), and change the thromboelastography (TEG) parameters of whole blood coagulation. Furthermore, confocal laser scanning microscopy and flow cytometry analysis were used to test intracellular uptake by macrophages, and the cellular uptake of HNTs in the RAW 264.7 was found to be content-dependent, but not time-dependent. These findings provide insight for the potential use of HNTs as biofriendly nanocontainers for biomaterials in vivo.

The clinical and laboratory features of Chinese Han anti-factor H autoantibody-associated hemolytic uremic syndrome.

Anti-complement factor H (CFH) autoantibody-associated hemolytic uremic syndrome (HUS) is a severe sub-type of HUS. We assessed the clinical and renal pathological features, circulating complement levels, and genetic background of Chinese pediatric patients with this sub-type of HUS. Thirty-three consecutive patients with acute kidney injury who tested positive for serum anti-CFH autoantibodies were enrolled in this study. All of the eight patients who underwent renal biopsies presented with changes typical of thrombotic microangiopathy, especially changes in chronic characteristics. Compared to patients in remission and normal control subjects, patients with acute disease had significantly lower plasma CFH levels and significantly higher plasma complement 3a (C3a), C5a, and terminal complement complex (SC5b-9) levels. The CFH-anti-CFH immunoglobin G (IgG) circulating immunocomplex (CFH-CIC) titers were more closely correlated with CFH plasma levels than anti-CFH IgG levels. Of the 22 patients, four (18%) were homozygous for CFHR3-1Δ and ten were heterozygous for CFHR1 or CFHR3 deletions. Most patients responded well to a combination of plasma and immunosuppressive therapies, with a remission rate of 87%. At the end of the follow-up, nine patients reached the combined end-points, including two with end-stage renal disease and seven with relapses. Plasma C3a, C5a, and SC5b-9 levels predicted disease activity in anti-CFH autoantibody-associated HUS patients enrolled in this study. These patients responded well to plasma therapy combined with immunosuppression.

Bacteremia, Not Coagulation Proteases Contribute to In Vivo Complement Activation in Sepsis

Bacterial sepsis induces strong activation of coagulation, complement and fibrinolytic systems that contribute to disseminated intravascular coagulation, organ damage and death. While the contact of the blood with pathogens or pathogen-associated molecular patterns (PAMPs) can trigger the activation of both systems, a bidirectional complement-coagulation crosstalk is believed to occur. Although the role of complement activation products as positive regulators of coagulation is documented, direct activation of the complement proteins by thrombin or other hemostatic proteases was alluded but not demonstrated in vivo. Here we aimed to: (i) determine if in vivo generation of thrombin and other hemostatic proteases can activate the complement proteins and (ii) discriminate between the direct effect of the pathogen/PAMPs vs. hemostatic proteases on complement activation in a clinically relevant model of sepsis. We have compared the time-course of complement activation markers (C3b, C5a and C5b-9 terminal complex) in plasma of baboons exposed to 1010 cfu/kg (LD100) E. coli vs. intravenous infusion of factor Xa/PC:PS, a potent procoagulant stimulus. In baboons challenged with LD100 E. coli, complement activation markers C3b, C5a and C5b-9 reached maximum levels after 2 hrs (see figure). Complement activation coincided with the peak of bacteremia and LPS, but not with markers of thrombin generation (TAT and fibrinogen consumption; see figure) or fibrinolysis (FDP, D-dimers), which reached peak levels after 6 hours. Differently, infusion of FXa/PC:PS (36.6 pmol/L FXa and 56.3 nmol/L PC/PS per kg body weight) induced a rapid burst of thrombin and almost full consumption of fibrinogen during the first 10 min post-infusion, with no increase of complement activation markers. Based on these data we conclude that in vivo activation of the coagulation cascade does not support complement activation as was postulated by previous in vitro studies. Therefore, we conclude that pathogens and PAMPs are the main activators of the complement during sepsis while direct activation by hemostatic proteases is minor or absent. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Complement Activation in a Murine Model of Sickle Cell Disease: Inhibition of Vaso-Occlusion By Blocking C5 Activation

▪IntroductionInnate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. The alternative complement pathway is abnormally activated in SCD and is additionally activated by phosphatidylserine (PS) on the outer leaflet of SS-red blood cells (SS-RBC). PS on the surface of SS-RBC and activated platelets accelerates the assembly of prothrombinase complexes leading to generation of thrombin, which can cleave circulating C5 protein into two biologically active fragments, C5a and C5b. C5a is an anaphylatoxin, a potent pro-inflammatory mediator, that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion (VO). Active C5b fragments stimulate the formation of membrane attack complexes (MAC) on SS-RBC that increases their susceptibility to lysis. We hypothesize that complement activation on the surface of SS-RBC may stimulate VO and RBC turnover in SCD.Methods and ResultsWhole blood from Townes-AA, -AS, and -SS mice (n=4) was collected in EDTA and RBC were immunostained with erythroid specific anti-Ter119 (Ly-76) IgG conjugated to PE-Cy7, anti-C5b-9 (MAC) IgG conjugated to Alexa Fluor 647, and anti-C3 (also specific for activation fragments C3b, iC3b, C3d and C3dg) IgG conjugated to PE. The percentages of Ter119-positive RBC that were positive for MAC were 6.3%, 6.5%, and 26.0% for AA-, AS-, and SS-RBCs, respectively (p<0.01 SS vs. AA and AS) suggesting enhanced C5 activation in Townes-SS mice. However, EDTA plasma C5a levels measured by ELISA were not significantly different between Townes-AA, -AS and -SS mice. C3 activation fragments were also found on a subset of MAC positive RBC. We used a mouse model of hypoxia/reoxygenation (H/R)-induced stasis to investigate the role of complement in VO. Townes-SS mice with implanted dorsal skin-fold chambers were infused with 30 µg of either anti-C5 IgG mAb BB5.1 (which blocks murine C5 cleavage ) (n=4), annexin V (n=2), or with isotype control IgG (n=4) 30 minutes prior to H/R (1 hour of hypoxia at 7% O2, followed by reoxygenation for 4 hours in room air). Percent microvascular stasis (% non-flowing venules) in the subcutaneous venules in the dorsal skin fold chamber window was measured using intravital microscopy at 1 and 4 hours post-hypoxia. Percent stasis was significantly lower in the Townes-SS mice receiving anti-C5 IgG or annexin V than the mice receiving the isotype control IgG at 1 and 4 hours post-hypoxia (Figure 1, means ± SD, *p<0.01 anti-C5 IgG or annexin V vs. control IgG). This finding implies that H/R may acutely activate C5 , thus leading to stasis. Initial studies suggest increased plasma C5a levels in Towne-SS mice treated with isotype control IgG compared to mice treated with anti-C5 IgG in response to H/R.ConclusionsThese results demonstrate an increased percentage of SS-RBC expressing MAC on their surface and inhibition of H/R-induced stasis with anti-C5 IgG. However, circulating C5a levels were not different between untreated Townes-AA, -AS, or -SS mice suggesting that C5a is not chronically elevated in SCD. Rather, the generation of C5a may occur locally and in the acute setting. Current studies underway are examining the effects of anti-C5 IgG on MAC deposition and RBC half-lives; the mechanism(s) of complement activation in SCD mice (classical, alternative, tic-over, etc); the ability of recombinant murine C5a to induce stasis; and complement activation markers on RBC and in plasma from SCD patients and controls. [Display omitted] DisclosuresChen:Imara: Research Funding. Belcher:Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:Imara: Research Funding; CSL-Behring: Research Funding.

Effectiveness of sulodexide might be associated with inhibition of complement system in hepatitis B virus-associated membranous nephropathy: An inspiration from a pilot trial

BackgroundThe activation of complement system is associated with the development of hepatitis B virus-associated membranous nephropathy (HBV-MN) and heparin could inhibit the activation of complement system. MethodsThis was a three-center trial. Seventy-nine patients with HBV-MN participated in the study. The follow-up of the study consisted of two periods: Stage 1 (S1) and Stage 2 (S2). All patients received 0.5mg entecavir plus 150–300mg/day of irbesartan but sulodexide was prescribed during S1. They were randomized into 4 groups according to sulodexide dose: blank (Group 1), 250 lipasemic unit (lsu)/day for 1year (Group 2), 500 lsu/day for 1year (Group 3) and 1000 lsu/day for 6months followed by 250 lsu/day for 6months (Group 4). Major clinical outcomes were valid remission (VR): (1) urine albumin/creatinine ratio (UACR) <150mg/mmol and >50% decline of baseline; (2) albumin >35g/L; (3) glomerular filtration rate (GFR) >90ml/(min*1.73m2). Results(1) Groups 3 and 4 had significantly lower UACR and higher albumin than did Groups 1 and 2 at major visits; (2) Groups 3 and 4 achieved more VR compared with Group 1 (42.1% and 60.0% vs. 9.1%, p both<0.05); (3) in Groups 3 and 4, instead of Groups 1 and 2, more C3 deposition in the kidney was observed in those achieving VR; (4) plasma C3a, C5a and C5b-9 decreased significantly in Groups 3 and 4 during S1. Conclusions(1) The prescription of both sulodexide and entecavir could improve the prognosis of patients with HBV-MN but their mechanisms might be different; (2) the renoprotection of sulodexide in HBV-MN might probably relate to the inhibition of complement system.

Splanchnic blood flow and vasoactive mediators in acute pancreatitis

Introduction: Changes of microcirculation and endothelial dysfunction are an important step from mild to severe disease. Changes in splanchnic arteries in acute necrotizing pancreatitis are the consequence of the release of vasoactive mediators. Materials and methods: The study involved 53 acute pancreatitis patients (28 – severe, 25 – mild). We measured interleukin-6, C3a and endothelin-1 in the blood plasma. Flow in the visceral arteries was assessed with the help of the Doppler sonography. Results: Mild acute pancreatitis often not accompanied by hemodynamic disorders according to Doppler ultrasound during the first week of illness. Pancreatic necrosis is noted a steady increase in peak systolic blood flow velocity and resistance index in the common hepatic, splenic and superior mesenteric arteries during the first week. In patients with acute pancreatitis on admission identified increase the levels endothelin-1 and C3a. A significant increase of both substances determined only in severe acute pancreatitis. The IL-6 levels were increased in all patients. There was a significant direct correlation concentration of IL-6 with peak systolic velocity in the superior mesenteric artery, with an index of resistance in common hepatic and superior mesenteric arteries. There was a significant direct correlation concentrations of C3a with peak systolic in the common hepatic and splenic arteries, with an index of resistance in superior mesenteric artery. Conclusion: Changes in the quantitative and qualitative detection of Doppler spectrum are ahead echographic signs of necrotizing forms of pancreatitis in B-mode ultrasound. IL-6, endothelin-1, and C3a levels of correlates with disturbances in splanchnic blood flow.

Platelet bound oxLDL shows an inverse correlation with plasma anaphylatoxin C5a in patients with coronary artery disease

Both oxidized lipids as well as the complement system contribute to atherothrombosis. The expression of complement receptors correlates with the expression of platelet activation markers, and platelet bound oxidized low-density lipoprotein (oxLDL) modulates platelet function. In the present study, we investigated the relationship of markers of complement activation, the anaphylatoxins C5a and C3a, and oxidized low-density lipoprotein.Two hundred and seven patients with coronary artery disease (CAD) were analyzed in this study. Using enzyme-linked immunosorbent assays, plasma levels of oxLDL, C3a, and C5a were measured. Moreover, we assessed platelet bound oxLDL by flow cytometry. The overall level of C5a in the troponin negative group (stable angina (SA) and unstable angina (UA)) compared to the troponin positive group (non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI)) did not differ significantly (62.7 ± 32.4 ng/ml versus 65.8 ± 40.3 ng/ml). While C5a and C3a showed a significant correlation with each other (r = 0.25, p < 0.001), there was no statistically significant relationship between C3a and platelet bound oxLDL (r = 0.06, p = 0.37). Furthermore, plasma oxLDL did not correlate with either C3a or C5a. However, we observed a moderate, yet significant negative correlation between plasma C5a and platelet bound oxLDL (r = −0.15, p = 0.04). Partial correlation analysis correcting for the presence of acute coronary syndrome (ACS), troponin status or the subgroups SA, UA, NSTEMI, or STEMI did not alter this correlation substantially. Interestingly, flow cytometric analysis of human platelets showed increased expression of C5aR and P-selectin after in vitro stimulation with oxLDL.In conclusion, the complement anaphylatoxin C5a shows an inverse correlation with platelet bound oxLDL. The relationship of oxidized lipids to particular complement components may add to the platelet–lipid interplay in atherogenesis and trigger future clinical and mechanistic studies.

Improving blood-compatibility via surface heparin-immobilization based on a liquid crystalline matrix

Blood compatibility is of considerable importance in developing medical materials and devices that are in contact with blood. In this work, we successfully developed a novel liquid crystalline heparin-immobilized material (Hep-OPPC) by two-step modification for further improvement of hydrophilicity and hemocompatibility of the liquid crystalline hydroxypropyl cellulose ester (OPCL). The results showed that Hep-immobilization on the OPCL led to dramatic changes in the surface morphology and crystallinity, whereas, the Hep-OPPCs also maintained the liquid crystalline feature at room temperature after heparinization. Furthermore, the hemocompatibility of the Hep-OPPCs was markedly enhanced at low levels of hemolysis assay (HR) with unimpaired erythrocytomorphology, significantly lower concentrations of C3a in blood plasma and remarkable increases in plasma re-calcification time (PRT). This suggests that the heparinized surface could restrict the transformation of fibrinogen with less activation of the intrinsic coagulation system. Moreover, the activated partial thromboplastin time (APTT) and prothrombin time (PT) values of the Hep-OPPCs with low heparin density could also be prolonged in this study suggesting that the liquid crystal feature of the matrix might be blocking the clotting factors. We concluded that the heparin-immobilized liquid crystalline material has the potential to be used in blood-contact materials.

Absence of inflammatory lung injury in rabbits challenged intravascularly with complement-derived chemotactic factors.

Systemic activation of the complement system results in the generation of chemotactic factors that have been suggested to play a role in the pathogenesis of inflammatory pulmonary diseases such as the adult respiratory distress syndrome. This led us to ask whether systemic complement activation by cobra venom factor (CVF) or intravascularly administered zymosan-activated rabbit plasma (ZAP) or rabbit C5a would result in lung injury. As had been described previously for CVF and ZAP, intravenously administered rabbit C5a also caused an acute neutropenia along with sequestration of neutrophils within the pulmonary vasculature. However, no significant lung inflammation as measured by neutrophil emigration or increased vascular permeability occurred with any of the three stimuli. Only when these agents were combined with anesthesia, surgical manipulation, and intubation did significant neutrophil emigration into alveoli occur, but again without any change in vascular permeability. After administration of ZAP, a decrease in dynamic compliance and an increase in pulmonary resistance as well as a transient period of hypoxemia occurred that was not observed after CVF or rabbit C5a treatment. Thus, our studies suggest that changes in lung function after ZAP instillation may not represent changes from complement activation alone in that they are not reproduced with CVF or rabbit C5a. We conclude that complement activation, as an isolated event, may be an insufficient insult in the lung to produce significant lung injury.

Functional analysis of a complement polymorphism (rs17611) associated with rheumatoid arthritis.

Complement is implicated in the pathogenesis of rheumatoid arthritis (RA); elevated levels of complement activation products have been measured in plasma, synovial fluid, and synovial tissues of patients. Complement polymorphisms are associated with RA in genome-wide association studies. Coding-region polymorphisms may directly impact protein activity; indeed, we have shown that complement polymorphisms affecting a single amino acid change cause subtle changes in individual component function that in combination have dramatic effects on complement activity and disease risk. In this study, we explore the functional consequences of a single nucleotide polymorphism (SNP) (rs17611) encoding a V802I polymorphism in C5 and propose a mechanism for its link to RA pathology. Plasma levels of C5, C5a, and terminal complement complex were measured in healthy and RA donors and correlated to rs17611 polymorphic status. Impact of the SNP on C5 functionality was assessed. Plasma C5a levels were significantly increased and C5 levels significantly lower with higher copy number of the RA risk allele for rs17611, suggesting increased turnover of C5 V802. Functional assays using purified C5 variants revealed no significant differences in lytic activity, suggesting that increased C5 V802 turnover was not mediated by complement convertase enzymes. C5 is also cleaved in vivo by proteases; the C5 V802 variant was more sensitive to cleavage with elastase and the “C5a” generated was biologically active. We hypothesize that this SNP in C5 alters the rate at which elastase generates active C5a in rheumatoid joints, hence recruiting neutrophils to the site thus maintaining a state of inflammation in arthritic joints.

Spontaneous abortion is associated with elevated systemic C5a and reduced mRNA of complement inhibitory proteins in placenta.

Spontaneous abortion in early pregnancy due to unknown reasons is a common problem. The excess complement activation and consequent placental inflammation and anti-angiogenic milieu is emerging as an important associated factor in many pregnancy-related complications. In the present study we sought to examine the expression of complement inhibitory proteins at the feto-maternal interface and levels of complement split products in the circulation to understand their role in spontaneous abortion. Consenting pregnant women who either underwent elective abortion due to non-clinical reasons (n = 13) or suffered miscarriage (n = 14) were recruited for the study. Systemic levels of complement factors C3a and C5a were measured by enzyme-linked immunosorbent assay (ELISA). Plasma C5 and C3 protein levels were examined by Western blot. Expressions of complement regulatory proteins such as CD46 and CD55 in the decidua were investigated by quantitative polymerase chain reaction (PCR) and Western blot. The median of plasma C3a level was 82·83 ng/ml and 66·17 ng/ml in elective and spontaneous abortion patients, respectively. Medians of plasma C5a levels in elective and spontaneous abortion patients were 0·96 ng/ml and 1·14 ng/ml, respectively. Only plasma C5a levels but not C3a levels showed significant elevation in spontaneous abortion patients compared to elective abortion patients. Further, there was a threefold decrease in the mRNA expressions of complement inhibitory proteins CD46 and CD55 in the decidua obtained from spontaneous abortion patients compared to that of elective abortion patients. These data suggested that dysregulated complement cascade may be associated with spontaneous abortion.