ABSTRACT
INTRODUCTION/AIMS: There are no blood biomarkers to monitor treatment effects in myasthenia gravis (MG) or studies visualizing the acetylcholine receptor (AChR) antibody-induced membrane attack complex (MAC) at the human muscle membrane. This study aimed to compare levels of complement activation products and native complement components in MG patients and healthy controls (HCs) and to model the AChR antibody-mediated attacks in human muscle cells. METHODS: We assessed the complement components and activation product levels with enzyme-linked immunosorbent assay and magnetic bead-based sandwich assays in plasma and sera of 23 MG patients and matched HCs. Receiver operator characteristic (ROC) curve analysis evaluated the diagnostic accuracy. Complement levels were correlated with the myasthenia gravis composite (MGC) scores. AChR+ MG modeling in human muscle cells used sera from nine MG patients and three HCs. RESULTS: MG patients had significantly higher plasma levels of C3a (p < .0001), C5 (p = .0003), and soluble C5b-9 (sC5b-9; p < .0001) than HCs. The ROC curve analysis showed a clear separation between MG patients and HCs for plasma C3a (AUC = 0.9720; p < .0001) and sC5b-9 (AUC = 0.8917, p < .0001). MG patients had higher levels of plasma complement Factor I (FI; p = .0002) and lower properdin levels (p < .0001). The MGC had moderate correlations with plasma Factor B (FB), FI, and Factor H. AChR+ MG patient sera triggered the deposition of MAC and reduced AChRs. DISCUSSION: We suggest validating plasma C3a and sC5b-9 as blood biomarkers for complement activation in MG. Further, the in vitro study allowed visualization of MAC deposition after applying AChR+ MG sera on human muscle cells.
ABSTRACT
Lyme borreliosis (LB) is caused by Borrelia burgdorferi and infection may lead to not only a large variety of clinical manifestations but also a subclinical outcome. The aim of the present study was to investigate if there is a constitutional difference in complement activation between individuals with previous subclinical Lyme borreliosis (SB) and patients previously diagnosed with Lyme neuroborreliosis (LNB).Lepirudin plasma for activation studies was collected from 60 SB individuals and from 22 patients pre-diagnosed with LNB. The plasma was incubated with live Borrelia spirochetes of two strains (complement sensitive B. garinii Lu59 and complement resistant B. afzelii ACA1).Complement factor C3 was measured in non-activated lepirudin plasma with immune-nephelometry and C3a and sC5b-9 generated during complement activation were measured by enzyme-linked immunosorbent assay.We found that the complement sensitive Lu59 induced higher complement activation than the complement resistant ACA1 when measuring activation products C3a and sC5b-9 in SB and LNB patients, p < 0.0001. No significant difference was found between SB and LNB patients in systemic levels of C3. Furthermore, SB individuals generated a higher activation of C3 cleavage to C3a (C3a/C3 ratio) than LNB patients after activation with ACA1, p < 0.001, but no significant differences were found in response to Lu59. In conclusion, Lu59 induced higher complement activation than ACA1 and individuals with previous SB showed increased generation of C3a compared with patients with previous LNB. In our study population, this mechanism could lead to less elimination of spirochetes in LNB patients and thereby be a factor contributing to the clinical outcome.
Subject(s)
Complement Activation , Lyme Disease/immunology , Lyme Neuroborreliosis/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Asymptomatic Infections , Borrelia burgdorferi , Complement C3/analysis , Humans , Lyme Disease/complications , Lyme Neuroborreliosis/complications , Lyme Neuroborreliosis/diagnosis , Male , Middle Aged , Young AdultABSTRACT
Individuals with celiac disease (CD) are at increased risk of invasive pneumococcal disease (IPD). The aim of this study was to explore whether the complement response to Streptococcus pneumoniae differed according to CD status, and could serve as an explanation for the excess risk of IPD in CD. Twenty-two children with CD and 18 controls, born 1999-2008, were included at Kalmar County Hospital, Sweden. The degree of complement activation was evaluated by comparing levels of activation products C3a and sC5b-9 in plasma incubated for 30 min with Streptococcus pneumoniae and in non-incubated plasma. Complement analyses were performed with enzyme-linked immunosorbent assay (ELISA). Pneumococcal stimulation caused a statistically significant increase in C3a as well as sC5b-9 in both children with CD and controls but there was no difference in response between the groups. After incubation, C3a increased on average 4.6 times and sC5b-9 22 times in both the CD and the control group (p = 0.497 and p = 0.724 respectively).Conclusion: Complement response to Streptococcus pneumoniae seems to be similar in children with and without CD and is thus unlikely to contribute to the increased susceptibility to invasive pneumococcal disease in CD.What is Known:⢠An excess risk of pneumococcal infections has been demonstrated in individuals with celiac disease.⢠Infectious complications can depend on hyposplenism but alternative mechanisms are sparsely examined.What is New:⢠Complement activation in response to Streptococcus pneumoniae was examined in children with and without celiac disease but no differences could be demonstrated.
Subject(s)
Celiac Disease/complications , Complement Activation , Pneumococcal Infections/etiology , Adolescent , Biomarkers/blood , Case-Control Studies , Celiac Disease/immunology , Celiac Disease/microbiology , Child , Complement C3a/metabolism , Complement Membrane Attack Complex/metabolism , Disease Susceptibility , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Pneumococcal Infections/immunology , Risk FactorsABSTRACT
Besides its vital role in immunity, the complement system also contributes to the shaping of the synaptic circuitry of the brain. We recently described that soluble Complement Receptor 2 (sCR2) is part of the nerve injury response in rodents. We here study CR2 in context of multiple sclerosis (MS) and explore the molecular effects of CR2 on C3 activation. Significant increases in sCR2 levels were evident in cerebrospinal fluid (CSF) from both patients with relapsing-remitting MS (n=33; 6.2ng/mL) and secondary-progressive MS (n=9; 7.0ng/mL) as compared to controls (n=18; 4.1ng/mL). Furthermore, CSF sCR2 levels correlated significantly both with CSF C3 and C1q as well as to a disease severity measure. In vitro, sCR2 inhibited the cleavage and down regulation of C3b to iC3b, suggesting that it exerts a modulatory role in complement activation downstream of C3. These results propose a novel function for CR2/sCR2 in human neuroinflammatory conditions.
Subject(s)
Complement C3/immunology , Multiple Sclerosis, Relapsing-Remitting/immunology , Multiple Sclerosis/immunology , Receptors, Complement 3d/immunology , Adult , Complement Activation/immunology , Complement C1q/cerebrospinal fluid , Complement C1q/immunology , Complement C3/cerebrospinal fluid , Electrophoresis, Polyacrylamide Gel , Female , Humans , Linear Models , Male , Middle Aged , Multiple Sclerosis/cerebrospinal fluid , Multiple Sclerosis/pathology , Multiple Sclerosis, Relapsing-Remitting/cerebrospinal fluid , Multiple Sclerosis, Relapsing-Remitting/pathology , Severity of Illness Index , Young AdultSubject(s)
Complement System Proteins/immunology , Immunoassay/methods , Plasma/immunology , Adult , Aged , Female , Humans , Magnetics/methods , Male , Middle Aged , Reference Values , Young AdultSubject(s)
Anti-Bacterial Agents/pharmacology , Antifungal Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Bacteria/drug effects , Candida/drug effects , Diketopiperazines/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Antifungal Agents/chemical synthesis , Antifungal Agents/chemistry , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/chemistry , Diketopiperazines/chemical synthesis , Diketopiperazines/chemistry , Humans , Microbial Sensitivity TestsABSTRACT
Hemolysis, as a result of disease or exposure to biomaterials, is characterized by excess amounts of cell-free heme intravascularly and consumption of the protective heme-scavenger proteins in plasma. The liberation of heme has been linked to the activation of inflammatory systems, including the complement system, through alternative pathway activation. Here, we investigated the impact of heme on the regulatory function of the complement system. Heme dose-dependently inhibited factor I-mediated degradation of soluble and surface-bound C3b, when incubated in plasma or buffer with complement regulatory proteins. Inhibition occurred with factor H and soluble complement receptor 1 as co-factors, and the mechanism was linked to the direct heme-interaction with factor I. The heme-scavenger protein hemopexin was the main contaminant in purified factor I preparations. This led us to identify that hemopexin formed a complex with factor I in normal human plasma. These complexes were significantly reduced during acute vasoocclusive pain crisis in patients with sickle cell disease, but the complexes were normalized at their baseline outpatient clinic visit. Hemopexin exposed a protective function of factor I activity in vitro, but only when it was present before the addition of heme. In conclusion, we present a mechanistic explanation of how heme promotes uncontrolled complement alternative pathway amplification by interfering with the regulatory capacity of factor I. Reduced levels of hemopexin and hemopexin-factor I complexes during an acute hemolytic crisis is a risk factor for heme-mediated factor I inhibition.
Subject(s)
Anemia, Sickle Cell , Hemopexin , Anemia, Sickle Cell/metabolism , Complement Factor I , Fibrinogen , Heme/metabolism , Hemopexin/pharmacology , HumansABSTRACT
Accurate determination of complement component C1q is hampered by the fact that C1q is an immune complex binding protein. Consequently, immunochemical techniques which rely on immune complex formation in fluid phase such as nephelometry and turbidimetry tend to give results which differ from those obtained by, for example, ELISA and other solid phase-based assays. In this chapter, we discuss the pros and cons of different techniques for the quantification of C1q and present a comprehensive protocol for a newly developed magnetic bead-based sandwich immunoassay which has replaced nephelometry in our complement diagnostic laboratory at the University Hospital in Uppsala.
Subject(s)
Complement C1q/analysis , Complement System Proteins/analysis , Immunoelectrophoresis/methods , Nephelometry and Turbidimetry/methods , Blood Protein Electrophoresis/methods , Diagnostic Tests, Routine/methods , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunomagnetic Separation/methodsABSTRACT
BACKGROUND: Low levels of total C4b-binding protein (C4BPt), a circulating inhibitor of the classical/lectin complement pathways, were observed in patients with antiphospholipid antibodies (aPLs) and during warfarin treatment. OBJECTIVES: To investigate the associations between aPL and C4BPt in patients with persistently positive (++) aPL, with/without clinical manifestations and systemic lupus erythematosus (SLE), and in controls. Furthermore, we explored the impact of anticoagulation on C4BPt and in relation to complement activation. METHODS: In a cross-sectional design we investigated defined subgroups: primary (p) antiphospholipid syndrome (APS, N = 67), aPL++ individuals without clinical manifestations (aPL carriers, N = 15), SLE-aPL++ (N = 118, among them, secondary [s] APS, N = 56), aPL negative (-) SLE (SLE-aPL-, N = 291), and 322 controls. Clinical characteristics, including treatment, were tabulated. C4BPt was determined with a magnetic bead method. Complement proteins (C1q, C2, C3, C4, C3a, C3dg, sC5b-9, factor I [FI]) were measured. A mediation analysis was performed to decompose the total effect of aPL++ on C4BPt into the direct and indirect effects of aPL++ through warfarin. RESULTS: Overall, C4BPt is 20% decreased in aPL++ patients, regardless of SLE, APS, clinical manifestations, and aPL profile. C4BPt levels associate positively with complement proteins C1q, C2, C3, and C4, and negatively with complement activation product C3dg. In the SLE group, warfarin treatment contributes to approximately half of the C4BPt reduction (9%) CONCLUSION: Both aPLs and warfarin are associated with C4BPt reduction. Complement activation in aPL++ patients may partly be explained by impaired inhibition through depressed C4BPt levels. Further studies are needed to understand the clinical implications.
Subject(s)
Antibodies, Antiphospholipid/blood , Anticoagulants/therapeutic use , Antiphospholipid Syndrome/drug therapy , Blood Coagulation/drug effects , Complement Activation/drug effects , Complement C4b-Binding Protein/metabolism , Warfarin/therapeutic use , Adult , Antiphospholipid Syndrome/blood , Antiphospholipid Syndrome/immunology , Biomarkers/blood , Case-Control Studies , Cross-Sectional Studies , Down-Regulation , Female , Humans , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/immunology , Male , Middle AgedABSTRACT
In this study we investigate the hydrolysis of C3 to C3(H2O) and its ability to initiate activation via the alternative pathway (AP) of the complement system. The internal thioester bond within C3 is hydrolyzed by water in plasma because of its inherent lability. This results in the formation of non-proteolytically activated C3(H2O) which is believed have C3b-like properties and be able to form an active initial fluid phase C3 convertase together with Factor B (FB). The generation of C3(H2O) occurs at a low but constant rate in blood, but the formation can be greatly accelerated by the interaction with various surfaces or nucleophilic and chaotropic agents. In order to more specifically elucidate the relevance of the C3(H2O) for AP activation, formation was induced in solution by repeated freeze/thawing, methylamine or KCSN treatment and named C3(x) where the x can be any of the reactive nucleophilic or chaotropic agents. Isolation and characterization of C3(x) showed that it exists in several forms with varying attributes, where some have more C3b-like properties and can be cleaved by Factor I in the presence of Factor H. However, in common for all these variants is that they are less active partners in initial formation of the AP convertase compared with the corresponding activity of C3b. These observations support the idea that formation of C3(x) in the fluid phase is not a strong initiator of the AP. It is rather likely that the AP mainly acts as an amplification mechanism of complement activation that is triggered by deposition of target-bound C3b molecules generated by other means.
Subject(s)
Complement Activation/physiology , Complement C3/metabolism , Complement Pathway, Alternative/physiology , Complement C3/chemistry , Humans , HydrolysisABSTRACT
Objectives: C1q is a valuable biomarker of disease activity in systemic lupus erythematosus (SLE). The "gold standard" assay, rocket immunoelectrophoresis (RIE), is time-consuming, and thus a shift to soluble immune precipitation techniques such as nephelometry has occurred. However, quantification of C1q with these techniques has been questioned as a result of the antibody binding properties of C1q. In the present work, we have compared results using various techniques (RIE, nephelometry, and ELISA) and have developed and validated a new magnetic bead-based sandwich immunoassay (MBSI). Methods: C1q was quantified by nephelometry and the new sandwich immunoassay in 45 serum samples analyzed using RIE. C1q was also assessed in plasma using RIE and sandwich immunoassay in samples from SLE patients with nephritis (n = 69), SLE patients without nephritis (n = 310) as classified by BILAG score, and matched controls (n = 322). In addition, cerebrospinal fluid (CSF) samples from 31 patients, previously analyzed with ELISA, were also analyzed with the MBSI to test the behavior of this new assay in the lower detection range. Results: We found a strong correlation between the new MBSI, RIE, and ELISA, but not with nephelometry. The MBSI demonstrated lower levels of C1q in SLE patients than in matched controls (p < 0.0001), and patients with nephritis had lower levels than patients without nephritis (p < 0.01). Similarily, RIE showed significant differences between the patient groups (p < 0.0001). An association was also found between the levels of C1q and the SLE disease activity index (SLEDAI). Furthermore, there was good correlation between the values obtained by MBSI and ELISA, in both serum (r = 0.960) and CSF (r = 0.786), underscoring the ability of both techniques to measure low concentrations of C1q with high accuracy. Conclusion: The sandwich immunoassay correlated well with RIE, but soluble immune precipitation techniques, such as nephelometry, did not appear suitable alternatives, since C1q itself, and possibly anti-C1q antibodies, interfered with the measurements. The new sandwich immunoassay is therefore a good replacement for RIE in monitoring SLE disease activity.
Subject(s)
Complement C1q/analysis , Complement C1q/cerebrospinal fluid , Immunomagnetic Separation/methods , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/cerebrospinal fluid , Antibodies, Monoclonal , Autoantibodies/blood , Biomarkers/analysis , Biomarkers/cerebrospinal fluid , Complement C1q/immunology , Data Accuracy , Edetic Acid , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoelectrophoresis/methods , Lupus Nephritis/blood , Lupus Nephritis/cerebrospinal fluid , Magnetic Fields , Nephelometry and Turbidimetry/methods , Severity of Illness IndexABSTRACT
Aberrations in complement system functions have been identified as either direct or indirect pathophysiological mechanisms in many diseases and pathological conditions, such as infections, autoimmune diseases, inflammation, malignancies, and allogeneic transplantation. Currently available techniques to study complement include quantification of (a) individual complement components, (b) complement activation products, and (c) molecular mechanisms/function. An emerging area of major interest in translational studies aims to study and monitor patients on complement regulatory drugs for efficacy as well as adverse events. This area is progressing rapidly with several anti-complement therapeutics under development, in clinical trials, or already in clinical use. In this review, we summarized the appropriate indications, techniques, and interpretations of basic complement analyses, exemplified by a number of clinical disorders.
Subject(s)
Complement Activation/drug effects , Clinical Trials as Topic , Complement Activation/immunology , Complement C1 Inhibitor Protein/therapeutic use , Complement C5/antagonists & inhibitors , Complement System Proteins/immunology , Humans , Receptors, Complement/immunology , Signal Transduction/immunologyABSTRACT
In the alternative pathway (AP) an amplification loop is formed, which is strictly controlled by various fluid-phase and cell-bound regulators resulting in a state of homeostasis. Generation of the "C3b-like" C3(H2O) has been described as essential for AP activation, since it conveniently explains how the initial fluid-phase AP convertase of the amplification loop is generated. Also, the AP has a status of being an unspecific pathway despite thorough regulation at different surfaces. During complement attack in pathological conditions and inflammation, large amounts of C3b are formed by the classical/lectin pathway (CP/LP) convertases. After the discovery of LP´s recognition molecules and its tight interaction with the AP, it is increasingly likely that the AP acts in vivo mainly as a powerful amplification mechanism of complement activation that is triggered by previously generated C3b molecules initiated by the binding of specific recognition molecules. Also in many pathological conditions caused by a dysregulated AP amplification loop such as paroxysmal nocturnal hemoglobulinuria (PNH) and atypical hemolytic uremic syndrome (aHUS), C3b is available due to minute LP and CP activation and/or generated by non-complement proteases. Therefore, C3(H2O) generation in vivo may be less important for AP activation during specific attack or dysregulated homeostasis, but may be an important ligand for C3 receptors in cell-cell interactions and a source of C3 for the intracellular complement reservoir.
Subject(s)
Complement Activation/immunology , Complement C3b/immunology , Complement Pathway, Alternative/immunology , Animals , Homeostasis , HumansABSTRACT
Complement system aberrations have been identified as pathophysiological mechanisms in a number of diseases and pathological conditions either directly or indirectly. Examples of such conditions include infections, inflammation, autoimmune disease, as well as allogeneic and xenogenic transplantation. Both prospective and retrospective studies have demonstrated significant complement-related differences between patient groups and controls. However, due to the low degree of specificity and sensitivity of some of the assays used, it is not always possible to make predictions regarding the complement status of individual patients. Today, there are three main indications for determination of a patient's complement status: (1) complement deficiencies (acquired or inherited); (2) disorders with aberrant complement activation; and (3) C1 inhibitor deficiencies (acquired or inherited). An additional indication is to monitor patients on complement-regulating drugs, an indication which may be expected to increase in the near future since there is now a number of such drugs either under development, already in clinical trials or in clinical use. Available techniques to study complement include quantification of: (1) individual components; (2) activation products, (3) function, and (4) autoantibodies to complement proteins. In this review, we summarize the appropriate indications, techniques, and interpretations of basic serological complement analyses, exemplified by a number of clinical disorders.
Subject(s)
Angioedemas, Hereditary/immunology , Biomarkers/analysis , Complement Activation/immunology , Complement System Proteins/immunology , Immunologic Deficiency Syndromes/immunology , Angioedemas, Hereditary/diagnosis , Autoantibodies/immunology , Autoimmune Diseases/diagnosis , Autoimmune Diseases/immunology , Complement System Proteins/deficiency , Humans , Immunologic Deficiency Syndromes/diagnosis , Prospective Studies , Retrospective StudiesABSTRACT
BACKGROUND: The complement system (CS) plays a role in the pathogenesis of a number of ocular diseases, including diabetic retinopathy (DR), glaucoma, uveitis, and age-related macular degeneration (AMD). Given that many of the complex eye-related degenerative diseases have limited treatment opportunities, we aimed to mimic the in vivo retinal degenerative process by developing a relevant co-culture system. METHOD AND MATERIALS: The adult porcine retina was co-cultured with the spontaneously arising human retinal pigment epithelial cells-19 (ARPE-19). RESULTS: Inflammatory activity was found after culture and included migrating microglial cells, gliosis, cell death, and CS activation (demonstrated by a minor increase in the secreted anaphylotoxin C3a in co-culture). CS components, including C1q, C3, C4, soluble C5b-9, and the C5a receptor, were expressed in the retina and/or ARPE cells after culture. C1q, C3, and CS regulators such as C4 binding protein (C4BP), factor H (CFH), and factor I (CFI) were secreted after culture. DISCUSSION: Thus, our research indicates that this co-culturing system may be useful for investigations of the CS and its involvement in experimental neurodegenerative diseases.
Subject(s)
Complement System Proteins/physiology , Retinal Degeneration/etiology , Animals , Cell Death , Coculture Techniques , Complement C3/analysis , Complement Membrane Attack Complex/analysis , Humans , Immunohistochemistry , Retinal Degeneration/immunology , Retinal Pigment Epithelium/cytology , SwineABSTRACT
A strong initial inflammatory response is important in neuroborreliosis. Since complement is a main player in early inflammation, we monitored the concentration and activation of complement in plasma and cerebrospinal fluid from 298 patients, of whom 23 were diagnosed with neuroborreliosis. Using sandwich ELISAs, we found significantly elevated levels of C1q, C4, C3, and C3a in cerebrospinal fluid, but not in plasma, in patients with neuroborreliosis. This finding indicates that complement plays a role in the human immune response in neuroborreliosis, that the immunologic process is compartmentalized to the CNS, and that complement activation may occur via the classical pathway.
Subject(s)
Central Nervous System/immunology , Complement Activation , Complement C1q/cerebrospinal fluid , Complement C3/cerebrospinal fluid , Lyme Neuroborreliosis/cerebrospinal fluid , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Lyme Neuroborreliosis/immunology , Lyme Neuroborreliosis/pathology , Male , Middle Aged , Statistics, NonparametricABSTRACT
The complement system is a vital component of the immune-priveliged human eye that is always active at a low-grade level, preventing harmful intraocular injuries caused by accumulation of turnover products and controlling pathogens to preserve eye homeostasis and vision. The complement system is a double-edged sword that is essential for protection but may also become harmful and contribute to eye pathology. Here, we review the evidence for the involvement of complement system dysregulation in age-related macular degeneration, glaucoma, uveitis, and neuromyelitis optica, highlighting the relationship between morphogical changes and complement system protein expression and regulation in these diseases. The potential benefits of complement inhibition in age-related macular degeneration, glaucoma, uveitis, and neuromyelitis optica are abundant, as are those of further research to improve our understanding of complement-mediated injury in these diseases.
Subject(s)
Complement Activation/immunology , Complement System Proteins/immunology , Eye Diseases/immunology , Immunity, Innate/immunology , Eye Diseases/pathology , Glaucoma/immunology , Glaucoma/pathology , Humans , Macular Degeneration/immunology , Macular Degeneration/pathology , Models, Immunological , Neuromyelitis Optica/immunology , Neuromyelitis Optica/pathology , Uveitis/immunology , Uveitis/pathologyABSTRACT
BACKGROUND: Inappropriate and uncontrolled activation of the cascade systems in the blood is a driving force in adverse inflammatory and thrombotic reactions elicited by biomaterials, but limited data are available on the activation of the contact system by polymers and the present study was undertaken to investigate these mechanisms in established models. METHODS: Polymer particles were incubated in (1) EDTA-plasma (10 mM) to monitor the adsorption of 20 selected proteins; (2) lepirudin-anticoagulated plasma to evaluate contact system activation, monitored by the formation of complexes between the generated proteases factor[F]XIIa, FXIa and kallikrein and the serpins C1-inhibitor [C1INH] and antithrombin [AT]; (3) lepirudin-anticoagulated whole blood to determine cytokine release. RESULTS: Strong negative correlations were found between 10 cytokines and the ratio of deposited FXII/C1INH, generated FXIIa-C1INH complexes, and kallikrein-C1INH complexes. Formation of FXIIa-C1INH complexes correlated negatively with the amount of C3a and positively with deposited IgG. CONCLUSIONS: A reciprocal relationship was found between activation of the contact system and the complement system induced by the polymers studied here. The ratios of FXII/C1INH or C4/C4BP, adsorbed from EDTA-plasma are useful surrogate markers for cytokine release and inflammatory response to materials intended for blood contact.
Subject(s)
Biocompatible Materials , Complement Activation , Polymers , Adsorption , Antithrombins/metabolism , Blood Coagulation/drug effects , Blood Proteins/chemistry , Complement C1 Inactivator Proteins/metabolism , Complement C1 Inhibitor Protein , Complement C3a/analysis , Cytokines/metabolism , Enzyme Activation , Factor XIIa/metabolism , Factor XIa/metabolism , Glass , Hirudins/pharmacology , Humans , Hydrophobic and Hydrophilic Interactions , Immunoglobulin G/analysis , Kallikreins/metabolism , Particle Size , Polystyrenes , Polyvinyl Chloride , Recombinant Proteins/pharmacologyABSTRACT
There is a compelling need to understand and assess the toxicity of industrially produced nanoparticles (NPs). In order to appreciate the long-term effects of NPs, sensitive human-based screening tests that comprehensively map the NP properties are needed to detect possible toxic mechanisms. Animal models can only be used in a limited number of test applications and are subject to ethical concerns, and the interpretation of experiments in animals is also distorted by the species differences. Here, we present a novel easy-to-perform highly sensitive whole-blood model using fresh non-anticoagulated human blood, which most justly reflects complex biological cross talks in a human system. As a demonstrator of the tests versatility, we evaluated the toxicity of TiO2 NPs that are widely used in various applications and otherwise considered to have relatively low toxic properties. We show that TiO2 NPs at very low concentrations (50 ng/mL) induce strong activation of the contact system, which in this model elicits thromboinflammation. These data are in line with the finding of components of the contact system in the protein corona of the TiO2 NPs after exposure to blood. The contact system activation may lead to both thrombotic reactions and generation of bradykinin, thereby representing fuel for chronic inflammation in vivo and potentially long-term risk of autoimmunity, arteriosclerosis and cancer. These results support the notion that this novel whole-blood model represents an important contribution to testing of NP toxicity.
Subject(s)
Kallikreins/blood , Models, Biological , Nanoparticles/toxicity , Titanium/toxicity , Adsorption , Antithrombin III/metabolism , Blood Coagulation/drug effects , Blood Proteins/metabolism , Complement System Proteins/metabolism , Humans , Inflammation/pathology , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Peptide Hydrolases/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time Factors , Titanium/chemistryABSTRACT
BACKGROUND: The use of acetate in haemodialysis fluids may induce negative effects in patients including nausea and increased inflammation. Therefore, haemodialysis fluids where acetate is substituted with citrate have recently been developed. In this study, we investigated the biocompatibility of citrate employing concentrations used in haemodialysis. METHODS: The effects of citrate and acetate were investigated in human whole blood in vitro under conditions promoting biomaterial-induced activation. Complement activation was measured as generation of C3a, C5a and the sC5b-9 complex, and granulocyte activation as up-regulation of CD11b expression. For the experimental set-up, a mathematical model was created to calculate the concentrations of acetate and citrate attained during haemodialysis. RESULTS: Citrate reduced granulocyte activation and did not induce higher complement activation compared with acetate at concentrations attained during haemodialysis. Investigating different citrate concentrations clearly showed that citrate is a potent complement inhibitor already at low concentrations, i.e. 0.25 mM, which is comparable with concentrations detected in the blood of patients during dialysis with citrate-containing fluids. Increased citrate concentration up to 6 mM further reduced the activation of C3a, C5a and sC5b-9, as well as the expression of CD11b. CONCLUSIONS: Our results suggest that citrate is a promising substitute for acetate for a more biocompatible dialysis, most likely resulting in less adverse effects for the patients.