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Sample records for complement component c3

  1. Functional Characterization of Autoantibodies against Complement Component C3 in Patients with Lupus Nephritis*

    PubMed Central

    Vasilev, Vasil V.; Noe, Remi; Dragon-Durey, Marie-Agnes; Chauvet, Sophie; Lazarov, Valentin J.; Deliyska, Boriana P.; Fremeaux-Bacchi, Veronique; Dimitrov, Jordan D.; Roumenina, Lubka T.

    2015-01-01

    Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Because the complement system plays a critical role in orchestrating inflammatory and immune responses as well as in the clearance of immune complexes, autoreactivity to complement components may have considerable pathological consequences. Autoantibodies against the central complement component C3 have been reported in systemic lupus erythematosus, but their molecular mechanism and functional relevance are not well understood. The objective of this study was to evaluate the frequency and the functional properties of the anti-C3 autoantibodies. Anti-C3 autoantibodies were measured in plasma of 39 LN patients, and identification of their epitopes on the C3 molecule was performed. By using surface plasmon resonance, we analyzed the influence of patient-derived IgG antibodies on the interaction of C3b with Factor B, Factor H, and complement receptor 1. The capacity of these antibodies to dysregulate the C3 convertase on the surface of endothelial cell was measured by flow cytometry. Here we report that the frequency of anti-C3 autoantibodies in LN is ∼30%. They inhibited interactions of the negative complement regulators Factor H and complement receptor 1 with C3b. An enhanced C3 deposition was also observed on human endothelial cells in the presence of C3 autoantibodies. In addition, anti-C3 autoantibody levels correlated with disease activity. In conclusion, the anti-C3 autoantibodies in LN may contribute to the autoimmune pathology by their capacity to overactivate the complement system. PMID:26245903

  2. Characterization of the third component of complement (C3) after activation by cigarette smoke

    SciTech Connect

    Kew, R.R.; Ghebrehiwet, B.; Janoff, A.

    1987-08-01

    Activation of lung complement by tobacco smoke may be an important pathogenetic factor in the development of pulmonary emphysema in smokers. We previously showed that cigarette smoke can modify C3 and activate the alternative pathway of complement in vitro. However, the mechanism of C3 activation was not fully delineated in these earlier studies. In the present report, we show that smoke-treated C3 induces cleavage of the alternative pathway protein, Factor B, when added to serum containing Mg-EGTA. This effect of cigarette smoke is specific for C3 since smoke-treated C4, when added to Mg-EGTA-treated serum, fails to activate the alternative pathway and fails to induce Factor B cleavage. Smoke-modified C3 no longer binds significant amounts of (/sup 14/C)methylamine (as does native C3), and relatively little (/sup 14/C)methylamine is incorporated into its alpha-chain. Thus, prior internal thiolester bond cleavage appears to have occurred in C3 activated by cigarette smoke. Cigarette smoke components also induce formation of noncovalently associated, soluble C3 multimers, with a Mr ranging from 1 to 10 million. However, prior cleavage of the thiolester bond in C3 with methylamine prevents the subsequent formation of these smoke-induced aggregates. These data indicate that cigarette smoke activates the alternative pathway of complement by specifically modifying C3 and that these modifications include cleavage of the thiolester bond in C3 and formation of noncovalently linked C3 multimers.

  3. Functions of the complement components C3 and C5 during sepsis

    PubMed Central

    Flierl, Michael A.; Rittirsch, Daniel; Nadeau, Brian A.; Day, Danielle E.; Zetoune, Firas S.; Sarma, J. Vidya; Huber-Lang, Markus S.; Ward, Peter A.

    2008-01-01

    Activation of the complement system is a key event in the pathogenesis of sepsis. Nevertheless, the exact mechanisms remain inadequately understood. In the current study, we examined the role of complement C3 and C5 in sepsis in wild-type and C3- or C5-deficient mice induced by cecal ligation and puncture. When compared to wild-type mice, C5−/− showed identical survival, and C3−/− presented significantly reduced survival. Interestingly, this was associated with significant decreases in plasma levels of proinflammatory mediators. Moreover, although septic C3−/− animals displayed a 10-fold increase of blood-borne bacteria, C5−/− animals exhibited a 400-fold increase in bacteremia when compared to wild-type mice. These effects were linked to the inability of C5−/− mice to assemble the terminal membrane attack complex (MAC), as determined by complement hemolytic activity (CH-50). Surprisingly, although negative control C3−/− mice failed to generate the MAC, significant increases of MAC formation was found in septic C3−/− mice. In conclusion, our data corroborate that hemolytic complement activity is essential for control of bacteremia in septic mice. Thus, during sepsis, blockade of C5a or its receptors (rather than C5) seems a more promising strategy, because C5a-blockade still allows for MAC formation while the adverse effects of C5a are prevented.—Flierl, M. A., Rittirsch, D., Nadeau, B. A., Day, D. E., Zetoune, F. S., Sarma, J. V., Huber-Lang, M. S., Ward, P. A. Functions of the complement components C3 and C5 during sepsis. PMID:18587006

  4. [Immunoturbidimetric determination of C-3 components of complement systems using nonlinear calibration].

    PubMed

    Rohácek, J; Semrád, V; Klierová, E; Zápotocná, M

    1991-01-01

    A method for the immunoturbidimetric analysis of the C-3-component in the complement system was elaborated by means of the antiserum Q-SwAHu/C3 USOL (SEVAG) Praha. A diluted human control serum USOL (SEVAG) Praha with declared values of plasma proteins was applied as a standard solution. The relation between concentration and absorption in an eight step calibration series is well described by a parabola of the 2nd degree. The precision in series and the accuracy of the method are mentioned. The proposed technique is in a relatively good correlation with the radial immunodiffusion according to MANCINI.

  5. Drusen complement components C3a and C5a promote choroidal neovascularization

    PubMed Central

    Nozaki, Miho; Raisler, Brian J.; Sakurai, Eiji; Sarma, J. Vidya; Barnum, Scott R.; Lambris, John D.; Chen, Yali; Zhang, Kang; Ambati, Balamurali K.; Baffi, Judit Z.; Ambati, Jayakrishna

    2006-01-01

    Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30–50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD. PMID:16452172

  6. Derivatives of human complement component C3 for therapeutic complement depletion: a novel class of therapeutic agents.

    PubMed

    Fritzinger, David C; Hew, Brian E; Lee, June Q; Newhouse, James; Alam, Maqsudul; Ciallella, John R; Bowers, Mallory; Gorsuch, William B; Guikema, Benjamin J; Stahl, Gregory L; Vogel, Carl-Wilhelm

    2008-01-01

    To obtain proteins with the complement-depleting activity of Cobra Venom Factor (CVF), but with less immunogenicity, we have prepared human C3/CVF hybrid proteins, in which the C-terminus of the alpha-chain of human C3 is exchanged with homologous regions of the C-terminus of the beta-chain of CVF. We show that these hybrid proteins are able to deplete complement, both in vitro and in vivo. One hybrid protein, HC3-1496, is shown to be effective in reducing complement-mediated damage in two disease models in mice, collagen-induced arthritis and myocardial ischemia/reperfusion injury. Human C3/CVF hybrid proteins represent a novel class ofbiologicals as potential therapeutic agents in many diseases where complement is involved in the pathogenesis.

  7. Halogenation and proteolysis of complement component C3 on Salmonella typhimurium during phagocytosis by human neutrophils

    SciTech Connect

    Joiner, K.A.; Schweinle, J.E.

    1989-05-01

    We examined the fate of C component C3 on the surface of Salmonella typhimurium during ingestion by human neutrophils. Initial experiments showed that C3 fragments and C3-acceptor complexes were the major serum ligands which were surface iodinated by canine myeloperoxidase on serum-incubated rough and smooth isolates of S. typhimurium. In contrast, labeled C3 was not identified when the same organisms were ingested by neutrophils in the presence of 125I-Na, a situation previously shown to iodinate particulate targets via the neutrophil myeloperoxidase-halide-H2O2 system. Pretreatment of neutrophils before phagocytosis with the lipid-soluble protease inhibitor diisopropylfluorophosphate (DFP), but not with other protease inhibitors (p-nitrophenylguanidinobenzoate, leupeptin, pepstatin), substantially blocked proteolysis of 125I-C3 on S. typhimurium strain RG108 during ingestion by neutrophils. Purification of neutrophil phagosomes containing S. typhimurium-bearing 125I-C3 showed that DFP but no other protease inhibitors blocked proteolysis of 125I-C3 within phagosomes. Iodinated C3-acceptor complexes were identified by immunoprecipitation from the detergent-insoluble fraction of phagosomes prepared from DFP-treated cells ingesting S. typhimurium in the presence of 125I-Na. These results show that C3 fragments on the surface of S. typhimurium are the major serum ligands which are halogenated and degraded by proteolysis during phagocytosis by human neutrophils, and suggest that the majority of proteolysis on the ingested target occurs within the neutrophil phagosome.

  8. Comparison of a fluorometric method with radial immunodiffusion assays for determination of complement components C3 and C4.

    PubMed Central

    Koelle, M; Bartholomew, W R

    1982-01-01

    Measurements of patient serum complement components C3 and C4 are useful indicators of complement consumption in immune complex diseases. A fluorometric quantitative immunofluorescence system was evaluated in terms of measuring these complement components, and the results were compared with those of radial immunodiffusion assays. For comparison of the two systems, 232 patient sera were evaluated for C3, and 202 specimens were tested for C4. Analysis of the data by linear regression indicated a proportional difference between the methods. C3 and C4 concentrations measured by the fluorometric method were lower than those measured by radial immunodiffusion, especially concentrations exceeding the normal ranges. In detecting lower concentrations (less than 120 mg/dl for C3 and less than 25 mg/dl for C4), the two methods showed better agreement. Each assay system was reproducible and could be used to evaluate changes that occur in concentrations of complement components during therapeutic treatment. However, the ease in processing a large volume of specimens and the short time needed to complete the assay are advantages that make the fluorometric method more suitable than radial immunodiffusion for use in a large clinical laboratory. PMID:6811611

  9. Comparison of a fluorometric method with radial immunodiffusion assays for determination of complement components C3 and C4.

    PubMed

    Koelle, M; Bartholomew, W R

    1982-08-01

    Measurements of patient serum complement components C3 and C4 are useful indicators of complement consumption in immune complex diseases. A fluorometric quantitative immunofluorescence system was evaluated in terms of measuring these complement components, and the results were compared with those of radial immunodiffusion assays. For comparison of the two systems, 232 patient sera were evaluated for C3, and 202 specimens were tested for C4. Analysis of the data by linear regression indicated a proportional difference between the methods. C3 and C4 concentrations measured by the fluorometric method were lower than those measured by radial immunodiffusion, especially concentrations exceeding the normal ranges. In detecting lower concentrations (less than 120 mg/dl for C3 and less than 25 mg/dl for C4), the two methods showed better agreement. Each assay system was reproducible and could be used to evaluate changes that occur in concentrations of complement components during therapeutic treatment. However, the ease in processing a large volume of specimens and the short time needed to complete the assay are advantages that make the fluorometric method more suitable than radial immunodiffusion for use in a large clinical laboratory.

  10. Myeloperoxidase reduces the opsonizing activity of immunoglobulin G and complement component C3b.

    PubMed

    Coble, B I; Dahlgren, C; Hed, J; Stendahl, O

    1984-12-20

    The effect of myeloperoxidase, hydrogen peroxide (H2O2) and a halide (Cl) on the opsonizing molecules in immunoglobulin G (IgG) and complement factor C3b was assayed. At concentrations of the enzyme (1 microgram/ml) that can be found in the extracellular fluid during inflammation, the myeloperoxidase-H2O2-Cl system inhibited the opsonizing effect of IgG and C3b measured as phagocytic uptake and superoxide generation. The effect was related to the enzymatic peroxidative activity of the protein. The presence of albumin (10 mg/ml) reduced the effect of myeloperoxidase with 10-20%. Taurine, which in the presence of myeloperoxidase-H2O2-Cl forms hydrophilic chloramines, and D-penicillamine, which scavenges HOCl, neutralize the inhibitory effect of myeloperoxidase. This suggests that either hypochlorous acid or lipophilic chloramines may exert its effect by oxidizing free sulphydryl groups exposed on the opsonizing ligands. Since the myeloperoxidase-H2O2-halide system also affects chemotactic factors, leukotrienes, proteinases and membrane receptors, the system may in several ways affect the development of the inflammatory response.

  11. The structure of C2b, a fragment of complement component C2 produced during C3 convertase formation

    SciTech Connect

    Krishnan, Vengadesan; Xu, Yuanyuan; Macon, Kevin; Volanakis, John E.; Narayana, Sthanam V. L.

    2009-03-01

    The crystal structure of C2b has been determined at 1.8 Å resolution, which reveals the arrangement of its three complement control protein (CCP) modules. A model for complement component C2 is presented and its conformational changes during the C3-convertase formation are also discussed. The second component of complement (C2) is a multi-domain serine protease that provides catalytic activity for the C3 and C5 convertases of the classical and lectin pathways of human complement. The formation of these convertases requires the Mg{sup 2+}-dependent binding of C2 to C4b and the subsequent cleavage of C2 by C1s or MASP2, respectively. The crystal structure of full-length C2 is not yet available, although the structure of its C-terminal catalytic segment C2a has been determined. The crystal structure of the N-terminal segment C2b of C2 determined to 1.8 Å resolution presented here reveals the arrangement of its three CCP domains. The domains are arranged differently compared with most other CCP-domain assemblies, but their arrangement is similar to that found in the Ba part of the full-length factor B structure. The crystal structures of C2a, C2b and full-length factor B are used to generate a model for C2 and a discussion of the domain association and possible interactions with C4b during formation of the C4b–C2 complex is presented. The results of this study also suggest that upon cleavage by C1s, C2a domains undergo conformational rotation while bound to C4b and the released C2b domains may remain folded together similar to as observed in the intact protein.

  12. In Vitro and In Vivo Differences in Murine Third Complement Component (C3) Opsonization and Macrophage/Leukocyte Responses to Antibody-Functionalized Iron Oxide Nanoworms

    PubMed Central

    Wang, Guankui; Griffin, James I.; Inturi, Swetha; Brenneman, Barbara; Banda, Nirmal K.; Holers, V. Michael; Moghimi, Seyed Moein; Simberg, Dmitri

    2017-01-01

    Balancing surface functionalization and low immune recognition of nanomedicines is a major challenge. Opsonization with the third component of the complement protein (C3) plays a major role in immune cell recognition of nanomedicines. We used dextran-coated superparamagnetic iron oxide nanoworms (SPIO NWs) to study the effect of surface functionalization on C3 opsonization in mouse serum and subsequent macrophage/leukocyte recognition in vitro as well as on intravenous injection into mice. Previously, we found that in mouse serum, SPIO NWs became opsonized with C3 via complement lectin pathway. Crosslinking the dextran shell with epichlorohydrin significantly decreased C3 opsonization and uptake by mouse peritoneal macrophages. Crosslinked nanoworms (NWs) further functionalized with polyethylene glycol (PEG) or with PEG-antibody (Ab) (~160 IgG molecules/particle) did not show an increase in C3 opsonization and peritoneal macrophage uptake in vitro. Following tail vein injection into mice, plain crosslinked NWs and PEGylated crosslinked NWs showed very low C3 opsonization and mouse leukocyte uptake. However, Ab-decorated crosslinked NWs showed significant C3 opsonization and high level of complement-dependent uptake by leukocytes in mice. Decreasing the number of conjugated Abs to 46 IgG molecules/particle significantly reduced C3 opsonization and leukocyte uptake. Using fresh mouse lepirudin plasma rather than serum showed better correlation with C3 opsonization in vivo. The reason for this difference could be related to the known instability of complement classical pathway in mouse sera. Our data illustrate that fine-tuning in nanoparticle surface functionalization with Abs is required to avoid excessive complement activation and complement-mediated immune uptake in mice, and raise issues with in vitro immunological assays of nanomedicines intended to mimic in vivo conditions. PMID:28239384

  13. Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium.

    PubMed

    Li, Yafeng; Song, Delu; Song, Ying; Zhao, Liangliang; Wolkow, Natalie; Tobias, John W; Song, Wenchao; Dunaief, Joshua L

    2015-05-08

    Dysregulation of iron homeostasis may be a pathogenic factor in age-related macular degeneration (AMD). Meanwhile, the formation of complement-containing deposits under the retinal pigment epithelial (RPE) cell layer is a pathognomonic feature of AMD. In this study, we investigated the molecular mechanisms by which complement component 3 (C3), a central protein in the complement cascade, is up-regulated by iron in RPE cells. Modulation of TGF-β signaling, involving ERK1/2, SMAD3, and CCAAT/enhancer-binding protein-δ, is responsible for iron-induced C3 expression. The differential effects of spatially distinct SMAD3 phosphorylation sites at the linker region and at the C terminus determined the up-regulation of C3. Pharmacologic inhibition of either ERK1/2 or SMAD3 phosphorylation decreased iron-induced C3 expression levels. Knockdown of SMAD3 blocked the iron-induced up-regulation and nuclear accumulation of CCAAT/enhancer-binding protein-δ, a transcription factor that has been shown previously to bind the basic leucine zipper 1 domain in the C3 promoter. We show herein that mutation of this domain reduced iron-induced C3 promoter activity. In vivo studies support our in vitro finding of iron-induced C3 up-regulation. Mice with a mosaic pattern of RPE-specific iron overload demonstrated co-localization of iron-induced ferritin and C3d deposits. Humans with aceruloplasminemia causing RPE iron overload had increased RPE C3d deposition. The molecular events in the iron-C3 pathway represent therapeutic targets for AMD or other diseases exacerbated by iron-induced local complement dysregulation.

  14. Mapping of the C3d receptor (CR2)-binding site and a neoantigenic site in the C3d domain of the third component of complement.

    PubMed Central

    Lambris, J D; Ganu, V S; Hirani, S; Müller-Eberhard, H J

    1985-01-01

    The C3d domain of C3 contains the site that binds to the C3d receptor (CR2) which is expressed on B lymphocytes. It also contains a neoantigenic determinant that is recognized by monoclonal antibody (mAb) 130 and is expressed when C3b is cleaved to iC3b and subsequently to C3dg or C3d. mAb 130 inhibits the binding of C3d to CR2. In this study, the locations of the CR2-binding site and of the neoantigen recognized by mAb 130 within the C3d domain were investigated. Treatment of human C3d with CNBr generated two major fragments with Mrs of 12,500 and 8600. Binding studies showed that only the Mr 8600 fragment was capable of binding to both CR2 and mAb 130. Amino-terminal sequence analysis of the Mr 8600 fragment and comparison with the amino acid sequence derived from human C3 cDNA [de Bruijn, M. H. L. & Fey, G. H. (1985) Proc. Natl. Acad. Sci. USA 82, 708-712] placed it between residues 1199 and 1274 of the C3 sequence. Several peptides were synthesized according to the derived C3 sequence of amino acid residues 1209-1236. Based on their differential binding to CR2 and mAb 130, we localized the CR2-binding site and mAb 130 neoantigenic site, respectively, to residues 1227-1232 and 1217-1232 of the C3 sequence. PMID:2408276

  15. Deficiency of complement component C3 is associated with accelerated removal of soluble 123I-labelled aggregates of IgG from the circulation.

    PubMed Central

    Halma, C; Daha, M R; Camps, J A; Evers-Schouten, J H; Pauwels, E K; Van, E s

    1992-01-01

    Complement and erythrocyte complement receptors CR1 (CD35) play an important role in the clearance of immune complexes. We studied the elimination of soluble 123I-labelled aggregates of human immunoglobulin G (123I-AIgG), used as a model for immune complexes, in two patients with a congenital and two patients with an acquired deficiency of complement component C3, and compared these with 10 healthy controls. The first disappearance halflife of 123I-AIgG was shorter (3.3 +/- 0.4 versus 7.0 +/- 0.4 min in the controls, P = 0.005) and maximal hepatic uptake of aggregates was increased in the C3 deficient patients (maximal liver/background ratio 3.6 +/- 0.4 versus 2.7 +/- 0.2 in controls, P = 0.04). Apparently, in the absence of C3, removal of circulating immune complexes by the liver is accelerated, probably through Fc receptor-dependent mechanisms. PMID:1458675

  16. Complement components C2, C3, and C4 (C4A and C4B) and BF polymorphisms in populations of the Indian subcontinent.

    PubMed

    Ad'hiah, A H; Papiha, S S

    1996-10-01

    Genetic polymorphisms of the complement components (five loci: C2, C3, C4A, C4B, and BF) have been investigated in the Telugu-speaking Hindu population of Hyderabad, Andhra Pradesh, India, and the Bangali-speaking Muslim population of Dacca, Bangladesh. The available data are compared to understand the genetic variation of complement components in populations of the Indian subcontinent. The C3*F and BF*F alleles show wide frequency variations in different ethnic groups of India. The range of variation in the C3*F allele is intermediate between European whites and southeast Asian populations, whereas the BF*F allele places the Indian frequencies between European whites and African blacks. This is the first population study to investigate the C2 and C4 (C4A and C4B) polymorphisms in two distinct groups of the Indian subcontinent. For the C2 polymorphism only the C2*B variant allele was observed, and its frequency was slightly higher than in European populations. In both populations the C4A and C4B loci were highly polymorphic, with a high frequency of the null alleles C4A*QO and C4B*QO, which may account for the greater susceptibility to certain autoimmune diseases in populations of South Asia.

  17. Effect of analytical factors on immunochemical reference limits for complement component C3 in serum of a reference pediatric population.

    PubMed

    Buffone, G J; Lewis, S A

    1977-06-01

    We evaluated analytical factors such as antibody specificity, standard materials, and methodology for the measurement of C3. Mancini-type radial immunodiffusion and immunonephelometry were shown to give comparable data if variables other than procedural variables are eliminated. The most significant analytical factors affecting the measurement were antiserum specificity and source of standard material.

  18. Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates.

    PubMed Central

    Friedman, H M; Glorioso, J C; Cohen, G H; Hastings, J C; Harris, S L; Eisenberg, R J

    1986-01-01

    The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigenic group on gC could be assigned as the C3b-binding region. Next, 21 gC mutants were evaluated for C3b binding, including 1 which failed to synthesize gC, 4 which synthesized truncated forms of the glycoprotein such that gC did not insert into the cell's membrane, and 16 which expressed gC on the cell's surface but which had mutations in various antigenic groups. Eleven strains did not bind C3b. This included the 1 strain which did not synthesize gC, the 4 strains which secreted gC without inserting the glycoprotein into the cell membrane, and 6 of 16 strains which expressed gC on the cell surface. In these six strains, the mutations were at three different antigenic sites. One hypothesis to explain these findings is that C3b binding is modified by changes in the conformation of gC which develop either after antibodies bind to gC or as a result of mutations in the gC gene. Attachment of C3b to gC was also evaluated in 31 low-passage clinical isolates of HSV-1. Binding was detected with each HSV-1 isolate, but not with nine HSV-2 isolates. Therefore, although mutants that lack C3b binding are readily selected in vitro, the C3b-binding function of gC is maintained in vivo. These results indicate that the sites on gC that bind C3b are different from those that bind monoclonal antibodies, that antibodies directed against all sites on gC block C3b binding, and that C3b binding is a conserved function of gC in vivo. PMID:3021981

  19. Crystallization and X-ray diffraction analysis of the complement component-3 (C3) inhibitory domain of Efb from Staphylococcus aureus

    SciTech Connect

    Hammel, Michal; Ramyar, Kasra X.; Spencer, Charles T.; Geisbrecht, Brian V.

    2006-03-01

    The crystallization and results of multiwavelength anomalous diffraction studies of a recombinant C3-inhibitory fragment of Efb from S. aureus are reported. The extracellular fibrinogen-binding protein (Efb) of Staphylococcus aureus is a multifunctional virulence factor capable of potent inhibition of complement component-3 (C3) activity in addition to its previously described fibrinogen-binding properties. A truncated recombinant form of Efb (Efb-C) that binds C3 has been overexpressed and purified and has been crystallized using the hanging-drop vapor-diffusion technique. Crystals of native Efb-C grew in the tetragonal space group P4{sub 3} (unit-cell parameters a = b = 59.53, c = 46.63 Å) with two molecules in the asymmetric unit and diffracted well beyond 1.25 Å limiting Bragg spacing. To facilitate de novo phasing of the Efb-C crystals, two independent site-directed mutants were engineered in which either residue Ile112 or Val140 was replaced with methionine and crystals isomorphous to those of native Efb-C were reproduced using a seleno-l-methionine-labeled form of each mutant protein. Multiwavelength anomalous diffraction (MAD) data were collected on both mutants and analyzed for their phasing power toward solution and refinement of a high-resolution Efb-C crystal structure.

  20. Complement component 3 (C3)

    MedlinePlus

    ... infections Gram negative septicemia Parasitic infections, such as malaria Paroxysmal nocturnal hemoglobinuria (PNH) Shock Normal Results The ... Glomerulonephritis Hepatitis Hereditary angioedema Kidney transplant Lupus ... nocturnal hemoglobinuria (PNH) Septicemia Shock Systemic lupus ...

  1. The role of complement in C3 glomerulopathy.

    PubMed

    Zipfel, Peter F; Skerka, Christine; Chen, Qian; Wiech, Thorsten; Goodship, Tim; Johnson, Sally; Fremeaux-Bacchi, Veronique; Nester, Clara; de Córdoba, Santiago Rodríguez; Noris, Marina; Pickering, Matthew; Smith, Richard

    2015-09-01

    C3 glomerulopathy describes a spectrum of disorders with glomerular pathology associated with C3 cleavage product deposition and with defective complement action and regulation (Fakhouri et al., 2010; Sethi et al., 2012b). Kidney biopsies from these patients show glomerular accumulation or deposition of C3 cleavage fragments, but no or minor deposition of immunoglobulins (Appel et al., 2005; D'Agati and Bomback, 2012; Servais et al., 2007; Sethi and Fervenza, 2011). At present the current situation asks for a better definition of the underlining disease mechanisms, for precise biomarkers, and for a treatment for this disease. The complement system is a self activating and propelling enzymatic cascade type system in which inactive, soluble plasma components are activated spontaneously and lead into an amplification loop (Zipfel and Skerka, 2009). Activation of the alternative pathway is spontaneous, occurs by default, and cascade progression leads to amplification by complement activators. The system however is self-controlled by multiple regulators and inhibitors, like Factor H that control cascade progression in fluid phase and on surfaces. The activated complement system generates a series of potent effector components and activation products, which damage foreign-, as well as modified self cells, recruit innate immune cells to the site of action, coordinate inflammation and the response of the adaptive immune system in form of B cells and T lymphocytes (Kohl, 2006; Medzhitov and Janeway, 2002; Ogden and Elkon, 2006; Carroll, 2004; Kemper and Atkinson, 2007; Morgan, 1999; Muller-Eberhard, 1986; Ricklin et al., 2010). Complement controls homeostasis and multiple reactions in the vertebrate organism including defense against microbial infections (Diaz-Guillen et al., 1999; Mastellos and Lambris, 2002; Nordahl et al., 2004; Ricklin et al., 2010). In consequence defective control of the spontaneous self amplifying cascade or regulation is associated with numerous

  2. Molecular Basis for Complement Recognition and Inhibition Determined by Crystallographic Studies of the Staphylococcal Complement Inhibitor (SCIN) Bound to C3c and C3b

    SciTech Connect

    Garcia, Brandon L.; Ramyar, Kasra X.; Tzekou, Apostolia; Ricklin, Daniel; McWhorter, William J.; Lambris, John D.; Geisbrecht, Brian V.

    2010-10-22

    The human complement system plays an essential role in innate and adaptive immunity by marking and eliminating microbial intruders. Activation of complement on foreign surfaces results in proteolytic cleavage of complement component 3 (C3) into the potent opsonin C3b, which triggers a variety of immune responses and participates in a self-amplification loop mediated by a multi-protein assembly known as the C3 convertase. The human pathogen Staphylococcus aureus has evolved a sophisticated and potent complement evasion strategy, which is predicated upon an arsenal of potent inhibitory proteins. One of these, the staphylococcal complement inhibitor (SCIN), acts at the level of the C3 convertase (C3bBb) and impairs downstream complement function by trapping the convertase in a stable but inactive state. Previously, we have shown that SCIN binds C3b directly and competitively inhibits binding of human factor H and, to a lesser degree, that of factor B to C3b. Here, we report the co-crystal structures of SCIN bound to C3b and C3c at 7.5 and 3.5 {angstrom} limiting resolution, respectively, and show that SCIN binds a critical functional area on C3b. Most significantly, the SCIN binding site sterically occludes the binding sites of both factor H and factor B. Our results give insight into SCIN binding to activated derivatives of C3, explain how SCIN can recognize C3b in the absence of other complement components, and provide a structural basis for the competitive C3b-binding properties of SCIN. In the future, this may suggest templates for the design of novel complement inhibitors based upon the SCIN structure.

  3. A Revised Mechanism for the Activation of Complement C3 to C3b

    PubMed Central

    Rodriguez, Elizabeth; Nan, Ruodan; Li, Keying; Gor, Jayesh; Perkins, Stephen J.

    2015-01-01

    The solution structure of complement C3b is crucial for the understanding of complement activation and regulation. C3b is generated by the removal of C3a from C3. Hydrolysis of the C3 thioester produces C3u, an analog of C3b. C3b cleavage results in C3c and C3d (thioester-containing domain; TED). To resolve functional questions in relation to C3b and C3u, analytical ultracentrifugation and x-ray and neutron scattering studies were used with C3, C3b, C3u, C3c, and C3d, using the wild-type allotype with Arg102. In 50 mm NaCl buffer, atomistic scattering modeling showed that both C3b and C3u adopted a compact structure, similar to the C3b crystal structure in which its TED and macroglobulin 1 (MG1) domains were connected through the Arg102–Glu1032 salt bridge. In physiological 137 mm NaCl, scattering modeling showed that C3b and C3u were both extended in structure, with the TED and MG1 domains now separated by up to 6 nm. The importance of the Arg102–Glu1032 salt bridge was determined using surface plasmon resonance to monitor the binding of wild-type C3d(E1032) and mutant C3d(A1032) to immobilized C3c. The mutant did not bind, whereas the wild-type form did. The high conformational variability of TED in C3b in physiological buffer showed that C3b is more reactive than previously thought. Because the Arg102-Glu1032 salt bridge is essential for the C3b-Factor H complex during the regulatory control of C3b, the known clinical associations of the major C3S (Arg102) and disease-linked C3F (Gly102) allotypes of C3b were experimentally explained for the first time. PMID:25488663

  4. Mapping the Complement Factor H-Related Protein 1 (CFHR1):C3b/C3d Interactions

    PubMed Central

    Laskowski, Jennifer; Thurman, Joshua M.; Hageman, Gregory S.; Holers, V. Michael

    2016-01-01

    Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation. PMID:27814381

  5. Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome

    PubMed Central

    Miller, Elizabeth C.; Liszewski, M. Kathryn; Strain, Lisa; Blouin, Jacques; Brown, Alison L.; Moghal, Nadeem; Kaplan, Bernard S.; Weiss, Robert A.; Lhotta, Karl; Kapur, Gaurav; Mattoo, Tej; Nivet, Hubert; Wong, William; Gie, Sophie; de Ligny, Bruno Hurault; Fischbach, Michel; Gupta, Ritu; Hauhart, Richard; Meunier, Vincent; Loirat, Chantal; Dragon-Durey, Marie-Agnès; Fridman, Wolf H.; Janssen, Bert J. C.

    2008-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a disease of complement dysregulation. In approximately 50% of patients, mutations have been described in the genes encoding the complement regulators factor H, MCP, and factor I or the activator factor B. We report here mutations in the central component of the complement cascade, C3, in association with aHUS. We describe 9 novel C3 mutations in 14 aHUS patients with a persistently low serum C3 level. We have demonstrated that 5 of these mutations are gain-of-function and 2 are inactivating. This establishes C3 as a susceptibility factor for aHUS. PMID:18796626

  6. Serum immunoglobulins and complement (C'3) in oral lichen planus.

    PubMed

    Sklavounou, A D; Laskaris, G; Angelopoulos, A P

    1983-01-01

    Serum immunoglobulins and complement (C'3) were determined by single radial immunodiffusion according to the method of Mancini and co-workers in fifty patients with oral lichen planus and twenty persons with clinically normal oral mucosa. Significantly increased levels of serum IgG (p less than 0.05) and a significant reduction of serum IgA concentration (p less than 0.05) in the experimental group as compared with normal controls were observed. Mean serum IgM and complement (C'3) levels were similar in patients and controls. No correlation between disease variety or extensiveness and immunoglobulin or complement levels was noticed. These results suggest that patients with oral lichen planus may have a generalized immunologic disorder in which humoral immunity is disturbed. Whether humoral immunity is of etiologic significance, contributes to the disease process, or, finally, represents an event secondary to the pathologic changes seen in the disease remains to be determined.

  7. Factor C acts as a lipopolysaccharide-responsive C3 convertase in horseshoe crab complement activation.

    PubMed

    Ariki, Shigeru; Takahara, Shusaku; Shibata, Toshio; Fukuoka, Takaaki; Ozaki, Aya; Endo, Yuichi; Fujita, Teizo; Koshiba, Takumi; Kawabata, Shun-ichiro

    2008-12-01

    The complement system in vertebrates plays an important role in host defense against and clearance of invading microbes, in which complement component C3 plays an essential role in the opsonization of pathogens, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. In an effort to understand the molecular activation mechanism of invertebrate C3, we isolated and characterized an ortholog of C3 (designated TtC3) from the horseshoe crab Tachypleus tridentatus. Flow cytometric analysis using an Ab against TtC3 revealed that the horseshoe crab complement system opsonizes both Gram-negative and Gram-positive bacteria. Evaluation of the ability of various pathogen-associated molecular patterns to promote the proteolytic conversion of TtC3 to TtC3b in hemocyanin-depleted plasma indicated that LPS, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to LPS stimulation. Although originally characterized as an LPS-sensitive initiator of hemolymph coagulation stored within hemocytes, we identified factor C in hemolymph plasma. An anti-factor C Ab inhibited various LPS-induced phenomena, including plasma amidase activity, the proteolytic activation of TtC3, and the deposition of TtC3b on the surface of Gram-negative bacteria. Moreover, activated factor C present on the surface of Gram-negative bacteria directly catalyzed the proteolytic conversion of the purified TtC3, thereby promoting TtC3b deposition. We conclude that factor C acts as an LPS-responsive C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of horseshoe crab complement activation.

  8. Complement therapeutics in inflammatory diseases: promising drug candidates for C3-targeted intervention

    PubMed Central

    Mastellos, Dimitrios C.; Ricklin, Daniel; Hajishengallis, Evlambia; Hajishengallis, George; Lambris, John D.

    2015-01-01

    SUMMARY There is an increasing appreciation that complement dysregulation lies at the heart of numerous immune-mediated and inflammatory disorders. Complement inhibitors are therefore being evaluated as new therapeutic options in various clinical translation programs and the first clinically approved complement-targeted drugs have profoundly impacted the management of certain complement-mediated diseases. Among the many members of the intricate protein network of complement, the central component C3 represents a ‘hot-spot’ for complement-targeted therapeutic intervention. C3 modulates both innate and adaptive immune responses and is linked to diverse immunomodulatory systems and biological processes that affect human pathophysiology. Compelling evidence from preclinical disease models has shown that C3 interception may offer multiple benefits over existing therapies or even reveal novel therapeutic avenues in disorders that are not commonly regarded complement-driven, such as periodontal disease. Using the clinically-developed compstatin family of C3 inhibitors and periodontitis as illustrative examples, this review highlights emerging therapeutic concepts and developments in the design of C3-targeted drug candidates as novel immunotherapeutics for oral and systemic inflammatory diseases. PMID:26332138

  9. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...

  10. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...

  11. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...

  12. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...

  13. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... immunochemical techniques the complement C3b inactivator (a plasma protein) in serum. Complement is a group of serum proteins that destroy infectious agents. Measurement of complement C3b inactivator aids in...

  14. Complement C3 gene: Expression characterization and innate immune response in razor clam Sinonovacula constricta.

    PubMed

    Peng, Maoxiao; Niu, Donghong; Wang, Fei; Chen, Zhiyi; Li, Jiale

    2016-08-01

    Complement component 3 (C3) is central to the complement system, playing an important role in immune defense, immune regulation and immune pathology. Several C3 genes have been characterized in invertebrates but very few in shellfish. The C3 gene was identified from the razor clam Sinonovacula constricta, referred to here as Sc-C3. It was found to be highly homologous with the C3 gene of Ruditapes decussatus. All eight model motifs of the C3 gene were found to be included in the thiolester bond and the C345C region. Sc-C3 was widely expressed in all healthy tissues with expression being highest in hemolymph. A significant difference in expression was revealed at the umbo larvae development stage. The expression of Sc-C3 was highly regulated in the hemolymph and liver, with a distinct response pattern being noted after a challenge with Micrococcus lysodeikticus and Vibrio parahemolyticus. It is therefore suggested that a complicated and unique response pathway may be present in S. constricta. Further, serum of S. constricta containing Sc-C3 was extracted. This was activated by LPS or bacterium for verification for function. The more obvious immune function of Sc-C3 was described as an effective membrane rupture in hemocyte cells of rabbit, V. parahemolyticus and Vibrio anguillarum. Thus, Sc-C3 plays an essential role in the immune defense of S. constricta.

  15. Dynamic Structural Changes During Complement C3 Activation Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry

    PubMed Central

    Schuster, Michael C.; Ricklin, Daniel; Papp, Krisztián; Molnar, Kathleen S.; Coales, Stephen J.; Hamuro, Yoshitomo; Sfyroera, Georgia; Chen, Hui; Winters, Michael S.; Lambris, John D.

    2008-01-01

    Proteolytic cleavage of component C3 to C3b is a central step in the activation of complement. Whereas C3 is largely biologically inactive, C3b is directly involved in various complement activities. While the recently described crystal structures of C3 and C3b provide a molecular basis of complement activation, they do not reflect the dynamic changes that occur in solution. In addition, the available C3b structures diverge in some important aspects. Here we have utilized hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) to investigate relative changes in the solution-phase structures of C3 and C3b. By combining two forms of mass spectrometry we could maximize the primary sequence coverage of C3b and demonstrate the feasibility of this method for large plasma proteins. While the majority of the 82 peptides that could be followed over time showed only minor alterations in HDX, we observed clear changes in solvent accessibility for 16 peptides, primarily in the α-chain (α’NT, MG6-8, CUB, TED, C345C domains). Most of these peptides could be directly linked to the structural transitions visible in the crystal structures and revealed additional information about the probability of the structural variants of C3b. In addition, a discontinuous cluster of seven peptides in the MG3, MG6, LNK and α’NT domains showed a decreased accessibility after activation to C3b. Although no gross conformational changes are detected in the crystal structure, this area may reflect a structurally flexible region in solution that contributes to C3 activation and function. PMID:18456336

  16. Ionic tethering contributes to the conformational stability and function of complement C3b.

    PubMed

    López-Perrote, Andrés; Harrison, Reed E S; Subías, Marta; Alcorlo, Martín; Rodríguez de Córdoba, Santiago; Morikis, Dimitrios; Llorca, Oscar

    2017-02-27

    C3b, the central component of the alternative pathway (AP) of the complement system, coexists as a mixture of conformations in solution. These conformational changes can affect interactions with other proteins and complement regulators. Here we combine a computational model for electrostatic interactions within C3b with molecular imaging to study the conformation of C3b. The computational analysis shows that the TED domain in C3b is tethered ionically to the macroglobulin (MG) ring. Monovalent counterion concentration affects the magnitude of electrostatic forces anchoring the TED domain to the rest of the C3b molecule in a thermodynamic model. This is confirmed by observing NaCl concentration dependent conformational changes using single molecule electron microscopy (EM). We show that the displacement of the TED domain is compatible with C3b binding to Factor B (FB), suggesting that the regulation of the C3bBb convertase could be affected by conditions that promote movement in the TED domain. Our molecular model also predicts mutations that could alter the positioning of the TED domain, including the common R102G polymorphism, a risk variant for developing age-related macular degeneration. The common C3b isoform, C3bS, and the risk isoform, C3bF, show distinct energetic barriers to displacement in the TED that are related to a network of electrostatic interactions at the interface of the TED and MG-ring domains of C3b. These computational predictions agree with experimental evidence that shows differences in conformation observed in C3b isoforms purified from homozygous donors. Altogether, we reveal an ionic, reversible attachment of the TED domain to the MG ring that may influence complement regulation in some mutations and polymorphisms of C3b.

  17. Polymorphism of the complement receptor for C3bi.

    PubMed Central

    Russ, G R; Haddad, A P; Tait, B D; d'Apice, A J

    1985-01-01

    RM2.184, a mouse IgG2a monoclonal antibody, recognizes a polymorphic determinant on the complement receptor for C3bi which is present on granulocytes and monocytes. The RM2.184 epitope is distinct from the monomorphic determinant recognized by the monoclonal antibody OKM1. The RM2.184 epitope is probably on the alpha subunit and dependent on the association of the alpha and beta subunits for its configuration, as it can not be detected after the subunits have been dissociated. The phenotypic frequency of the RM2.184 antigen is approximately 14%, and its segregation in families is independent of HLA and consistent with an autosomal co-dominant mode of inheritance. Images PMID:2414326

  18. Complement components in Nigerians with bronchial asthma.

    PubMed

    Onyemelukwe, G C

    1989-10-01

    Serum complement components C1q, C3, C4, factor B, and C3d breakdown products were measured in asthmatic Nigerians and in age-matched and sex-matched controls. C3 mean level was higher than in controls while C1q and C4 mean levels were lower than in controls. High levels of C3d in asthmatic patients suggest the possible role of C3a and C5a anaphylatoxins in the etiopathogenesis of perennial asthma in Nigerian patients in a tropical environment with ubiquitous airborne allergens and infective agents. The significantly elevated levels of IgM and IgG may suggest recurrent respiratory challenge of perennial antigens in our environment.

  19. Functional and structural characterization of four mouse monoclonal antibodies to complement C3 with potential therapeutic and diagnostic applications.

    PubMed

    Subías Hidalgo, Marta; Yébenes, Hugo; Rodríguez-Gallego, César; Martín-Ambrosio, Adrián; Domínguez, Mercedes; Tortajada, Agustin; Rodríguez de Córdoba, Santiago; Llorca, Oscar

    2017-03-01

    C3 is the central component of the complement system. Upon activation, C3 sequentially generates various proteolytic fragments, C3a, C3b, iC3b, C3dg, each of them exposing novel surfaces, which are sites of interaction with other proteins. C3 and its fragments are therapeutic targets and markers of complement activation. We report the structural and functional characterization of four monoclonal antibodies (mAbs) generated by immunizing C3-deficient mice with a mixture of human C3b, iC3b and C3dg fragments, and discuss their potential applications. This collection includes three mAbs interacting with native C3 and inhibiting AP complement activation; two of them by blocking the cleavage of C3 by the AP C3-converase and one by impeding formation of the AP C3-convertase. The interaction sites of these mAbs in the target molecules were determined by resolving the structures of Fab fragments bound to C3b and/or iC3b using electron microscopy. A fourth mAb specifically recognizes the iC3b, C3dg, and C3d fragments. It binds to an evolutionary-conserved neoepitope generated after C3b cleavage by FI, detecting iC3b/C3dg deposition over opsonized surfaces by flow cytometry and immunohistochemistry in human and other species. Because well-characterized anti-complement mAbs are uncommon, the mAbs reported here may offer interesting therapeutic and diagnostic opportunities.

  20. Interleukin 2 mediates stimulation of complement C3 biosynthesis in human proximal tubular epithelial cells.

    PubMed Central

    Brooimans, R A; Stegmann, A P; van Dorp, W T; van der Ark, A A; van der Woude, F J; van Es, L A; Daha, M R

    1991-01-01

    Previous reports have suggested the production of complement components C4, C2, and factor B by renal tissue. However, the cells involved in production of complement have not been identified. In this study metabolic labeling experiments demonstrated that human proximal tubular epithelial cells (PTEC) synthesize a 180-kD precursor of C3 that is secreted after proteolytic cleavage into a disulphide linked two-chain molecule as found in plasma. C3 present in culture supernatants of PTEC was functionally active, however, during the culture period there was a partial inactivation of the C3 molecule as assessed by hemolytic titration. Recombinant IL-2 enhances the rate of C3 synthesis in a dose-dependent manner reaching maximal stimulation at doses of 200-400 U/ml IL-2. Northern blot analysis demonstrated a 5.2-kb C3 mRNA species present in PTEC that was increased within 24 h of IL-2 treatment. IL-2-induced enhancement of C3 production by PTEC could be neutralized with specific antibodies to IL-2. This study demonstrates that C3 synthesis in PTEC is upregulated by IL-2, the major cytokine produced by activated T cells. Images PMID:1864952

  1. Bypass-activation of the complement system starting with C3

    PubMed Central

    Bitter-Suermann, D.; Dierich, M.; König, W.; Hadding, U.

    1972-01-01

    Antibody independent activation of the complement system starting with C3 can be achieved by means of a purified factor from cobra venom (VF), which interacts with a purified serum factor (SF). The latter is a normal constituent of guinea-pig and human serum (C3-proactivator). The interaction between VF and SF is Mg+ + dependent and leads to the formation of a complex. Immunological analysis reveals that both VF- and SF-antigens are contained in the complex. The VF—SF complex activates enzymatically isolated C3, which in the presence of the subsequent components yields all effects of the normal complement sequence. Purified C5 is not affected by the complex. Its activation is mediated by activated C3. The VF—SF system represents a model for direct activation of C3 to C9 independent of antibody, C1, C4 and C2. An analogous pathway of alternate complement activation might be used by other substances, e.g. endotoxin, guinea-pig γ1-immune aggregates and zymosan. The corresponding serum factors are under investigation. ImagesFIG. 5FIG. 6 PMID:4214761

  2. Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3.

    PubMed

    Potempa, Michal; Potempa, Jan; Kantyka, Tomasz; Nguyen, Ky-Anh; Wawrzonek, Katarzyna; Manandhar, Surya P; Popadiak, Katarzyna; Riesbeck, Kristian; Eick, Sigrun; Blom, Anna M

    2009-02-01

    Periodontitis is an inflammatory disease of the supporting structures of the teeth caused by, among other pathogens, Prevotella intermedia. Many strains of P. intermedia are resistant to killing by the human complement system, which is present at up to 70% of serum concentration in gingival crevicular fluid. Incubation of human serum with recombinant cysteine protease of P. intermedia (interpain A) resulted in a drastic decrease in bactericidal activity of the serum. Furthermore, a clinical strain 59 expressing interpain A was more serum-resistant than another clinical strain 57, which did not express interpain A, as determined by Western blotting. Moreover, in the presence of the cysteine protease inhibitor E64, the killing of strain 59 by human serum was enhanced. Importantly, we found that the majority of P. intermedia strains isolated from chronic and aggressive periodontitis carry and express the interpain A gene. The protective effect of interpain A against serum bactericidal activity was found to be attributable to its ability to inhibit all three complement pathways through the efficient degradation of the alpha-chain of C3 -- the major complement factor common to all three pathways. P. intermedia has been known to co-aggregate with P. gingivalis, which produce gingipains to efficiently degrade complement factors. Here, interpain A was found to have a synergistic effect with gingipains on complement degradation. In addition, interpain A was able to activate the C1 complex in serum, causing deposition of C1q on inert and bacterial surfaces, which may be important at initial stages of infection when local inflammatory reaction may be beneficial for a pathogen. Taken together, the newly characterized interpain A proteinase appears to be an important virulence factor of P. intermedia.

  3. Surface complement C3 fragments and cellular binding of microparticles in patients with SLE

    PubMed Central

    Winberg, Line Kjær; Nielsen, Claus Henrik; Jacobsen, Søren

    2017-01-01

    Objectives To examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing the ability of MPs to bind to circulating phagocytes and erythrocytes. These features may be relevant for clearance of MPs in SLE pathogenesis. Methods Attached C3 fragments (C3b, iC3b, C3d), membrane integrity and cell surface markers of MPs from 18 patients with SLE and 11 HCs were measured by adding specific antibodies, 7-aminoactinomycin D (7AAD) and annexin V. MPs from all subjects were labelled with carboxyfluorescein diacetate succinimidyl ester and allowed to bind to autologous phagocytes and erythrocytes in the presence of autologous serum, and the binding to individual cell populations was assessed by flow cytometry. Results The proportion of MPs bearing C3 fragments was higher in patients with SLE than in HCs (p=0.026), but the amount of opsonising C3b/iC3b molecules was lower (p=0.004). The C3b/iC3b level correlated with the concentration of circulating C3 (rs=0.53, p=0.036). Phagocytes and erythrocytes from patients and HCs bound autologous MPs, and granulocytes from patients bound 13% more MPs than those from HCs (p=0.043). The presence of erythrocytes inhibited the MP binding to granulocytes by approximately 50%. Conclusions Our demonstration of altered composition of C3 fragments on MPs from patients with SLE, including decreased numbers of opsonising C3 fragments, and competitive binding of MPs to circulating phagocytes and erythrocytes corroborates the hypothesis of defective clearance of apoptotic material in SLE, and indicates that differences in both MP opsonisation and binding of MPs to cells are important in the pathogenesis of SLE.

  4. Platelet-associated complement C3 in immune thrombocytopenic purpura

    SciTech Connect

    Myers, T.J.; Kim, B.K.; Steiner, M.; Baldini, M.G.

    1982-05-01

    Platelet-associated C3 (PA-C3) was measured with a quantitative immunofluorescence assay. With this assay, PA-C3 levels were determined for 78 normal volunteers, 30 patients with immune thrombocytopenic purpura (ITP), and 20 patients with nonimmune thrombocytopenias. Platelet-associatd IgG (PA-lgG) levels were also measured with our standard quantitative immunofluorescence assay. All patients with nonimmune thrombocytopenias and ITP in remission had normal PA-C3 levels. Twenty-four patients with active ITP wre classified into 3 groups: 9 (38%) with increased PA-IgG and normal PA-C3 levels, 10 (42%) with elevated PA-C3 and PA-IgG levels, and 5 (20%) with increased PA-C3 values only. A direct correlation was found between PA-C3 and PA-IgG levels. PA-IgG levels were higher in the group of patients with elevated PA-C3 levels than in those with normal values. Platelet survival studies showed reduced survival times of 1.5-2.5 days for the 5 patients with elevated PA-C3 levels only. Elevated PA-C3 levels returned to normal in 7 ITP patients whose platelet counts increased in response to corticosteriod therapy or to splenectomy. Therefore, PA-C3 and PA-IgG assays can be used to identify patients with ITP, to follow their response to therapy, and to classify them into immunologic subgroups similar to red cell classifiation by Coombs' testing in immune hemolytic anemia.

  5. Renal cortical complement C3 gene expression in IgA nephropathy.

    PubMed

    Montinaro, V; Gesualdo, L; Ranieri, E; Monno, R; Grandaliano, G; Schena, F P

    1997-03-01

    Glomerular C3 deposits are commonly found in immunoglobulin A (IgA) nephropathy. Renal gene expression and protein synthesis of complement components have been shown in settings of tissue inflammation. In this study, the pathogenetic involvement of locally produced C3 in IgA nephropathy was analyzed. C3 gene expression was analyzed by reverse transcription, polymerase chain reaction, and in situ hybridization techniques. C3 mRNA was detected in 56% of cases, with a significantly higher percentage in patients with moderate-to-severe lesions than in those with mild lesions (P < 0.01). By in situ hybridization, C3 transcript was predominantly expressed by tubular cells and some interstitial cells. C3 mRNA was also observed on glomerular parietal epithelial cells. Immunoreactive native C3 was detected on cortical tubuli by an anti-C3c immunoalkaline-phosphatase technique. A significant correlation was found between renal C3 transcription and glomerulosclerosis, intracapillary proliferation (both P < 0.005) and markers of interstitial damage, including tubular atrophy (P < 0.05), interstitial infiltration (P < 0.05), and fibrosis (P < 0.005). Proteinuria (P < 0.05), but not serum creatinine, at the time of renal biopsy correlated with C3 mRNA. In conclusion, it was demonstrated that the C3 gene was expressed primarily in proximal tubular cells and occasionally in glomerular crescents, and that its expression correlated with clinical and histologic markers of severity and poor outcome of IgA nephropathy. Thus, a pathogenetic involvement of the local transcription and translation of the C3 gene in IgA nephropathy was suggested.

  6. Receptor for complement peptide C3a: a therapeutic target for neonatal hypoxic-ischemic brain injury.

    PubMed

    Järlestedt, Katarina; Rousset, Catherine I; Ståhlberg, Anders; Sourkova, Hana; Atkins, Alison L; Thornton, Claire; Barnum, Scott R; Wetsel, Rick A; Dragunow, Mike; Pekny, Milos; Mallard, Carina; Hagberg, Henrik; Pekna, Marcela

    2013-09-01

    Complement is an essential component of inflammation that plays a role in ischemic brain injury. Recent reports demonstrate novel functions of complement in normal and diseased CNS, such as regulation of neurogenesis and synapse elimination. Here, we examined the role of complement-derived peptide C3a in unilateral hypoxia-ischemia (HI), a model of neonatal HI encephalopathy. HI injury was induced at postnatal day 9 (P9), and loss of hippocampal tissue was determined on P31. We compared WT mice with transgenic mice expressing C3a under the control of glial fibrillary acidic protein promoter, which express biologically active C3a only in CNS and without the requirement of a priori complement activation. Further, we injected C3a peptide into the lateral cerebral ventricle of mice lacking the C3a receptor (C3aR) and WT mice and assessed HI-induced memory impairment 41 d later. We found that HI-induced tissue loss in C3a overexpressing mice was reduced by 50% compared with WT mice. C3a peptide injected 1 h after HI protected WT but not C3aR-deficient mice against HI-induced memory impairment. Thus, C3a acting through its canonical receptor ameliorates behavioral deficits after HI injury, and C3aR is a novel therapeutic target for the treatment of neonatal HI encephalopathy.

  7. Glomerular C3c localization indicates ongoing immune deposit formation and complement activation in experimental glomerulonephritis.

    PubMed Central

    Schulze, M.; Pruchno, C. J.; Burns, M.; Baker, P. J.; Johnson, R. J.; Couser, W. G.

    1993-01-01

    In antibody-mediated glomerular disease, deposits of C3 (C3b) are common and are degraded by factor I to C3c and C3d. However, the kinetics of C3b degradation in glomerulonephritis have not been defined. To do this, we studied three models of complement-dependent glomerulonephritis with established C3 deposits (passive Heymann nephritis, cationized immunoglobulin G membranous nephropathy, and concanavalin A-anticoncanavalin A glomerulonephritis). C3b deposition was halted by administration of cobra venom factor, and the disappearance of C3c and C3d from glomeruli was measured with specific antibodies and quantitative fluorescence densitometry. Results showed that C3c deposits were reduced by over 85% within 24 hours in all three models. C3c clearance was unaffected by site or mechanism of deposit formation. C3d deposits persisted despite lack of ongoing complement activation. In passive Heymann nephritis when disease activity was monitored by urinary C5b-9 excretion, C3c was cleared in parallel with return of urine C5b-9 excretion to normal values. We conclude that glomerular deposits of C3c are cleared within 24 hours of cessation of complement activation. Positive staining for C3 utilizing antibody specific for the C3c portion documents recent complement activation usually reflecting new immune deposit formation. Images Figure 1 Figure 2 Figure 3 PMID:7678717

  8. The evolutionary analysis on complement genes reveals that fishes C3 and C9 experience different evolutionary patterns.

    PubMed

    Wang, Shanchen; Wang, Rixin; Xu, Tianjun

    2013-12-01

    Complement is a humoral factor of innate immunity and plays an essential role in altering the host of the presence of potential pathogens and clearing of invading microorganisms. The third complement component (C3) not only is regarded as the crossing of the three pathways of complement activation, but also serves one of the bridges linking innate and acquired immunity. The nine complement component (C9) can combine with C5b, C6, C7 and C8 to form MAC which bounds to the surface of microorganisms to kill them. The evidence of evolution on C3 genes which have multiple functions and plays central role in innate immunity was documented in our previous study. Now we were interested in the evolution of C9 genes which were the terminal complement components. For these reasons, we want to explore the evolutionary patterns of C9 and whether C3 and C9 experience different evolutionary patterns. In our study, we used the sliding window method to separately calculate the values of ω among fishes and mammals of C3 and C9 codons. In order to detect the positive selection sites, we used the maximum likelihood (ML) method to study the evolutionary pattern on C3 and C9 genes. Positive selection sites were detected in mammalian C9 genes and no positive selection sites were detected in fishes C9 genes. However, no positive selection sites were detected in mammalian C3 genes and positive selection sites were detected in fishes C3 genes. The result indicated that C3 and C9 had different evolutionary patterns on mammals and fishes. In conclusion, different living environments lead to different evolutionary patterns on C3 and C9 in mammals and fishes. Besides, different complement components may have different evolutionary patterns on mammals and fishes.

  9. Genetics Home Reference: complement component 8 deficiency

    MedlinePlus

    ... the membranes surrounding the brain and spinal cord (meningitis). Although meningitis can be life-threatening, individuals with complement component ... leaves affected individuals prone to recurrent episodes of meningitis. Learn more about the genes associated with complement ...

  10. Preliminary characterization of complement in a colonial tunicate: C3, Bf and inhibition of C3 opsonic activity by compstatin.

    PubMed

    Franchi, Nicola; Ballarin, Loriano

    2014-10-01

    The complement system is a fundamental effector mechanism of the innate immunity in both vertebrates and invertebrates. The comprehension of its roots in the evolution is a useful step to understand how the main complement-related proteins had changed in order to adapt to new environmental conditions and life-cycles or, in the case of vertebrates, to interact with the adaptive immunity. Data on organisms evolutionary close to vertebrates, such as tunicates, are of primary importance for a better understanding of the changes in immune responses associated with the invertebrate-vertebrate transition. Here we report on the characterization of C3 and Bf transcripts from the colonial ascidian Botryllus schlosseri (BsC3 and BsBf, respectively), a reliable model organism for immunobiological research, and present a comparative analysis of amino acid sequences of C3s and Bfs suggesting that, in deuterostomes, the structure of these proteins remained largely unchanged. We also present new data on the cells responsible of the expression of BsC3 and BsBf showing that cytotoxic immunocytes are the sole cells where the relative transcripts can be found. Finally, using the C3 specific inhibitor compstatin, we demonstrate the opsonic role of BsC3 in accordance with the idea that promotion of phagocytosis is one of the main function of C3 in metazoans.

  11. Zinc-induced Self-association of Complement C3b and Factor H

    PubMed Central

    Nan, Ruodan; Tetchner, Stuart; Rodriguez, Elizabeth; Pao, Po-Jung; Gor, Jayesh; Lengyel, Imre; Perkins, Stephen J.

    2013-01-01

    The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3. During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100 μm zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions showed that putative weak zinc binding sites with different capacities exist in all five proteins, in agreement with experiments. Factor H forms large oligomers in >10 μm zinc. In contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much reduced at 60 μm zinc and even more so at >100 μm zinc. The removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc. Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment epithelial deposits in the retina as well as reducing the progression to advanced age-related macular degeneration in higher risk patients. PMID:23661701

  12. Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.

    PubMed

    van Vuuren, B Jansen; Bergseth, G; Mollnes, T E; Shaw, A M

    2014-01-15

    Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade.

  13. Detection of complement activation using monoclonal antibodies against C3d

    PubMed Central

    Thurman, Joshua M.; Kulik, Liudmila; Orth, Heather; Wong, Maria; Renner, Brandon; Sargsyan, Siranush A.; Mitchell, Lynne M.; Hourcade, Dennis E.; Hannan, Jonathan P.; Kovacs, James M.; Coughlin, Beth; Woodell, Alex S.; Pickering, Matthew C.; Rohrer, Bärbel; Holers, V. Michael

    2013-01-01

    During complement activation the C3 protein is cleaved, and C3 activation fragments are covalently fixed to tissues. Tissue-bound C3 fragments are a durable biomarker of tissue inflammation, and these fragments have been exploited as addressable binding ligands for targeted therapeutics and diagnostic agents. We have generated cross-reactive murine monoclonal antibodies against human and mouse C3d, the final C3 degradation fragment generated during complement activation. We developed 3 monoclonal antibodies (3d8b, 3d9a, and 3d29) that preferentially bind to the iC3b, C3dg, and C3d fragments in solution, but do not bind to intact C3 or C3b. The same 3 clones also bind to tissue-bound C3 activation fragments when injected systemically. Using mouse models of renal and ocular disease, we confirmed that, following systemic injection, the antibodies accumulated at sites of C3 fragment deposition within the glomerulus, the renal tubulointerstitium, and the posterior pole of the eye. To detect antibodies bound within the eye, we used optical imaging and observed accumulation of the antibodies within retinal lesions in a model of choroidal neovascularization (CNV). Our results demonstrate that imaging methods that use these antibodies may provide a sensitive means of detecting and monitoring complement activation–associated tissue inflammation. PMID:23619360

  14. Identification of C3b-Binding Small-Molecule Complement Inhibitors Using Cheminformatics.

    PubMed

    Garcia, Brandon L; Skaff, D Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K; Wyckoff, Gerald J; Geisbrecht, Brian V

    2017-03-15

    The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of more than two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology, such as acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases, which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small-molecule inhibitors, small-molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study, we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies, we identified 45 small molecules that putatively bind C3b near ligand-guided functional hot spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand that guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small-molecule complement inhibitors and, to our knowledge, provides the first demonstration of cheminformatics-based, complement-directed drug discovery.

  15. C3 glomerulopathy–associated CFHR1 mutation alters FHR oligomerization and complement regulation

    PubMed Central

    Tortajada, Agustín; Yébenes, Hugo; Abarrategui-Garrido, Cynthia; Anter, Jaouad; García-Fernández, Jesús M.; Martínez-Barricarte, Rubén; Alba-Domínguez, María; Malik, Talat H.; Bedoya, Rafael; Pérez, Rocío Cabrera; Trascasa, Margarita López; Pickering, Matthew C.; Harris, Claire L.; Sánchez-Corral, Pilar; Llorca, Oscar; Rodríguez de Córdoba, Santiago

    2013-01-01

    C3 glomerulopathies (C3G) are a group of severe renal diseases with distinct patterns of glomerular inflammation and C3 deposition caused by complement dysregulation. Here we report the identification of a familial C3G-associated genomic mutation in the gene complement factor H–related 1 (CFHR1), which encodes FHR1. The mutation resulted in the duplication of the N-terminal short consensus repeats (SCRs) that are conserved in FHR2 and FHR5. We determined that native FHR1, FHR2, and FHR5 circulate in plasma as homo- and hetero-oligomeric complexes, the formation of which is likely mediated by the conserved N-terminal domain. In mutant FHR1, duplication of the N-terminal domain resulted in the formation of unusually large multimeric FHR complexes that exhibited increased avidity for the FHR1 ligands C3b, iC3b, and C3dg and enhanced competition with complement factor H (FH) in surface plasmon resonance (SPR) studies and hemolytic assays. These data revealed that FHR1, FHR2, and FHR5 organize a combinatorial repertoire of oligomeric complexes and demonstrated that changes in FHR oligomerization influence the regulation of complement activation. In summary, our identification and characterization of a unique CFHR1 mutation provides insights into the biology of the FHRs and contributes to our understanding of the pathogenic mechanisms underlying C3G. PMID:23728178

  16. Genetic and intervention studies implicating complement C3 as a major target for the treatment of periodontitis1

    PubMed Central

    Maekawa, Tomoki; Abe, Toshiharu; Hajishengallis, Evlambia; Hosur, Kavita B.; DeAngelis, Robert A.; Ricklin, Daniel; Lambris, John D.; Hajishengallis, George

    2014-01-01

    Chronic periodontitis is induced by a dysbiotic microbiota and leads to inflammatory destruction of tooth-supporting connective tissue and bone. The third component of complement, C3, is a point of convergence of distinct complement activation mechanisms but its involvement in periodontitis was not previously addressed. We investigated this question using two animal species models, namely, C3-deficient or wild-type mice and non-human primates (NHP) locally treated with a potent C3 inhibitor (the compstatin analog Cp40) or an inactive peptide control. In mice, C3 was required for maximal periodontal inflammation and bone loss and for the sustenance of the dysbiotic microbiota. The effect of C3 on the microbiota was therefore different from that reported for the C5a receptor, which is required for the initial induction of dysbiosis. C3-dependent bone loss was demonstrated in distinct models, including Porphyromonas gingivalis-induced periodontitis, ligature-induced periodontitis, and aging-associated periodontitis. Importantly, local treatment of NHP with Cp40 inhibited ligature-induced periodontal inflammation and bone loss, which correlated with lower gingival crevicular fluid levels of proinflammatory mediators (e.g., IL-17 and RANKL) and decreased osteoclastogenesis in bone biopsy specimens, as compared to control treatment. This is the first time, for any disease, that complement inhibition in NHP was shown to inhibit inflammatory processes that lead to osteoclastogenesis and bone loss. These data strongly support the feasibility of C3-targeted intervention for the treatment of human periodontitis. PMID:24808362

  17. Secreted Aspergillus fumigatus Protease Alp1 Degrades Human Complement Proteins C3, C4, and C5▿

    PubMed Central

    Behnsen, Judith; Lessing, Franziska; Schindler, Susann; Wartenberg, Dirk; Jacobsen, Ilse D.; Thoen, Marcel; Zipfel, Peter F.; Brakhage, Axel A.

    2010-01-01

    The opportunistic human pathogenic fungus Aspergillus fumigatus is a major cause of fungal infections in immunocompromised patients. Innate immunity plays an important role in the defense against infections. The complement system represents an essential part of the innate immune system. This cascade system is activated on the surface of A. fumigatus conidia and hyphae and enhances phagocytosis of conidia. A. fumigatus conidia but not hyphae bind to their surface host complement regulators factor H, FHL-1, and CFHR1, which control complement activation. Here, we show that A. fumigatus hyphae possess an additional endogenous activity to control complement activation. A. fumigatus culture supernatant efficiently cleaved complement components C3, C4, C5, and C1q as well as immunoglobulin G. Secretome analysis and protease inhibitor studies identified the secreted alkaline protease Alp1, which is present in large amounts in the culture supernatant, as the central molecule responsible for this cleavage. An alp1 deletion strain was generated, and the culture supernatant possessed minimal complement-degrading activity. Moreover, protein extract derived from an Escherichia coli strain overproducing Alp1 cleaved C3b, C4b, and C5. Thus, the protease Alp1 is responsible for the observed cleavage and degrades a broad range of different substrates. In summary, we identified a novel mechanism in A. fumigatus that contributes to evasion from the host complement attack. PMID:20498262

  18. Complement in Experimental Autoimmune Encephalomyelitis Revisited: C3 is Required for Development of Maximal Disease

    PubMed Central

    Szalai, Alexander J.; Hu, Xianzhen; Adams, Jillian E.; Barnum, Scott R.

    2007-01-01

    Complement per se has been shown to play an important role in demyelinating disease but controversy remains regarding the role of C3 in the development and progression of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. In this study we used C3-/- mice to confirm previous findings that C3 is required for full development of EAE. Furthermore, C3+/- mice (with serum C3 levels 50% that of wild type mice) developed EAE with a severity intermediate between wild type and C3-/- mice. Importantly transfer of wild type encephalitogenic T cells to C3-/- mice resulted in attenuated EAE. C3-/- mice with EAE had fewer CD4+ and CD8+ T cells in the CNS and 50% fewer of these cells produced IFN-γ compared to wild type mice. When treated with anti-CD3 antibody, CD4+ T cell from wild type and C3-/- mice had similar activation profiles as judged by IFN-γ production and CD25 and CD69 expression, indicating there is no gross or intrinsic defect in T cells from C3-/- mice. T cells from primed C3-/- mice proliferated comparably to that of control T cells on re-stimulation with MOG peptide. Our results confirm a requirement for C3 for maximal development of EAE and suggest that receptors for C3-derived activation fragments might be a viable therapeutic target for prevention and treatment demyelinating disease. PMID:17353050

  19. Potent complement C3a receptor agonists derived from oxazole amino acids: Structure-activity relationships.

    PubMed

    Singh, Ranee; Reed, Anthony N; Chu, Peifei; Scully, Conor C G; Yau, Mei-Kwan; Suen, Jacky Y; Durek, Thomas; Reid, Robert C; Fairlie, David P

    2015-12-01

    Potent ligands for the human complement C3a receptor (C3aR) were developed from the almost inactive tripeptide Leu-Ala-Arg corresponding to the three C-terminal residues of the endogenous peptide agonist C3a. The analogous Leu-Ser-Arg was modified by condensing the serine side chain with the leucine carbonyl with elimination of water to form leucine-oxazole-arginine. Subsequent elaboration with a variety of N-terminal amide capping groups produced agonists as potent as human C3a itself in stimulating Ca(2+) release from human macrophages. Structure-activity relationships are discussed.

  20. Compstatin: a C3-targeted complement inhibitor reaching its prime for bedside intervention.

    PubMed

    Mastellos, Dimitrios C; Yancopoulou, Despina; Kokkinos, Petros; Huber-Lang, Markus; Hajishengallis, George; Biglarnia, Ali R; Lupu, Florea; Nilsson, Bo; Risitano, Antonio M; Ricklin, Daniel; Lambris, John D

    2015-04-01

    There is a growing awareness that complement plays an integral role in human physiology and disease, transcending its traditional perception as an accessory system for pathogen clearance and opsonic cell killing. As the list of pathologies linked to dysregulated complement activation grows longer, it has become clear that targeted modulation of this innate immune system opens new windows of therapeutic opportunity for anti-inflammatory drug design. Indeed, the introduction of the first complement-targeting drugs has reignited a vibrant interest in the clinical translation of complement-based inhibitors. Compstatin was discovered as a cyclic peptide that inhibits complement activation by binding C3 and interfering with convertase formation and C3 cleavage. As the convergence point of all activation pathways and a molecular hub for crosstalk with multiple pathogenic pathways, C3 represents an attractive target for therapeutic modulation of the complement cascade. A multidisciplinary drug optimization effort encompassing rational 'wet' and in silico synthetic approaches and an array of biophysical, structural and analytical tools has culminated in an impressive structure-function refinement of compstatin, yielding a series of analogues that show promise for a wide spectrum of clinical applications. These new derivatives have improved inhibitory potency and pharmacokinetic profiles and show efficacy in clinically relevant primate models of disease. This review provides an up-to-date survey of the drug design effort placed on the compstatin family of C3 inhibitors, highlighting the most promising drug candidates. It also discusses translational challenges in complement drug discovery and peptide drug development and reviews concerns related to systemic C3 interception.

  1. Polymorphism of C3 complement in association with myocardial infarction in a sample of central Tunisia

    PubMed Central

    2013-01-01

    Background Myocardial infarction (MI) is a major clinical problem because of its large contribution to mortality. The genetic bases of this disease have been widely studied in recent years to find a clear association with some genetic markers that increase the risk of its occurrence. In the present investigation, the correlation between MI and the C3 complement polymorphism was analyzed using a case–control study. Methods Our study ported on one hundred seventy survived myocardial infarction patients and ninety five healthy controls. The C3 allele identification was investigated using the amplification refractory mutation system PCR to determine the C3*S and the C3*F alleles of the C3 polymorphism. Results Frequencies of C3*S and C3*F in patients are 0.59 and 0.41 respectively. Fisher test results showed a significant increase of C3*F allele in the sample of patients (0.41; odds ratio: 2.616; C.I [1.738-3.938]) compared to controls (0.21; odds ratio: 0.382; 95% CI [0.254-0.575]), p = 2.742 × 10-6. Conclusion A strong positive correlation was found between C3 polymorphism and MI estimating that the risk of myocardial infarction is significantly increased among patients with C3*F allele of this polymorphism. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1190484203893646 PMID:23764154

  2. Complement peptide C3a stimulates neural plasticity after experimental brain ischaemia.

    PubMed

    Stokowska, Anna; Atkins, Alison L; Morán, Javier; Pekny, Tulen; Bulmer, Linda; Pascoe, Michaela C; Barnum, Scott R; Wetsel, Rick A; Nilsson, Jonas A; Dragunow, Mike; Pekna, Marcela

    2017-02-01

    Ischaemic stroke induces endogenous repair processes that include proliferation and differentiation of neural stem cells and extensive rewiring of the remaining neural connections, yet about 50% of stroke survivors live with severe long-term disability. There is an unmet need for drug therapies to improve recovery by promoting brain plasticity in the subacute to chronic phase after ischaemic stroke. We previously showed that complement-derived peptide C3a regulates neural progenitor cell migration and differentiation in vitro and that C3a receptor signalling stimulates neurogenesis in unchallenged adult mice. To determine the role of C3a-C3a receptor signalling in ischaemia-induced neural plasticity, we subjected C3a receptor-deficient mice, GFAP-C3a transgenic mice expressing biologically active C3a in the central nervous system, and their respective wild-type controls to photothrombotic stroke. We found that C3a overexpression increased, whereas C3a receptor deficiency decreased post-stroke expression of GAP43 (P < 0.01), a marker of axonal sprouting and plasticity, in the peri-infarct cortex. To verify the translational potential of these findings, we used a pharmacological approach. Daily intranasal treatment of wild-type mice with C3a beginning 7 days after stroke induction robustly increased synaptic density (P < 0.01) and expression of GAP43 in peri-infarct cortex (P < 0.05). Importantly, the C3a treatment led to faster and more complete recovery of forepaw motor function (P < 0.05). We conclude that C3a-C3a receptor signalling stimulates post-ischaemic neural plasticity and intranasal treatment with C3a receptor agonists is an attractive approach to improve functional recovery after ischaemic brain injury.

  3. Complement C3 is expressed by mast cells in cutaneous vasculitis and is degraded by chymase.

    PubMed

    Lipitsä, Tiina; Naukkarinen, Anita; Laitala, Joel; Harvima, Ilkka T

    2016-10-01

    The complement factor C3 and chymase released from tryptase(+), chymase(+) mast cells may be involved in the pathogenesis of cutaneous leukocytoclastic vasculitis. To study whether mast cells contain C3 in vasculitis and whether chymase interacts with C3, cryosections from vasculitis biopsies were double-stained histochemically for C3c in tryptase(+) mast cells, as well as for chymase and vessel wall C3c, or they were treated with 5 µg/ml rh-chymase for 24 h followed by immunofluorescence (IF) analysis of C3c, IgG, IgM and IgA. The effect of rh-chymase on purified human C3, C3a and IgG was studied using SDS-PAGE electrophoresis and LAD2 mast cell cultures. The results show that 34.2 ± 17.9, 37.4 ± 15.5 and 43.4 ± 18.6 % (mean ± SD) of the mast cells express C3c immunoreactivity in the healthy skin, initial petechial (IP) and palpable purpura (PP) lesions, respectively. About 9.4-12.1 % of the chymase(+) mast cells were in apparent contact with C3c(+) vessels in IP and PP. The treatment of cryosections with rh-chymase decreased the IF staining of C3c, but not that of immunoglobulins. In SDS-PAGE, 1-10 µg/ml rh-chymase degraded the alpha- and beta-chains of C3, but did not degrade IgG. Unexpectedly, the rh-chymase treatment of C3 produced fragments that resulted in the release of tryptase and histamine from LAD2 cells. However, rh-chymase degraded C3a and consequently inhibited C3a activity on LAD2. In conclusion, mast cells can be one source for C3 in the early and late phases of vasculitis pathogenesis. However, rh-chymase degraded native C3, vessel wall C3c, and biologically active C3a. Therefore, chymase may control C3-related pathology.

  4. Complement C3 Is the Strongest Predictor of Whole-Body Insulin Sensitivity in Psoriatic Arthritis

    PubMed Central

    D’Angelo, Salvatore; Russo, Emilio; Nicolosi, Kassandra; Gallucci, Antonio; Chiaravalloti, Agostino; Bruno, Caterina; Naty, Saverio; De Sarro, Giovambattista; Olivieri, Ignazio; Grembiale, Rosa Daniela

    2016-01-01

    Objectives To evaluate the correlation between inflammatory measures and whole-body insulin sensitivity in psoriatic arthritis (PsA) patients. Methods For the present study, 40 nondiabetic PsA patients were recruited. A standard oral glucose tolerance test (OGTT) was performed. The insulin sensitivity index (ISI), insulinogenic index (IGI) and oral disposition index (ODI) were calculated from dynamic values of glucose and insulin obtained during OGTT. Results In our study population, mean ISI was 3.5 ± 2.5, median IGI was 1.2 (0.7–1.8), mean ODI 4.5 ± 4.5. In univariate correlation analysis, ISI correlated inversely with systolic blood pressure (sBP) (R = -0.52, p = 0.001), diastolic blood pressure (dBP) (R = -0.45, p = 0.004) and complement C3 (R = -0.43, p = 0.006) and ODI correlated inversely with sBP (R = -0.38, p = 0.02), dBP (R = -0.35, p = 0.03) and complement C3 (R = -0.37, p = 0.02). No significant correlations were found between analyzed variables and IGI. In a stepwise multiple regression, only complement C3 entered in the regression equation and accounted for approximately 50% of the variance of ISI. Using a receiver operating characteristic (ROC) curve we identified the best cut-off for complement C3 of 1.32 g/L that yielded a sensitivity of 56% and a specificity of 96% for classification of insulin resistant patients. Conclusions In conclusion, our data suggest that serum complement C3 could represent a useful marker of whole-body insulin sensitivity in PsA patients. PMID:27656896

  5. Properdin binding to complement activating surfaces depends on initial C3b deposition

    PubMed Central

    Harboe, Morten; Johnson, Christina; Nymo, Stig; Ekholt, Karin; Schjalm, Camilla; Lindstad, Julie K.; Pharo, Anne; Hellerud, Bernt Christian; Nilsson Ekdahl, Kristina; Mollnes, Tom Eirik

    2017-01-01

    Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions. PMID:28069958

  6. Elevated serum complement C3 levels are associated with prehypertension in an adult population.

    PubMed

    Bao, Xue; Meng, Ge; Zhang, Qing; Liu, Li; Wu, Hongmei; Du, Huanmin; Shi, Hongbin; Xia, Yang; Guo, Xiaoyan; Liu, Xing; Han, Peipei; Dong, Renwei; Wang, Xiuyang; Li, Chunlei; Su, Qian; Gu, Yeqing; Fang, Liyun; Yu, Fei; Yang, Huijun; Kang, Li; Ma, Yixuan; Yu, Bin; Ma, Xinyu; Sun, Shaomei; Wang, Xing; Zhou, Ming; Jia, Qiyu; Guo, Qi; Song, Kun; Wang, GuoLin; Huang, Guowei; Niu, Kaijun

    2017-01-01

    Prehypertension is a public health epidemic associated with various adverse outcomes, but can be reversed by timely intervention. However, little attention has been paid to prehypertension. Complement C3 is a central hub of complement-related immune system. We examined the association between C3 and prehypertension in an adult population for the first time, aiming to investigate whether pro-inflammatory immune response is involved in the prehypertensive state. About 7820 Tianjin residents without hypertension were categorized into sex-specific quintiles based on their serum concentration of complement C3. Adjusted logistic regression models were used separately by gender to assess the association between C3 quintiles and the prevalence of prehypertension. After multiple adjustment, the odds ratios (95% confidence interval) for prehypertension across increasing quintiles of C3 were 1.00 (reference), 1.02 (0.84, 1.25), 1.15 (0.94, 1.42), 1.25 (1.01, 1.54), and 1.71 (1.35, 2.17) (p for trend < 0.0001) among men and were 1.00 (reference), 1.17 (0.92, 1.49), 1.13 (0.88, 1.44), 1.15 (0.89, 1.48), and 1.40 (1.07, 1.84) (p for trend = 0.03) among women. The findings suggested that elevated serum C3 levels are associated with prehypertension. Reducing inflammation may be a potential therapeutic strategy for prehypertension and hypertension that is worthy of further studies and discussion.

  7. A revised mechanism for the activation of complement C3 to C3b: a molecular explanation of a disease-associated polymorphism.

    PubMed

    Rodriguez, Elizabeth; Nan, Ruodan; Li, Keying; Gor, Jayesh; Perkins, Stephen J

    2015-01-23

    The solution structure of complement C3b is crucial for the understanding of complement activation and regulation. C3b is generated by the removal of C3a from C3. Hydrolysis of the C3 thioester produces C3u, an analog of C3b. C3b cleavage results in C3c and C3d (thioester-containing domain; TED). To resolve functional questions in relation to C3b and C3u, analytical ultracentrifugation and x-ray and neutron scattering studies were used with C3, C3b, C3u, C3c, and C3d, using the wild-type allotype with Arg(102). In 50 mm NaCl buffer, atomistic scattering modeling showed that both C3b and C3u adopted a compact structure, similar to the C3b crystal structure in which its TED and macroglobulin 1 (MG1) domains were connected through the Arg(102)-Glu(1032) salt bridge. In physiological 137 mm NaCl, scattering modeling showed that C3b and C3u were both extended in structure, with the TED and MG1 domains now separated by up to 6 nm. The importance of the Arg(102)-Glu(1032) salt bridge was determined using surface plasmon resonance to monitor the binding of wild-type C3d(E1032) and mutant C3d(A1032) to immobilized C3c. The mutant did not bind, whereas the wild-type form did. The high conformational variability of TED in C3b in physiological buffer showed that C3b is more reactive than previously thought. Because the Arg(102)-Glu(1032) salt bridge is essential for the C3b-Factor H complex during the regulatory control of C3b, the known clinical associations of the major C3S (Arg(102)) and disease-linked C3F (Gly(102)) allotypes of C3b were experimentally explained for the first time.

  8. Serum immunoglobulin, complement C3, and salivary IgA levels in lead workers

    SciTech Connect

    Ewers, U.; Stiller-Winkler, R.; Idel, H.

    1982-12-01

    Sera of 72 lead workers and of 53 reference subjects were examined for levels of immunoglobulins IgM, IgG, and IgA, and complement C3 by radial immunodiffusion. Salivary IgA levels were determined in 33 lead workers and 40 reference subjects. On the average the lead workers had lower serum complement C3 and immunoglobulin levels, as well as lower salivary IgA levels, than the reference subjects. A significant negative correlation was found between blood lead concentrations (PbB) and the serum levels of complement C3 and IgG in the group of lead workers, as well as in the total population examined. However, a significant positive correlation was observed between PbB and serum IgA in the group of lead workers. The results obtained in this study are discussed in relation to numerous reports in the literature showing that lead exerts adverse effects on the immune system in animals.

  9. Functional analysis and quantification of the complement C3 derived anaphylatoxin C3a with a monoclonal antibody.

    PubMed Central

    Burger, R; Bader, A; Kirschfink, M; Rother, U; Schrod, L; Wörner, I; Zilow, G

    1987-01-01

    The C3 fragment C3a belongs to the anaphylatoxins. It has immune regulatory activity and contributes to the pathogenesis of the adult respiratory distress syndrome (ARDS). The low molecular weight (9 kD) of C3a complicates the production of antibodies to C3a. We obtained a monoclonal antibody (designated H13) to human C3a. It reacts with C3a or C3a-desArg and with native C3 but not with C5 or C5a. In immunoblot analysis it reacts with the alpha- but not with beta-chain of C3 and binds to a protein with a mol. wt of about 10 kD present in zymosan-activated sera which is only marginally detectable in nonactivated serum and absent in plasma. H13 crossreacts with the analogous proteins of rabbit, guinea pig and sheep. H13 has the capacity to bind 125I-radiolabelled C3a efficiently but fails totally to react with 125I-C5a or with other C3 alpha-chain fragments. H13 blocks C3a functional activity. It markedly inhibits C3a-induced 3H-serotonin release from platelets in vitro and similarly inhibits the C3a-induced extravasation of Evans blue into the skin in vivo. H13 does not interfere with the haemolytic activity of C3. An ELISA system was established using H13 which permits quantification of C3a in sera of polytrauma patients. The antibody H13 should facilitate further functional analysis of C3a in experimental systems. It should be useful for quantification of C3a in diagnostic assays and also for application in immunopathology. Images Fig. 3 PMID:3498585

  10. Liver ubiquitome uncovers nutrient-stress-mediated trafficking and secretion of complement C3

    PubMed Central

    Magliarelli, Helena de Fatima; Matondo, Mariette; Mészáros, Gergő; Goginashvili, Alexander; Erbs, Eric; Zhang, Zhirong; Mihlan, Michael; Wolfrum, Christian; Aebersold, Ruedi; Sumara, Izabela; Ricci, Romeo

    2016-01-01

    Adaptation to changes in nutrient availability is crucial for cells and organisms. Posttranslational modifications of signaling proteins are very dynamic and are therefore key to promptly respond to nutrient deprivation or overload. Herein we screened for ubiquitylation of proteins in the livers of fasted and refed mice using a comprehensive systemic proteomic approach. Among 1641 identified proteins, 117 were differentially ubiquitylated upon fasting or refeeding. Endoplasmic reticulum (ER) and secretory proteins were enriched in the livers of refed mice in part owing to an ER-stress-mediated response engaging retro-translocation and ubiquitylation of proteins from the ER. Complement C3, an innate immune factor, emerged as the most prominent ER-related hit of our screen. Accordingly, we found that secretion of C3 from the liver and primary hepatocytes as well as its dynamic trafficking are nutrient dependent. Finally, obese mice with a chronic nutrient overload show constitutive trafficking of C3 in the livers despite acute changes in nutrition, which goes in line with increased C3 levels and low-grade inflammation reported for obese patients. Our study thus suggests that nutrient sensing in the liver is coupled to release of C3 and potentially its metabolic and inflammatory functions. PMID:27735945

  11. Complement C3a enhances CXCL12 (SDF-1)-mediated chemotaxis of bone marrow hematopoietic cells independently of C3a receptor.

    PubMed

    Honczarenko, Marek; Ratajczak, Mariusz Z; Nicholson-Weller, Anne; Silberstein, Leslie E

    2005-09-15

    Complement C3a promotes CXCL12-induced migration and engraftment of human and murine hemopoietic progenitor cells, suggesting a cross-influence between anaphylatoxin and chemokine axes. Here we have explored the underlying mechanism(s) of complement anaphylatoxin and chemokine cooperation. In addition to C3a, C3a-desArg and C4a but not C5a, are potent enhancers of CXCL12-induced chemotaxis of human and murine bone marrow (BM) stem/progenitor cells and B lineage cells. C3a enhancement of chemotaxis is chemokine specific because it is also observed for chemotaxis to CCL19 but not to CXCL13. The potentiating effect of C3a on CXCL12 is independent of the classical C3a receptor (C3aR). First, human BM CD34(+) and B lineage cells do not express C3aR by flow cytometry. Second, the competitive C3aR inhibitor SB290157 does not affect C3a-mediated enhancement of CXCL12-induced chemotaxis. Third, enhancement of chemotaxis of hemopoietic cells is also mediated by C3a-desArg, which does not bind to C3aR. Finally, C3a enhances CXCL12-induced chemotaxis of BM cells from C3aR knockout mice similar to BM cells from wild-type mice. Subsequent studies revealed that C3a increased the binding affinity of CXCL12 to human CXCR4(+)/C3aR(-), REH pro-B cells, which is compatible with a direct interaction between C3a and CXCL12. BM stromal cells were able to generate C3a, C3a-desArg, C4a, as well as CXCL12, suggesting that this pathway could function in vivo. Taken together, we demonstrate a C3a-CXCL12 interaction independent of the C3aR, which may provide a mechanism to modulate the function of CXCL12 in the BM microenvironment.

  12. The Effect of Lutein Supplementation on Blood Plasma Levels of Complement Factor D, C5a and C3d

    PubMed Central

    Tian, Yuan; Kijlstra, Aize; van der Veen, Rob L. P.; Makridaki, Maria; Murray, Ian J.; Berendschot, Tos T. J. M.

    2013-01-01

    Lutein is selectively taken up by the primate retina and plays an important role as a filter for harmful blue light and as an antioxidant. Recent studies have shown that lutein has systemic anti-inflammatory properties. Dietary lutein has been associated with reduced circulating levels of inflammatory biomarkers such as CRP and sICAM. Whether lutein also affects activation of the complement system has not yet been addressed and was the purpose of the study described here. Seventy-two subjects with signs of early macular degeneration were randomly assigned to receive either a 10 mg lutein supplement or a placebo during one year. EDTA blood samples were collected at 0, 4, 8 and 12 months. Complement factor D (CFD), a rate limiting component of the alternative pathway of complement activation and the complement activation products C5a and C3d were determined in the plasma samples by ELISA. A significant 0.11 µg/ml monthly decrease in plasma CFD concentration was observed in the lutein group (p<0.001), resulting in a 51% decrease from 2.3 µg/ml at baseline to 1.0 µg/ml at 12 months. The C5a concentration showed a significant 0.063ng/ml monthly decrease in the lutein group (p<0.001) resulting in a 36% decrease from 2.2ng/ml at baseline to 1.6ng/ml at 12 months. The C3d concentration showed a significant 0.19µg/ml monthly decrease in the lutein group (p=0.004) that gave rise to a 9% decrease from 15.4µg/ml at baseline to 14.4µg/ml at 12 months. In the placebo group we found a significant 0.04 µg/ml monthly decrease in plasma CFD concentration, whereas no changes were observed for C5a and C3d. Lutein supplementation markedly decreases circulating levels of the complement factors CFD, C5a and C3d levels, which might allow a simple method to control this inflammatory pathway of the innate immune system. PMID:24009749

  13. Molecular dynamics simulations of wild type and mutants of human complement receptor 2 complexed with C3d.

    PubMed

    Wan, Hua; Hu, Jian-ping; Tian, Xu-hong; Chang, Shan

    2013-01-28

    The interaction between human complement receptor type 2 (CR2) and antigen-bound C3d can bridge the innate and adaptive immune systems. The recently determined structure of the CR2(SCR1-2):C3d complex has revealed the expected binding interface of CR2-C3d. In this article, wild type (WT) and three mutants of the new structure are studied by molecular dynamics (MD) simulations. The differently decreased structural stabilities of the mutants relative to WT are shown to be consistent with the experimental data, which can be explained by the different hydrogen bond patterns at the interfaces. It is also found that two clusters of residues (D36/E37/E39 and E160/D163/E166) in the acidic pocket of C3d are important for CR2-C3d interactions, which is in good agreement with previous mutagenesis study. In addition, functional dynamics and the conformational change of CR2 are explored by using domain cross-correlation map (DCCM), principal component analysis (PCA), and free energy landscape (FEL) methods. The conformational change mainly corresponds to the opening of a V-shaped structure of CR2, which is consistent with the previously reported high interdomain flexibility of CR2. We further suppose that the opening of a V-shaped structure of CR2 may favor the binding stability of CR2(SCR1-2):C3d. This study would provide some new insights into the understanding of the CR2-C3d interaction mechanism.

  14. Structure of Complement C3(H2O) Revealed By Quantitative Cross-Linking/Mass Spectrometry And Modeling*

    PubMed Central

    Pellarin, Riccardo; Sali, Andrej; Barlow, Paul N.

    2016-01-01

    The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation. PMID:27250206

  15. Immunofluorescence staining for the detection of immunoglobulins and complement (C3) in dogs with renal disease.

    PubMed

    Aresu, L; Pregel, P; Bollo, E; Palmerini, D; Sereno, A; Valenza, F

    2008-12-06

    Renal cortical biopsies from 74 dogs with different degrees of renal failure were studied by immunofluorescence to assess the frequency and extent of the deposition of immunoglobulins G, M and A (IgG, IgM, IgA) and complement C3. The dogs were divided into two groups on the basis of their clinical signs, and standard histological and electron microscopical examinations, according to whether their disease was an immune-mediated nephropathy (IMN) or a non-immune-mediated nephropathy (NIMN). In the dogs with an imn there was strong immunofluorescence due to IgG in the mesangium and the glomerular basement membrane and to IgM in the mesangium. The mechanism of immune complex trapping in the glomerulus also resulted in positive reactions to IgM in the dogs with an NIMN.

  16. Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease

    PubMed Central

    Seignez, Antoine; Joly, Anne-Laure; Chaumonnot, Killian; Hazoumé, Adonis; Sanka, Michel; Boudesco, Christophe; Hammann, Arlette; Seigneuric, Renaud; Jégo, Gaetan; Ducoroy, Patrick; Delarue, Patrice; Senet, Patrick; Castilla-Llorente, Cristina; Solary, Eric; Durey, Marie-Agnès; Rubio, Marie-Thérèse; Hermine, Olivier; Kohli, Evelyne

    2017-01-01

    Better identification of severe acute graft-versus-host disease (GvHD) may improve the outcome of this life-threatening complication of allogeneic hematopoietic stem cell transplantation. GvHD induces tissue damage and the release of damage-associated molecular pattern (DAMP) molecules. Here, we analyzed GvHD patients (n = 39) to show that serum heat shock protein glycoprotein 96 (Gp96) could be such a DAMP molecule. We demonstrate that serum Gp96 increases in gastrointestinal GvHD patients and its level correlates with disease severity. An increase in Gp96 serum level was also observed in a mouse model of acute GvHD. This model was used to identify complement C3 as a main partner of Gp96 in the serum. Our biolayer interferometry, yeast two-hybrid and in silico modeling data allowed us to determine that Gp96 binds to a complement C3 fragment encompassing amino acids 749–954, a functional complement C3 hot spot important for binding of different regulators. Accordingly, in vitro experiments with purified proteins demonstrate that Gp96 downregulates several complement C3 functions. Finally, experimental induction of GvHD in complement C3–deficient mice confirms the link between Gp96 and complement C3 in the serum and with the severity of the disease. PMID:28352659

  17. Serum Gp96 is a chaperone of complement-C3 during graft-versus-host disease.

    PubMed

    Seignez, Antoine; Joly, Anne-Laure; Chaumonnot, Killian; Hazoumé, Adonis; Sanka, Michel; Marcion, Guillaume; Boudesco, Christophe; Hammann, Arlette; Seigneuric, Renaud; Jégo, Gaetan; Ducoroy, Patrick; Delarue, Patrice; Senet, Patrick; Castilla-Llorente, Cristina; Solary, Eric; Durey, Marie-Agnès; Rubio, Marie-Thérèse; Hermine, Olivier; Kohli, Evelyne; Garrido, Carmen

    2017-03-23

    Better identification of severe acute graft-versus-host disease (GvHD) may improve the outcome of this life-threatening complication of allogeneic hematopoietic stem cell transplantation. GvHD induces tissue damage and the release of damage-associated molecular pattern (DAMP) molecules. Here, we analyzed GvHD patients (n = 39) to show that serum heat shock protein glycoprotein 96 (Gp96) could be such a DAMP molecule. We demonstrate that serum Gp96 increases in gastrointestinal GvHD patients and its level correlates with disease severity. An increase in Gp96 serum level was also observed in a mouse model of acute GvHD. This model was used to identify complement C3 as a main partner of Gp96 in the serum. Our biolayer interferometry, yeast two-hybrid and in silico modeling data allowed us to determine that Gp96 binds to a complement C3 fragment encompassing amino acids 749-954, a functional complement C3 hot spot important for binding of different regulators. Accordingly, in vitro experiments with purified proteins demonstrate that Gp96 downregulates several complement C3 functions. Finally, experimental induction of GvHD in complement C3-deficient mice confirms the link between Gp96 and complement C3 in the serum and with the severity of the disease.

  18. Activation of the endothelium by IL-1 alpha and glucocorticoids results in major increase of complement C3 and factor B production and generation of C3a.

    PubMed Central

    Coulpier, M; Andreev, S; Lemercier, C; Dauchel, H; Lees, O; Fontaine, M; Ripoche, J

    1995-01-01

    Constitutive secretion of complement C3 and factor B by the endothelial cell (EC) is lowered by therapeutic concentrations of glucocorticoids such as hydrocortisone or dexamethasone, whereas regulatory protein factor H production is increased by these hormones. In contrast, the proinflammatory cytokine IL-1 alpha has a stimulatory effect on C3 and factor B secretion by the endothelium and an inhibitory effect on factor H secretion. In this study, we examined the combined effect of IL-1 alpha and glucocorticoids on C3 and factor B expression by the endothelial cell. When dexamethasone or hydrocortisone were added to IL-1 alpha, significant potentialization of IL-1 alpha-induced stimulation of C3 and factor B production was observed, occurring at various concentrations of either stimuli. Dose-response experiments indicate that, in vitro, optimal concentrations are in the range of 10(-7) to 10(-5) M for dexamethasone and 50-200 U for IL-1 alpha. In contrast, dexamethasone counteracts, in an additive way, the inhibitory effect of IL-1 alpha on regulatory complement protein factor H production by EC. Such a potentialization between glucocorticoids and IL-1 alpha was not observed for another marker of endothelial activation, IL-1 alpha-induced stimulation of coagulation tissue factor expression. The association of glucocorticoids and IL-1 alpha therefore appears to be a specific and major stimulus for the secretion of complement C3 and factor B, two acute-phase proteins, by the endothelium. As a result of the in vitro endothelium stimulation by glucocorticoids and IL-1 alpha, C3a is generated in the vicinity of the endothelial cell. This study further suggests that complement activation, with its deleterious consequences, may result from the stimulation of endothelium in situations where high levels of IL-1 alpha and endogenous glucocorticoids coexist, such as in septic shock. Images Fig. 4 Fig. 6 PMID:7621583

  19. Complement component 5 promotes lethal thrombosis

    PubMed Central

    Mizuno, Tomohiro; Yoshioka, Kengo; Mizuno, Masashi; Shimizu, Mie; Nagano, Fumihiko; Okuda, Tomoyuki; Tsuboi, Naotake; Maruyama, Shoichi; Nagamatsu, Tadashi; Imai, Masaki

    2017-01-01

    Extracellular histones promote platelet aggregation and thrombosis; this is followed by induction of coagulation disorder, which results in exhaustion of coagulation factors. Complement component 5 (C5) is known to be associated with platelet aggregation and coagulation system activation. To date, the pathological mechanism underlying liver injury has remained unclear. Here, we investigated whether C5 promotes liver injury associated with histone-induced lethal thrombosis. C5-sufficient and C5-deficient mice received single tail vein injections of purified, unfractionated histones obtained from calf thymus (45–75 μg/g). Subsequently, the mice were monitored for survival for up to 72 h. Based on the survival data, the 45 μg/g dose was used for analysis of blood cell count, liver function, blood coagulation ability, and promotion of platelet aggregation and platelet/leukocyte aggregate (PLA) production by extracellular histones. C5-deficient mice were protected from lethal thrombosis and had milder thrombocytopenia, consumptive coagulopathy, and liver injury with embolism and lower PLA production than C5-sufficient mice. These results indicate that C5 is associated with coagulation disorders, PLA production, and embolism-induced liver injury. In conclusion, C5 promotes liver injury associated with histone-induced lethal thrombosis. PMID:28205538

  20. Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation

    PubMed Central

    Martínez-Barricarte, Rubén; Heurich, Meike; Valdes-Cañedo, Francisco; Vazquez-Martul, Eduardo; Torreira, Eva; Montes, Tamara; Tortajada, Agustín; Pinto, Sheila; Lopez-Trascasa, Margarita; Morgan, B. Paul; Llorca, Oscar; Harris, Claire L.; Rodríguez de Córdoba, Santiago

    2010-01-01

    Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C3923ΔDG, which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H2O) by spontaneous thioester hydrolysis, C3923ΔDG generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C3b923ΔDG and C3(H2O)923ΔDG were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase–restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments. PMID:20852386

  1. Delineation of the complement receptor type 2-C3d complex by site-directed mutagenesis and molecular docking.

    PubMed

    Shaw, Craig D; Storek, Michael J; Young, Kendra A; Kovacs, James M; Thurman, Joshua M; Holers, V Michael; Hannan, Jonathan P

    2010-12-10

    The interactions between the complement receptor type 2 (CR2) and the C3 complement fragments C3d, C3dg, and iC3b are essential for the initiation of a normal immune response. A crystal-derived structure of the two N-terminal short consensus repeat (SCR1-2) domains of CR2 in complex with C3d has previously been elucidated. However, a number of biochemical and biophysical studies targeting both CR2 and C3d appear to be in conflict with these structural data. Previous mutagenesis and heteronuclear NMR spectroscopy studies directed toward the C3d-binding site on CR2 have indicated that the CR2-C3d cocrystal structure may represent an encounter/intermediate or nonphysiological complex. With regard to the CR2-binding site on C3d, mutagenesis studies by Isenman and coworkers [Isenman, D. E., Leung, E., Mackay, J. D., Bagby, S. & van den Elsen, J. M. H. (2010). Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. J. Immunol. 184, 1946-1955] have implicated an electronegative "concave" surface on C3d in the binding process. This surface is discrete from the CR2-C3d interface identified in the crystal structure. We generated a total of 18 mutations targeting the two (X-ray crystallographic- and mutagenesis-based) proposed CR2 SCR1-2 binding sites on C3d. Using ELISA analyses, we were able to assess binding of mutant forms of C3d to CR2. Mutations directed toward the concave surface of C3d result in substantially compromised CR2 binding. By contrast, targeting the CR2-C3d interface identified in the cocrystal structure and the surrounding area results in significantly lower levels of disruption in binding. Molecular modeling approaches used to investigate disparities between the biochemical data and the X-ray structure of the CR2-C3d cocrystal result in highest-scoring solutions in which CR2 SCR1-2 is

  2. Complement-activated oligodendroglia: a new pathogenic entity identified by immunostaining with antibodies to human complement proteins C3d and C4d.

    PubMed

    Yamada, T; Akiyama, H; McGeer, P L

    1990-05-04

    Clusters of oligodendroglial fibers were identified immunohistochemically in human brain tissue with antibodies to the complement proteins C3d and C4d in several neurological disorders. These included Pick's, Huntington's, Parkinson's and Alzheimer's disease, amyotrophic lateral sclerosis, progressive supranuclear palsy and Shy-Drager syndrome. These complement-activated oligodendroglia occurred in selected areas of gray and white matter. They were rarely observed in control tissue. Immunogold electron microscopy established that the C4d antibody was attached to degenerating myelin sheaths. These data indicate attachment of classical complement pathway proteins to selective oligodendroglia in several neurological disorders.

  3. Genetics Home Reference: complement component 2 deficiency

    MedlinePlus

    ... Page Jönsson G, Sjöholm AG, Truedsson L, Bengtsson AA, Braconier JH, Sturfelt G. Rheumatological manifestations, organ damage ... 31. Review. Citation on PubMed Truedsson L, Bengtsson AA, Sturfelt G. Complement deficiencies and systemic lupus erythematosus. ...

  4. Mutational analysis of the complement receptor type 2 (CR2/CD21)-C3d interaction reveals a putative charged SCR1 binding site for C3d.

    PubMed

    Hannan, Jonathan P; Young, Kendra A; Guthridge, Joel M; Asokan, Rengasamy; Szakonyi, Gerda; Chen, Xiaojiang S; Holers, V Michael

    2005-02-25

    We have characterized the interaction between the first two short consensus repeats (SCR1-2) of complement receptor type 2 (CR2, CD21) and C3d in solution, by utilising the available crystal structures of free and C3d-bound forms of CR2 to create a series of informative mutations targeting specific areas of the CR2-C3d complex. Wild-type and mutant forms of CR2 were expressed on the surface of K562 erythroleukemia cells and their binding ability assessed using C3dg-biotin tetramers complexed to fluorochrome conjugated streptavidin and measured by flow cytometry. Mutations directed at the SCR2-C3d interface (R83A, R83E, G84Y) were found to strongly disrupt C3dg binding, supporting the conclusion that the SCR2 interface reflected in the crystal structure is correct. Previous epitope and peptide mapping studies have also indicated that the PILN11GR13IS sequence of the first inter-cysteine region of SCR1 is essential for the binding of iC3b. Mutations targeting residues within or in close spatial proximity to this area (N11A, N11E, R13A, R13E, Y16A, S32A, S32E), and a number of other positively charged residues located primarily on a contiguous face of SCR1 (R28A, R28E, R36A, R36E, K41A, K41E, K50A, K50E, K57A, K57E, K67A, K67E), have allowed us to reassess those regions on SCR1 that are essential for CR2-C3d binding. The nature of this interaction and the possibility of a direct SCR1-C3d association are discussed extensively. Finally, a D52N mutant was constructed introducing an N-glycosylation sequence at an area central to the CR2 dimer interface. This mutation was designed to disrupt the CR2-C3d interaction, either directly through steric inhibition, or indirectly through disruption of a physiological dimer. However, no difference in C3dg binding relative to wild-type CR2 could be observed for this mutant, suggesting that the dimer may only be found in the crystal form of CR2.

  5. Differential effects of complement activation products c3a and c5a on cardiovascular function in hypertensive pregnant rats.

    PubMed

    Lillegard, Kathryn E; Loeks-Johnson, Alex C; Opacich, Jonathan W; Peterson, Jenna M; Bauer, Ashley J; Elmquist, Barbara J; Regal, Ronald R; Gilbert, Jeffrey S; Regal, Jean F

    2014-11-01

    Early-onset pre-eclampsia is characterized by decreased placental perfusion, new-onset hypertension, angiogenic imbalance, and endothelial dysfunction associated with excessive activation of the innate immune complement system. Although our previous studies demonstrated that inhibition of complement activation attenuates placental ischemia-induced hypertension using the rat reduced uterine perfusion pressure (RUPP) model, the important product(s) of complement activation has yet to be identified. We hypothesized that antagonism of receptors for complement activation products C3a and C5a would improve vascular function and attenuate RUPP hypertension. On gestational day (GD) 14, rats underwent sham surgery or vascular clip placement on ovarian arteries and abdominal aorta (RUPP). Rats were treated once daily with the C5a receptor antagonist (C5aRA), PMX51 (acetyl-F-[Orn-P-(D-Cha)-WR]), the C3a receptor antagonist (C3aRA), SB290157 (N(2)-[(2,2-diphenylethoxy)acetyl]-l-arginine), or vehicle from GD 14-18. Both the C3aRA and C5aRA attenuated placental ischemia-induced hypertension without affecting the decreased fetal weight or decreased concentration of free circulating vascular endothelial growth factor (VEGF) also present in this model. The C5aRA, but not the C3aRA, attenuated placental ischemia-induced increase in heart rate and impaired endothelial-dependent relaxation. The C3aRA abrogated the acute pressor response to C3a peptide injection, but it also unexpectedly attenuated the placental ischemia-induced increase in C3a, suggesting nonreceptor-mediated effects. Overall, these results indicate that both C3a and C5a are important products of complement activation that mediate the hypertension regardless of the reduction in free plasma VEGF. The mechanism by which C3a contributes to placental ischemia-induced hypertension appears to be distinct from that of C5a, and management of pregnancy-induced hypertension is likely to require a broad anti

  6. Electrostatic Contributions Drive the Interaction Between Staphylococcus aureus Protein Efb-C and its Complement Target C3d

    SciTech Connect

    Haspel, N.; Ricklin, D.; Geisbrecht, B.V.; Kavraki, L.E.; Lambris, J.D.

    2008-11-13

    The C3-inhibitory domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb-C) defines a novel three-helix bundle motif that regulates complement activation. Previous crystallographic studies of Efb-C bound to its cognate subdomain of human C3 (C3d) identified Arg-131 and Asn-138 of Efb-C as key residues for its activity. In order to characterize more completely the physical and chemical driving forces behind this important interaction, we employed in this study a combination of structural, biophysical, and computational methods to analyze the interaction of C3d with Efb-C and the single-point mutants R131A and N138A. Our results show that while these mutations do not drastically affect the structure of the Efb-C/C3d recognition complex, they have significant adverse effects on both the thermodynamic and kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties.

  7. Electrostatic contributions drive the interaction between Staphylococcus aureus protein Efb-C and its complement target C3d.

    PubMed

    Haspel, Nurit; Ricklin, Daniel; Geisbrecht, Brian V; Kavraki, Lydia E; Lambris, John D

    2008-11-01

    The C3-inhibitory domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb-C) defines a novel three-helix bundle motif that regulates complement activation. Previous crystallographic studies of Efb-C bound to its cognate subdomain of human C3 (C3d) identified Arg-131 and Asn-138 of Efb-C as key residues for its activity. In order to characterize more completely the physical and chemical driving forces behind this important interaction, we employed in this study a combination of structural, biophysical, and computational methods to analyze the interaction of C3d with Efb-C and the single-point mutants R131A and N138A. Our results show that while these mutations do not drastically affect the structure of the Efb-C/C3d recognition complex, they have significant adverse effects on both the thermodynamic and kinetic profiles of the resulting complexes. We also characterized other key interactions along the Efb-C/C3d binding interface and found an intricate network of salt bridges and hydrogen bonds that anchor Efb-C to C3d, resulting in its potent complement inhibitory properties.

  8. Complement Receptor 2 is increased in cerebrospinal fluid of multiple sclerosis patients and regulates C3 function.

    PubMed

    Lindblom, Rickard P F; Aeinehband, Shahin; Ström, Mikael; Al Nimer, Faiez; Sandholm, Kerstin; Khademi, Mohsen; Nilsson, Bo; Piehl, Fredrik; Ekdahl, Kristina N

    2016-05-01

    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.

  9. Distinct recognition of complement iC3b by integrins αXβ2 and αMβ2.

    PubMed

    Xu, Shutong; Wang, Jianchuan; Wang, Jia-Huai; Springer, Timothy A

    2017-03-28

    Recognition by the leukocyte integrins αXβ2 and αMβ2 of complement iC3b-opsonized targets is essential for effector functions including phagocytosis. The integrin-binding sites on iC3b remain incompletely characterized. Here, we describe negative-stain electron microscopy and biochemical studies of αXβ2 and αMβ2 in complex with iC3b. Despite high homology, the two integrins bind iC3b at multiple distinct sites. αXβ2 uses the αX αI domain to bind iC3b on its C3c moiety at one of two sites: a major site at the interface between macroglobulin (MG) 3 and MG4 domains, and a less frequently used site near the C345C domain. In contrast, αMβ2 uses its αI domain to bind iC3b at the thioester domain and simultaneously interacts through a region near the αM β-propeller and β2 βI domain with a region of the C3c moiety near the C345C domain. Remarkably, there is no overlap between the primary binding site of αXβ2 and the binding site of αMβ2 on iC3b. Distinctive binding sites on iC3b by integrins αXβ2 and αMβ2 may be biologically beneficial for leukocytes to more efficiently capture opsonized pathogens and to avoid subversion by pathogen factors.

  10. Mesenchymal stem cells infected with Mycoplasma arginini secrete complement C3 to regulate immunoglobulin production in B lymphocytes.

    PubMed

    Lee, D-S; Yi, T G; Lee, H-J; Kim, S-N; Park, S; Jeon, M-S; Song, S U

    2014-04-24

    Mesenchymal stem cells (MSCs) have immunomodulatory functions such as the suppression of T and B cells. MSCs suppress immunoglobulin (Ig) production by B cells via cell-cell contact as well as via secretion of soluble factors. Our study showed that the conditioned medium (CM) of MSCs infected with a mycoplasma strain, Mycoplasma arginini, has marked inhibitory effects on Ig production by lipopolysaccharide/interleukin-4-induced B cells compared with mycoplasma-free MSC-CM. We analyzed mycoplasma-infected MSC-CM by fast protein liquid chromatography and liquid chromatography to screen the molecules responsible for Ig inhibition. Complement C3 (C3) was the most critical molecule among the candidates identified. C3 was shown to be involved in the suppression of the Ig production of B cells. C3 was secreted by mycoplasma-infected MSCs, but not by mycoplasma-free MSCs or B cells. It was able to directly inhibit Ig production by B cells. In the presence of a C3 inhibitor, Ig inhibition by MSC-CM was abrogated. This inhibitory effect was concomitant with the downregulation of B-cell-induced maturation protein-1, which is a regulator of the differentiation of antibody-secreting plasma cells. These results suggest that C3 secreted from mycoplasma-infected MSCs has an important role in the immunomodulatory functions of MSCs. However, its role in vivo needs to be explored.

  11. Localization of the expression of complement component 3 in the human endometrium by in situ hybridization

    SciTech Connect

    Sayegh, R.A.; Tao, Xiao Jing; Awwad, J.T.

    1996-04-01

    C3 production by the human endometrium has been previously described. The objective of the current study was to localize the site of expression and regulation of the third component of complement, C3, in the endometrium. Eight secretory and eight proliferative archival endometrial samples from hysterectomy and endometrial biopsy specimens were used for in situ hybridization analysis. This analysis was performed with a radiolabeled riboprobe synthesized from a 736-bp template representing sequence 1944-2680 of the human C3 complementary DNA. Duplicate sections were hybridized with sense and antisense riboprobes. Resultant autoradiograms were analyzed qualitatively by light- and darkfield microscopy. In proliferative endometrium, minimal expression of C3 was observed and was limited to a few stromal patches and glands throughout the section. In the secretory samples, prominent C3 expression was observed in both the glands and stroma of the basalis layer. Endometrial lymphocytes did not express C3. Endometrial stromal and glandular cells express the C3 gene. Endometrial lymphocytes did not express C3, but other nondistinct lymphoid elements scattered in the stroma may be expressing C3. There was a visibly more intense expression of C3 in the basalis layer of the secretory endometrium than in proliferative endometrium. The spatial and temporal pattern of C3 expression may have implications in normal menstrual physiology and in the immunological response of the endometrium to the invading trophoblast during placentation. 23 refs., 4 figs., 1 tab.

  12. Acylation Stimulating Protein, Complement C3 and Lipid Metabolism in Ketosis-Prone Diabetic Subjects

    PubMed Central

    Liu, Yan; Gupta, Priyanka; Lapointe, Marc; Yotsapon, Thewjitcharoen; Sarat, Sunthornyothin; Cianflone, Katherine

    2014-01-01

    Background Ketosis-prone diabetes (KPDM) is new-onset diabetic ketoacidosis without precipitating factors in non-type 1 diabetic patients; after management, some are withdrawn from exogenous insulin, although determining factors remain unclear. Methods Twenty KPDM patients and twelve type 1 diabetic patients (T1DM), evaluated at baseline, 12 and 24 months with/without insulin maintenance underwent a standardized mixed-meal tolerance test (MMTT) for 2 h. Results At baseline, triglyceride and C3 were higher during MMTT in KPDM vs. T1DM (p<0.0001) with no differences in non-esterified fatty acids (NEFA) while Acylation Stimulating Protein (ASP) tended to be higher. Within 12 months, 11 KPDM were withdrawn from insulin treatment (KPDM-ins), while 9 were maintained (KPDM+ins). NEFA was lower in KPDM-ins vs. KPDM+ins at baseline (p = 0.0006), 12 months (p<0.0001) and 24 months (p<0.0001) during MMTT. NEFA in KPDM-ins decreased over 30–120 minutes (p<0.05), but not in KPDM+ins. Overall, C3 was higher in KPDM-ins vs KPDM+ins at 12 months (p = 0.0081) and 24 months (p = 0.0019), while ASP was lower at baseline (p = 0.0024) and 12 months (p = 0.0281), with a decrease in ASP/C3 ratio. Conclusions Notwithstanding greater adiposity in KPDM-ins, greater NEFA decreases and lower ASP levels during MMTT suggest better insulin and ASP sensitivity in these patients. PMID:25275325

  13. [Levels of total hemolytic complement, C3, C4 and antibodies against the myocardium in rheumatic fever].

    PubMed

    Martinez, R D

    1978-01-01

    The levels of the hemolytic complement (UH 50%), C3, C4 and the antibodies against myocardium and against the antigenic fractions of myocardium precipitated with ammonium sulphate were studied in 8 patients with active rehumatic fever (ARF), 28 with inactive rheumatic fever (IRF) and 26 people without cardiopaties (NI). The UH 50% was low in 2 out of 36 patients with rheumatic fever (RF). C3 was normal and C4 low in 12.5% of the ARF patients. C3 had subnormal values in 25% and C4 in 33% of IRF patients, this last value had a stadistic significant decrease with respect to the values of C4 in normal people. The 36 patients with RF had antibodies against the myocardium and also against the heart antigenic fractions precipitated with 10% ammonium sulphate. 11.5% of the normal group had anti-myocardial antibodies and none had antibodies against the fractions. The levels of anti-streptolysin-O and C-reactive protein were higher in the ARF group than in the patients with IRF or the normal people. The participation of the hemolytic complement, the anti-myocardium antibodies, the anti-streptococcus antibodies and the cytophilic activity in the etiopathogeny of rheumatic fever is discussed.

  14. Deficiency of the Complement Component 3 but Not Factor B Aggravates Staphylococcus aureus Septic Arthritis in Mice.

    PubMed

    Na, Manli; Jarneborn, Anders; Ali, Abukar; Welin, Amanda; Magnusson, Malin; Stokowska, Anna; Pekna, Marcela; Jin, Tao

    2016-04-01

    The complement system plays an essential role in the innate immune response and protection against bacterial infections. However, detailed knowledge regarding the role of complement in Staphylococcus aureus septic arthritis is still largely missing. In this study, we elucidated the roles of selected complement proteins in S. aureus septic arthritis. Mice lacking the complement component 3 (C3(-/-)), complement factor B (fB(-/-)), and receptor for C3-derived anaphylatoxin C3a (C3aR(-/-)) and wild-type (WT) control mice were intravenously or intra-articularly inoculated with S. aureus strain Newman. The clinical course of septic arthritis, as well as histopathological and radiological changes in joints, was assessed. After intravenous inoculation, arthritis severity and frequency were significantly higher in C3(-/-)mice than in WT controls, whereas fB(-/-)mice displayed intermediate arthritis severity and frequency. This was in accordance with both histopathological and radiological findings. C3, but not fB, deficiency was associated with greater weight loss, more frequent kidney abscesses, and higher bacterial burden in kidneys. S. aureus opsonized with C3(-/-)sera displayed decreased uptake by mouse peritoneal macrophages compared with bacteria opsonized with WT or fB(-/-)sera. C3aR deficiency had no effect on the course of hematogenous S. aureus septic arthritis. We conclude that C3 deficiency increases susceptibility to hematogenous S. aureus septic arthritis and impairs host bacterial clearance, conceivably due to diminished opsonization and phagocytosis of S. aureus.

  15. Complement C3 on microglial clusters in multiple sclerosis occur in chronic but not acute disease: Implication for disease pathogenesis

    PubMed Central

    Michailidou, Iliana; Naessens, Daphne M. P.; Hametner, Simon; Guldenaar, Willemijn; Kooi, Evert‐Jan; Geurts, Jeroen J. G.; Baas, Frank; Lassmann, Hans

    2016-01-01

    Microglial clusters with C3d deposits are observed in the periplaque of multiple sclerosis (MS) brains and were proposed as early stage of lesion formation. As such they should appear in the brain of MS donors with acute disease but thus far this has not been shown. Using postmortem brain tissue from acute (n = 10) and chronic (n = 15) MS cases we investigated whether C3d+ microglial clusters are part of an acute attack against myelinated axons, which could have implications for disease pathogenesis. The specificity of our findings to MS was tested in ischemic stroke cases (n = 8) with initial or advanced lesions and further analyzed in experimental traumatic brain injury (TBI, n = 26), as both conditions are primarily nondemyelinating but share essential features of neurodegeneration with MS lesions. C3d+ microglial clusters were found in chronic but not acute MS. They were not associated with antibody deposits or terminal complement activation. They were linked to slowly expanding lesions, localized on axons with impaired transport and associated with neuronal C3 production. C3d+ microglial clusters were not specific to MS as they were also found in stroke and experimental TBI. We conclude that C3d+ microglial clusters in MS are not part of an acute attack against myelinated axons. As such it is unlikely that they drive formation of new lesions but could represent a physiological mechanism to remove irreversibly damaged axons in chronic disease. GLIA 2017;65:264–277 PMID:27778395

  16. Complement effectors, C5a and C3a, in cystic fibrosis lung fluid correlate with disease severity

    PubMed Central

    Hair, Pamela S.; Sass, Laura A.; Vazifedan, Turaj; Shah, Tushar A.; Krishna, Neel K.

    2017-01-01

    In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, inflammation and tissue destruction. The complement system is a major mediator of inflammation for many diseases with the effectors C5a and C3a often playing important roles. We have previously shown in a small pilot study that CF sputum soluble fraction concentrations of C5a and C3a were associated with clinical measures of CF disease. Here we report a much larger study of 34 CF subjects providing 169 testable sputum samples allowing longitudinal evaluation comparing C5a and C3a with clinical markers. Levels of the strongly pro-inflammatory C5a correlated negatively with FEV1% predicted (P < 0.001), whereas the often anti-inflammatory C3a correlated positively with FEV1% predicted (P = 0.01). C5a concentrations correlated negatively with BMI percentile (P = 0.017), positively with worsening of an acute pulmonary exacerbation score (P = 0.007) and positively with P. aeruginosa growth in sputum (P = 0.002). C5a levels also correlated positively with concentrations of other sputum markers associated with worse CF lung disease including neutrophil elastase (P < 0.001), myeloperoxidase activity (P = 0.006) and DNA concentration (P < 0.001). In contrast to C5a, C3a levels correlated negatively with worse acute pulmonary exacerbation score and correlated negatively with sputum concentrations of neutrophil elastase, myeloperoxidase activity and DNA concentration. In summary, these data suggest that in CF sputum, increased C5a is associated with increased inflammation and poorer clinical measures, whereas increased C3a appears to be associated with less inflammation and improved clinical measures. PMID:28278205

  17. Glomeruli of Dense Deposit Disease contain components of the alternative and terminal complement pathway

    PubMed Central

    Sethi, Sanjeev; Gamez, Jeffrey D.; Vrana, Julie A.; Theis, Jason D.; Bergen, H. Robert; Zipfel, Peter F.; Dogan, Ahmet; Smith, Richard J. H.

    2009-01-01

    Dense Deposit Disease (DDD), or membranoproliferative glomerulonephritis type II, is a rare renal disease characterized by dense deposits in the mesangium and along the glomerular basement membranes that can be seen by electron microscopy. Although these deposits contain complement factor C3, as determined by immunofluorescence microscopy, their precise composition remains unknown. To address this question, we used mass spectrometry to identify the proteins in laser microdissected glomeruli isolated from paraffin-embedded tissue of eight confirmed cases of DDD. Compared to glomeruli from five control patients, we found that all of the glomeruli from patients with DDD contain components of the alternative pathway and terminal complement complex. Factor C9 was uniformly present as well as the two fluid-phase regulators of terminal complement complex clusterin and vitronectin. In contrast, in nine patients with immune complex–mediated membranoproliferative glomerulonephritis, glomerular samples contained mainly immunoglobulins and complement factors C3 and C4. Our study shows that in addition to fluid-phase dysregulation of the alternative pathway, soluble components of the terminal complement complex contribute to glomerular lesions found in DDD. PMID:19177158

  18. Antibody-mediated complement C3b/iC3b binding to group B Streptococcus in paired mother and baby serum samples in a refugee population on the Thailand-Myanmar border.

    PubMed

    Herbert, Jenny; Thomas, Stephen; Brookes, Charlotte; Turner, Claudia; Turner, Paul; Nosten, Francois; Le Doare, Kirsty; Hudson, Michael; Heath, Paul T; Gorringe, Andrew; Taylor, Stephen

    2015-03-01

    Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal sepsis and meningitis. In this study, we determined antibody-mediated deposition of complement C3b/iC3b onto the bacterial cell surface of GBS serotypes Ia, Ib, II, III, and V. This was determined for 520 mother and umbilical cord serum sample pairs obtained at the time of birth from a population on the Thailand-Myanmar border. Antibody-mediated deposition of complement C3b/iC3b was detected to at least one serotype in 91% of mothers, despite a known carriage rate in this population of only 12%. Antibody-mediated C3b/iC3b deposition corresponded to known carriage rates, with the highest levels of complement deposition observed onto the most prevalent serotype (serotype II) followed by serotypes Ia, III, V, and Ib. Finally, neonates born to mothers carrying serotype II GBS at the time of birth showed higher antibody-mediated C3b/iC3b deposition against serotype II GBS than neonates born to mothers with no serotype II carriage. Assessment of antibody-mediated C3b/iC3b deposition against GBS may provide insights into the seroepidemiology of anti-GBS antibodies in mothers and infants in different populations.

  19. Antibody-Mediated Complement C3b/iC3b Binding to Group B Streptococcus in Paired Mother and Baby Serum Samples in a Refugee Population on the Thailand-Myanmar Border

    PubMed Central

    Herbert, Jenny; Thomas, Stephen; Brookes, Charlotte; Turner, Claudia; Turner, Paul; Nosten, Francois; Le Doare, Kirsty; Hudson, Michael; Heath, Paul T.; Gorringe, Andrew

    2015-01-01

    Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal sepsis and meningitis. In this study, we determined antibody-mediated deposition of complement C3b/iC3b onto the bacterial cell surface of GBS serotypes Ia, Ib, II, III, and V. This was determined for 520 mother and umbilical cord serum sample pairs obtained at the time of birth from a population on the Thailand-Myanmar border. Antibody-mediated deposition of complement C3b/iC3b was detected to at least one serotype in 91% of mothers, despite a known carriage rate in this population of only 12%. Antibody-mediated C3b/iC3b deposition corresponded to known carriage rates, with the highest levels of complement deposition observed onto the most prevalent serotype (serotype II) followed by serotypes Ia, III, V, and Ib. Finally, neonates born to mothers carrying serotype II GBS at the time of birth showed higher antibody-mediated C3b/iC3b deposition against serotype II GBS than neonates born to mothers with no serotype II carriage. Assessment of antibody-mediated C3b/iC3b deposition against GBS may provide insights into the seroepidemiology of anti-GBS antibodies in mothers and infants in different populations. PMID:25589553

  20. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Complement components immunological test system. 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  1. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Complement components immunological test system. 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  2. Complement activation pathways in murine immune complex-induced arthritis and in C3a and C5a generation in vitro

    PubMed Central

    Banda, N K; Levitt, B; Wood, A K; Takahashi, K; Stahl, G L; Holers, V M; Arend, W P

    2010-01-01

    The alternative pathway (AP) of complement alone is capable of mediating immune complex-induced arthritis in the collagen antibody-induced arthritis (CAIA) model in mice. Whether the classical pathway (CP) or lectin pathway (LP) alone can mediate CAIA is not known. Using mice genetically deficient in different complement components, our results reported herein establish that the CP and LP alone are each incapable of mediating CAIA. A lower level or absence of C3 and/or C5 activation by the CP may be possible explanations for the importance of the AP in CAIA and in many murine models of disease. In addition, other investigators have reported that CP C5 convertase activity is absent in mouse sera. To address these questions, we employed an in vitro system of adherent immunoglobulin (Ig)G-induced complement activation using plates coated with murine anti-collagen monoclonal antibody (mAb). These experiments used complement-deficient mouse sera and wild-type mouse or normal human sera under conditions inactivating either the CP (Ca++ deficiency) or the AP (mAb inhibitory to factor B). Robust generation of both C3a and C5a by either the AP or CP alone were observed with both mouse and human sera, although there were some small differences between the species of sera. We conclude that neither the CP nor LP alone is capable of mediating CAIA in vivo and that mouse sera exhibits a high level of IgG-induced C5a generation in vitro through either the CP or AP. PMID:19843088

  3. Solution structure of the complex between CR2 SCR 1-2 and C3d of human complement: an X-ray scattering and sedimentation modelling study.

    PubMed

    Gilbert, Hannah E; Eaton, Julian T; Hannan, Jonathan P; Holers, V Michael; Perkins, Stephen J

    2005-02-25

    Complement receptor type 2 (CR2, CD21) forms a tight complex with C3d, a fragment of C3, the major complement component. Previous crystal structures of the C3d-CR2 SCR 1-2 complex and free CR2 SCR 1-2 showed that the two SCR domains of CR2 form contact with each other in a closed V-shaped structure. SCR 1 and SCR 2 are connected by an unusually long eight-residue linker peptide. Medium-resolution solution structures for CR2 SCR 1-2, C3d, and their complex were determined by X-ray scattering and analytical ultracentrifugation. CR2 SCR 1-2 is monomeric. For CR2 SCR 1-2, its radius of gyration R(G) of 2.12(+/-0.05) nm, its maximum length of 10nm and its sedimentation coefficient s20,w(o) of 1.40(+/-0.03) S do not agree with those calculated from the crystal structures, and instead suggest an open structure. Computer modelling of the CR2 SCR1-2 solution structure was based on the structural randomisation of the eight-residue linker peptide joining SCR 1 and SCR 2 to give 9950 trial models. Comparisons with the X-ray scattering curve indicated that the most favoured arrangements for the two SCR domains corresponded to an open V-shaped structure with no contacts between the SCR domains. For C3d, X-ray scattering and sedimentation velocity experiments showed that it exists as a monomer-dimer equilibrium with a dissociation constant of 40 microM. The X-ray scattering curve for monomeric C3d gave an R(G) value of 1.95 nm, and this together with its s20,w(o) value of 3.17 S gave good agreement with the monomeric C3d crystal structure. Modelling of the C3d dimer gave good agreements with its scattering and ultracentrifugation parameters. For the complex, scattering and ultracentrifugation experiments showed that there was no dimerisation, indicating that the C3d dimerisation site was located close to the CR2 SCR 1-2 binding site. The R(G) value of 2.44(+/-0.1) nm, its length of 9 nm and its s20,w(o) value of 3.45(+/-0.01) S showed that its structure was not much more

  4. Plasma Complement Components and Activation Fragments: Associations with Age-Related Macular Degeneration Genotypes and Phenotypes

    PubMed Central

    Reynolds, Robyn; Hartnett, M. Elizabeth; Atkinson, John P.; Giclas, Patricia C.; Rosner, Bernard; Seddon, Johanna M.

    2010-01-01

    Purpose Several genes encoding complement system components and fragments are associated with age-related macular degeneration (AMD). This study was conducted to determine whether alterations in circulating levels of these markers of complement activation and regulation are also independently associated with advanced AMD and whether they are related to AMD genotypes. Methods Plasma and DNA samples were selected from individuals in our AMD registry who had progressed to or developed the advanced stages of AMD, including 58 with geographic atrophy and 62 with neovascular disease. Subjects of similar age and sex, but without AMD, and who did not progress were included as controls (n = 60). Plasma complment components (C3, CFB, CFI, CFH, and factor D) and activation fragments (Bb, C3a, C5a, iC3b, and SC5b-9) were analyzed. DNA samples were genotyped for seven single-nucleotide polymorphisms in six genes previously shown to be associated with AMD: CFB, CFH, C2, C3, and CFI and the LOC387715/ARMS2 gene region. The association between AMD and each complement biomarker was assessed by using logistic regression, controlling for age, sex, and proinflammatory risk factors: smoking and body mass index (BMI). Functional genomic analyses were performed to assess the relationship between the complement markers and genotypes. Concordance, or C, statistics were calculated to assess the effect of complement components and activation fragments in an AMD gene-environment prediction model. Results The highest quartiles of Bb and C5a were significantly associated with advanced AMD, when compared with the lowest quartiles. In multivariate models without genetic variants, the odds ratio (OR) for Bb was 3.3 (95% confidence interval [CI] = 1.3-8.6), and the OR for C5a was 3.6 (95% CI = 1.2-10.3). With adjustment for genetic variants, these ORs were substantially higher. The alternative pathway regulator CFH was inversely associated with AMD in the model without genotypes (OR = 0.3; P = 0

  5. Complement component 3 is necessary to preserve myocardium and myocardial function in chronic myocardial infarction.

    PubMed

    Wysoczynski, Marcin; Solanki, Mitesh; Borkowska, Sylwia; van Hoose, Patrick; Brittian, Kenneth R; Prabhu, Sumanth D; Ratajczak, Mariusz Z; Rokosh, Gregg

    2014-09-01

    Activation of the complement cascade (CC) with myocardial infarction (MI) acutely initiates immune cell infiltration, membrane attack complex formation on injured myocytes, and exacerbates myocardial injury. Recent studies implicate the CC in mobilization of stem/progenitor cells and tissue regeneration. Its role in chronic MI is unknown. Here, we consider complement component C3, in the chronic response to MI. C3 knockout (KO) mice were studied after permanent coronary artery ligation. C3 deficiency exacerbated myocardial dysfunction 28 days after MI compared to WT with further impaired systolic function and LV dilation despite similar infarct size 24 hours post-MI. Morphometric analysis 28 days post-MI showed C3 KO mice had more scar tissue with less viable myocardium within the infarct zone which correlated with decreased c-kit(pos) cardiac stem/progenitor cells (CPSC), decreased proliferating Ki67(pos) CSPCs and decreased formation of new BrdU(pos) /α-sarcomeric actin(pos) myocytes, and increased apoptosis compared to WT. Decreased CSPCs and increased apoptosis were evident 7 days post-MI in C3 KO hearts. The inflammatory response with MI was attenuated in the C3 KO and was accompanied by attenuated hematopoietic, pluripotent, and cardiac stem/progenitor cell mobilization into the peripheral blood 72 hours post-MI. These results are the first to demonstrate that CC, through C3, contributes to myocardial preservation and regeneration in response to chronic MI. Responses in the C3 KO infer that C3 activation in response to MI expands the resident CSPC population, increases new myocyte formation, increases and preserves myocardium, inflammatory response, and bone marrow stem/progenitor cell mobilization to preserve myocardial function.

  6. Sites within the complement C3b/C4b receptor important for the specificity of ligand binding.

    PubMed Central

    Krych, M; Hourcade, D; Atkinson, J P

    1991-01-01

    Cysteine-rich repeated units of 40-70 amino acids are building blocks of many mammalian proteins, including 12 proteins of the complement system. Human complement arranged motifs, designated short consensus repeats (SCRs), which constitute the entire extracellular portion of this protein. Klickstein et al. [Klickstein, L. B., Bartow, T. J., Miletic, V., Rabson, L. D., Smith, J. A. & Fearon, D. T. (1988) J. Exp. Med. 168, 1699-1717 (abstr.)] localized a C4b binding domain to SCR-1 and/or SCR-2 and a C3b binding domain to SCR-8 and/or SCR-9. These SCRs bind different ligands, although SCR-1 and SCR-8 are 55% homologous and SCR-2 and SCR-9 are 70% homologous. To examine if one or two SCRs are required for ligand binding and to define sites within the SCRs that determine specificity of binding, mutagenesis analysis of a truncated, secreted form of CR1, called CR1-4 by Hourcade et al. [Hourcade, D., Meisner, D. R., Atkinson, J. P. & Holers, V. M. (1988) J. Exp. Med. 168, 1255-1270], was undertaken. The latter, composed of the first eight and one-half amino-terminal SCRs of CR1, efficiently bound C4b but not iC3. SCR-1 and SCR-2 were necessary for this interaction. Analysis of the mutant CR1-4 proteins, in which amino acids in SCR-1 and SCR-2 were substituted a few at a time with the homologous amino acids of SCR-8 and SCR-9, led to the identification of one amino acid in SCR-1 and three amino acids in SCR-2 important for C4b binding. Furthermore, five amino acids at the end of SCR-9, if placed in the homologous positions of SCR-2, conferred iC3 binding and are likely essential for ligand binding activity of SCR-8 and SCR-9. This iC3 binding occurred only if SCR-1 was present, indicating that two contiguous SCRs are necessary for this interaction. These results provide identification of amino acids within SCRs that are important for ligand binding. Images PMID:1827918

  7. NFκB-activated Astroglial Release of Complement C3 Compromises Neuronal Morphology and Function Associated with Alzheimer’s Disease

    PubMed Central

    Lian, Hong; Yang, Li; Cole, Allysa; Sun, Lu; Chiang, Angie C.-A.; Fowler, Stephanie W.; Shim, David J.; Rodriguez-Rivera, Jennifer; Taglialatela, Giulio; Jankowsky, Joanna L.; Lu, Hui-Chen; Zheng, Hui

    2014-01-01

    SUMMARY Abnormal NFκB activation has been implicated in Alzheimer’s disease (AD). However, the signaling pathways governing NFκB regulation and function in the brain are poorly understood. We identify complement protein C3 as an astroglial target of NFκB, and show that C3 release acts through neuronal C3aR to disrupt dendritic morphology and network function. Exposure to Aβ activates astroglial NFκB and C3 release, consistent with the high levels of C3 expression in brain tissue from AD patients and APP transgenic mice, where C3aR antagonist treatment rescues cognitive impairment. Thus, dysregulation of neuron-glia interaction through NFκB/C3/C3aR signaling may contribute to synaptic dysfunction in AD and C3aR antagonists may be therapeutically beneficial. PMID:25533482

  8. Cigarette smoke can activate the alternative pathway of complement in vitro by modifying the third component of complement.

    PubMed Central

    Kew, R R; Ghebrehiwet, B; Janoff, A

    1985-01-01

    Cigarette smoking is associated with significant increases in the number of pulmonary mononuclear phagocytes and neutrophils. A potent chemoattractant for these cells is C5a, a peptide generated during complement (C) activation. We, therefore, investigated the possibility that cigarette smoke could activate the complement system in vitro. Our results show that factor(s) (mol wt less than 1,000) present in an aqueous solution of whole, unfiltered cigarette smoke can deplete the hemolytic capacity of whole human serum in a dose-dependent manner. The particle-free, filtered gas phase of cigarette smoke is inactive. The smoke factor(s) do not activate serum C1, but do deplete serum C4 activity. Treatment of purified human C3 with whole smoke solution modifies the molecule such that its subsequent addition to serum (containing Mg/EGTA to block the classical pathway) results in consumption of hemolytic complement by activation of the alternative pathway. Smoke-modified C3 shows increased anodal migration in agarose electrophoresis, but this is not due to proteolytic cleavage of the molecule as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to methylamine-treated C3, C3 treated with smoke is only partially susceptible to the action of the complement regulatory proteins Factors H and I. In addition, smoke-modified C3 has diminished binding to Factor H as compared with methylamine-treated C3. Finally, smoke-modified C3 incorporates [14C]methylamine which suggests that the thiolester bond may be intact. These data indicate that aqueous whole cigarette smoke solution can modify C3 and activate the alternative pathway of complement, perhaps by a previously unrecognized mechanism. Should this occur in vivo, complement activation might partly account for the extensive pulmonary leukocyte recruitment observed in smokers. Images PMID:3156879

  9. Schizophrenia risk from complex variation of complement component 4

    PubMed Central

    Sekar, Aswin; Bialas, Allison R.; de Rivera, Heather; Davis, Avery; Hammond, Timothy R.; Kamitaki, Nolan; Tooley, Katherine; Presumey, Jessy; Baum, Matthew; Van Doren, Vanessa; Genovese, Giulio; Rose, Samuel A.; Handsaker, Robert E.; Daly, Mark J.; Carroll, Michael C.; Stevens, Beth; McCarroll, Steven A.

    2016-01-01

    Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia’s strongest genetic association at a population level involves variation in the Major Histocompatibility Complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to recognize. We show here that schizophrenia’s association with the MHC locus arises in substantial part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles promoted widely varying levels of C4A and C4B expression and associated with schizophrenia in proportion to their tendency to promote greater expression of C4A in the brain. Human C4 protein localized at neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals affected with schizophrenia. PMID:26814963

  10. Schizophrenia risk from complex variation of complement component 4.

    PubMed

    Sekar, Aswin; Bialas, Allison R; de Rivera, Heather; Davis, Avery; Hammond, Timothy R; Kamitaki, Nolan; Tooley, Katherine; Presumey, Jessy; Baum, Matthew; Van Doren, Vanessa; Genovese, Giulio; Rose, Samuel A; Handsaker, Robert E; Daly, Mark J; Carroll, Michael C; Stevens, Beth; McCarroll, Steven A

    2016-02-11

    Schizophrenia is a heritable brain illness with unknown pathogenic mechanisms. Schizophrenia's strongest genetic association at a population level involves variation in the major histocompatibility complex (MHC) locus, but the genes and molecular mechanisms accounting for this have been challenging to identify. Here we show that this association arises in part from many structurally diverse alleles of the complement component 4 (C4) genes. We found that these alleles generated widely varying levels of C4A and C4B expression in the brain, with each common C4 allele associating with schizophrenia in proportion to its tendency to generate greater expression of C4A. Human C4 protein localized to neuronal synapses, dendrites, axons, and cell bodies. In mice, C4 mediated synapse elimination during postnatal development. These results implicate excessive complement activity in the development of schizophrenia and may help explain the reduced numbers of synapses in the brains of individuals with schizophrenia.

  11. Levels of complement components, immunoglobulins and acute phase proteins in plasma during aging in Nigeria.

    PubMed

    Oyeyinka, G O; Salimonu, L S

    1999-01-01

    Plasma samples from Nigerians aged 6-95 years were examined for their content of complement components (C3, C4, factor B-Bf), immuloglobins (IgG, IgA, IgM IgD) and acute phase proteins (transferrin, albumin, C-reactive protein--CRP, alpha-2-macroglobulin). Albumin, was estimated colorimetrically and the other components by the single radial immunodiffusion techniques. No significant age-related changes in mean values of the four immunobulins and the four acute phase proteins could be demonstrated. Also, the mean values for C3 and Bf did not change significantly with age but C4 values rose significantly with increasing age (r -0.232: P < 0.01).

  12. Genetic Deficiency of Complement Component 3 Does Not Alter Disease Progression in a Mouse Model of Huntington's Disease.

    PubMed

    Larkin, Paul B; Muchowski, Paul J

    2012-01-01

    Several genes and proteins of the complement cascade are present at elevated levels in brains of patients with Huntington's disease (HD). The complement cascade is well characterized as an effector arm of the immune system, and in the brain it is important for developmental synapse elimination. We hypothesized that increased levels of complement in HD brains contributes to disease progression, perhaps by contributing to synapse elimination or inflammatory signaling. We tested this hypothesis in the R6/2 mouse model of HD by crossing mice deficient in complement component 3 (C3), a crucial complement protein found at increased levels in HD brains, to R6/2 mice and monitoring behavioral and neuropathological disease progression. We found no alterations in multiple behavioral assays, weight or survival in R6/2 mice lacking C3. We also quantified the expression of several complement cascade genes in R6/2 brains and found that the large scale upregulation of complement genes observed in HD brains is not mirrored in R6/2 brains. These data show that C3 deficiency does not alter disease progression in the R6/2 mouse model of HD.

  13. Resistance to Streptozotocin-Induced Autoimmune Diabetes in Absence of Complement C3: Myeloid-Derived Suppressor Cells Play a Role.

    PubMed

    Gao, Xiaogang; Liu, Huanhai; He, Bin; Fu, Zhiren

    2013-01-01

    The contribution of complement to the development of autoimmune diabetes has been proposed recently. The underlying mechanisms, however, remain poorly understood. We hypothesize that myeloid-derived suppressor cells (MDSC), which act as regulators in autoimmunity, play a role in resistance to diabetes in absence of complement C3. Indeed, MDSC number was increased significantly in STZ-treated C3-/- mice. These cells highly expressed arginase I and inducible nitric oxide synthase (iNOS). Importantly, depletion of MDSC led to the occurrence of overt diabetes in C3-/- mice after STZ. Furthermore, C3-/- MDSC actively suppressed diabetogenic T cell proliferation and prevented/delayed the development of diabetes in arginase and/or iNOS-dependent manner. Both Tregs and transforming growth factor-β (TGF-β) are crucial for MDSC induction in STZ-treated C3-/- mice as depletion of Tregs or blocking TGF-β bioactivity dramatically decreased MDSC number. These findings indicate that MDSC are implicated in resistance to STZ-induced diabetes in the absence of complement C3, which may be helpful for understanding of mechanisms underlying preventive effects of complement deficiency on autoimmune diseases.

  14. C3 — EDRN Public Portal

    Cancer.gov

    Complement component C3, a secreted protein found in plasma, plays a central role in the activation of complement system. Its activation is required for both classical and alternative complement activation pathways. People with C3 deficiency are susceptible to bacterial infection.

  15. C3 glomerulopathy

    PubMed Central

    Cook, H. Terence

    2017-01-01

    C3 glomerulopathy is a recently defined entity that encompasses a group of kidney diseases caused by abnormal control of complement activation with deposition of complement component C3 in glomeruli leading to variable glomerular inflammation. Before the recognition of the unique pathogenesis of these cases, they were variably classified according to their morphological features. C3 glomerulopathy accounts for roughly 1% of all renal biopsies. Clear definition of this entity has allowed a better understanding of its pathogenesis and clinical course and is likely to lead to the design of rational therapies over the next few years. PMID:28357053

  16. Enhanced recognition of plasma proteins in a non-native state by complement C3b. A possible clearance mechanism for damaged proteins in blood.

    PubMed

    Ramadass, Mahalakshmi; Ghebrehiwet, Berhane; Kew, Richard R

    2015-03-01

    Complement C3 is a key fluid-phase protein of the immune system that covalently tags pathogenic cells and molecules for subsequent clearance. Previously, we reported that complement activation results in the formation of multiple C3b:plasma protein complexes in serum. However, it is not known if C3b attaches to any plasma protein in close proximity or preferentially binds damaged proteins. The objective of this study was to determine if C3b couples to plasma proteins in a non-native state and if this could be a potential mechanism to detect and clear damaged proteins from the blood. Using a purified in vitro system with alternative pathway proteins C3, factors B and D it was observed that guanidinium-HCl denaturation of three purified plasma proteins (albumin, alpha-1 proteinase inhibitor, vitamin D binding protein) greatly increased their capacity to form covalent complexes with C3b. However, native vitamin D binding protein, covalently attached to C3b, still retained the ability to bind its natural ligand G-actin, indicating that C3b links to plasma proteins in their native configuration but denaturation substantially increases this interaction. Serum complement activation generated a large number of C3b:plasma protein complexes that bound red blood cell membranes, suggesting a CR1-mediated clearance mechanism. Thermally denatured (60°C) serum activated the alternative pathway when added to fresh serum as evidenced by factor B cleavage and iC3b generation, but this heat-treated serum could not generate the pro-inflammatory peptide C5a. These results show that C3 recognizes and tags damaged plasma proteins for subsequent removal from the blood without triggering proinflammatory functions.

  17. Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner.

    PubMed

    Tagawa, Keisuke; Yoshihara, Toyoki; Shibata, Toshio; Kitazaki, Kazuki; Endo, Yuichi; Fujita, Teizo; Koshiba, Takumi; Kawabata, Shun-ichiro

    2012-01-01

    Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.

  18. Altered erythrocyte C3b receptor expression, immune complexes, and complement activation in homosexual men in varying risk groups for acquired immune deficiency syndrome.

    PubMed Central

    Tausk, F A; McCutchan, A; Spechko, P; Schreiber, R D; Gigli, I

    1986-01-01

    We studied levels of erythrocyte C3b receptors (E-CR1) and correlated them to the level of circulating immune complexes (CIC) and complement activation in patients with or at risk for acquired immunodeficiency syndrome (AIDS). A significant reduction was found in patients with AIDS (185 +/- 93 CR1/cell), AIDS-related complex, and generalized lymphadenopathy, whereas healthy male homosexuals or normal controls had 434 +/- 193 and 509 +/- 140 CR1/cell, respectively (P less than 0.001). Family studies indicate that this defect is acquired. Reduction in E-CR1 was associated with increased levels of CIC when assayed by binding to Raji cells, but not when tested by C1q binding. Complement activation was assessed by levels of C3bi/C3d-g in plasma, measured with a monoclonal antibody specific for a neoantigen in C3d. AIDS patients had increased C3 activation (2.68 +/- 1.67%) when compared with normal controls (0.9 +/- 0.22%) (P less than 0.01). The decreased E-CR1, the presence of CIC, and C3 activation suggest that complement activation by immune complexes may play a role in the clinical expression of the disease. PMID:2944915

  19. Evidence for presence of an internal thiolester bond in third component of human complement.

    PubMed Central

    Tack, B F; Harrison, R A; Janatova, J; Thomas, M L; Prahl, J W

    1980-01-01

    Treatment of the third component of human complement (C3) with methylamine results in a loss of hemolytic function and the appearance of a thiol group. Studies with [14C]methylamine have indicatd a stoichiometric and covalent reaction with the native protein. Hydrazine-inactivated C3 and C3b prepared with bovine trypsin were unreactive with [14C]-methylamine. Alkylation experiments with [1-14C]iodoacetamide have further established a 1:1 correspondence between methylamine incorporation and expression of the reactive thiol. Autoradiographic analyses of [14C]methylamine-treated C3 and methylamine-inactivated [1-14C]carboxyamidomethylated C3 after NaDodSO4/polyacrylamide gel electrophoresis have shown a specific incorporation of each radiolabel into the alpha polypeptide chain. [14C]Methylamine-treated C3 was immobilized on Sepharose 4B by reaction of the protein thiol with a mixed disulfide. Digestion with bovine trypsin in 4 M urea released 96% of the bound absorbance (at 280 nm) units; the radiolabel remained associated with the Sepharose beads. Peptide material labeled with 14C was eluted with dithiothreitol, carboxymethylated with [3H]iodoacetic acid, and chromatographed on Sephadex G-75. On Edman degradation S-[3H]carboxymethylcysteine was released at step 9 and gamma-glutamyl[14C]-methylamide was released at step 12. We interpret these data to indicate the presence of an internal thiolester bond in native C3. In addition, evidence is presented for an identical reactive site in alpha 2-macroglobulin. Images PMID:6934510

  20. Aurin tricarboxylic acid self-protects by inhibiting aberrant complement activation at the C3 convertase and C9 binding stages.

    PubMed

    Lee, Moonhee; Guo, Jian-Ping; McGeer, Edith G; McGeer, Patrick L

    2013-05-01

    Aberrant complement activation is known to exacerbate the pathology in a spectrum of degenerative diseases of aging. We previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent which prevents formation of the membrane attack complex of complement. It inhibits C9 attachment to tissue bound C5b678 and thus prevents bystander lysis of host cells. In this study, we investigated the effects of ATA on the alternative complement pathway. We found that ATA prevented cleavage of the tissue bound properdin-C3b-Factor B complex into the active C3 convertase enzyme properdin-C3b-Factor Bb. This inhibition was reversed by adding Factor D to the serum. Using enzyme-linked immunosorbent type assays, we established that ATA binds directly to Factor D and C9 but not to properdin or other complement proteins. We conclude that ATA, by inhibiting at two stages of the alternative pathway, might be a particularly effective therapeutic agent in conditions such as macular degeneration, paroxysmal nocturnal hemoglobinemia, and rheumatoid arthritis, in which activation of the alternative complement pathway initiates self damage.

  1. Complement Component 3 Is Associated with Metabolic Comorbidities in Older HIV-Positive Adults

    PubMed Central

    Bryant, Alex K.; Fazeli, Pariya L.; Letendre, Scott L.; Ellis, Ronald J.; Potter, Michael; Burdo, Tricia H.; Singh, Kumud K.; Jeste, Dilip V.; Grant, Igor

    2016-01-01

    Abstract Our objective was to evaluate the association of plasma inflammatory biomarkers with MetS in an older population of treated HIV-infected (HIV+) as compared to age-matched HIV-negative (HIV−) adults. This was done in a retrospective observational study. Plasma concentrations of complement component 3 (C3), cystatin C, fibroblast growth factor 1, interleukin 6, oxidized LDL, soluble RAGE, soluble CD163, soluble CD14, and osteopontin were measured in 79 HIV+ participants on combination antiretroviral treatment (cART) with a suppressed HIV viral load and 47 HIV− participants with a median age of 59 (range 50 to 79). Outcomes were individual MetS components (hypertension, type II diabetes, dyslipidemia, and obesity) and MetS. Covariates were screened for inclusion in multivariable models. Odds ratios are reported per 50 mg/dl increase in C3. In the HIV+ group, higher C3 levels were associated with MetS (OR 3.19, p = 0.004), obesity (OR 2.02, p = 0.01), type II diabetes (OR 1.93, p = 0.02), and at a trend level with dyslipidemia (OR 1.87, p = 0.07) and hypertension (OR 1.66, p = 0.09). C3 levels were significantly higher in HIV+ participants with MetS compared to those without MetS (p = 0.002). C3 was higher among HIV+ patients with three or four MetS components as compared to those with one or two (p = 0.04) and those with none (p = 0.002). No associations were found between C3 and the outcomes for HIV− participants. C3 is strongly associated with both MetS and MetS components in an older HIV+ sample on cART compared to HIV− controls. C3 warrants further investigation as a marker of cardiometabolic risk among persons aging with HIV. PMID:26499082

  2. Identification of a C3bi-specific membrane complement receptor that is expressed on lymphocytes, monocytes, neutrophils, and erythrocytes

    PubMed Central

    Ross, GD; Lambris, JD

    1982-01-01

    Cells expressing a membrane C receptor (CR(3)) specific for C3b-inactivator- cleaved C3b (C3bi) were identified by rosette assay with C3bi-coated sheep erythrocytes (EC3bi) or C3bi-coated fluorescent microspheres (C3bi-ms). C3bi- ms, probably because of their smaller size, bound to a higher proportion of cells than did EC3bi. C3bi-ms bound to greater than 90 percent of mature neutrophils, 85 percent of monocytes, 92 percent of erythrocytes, and 12 percent of peripheral blood lymphocytes. Binding of C3bi-ms to neutrophils, monocytes, and erythrocytes was inhibited by fluid-phase C3bi, Fab anti-C3c, or Fab anti-C3d but was not inhibited by F(ab’)(2) anti-CR(1) (C3b receptor) or F(ab’)(2) anti-CR(2) (C3d receptor) nor by fluid-phase C3b, C3c, or C3d. This indicated that monocytes, neutrophils, and erythrocytes expressed C3bi receptors (CR(3)) that were separate and distinct from CR(1) and CR(2) and specific for a site in the C3 molecule that was only exposed subsequently to cleavage of C3b by C3b inactivator and that was either destroyed, covered, or liberated by cleavage of C3bi into C3c and C3d fragments. Lymphocytes differed from these other cell types in that they expressed CR2 in addition to CRa. Lymphocyte C3bi-ms rosettes were inhibited from 50 to 84 percent by F(ab’)(2)-anti-CR(2) or fluid-phase C3d, whereas C3d-ms rosettes were inhibited completely by F(ab’)(2) anti-CR(2), fluid-phase C3bi, or fluid- phase C3d. Thus, with lymphocytes, C3bi was bound to CR(3), and in addition was bound to CR(2) by way of the intact d region of the C3bi molecule. In studies of the acquisition of C receptors occurring during myeloid cell maturation, the ability to rosette with C3bi-coated particles was detected readily with immature low-density cells, whereas this ability was nearly undetectable with high density mature polymorphonuclear cells. This absence of C3bi binding to polymorphs was not due to a loss of the CR(3) but instead was due to the maturation

  3. Complement

    MedlinePlus

    ... fungal infections and some parasitic infections such as malaria . Normal Results Total blood complement level: 41 to ... Glomerulonephritis Hepatitis Hereditary angioedema Kidney transplant Lupus nephritis Malaria Protein in diet Rheumatoid arthritis Septicemia Shock Systemic ...

  4. Serum amyloid P component bound to gram-negative bacteria prevents lipopolysaccharide-mediated classical pathway complement activation.

    PubMed

    de Haas, C J; van Leeuwen, E M; van Bommel, T; Verhoef, J; van Kessel, K P; van Strijp, J A

    2000-04-01

    Although serum amyloid P component (SAP) is known to bind many ligands, its biological function is not yet clear. Recently, it was demonstrated that SAP binds to lipopolysaccharide (LPS). In the present study, SAP was shown to bind to gram-negative bacteria expressing short types of LPS or lipo-oligosaccharide (LOS), such as Salmonella enterica serovar Copenhagen Re and Escherichia coli J5, and also to clinical isolates of Haemophilus influenzae. It was hypothesized that SAP binds to the bacteria via the lipid A part of LPS or LOS, since the htrB mutant of the nontypeable H. influenzae strain NTHi 2019-B29-3, which expresses a nonacetylated lipid A, did not bind SAP. This was in contrast to the parental strain NTHi 2019. The binding of SAP resulted in a clear inhibition of the deposition of complement component C3 on the bacteria. SAP inhibited only the activation of the classical complement pathway; the alternative route remained unaffected. In the classical route, SAP prevented the deposition of the first complement component, Clq, probably by interfering with the binding of Clq to LPS. Since antibody-mediated Clq activation was not inhibited by SAP, SAP seems to inhibit only the LPS-induced classical complement pathway activation. The SAP-induced inhibition of C3 deposition strongly diminished the complement-mediated lysis as well as the phagocytosis of the bacteria. The binding of SAP to gram-negative bacteria, therefore, might influence the pathophysiology of an infection with such bacteria.

  5. Control of the collective migration of enteric neural crest cells by the Complement anaphylatoxin C3a and N-cadherin.

    PubMed

    Broders-Bondon, Florence; Paul-Gilloteaux, Perrine; Gazquez, Elodie; Heysch, Julie; Piel, Matthieu; Mayor, Roberto; Lambris, John D; Dufour, Sylvie

    2016-06-01

    We analyzed the cellular and molecular mechanisms governing the adhesive and migratory behavior of enteric neural crest cells (ENCCs) during their collective migration within the developing mouse gut. We aimed to decipher the role of the complement anaphylatoxin C3a during this process, because this well-known immune system attractant has been implicated in cephalic NCC co-attraction, a process controlling directional migration. We used the conditional Ht-PA-cre transgenic mouse model allowing a specific ablation of the N-cadherin gene and the expression of a fluorescent reporter in migratory ENCCs without affecting the central nervous system. We performed time-lapse videomicroscopy of ENCCs from control and N-cadherin mutant gut explants cultured on fibronectin (FN) and micropatterned FN-stripes with C3a or C3aR antagonist, and studied cell migration behavior with the use of triangulation analysis to quantify cell dispersion. We performed ex vivo gut cultures with or without C3aR antagonist to determine the effect on ENCC behavior. Confocal microscopy was used to analyze the cell-matrix adhesion properties. We provide the first demonstration of the localization of the complement anaphylatoxin C3a and its receptor on ENCCs during their migration in the embryonic gut. C3aR receptor inhibition alters ENCC adhesion and migration, perturbing directionality and increasing cell dispersion both in vitro and ex vivo. N-cadherin-null ENCCs do not respond to C3a co-attraction. These findings indicate that C3a regulates cell migration in a N-cadherin-dependent process. Our results shed light on the role of C3a in regulating collective and directional cell migration, and in ganglia network organization during enteric nervous system ontogenesis. The detection of an immune system chemokine in ENCCs during ENS development may also shed light on new mechanisms for gastrointestinal disorders.

  6. Complement C3a binding to its receptor as a negative modulator of Th2 response in liver injury in trichloroethylene-sensitized mice.

    PubMed

    Wang, Feng; Zha, Wan-sheng; Zhang, Jia-xiang; Li, Shu-long; Wang, Hui; Ye, Liang-ping; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

    2014-08-17

    Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure.

  7. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these proteins aids in the diagnosis of immunologic disorders, especially those associated with deficiencies...

  8. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these proteins aids in the diagnosis of immunologic disorders, especially those associated with deficiencies...

  9. 21 CFR 866.5240 - Complement components immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these proteins aids in the diagnosis of immunologic disorders, especially those associated with deficiencies...

  10. The sequence and topology of human complement component C9.

    PubMed Central

    Stanley, K K; Kocher, H P; Luzio, J P; Jackson, P; Tschopp, J

    1985-01-01

    A partial nucleotide sequence of human complement component C9 cDNA representing 94% of the coding region of the mature protein is presented. The amino acid sequence predicted from the open reading frame of this cDNA concurs with the amino acid sequence at the amino-terminal end of three proteolytic fragments of purified C9 protein. No long stretches of hydrophobic residues are present, even in the carboxy-terminal half of the molecule which reacts with lipid-soluble photoaffinity probes. Monoclonal antibody epitopes have been mapped by comparing overlapping fragments of C9 molecule to which the antibodies bind on Western blots. Several of these epitopes map to small regions containing other surface features (e.g., proteolytic cleavage sites and N-linked oligosaccharide). The amino-terminal half of C9 is rich in cysteine residues and contains a region with a high level of homology to the LDL receptor cysteine-rich domains. A model for C9 topology based on these findings is proposed. Images Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:4018030

  11. Molecular genetics of the fourth component of human complement

    SciTech Connect

    Carroll, M.C.

    1987-05-15

    The fourth component of complement in humans is coded for by two closely linked loci, i.e., C4A and C4B, that have been positioned within the class III region of the human major histocompatibility complex along with the genes for C2, Bf, and steroid 21-OH. Both C4 loci are highly polymorphic and certain alleles, particularly the nulls, are associated with susceptibility to autoimmune disease. About one-half of the null alleles are due to a large deletion that includes both a C4 and flanking 21-OH gene. Despite the near identity of the products of the two loci, the proteins differ dramatically in their efficiency of covalent binding to antigen. The amino acid substitutions responsible for the functional differences have been identified and they are clustered relatively near the covalent binding site within the C4d region of the ..cap alpha.. chain. These observations support the hypothesis that the susceptibility to autoimmune disease is related to the structural variation of the C4 protein.

  12. Complement C3 and Decay-Accelerating Factor Expression Levels Are Modulated by Human Chorionic Gonadotropin in Endometrial Compartments During the Implantation Window

    PubMed Central

    Argandoña, Felipe; Azúa, Rodrigo; Kohen, Paulina; Devoto, Luigi

    2013-01-01

    The control of complement activation in the embryo–maternal environment has been demonstrated to be critical for embryo survival. Complement proteins are expressed in the human endometrium; however, the modulation of this expression by embryo signals has not been explored. To assess the expression of complement proteins in response to human chorionic gonadotropin (hCG), we designed an experimental study using in vivo and in vitro models. Twelve fertile women were treated with hCG or left untreated during the mid-luteal phase, and an endometrial biopsy was performed 24 hours later. The localizations of C3, membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55), and protectin (CD59) were assessed by immunohistochemistry, and the messenger RNA (mRNA) levels of these proteins were quantified by real-time reverse transcriptase–polymerase chain reaction (RT-PCR) in cells harvested from endometrial compartments using laser capture microdissection. Endometrial explants were cultured with or without hCG for 24 hours, and the C3 and DAF protein levels were measured by Western blotting. Elevated C3 mRNA levels in stromal cells and elevated DAF levels in epithelial luminal cells were detected after hCG treatment. In the endometrial explant model, the progesterone receptor antagonist RU486 inhibited the increases in the levels of C3 and DAF in response to hCG. The findings of this study indicate that hCG plays a role in embryo–endometrium communication and affects the expression of complement proteins in endometrial compartments during the implantation window. PMID:23427180

  13. Complement anaphylatoxin receptors C3aR and C5aR are required in the pathogenesis of experimental autoimmune uveitis.

    PubMed

    Zhang, Lingjun; Bell, Brent A; Yu, Minzhong; Chan, Chi-Chao; Peachey, Neal S; Fung, John; Zhang, Xiaoming; Caspi, Rachel R; Lin, Feng

    2016-03-01

    Recent studies have suggested that reagents inhibiting complement activation could be effective in treating T cell mediated autoimmune diseases such as autoimmune uveitis. However, the precise role of the complement anaphylatoxin receptors (C3a and C5a receptors) in the pathogenesis of autoimmune uveitis remains elusive and controversial. We induced experimental autoimmune uveitis in mice deficient or sufficient in both C3a and C5a receptors and rigorously compared their retinal phenotype using various imaging techniques, including indirect ophthalmoscopy, confocal scanning laser ophthalmoscopy, spectral domain optical coherence tomography, topical endoscopic fundus imaging, and histopathological analysis. We also assessed retinal function using electroretinography. Moreover, we performed Ag-specific T cell recall assays and T cell adoptive transfer experiments to compare pathogenic T cell activity between wild-type and knockout mice with experimental autoimmune uveitis. These experiments showed that C3a receptor/C5a receptor-deficient mice developed much less severe uveitis than did control mice using all retinal examination methods and that these mice had reduced pathogenic T cell responses. Our data demonstrate that both complement anaphylatoxin receptors are important for the development of experimental autoimmune uveitis, suggesting that targeting these receptors could be a valid approach for treating patients with autoimmune uveitis.

  14. Complement component C7 deficiency in two Spanish families.

    PubMed

    Barroso, Sonia; Sánchez, Berta; Alvarez, Antonia José; López-Trascasa, Margarita; Lanuza, Amparo; Luque, Rafael; Wichmann, Ingeborg; Núñez-Roldán, Antonio

    2004-12-01

    Different genetic mutations have been described in complement component C7 deficiency, a molecular defect clinically associated with an increased susceptibility to neisserial recurrent infections. In this work we report the genetic basis of C7 deficiency in two different Spanish families (family 1 and family 2). In family 1, of Gypsy ethnical background, exon-specific polymerase chain reaction and sequencing revealed a not previously described single base deletion of nucleotide 1309 (exon 10) in the patient, as well as in her father, leading to a stop codon that causes the premature truncation of the C7 protein (K416 X 419). Additionally, the patient and her mother displayed a missense mutation at position 1135 (exon 9) located in the first nucleotide of the codon GGG (CGG), resulting in a change of amino acid (G357R). This mutation was firstly described in individuals of Moroccan Sephardic Jewish ancestry and has been also reported among Spaniards. In family 2, another novel mutation was found in homozygosity in two siblings; a two base-pair deletion of nucleotides 1922 and 1923 in exon 14 leading to the generation of a downstream stop codon causing the truncation of the C7 protein product (S620 X 630). Our results provide more evidence for the heterogeneous molecular basis of C7 deficiency as well as for the subsequent susceptibility to meningococcal disease, since different families carry different molecular defects. On the other hand, certain C7 defects appear to be prevalent in individuals from certain populations or living in defined geographical areas.

  15. Characterization and expression analysis of a complement component gene in sea cucumber ( Apostichopus japonicus)

    NASA Astrophysics Data System (ADS)

    Chen, Zhong; Zhou, Zunchun; Yang, Aifu; Dong, Ying; Guan, Xiaoyan; Jiang, Bei; Wang, Bai

    2015-12-01

    The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B (Bf) serves as the catalytic subunit of complement component 3 (C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber ( Apostichopus japonicus), termed AjBf, was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA of AjBf was 3231 bp in length barring the poly (A) tail. It contained an open reading frame (ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5'-UTR (5'-terminal untranslated region) and a 384 bp 3'-UTR. AjBf was a mosaic protein with six CCP (complement control protein) domains, a VWA (von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of AjBf protein was 101 kDa. Quantitative real time PCR (qRT-PCR) analysis indicated that the expression level of AjBf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that AjBf expressed higher in coelomocytes than in other four tissues. In addation, AjBf expression in different tissues was induced significantly after LPS or PolyI:C challenge. These results indicated that AjBf plays an important role in immune responses to pathogen infection.

  16. The extracellular adherence protein from Staphylococcus aureus inhibits the classical and lectin pathways of complement by blocking formation of the C3 proconvertase.

    PubMed

    Woehl, Jordan L; Stapels, Daphne A C; Garcia, Brandon L; Ramyar, Kasra X; Keightley, Andrew; Ruyken, Maartje; Syriga, Maria; Sfyroera, Georgia; Weber, Alexander B; Zolkiewski, Michal; Ricklin, Daniel; Lambris, John D; Rooijakkers, Suzan H M; Geisbrecht, Brian V

    2014-12-15

    The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. Although the majority of staphylococcal complement inhibitors act on the alternative pathway to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical pathway (CP) and lectin pathway (LP). We screened a collection of recombinant, secreted staphylococcal proteins to determine whether S. aureus produces other molecules that inhibit the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 proconvertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.

  17. Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component.

    PubMed

    Szalai, A J; van Ginkel, F W; Wang, Y; McGhee, J R; Volanakis, J E

    2000-07-15

    The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.

  18. Complement component 3 deficiency prolongs MHC-II disparate skin allograft survival by increasing the CD4+ CD25+ regulatory T cells population

    PubMed Central

    Zheng, Quan-you; Liang, Shen-ju; Li, Gui-qing; Lv, Yan-bo; Li, You; Tang, Ming; Zhang, Kun; Xu, Gui-lian; Zhang, Ke-qin

    2016-01-01

    Recent reports suggest that complement system contributes to allograft rejection. However, its underlying mechanism is poorly understood. Herein, we investigate the role of complement component 3 (C3) in a single MHC-II molecule mismatched murine model of allograft rejection using C3 deficient mice (C3−/−) as skin graft donors or recipients. Compared with C3+/+ B6 allografts, C3−/− B6 grafts dramatically prolonged survival in MHC-II molecule mismatched H-2bm12 B6 recipients, indicating that C3 plays a critical role in allograft rejection. Compared with C3+/+ allografts, both Th17 cell infiltration and Th1/Th17 associated cytokine mRNA levels were clearly reduced in C3−/− allografts. Moreover, C3−/− allografts caused attenuated Th1/Th17 responses, but increased CD4+CD25+Foxp3+ regulatory T (Treg) cell expression markedly in local intragraft and H-2bm12 recipients. Depletion of Treg cells by anti-CD25 monoclonal antibody (mAb) negated the survival advantages conferred by C3 deficiency. Our results indicate for the first time that C3 deficiency can prolong MHC-II molecule mismatched skin allograft survival, which is further confirmed to be associated with increased CD4+ CD25+ Treg cell population expansion and attenuated Th1/Th17 response. PMID:27641978

  19. Current concepts in C3 glomerulopathy.

    PubMed

    Thomas, S; Ranganathan, D; Francis, L; Madhan, K; John, G T

    2014-11-01

    Complement component 3 glomerulopathy (C3G) is a recently defined entity comprising of dense deposit disease and C3 glomerulonephritis. The key histological feature is the presence of isolated C3 deposits without immunoglobulins. Often masqueradng as some of the common glomerulonephritides this is a prototype disorder occurring from dysregulated alternate complement pathway with recently identified genetic defects and autoantibodies. We review the pathophysiology, clinical features, and diagnostic and treatment strategies.

  20. Mapping of the C3d ligand binding site on complement receptor 2 (CR2/CD21) using nuclear magnetic resonance and chemical shift analysis.

    PubMed

    Kovacs, James M; Hannan, Jonathan P; Eisenmesser, Elan Z; Holers, V Michael

    2009-04-03

    Complement receptor 2 (CR2, CD21) is a cell membrane protein, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs (SCR1-2) mediate the interaction of CR2 with its four known ligands (C3d, Epstein-Barr virus gp350, interferon-alpha, and CD23). Inhibitory monoclonal antibodies against SCR1-2 block binding of all ligands. To develop ligand-specific inhibitors that would also assist in identifying residues unique to each receptor-ligand interaction, phage were selected from randomly generated libraries by panning with recombinant SCR1-2, followed by specific ligand-driven elution. Derived peptides were tested by competition ELISA. One peptide, C3dp1 (APQHLSSQYSRT) exhibited ligand-specific inhibition at midmicromolar IC(50). C3d was titrated into (15)N-labeled SCR1-2, which revealed chemical shift changes indicative of specific intermolecular interactions. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to C3d, the binding surface includes regions of SCR1, SCR2, and the inter-SCR linker, specifically residues Arg(13), Tyr(16), Arg(28), Tyr(29), Ser(32), Thr(34), Lys(48), Asp(56), Lys(57), Tyr(68), Arg(83), Gly(84), Asn(101), Asn(105), and Ser(109). SCR1 and SCR2 demonstrated distinct binding modes. The CR2 binding surface incorporating SCR1 is inconsistent with a previous x-ray CR2-C3d co-crystal analysis but consistent with mutagenesis, x-ray neutron scattering, and inhibitory monoclonal antibody epitope mapping. Titration with C3dp1 yielded chemical shift changes (Arg(13), Tyr(16), Thr(34), Lys(48), Asp(56), Lys(57), Tyr(68), Arg(83), Gly(84), Asn(105), and Ser(109)) overlapping with C3d, indicating that C3dp1 interacts at the same CR2 site as C3d.

  1. Molecular cloning of the cDNA encoding the Epstein-Barr virus/C3d receptor (complement receptor type 2) of human B lymphocytes

    SciTech Connect

    Moore, M.D.; Cooper, N.R.; Tack, B.F.; Nemerow, G.R.

    1987-12-01

    Complementary DNA clones for complement receptor type 2 (CR2), the B-lymphocyte membrane protein that serves as the receptor for Epstein-Barr virus and the C3d complement fragment, were obtained by screening a lambda gt11 library generated from Raji B lymphoblastoid cell mRNA. A 4.2-kilobase (kb) clone, representing the entire coding sequence of the protein plus untranslated 5' and 3' nucleotide sequences was obtained and sequenced. The 4.2-kb clone, which contains all but about 500 base pairs (bp) of the 5' untranslated region of the full-length CR2 mRNA, consists of 63 bp of 5' untranslated nucleotide sequence followed successively by a start codon, a 20-amino acid hydrophobic signal peptide, 1005 amino acids having a repeating motif, a 28-amino acid probable transmembrane domain, and a 34-amino acid cytoplasmic tail. The deduced amino acid sequence of the protein indicates that the extracellular domain consists entirely of 16 tandemly arranged repeating elements, each 60-75 amino acids in length, which are identified by multiple conserved residues. This repeating motif also occurs in the C3b/C4b receptor, several complement proteins, and a number of noncomplement proteins. In CR2, the 16 repeats occur in four clusters of four repeats each. Approximately 10% of the deduced amino acid sequence, including the amino and carboxyl termini, was confirmed by amino acid sequencing of tryptic peptides derived from purified CR2. The nucleotide and derived amino acid sequence of CR2 and related studies are presented here.

  2. Identification and comparative analysis of complement C3-associated microRNAs in immune response of Apostichopus japonicus by high-throughput sequencing

    PubMed Central

    Zhong, Lei; Zhang, Feng; Zhai, Yu; Cao, Yanhui; Zhang, Si; Chang, Yaqing

    2015-01-01

    MicroRNAs (miRNAs) are important effectors in mediating host–pathogen interaction. In this report, coelomocytes miRNA libraries of three Japanese sea cucumbers Apostichopus japonicus were built by Illumina® Hiseq2000 from different time points after lipopolysaccharide challenge (at time 0 h, 6 h and 12 h). The clean data received from high throughput sequencing were used to sequences analysis. Referenced to the Strongylocentrotus purpuratus genome, 38 conserved miRNAs were found, and three miRNA candidates were predicted by software. According to the evidence resulting from the expression of AjC3, expressing levels of spu-miR-133, spu-miR-137 and spu-miR-2004 altered along with the expression of AjC3 changing at different time points after LPS injection. Thus, we speculated that the three miRNAs may have influence on A. japonicus complement C3. The spu-miR-137 and miR-137 gene family in miRBase were analyzed by bioinformatics. There is an obvious discrepancy between invertebrates and vertebrates. The first and ninth nucleotides in invertebrate miR-137 are offset compared vertebrate miR-137. Importantly, this is the first attempt to map the stage of immune response regulome in echinoderms, which might be considered as information for elucidating the intrinsic mechanism underlying the immune system in this species. PMID:26634300

  3. Characterization of a Gene Coding for the Complement System Component FB from Loxosceles laeta Spider Venom Glands

    PubMed Central

    Myamoto, Daniela Tiemi; Pidde-Queiroz, Giselle; Gonçalves-de-Andrade, Rute Maria; Pedroso, Aurélio; van den Berg, Carmen W.; Tambourgi, Denise V.

    2016-01-01

    The human complement system is composed of more than 30 proteins and many of these have conserved domains that allow tracing the phylogenetic evolution. The complement system seems to be initiated with the appearance of C3 and factor B (FB), the only components found in some protostomes and cnidarians, suggesting that the alternative pathway is the most ancient. Here, we present the characterization of an arachnid homologue of the human complement component FB from the spider Loxosceles laeta. This homologue, named Lox-FB, was identified from a total RNA L. laeta spider venom gland library and was amplified using RACE-PCR techniques and specific primers. Analysis of the deduced amino acid sequence and the domain structure showed significant similarity to the vertebrate and invertebrate FB/C2 family proteins. Lox-FB has a classical domain organization composed of a control complement protein domain (CCP), a von Willebrand Factor domain (vWFA), and a serine protease domain (SP). The amino acids involved in Mg2+ metal ion dependent adhesion site (MIDAS) found in the vWFA domain in the vertebrate C2/FB proteins are well conserved; however, the classic catalytic triad present in the serine protease domain is not conserved in Lox-FB. Similarity and phylogenetic analyses indicated that Lox-FB shares a major identity (43%) and has a close evolutionary relationship with the third isoform of FB-like protein (FB-3) from the jumping spider Hasarius adansoni belonging to the Family Salcitidae. PMID:26771533

  4. Consequences of synovectomy of the knee joint: clinical, histopathological, and enzymatic changes and changes in 2 components of complement

    PubMed Central

    Myllylä, Timo; Peltonen, Leena; Puranen, Jaakko; Korhonen, L. Kalevi

    1983-01-01

    The state of 36 knee joints of 32 rheumatic patients was evaluated after surgical synovectomy, the follow-up period varying from 2 to 39 months. Synovitis was suspected by arthroscopy in 67% and verified histologically in 52%, although in a milder form than at the time of synovectomy. The regenerated synovial tissue was less permeable to serum proteins and contained lower activities of lysosomal enzymes than the excised synovial tissue. Although the number of leucocytes decreased in the joint fluid after synovectomy, the activities of lysosomal enzymes were lowered only slightly, and there was no change in the amount of C3 and C4 components of complement pathway. PMID:6830323

  5. Commercially Available Complement Component-Depleted Sera Are Unexpectedly Codepleted of Ficolin-2

    PubMed Central

    Brady, Allison M.; Geno, K. Aaron; Dalecki, Alex G.; Cheng, Xiaogang

    2014-01-01

    The ficolins are a family of innate pattern recognition molecules that are known to bind acetylated compounds and activate complement through the association of mannose binding lectin (MBL)/ficolin-associated serine proteases (MASPs). Their importance has more recently become appreciated, as they have been shown to play a role in a variety of disease processes from infection to autoimmunity. While studying ficolin-2-mediated complement deposition on Streptococcus pneumoniae, we found that sera depleted of C1q or other complement components were also codepleted of ficolin-2 but not ficolin-1, ficolin-3, or MBL. MBL present in C1q-depleted sera was able to mediate complement deposition on Saccharomyces cerevisiae, suggesting the presence of MASPs. We found that complement was activated on pneumococci in C1q-depleted serum only after opsonization with exogenous recombinant ficolin-2 (rFicolin-2). Also, no complement deposition was observed in C1q-depleted serum when pneumococci were opsonized with rFicolin-2 mutated at its lysine-57 residue, where MASPs are known to associate. Thus, these depleted sera are a unique tool to study ficolin-2-mediated complement pathways; however, one should be aware that ficolin-2 is absent from complement component-depleted sera. PMID:25030054

  6. Inhibition of pre-existing natural periodontitis in non-human primates by a locally administered peptide inhibitor of complement C3

    PubMed Central

    Maekawa, Tomoki; Briones, Ruel A.; Resuello, Ranillo R.G.; Tuplano, Joel V.; Hajishengallis, Evlambia; Kajikawa, Tetsuhiro; Koutsogiannaki, Sophia; Garcia, Cristina A.G.; Ricklin, Daniel; Lambris, John D.; Hajishengallis, George

    2016-01-01

    Aim Human periodontitis is associated with overactivation of complement, which is triggered by different mechanisms converging on C3, the central hub of the system. We assessed whether the C3 inhibitor Cp40 inhibits naturally-occurring periodontitis in non-human primates. Materials and Methods Non-human primates with chronic periodontitis were intra-gingivally injected with Cp40 either once (5 animals) or three times (10 animals) weekly for six weeks followed by a 6-week follow-up period. Clinical periodontal examinations and collection of gingival crevicular fluid and biopsies of gingiva and bone were performed at baseline and during the study. A one-way repeated measures ANOVA was used for data analysis. Results Whether administered once or three times weekly, Cp40 caused a significant reduction in clinical indices that measure periodontal inflammation (gingival index and bleeding on probing), tissue destruction (probing pocket depth and clinical attachment level) or tooth mobility. These clinical changes were associated with significantly reduced levels of pro-inflammatory mediators and decreased numbers of osteoclasts in bone biopsies. The protective effects of Cp40 persisted, albeit at reduced efficacy, for at least six weeks following drug discontinuation. Conclusion Cp40 inhibits pre-existing chronic periodontal inflammation and osteoclastogenesis in non-human primates, suggesting a novel adjunctive anti-inflammatory therapy for treating human periodontitis. PMID:26728318

  7. Expression of complement components and regulators by different subtypes of bone marrow-derived macrophages.

    PubMed

    Luo, Chang; Chen, Mei; Madden, Angelina; Xu, Heping

    2012-08-01

    Under inflammatory conditions, macrophages can differentiate into different functional subtypes. We show that bone marrow-derived macrophages constitutively express different levels of various complement-related genes. The relative expression levels are C1qb > Crry > CFH > C3 > C1r > CFB > DAF1 > CD59a > C2 > C1INH > C1s > C4. Upon activation, the expression of C1r, C1s, C3, C2, CFB, and C1INH was up-regulated, and CFH, CD59a, and DAF1, down-regulated in M1 (induced by interferon-γ + lipopolysaccharides (LPS)) and M2b (induced by immune complex + LPS) macrophages. The expression of C4 and CFH was slightly up-regulated in interleukin (IL)-10-induced M2c macrophages. Complement gene expression in IL-4-induced M2a macrophages was weakly down-regulated as compared to resting M0 macrophages. Higher levels of C3, C1INH, and CFB but lower levels of CFH expression in M1 and M2b macrophage suggests that they may be involved in the alternative pathway of complement activation during inflammation.

  8. Human complement C3b/C4b receptor (CR1) mRNA polymorphism that correlates with the CR1 allelic molecular weight polymorphism

    SciTech Connect

    Holers, V.M.; Chaplin, D.D.; Leykam, J.F.; Gruner, B.A.; Kumar, V.; Atkinson, J.P.

    1987-04-01

    The human C3b/C4b receptor (CR1) is a M/sub r/ approx. = 200,000 single-chain integral membrane glycoprotein of human erythrocytes and leukocytes. It functions both as a receptor for C3b- and C4b-coated ligands and as a regulator of complement activation. Prior structural studies have defined an unusual molecular weight allelic polymorphism in which the allelic products differ in molecular weight by as much as 90,000. On peripheral blood cells there is codominant expression of CR1 gene products of M/sub r/ 190,000 (A), 220,000 (B), 160,000 (C), and 250,000 (D). Results of prior biosynthetic and tryptic peptide mapping experiments have suggested that the most likely basis for the allelic molecular weight differences if at the polypeptide level. In order to define further the molecular basis for these molecular weight differences, human CR1 was purified to homogeneity, tryptic peptide fragments were isolated by HPLC and sequenced, oligonucleotide probes were prepared, and a CR1 cDNA was identified. A subclone of this CR1 cDNA was used as a probe of RNA blots of Epstein-Barr virus-transformed cell lines expressing the allelic variants. Each allelic variant encodes two distinct transcripts. A mRNA size polymorphism was identified that correlated with the gene product molecular weight polymorphism. This finding, in addition to a prior report of several homologous repeats in CR1, is consistent with the hypothesis that the molecular weight polymorphism is determined at the genomic level and may have been generated by unequal crossing-over.

  9. Complement Component 3 Regulates IFN-α Production by Plasmacytoid Dendritic Cells following TLR7 Activation by a Plant Virus-like Nanoparticle.

    PubMed

    Lebel, Marie-Ève; Langlois, Marie-Pierre; Daudelin, Jean-François; Tarrab, Esther; Savard, Pierre; Leclerc, Denis; Lamarre, Alain

    2017-01-01

    The increasing use of plant viruses for the development of new vaccines and immunotherapy approaches poses questions regarding the mechanism by which the mammalian immune system recognizes these viruses. For example, although natural Abs (NA) and complement are key components of the innate immune system involved in the opsonization, phagocytosis, and destruction of microorganisms infecting mammals, their implication in plant virus recognition and immunogenicity is not well defined. In this study, we address the involvement of NA and the complement system in the activation of innate immunity through engagement of TLR7 with papaya mosaic virus (PapMV)-like nanoparticles. We demonstrate that NA, although binding to PapMV, are not involved in its recognition by the immune system. On the other hand, C3 strongly binds to PapMV nanoparticles and its depletion significantly reduces PapMV's interaction with immune cells. Unexpectedly, however, we observed increased immune cell activation following administration of PapMV to complement-depleted mice. TLR7 activation by PapMV in the absence of C3 induced higher IFN-α production, resulting in superior immune cell activation and increased immunotherapeutic properties. In conclusion, in this study we established the involvement of the complement system in the recognition and the phagocytosis of PapMV nanoparticles and identified an unsuspected role for C3 in regulating the production of IFN-α following TLR7 activation.

  10. Systemic Lupus Erythematosus and Deficiencies of Early Components of the Complement Classical Pathway

    PubMed Central

    Macedo, Ana Catarina Lunz; Isaac, Lourdes

    2016-01-01

    The complement system plays an important role in the innate and acquired immune response against pathogens. It consists of more than 30 proteins found in soluble form or attached to cell membranes. Most complement proteins circulate in inactive forms and can be sequentially activated by the classical, alternative, or lectin pathways. Biological functions, such as opsonization, removal of apoptotic cells, adjuvant function, activation of B lymphocytes, degranulation of mast cells and basophils, and solubilization and clearance of immune complex and cell lysis, are dependent on complement activation. Although the activation of the complement system is important to avoid infections, it also can contribute to the inflammatory response triggered by immune complex deposition in tissues in autoimmune diseases. Paradoxically, the deficiency of early complement proteins from the classical pathway (CP) is strongly associated with development of systemic lupus erythematous (SLE) – mainly C1q deficiency (93%) and C4 deficiency (75%). The aim of this review is to focus on the deficiencies of early components of the CP (C1q, C1r, C1s, C4, and C2) proteins in SLE patients. PMID:26941740

  11. In vitro synthesis of immunoglobulins, secretory component and complement in normal and pathological skin and the adjacent mucous membranes

    PubMed Central

    Lai A Fat, R. F. M.; Suurmond, D.; Van Furth, R.

    1973-01-01

    A study on the synthesis of immunoglobulins (IgG, IgA, IgM, IgD, and IgE), secretory component and complement in normal and pathological skin and in the adjacent mucous membranes (i.e. conjunctiva, nasal, oral and vaginal mucosa) is reported. The results are based on the culture of tissue samples in a medium with two radioactive amino acids and the detection of synthesized proteins by autoradiography of the immunoelectrophoretic pattern of the culture fluid, except in the case of IgE for which the Ouchterlony technique was used. The results indicate that the normal skin does not synthesize immunoglobulins, whereas normal mucous membranes produce IgG and IgA. In the lesions of various skin diseases immunoglobulins are synthesized, mainly IgG but sometimes also IgA and IgE. The cells responsible for the production of immunoglobulins are plasma cells and lymphoid cells present in the skin lesions and mucous membranes. Synthesis of the free secretory component could be demonstrated only in certain mucous membranes (i.e. conjunctiva, nasal mucosa, and oral mucosa). Complement (C3) synthesis was found in normal skin, mucous membranes (i.e. conjunctiva, nasal and oral mucosa), and in the lesions of such skin diseases as discoid lupus erythematosus, (bullous) pemphigoid, dermatitis herpetiformis, malignant reticulosis, eczema and lichen planus. Complement production was also demonstrated in allergic skin reactions (i.e. tissue from allergic-positive patch tests, positive Mantoux tests and drug eruptions), but no immunoglobulin synthesis was detected in these lesions. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5 PMID:4199092

  12. Amastigotes of Trypanosoma cruzi escape destruction by the terminal complement components

    SciTech Connect

    Iida, K.; Whitlow, M.B.; Nussenzweig, V.

    1989-03-01

    We studied the effect of complement on two life cycle stages of the protozoan parasite Trypanosoma cruzi: epimastigotes, found in the insect vector, and amastigotes, found in the mammalian host. We found that while both stages activate vigorously the alternative pathway, only epimastigotes are destroyed. The amounts of C3 and C5b-7 deposited on the amastigotes were similar to those bound to the much larger epimastigotes. Binding of C9 to amastigotes was four to six times less than binding to epimastigotes, resulting in a lower C9/C5b-7 ratio. Although a fairly large amount of C9 bound stably to amastigotes, no functional channels were formed as measured by release of incorporated /sup 86/Rb. The bound C9 had the characteristic properties of poly-C9, that is, it expressed a neo-antigen unique to poly-C9, and migrated in SDS-PAGE with an apparent Mr greater than 10(5). The poly-C9 was removed from the surface of amastigotes by treatment with trypsin, indicating that it was not inserted in the lipid bilayer. Modification of amastigote surface by pronase treatment rendered the parasites susceptible to complement attack. These results suggest that amastigotes have a surface protein that binds to the C5b-9 complex and inhibits membrane insertion, thus protecting the parasites from complement-mediated lysis.

  13. The partly folded back solution structure arrangement of the 30 SCR domains in human complement receptor type 1 (CR1) permits access to its C3b and C4b ligands.

    PubMed

    Furtado, Patricia B; Huang, Chen Y; Ihyembe, Demvihin; Hammond, Russell A; Marsh, Henry C; Perkins, Stephen J

    2008-01-04

    Human complement receptor type 1 (CR1, CD35) is a type I membrane-bound glycoprotein that belongs to the regulators of complement activity (RCA) family. The extra-cellular component of CR1 is comprised of 30 short complement regulator (SCR) domains, whereas complement receptor type 2 (CR2) has 15 SCR domains and factor H (FH) has 20 SCR domains. The domain arrangement of a soluble form of CR1 (sCR1) was studied by X-ray scattering and analytical ultracentrifugation. The radius of gyration R(G) of sCR1 of 13.4(+/-1.1) nm is not much greater than those for CR2 and FH, and its R(G)/R(0) anisotropy ratio is 3.76, compared to ratios of 3.67 for FH and 4.1 for CR2. Unlike CR2, but similar to FH, two cross-sectional R(G) ranges were identified that gave R(XS) values of 4.7(+/-0.2) nm and 1.2(+/-0.7) nm, respectively, showing that the SCR domains adopt a range of conformations including folded-back ones. The distance distribution function P(r) showed that the most commonly occurring distance in sCR1 is at 11.5 nm. Its maximum length of 55 nm is less than double those for CR2 or FH, even though sCR1 has twice the number of SCR domains compared to CR2 Sedimentation equilibrium experiments gave a mean molecular weight of 235 kDa for sCR1. This is consistent with the value of 245 kDa calculated from its composition including 14 N-linked oligosaccharide sites, and confirmed that sCR1 is a monomer in solution. Sedimentation velocity experiments gave a sedimentation coefficient of 5.8 S. From this, the frictional ratio (f/f(0)) of sCR1 was calculated to be 2.29, which is greater than those of 1.96 for CR2 and 1.77 for FH. The constrained scattering modelling of the sCR1 solution structure starting from homologous SCR domain structures generated 5000 trial conformationally randomised models, 43 of which gave good scattering fits to show that sCR1 has a partly folded-back structure. We conclude that the inter-SCR linkers show structural features in common with those in FH, but

  14. Resistance to listeriosis in mice that are deficient in the fifth component of complement.

    PubMed Central

    Petit, J C

    1980-01-01

    Infection with Listeria monocytogenes was studied in strains of mice with genetic absence of the fifth component of complement (C5). Mice deficient in C5 consistently showed an increased growth of Listeria in their spleens as compared to normal mice. This increased growth was not corrected by administration of plasma containing C5. Furthermore, depletion of C5 and terminal complement components by administration of cobra venom factor did not impair the resistance to Listeria infection of normal mice. No phagocytic defect could be detected in macrophages from strains lacking C5. Transfer of bone marrow cells from C5+ but not from C5- mice corrected the marked increase of Listeria growth in mice having blockade of the reticuloendothelial system. We hypothesize that the defect of mice lacking C5 lies not in the absence of serum C5 but somewhere at the level of the macrophage. PMID:6766905

  15. Complement in autoimmune diseases.

    PubMed

    Vignesh, Pandiarajan; Rawat, Amit; Sharma, Madhubala; Singh, Surjit

    2017-02-01

    The complement system is an ancient and evolutionary conserved element of the innate immune mechanism. It comprises of more than 20 serum proteins most of which are synthesized in the liver. These proteins are synthesized as inactive precursor proteins which are activated by appropriate stimuli. The activated forms of these proteins act as proteases and cleave other components successively in amplification pathways leading to exponential generation of final effectors. Three major pathways of complement pathways have been described, namely the classical, alternative and lectin pathways which are activated by different stimuli. However, all the 3 pathways converge on Complement C3. Cleavage of C3 and C5 successively leads to the production of the membrane attack complex which is final common effector. Excessive and uncontrolled activation of the complement has been implicated in the host of autoimmune diseases. But the complement has also been bemusedly described as the proverbial "double edged sword". On one hand, complement is the final effector of tissue injury in autoimmune diseases and on the other, deficiencies of some components of the complement can result in autoimmune diseases. Currently available tools such as enzyme based immunoassays for functional assessment of complement pathways, flow cytometry, next generation sequencing and proteomics-based approaches provide an exciting opportunity to study this ancient yet mysterious element of innate immunity.

  16. The Serum Complement System: A Simplified Laboratory Exercise to Measure the Activity of an Important Component of the Immune System

    ERIC Educational Resources Information Center

    Inglis, Jordan E.; Radziwon, Kimberly A.; Maniero, Gregory D.

    2008-01-01

    The immune system is a vital physiological component that affords animals protection from disease and is composed of innate and adaptive mechanisms that rely on cellular and dissolved components. The serum complement system is a series of dissolved proteins that protect against a variety of pathogens. The activity of complement in serum can be…

  17. [Serum immunoglobulins and third complement component in and after acute non-A/non-B hepatitis (author's transl)].

    PubMed

    Storch, W; Sauer, I; Trautmann, B; Richter, H; Hagert, M

    1982-01-01

    The serum levels of IgG, IgA, IgM, IgD and IgE and of the third complement component (C 3) were determined by the single radial immunodiffusion method in 69 serum samples of 34 female patients during (2nd and 4th week) acute non-A/non-B hepatitis and 2 years after infection. The levels were compared with those of circulating immune complexes measured by polyethylene glycol precipitation method. The levels of immunoglobulins and C 3 were similar to those of healthy persons. During the course of disease there were no significant relations with exception of an increase of IgD levels in patients with chronic course. The positive correlation of the levels of IgM and immune complexes at the first and second serum sample (r = 0,5914, r = 0,6366 respectively, p less than 0,001) could not be verified in patients with noncomplicated course (r = 0,203 8, n. s.) but it was highly significant in patients with chronic course (r = 0,9429, p less than 0,001). Determinations of immunoglobulins and immune complexes may therefore be prognostically helpful in patients with non-A/non-B hepatitis.

  18. The purification and properties of the second component of human complement.

    PubMed Central

    Kerr, M A; Porter, R R

    1978-01-01

    The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared. Images PLATE 1 PLATE 2 Fig. 3. Fig. 5. PMID:417728

  19. A new, simplified method for oxidation of human complement component C2.

    PubMed

    Brickman, C M; Atherton, J P; Kantor, N L

    1990-09-14

    Oxy C2 is a valuable immunologic reagent, particularly for investigators whose research or clinical assays require a stable classical pathway C3 and/or C5 convertase or a highly active plasma complement source. To date only one method for the conversion of serum C2 or purified C2 to their respective oxy C2 forms has been published. However, this method has several disadvantages. For example, handling and dissolving the iodine crystals required in this process are difficult and time consuming. Also, the enhancement procedure results in a significant dilution of the original C2 sample. We have, therefore, developed a new, simplified method for oxidation of plasma, serum, or pure C2 which circumvents the difficulties associated with the earlier method. Moreover, this method offers additional flexibility with regard to oxidative conditions (i.e., buffer pH, temperature, and C2 concentrations) and reagent handling and final C2 product stability.

  20. C3 glomerulopathy: consensus report.

    PubMed

    Pickering, Matthew C; D'Agati, Vivette D; Nester, Carla M; Smith, Richard J; Haas, Mark; Appel, Gerald B; Alpers, Charles E; Bajema, Ingeborg M; Bedrosian, Camille; Braun, Michael; Doyle, Mittie; Fakhouri, Fadi; Fervenza, Fernando C; Fogo, Agnes B; Frémeaux-Bacchi, Véronique; Gale, Daniel P; Goicoechea de Jorge, Elena; Griffin, Gene; Harris, Claire L; Holers, V Michael; Johnson, Sally; Lavin, Peter J; Medjeral-Thomas, Nicholas; Paul Morgan, B; Nast, Cynthia C; Noel, Laure-Hélène; Peters, D Keith; Rodríguez de Córdoba, Santiago; Servais, Aude; Sethi, Sanjeev; Song, Wen-Chao; Tamburini, Paul; Thurman, Joshua M; Zavros, Michael; Cook, H Terence

    2013-12-01

    C3 glomerulopathy is a recently introduced pathological entity whose original definition was glomerular pathology characterized by C3 accumulation with absent or scanty immunoglobulin deposition. In August 2012, an invited group of experts (comprising the authors of this document) in renal pathology, nephrology, complement biology, and complement therapeutics met to discuss C3 glomerulopathy in the first C3 Glomerulopathy Meeting. The objectives were to reach a consensus on: the definition of C3 glomerulopathy, appropriate complement investigations that should be performed in these patients, and how complement therapeutics should be explored in the condition. This meeting report represents the current consensus view of the group.

  1. C1q complement component and -antibodies reflect SLE activity and kidney involvement.

    PubMed

    Horák, P; Hermanová, Z; Zadrazil, J; Ciferská, H; Ordeltová, M; Kusá, L; Zurek, M; Tichý, T

    2006-07-01

    The role of the complement system in the pathogenesis of systemic diseases is very ambivalent. In systemic lupus erythematosus (SLE), many abnormalities in the activation of the complement system have been reported. The most important antibodies formed against the complement system in SLE are the ones associated with the C1q component. The aim of this study was to assess separately the anti-C1q antibodies and C1q component in the serum from 65 patients with SLE, then in individuals with (n=33) and without (n=32) lupus nephritis and with active (n=36) and nonactive (n=29) form of the disease (European Consensus Lupus Activity Measurement, ECLAM>3, ECLAMcomplement component. The mean serum levels were 90.89+/-13 IU/ml for anti-C1q antibodies and 145+/-52 mg/l for C1q. The significant difference in C1q antibodies levels was found between individuals with and without lupus nephritis (117.5+/-52 IU/ml vs. 28.2+/-12.2 IU/ml, p=0.0001) and between those with active and nonactive SLE (154.6+/-115 IU/ml vs. 50.6+/-73, p=0.001). C1q complement component was statistically lower in patients with lupus nephritis (144+/-30 mg/l vs. 175+/-50 mg/ml, p=0.002) and in active patients (138+/-40 mg/l vs. 202+/-20 mg/l, p=0.001). If the two parameters are measured together, they seem to have a mirror-like pattern of serum concentration, and they are potential markers of SLE activity and of the presence of lupus nephritis.

  2. Structural insights on complement activation.

    PubMed

    Alcorlo, Martín; López-Perrote, Andrés; Delgado, Sandra; Yébenes, Hugo; Subías, Marta; Rodríguez-Gallego, César; Rodríguez de Córdoba, Santiago; Llorca, Oscar

    2015-10-01

    The proteolytic cleavage of C3 to generate C3b is the central and most important step in the activation of complement, a major component of innate immunity. The comparison of the crystal structures of C3 and C3b illustrates large conformational changes during the transition from C3 to C3b. Exposure of a reactive thio-ester group allows C3b to bind covalently to surfaces such as pathogens or apoptotic cellular debris. The displacement of the thio-ester-containing domain (TED) exposes hidden surfaces that mediate the interaction with complement factor B to assemble the C3-convertase of the alternative pathway (AP). In addition, the displacement of the TED and its interaction with the macroglobulin 1 (MG1) domain generates an extended surface in C3b where the complement regulators factor H (FH), decay accelerating factor (DAF), membrane cofactor protein (MCP) and complement receptor 1 (CR1) can bind, mediating accelerated decay of the AP C3-convertase and proteolytic inactivation of C3b. In the last few years, evidence has accumulated revealing that the structure of C3b in solution is significantly more flexible than anticipated. We review our current knowledge on C3b structural flexibility to propose a general model where the TED can display a collection of conformations around the MG ring, as well as a few specialized positions where the TED is held in one of several fixed locations. Importantly, this conformational heterogeneity in C3b impacts complement regulation by affecting the interaction with regulators.

  3. Early Components of the Complement Classical Activation Pathway in Human Systemic Autoimmune Diseases

    PubMed Central

    Lintner, Katherine E.; Wu, Yee Ling; Yang, Yan; Spencer, Charles H.; Hauptmann, Georges; Hebert, Lee A.; Atkinson, John P.; Yu, C. Yung

    2016-01-01

    The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases. PMID:26913032

  4. Complement component 5 contributes to poor disease outcome in humans and mice with pneumococcal meningitis.

    PubMed

    Woehrl, Bianca; Brouwer, Matthijs C; Murr, Carmen; Heckenberg, Sebastiaan G B; Baas, Frank; Pfister, Hans W; Zwinderman, Aeilko H; Morgan, B Paul; Barnum, Scott R; van der Ende, Arie; Koedel, Uwe; van de Beek, Diederik

    2011-10-01

    Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Disease outcome has been related to the severity of the proinflammatory response in the subarachnoid space. The complement system, which mediates key inflammatory processes, has been implicated as a modulator of pneumococcal meningitis disease severity in animal studies. Additionally, SNPs in genes encoding complement pathway proteins have been linked to susceptibility to pneumococcal infection, although no associations with disease severity or outcome have been established. Here, we have performed a robust prospective nationwide genetic association study in patients with bacterial meningitis and found that a common nonsynonymous complement component 5 (C5) SNP (rs17611) is associated with unfavorable disease outcome. C5 fragment levels in cerebrospinal fluid (CSF) of patients with bacterial meningitis correlated with several clinical indicators of poor prognosis. Consistent with these human data, C5a receptor-deficient mice with pneumococcal meningitis had lower CSF wbc counts and decreased brain damage compared with WT mice. Adjuvant treatment with C5-specific monoclonal antibodies prevented death in all mice with pneumococcal meningitis. Thus, our results suggest C5-specific monoclonal antibodies could be a promising new antiinflammatory adjuvant therapy for pneumococcal meningitis.

  5. Complement component 5 contributes to poor disease outcome in humans and mice with pneumococcal meningitis

    PubMed Central

    Woehrl, Bianca; Brouwer, Matthijs C.; Murr, Carmen; Heckenberg, Sebastiaan G.B.; Baas, Frank; Pfister, Hans W.; Zwinderman, Aeilko H.; Morgan, B. Paul; Barnum, Scott R.; van der Ende, Arie; Koedel, Uwe; van de Beek, Diederik

    2011-01-01

    Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Disease outcome has been related to the severity of the proinflammatory response in the subarachnoid space. The complement system, which mediates key inflammatory processes, has been implicated as a modulator of pneumococcal meningitis disease severity in animal studies. Additionally, SNPs in genes encoding complement pathway proteins have been linked to susceptibility to pneumococcal infection, although no associations with disease severity or outcome have been established. Here, we have performed a robust prospective nationwide genetic association study in patients with bacterial meningitis and found that a common nonsynonymous complement component 5 (C5) SNP (rs17611) is associated with unfavorable disease outcome. C5 fragment levels in cerebrospinal fluid (CSF) of patients with bacterial meningitis correlated with several clinical indicators of poor prognosis. Consistent with these human data, C5a receptor–deficient mice with pneumococcal meningitis had lower CSF wbc counts and decreased brain damage compared with WT mice. Adjuvant treatment with C5-specific monoclonal antibodies prevented death in all mice with pneumococcal meningitis. Thus, our results suggest C5-specific monoclonal antibodies could be a promising new antiinflammatory adjuvant therapy for pneumococcal meningitis. PMID:21926466

  6. Complement component C5 deficiency reduces edema formation in murine ligation-induced acute pancreatitis.

    PubMed

    Merriam, L T; Webster, C; Joehl, R J

    1997-01-01

    The complement cascade is activated in humans and animals with acute pancreatitis. Activation of complement component C5 liberates C5a, C5a-desarg, and terminal complement complexes (TCCs) that increase capillary permeability, edema, and leukocyte chemotaxis at injured sites. Complement activation plays a major role in pathogenesis of capillary leak and edema formation in severe acute pancreatitis; however, the contribution of C5 (C5a/C5a-desarg, TCCs) has not been defined. Using He gene mutant mice lacking circulating C5, the role of C5 in ligation-induced acute pancreatitis was evaluated. We performed the following experiments: C5-sufficient (Hc1/Hc1) and C5-deficient (Hc0/Hc0) mice had bile and pancreatic ducts ligated. Sham-operated mice had ducts dissected but not ligated. Mice were killed at 4, 8, and 24 hr after bilepancreatic duct ligation. Serologic and morphologic evidences of acute pancreatitis were evaluated. Pancreatic edema was assessed using analysis of pancreatic water content, histologic edema score, and determination of wet weight ratio. After 4, 8, and 24 hr of bile-pancreatic duct ligation, hyperamylasemia and histologic changes of acute pancreatitis were observed in both C5-deficient and C5-sufficient mice. Edema developed in all mice with acute pancreatitis. However, when compared to C5-sufficient mice, mice deficient in C5 developed significantly less pancreatic edema at both 8 and 24 hr of bile-pancreatic duct ligation. This difference was not observed 4 hr after induction of acute pancreatitis. We conclude that C5 contributes to edema formation in murine ligation-induced acute pancreatitis. The presence of an early C5-independent phase, in conjunction with the observation of significant edema in mice deficient in C5, suggests there are other mediators of edema formation in this acute pancreatitis model.

  7. Enhancement of growth, photosynthetic performance and yield by exclusion of ambient UV components in C3 and C4 plants.

    PubMed

    Kataria, Sunita; Guruprasad, K N; Ahuja, Sumedha; Singh, Bupinder

    2013-10-05

    A field experiment was conducted under tropical climate for assessing the effect of ambient UV-B and UV-A by exclusion of UV components on the growth, photosynthetic performance and yield of C3 (cotton, wheat) and C4 (amaranthus, sorghum) plants. The plants were grown in specially designed UV exclusion chambers, wrapped with filters that excluded UV-B (<315nm), UV-A+B (<400nm), transmitted all the UV (280-400nm) or without filters. All the four plant species responded to UV exclusion by a significant increase in plant height, leaf area, leaf biomass, total biomass accumulation and yield. Measurements of the chlorophyll, chlorophyll fluorescence parameters, gas exchange parameters and the activity of Ribulose-1,5-bisphosphate carboxylase (Rubisco) by fixation of (14)CO2 indicated a direct relationship between enhanced rate of photosynthesis and yield of the plants. Quantum yield of electron transport was enhanced by the exclusion of UV indicating better utilization of PAR assimilation and enhancement in reducing power in all the four plant species. Exclusion of UV-B in particular significantly enhanced the net photosynthetic rate, stomatal conductance and activity of Rubisco. Additional fixation of carbon due to exclusion of ambient UV-B was channeled towards yield as there was a decrease in the level of UV-B absorbing substances and an increase in soluble proteins in all the four plant species. The magnitude of the promotion in all the parameters studied was higher in dicots (cotton, amaranthus) compared to monocots (wheat, sorghum) after UV exclusion. The results indicated a suppressive action of ambient UV-B on growth and photosynthesis; dicots were more sensitive than monocots in this suppression while no great difference in sensitivity was found between C3 and C4 plants. Experiments indicated the suppressive action of ambient UV on carbon fixation and yield of C3 and C4 plants. Exclusion of solar UV-B will have agricultural benefits in both C3 and C4 plants

  8. Renal transplantation in a patient with hereditary deficiency of the second component of complement.

    PubMed Central

    Zeitz, H J; Gewurz, A; Jonasson, O; Geis, W P; Gewurz, H

    1981-01-01

    The HLA haplotype A 10,B18 has been associated with hereditary deficiency of the second component of complement(C2). In an effort to detect individuals homozygous for C2 deficiency, a thorough audit of HLA serotyping results in 3,100 individuals was performed, and a single patient homozygous for the A10, B18 haplotype was identified. Detailed complement studies in this patient's serum and plasma revealed previously undetected selective absence of C2 antigen and haemolytic activity, and a hereditary basis for this deficiency was indicated by half-normal levels of C2 haemolytic activity in both of his children. The patient was of special interest in that he had previously developed renal failure which was treated by cadaver kidney transplantation. C2 antigen was undetectable in serum and plasma samples taken prior to and up to 9 months following transplantation. This experience suggests that HLA serotyping can be a valuable screening technique for the detection of individuals with C2 deficiency, and that renal transplantation does not reconstitute normal levels of C2. PMID:7039890

  9. Dual inhibition of complement and Toll-like receptors as a novel approach to treat inflammatory diseases—C3 or C5 emerge together with CD14 as promising targets

    PubMed Central

    Barratt-Due, Andreas; Pischke, Søren Erik; Nilsson, Per H.; Espevik, Terje; Mollnes, Tom Eirik

    2017-01-01

    The host is protected by pattern recognition systems, including complement and TLRs, which are closely cross-talking. If improperly activated, these systems might induce tissue damage and disease. Inhibition of single downstream proinflammatory cytokines, such as TNF, IL-1β, and IL-6, have failed in clinical sepsis trials, which might not be unexpected, given the substantial amounts of mediators involved in the pathogenesis of this condition. Instead, we have put forward a hypothesis of inhibition at the recognition phase by “dual blockade” of bottleneck molecules of complement and TLRs. By acting upstream and broadly, the dual blockade could be beneficial in conditions with improper or uncontrolled innate immune activation threatening the host. Key bottleneck molecules in these systems that could be targets for inhibition are the central complement molecules C3 and C5 and the important CD14 molecule, which is a coreceptor for several TLRs, including TLR4 and TLR2. This review summarizes current knowledge of inhibition of complement and TLRs alone and in combination, in both sterile and nonsterile inflammatory processes, where activation of these systems is of crucial importance for tissue damage and disease. Thus, dual blockade might provide a general, broad-acting therapeutic regimen against a number of diseases where innate immunity is improperly activated. PMID:27581539

  10. Structure of the poly-C9 component of the complement membrane attack complex

    PubMed Central

    Dudkina, Natalya V.; Spicer, Bradley A.; Reboul, Cyril F.; Conroy, Paul J.; Lukoyanova, Natalya; Elmlund, Hans; Law, Ruby H. P.; Ekkel, Susan M.; Kondos, Stephanie C.; Goode, Robert J. A.; Ramm, Georg; Whisstock, James C.; Saibil, Helen R.; Dunstone, Michelle A.

    2016-01-01

    The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion. PMID:26841934

  11. Structure of the poly-C9 component of the complement membrane attack complex.

    PubMed

    Dudkina, Natalya V; Spicer, Bradley A; Reboul, Cyril F; Conroy, Paul J; Lukoyanova, Natalya; Elmlund, Hans; Law, Ruby H P; Ekkel, Susan M; Kondos, Stephanie C; Goode, Robert J A; Ramm, Georg; Whisstock, James C; Saibil, Helen R; Dunstone, Michelle A

    2016-02-04

    The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.

  12. The interaction of soluble human complement receptor type 1 (sCR1, BRL55730) with human complement component C4.

    PubMed

    Gibb, A L; Freeman, A M; Smith, R A; Edmonds, S; Sim, E

    1993-01-22

    Human CR1 is a membrane-bound protein which plays an important role in the control of the human complement system. In addition to its involvement in the processing and clearance of immune complexes with C3b or C4b on their surface, CR1 acts as a cofactor for the proteolysis of C3b and C4b by Factor I. sCR1 is a recombinant, soluble form of CR1 which retains the cofactor activities of CR1, and is of potential therapeutic value for the suppression of complement-mediated tissue damage in vivo. An assay has been established using microtitre plates to explore the binding of sCR1 to the two isotypes of C4, C4A and C4B, and to C4 fragments. Specific binding of 125I-sCR1 to C4b and ammonia-treated C4 has been demonstrated. The binding of 125I-sCR1 to ammonia-treated C4 is dependent on pH and ionic strength, decreasing with an increase in pH and with an increase in ionic strength. At physiological ionic strength, up to twice as much 125I-sCR1 bound to ammonia-treated C4A as bound to ammonia-treated C4B. This preference of sCR1 for binding to the C4A isotype has implications for the clinical association of immune complex disease with C4A null alleles.

  13. Genetic association of complement component 2 polymorphism with systemic lupus erythematosus.

    PubMed

    Chen, H-H; Tsai, L-J; Lee, K-R; Chen, Y-M; Hung, W-T; Chen, D-Y

    2015-08-01

    Complement component 2 (C2), an early member of the classical pathway, mainly participates in apoptotic cell clearance. We hypothesize that C2 polymorphism may confer genetic susceptibility to complement dysfunction in systemic lupus erythematosus (SLE). The major aim of our study was to investigate the clinical and serological associations of C2 variants in Chinese patients with SLE. The single-nucleotide polymorphism (rs2844455, G/A SNP) located in the intron region of C2 gene was genotyped by direct sequencing in 95 SLE patients and 95 matched normal control subjects. The gene expression profiles were generated by quantitative real-time polymerase chain reaction (PCR) and reverse transcription PCR. Our results showed that the AA genotype was observed more frequently in SLE patients than in normal control subjects (22.1% vs 9.5%, P < 0.05). The A allele was strongly associated with the occurrence of hair loss, photosensitivity and anti-cardiolipin antibodies; whereas, the G allele was associated with lower frequencies of these clinical presentations. Relative expression levels were significantly lower in patients with the AA genotype [median: 18.86, interquartile range (IQR) 11.36-22.43, P = 0.002] than in those with the GG genotype (35.76, IQR: 19.33-49.71). As expected, we confirmed the A allele as a risk factor for SLE development in a Chinese population, in contrast, the G allele might be a protective factor against the pathogenic autoantibody formation and cutaneous manifestations in SLE patients.

  14. Production and interferon-gamma-mediated regulation of complement component C2 and factors B and D by the astroglioma cell line U105-MG.

    PubMed Central

    Barnum, S R; Ishii, Y; Agrawal, A; Volanakis, J E

    1992-01-01

    In this paper, we demonstrate the synthesis of the complement component C2 and factors B and D by the human astroglioma cell line U105-MG. All three components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of complement proteins, and particularly of alternative pathway activation components (C3, factors B and D), may play an important role in host defence in the central nervous system. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:1445220

  15. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.

    PubMed

    Hofsteenge, J; Blommers, M; Hess, D; Furmanek, A; Miroshnichenko, O

    1999-11-12

    C-Mannosylation is a unique form of protein glycosylation, involving the C-glycosidic attachment of a mannosyl residue to the indole moiety of Trp. In the two examples found so far, human RNase 2 and interleukin-12, only the first Trp in the recognition motif WXXW is specifically C-mannosylated. To establish the generality of protein C-mannosylation, and to learn more about its mechanism, the terminal components of the human complement system (C6, C7, C8,and C9), which contain multiple and complex recognition motifs, were examined. Together with C5b they form the cytolytic agent, the membrane attack complex. These are the first proteins that are C-mannosylated on more than one Trp residue as follows: six in C6, four in C7, C8alpha, and C8beta, and two in C9. Thus, from the 113 Trp residues in the complete membrane attack complex, 50 were found to undergo C-mannosylation. The other important finding is that in C6, C7, C8, and C9 Trp residues without a second Trp (or another aromatic residue) at the +3 position can be C-mannosylated. This shows that they must contain an additional C-mannosylation signal. Whether this is encoded in the primary or tertiary structure is presently unknown. Finally, all modified Trp residues are part of the highly conserved core of the thrombospondin type 1 repeats present in these proteins. Since this module has been found in a large number of other proteins, the results suggest further candidates for C-mannosylation.

  16. Inherited deficiency of the second component of complement. Rheumatic disease associations.

    PubMed Central

    Glass, D; Raum, D; Gibson, D; Stillman, J S; Schur, P H

    1976-01-01

    The prevalence of homozygous and heterozygous deficiency of the second component of complement (C2) was determined in patients with rheumatic disease including 137 with systemic lupus erythematosus (SLE), 274 with juvenile rheumatoid arthritis, and 134 with rheumatoid arthritis. 1 C2 homozygous deficient and 19 possible heterozygous deficient individuals were identified by using both immunochemical and functional assays to determine C2 levels. Of the 20, 8 had SLE (5.9%), 10 had juvenile rheumatoid arthritis (3.7%), and 2 had rheumatoid arthritis (1.4%), the homozygous deficient individual having SLE. The prevalence of C2 deficiency in the SLE and juvenile rheumatoid arthritis patients was significantly increased (P = 0.0009 and P = 0.02, respectively) when compared with controls, 6 (1.2%) of 509 blood donors having C2 levels consistent with heterozygous deficiency. 15 of the 20 C2 deficient patients were HLA typed and found to have antigens A10(Aw25), B18, or both. The patients with C2 deficiency and SLE had earlier age of onset of disease and less antinuclear antibody when compared with the C2 normal SLE patients. 11 families of the propositi were studied and found to have one or more C2 heterozygous deficient individuals. The family members had an equal distribution of rheumatic disease and antinuclear antibody in the C2 deficient and C2 normal groups. C2 deficient individuals were found to have significantly lower levels of properdin Factor B (242 mug/ml+/-54) when compared with the non-C2 deficient family members (282 mug/ml+/-73). These data support the concept that inherited deficiency of C2 is significantly associated with both SLE and juvenile rheumatoid arthritis. PMID:965492

  17. Complement component 1, q subcomponent binding protein is a marker for proliferation in breast cancer.

    PubMed

    Scully, Olivia Jane; Yu, Yingnan; Salim, Agus; Thike, Aye Aye; Yip, George Wai-Cheong; Baeg, Gyeong Hun; Tan, Puay-Hoon; Matsumoto, Ken; Bay, Boon Huat

    2015-07-01

    Complement component 1, q subcomponent binding protein (C1QBP), is a multi-compartmental protein with higher mRNA expression reported in breast cancer tissues. This study evaluated the association between immunohistochemical expression of the C1QBP protein in breast cancer tissue microarrays (TMAs) and clinicopathological parameters, in particular tumor size. In addition, an in vitro study was conducted to substantiate the breast cancer TMA findings. Breast cancer TMAs were constructed from pathological specimens of patients diagnosed with invasive ductal carcinoma. C1QBP protein and proliferating cell nuclear antigen (PCNA) immunohistochemical analyses were subsequently performed in the TMAs. C1QBP immunostaining was detected in 131 out of 132 samples examined. The C1QBP protein was predominantly localized in the cytoplasm of the breast cancer cells. Univariate analysis revealed that a higher C1QBP protein expression was significantly associated with older patients (P = 0.001) and increased tumor size (P = 0.002). Multivariate analysis showed that C1QBP is an independent predictor of tumor size in progesterone-positive tumors. Furthermore, C1QBP was also significantly correlated with expression of PCNA, a known marker of proliferation. Inhibition of C1QBP expression was performed by transfecting C1QBP siRNA into T47D breast cancer cells, a progesterone receptor-positive breast cancer cell line. C1QBP gene expression was analyzed by real-time RT-PCR, and protein expression by Western blot. Cell proliferation assays were also performed by commercially available assays. Down-regulation of C1QBP expression significantly decreased cell proliferation and growth in T47D cells. Taken together, our findings suggest that the C1QBP protein could be a potential proliferative marker in breast cancer.

  18. Complement component 7 (C7), a potential tumor suppressor, is correlated with tumor progression and prognosis

    PubMed Central

    Pan, Xiaodan; Chen, Kaiyan; Zhang, Nan; Jin, Jiaoyue; Wu, Junzhou; Feng, Jianguo; Yu, Herbert; Jin, Hongchuan; Su, Dan

    2016-01-01

    Our previous study found copy number variation of chromosome fragment 5p13.1-13.3 might involve in the progression of ovarian cancer. In the current study, the alteration was validated and complement component 7 (C7), located on 5p13.1, was identified. To further explore the clinical value of C7 in tumors, 156 malignant, 22 borderline, 33 benign and 24 normal ovarian tissues, as well as 173 non-small cell lung cancer (NSCLC) tissues along with corresponding adjacent and normal tissues from the tissue bank of Zhejiang Cancer Hospital were collected. The expression of C7 was analyzed using reverse transcriptase quantitative polymerase chain reaction. As a result, the C7 expression displayed a gradual downward trend in normal, benign, borderline and malignant ovarian tissues, and the decreased expression of C7 was correlative to poor differentiation in patients with ovarian cancer. Interestingly, a similar change of expression of C7 was found in normal, adjacent and malignant tissues in patients with NSCLC, and low expression of C7 was associated with worse grade and advanced clinical stage. Both results from this cohort and the public database indicated that NSCLC patients with low expression of C7 had a worse outcome. Furthermore, multivariate cox regression analysis showed NSCLC patients with low C7 had a 3.09 or 5.65-fold higher risk for relapse or death than those with high C7 respectively, suggesting C7 was an independent prognostic predictor for prognoses of patients with NSCLC. Additionally, overexpression of C7 inhibited colony formation of NSCLC cells, which hints C7 might be a potential tumor suppressor. PMID:27852032

  19. Inhibition of miR-92d-3p enhances inflammation responses in genetically improved farmed tilapia (GIFT, Oreochromis niloticus) with Streptococcus iniae infection by modulating complement C3.

    PubMed

    Qiang, Jun; Tao, Yi-Fan; He, Jie; Li, Hong-Xia; Xu, Pao; Bao, Jin-Wen; Sun, Yi-Lan

    2017-04-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that regulate target gene expression by binding to the 3'-untranslated regions (3'-UTRs) of their target mRNAs. The miR-92 family is an important miRNA family, which was discovered to be related to regulation of tumor proliferation, apoptosis, invasion, and metastasis. Inhibition of miR-92d-3p was found previously in head kidney of genetically improved farmed tilapia (GIFT, Oreochromis niloticus) exposed to Streptococcus iniae infection. In this study, we found that miR-92d-3p regulated complement C3 mRNA levels by binding to its 3'-UTR by 3'-UTR luciferase reporter assay, and reduced miR-92d-3p expression resulted in increased C3 mRNA levels. We detected a negative relationship between the expression levels of miR-92d-3p and C3 in GIFT injected with miRNA antagomir. We performed in vivo functional analysis by miR-92d-3p silencing. Inhibition of miR-92d-3p levels in GIFT head kidney caused a significant increase in C3 expression, which consequently increased the white blood cell counts and interleukin-1β, tumor necrosis factor-α, and interferon-γ mRNA levels, all of which may help to activate the inflammatory response in GIFT post-infection with S. iniae. Our findings indicate that miR-92d-3p regulated C3 levels by binding with the C3 mRNA 3'-UTR, and this interaction affected S. iniae infection induction and the immune response in GIFT. We concluded that miR-92d-3p plays an important role in modulating the inflammatory response in GIFT head kidney. Our findings may contribute to understanding the mechanisms of miRNA-mediated gene regulation in tilapia in response to S. iniae infection.

  20. Long-term sublingual immunotherapy for Japanese cedar pollinosis and the levels of IL-17A and complement components 3a and 5a.

    PubMed

    Sakashita, Masafumi; Yamada, Takechiyo; Imoto, Yoshimasa; Hirota, Tomomitsu; Tamari, Mayumi; Ito, Yumi; Kubo, Seita; Osawa, Yoko; Takahashi, Noboru; Fujieda, Shigeharu

    2015-09-01

    Allergen-specific immunotherapy is the only treatment that can alter the natural course of allergic disease. We performed long-term sublingual immunotherapy (SLIT) for patients with seasonal allergic rhinitis caused by Japanese cedar pollen (SAR-JCP), screened molecules as candidate biomarkers, and investigated serum IL-17A and complement components 3a (C3a) and C5a in order to evaluate whether these molecules show changes correlated to symptom scores. In this study, we found that the long-term SLIT reduced the serum levels of IL-17A and C3a and C5a. The levels of C3a in the patients significantly decreased from year 1 compared with those at the baseline, and their levels of IL-17A significantly decreased from year 2 compared with those at baseline. The levels of IL-17A, C3a, and C5a at year 4 of SLIT were significantly lower than not only those at baseline, but also those at year 1. A significant positive correlation was found between the symptom medication scores and the levels of IL-17A at year 4. The symptom medication scores in the group in which IL-17A levels decreased at year 4 were significantly lower than those in the group without such a decrease. The serum level of IL-17A might prove useful as a biological parameter to ascertain the effectiveness of SLIT for patients with SAR-JCP. It is necessary to produce new therapeutics for non-responders in whom serum IL-17A levels are still higher against long-term SLIT.

  1. Atomic resolution model of the antibody Fc interaction with the complement C1q component.

    PubMed

    Schneider, Sebastian; Zacharias, Martin

    2012-05-01

    The globular C1q heterotrimer is a subunit of the C1 complement factor. Binding of the C1q subunit to the constant (Fc) part of antibody molecules is a first step and key event of complement activation. Although three-dimensional structures of C1q and antibody Fc subunits have been determined experimentally no atomic resolution structure of the C1q-Fc complex is known so far. Based on systematic protein-protein docking searches and Molecular Dynamics simulations a structural model of the C1q-IgG1-Fc-binding geometry has been obtained. The structural model is compatible with available experimental data on the interaction between the two partner proteins. It predicts a binding geometry that involves mainly the B-subunit of the C1q-trimer and both subunits of the IgG1-Fc-dimer with small conformational adjustments with respect to the unbound partners to achieve high surface complementarity. In addition to several charge-charge and polar contacts in the rim region of the interface it also involves nonpolar contacts between the two proteins and is compatible with the carbohydrate moiety of the Fc subunit. The model for the complex structure provides a working model for rationalizing available biochemical data on this important interaction and can form the basis for the design of Fc variants with a greater capacity to activate the complement system for example on binding to cancer cells or other target structures.

  2. Detection of complement activation by counterimmunoelectrophoresis (CIE).

    PubMed

    Arroyave, C M; Tan, E M

    1976-01-01

    Counterimmunoelectrophoresis (CIE) was used as a method of detecting activation of the third component of the complement system (C3). Highly purified C3, normal human serum (NHS), EDTA-treated plasma and serum activated with aggregated human immunoglobulin (agg-IgG) or inulin were used as sources of C3 and/or C3 split products. Activation of the alternative pathway of complement was assayed in the presence of EGTA (10 mM) and MgCl2 (0.3 mM), conditions which block activation of the classical pathway. When purified native C3, fresh NHS and fresh EDTA-plasma were tested in CIE against either antisera to whole C3 or to C3 split products, only one precipitin line was found, which was identified as native C3. However, when serum activated with agg-IgG or inulin were tested against the same reagents, two precipitin lines were seen. The first, with more cathodal mobility was identical to that of native C3. The second line had a more anodal mobility, was distinctly separated from the first and contained C3c and C3d as shown immunochemically with specific antisera. Native C3 and split products of C3 were identified by this CIE method in patients showing evidence of activated complement by having subnormal total complement (CH50) levels. When C3 split products were identified, the C3c-C3d precipitin line could always be distinguished from native C3 by its different electrophoretic mobility, even when C3 concentrations in serum varied from 0.25 mg/ml to 1.5 mg/ml. The sensitivity of CIE was compared to that of CH50 by asssaying at different time intervals after agg-IgG was added to fresh NHS. C3c-C3d split products were detected by CIE before any fall in CH50 and at all times when a significant decrease in CH50 was present. This study shows that the CIE technique is a highly sensitive, specific and rapid method for detecting activation of the complement system via classical or alternative pathways in human disease.

  3. Complement Component C1q Mediates Mitochondria-Driven Oxidative Stress in Neonatal Hypoxic–Ischemic Brain Injury

    PubMed Central

    Ten, Vadim S.; Yao, Jun; Ratner, Veniamin; Sosunov, Sergey; Fraser, Deborah A.; Botto, Marina; Baalasubramanian, Sivasankar; Morgan, B. Paul; Silverstein, Samuel; Stark, Raymond; Polin, Richard; Vannucci, Susan J.; Pinsky, David; Starkov, Anatoly A.

    2010-01-01

    Hypoxic–ischemic (HI) brain injury in infants is a leading cause of lifelong disability. We report a novel pathway mediating oxidative brain injury after hypoxia–ischemia in which C1q plays a central role. Neonatal mice incapable of classical or terminal complement activation because of C1q or C6 deficiency or pharmacologically inhibited assembly of membrane attack complex were subjected to hypoxia–ischemia. Only C1q−/− mice exhibited neuroprotection coupled with attenuated oxidative brain injury. This was associated with reduced production of reactive oxygen species (ROS) in C1q−/− brain mitochondria and preserved activity of the respiratory chain. Compared with C1q+/+ neurons, cortical C1q−/− neurons exhibited resistance to oxygen– glucose deprivation. However, postischemic exposure to exogenous C1q increased both mitochondrial ROS production and mortality of C1q−/− neurons. This C1q toxicity was abolished by coexposure to antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thus, the C1q component of complement, accelerating mitochondrial ROS emission, exacerbates oxidative injury in the developing HI brain. The terminal complement complex is activated in the HI neonatal brain but appeared to be nonpathogenic. These findings have important implications for design of the proper therapeutic interventions against HI neonatal brain injury by highlighting a pathogenic priority of C1q-mediated mitochondrial oxidative stress over the C1q deposition-triggered terminal complement activation. PMID:20147536

  4. Complement depletion with humanised cobra venom factor: efficacy in preclinical models of vascular diseases.

    PubMed

    Vogel, Carl-Wilhelm; Fritzinger, David C; Gorsuch, W Brian; Stahl, Gregory L

    2015-03-01

    The complement system is an intrinsic part of the immune system and has important functions in both innate and adaptive immunity. On the other hand, inadvertent or misdirected complement activation is also involved in the pathogenesis of many diseases, contributing solely or significantly to tissue injury and disease development. Multiple approaches to develop pharmacological agents to inhibit complement are currently being pursued. We have developed a conceptually different approach of not inhibiting but depleting complement, based on the complement-depleting activities of cobra venom factor (CVF), a non-toxic cobra venom component with structural and functional homology to complement component C3. We developed a humanised version of CVF by creating human complement component C3 derivatives with complement-depleting activities of CVF (humanised CVF) as a promising therapeutic agent for diseases with complement pathogenesis. Here we review the beneficial therapeutic effect of humanised CVF in several murine models of vascular diseases such as reperfusion injury.

  5. Complement activation in chronic liver disease.

    PubMed Central

    Munoz, L E; De Villiers, D; Markham, D; Whaley, K; Thomas, H C

    1982-01-01

    Patients with HBsAg positive chronic active liver disease (CALD) and primary biliary cirrhosis (PBC) exhibit increased C3d concentrations and changes in the serum concentrations of the complement components consistent with activation of the classical and alternative pathways. In these patients the concentrations of the regulatory proteins, C3b inactivator (C3bINA) and beta IH globulin, are normal. Patients with HBsAg negative CALD and alcohol induced liver disease (ALD) exhibit no evidence of an increased level of complement system activation. In these patients diminished serum concentrations of complement components appear to be related to diminished hepatic synthetic function. C4 synthesis may be specifically reduced in autoimmune chronic active liver disease. PMID:7083631

  6. Third component of complement, immunoglobulin deposition, and leucocyte attachment related to surface sulfate on larval Taenia taeniaeformis.

    PubMed

    Letonja, T; Hammerberg, B

    1983-08-01

    Cysticerci and strobilocerci of Taenia taeniaeformis were incubated with leucocytes from peritoneal washings of normal and T. taeniaeformis-infected rats in the presence of either normal sera or sera from infected rats. Leucocytes from infected and normal rats attached exclusively to the scolices but not the bladders of the larvae in the presence of serum from normal or infected rats. Heat inactivation at 56 C for 30 min destroyed the serum-mediated cell attachment. Histochemical staining of the larval taeniids with acid Alcian Blue demonstrated high concentrations of sulfated mucopolysaccharides on bladders that were not present on scolices. Immunofluorescent staining detected no difference in IgG deposition on the surfaces of bladders and scolices after incubation with rat sera in contrast to the markedly greater amounts of complement protein C3 found on scolices versus bladders. These results indicate that polysulfated substances on the bladder of this larval taeniid are associated with regional resistance to C3 deposition and leucocyte attachment.

  7. Genetics of the complement system.

    PubMed Central

    Lachmann, P

    1975-01-01

    The complement system, unlike the coagulation system, was largely characterized by in-vitro techniques which did not make use of genetically deficient plasmas. The existence of the genetically deficient plasmas. The existence of the genetically deficient subjects therefore has served largely to increase our knowledge of the in-vivo role of complement. At the present time its clearest role is in the resistance to infection; obviously in the case of C3 deficiency and bacterial infection and possibly more subtly in the case of deficiency of the early active complement components and low virulence organisms. There is so far no evidence that genetic complement deficiency interferes with antibody formation or with the generation of tolerance as has been suggested in the pas (Azar et al, 1968; Dukor and Hartmann, 1973). PMID:768477

  8. Altered cognitive performance and synaptic function in the hippocampus of mice lacking C3.

    PubMed

    Perez-Alcazar, Marta; Daborg, Jonny; Stokowska, Anna; Wasling, Pontus; Björefeldt, Andreas; Kalm, Marie; Zetterberg, Henrik; Carlström, Karl E; Blomgren, Klas; Ekdahl, Christine T; Hanse, Eric; Pekna, Marcela

    2014-03-01

    Previous work implicated the complement system in adult neurogenesis as well as elimination of synapses in the developing and injured CNS. In the present study, we used mice lacking the third complement component (C3) to elucidate the role the complement system plays in hippocampus-dependent learning and synaptic function. We found that the constitutive absence of C3 is associated with enhanced place and reversal learning in adult mice. Our findings of lower release probability at CA3-CA1 glutamatergic synapses in combination with unaltered overall efficacy of these synapses in C3 deficient mice implicate C3 as a negative regulator of the number of functional glutamatergic synapses in the hippocampus. The C3 deficient mice showed no signs of spontaneous epileptiform activity in the hippocampus. We conclude that C3 plays a role in the regulation of the number and function of glutamatergic synapses in the hippocampus and exerts negative effects on hippocampus-dependent cognitive performance.

  9. Molecular analysis of human complement component C5: localization of the structural gene to chromosome 9

    SciTech Connect

    Wetsel, R.A.; Lemons, R.S.; Le Beau, M.M.; Barnum, S.R.; Noack, D.; Tack, B.F.

    1988-03-08

    A human C5 clone (pC5HG2) was isolated from a cDNA library constructed from Hep G2 mRNA. he DNA sequence showed that the pC5HG2 insert was comprised of 3309 base pairs of pro-C5 coding sequence and 404 base pairs of 3'-untranslated sequence. The derived amino acid sequence contained the entire coding sequence of the C5 ..cap alpha..-chain, the ..beta..-..cap alpha..-chain junction region, and 100 amino acids (approximately 50%) of the ..beta..-chain. Protein sequences of four C5 tryptic peptides were aligned exactly to this sequence and demonstrated that C5 synthesized and secreted by Hep G2 cells is probably identical with plasma-derived C5. Coding sequence alignment of the human C5 sequences with those of murine C5 indicated that 80% of the nucleotides and 79% of the amino acids were placed identically in the two species. Amino acid sequence alignment of the homologous family members C3, C4, and ..cap alpha../sub 2/-macroglobulin with that of C5 demonstrated 27%, 25%, and 19% identity, respectively. As was found in murine C5, the corresponding thiol ester region of human C5 contained several conserved amino acids, but the critical cysteine and glutamine residues which give rise to the intramolecular thiol ester bond in C3, C4, and ..cap alpha../sub 2/-macroglobulin were absent in C5, having been replaced by serine and alanine, respectively. With the use of a panel of hamster-human somatic cell hybrids, the C5 gene was mapped to human chromosome 9. In situ chromosomal hybridization studies employing metaphase cells further localized the gene to bands 9q32-34, with the largest cluster of grains at 9q34.1.

  10. Update on C3 glomerulopathy

    PubMed Central

    Barbour, Thomas D.; Ruseva, Marieta M.; Pickering, Matthew C.

    2016-01-01

    C3 glomerulopathy refers to a disease process in which abnormal control of complement activation, degradation or deposition results in predominant C3 fragment deposition within the glomerulus and glomerular damage. Recent studies have improved our understanding of its pathogenesis. The key abnormality is uncontrolled C3b amplification in the circulation and/or along the glomerular basement membrane. Family studies in which disease segregates with structurally abnormal complement factor H-related (CFHR) proteins demonstrate that abnormal CFHR proteins are important in some types of C3 glomerulopathy. This is currently thought to be due to the ability of these proteins to antagonize the major negative regulator of C3 activation, complement factor H (CFH), a process termed ‘CFH de-regulation’. Recent clinicopathological cohort studies have led to further refinements in case definition, culminating in a 2013 consensus report, which provides recommendations regarding investigation and treatment. Early clinical experience with complement-targeted therapeutics, notably C5 inhibitors, has also now been published. Here, we summarize the latest developments in C3 glomerulopathy. PMID:25326473

  11. Phenotyping of human complement component C4, a class-III HLA antigen.

    PubMed Central

    Sim, E; Cross, S J

    1986-01-01

    The plasma complement protein C4 is encoded at two highly polymorphic loci, A and B, within the class-III region of the major histocompatibility complex. At least 34 different polymorphic variants of human C4 have been identified, including non-expressed or 'null' alleles. The main method of identification of C4 polymorphic allotypes is separation on the basis of charge by agarose-gel electrophoresis of plasma. On staining by immunofixation with anti-C4 antibodies, each C4 type gives three major bands, but, since individuals can have up to five allotypes, the overlapping banding pattern is difficult to interpret. We show that digestion of plasma samples with carboxypeptidase B, which removes C-terminal basic amino acids, before electrophoresis, produces a single, sharp, distinct band for each allotype and allows identification of the biochemical basis of the multiple banding pattern previously observed in C4 phenotype determination. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3103606

  12. The production and secretion of complement component C1q by human mast cells.

    PubMed

    van Schaarenburg, Rosanne A; Suurmond, Jolien; Habets, Kim L L; Brouwer, Mieke C; Wouters, Diana; Kurreeman, Fina A S; Huizinga, Tom W J; Toes, René E M; Trouw, Leendert A

    2016-10-01

    C1q is the initiation molecule of the classical pathway of the complement system and is produced by macrophages and immature dendritic cells. As mast cells share the same myeloid progenitor cells, we have studied whether also mast cells can produce and secrete C1q. Mast cells were generated in vitro from CD34+ progenitor cells from buffy coats or cord blood. Fully differentiated mast cells were shown by both RNA sequencing and qPCR to express C1QA, C1QB and C1QC. C1q produced by mast cells has a similar molecular make-up as serum C1q. Reconstituting C1q depleted serum with mast cell supernatant in haemolytic assays, indicated that C1q secreted by mast cells is functionally active. The level of C1q in supernatants produced under basal conditions was considerably enhanced upon stimulation with LPS, dexamethasone in combination with IFN- γ or via FcεRI triggering. Mast cells in human tissues stained positive for C1q in both healthy and in inflamed tissue. Moreover, mast cells in healthy and diseased skin appear to be the predominant C1q positive cells. Together, our data reveal that mast cells are able to produce and secrete functional active C1q and indicate mast cells as a local source of C1q in human tissue.

  13. High prevalence of complement component C6 deficiency among African-Americans in the South-eastern USA

    PubMed Central

    Zhu, Z-B; Atkinson, T P; Hovanky, K T; Boppana, S B; Dai, Y L; Densen, P; Go, R C P; Jablecki, J S; Volanakis, J E

    2000-01-01

    Complement component C6 is a part of the membrane attack complex that forms a pore-like structure in cell membranes following complement activation. Deficiency of terminal complement components including C6 predisposes individuals to infection with Neisseriae. Using polymerase chain reaction/single-strand conformation polymorphism analysis followed by DNA sequencing, we screened genomic DNA from 200 randomly chosen blacks and an equal number from whites for three loss-of-function C6 mutations. Ten blacks and two whites were found to be heterozygous for one of the mutations. Two of the mutations, 1195delC and 1936delG, were found exclusively in black individuals. A third previously undescribed mutation, 878delA, was found at equal frequency among the two groups. The difference between the two groups was significant (P = 0.027), indicating that C6 deficiency due to these three mutations is more common among blacks than whites in the local area, principally Jefferson County, Alabama. In addition, three previously undescribed point mutations, two of which result in amino acid substitutions, were identified within exon 6. A review of the county health department records over the past 6 years revealed a higher incidence of meningococcal meningitis in blacks due to serogroups Y and W-135 which paralleled the difference in the estimated prevalence of C6 deficiency. Among black residents of the county (n = 235 598) there were 15 cases of meningitis due to these two serogroups, compared with two cases in the white population (n = 422 604) (P = 0.002). We conclude that C6 deficiency is more common among blacks than whites in the south-eastern United States, with a frequency approaching 1 in 1600 black individuals. PMID:10632667

  14. Interactions of PLGA nanoparticles with blood components: protein adsorption, coagulation, activation of the complement system and hemolysis studies.

    PubMed

    Fornaguera, Cristina; Calderó, Gabriela; Mitjans, Montserrat; Vinardell, Maria Pilar; Solans, Conxita; Vauthier, Christine

    2015-04-14

    The intravenous administration of poly(lactic-co-glycolic) acid (PLGA) nanoparticles has been widely reported as a promising alternative for delivery of drugs to specific cells. However, studies on their interaction with diverse blood components using different techniques are still lacking. Therefore, in the present work, the interaction of PLGA nanoparticles with blood components was described using different complementary techniques. The influence of different encapsulated compounds/functionalizing agents on these interactions was also reported. It is worth noting that all these techniques can be simply performed, without the need for highly sophisticated apparatus or skills. Moreover, their transference to industries and application of quality control could be easily performed. Serum albumin was adsorbed onto all types of tested nanoparticles. The saturation concentration was dependent on the nanoparticle size. In contrast, fibrinogen aggregation was dependent on nanoparticle surface charge. The complement activation was also influenced by the nanoparticle functionalization; the presence of a functionalizing agent increased complement activation, while the addition of an encapsulated compound only caused a slight increase. None of the nanoparticles influenced the coagulation cascade at low concentrations. However, at high concentrations, cationized nanoparticles did activate the coagulation cascade. Interactions of nanoparticles with erythrocytes did not reveal any hemolysis. Interactions of PLGA nanoparticles with blood proteins depended both on the nanoparticle properties and the protein studied. Independent of their loading/surface functionalization, PLGA nanoparticles did not influence the coagulation cascade and did not induce hemolysis of erythrocytes; they could be defined as safe concerning induction of embolization and cell lysis.

  15. Functional analysis of Cobra Venom Factor/human C3 chimeras transiently expressed in mammalian cells.

    PubMed

    Kölln, Johanna; Matzas, Mark; Jänner, Nathalie; Mix, Thorsten; Klensang, Katrin; Bredehorst, Reinhard; Spillner, Edzard

    2004-05-01

    The complement activating venom component Cobra Venom Factor (CVF), a functional and structural homologue of the human complement component C3, forms a stable CVF-dependent C3 convertase complex, which, in contrast to C3-dependent convertase effects continuous activation of the complement and, thereby, decomplementation. In order to elucidate the mechanism underlying the enhanced activity of CVF compared to human C3, we generated two CVF/C3 chimeras and established different affinity-based assay systems for functional analysis of these constructs. To allow for convenient expression and subsequent functional characterisation, the CVF/C3 chimeras as well as CVF and C3 were transiently expressed in mammalian cells. Problems due to the low concentration of the recombinant proteins in the supernatants of transient expressions were circumvented by fusion to peptide tags enabling their efficient immobilisation onto suitable surfaces and subsequent characterisation. In an alternative approach monoclonal antibody fragments generated from a semisynthetic phage display scFv library were employed for concentrating the recombinant proteins by immunoprecipitation. Utilising both approaches all transiently expressed proteins could be characterised for their complement consumption activity. The data obtained with the CVF/C3 chimeras demonstrate that the increased stability of the CVFBb complex is independent of the domains in CVF corresponding to binding sites of factor B and H and the cleavage sites of factor I in the human C3 molecule.

  16. Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a

    PubMed Central

    McCaffary, David; Iqbal, Asif Jilani; Greaves, David R.

    2016-01-01

    Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of

  17. Netrin-1 Reduces Monocyte and Macrophage Chemotaxis towards the Complement Component C5a.

    PubMed

    Taylor, Lewis; Brodermann, Maximillian Hugo; McCaffary, David; Iqbal, Asif Jilani; Greaves, David R

    2016-01-01

    Netrin-1, acting at its cognate receptor UNC5b, has been previously demonstrated to inhibit CC chemokine-induced immune cell migration. In line with this, we found that netrin-1 was able to inhibit CCL2-induced migration of bone marrow derived macrophages (BMDMs). However, whether netrin-1 is capable of inhibiting chemotaxis to a broader range of chemoattractants remains largely unexplored. As our initial experiments demonstrated that RAW264.7 and BMDMs expressed high levels of C5a receptor 1 (C5aR1) on their surface, we aimed to determine the effect of netrin-1 exposure on monocyte/macrophage cell migration induced by C5a, a complement peptide that plays a major role in multiple inflammatory pathologies. Treatment of RAW264.7 macrophages, BMDMs and human monocytes with netrin-1 inhibited their chemotaxis towards C5a, as measured using two different real-time methods. This inhibitory effect was found to be dependent on netrin-1 receptor signalling, as an UNC5b blocking antibody was able to reverse netrin-1 inhibition of C5a induced BMDM migration. Treatment of BMDMs with netrin-1 had no effect on C5aR1 proximal signalling events, as surface C5aR1 expression, internalisation and intracellular Ca2+ release following C5aR1 ligation remained unaffected after netrin-1 exposure. We next examined receptor distal events that occur following C5aR1 activation, but found that netrin-1 was unable to inhibit C5a induced phosphorylation of ERK1/2, Akt and p38, pathways important for cellular migration. Furthermore, netrin-1 treatment had no effect on BMDM cytoskeletal rearrangement following C5a stimulation as determined by microscopy and real-time electrical impedance sensing. Taken together these data highlight that netrin-1 inhibits monocyte and macrophage cell migration, but that the mechanism behind this effect remains unresolved. Nevertheless, netrin-1 and its cognate receptors warrant further investigation as they may represent a potential avenue for the development of

  18. Infectious diseases associated with complement deficiencies.

    PubMed Central

    Figueroa, J E; Densen, P

    1991-01-01

    The complement system consists of both plasma and membrane proteins. The former influence the inflammatory response, immune modulation, and host defense. The latter are complement receptors, which mediate the cellular effects of complement activation, and regulatory proteins, which protect host cells from complement-mediated injury. Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components. Four aspects of the complement cascade are critical to its function and regulation: (i) activation of the classical pathway, (ii) activation of the alternative pathway, (iii) C3 convertase formation and C3 deposition, and (iv) membrane attack complex assembly and insertion. In general, mechanisms evolved by pathogenic microbes to resist the effects of complement are targeted to these four steps. Because individual complement proteins subserve unique functional activities and are activated in a sequential manner, complement deficiency states are associated with predictable defects in complement-dependent functions. These deficiency states can be grouped by which of the above four mechanisms they disrupt. They are distinguished by unique epidemiologic, clinical, and microbiologic features and are most prevalent in patients with certain rheumatologic and infectious diseases. Ethnic background and the incidence of infection are important cofactors determining this prevalence. Although complement undoubtedly plays a role in host defense against many microbial pathogens, it appears most important in protection against encapsulated bacteria, especially Neisseria meningitidis but also Streptococcus pneumoniae, Haemophilus influenzae, and, to a lesser extent, Neisseria gonorrhoeae. The availability of effective polysaccharide vaccines and antibiotics provides an immunologic and chemotherapeutic rationale for preventing and treating infection in patients with these deficiencies. PMID

  19. Functional cooperation of xenoproteins after hamster-to-rat liver transplantation: With particular reference to hamster C3 and secretory component for rat IgA

    PubMed Central

    Celli, S.; Valdivia, L.A.; Kelly, R.H.; Demetris, A.J.; Fung, J.J.; Rao, A.S.; Pan, F.; Tsugita, M.; Starzl, T.E.

    2010-01-01

    Long-term survival after hamster-to-rat liver xenotransplantation has provided the opportunity to study the posttransplantation source of major serum proteins and the functional consequences of several different receptor-ligand interactions, where one or the other is a xenogeneic protein. We report here that serum albumin, α-1-antitrypsin, complement component 3, and other acute phase reactants switch from recipient to donor origin during the first week after transplantation while serum immunoglobulins remain largely that of recipient. Despite the disparate source of complement (hamster) and immunoglobulins (rat), these two proteins were able to cooperate effectively to produce lysis of sheep red blood cells. Moreover, rat IgA was successfully processed by hamster hepatocytes and biliary epithelial cells, being present in the bile of successful liver xenograft recipients within one day after transplantation. The ability of these liver xenograft recipients to survive long-term in conventional and viral-free animal facilities without grossly obvious morbidity or unusual susceptibility to stress, suggests that xenogeneic proteins are able to successfully interact with several different physiologic systems in the hamster-to-rat combination. PMID:21318076

  20. Identification and structural characterization of two incompletely processed forms of the fourth component of human complement.

    PubMed Central

    Chan, A C; Atkinson, J P

    1983-01-01

    Immunoprecipitates of human C4 from EDTA-plasma were incubated with [14C]methylamine and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and fluorography. In addition to finding label in the alpha-chains of the secreted (C4s) and predominant plasma (C4p) forms of C4, two additional molecules with apparent molecular weights of approximately 168,000 (p168) and approximately 125,000 (p125) covalently incorporated methylamine, indicating the presence of an internal thioester bond. These two molecules were present at a concentration of approximately 5% of total plasma C4 and were not immunoprecipitated by antisera to C3 or alpha 2-macroglobulin. A human hepatoma-derived cell line (HepG2), in addition to synthesizing C4s and small quantities of the polypeptide precursor of C4 (pro-C4), was found to secrete p168 and p125 at concentrations of 14 +/- 4.8 and 21 +/- 9.2% (mean +/- SD), respectively, of total secreted C4. These molecules were not found intracellularly. Both molecules were present on reduced, but not nonreduced, SDS-polyacrylamide gels. Chido (C4B) and Rodgers' (C4A) alloantisera precipitated the C4A and C4B variants of pro-C4, p168, p125, and C4s. Both tryptic and Staphylococcus aureus V8 protease peptide analyses showed homology between p168 and the beta- and alpha-chains and between p125 and the alpha- and gamma-chains. Partial NH2-terminal sequencing revealed that the beta-chain was NH2-terminal in p168 and that the alpha-chain was NH2-terminal in p125. Taken together, these data indicate that p168 and p125 represent uncleaved beta-alpha- and alpha-gamma-fragments of pro-C4, respectively. Thus, in most individuals, plasma C4 consists of five structurally distinct molecules, the single polypeptide precursor (pro-C4), the three-subunit secreted (C4s) and predominant plasma (C4p) forms of C4, and two incompletely processed two-subunit molecules with uncleaved beta-alpha- (p168) or uncleaved alpha-gamma (p125)-subunits. In addition

  1. Principal Component Regression and Linear Mixed Model in Association Analysis of Structured Samples: Competitors or Complements?

    PubMed Central

    Zhang, Yiwei; Pan, Wei

    2014-01-01

    Genome-wide association studies (GWAS) have been established as a major tool to identify genetic variants associated with complex traits, such as common diseases. However, GWAS may suffer from false positives and false negatives due to confounding population structures, including known or unknown relatedness. Another important issue is unmeasured environmental risk factors. Among many methods for adjusting for population structures, two approaches stand out: one is principal component regression (PCR) based on principal component analysis (PCA), which is perhaps most popular due to its early appearance, simplicity and general effectiveness; the other is based on a linear mixed model (LMM) that has emerged recently as perhaps the most flexible and effective, especially for samples with complex structures as in model organisms. As shown previously, the PCR approach can be regarded as an approximation to a LMM; such an approximation depends on the number of the top principal components (PCs) used, the choice of which is often difficult in practice. Hence, in the presence of population structure, the LMM appears to outperform the PCR method. However, due to the different treatments of fixed versus random effects in the two approaches, we show an advantage of PCR over LMM: in the presence of an unknown but spatially confined environmental confounder (e.g. environmental pollution or life style), the PCs may be able to implicitly and effectively adjust for the confounder while the LMM cannot. Accordingly, to adjust for both population structures and non-genetic confounders, we propose a hybrid method combining the use and thus strengths of PCR and LMM. We use real genotype data and simulated phenotypes to confirm the above points, and establish the superior performance of the hybrid method across all scenarios. PMID:25536929

  2. Determination of the second component of complement (C2) by electroimmunoassay in sera from patients with systemic lupus erythematosus.

    PubMed Central

    Ueda, A; Kusaba, T; Yanase, T

    1983-01-01

    The concentration of C2 was determined by electroimmunoassay in sera from healthy controls, patients with systemic lupus erythematosus (SLE), their relatives and patients with other diseases. Monospecific anti-human C2 serum was obtained by immunizing rabbits with purified human C2 and then absorbing the rabbit serum with inactivated normal human serum that was made insoluble. In addition, it was shown that human C2 could be purified by means of affinity chromatography on anti-C2 antibody coupled Sepharose. The serum concentration of C2 was 37.8 +/- 5.0 (s.d.) micrograms/ml in healthy controls (n = 133). In patients with SLE, the values were below normal in the active phase and were within normal limits in the inactive phase, showing good correlations with other complement parameters such as CH50, C4, C3 and factor B. C2 concentration was well correlated with C2 haemolytic activity in the inactive phase of SLE, but there was no relationship between the two in the active phase. The mean value of C2 concentration in the relatives of patients with SLE showed no significant difference from that in healthy controls. C2 concentration tended to be high in patients with scleroderma, polymyositis, rheumatoid arthritis, Behçet's disease and aortitis syndrome. However, the values were often low in patients with chronic liver diseases, suggesting a decrease of C2 production in the liver. Images Fig. 2 Fig. 3 PMID:6409476

  3. Prosteatotic and Protective Components in a Unique Model of Fatty Liver: Gut Microbiota and Suppressed Complement System

    PubMed Central

    Liu, Long; Zhao, Xing; Wang, Qian; Sun, Xiaoxian; Xia, Lili; Wang, Qianqian; Yang, Biao; Zhang, Yihui; Montgomery, Sean; Meng, He; Geng, Tuoyu; Gong, Daoqing

    2016-01-01

    Goose can develop severe hepatic steatosis without overt injury, thus it may serve as a unique model for uncovering how steatosis-related injury is prevented. To identify the markedly prosteatotic and protective mechanisms, we performed an integrated analysis of liver transcriptomes and gut microbial metagenomes using samples collected from overfed and normally-fed geese at different time points. The results indicated that the fatty liver transcriptome, initially featuring a ‘metabolism’ pathway, was later joined by ‘cell growth and death’ and ‘immune diseases’ pathways. Gut microbiota played a synergistic role in the liver response as microbial and hepatic genes affected by overfeeding shared multiple pathways. Remarkably, the complement system, an inflammatory component, was comprehensively suppressed in fatty liver, which was partially due to increased blood lactic acid from enriched Lactobacillus. Data from in vitro studies suggested that lactic acid suppressed TNFα via the HNF1α/C5 pathway. In conclusion, gut microbes and their hosts respond to excess energy influx as an organic whole, severe steatosis and related tolerance of goose liver may be partially attributable to gut microbiotic products and suppressed complement system, and lactic acid from gut microbiota participates in the suppression of hepatic TNFα/inflammation through the HNF1α/C5 pathway. PMID:27550859

  4. Angioedema induced by a peptide derived from complement component C2

    PubMed Central

    1988-01-01

    Synthetic peptides that correspond to the COOH-terminal portion of C2b enhance vascular permeability in human and guinea pig skin. In human studies, 1 nmol of the most active peptide of 25-amino acid residues produced substantial local edema. A pentapeptide and a heptapeptide corresponding to the COOH-terminal sequence of C2b each induced contraction of estrous rat uterus in the micromole range; a peptide of 25 amino acids from this region induced a like contraction of rat uterus at a concentration 20-fold lower than the smaller peptides. The vascular permeability of guinea pig skin was enhanced by doses of these synthetic peptides in a similar fashion as that observed for the concentration of rat uterus. The induction of localized edema by intradermal injection in both the guinea pig and the human proceeds in the presence of antihistaminic drugs, suggesting that there is a histamine-independent component to the observed increase in vascular permeability. Cleavage of C2 with the enzymic subcomponent of C1, C1s, yields only C2a and C2b, and no small peptides, whereas cleavage of C2 with C1s and plasmin yields a set of small peptides. These plasmin- cleaved peptides are derived from the COOH terminus of C2b, and they induce the contraction of estrous rat uterus. PMID:2972793

  5. Interactions of PLGA nanoparticles with blood components: protein adsorption, coagulation, activation of the complement system and hemolysis studies

    NASA Astrophysics Data System (ADS)

    Fornaguera, Cristina; Calderó, Gabriela; Mitjans, Montserrat; Vinardell, Maria Pilar; Solans, Conxita; Vauthier, Christine

    2015-03-01

    The intravenous administration of poly(lactic-co-glycolic) acid (PLGA) nanoparticles has been widely reported as a promising alternative for delivery of drugs to specific cells. However, studies on their interaction with diverse blood components using different techniques are still lacking. Therefore, in the present work, the interaction of PLGA nanoparticles with blood components was described using different complementary techniques. The influence of different encapsulated compounds/functionalizing agents on these interactions was also reported. It is worth noting that all these techniques can be simply performed, without the need for highly sophisticated apparatus or skills. Moreover, their transference to industries and application of quality control could be easily performed. Serum albumin was adsorbed onto all types of tested nanoparticles. The saturation concentration was dependent on the nanoparticle size. In contrast, fibrinogen aggregation was dependent on nanoparticle surface charge. The complement activation was also influenced by the nanoparticle functionalization; the presence of a functionalizing agent increased complement activation, while the addition of an encapsulated compound only caused a slight increase. None of the nanoparticles influenced the coagulation cascade at low concentrations. However, at high concentrations, cationized nanoparticles did activate the coagulation cascade. Interactions of nanoparticles with erythrocytes did not reveal any hemolysis. Interactions of PLGA nanoparticles with blood proteins depended both on the nanoparticle properties and the protein studied. Independent of their loading/surface functionalization, PLGA nanoparticles did not influence the coagulation cascade and did not induce hemolysis of erythrocytes; they could be defined as safe concerning induction of embolization and cell lysis.The intravenous administration of poly(lactic-co-glycolic) acid (PLGA) nanoparticles has been widely reported as a promising

  6. Production of the second component of complement by human monocytes: stimulation by antigen-activated lymphocytes or lymphokines

    PubMed Central

    1977-01-01

    Human peripheral blood mononuclear cells cultured in the presence of antigen produced hemolytically active second complement component earlier and in larger amounts than did control cultures of the same cells without antigen. The increased amount of C2 in culture supernates came primarily from the adherent cell population and was due to increased synthesis as demonstrated by inhibition with 10(-4) M cycloheximide. Purified adherent monocytes produced more C2 when exposed to lymphokine-rich supernates from antigen-stimulated lymphocytes than when exposed to control supernates from unstimulated lymphocyte cultures. The increased synthesis of C2, which appeared to be mediated by a lymphokine, was partially inhibited specifically by 0.025 M alpha-L(-) fucose, a sugar which has previously been shown in inhibit the response of macrophages to migration inhibitory factor. PMID:858999

  7. Analysis of C3 Suggests Three Periods of Positive Selection Events and Different Evolutionary Patterns between Fish and Mammals

    PubMed Central

    Meng, Fanxing; Sun, Yuena; Liu, Xuezhu; Wang, Jianxin; Xu, Tianjun; Wang, Rixin

    2012-01-01

    Background The third complement component (C3) is a central protein of the complement system conserved from fish to mammals. It also showed distinct characteristics in different animal groups. Striking features of the fish complement system were unveiled, including prominent levels of extrahepatic expression and isotypic diversity of the complement components. The evidences of the involvement of complement system in the enhancement of B and T cell responses found in mammals indicated that the complement system also serves as a bridge between the innate and adaptive responses. For the reasons mentioned above, it is interesting to explore the evolutionary process of C3 genes and to investigate whether the huge differences between aquatic and terrestrial environments affected the C3 evolution between fish and mammals. Methodology/Principal Findings Analysis revealed that these two groups of animals had experienced different evolution patterns. The mammalian C3 genes were under purifying selection pressure while the positive selection pressure was detected in fish C3 genes. Three periods of positive selection events of C3 genes were also detected. Two happened on the ancestral lineages to all vertebrates and mammals, respectively, one happened on early period of fish evolutionary history. Conclusions/Significance Three periods of positive selection events had happened on C3 genes during history and the fish and mammals C3 genes experience different evolutionary patterns for their distinct living environments. PMID:22624039

  8. Cleavage of the second component of complement by plasma proteases: implications in hereditary C1-inhibitor deficiency.

    PubMed Central

    Smith, M A; Kerr, M A

    1985-01-01

    EDTA plasma from patients with hereditary angioedema (HAE), the genetic deficiency of C1-inhibitor, when incubated at 37 degrees produces a kinin-like activity which can induce contraction of oestrus rat uterus. The second component of complement (C2) has previously been suggested to be the source of this kinin-like activity, with the implication that C2-kinin is a normal product of complement activation. Our results show that purified human C2 is cleaved rapidly to C2a and C2b when added to HAE plasma, but not normal plasma or plasma from a danazol-treated HAE patient. However, the addition to HAE plasma of C2 at 20 X normal plasma concentration had no effect on the kinin activity generated on incubation at 37 degrees. In the presence of soya bean trypsin inhibitor, the rate of C2 cleavage and products were unaltered but no kinin activity was generated. C2 was cleaved by purified C1s to C2a and C2b. Incubation of C2 with trypsin resulted in cleavage to C2a and C2b followed by more extensive cleavage of both C2a and C2b. Kallikrein cleaved C2 to C2a and C2b but plasmin had no effect on C2. In no case was kinin activity generated. When C2 was cleaved by C1s to C2a and C2b then incubated with trypsin, kallikrein, or plasmin, no kinin activity was generated: only trypsin cleaved the C2 fragments further. The results suggest that C2 is not the source of the kinin-like activity generated in hereditary angioedema plasma. Images Figure 4 Figure 5 Figure 6 Figure 7 PMID:2934317

  9. Complement component 4

    MedlinePlus

    ... antiseptic). The health care provider wraps an elastic band around the upper arm to apply pressure to ... or tube attached to the needle. The elastic band is removed from your arm. Once the blood ...

  10. C3b deposition on human erythrocytes induces the formation of a membrane skeleton–linked protein complex

    PubMed Central

    Karnchanaphanurach, Pallop; Mirchev, Rossen; Ghiran, Ionita; Asara, John M.; Papahadjopoulos-Sternberg, Brigitte; Nicholson-Weller, Anne; Golan, David E.

    2009-01-01

    Decay-accelerating factor (DAF, also known as CD55), a glycosylphosphatidylinositol-linked (GPI-linked) plasma membrane protein, protects autologous cells from complement-mediated damage by inhibiting complement component 3 (C3) activation. An important physical property of GPI-anchored complement regulatory proteins such as DAF is their ability to translate laterally in the plasma membrane. Here, we used single-particle tracking and tether-pulling experiments to measure DAF lateral diffusion, lateral confinement, and membrane skeletal associations in human erythrocyte membranes. In native membranes, most DAF molecules exhibited Brownian lateral diffusion. Fluid-phase complement activation caused deposition of C3b, one of the products of C3 cleavage, onto erythrocyte glycophorin A (GPA). We then determined that DAF, C3b, GPA, and band 3 molecules were laterally immobilized in the membranes of complement-treated cells, and GPA was physically associated with the membrane skeleton. Mass spectrometry analysis further showed that band 3, α-spectrin, β-spectrin, and ankyrin were present in a complex with C3b and GPA in complement-treated cells. C3b deposition was also associated with a substantial increase in erythrocyte membrane stiffness and/or viscosity. We therefore suggest that complement activation stimulates the formation of a membrane skeleton–linked DAF-C3b-GPA–band 3 complex on the erythrocyte surface. This complex may promote the removal of senescent erythrocytes from the circulation. PMID:19258706

  11. High temperature stress and its effect on pollen development and morphological components of harvest index in the C3 model grass Brachypodium distachyon.

    PubMed

    Harsant, Jeffrey; Pavlovic, Lazar; Chiu, Greta; Sultmanis, Stefanie; Sage, Tammy L

    2013-07-01

    The effect of high temperatures on harvest index (HI) and morphological components that contribute to HI was investigated in two lines (Bd21 and Bd21-3) of Brachypodium distachyon, a C3 grass recognized as a tractable plant, to address critical issues associated with enhancing cereal crop yields in the presence of global climate change. The results demonstrated that temperatures ≥32 °C eliminated HI. Reductions in yield at 32 °C were due primarily to declines in pollen viability, retention of pollen in anthers, and pollen germination, while abortion of microspores by the uninucleate stage that was correlated with abnormal tapetal development resulted in yield failure at 36 °C. Increasing temperatures from 24 to 32 °C resulted in reductions in tiller numbers but had no impact on axillary branch numbers per tiller. Grain developed at 24 and 28 °C primarily in tiller spikes, although spikes on axillary branches also formed grain. Grain quantity decreased in tiller spikes but increased in axillary branch spikes as temperatures rose from 24 to 28 °C. Differential patterns of axillary branching and floret development within spikelets between Bd21 and Bd21-3 resulted in higher grain yield in axillary branches of Bd21-3 at 28 °C. The response of male reproductive development and tiller branching patterns in B. distachyon to increasing temperatures mirrors that in other cereal crops, providing support for the use of this C3 grass in assessing the molecular control of HI in the presence of global warming.

  12. Complement's hidden arsenal: New insights and novel functions inside the cell.

    PubMed

    Liszewski, M Kathryn; Elvington, Michelle; Kulkarni, Hrishikesh S; Atkinson, John P

    2017-04-01

    A key component of both innate and adaptive immunity, new understandings of the complement system are expanding its roles beyond that traditionally appreciated. Evidence is accumulating that complement has an intracellular arsenal of components that provide not only immune defense, but also assist in key interactions for host cell functions. Although early work has primarily centered on T cells, the intracellular complement system likely functions in many if not most cells of the body. Some of these functions may trace their origins to the primitive complement system that began as a primeval form of C3 likely tasked for protection from intracellular pathogen invasion. This later expanded to include extracellular defense as C3 became a secreted protein to patrol the vasculature. Other components were added to the growing system including regulators to protect host cells from the indiscriminate effects of this potent system. Contemporary cells may retain some of these vestigial remnants. We now know that a) C3 serves as a damage-associated molecular pattern (in particular by coating pathogens that translocate into cells), b) most cells store C3 and recycle C3(H2O) for immediate use, and c) C3 assists in cellular survival and metabolic reprogramming. Other components also are part of this hidden arsenal including C5, properdin, factors H and B, and complement receptors. Importantly, better definition of the intracellular complement system may translate into new target discovery to assist in creating the next generation of complement therapeutics.

  13. Detection of immunoglobulins and complement components in formalin fixed and paraffin embedded renal biopsy material by immunoflourescence technique

    PubMed Central

    Mubarak, Muhammed; Kazi Javed, I; Kulsoom, Umme; Ishaque, Muhammed

    2012-01-01

    Background The technique of direct immunoflourescence (IF) is essential in the accurate diagnosis of renal glomerular diseases. The optimal results are obtained when the procedure is done on fresh frozen tissue (IF-F). However, techniques are available for IF study on formalin fixed and paraffin embedded (FFPE) renal biopsy specimens with variable reported success rates. Objectives We evaluated three such techniques on FFPE tissue and compared the results with those obtained by IF-F from the same patients. Materials and Methods Heat treatment with Tris buffer and citrate buffer, and pronase treatment of the FFPE material was carried out. Direct IF was done for renal panel immunoglobulins and complement components on all biopsies and the results were compared with the historical IF-F study. Results When compared to the IF-F, the immunoflourescence staining on the paraffin sections was less sensitive and less intense in all immune complex-mediated renal diseases, but the diagnostic findings were detected in majority of the cases. Conclusions In conclusion, it is possible to establish the diagnosis in most cases of immune complex-mediated glomerular diseases with IF on paraffin embedded tissue specimens. PMID:24475396

  14. Isolation of cDNA clones specifying the fourth component of mouse complement and its isotype, sex-limited protein.

    PubMed Central

    Nonaka, M; Takahashi, M; Natsuume-Sakai, S; Nonaka, M; Tanaka, S; Shimizu, A; Honjo, T

    1984-01-01

    cDNA clones specific for the fourth component of mouse complement (C4) and its hormonally regulated isotype, sex-linked protein (Slp), were isolated using as a probe a 20-mer synthetic oligonucleotide corresponding to a known sequence of human C4 cDNA. Two types of clones, one specific for C4 (pFC4/10, with a 3.7 kilobase insert) and one specific for Slp (pFSlp/1, with a 4.7 kilobase insert), were isolated from liver cDNA libraries constructed from the Slp-producing FM mouse strain. The cDNA inserts of these clones shared 70% of the restriction sites determined. Only one type of clone was isolated from the Slp-negative DBA/1 strain; this type showed restriction maps indistinguishable from that of pFC4/10. pFC4/10 and pFSlp/1 displayed extensive homology: 94% nucleotide homology and 89% derived amino acid homology in the C4a region and 92% nucleotide homology and 89% derived amino acid homology in the thiol-ester region. An Arg-Gln-Lys-Arg sequence in the beta-alpha junction and a Cys-Ala-Glu-Gln sequence in the thiol-ester site were identified for both proteins. A remarkable divergency between C4 and Slp sequences was recognized in the region immediately following the C4a sequence. PMID:6208559

  15. The Complement Anaphylatoxins C5a and C3a Suppress IFN-β Production in Response to Listeria monocytogenes by Inhibition of the Cyclic Dinucleotide-Activated Cytosolic Surveillance Pathway.

    PubMed

    Mueller-Ortiz, Stacey L; Calame, Daniel G; Shenoi, Nancy; Li, Yi-Dong; Wetsel, Rick A

    2017-03-08

    Listeria monocytogenes is an intracellular Gram-positive bacterium that induces expression of type I IFNs (IFN-α/IFN-β) during infection. These cytokines are detrimental to the host during infection by priming leukocytes to undergo L. monocytogenes-mediated apoptosis. Our previous studies showed that C5aR1(-/-) and C3aR(-/-) mice are highly susceptible to L. monocytogenes infection as a result of increased IFN-β-mediated apoptosis of major leukocyte cell populations, including CD4(+) and CD8(+) T cells. However, the mechanisms by which C3a and C5a modulate IFN-β expression during L. monocytogenes infection were not examined in these initial investigations. Accordingly, we report in this article that C5a and C3a suppress IFN-β production in response to L. monocytogenes via cyclic di-AMP (c-di-AMP), a secondary messenger molecule of L. monocytogenes, in J774A.1 macrophage-like cells and in bone marrow-derived dendritic cells (BMDCs). Moreover, C5a and C3a suppress IFN-β production by acting through their respective receptors, because no inhibition was seen in C5aR1(-/-) or C3aR(-/-) BMDCs, respectively. C5a and C3a suppress IFN-β production in a manner that is dependent on Bruton's tyrosine kinase, p38 MAPK, and TANK-binding kinase 1 (TBK1), as demonstrated by the individual use of Bruton's tyrosine kinase, p38 MAPK, and TBK1 inhibitors. Pretreatment of cells with C5a and C3a reduced the expression of the IFN-β signaling molecules DDX41, STING, phosphorylated TBK1, and phosphorylated p38 MAPK in wild-type BMDCs following treatment with c-di-AMP. Collectively, these data demonstrate that C3a and C5a, via direct signaling through their specific receptors, suppress IFN-β expression by modulation of a distinct innate cytosolic surveillance pathway involving DDX41, STING, and other downstream molecular targets of L. monocytogenes-generated c-di-AMP.

  16. Risk Assessment via Metabolism and Cell Growth Inhibition in a HepG2/C3A Cell Line Upon Treatment with Arpadol and its Active Component Harpagoside.

    PubMed

    Biazi, Bruna Isabela; D'Epiro, Gláucia Fernanda Rocha; Zanetti, Thalita Alves; de Oliveira, Marcelo Tempesta; Ribeiro, Lucia Regina; Mantovani, Mário Sérgio

    2017-03-01

    Harpagophytum procumbens (Hp) has been used as antiinflammatory and analgesic agent for the treatment of rheumatic diseases. The principal active component of Hp is harpagoside (HA). We tested the toxicity of this new therapeutic agent in a hepatic cell line (HepG2/C3A). Hp was found to be cytotoxic, and HA was found to decrease the number of cells in S phase, increase the number of cells in G2/M phase and induce apoptosis. Neither Hp nor HA was genotoxic. The expression of CDK6 and CTP3A4 was reduced by Hp, and both HA and Hp caused a significant reduction of CYP1A2 and CYP3A4 expression. It is possible that the cytotoxicity caused by HA and Hp does not involve transcriptional regulation of the cyclins and CDKs tested but is instead related to the inhibition of metabolism. This is evidenced by the results of an MTT assay and changes in the expression of genes related to drug metabolism, leading to cell death. Indeed, the cells exhibited decreased proliferation upon exposure to Hp and HA. The data show that treatment with either Hp or HA can be cytotoxic, and this should be taken into consideration when balancing the risks and benefits of treatments for rheumatic diseases. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Membranoproliferative glomerulonephritis with isolated C3 deposits: case report and literature review.

    PubMed

    Darouich, Sihem; Goucha, Rym; Jaafoura, Mohamed Habib; Zekri, Semy; Kheder, Adel; Ben Maiz, Hédi

    2011-02-01

    Membranoproliferative glomerulonephritis with isolated C3 deposits (MPGNC3) is an uncommon condition characterized by overt glomerular C3 deposits in the absence of immunoglobulins and intramembranous dense deposits. Here the authors describe the clinical and morphological features of primary MPGNC3 in a 13-year-old boy and critically review the previously published cases. The patient presented with nephrotic syndrome and microscopic hematuria. Blood tests revealed very low circulating C3 levels. The renal biopsy exhibited subendothelial, subepithelial, and mesangial deposits, with C3 but not immunoglobulins seen on immunofluorescence. This case and the review of the literature indicate that the serum complement profile with decreased levels of C3 and normal levels of classical pathway components together with glomerular deposits containing exclusively complement C3 is highly suggestive of alternative pathway activation. The diagnosis of acquired and/or genetic complement abnormalities in some cases supports that complement dysregulation is implicated in the pathogenesis of MPGNC3. Such data show great promise to provide new therapy strategies based on modulation of the complement system activity.

  18. Amelioration of lupus-like autoimmune disease in NZB/WF1 mice after treatment with a blocking monoclonal antibody specific for complement component C5.

    PubMed Central

    Wang, Y; Hu, Q; Madri, J A; Rollins, S A; Chodera, A; Matis, L A

    1996-01-01

    New Zealand black x New Zealand white (NZB/W) F1 mice spontaneously develop an autoimmune syndrome with notable similarities to human systemic lupus erythematosus. Female NZB/WF1 mice produce high titers of antinuclear antibodies and invariably succumb to severe glomerulonephritis by 12 months of age. Although the development of the immune-complex nephritis is accompanied by abundant local and systemic complement activation, the role of proinflammatory complement components in disease progression has not been established. In this study we have examined the contribution of activated terminal complement proteins to the pathogenesis of the lupus-like autoimmune disease. Female NZB/W F1 mice were treated with a monoclonal antibody (mAb) specific for the C5 component of complement that blocks the cleavage of C5 and thus prevents the generation of the potent proinflammatory factors C5a and C5b-9. Continuous therapy with anti-C5 mAb for 6 months resulted in significant amelioration of the course of glomerulonephritis and in markedly increased survival. These findings demonstrate an important role for the terminal complement cascade in the progression of renal disease in NZB/W F1 mice, and suggest that mAb-mediated C5 inhibition may be a useful approach to the therapy of immune-complex glomerulonephritis in humans. Images Fig. 3 Fig. 5 PMID:8710910

  19. Complement Component 5 Mediates Development of Fibrosis, via Activation of Stellate Cells, in 2 Mouse Models of Chronic Pancreatitis

    PubMed Central

    Sendler, Matthias; Beyer, Georg; Mahajan, Ujjwal M.; Kauschke, Vivien; Maertin, Sandrina; Schurmann, Claudia; Homuth, Georg; Völker, Uwe; Völzke, Henry; Halangk, Walter; Wartmann, Thomas; Weiss, Frank-Ulrich; Hegyi, Peter; Lerch, Markus M.; Mayerle, Julia

    2015-01-01

    Background & Aims Little is known about the pathogenic mechanisms of chronic pancreatitis. We investigated the roles of complement component 5 (C5) in pancreatic fibrogenesis in mice and patients. Methods Chronic pancreatitis was induced by ligation of the midpancreatic duct, followed by a single supramaximal intraperitoneal injection of cerulein, in C57Bl6 (control) and C5-deficient mice. Some mice were given injections of 2 different antagonists of the receptor for C5a over 21 days. In a separate model, mice were given injections of cerulein for 10 weeks to induce chronic pancreatitis. Direct effects of C5 were studied in cultured primary cells. We performed genotype analysis for the single-nucleotide polymorphisms rs 17611 and rs 2300929 in C5 in patients with pancreatitis and healthy individuals (controls). Blood cells from 976 subjects were analyzed by transcriptional profiling. Results During the initial phase of pancreatitis, levels of pancreatic damage were similar between C5-deficient and control mice. During later stages of pancreatitis, C5-deficient mice and mice given injections of C5a-receptor antagonists developed significantly less pancreatic fibrosis than control mice. Primary pancreatic stellate cells were activated in vitro by C5a. There were no differences in the rs 2300929 SNP between subjects with or without pancreatitis, but the minor allele rs17611 was associated with a significant increase in levels of C5 in whole blood. Conclusions In mice, loss of C5 or injection of a C5a-receptor antagonist significantly reduced the level of fibrosis of chronic pancreatitis, but this was not a consequence of milder disease in early stages of pancreatitis. C5 might be a therapeutic target for chronic pancreatitis. PMID:26001927

  20. The role of complement in AMD.

    PubMed

    Zipfel, Peter F; Lauer, Nadine; Skerka, Christine

    2010-01-01

    Age related macular degeneration (AMD) is a common form of blindness in the western world and genetic variations of several complement genes, including the complement regulator Factor H, the central complement component C3, Factor B, C2, and also Factor I confer a risk for the disease. However deletion of a chromosomal segment in the Factor H gene cluster on human chromosome 1, which results in the deficiency of the terminal pathway regulator CFHR1, and of the putative complement regulator CFHR3 has a protective effect for development of AMD. The Factor H gene encodes two proteins Factor H and FHL1 which are derived from alternatively processed transcripts. In particular a sequence variation at position 402 of both Factor H and FHL1 is associated with a risk for AMD. A tyrosine residue at position 402 represents the protective and a histidine residue the risk variant. AMD is considered a chronic inflammatory disease, which can be caused by defective and inappropriate regulation of the continuously activated alternative complement pathway. This activation generates complement effector products and inflammatory mediators that stimulate further inflammatory reactions. Defective regulation can lead to formation of immune deposits, drusen and ultimately translate into damage of retinal pigment epithelial cells, rupture of the interface between these epithelial cells and the Bruch's membrane and vision loss. Here we describe the role of complement in the retina and summarize the current concept how defective or inappropriate local complement control contributes to inflammation and the pathophysiology of AMD.

  1. Monoclonal antibody OKB7, which identifies the 14OKd complement receptor type 2 (CR/sub 2/), also identifies a 72Kd secreted fragment of CR/sub 2/ that contains the C3d-binding site

    SciTech Connect

    Myones, B.L.; Ross, G.D.

    1986-03-05

    CR/sub 2/ is a 140-145Kd glycoprotein expressed on B lymphocytes which binds both C3d and Epstein-Barr virus (EBV). OKB7, an IgG/sub 2a/ monoclonal antibody to CR/sub 2/, blocks C3d and EBV binding, while HB-5, another monoclonal IgG/sub 2a/ anti-CR/sub 2/, does not. A 72Kd C3d-binding glycoprotein (gp72), isolated from Raji cell media, was previously thought to be CR/sub 2/ because a polyclonal rabbit anti-gp72 inhibited EC3d rosettes. ELISA assay demonstrated that OKB7, but not HB-5, bound to purified gp72 fixed to microtiter wells. Insoluble and soluble gp72 blocked Raji cell uptake of /sup 125/I-labeled OKB7, but not labeled anti-B2 or HB-5. Rabbit anti-gp72 immunoprecipitated bands at 140Kd and 72Kd from /sup 125/I-labelled and solubilized B cell membranes. Culture media from Raji cells grown in the presence /sup 3/H-labeled amino acids was sequentially immunoprecipitated by irrelevant antibody, OKB7, and HB-5. A single 72Kd radiolabeled band was demonstrated only with OKB7, and this was identical to that produced by the immunoprecipitation of /sup 125/I-labeled gp72 with rabbit anti-gp72. Thus, OKB7, which identifies the 140Kd CR/sub 2/ molecule, also identifies a 72Kd shed fragment of CR/sub 2/ isolated from Raji cell media, which contains the C3d-binding site.

  2. Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

    PubMed

    Moulton, Elizabeth A; Bertram, Paula; Chen, Nanhai; Buller, R Mark L; Atkinson, John P

    2010-09-01

    Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

  3. C3 Polymorphism Influences Circulating Levels of C3, ASP and Lipids in Schizophrenic Patients.

    PubMed

    Nsaiba, Mohamed Jalloul; Lapointe, Marc; Mabrouk, Hajer; Douki, Wahiba; Gaha, Lotfi; Pérusse, Louis; Bouchard, Claude; Jrad, Besma Bel Hadj; Cianflone, Katherine

    2015-05-01

    Excessive activation of complement is associated with many diseases including schizophrenia. Investigation of C3 polymorphisms, circulating C3, cleavage product ASP/C3adesArg, and lipid metabolism. Cross-sectional analysis. C3 genotyping (CC vs GG for R102L) was performed on 434 Tunisian people consisting of 272 schizophrenic (SZ) patients and 162 control subjects. In a age- and gender-matched subgroups of the three genotypes (131 SZ and 112 NOR), plasma triglycerides, total cholesterol (C), LDL-C, HDL-C, ASP, and complement C3 were measured. C3 gene polymorphism influences BMI and plasma C3, ASP, triglyceride, total cholesterol, LDL-C and HDL-C among SZ patients (p < 0.05-0.0001), with increasing values demonstrated from CC (common form) to CG (heterozygote form) to GG (rare homozygote) forms. Significant correlations between plasma C3 and BMI, triglyceride, HDL-C and ASP (p < 0.05-0.0001) were observed, while ASP correlated with BMI and LDL-C (p = 0.005, p = 0.001, respectively) in SZ patients. Further, proportional conversion of C3 to ASP (%ASP/C3) also increased (p < 0.0001, GG>CG>CC). C3 polymorphisms and plasma C3, ASP and %ASP/C3 correlated with lipid parameters in this SZ population, suggesting that factors predisposing patients to schizophrenia are permissive for complement pathway activation and dyslipidemic influences.

  4. Sundanese Complementation

    ERIC Educational Resources Information Center

    Kurniawan, Eri

    2013-01-01

    The focus of this thesis is the description and analysis of clausal complementation in Sundanese, an Austronesian language spoken in Indonesia. The thesis examined a range of clausal complement types in Sundanese, which consists of (i) "yen/(wi)rehna" "that" complements, (ii) "pikeun" "for" complements,…

  5. Intracellular complement activation sustains T cell homeostasis and mediates effector differentiation.

    PubMed

    Liszewski, M Kathryn; Kolev, Martin; Le Friec, Gaelle; Leung, Marilyn; Bertram, Paula G; Fara, Antonella F; Subias, Marta; Pickering, Matthew C; Drouet, Christian; Meri, Seppo; Arstila, T Petteri; Pekkarinen, Pirkka T; Ma, Margaret; Cope, Andrew; Reinheckel, Thomas; Rodriguez de Cordoba, Santiago; Afzali, Behdad; Atkinson, John P; Kemper, Claudia

    2013-12-12

    Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While "tonic" intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.

  6. The Interaction of Classical Complement Component C1 with Parasite and Host Calreticulin Mediates Trypanosoma cruzi Infection of Human Placenta

    PubMed Central

    Castillo, Christian; Ramírez, Galia; Valck, Carolina; Aguilar, Lorena; Maldonado, Ismael; Rosas, Carlos; Galanti, Norbel; Kemmerling, Ulrike; Ferreira, Arturo

    2013-01-01

    Background 9 million people are infected with Trypanosoma cruzi in Latin America, plus more than 300,000 in the United States, Canada, Europe, Australia, and Japan. Approximately 30% of infected individuals develop circulatory or digestive pathology. While in underdeveloped countries transmission is mainly through hematophagous arthropods, transplacental infection prevails in developed ones. Methodology/Principal Findings During infection, T. cruzi calreticulin (TcCRT) translocates from the endoplasmic reticulum to the area of flagellum emergence. There, TcCRT acts as virulence factor since it binds maternal classical complement component C1q that recognizes human calreticulin (HuCRT) in placenta, with increased parasite infectivity. As measured ex vivo by quantitative PCR in human placenta chorionic villi explants (HPCVE) (the closest available correlate of human congenital T. cruzi infection), C1q mediated up to a 3–5-fold increase in parasite load. Because anti-TcCRT and anti-HuCRT F(ab′)2 antibody fragments are devoid of their Fc-dependent capacity to recruit C1q, they reverted the C1q-mediated increase in parasite load by respectively preventing its interaction with cell-bound CRTs from both parasite and HPCVE origins. The use of competing fluid-phase recombinant HuCRT and F(ab′)2 antibody fragments anti-TcCRT corroborated this. These results are consistent with a high expression of fetal CRT on placental free chorionic villi. Increased C1q-mediated infection is paralleled by placental tissue damage, as evidenced by histopathology, a damage that is ameliorated by anti-TcCRT F(ab′)2 antibody fragments or fluid-phase HuCRT. Conclusions/Significance T. cruzi infection of HPCVE is importantly mediated by human and parasite CRTs and C1q. Most likely, C1q bridges CRT on the parasite surface with its receptor orthologue on human placental cells, thus facilitating the first encounter between the parasite and the fetal derived placental tissue. The results

  7. Simple Method To Distinguish between Primary and Secondary C3 Deficiencies

    PubMed Central

    Pereira de Carvalho Florido, Marlene; Ferreira de Paula, Patrícia; Isaac, Lourdes

    2003-01-01

    Due to the increasing numbers of reported clinical cases of complement deficiency in medical centers, clinicians are now more aware of the role of the complement system in the protection against infections caused by microorganisms. Therefore, clinical laboratories are now prepared to perform a number of diagnostic tests of the complement system other than the standard 50% hemolytic component assay. Deficiencies of alternative complement pathway proteins are related to severe and recurrent infections; and the application of easy, reliable, and low-cost methods for their detection and distinction are always welcome, notably in developing countries. When activation of the alternative complement pathway is evaluated in hemolytic agarose plates, some but not all human sera cross-react to form a late linear lysis. Since the formation of this linear lysis is dependent on C3 and factor B, it is possible to use late linear lysis to routinely screen for the presence of deficiencies of alternative human complement pathway proteins such as factor B. Furthermore, since linear lysis is observed between normal human serum and primary C3-deficient serum but not between normal human serum and secondary C3-deficient serum caused by the lack of factor H or factor I, this assay may also be used to discriminate between primary and secondary C3 deficiencies. PMID:12626445

  8. Deletion of both ICAM-1 and C3 Enhances Severity of Experimental Autoimmune Encephalomyelitis Compared to C3-Deficient Mice

    PubMed Central

    Smith, Sherry S.; Ludwig, Michael; Wohler, Jillian E.; Bullard, Daniel C.; Szalai, Alex J.; Barnum, Scott R.

    2008-01-01

    Multiple sclerosis (MS) is an autoimmune disease characterized by central nervous system (CNS) inflammation and leukocyte infiltration, demyelination of neurons, and blood-brain barrier breakdown. The development of experimental autoimmune encephalomyelitis (EAE), the animal model for MS is dependent on a number of components of the immune system including complement and adhesion molecules. Previous studies in our lab have examined the role of C3, the central complement component, and intercellular adhesion molecule-1 (ICAM-1) a key cell adhesion molecule involved in leukocyte trafficking to sites of inflammation including the CNS. In these studies we demonstrated that myelin oligodendrocyte glycoprotein (MOG)-induced EAE is markedly attenuated in both ICAM-1−/− and C3−/− mice. Given the pivotal role that these proteins play in EAE, we hypothesized that EAE in ICAM-1−/− and C3−/− double mutant mice would likely fail to develop. Unexpectedly, EAE in ICAM-1−/− × C3−/− mice was only modestly attenuated compared to wild type mice and significantly worse than C3−/− mice. Leukocyte infiltration was commensurate with disease severity between the three groups of mice. Spinal cord T cells from ICAM-1−/− × C3−/− mice produced the highest levels of IFN-γ and TNF-α, despite reduced disease severity compared to wild type mice. The mechanisms behind the elevated EAE severity in ICAM-1−/− × C3−/− mice may relate to altered homing of leukocytes or processing of self-antigens in the double mutant background. PMID:18634851

  9. Complement-mediated Opsonization of Invasive Group A Streptococcus pyogenes Strain AP53 Is Regulated by the Bacterial Two-component Cluster of Virulence Responder/Sensor (CovRS) System*

    PubMed Central

    Agrahari, Garima; Liang, Zhong; Mayfield, Jeffrey A.; Balsara, Rashna D.; Ploplis, Victoria A.; Castellino, Francis J.

    2013-01-01

    Group A Streptococcus pyogenes (GAS) strain AP53 is a primary isolate from a patient with necrotizing fasciitis. These AP53 cells contain an inactivating mutation in the sensor component of the cluster of virulence (cov) responder (R)/sensor (S) two-component gene regulatory system (covRS), which enhances the virulence of the primary strain, AP53/covR+S−. However, specific mechanisms by which the covRS system regulates the survival of GAS in humans are incomplete. Here, we show a key role for covRS in the regulation of opsonophagocytosis of AP53 by human neutrophils. AP53/covR+S− cells displayed potent binding of host complement inhibitors of C3 convertase, viz. Factor H (FH) and C4-binding protein (C4BP), which concomitantly led to minimal C3b deposition on AP53 cells, further showing that these plasma protein inhibitors are active on GAS cells. This resulted in weak killing of the bacteria by human neutrophils and a corresponding high death rate of mice after injection of these cells. After targeted allelic alteration of covS− to wild-type covS (covS+), a dramatic loss of FH and C4BP binding to the AP53/covR+S+ cells was observed. This resulted in elevated C3b deposition on AP53/covR+S+ cells, a high level of opsonophagocytosis by human neutrophils, and a very low death rate of mice infected with AP53/covR+S+. We show that covRS is a critical transcriptional regulator of genes directing AP53 killing by neutrophils and regulates the levels of the receptors for FH and C4BP, which we identify as the products of the fba and enn genes, respectively. PMID:23928307

  10. Genetic control of the alternative pathway of complement in humans and age-related macular degeneration.

    PubMed

    Hecker, Laura A; Edwards, Albert O; Ryu, Euijung; Tosakulwong, Nirubol; Baratz, Keith H; Brown, William L; Charbel Issa, Peter; Scholl, Hendrik P; Pollok-Kopp, Beatrix; Schmid-Kubista, Katharina E; Bailey, Kent R; Oppermann, Martin

    2010-01-01

    Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.

  11. Complement factor B expression profile in a spontaneous uveitis model.

    PubMed

    Zipplies, Johanna K; Kirschfink, Michael; Amann, Barbara; Hauck, Stefanie M; Stangassinger, Manfred; Deeg, Cornelia A

    2010-12-01

    Equine recurrent uveitis serves as a spontaneous model for human autoimmune uveitis. Unpredictable relapses and ongoing inflammation in the eyes of diseased horses as well as in humans lead to destruction of the retina and finally result in blindness. However, the molecular mechanisms leading to inflammation and retinal degeneration are not well understood. An initial screening for differentially regulated proteins in sera of uveitic cases compared to healthy controls revealed an increase of the alternative pathway complement component factor B in ERU cases. To determine the activation status of the complement system, sera were subsequently examined for complement split products. We could demonstrate a significant higher concentration of the activation products B/Ba, B/Bb, Bb neoantigen, iC3b and C3d in uveitic condition compared to healthy controls, whereas for C5b-9 no differences were detected. Additionally, we investigated complement activation directly in the retina by immunohistochemistry, since it is the main target organ of this autoimmune disease. Interestingly, infiltrating cells co-expressed activated factor Bb neoantigen, complement split product C3d as well as CD68, a macrophage marker. In this study, we could demonstrate activation of the complement system both systemically as well as in the eye, the target organ of spontaneous recurrent uveitis. Based on these novel findings, we postulate a novel role for macrophages in connection with complement synthesis at the site of inflammation.

  12. AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins.

    PubMed

    Foster, J A; Friday, B B; Maulit, M T; Blobel, C; Winfrey, V P; Olson, G E; Kim, K S; Gerton, G L

    1997-05-09

    The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in

  13. Incorporation of Host Complement Regulatory Proteins into Newcastle Disease Virus Enhances Complement Evasion

    PubMed Central

    Biswas, Moanaro; Johnson, John B.; Kumar, Sandeep R. P.; Parks, Griffith D.

    2012-01-01

    Newcastle disease virus (NDV), an avian paramyxovirus, is inherently tumor selective and is currently being considered as a clinical oncolytic virus and vaccine vector. In this study, we analyzed the effect of complement on the neutralization of NDV purified from embryonated chicken eggs, a common source for virus production. Fresh normal human serum (NHS) neutralized NDV by multiple pathways of complement activation, independent of neutralizing antibodies. Neutralization was associated with C3 deposition and the activation of C2, C3, C4, and C5 components. Interestingly, NDV grown in mammalian cell lines was resistant to complement neutralization by NHS. To confirm whether the incorporation of regulators of complement activity (RCA) into the viral envelope afforded complement resistance, we grew NDV in CHO cells stably transfected with CD46 or HeLa cells, which strongly express CD46 and CD55. NDV grown in RCA-expressing cells was resistant to complement by incorporating CD46 and CD55 on virions. Mammalian CD46 and CD55 molecules on virions exhibited homologous restriction, since chicken sera devoid of neutralizing antibodies to NDV were able to effectively neutralize these virions. The incorporation of chicken RCA into NDV produced in embryonated eggs similarly provided species specificity toward chicken sera. PMID:22973037

  14. Complement Evasion Mediated by Enhancement of Captured Factor H: Implications for Protection of Self-Surfaces from Complement

    PubMed Central

    Herbert, Andrew P.; Makou, Elisavet; Chen, Zhuo A.; Kerr, Heather; Richards, Anna; Rappsilber, Juri

    2015-01-01

    In an attempt to evade annihilation by the vertebrate complement system, many microbes capture factor H (FH), the key soluble complement-regulating protein in human plasma. However, FH is normally an active complement suppressor exclusively on self-surfaces and this selective action of FH is pivotal to self versus non-self discrimination by the complement system. We investigated whether the bacterially captured FH becomes functionally enhanced and, if so, how this is achieved at a structural level. We found, using site-directed and truncation mutagenesis, surface plasmon resonance, nuclear magnetic resonance spectroscopy, and cross-linking and mass spectrometry, that the N-terminal domain of Streptococcus pneumoniae protein PspC (PspCN) not only binds FH extraordinarily tightly but also holds it in a previously uncharacterized conformation. Functional enhancement arises from exposure of a C-terminal cryptic second binding site in FH for C3b, the activation-specific fragment of the pivotal complement component, C3. This conformational change of FH doubles its affinity for C3b and increases 5-fold its ability to accelerate decay of the binary enzyme (C3bBb) responsible for converting C3 to C3b in an amplification loop. Despite not sharing critical FH-binding residues, PspCNs from D39 and Tigr4 S. pneumoniae exhibit similar FH-anchoring and enhancing properties. We propose that these bacterial proteins mimic molecular markers of self-surfaces, providing a compelling hypothesis for how FH prevents complement-mediated injury to host tissue while lacking efficacy on virtually all other surfaces. In hemolysis assays with 2-aminoethylisothiouronium bromide–treated erythrocytes that recapitulate paroxysmal nocturnal hemoglobinuria, PspCN enhanced protection of cells by FH, suggesting a new paradigm for therapeutic complement suppression. PMID:26459349

  15. RECEPTOR FOR SOLUBLE C3 AND C3b ON HUMAN LYMPHOBLASTOID (RAJI) CELLS

    PubMed Central

    Theofilopoulos, Argyrios N.; Bokisch, Viktor A.; Dixon, Frank J.

    1974-01-01

    This study describes the presence of a receptor for fluid phase human C3 and C3b on Raji cell membranes. The binding of C3 and C3b was demonstrated indirectly by a fluoresceinated anti-C3 serum and directly by using radioiodinated proteins. No other complement proteins or serum factors were needed to mediate binding of C3 and C3b to the receptor. The possibility of enzymatic cleavage of C3 before or after its attachment on the cell membrane was ruled out by the demonstration of antigenically intact C3 on Raji cells. Inhibition and dissociation of Raji cell-EAC1423 rosettes by C3 and C3b indicated that both of these proteins bind to the same receptor site or closely associated receptor sites on Raji cells. C3b-bearing Raji cells were immune adherence negative, indicating that C3b binding to the receptor is brought about through the immune adherence region of the molecule and not the C3d portion. The C3 receptor on Raji cell membranes is uniformly distributed and can move on the membrane plane. Approximately 4 x 105 molecules of C3 or C3b bind per Raji cell. The receptor had a higher affinity for C3 than C3b, as was shown by uptake experiments and inhibition of Raji cell-EAC1423 rosette formation. Apart from the described receptor for C3 and C3b another specific receptor for C3b inactivator-cleaved C3b (C3d) bound to red cells was shown to be present on Raji cells. Raji cells cultured in medium containing fresh normal human serum and cobra venom factor were lysed. Similar results were obtained when C3b-bearing Raji cells were cultured in medium with fresh normal human serum. The lytic effect could be abolished by inactivating serum C3 proactivator (C3PA) and required C6. It was concluded that C3b bound to the Raji cell membrane activates the complement system through the alternate pathway and results in membrane damage and cytolysis. It is postulated that cell destruction by this mechanism may play an important role in vivo in controlling cell growth. PMID:4591176

  16. Target deletion of complement component 9 attenuates antibody-mediated hemolysis and lipopolysaccharide (LPS)-induced acute shock in mice

    PubMed Central

    Fu, Xiaoyan; Ju, Jiyu; Lin, Zhijuan; Xiao, Weiling; Li, Xiaofang; Zhuang, Baoxiang; Zhang, Tingting; Ma, Xiaojun; Li, Xiangyu; Ma, Chao; Su, Weiliang; Wang, Yuqi; Qin, Xuebin; Liang, Shujuan

    2016-01-01

    Terminal complement membrane attack complex (MAC) formation is induced initially by C5b, followed by the sequential condensation of the C6, C7, C8. Polymerization of C9 to the C5b-8 complex forms the C5b-9 (or MAC). The C5b-9 forms lytic or non lytic pores in the cell membrane destroys membrane integrity. The biological functionalities of MAC has been previously investigated by using either the mice deficient in C5 and C6, or MAC’s regulator CD59. However, there is no available C9 deficient mice (mC9−/−) for directly dissecting the role of C5b-9 in the pathogenesis of human diseases. Further, since C5b-7 and C5b-8 complexes form non lytic pore, it may also plays biological functionality. To better understand the role of terminal complement cascades, here we report a successful generation of mC9−/−. We demonstrated that lack of C9 attenuates anti-erythrocyte antibody-mediated hemolysis or LPS-induced acute shock. Further, the rescuing effect on the acute shock correlates with the less release of IL-1β in mC9−/−, which is associated with suppression of MAC-mediated inflammasome activation in mC9−/−. Taken together, these results not only confirm the critical role of C5b-9 in complement-mediated hemolysis and but also highlight the critical role of C5b-9 in inflammasome activation. PMID:27444648

  17. Complement c3 is inversely associated with habitual intake of provitamin A but not with dietary fat, fatty acids, or vitamin E in middle-aged to older white adults and positively associated with intake of retinol in middle-aged to older white women.

    PubMed

    van Greevenbroek, Marleen M J; Arts, Ilja C W; van der Kallen, Carla J H; Dagnelie, Pieter C; Ferreira, Isabel; Jansen, Eugene; Schalkwijk, Casper G; Feskens, Edith J M; Stehouwer, Coen D A

    2014-01-01

    Complement factor 3 (C3) has been identified as a novel risk factor for obesity-associated cardiometabolic diseases. Data in the literature suggest that C3 concentrations may be influenced by diet. Therefore, we investigated the associations of intake of total fat, specific fatty acids, and fat-soluble vitamin E (and individual tocopherols) and vitamin A (and its dietary precursors) with circulating C3. In a white cohort [Cohort on Diabetes and Atherosclerosis Maastricht (CODAM); n = 501; 59.4 ± 7.1 y; 61% men], associations of habitual nutrient intake (assessed by a food-frequency questionnaire) with circulating C3 were evaluated by using cross-sectional multiple linear regression analyses. Adjustments were first performed for age, sex, glucose metabolism status (i.e., impaired glucose metabolism or type 2 diabetes), and energy intake and subsequently for BMI, waist circumference, alcohol intake, smoking behavior, and season of blood collection. No associations with C3 were observed for total dietary fat intake or intake of specific fatty acids [saturated, monounsaturated, polyunsaturated, n-6 (ω-6), and n-3 (ω- 3) fatty acids], vitamin E, or individual tocopherols. We observed an inverse association with intake of provitamin A carotenoids α-carotene (in μg/d; regression coefficient β = -0.075; 95% CI: -0.140, -0.010; P = 0.025) and β-carotene (in μg/d; β = -0.021; 95% CI: -0.044, 0.002; P = 0.068) with C3 (in mg/L). In contrast, and only in women, dietary retinol intake (in μg/d) was positively associated with C3 (β = 0.116; 95% CI: 0.014, 0.218; P = 0.026; n = 196). In conclusion, these data suggest that fasting concentrations of C3 may, in a complex manner, be modifiable by variation in dietary provitamin A carotenoids and/or retinol content of the usual diet but most likely not by variations in fat composition and vitamin E content.

  18. The Complement System Component C5a Produces Thermal Hyperalgesia via Macrophage-to-Nociceptor Signaling That Requires NGF and TRPV1

    PubMed Central

    Shutov, Leonid P.; Warwick, Charles A.; Shi, Xiaoyu; Gnanasekaran, Aswini; Shepherd, Andrew J.; Mohapatra, Durga P.; Woodruff, Trent M.; Clark, J. David

    2016-01-01

    The complement cascade is a principal component of innate immunity. Recent studies have underscored the importance of C5a and other components of the complement system in inflammatory and neuropathic pain, although the underlying mechanisms are largely unknown. In particular, it is unclear how the complement system communicates with nociceptors and which ion channels and receptors are involved. Here we demonstrate that inflammatory thermal and mechanical hyperalgesia induced by complete Freund's adjuvant was accompanied by C5a upregulation and was markedly reduced by C5a receptor (C5aR1) knock-out or treatment with the C5aR1 antagonist PMX53. Direct administration of C5a into the mouse hindpaw produced strong thermal hyperalgesia, an effect that was absent in TRPV1 knock-out mice, and was blocked by the TRPV1 antagonist AMG9810. Immunohistochemistry of mouse plantar skin showed prominent expression of C5aR1 in macrophages. Additionally, C5a evoked strong Ca2+ mobilization in macrophages. Macrophage depletion in transgenic macrophage Fas-induced apoptosis mice abolished C5a-dependent thermal hyperalgesia. Examination of inflammatory mediators following C5a injection revealed a rapid upregulation of NGF, a mediator known to sensitize TRPV1. Preinjection of an NGF-neutralizing antibody or Trk inhibitor GNF-5837 prevented C5a-induced thermal hyperalgesia. Notably, NGF-induced thermal hyperalgesia was unaffected by macrophage depletion. Collectively, these results suggest that complement fragment C5a induces thermal hyperalgesia by triggering macrophage-dependent signaling that involves mobilization of NGF and NGF-dependent sensitization of TRPV1. Our findings highlight the importance of macrophage-to-neuron signaling in pain processing and identify C5a, NGF, and TRPV1 as key players in this cross-cellular communication. SIGNIFICANCE STATEMENT This study provides mechanistic insight into how the complement system, a key component of innate immunity, regulates the

  19. The alternative complement component factor B regulates UV-induced oedema, systemic suppression of contact and delayed hypersensitivity, and mast cell infiltration into the skin.

    PubMed

    Byrne, Scott N; Hammond, Kirsten J L; Chan, Carling Y-Y; Rogers, Linda J; Beaugie, Clare; Rana, Sabita; Marsh-Wakefield, Felix; Thurman, Joshua M; Halliday, Gary M

    2015-04-01

    Ultraviolet (UV) wavelengths in sunlight are the prime cause of skin cancer in humans with both the UVA and UVB wavebands making a contribution to photocarcinogenesis. UV has many different biological effects on the skin that contribute to carcinogenesis, including suppression of adaptive immunity, sunburn and altering the migration of mast cells into and away from irradiated skin. Many molecular mechanisms have been identified as contributing to skin responses to UV. Recently, using gene set enrichment analysis of microarray data, we identified the alternative complement pathway with a central role for factor B (fB) in UVA-induced immunosuppression. In the current study we used mice genetically deficient in fB (fB-/- mice) to study the functional role of the alternative complement pathway in skin responses to UV. We found that fB is required for not only UVA but also UVB-induced immunosuppression and solar-simulated UV induction of the oedemal component of sunburn. Factor B-/- mice had a larger number of resident skin mast cells than control mice, but unlike the controls did not respond to UV by increasing mast cell infiltration into the skin. This study provides evidence for a function role for fB in skin responses to UV radiation. Factor B regulates UVA and UVB induced immunosuppression, UV induced oedema and mast cell infiltration into the skin. The alternative complement pathway is therefore an important regulator of skin responses to UV.

  20. Novel Mechanistic Interplay between Products of Oxidative Stress and Components of the Complement System in AMD Pathogenesis.

    PubMed

    Du, Hongjun; Xiao, Xu; Stiles, Travis; Douglas, Christopher; Ho, Daisy; Shaw, Peter X

    2016-02-01

    Age-related macular degeneration (AMD) is a leading cause of vision loss affecting tens of millions of elderly worldwide. Early AMD includes soft drusen and pigmentary changes in the retinal pigment epithelium (RPE). As people age, such soft confluent drusen can progress into two forms of advanced AMD, geographic atrophy (GA, or dry AMD) or choroidal neovascularization (CNV, or wet AMD) and result in the loss of central vision. The exact mechanism for developing early AMD and progressing to advanced stage of disease is still largely unknown. However, significant evidence exists demonstrating a complex interplay of genetic and environmental factors as the cause of AMD progression. Together, complement factor H (CFH) and HTRA1/ARMS polymorphisms contribute to more than 50% of the genetic risk for AMD. Environmentally, oxidative stress from activities such as smoking has also demonstrated a powerful contribution to AMD progression. To extend our previous finding that genetic polymorphisms in CFH results in OxPLs and the risk-form of CFH (CFH Y402H) has reduced affinity for oxidized phospholipids, and subsequent diminished capacity which subsequently diminishes the capability to attenuate the inflammatory effects of these molecules, we compared the binding properties of CFH and CFH related protein 1 (CFHR1), which is also associated with disease risk, to OxPLs and their effects on modulating inflammation and lipids uptake. As both CFH-402H and CFHR1 are associated with increased risk to AMD, we hypothesized that like CFH-402H, CFHR1 contribution to AMD risk may also be due to its diminished affinity for OxPLs. Interestingly, we found that association of CFHR1 with OxPLs was not statistically different than CFH. However, binding of CFHR1 did not elicit the same protective benefits as CFH in that both inflammation and lipid uptake are unaffected by CFHR1 association with OxPLs. These findings demonstrate a novel and interesting complexity to the potential interplay

  1. On the Functional Overlap between Complement and Anti-Microbial Peptides

    PubMed Central

    Zimmer, Jana; Hobkirk, James; Mohamed, Fatima; Browning, Michael J.; Stover, Cordula M.

    2015-01-01

    Intriguingly, activated complement and anti-microbial peptides share certain functionalities; lytic, phagocytic, and chemo-attractant activities and each may, in addition, exert cell instructive roles. Each has been shown to have distinct LPS detoxifying activity and may play a role in the development of endotoxin tolerance. In search of the origin of complement, a functional homolog of complement C3 involved in opsonization has been identified in horseshoe crabs. Horseshoe crabs possess anti-microbial peptides able to bind to acyl chains or phosphate groups/saccharides of endotoxin, LPS. Complement activity as a whole is detectable in marine invertebrates. These are also a source of anti-microbial peptides with potential pharmaceutical applicability. Investigating the locality for the production of complement pathway proteins and their role in modulating cellular immune responses are emerging fields. The significance of local synthesis of complement components is becoming clearer from in vivo studies of parenchymatous disease involving specifically generated, complement-deficient mouse lines. Complement C3 is a central component of complement activation. Its provision by cells of the myeloid lineage varies. Their effector functions in turn are increased in the presence of anti-microbial peptides. This may point to a potentiating range of activities, which should serve the maintenance of health but may also cause disease. Because of the therapeutic implications, this review will consider closely studies dealing with complement activation and anti-microbial peptide activity in acute inflammation (e.g., dialysis-related peritonitis, appendicitis, and ischemia). PMID:25646095

  2. Regulatory components of the alternative complement pathway in endothelial cell cytoplasm, factor H and factor I, are not packaged in Weibel-Palade bodies.

    PubMed

    Turner, Nancy A; Sartain, Sarah E; Hui, Shiu-Ki; Moake, Joel L

    2015-01-01

    It was recently reported that factor H, a regulatory component of the alternative complement pathway, is stored with von Willebrand factor (VWF) in the Weibel-Palade bodies of endothelial cells. If this were to be the case, it would have therapeutic importance for patients with the atypical hemolytic-uremic syndrome that can be caused either by a heterozygous defect in the factor H gene or by the presence of an autoantibody against factor H. The in vivo Weibel-Palade body secretagogue, des-amino-D-arginine vasopressin (DDAVP), would be expected to increase transiently the circulating factor H levels, in addition to increasing the circulating levels of VWF. We describe experiments demonstrating that factor H is released from endothelial cell cytoplasm without a secondary storage site. These experiments showed that factor H is not stored with VWF in endothelial cell Weibel-Palade bodies, and is not secreted in response in vitro in response to the Weibel-Palade body secretagogue, histamine. Furthermore, the in vivo Weibel-Palade body secretagogue, DDAVP does not increase the circulating factor H levels concomitantly with DDAVP-induced increased VWF. Factor I, a regulatory component of the alternative complement pathway that is functionally related to factor H, is also located in endothelial cell cytoplasm, and is also not present in endothelial cell Weibel-Palade bodies. Our data demonstrate that the factor H and factor I regulatory proteins of the alternative complement pathway are not stored in Weibel-Palade bodies. DDAVP induces the secretion into human plasma of VWF--but not factor H.

  3. Alzheimer's β-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner

    PubMed Central

    Bergamaschini, L; Canziani, S; Bottasso, B; Cugno, M; Braidotti, P; Agostoni, A

    1999-01-01

    β-Amyloid (β-A) accumulates in the brain of patients with Alzheimer's disease (AD) and is presumably involved in the pathogenesis of this disease, on account of its neurotoxicity and complement-activating ability. Although assembly of β-A in particular aggregates seems to be crucial, soluble non-fibrillar β-A may also be involved. Non-fibrillar β-A does not bind C1q, so we investigated alternative mechanisms of β-A-dependent complement activation in vitro. On incubation with normal human plasma, non-fibrillar β-A 1-42, and truncated peptide 1–28, induced dose-dependent activation of C1s and C4, sparing C3, as assessed by densitometric analysis of immunostained membrane after SDS–PAGE and Western blotting. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar β-A can still activate C1s and C4 in plasma genetically deficient in C1q (C1qd). In Factor XII-deficient plasma (F.XIId) the amount of cleaved C4 was about 5–10% less that in C1qd and in normal EDTA plasma; the reconstitution of F.XIId plasma with physiologic concentrations of F.XII resulted in an increased (8–15%) β-A-dependent cleavage of C4. Thus our results indicate that the C1q-independent activation of C1 and C4 can be partially mediated by the activation products of contact system. Since the activation of contact system and of C4 leads to generation of several humoral inflammatory peptides, non-fibrillar β-A might play a role in initiating the early inflammatory reactions leading to a multistep cascade contributing to neuronal and clinical dysfunction of AD brain. PMID:10193429

  4. Alzheimer's beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner.

    PubMed

    Bergamaschini, L; Canziani, S; Bottasso, B; Cugno, M; Braidotti, P; Agostoni, A

    1999-03-01

    beta-Amyloid (beta-A) accumulates in the brain of patients with Alzheimer's disease (AD) and is presumably involved in the pathogenesis of this disease, on account of its neurotoxicity and complement-activating ability. Although assembly of beta-A in particular aggregates seems to be crucial, soluble non-fibrillar beta-A may also be involved. Non-fibrillar beta-A does not bind C1q, so we investigated alternative mechanisms of beta-A-dependent complement activation in vitro. On incubation with normal human plasma, non-fibrillar beta-A 1-42, and truncated peptide 1-28, induced dose-dependent activation of C1s and C4, sparing C3, as assessed by densitometric analysis of immunostained membrane after SDS-PAGE and Western blotting. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar beta-A can still activate C1s and C4 in plasma genetically deficient in C1q (C1qd). In Factor XII-deficient plasma (F.XIId) the amount of cleaved C4 was about 5-10% less that in C1qd and in normal EDTA plasma; the reconstitution of F.XIId plasma with physiologic concentrations of F.XII resulted in an increased (8-15%) beta-A-dependent cleavage of C4. Thus our results indicate that the C1q-independent activation of C1 and C4 can be partially mediated by the activation products of contact system. Since the activation of contact system and of C4 leads to generation of several humoral inflammatory peptides, non-fibrillar beta-A might play a role in initiating the early inflammatory reactions leading to a multistep cascade contributing to neuronal and clinical dysfunction of AD brain.

  5. Complement activation by Coccidioides immitis: in vitro and clinical studies.

    PubMed Central

    Galgiani, J N; Yam, P; Petz, L D; Williams, P L; Stevens, D A

    1980-01-01

    Mycelial- or spherule-phase derivatives of Coccidioides immitis caused a decrease in vitro of total hemolytic complement in serum from a nonsensitized person. Activation involved both classic and alternative pathways as shown by deprssion of hemolytic C4 and by generation of products of activation of components C3, C4, and factor B. In addition, functional complement activity or immunoreactive levels of complement components or both were measured in 23 patients with self-limited or disseminated coccidioidomycosis. Low total hemolytic complement was found in nine, usually during the early phase of primary illness, and was transient. Hemolytic C4 was low, and the effect of inulin to decrease complement levels was blunted, suggested both classic and alternative pathways may be deficient. However, associated depression of immunoreactive levels of components assayed (C3, C4, C5, factor B, and properdin) was not consistently found. This disparity raises the possibility of enhanced in vitro inactivation analogous to activation by immune complexes. Images Fig. 2 PMID:6901703

  6. Hostility, Anger and Depression Predict Increases in C3 over a 10-Year Period

    PubMed Central

    Boyle, Stephen H.; Jackson, William G.; Suarez, Edward C.

    2007-01-01

    We examined the relation of hostility, anger and depression to 10-year changes in the third (C3) and fourth (C4) complement in 313, apparently healthy male participants enrolled in the Air Force Health Study (AFHS), a 20-year study designed to evaluate the health consequences of dioxin exposure. Hostility, depression and anger were assessed using subscales from the Minnesota Multiphasic Personality Inventory (MMPI), which was administered in 1985. Given the high intercorrelations among these psychological scales, we used a principal component analysis to generate a composite score representing the linear combination of the hostility, anger and depression scales. The dependent variables, C3 and C4 levels, were determined from samples collected in 1992, 1997 and 2002. Regression analyses controlling for age, race, alcohol use, body mass index and cigarette use as well as onset of disease and use of lipid lowering and blood pressure medications during follow-up revealed a significant time X composite score interaction for C3 complement (p < .0003), but not C4. Post-hoc analyses revealed that high composite scores were associated with larger 10-year increases in C3. These observations suggest that men who are hostile and are prone to experience frequent and intense feelings of anger and depression show activation of the complement system, and specifically increases in C3, that may contribute to the development of coronary heart disease. PMID:17321106

  7. Hostility, anger, and depression predict increases in C3 over a 10-year period.

    PubMed

    Boyle, Stephen H; Jackson, William G; Suarez, Edward C

    2007-08-01

    We examined the relation of hostility, anger, and depression to 10-year changes in the third (C3), and fourth (C4) complement in 313, apparently healthy male participants enrolled in the Air Force Health Study (AFHS), a 20-year study designed to evaluate the health consequences of dioxin exposure. Hostility, depression, and anger were assessed using subscales from the Minnesota Multiphasic Personality Inventory (MMPI), which was administered in 1985. Given the high intercorrelations among these psychological scales, we used a principal component analysis to generate a composite score representing the linear combination of the hostility, anger, and depression scales. The dependent variables, C3 and C4 levels, were determined from samples collected in 1992, 1997, and 2002. Regression analyses controlling for age, race, alcohol use, body mass index, and cigarette use as well as onset of disease, and use of lipid lowering and blood pressure medications during follow-up revealed a significant timexcomposite score interaction for C3 complement (p<.0003), but not C4. Post-hoc analyses revealed that high composite scores were associated with larger 10-year increases in C3. These observations suggest that men who are hostile and are prone to experience frequent and intense feelings of anger, and depression show activation of the complement system, and specifically increases in C3, that may contribute to the development of coronary heart disease.

  8. Human inhibitor of the first component of complement, C1: characterization of cDNA clones and localization of the gene to chromosome 11.

    PubMed Central

    Davis, A E; Whitehead, A S; Harrison, R A; Dauphinais, A; Bruns, G A; Cicardi, M; Rosen, F S

    1986-01-01

    C1 inhibitor is a heavily glycosylated plasma protein that regulates the activity of the first component of complement (C1) by inactivation of the serine protease subcomponents, C1r and C1s. C1 inhibitor cDNA clones have been isolated, and one of these (pC1INH1, 950 base pairs) has been partially sequenced. Sequence analysis demonstrates that the C1 inhibitor is a member of the serpin "superfamily" of protease inhibitors. In the region sequenced, C1 inhibitor has 22% identity with antithrombin III, 26% with alpha 1-antitrypsin and alpha 1-antichymotrypsin, and 18% with human angiotensinogen. C1 inhibitor has a larger amino-terminal extension than do the other plasma protease inhibitors. In addition, inspection of residues that are invariant among the other protease inhibitors shows that C1 inhibitor differs at 14 of 41 of these positions. Thus, it appears that C1 inhibitor diverged from the group relatively early in evolution, although probably after the divergence of angiotensinogen. Southern blot analysis of BamHI-digested DNA from normal individuals and from rodent-human somatic cell hybrid cell lines (that contain a limited but varied human chromosome complement) was used to localize the human C1 inhibitor gene to chromosome 11. Images PMID:3458172

  9. The serine proteinase chain of human complement component C1s. Cyanogen bromide cleavage and N-terminal sequences of the fragments.

    PubMed Central

    Carter, P E; Dunbar, B; Fothergill, J E

    1983-01-01

    Human complement component C1s was purified from fresh blood by conventional methods of precipitation and chromatography. The single-chain zymogen form was activated by treatment with C1r. Reduction and carboxymethylation then allowed the light chain and heavy chain to be separated on DEAE-Sepharose CL-6B in 8 M-urea. Liquid-phase sequencing of the light chain determined 50 residues from the N-terminus. CNBr-cleavage fragments of the light chain were separated by high-pressure liquid chromatography on gel-permeation and reverse-phase columns. N-Terminal sequencing of these fragments determined the order of a further 138 residues, giving a total of 188 residues or about 75% of the light chain. Seven of these eight sequences could be readily aligned with the amino acid sequences of other serine proteinases. The typical serine proteinase active-site residues are clearly conserved in C1s, and the specificity-related side chain of the substrate-binding pocket is aspartic acid, as in trypsin, consistent with the proteolytic action of C1s on C4 at an arginine residue. Somewhat surprisingly, when the C1s sequence is compared with that of complement subcomponent C1r, the percentage difference (59%) is approximately the same as that found between the other mammalian serine proteinases (56-71%). PMID:6362661

  10. Evolution of the complement system in protostomes revealed by de novo transcriptome analysis of six species of Arthropoda.

    PubMed

    Sekiguchi, Reo; Nonaka, Masaru

    2015-05-01

    To elucidate the evolutionary history of the complement system in Arthropoda, de novo transcriptome analysis was performed with six species among the Chelicerata, Myriapoda, and Crustacea, and complement genes were identified based on their characteristic domain structures. Complement C3 and factor B (FB) were identified from a sea spider, a jumping spider, and a centipede, but not from a sea firefly or two millipede species. No additional complement components identifiable by their characteristic domain structures were found from any of these six species. These results together with genome sequence information for several species of the Hexapoda suggest that the common ancestor of the Arthropoda possessed a simple complement system comprising C3 and FB, and thus resembled the alternative pathway of the mammalian complement system. It was lost at least twice independently during the evolution of Arthropoda in the millipede lineage and in the common ancestor of Crustacea and Hexapoda.

  11. Hybrid proteins of Cobra Venom Factor and cobra C3: tools to identify functionally important regions in Cobra Venom Factor.

    PubMed

    Hew, Brian E; Wehrhahn, Daniel; Fritzinger, David C; Vogel, Carl-Wilhelm

    2012-09-15

    Cobra Venom Factor (CVF) is the complement-activating protein in cobra venom. CVF is structurally and functionally highly homologous to complement component C3. CVF, like C3b, the activated form of C3, forms a bimolecular complex with Factor B in serum, called C3/C5 convertase, an enzyme which activates complement components C3 and C5. Despite the high degree of homology, the two C3/C5 convertases exhibit significant functional differences. The most important difference is that the convertase formed with CVF (CVF,Bb) is physico-chemically far more stable than the convertase formed with C3b (C3b,Bb). In addition, the CVF,Bb convertase and CVF are completely resistant to inactivation by the complement regulatory proteins Factor H and Factor I. Furthermore, the CVF,Bb enzyme shows efficient C5-cleaving activity in fluid phase. In contrast, the C3b,Bb enzyme is essentially devoid of fluid-phase C5-cleaving activity. By taking advantage of the high degree of sequence identity at both the amino acid (85%) and DNA levels (93%) between CVF and cobra C3, we created hybrid proteins of CVF and cobra C3 where sections, or only a few amino acids, of the CVF sequence were replaced with the homologous amino acid sequence of cobra C3. In a first set of experiments, we created five hybrid proteins, termed H1 through H5, where the cobra C3 substitutions collectively spanned the entire length of the CVF protein. We also created three additional hybrid proteins where only four or five amino acid residues in CVF were exchanged with the corresponding amino acid residues from cobra C3. Collectively, these hybrid proteins, representing loss-of-function mutants of CVF, allowed the identification of regions and individual amino acid residues important for the CVF-specific functions. The results include the observation that the CVF β-chain is crucially important for forming a stable convertase, whereas the CVF α-chain appears to harbor no CVF-specific functions. Furthermore, the CVF

  12. Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway*

    PubMed Central

    Kang, Mingsong; Ko, Ya-Ping; Liang, Xiaowen; Ross, Caná L.; Liu, Qing; Murray, Barbara E.; Höök, Magnus

    2013-01-01

    Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r2C1s2 was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens. PMID:23720782

  13. Preimplantation embryos cooperate with oviductal cells to produce embryotrophic inactivated complement-3b.

    PubMed

    Tse, Pui-Keung; Lee, Yin-Lau; Chow, Wang-Ngai; Luk, John M C; Lee, Kai-Fai; Yeung, William S B

    2008-03-01

    Human oviductal epithelial (OE) cells produce complement protein 3 (C3) and its derivatives, C3b and inactivated complement-3b (iC3b). Among them, iC3b is the most potent embryotrophic molecule. We studied the production of iC3b in the oviductal cell/embryo culture system. In the immune system, C3 convertase converts C3 into C3b, and the conversion of C3b to iC3b requires factor I (fI) and its cofactors, such as factor H or membrane cofactor protein. Human oviductal epithelium and OE cells expressed mRNA and protein of the components of C3 convertase, including C2, C4, factor B, and factor D. The OE cell-conditioned medium contained active C3 convertase activity that was suppressed by C3 convertase inhibitor, H17 in a dose and time-dependent manner. Although the oviductal epithelium and OE cells produced fI, the production of its cofactor, factor H required for the conversion of C3b to iC3b, was weak. Thus, OE cell-conditioned medium was inefficient in producing iC3b from exogenous C3b. On the contrary, mouse embryos facilitated such conversion to iC3b, which was taken up by the embryos, resulting in the formation of more blastocysts of larger size. The facilitatory activity was mediated by complement receptor 1-related gene/protein Y (Crry) with known membrane cofactor protein activity on the trophectoderm of the embryos as anti-Crry antibody inhibited the conversion and embryotrophic activity of C3b in the presence of fI. In conclusion, human oviduct possesses C3 convertase activity converting C3 to C3b, and Crry of the preimplantation embryos may be involved in the production of embryotrophic iC3b on the surface of the embryos.

  14. Trypanosoma musculi Infections in Normocomplementemic, C5-Deficient, and C3-Depleted Mice

    PubMed Central

    Jarvinen, Julie A.; Dalmasso, Agustin P.

    1977-01-01

    The role of complement in host resistance to infection with Trypanosoma musculi was studied in normal, C5-deficient, and C3-depleted mice. Infections in normocomplementemic strains (CBA and B10.D2/n) were generally similar to those in strains genetically deficient in C5 (A and B10.D2/o). There were no differences in inhibition of reproduction, duration of infection, persistence of parasites in the kidneys, or resistance to reinfection. However, peak parasitemias in B10.D2/o mice were slightly greater than in B10.D2/n mice. In addition, B10.D2/o mice had slightly decreased serum levels of C1 early in the course of infection and of C3 early during the elimination of adult forms. These components were unchanged or increased in infections of B10.D2/n. Depletion of C3 and late-acting components in B10.D2/n mice by treatment with cobra venom factor during the reproductive stage of infection resulted in an increase of reproductive forms before the apparent development of ablastic immunity as well as slightly greater peak parasitemias when compared with those of untreated controls. Cobra venom factor treatment of B10.D2/o mice during the reproductive stage did not alter the course of infection. Cobra venom factor treatment of C3H mice during the adult stage prolonged infections by interfering with parasite elimination. It is concluded that complement-mediated lysis is not involved in control of T. musculi. It is not clear whether a C3-dependent function such as phagocytosis may facilitate elimination of the parasites. The major difference in degree of parasitemias among the various strains of mice studied is due to genetic factors rather than the levels of C3, C5, or late-acting complement components. PMID:863515

  15. Complement activity and pharmacological inhibition in cardiovascular disease

    PubMed Central

    Théroux, Pierre; Martel, Catherine

    2006-01-01

    While complement is the most important component of humoral autoimmunity, and inflammation plays a key role in atherosclerosis, relatively few studies have looked at complement implications in atherosclerosis and its complications. C-reactive protein is a marker of inflammation and is also involved in atherosclerosis; it activates complement and colocalizes with activated complement proteins within the infarcting myocardium and the active atherosclerotic plaques. As new agents capable of modulating complement activity are being developed, new targets for the management of atherosclerosis are emerging that are related to autoimmunity and inflammation. The present paper reviews the putative roles of the various complement activation pathways in the development of atherosclerosis, in ST segment elevation and non-ST segment elevation acute coronary syndromes, and in coronary artery bypass graft surgery. It also provides a perspective on new therapeutic interventions being developed to modulate complement activity. These interventions include the C1 esterase inhibitor, which may be consumed in some inflammatory states resulting in the loss of one of the mechanisms inhibiting activation of the classical and lectin pathways; TP10, a recombinant protein of the soluble complement receptor type 1 (sCR1) which inhibits the C3 and C5 convertases of the common pathway by binding C3b and C4b; a truncated version of the soluble complement receptor type 1 CRI lacking the C4b binding site which selectively inhibits the alternative pathway; and pexelizumab, a monoclonal antibody selectively blocking C5 to prevent the activation of the terminal pathway that is involved in excessive inflammation and autoimmune responses. PMID:16498508

  16. Amino acid residues 1101-1105 of the isotypic region of human C4B is important to the covalent binding activity of complement component C4.

    PubMed

    Reilly, B D; Levine, R P; Skanes, V M

    1991-11-01

    The C4A and C4B isotypes of human C4 show certain functional differences that stem from their relative preference for transacylation to amino (-NH2) vs hydroxyl (-OH) nucleophiles, respectively, on complement-activating surfaces. Comparison of amino acid sequences of the alpha-chain fragment of C4, C4d, has shown C4A- and C4B-specific sequences at residues 1101-1106 are the only consistent structural difference between isotype, i.e., Pro, Cys, Pro, Val, Leu, Asp in C4A and Leu, Ser, Pro, Val Ile, His in C4B. These residues may be responsible either in part or entirely for properties associated with isotype. To examine the functional role of residues 1101-1106 in C4B-mediated hemolysis, whole serum or immunopurified human C4 with allotypes, A3B1, A3, B2B1, or B1 were preincubated in the presence or absence of an antipeptide mAb (BII-1) specific for amino acid residues 1101-1105 of C4B. Sensitized sheep E and C4-deficient guinea pig serum was then added and lysis measured by absorbance at 415 nm. Our results show lysis of antibody-sensitized sheep E is inhibited by antibody and C4B2B1, C4B1, or C4A3B1 but not antibody and C4A3. The interference of hemolysis by BII-1 could not be explained by inhibition of activation of C4B or inhibition of C3 or C5 convertase activity. Furthermore, results from uptake experiments show that BII-1 interferes with the covalent binding activity of C4B, indicating residues 1101-1105 play a role in the covalent binding reaction of C4B to the target E-antibody complex.

  17. Molecular cloning of the alpha subunit of complement component C8 (CpC8α) of whitespotted bamboo shark (Chiloscyllium plagiosum).

    PubMed

    Wang, Ying; Zhang, Mengmeng; Wang, Conghui; Ye, Boping; Hua, Zichun

    2013-12-01

    Complement-mediated cytolysis is the important effect of immune response, which results from the assembly of terminal complement components (C5b-9). Among them, α subunit of C8 (C8α) is the first protein that traverses the lipid bilayer, and then initiates the recruitment of C9 molecules to form pore on target membranes. In this article, a full-length cDNA of C8α (CpC8α) is identified from the whitespotted bamboo shark (Chiloscyllium plagiosum) by RACE. The CpC8α cDNA is 2183 bp in length, encoding a protein of 591 amino acids. The deduced CpC8α exhibits 89%, 49% and 44% identity with nurse shark, frog and human orthologs, respectively. Sequence alignment indicates that the C8α is well conserved during the evolution process from sharks to mammals, with the same modular architecture as well as the identical cysteine composition in the mature protein. Phylogenetic analysis places CpC8α and nurse shark C8α in cartilaginous fish clade, in parallel with the teleost taxa, to form the C8α cluster with higher vertebrates. Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8α to mammals. Finally, expression analysis revealed CpC8α transcripts were constitutively highly expressed in shark liver, with much less expression in other tissues. The well conserved structure and properties suggests an analogous function of CpC8α to mammalian C8α, though it remains to be confirmed by further study.

  18. Blast-Induced Moderate Neurotrauma (BINT) Elicits Early Complement Activation and Tumor Necrosis Factor Alpha (TNFalpha) Release in a Rat Brain

    DTIC Science & Technology

    2012-04-25

    Morganti-Kossmann MC, Jones JL, Barnum SR. Elevated levels of the complement components C3 and factor B in ventricular cerebrospinal fluid of patients with...Ingersoll SA, Martin CB, Barnum SR, Martin BK. CNS-specific expression of C3a and C5a exacerbate demyelination severity in the cuprizone model. Mol Immunol

  19. Complement Evasion by Pathogenic Leptospira.

    PubMed

    Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

    2016-01-01

    Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira. Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira, have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host.

  20. Complement Evasion by Pathogenic Leptospira

    PubMed Central

    Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

    2016-01-01

    Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira. Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira, have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host. PMID:28066433

  1. Complement involvement in kidney diseases: From physiopathology to therapeutical targeting.

    PubMed

    Salvadori, Maurizio; Rosso, Giuseppina; Bertoni, Elisabetta

    2015-05-06

    Complement cascade is involved in several renal diseases and in renal transplantation. The different components of the complement cascade might represent an optimal target for innovative therapies. In the first section of the paper the authors review the physiopathology of complement involvement in renal diseases and transplantation. In some cases this led to a reclassification of renal diseases moving from a histopathological to a physiopathological classification. The principal issues afforded are: renal diseases with complement over activation, renal diseases with complement dysregulation, progression of renal diseases and renal transplantation. In the second section the authors discuss the several complement components that could represent a therapeutic target. Even if only the anti C5 monoclonal antibody is on the market, many targets as C1, C3, C5a and C5aR are the object of national or international trials. In addition, many molecules proved to be effective in vitro or in preclinical trials and are waiting to move to human trials in the future.

  2. Serum complement and immunoconglutinin in malnutrition.

    PubMed Central

    Chandra, R K

    1975-01-01

    Serum haemolytic complement activity and C3 were significantly decreased in 35 malnourished children. The changes were more pronounced in those with infection. Electrophoretically altered forms of complement C were detected in 14. There was an inverse correlation between C3 levels and immunoconglutinin titres. Nutritional rehabilitation and eradication of infection reversed the abnormalities. It is suggested that reduced complement function in malnutrition is the combined result of impaired synthesis, complement activation in vivo, and changes in plasma volume, and that it may contribute to an increased susceptibility to infection in undernourished individuals. PMID:807166

  3. A COMPARISON OF THE SPECIFICITY OF INHIBITION BY PHOSPHONATE ESTERS OF THE FIRST COMPONENT OF COMPLEMENT AND THE ANTIGEN-INDUCED RELEASE OF HISTAMINE FROM GUINEA PIG LUNG

    PubMed Central

    Becker, Elmer L.; Austen, K. Frank

    1964-01-01

    The ability of a number p-nitrophenylethyl alkyl, phenyl alkyl, chloroalkyl, and aminoalkyl phosphonates to inhibit the activated first component (C'1a) of guinea pig complement, and the antigen-induced release of histamine from sliced, perfused guinea pig lung has been compared. C'1a in its reactivity with these phosphonates is distinctly more similar to trypsin than to any of the other enzymes studied previously. It is suggested that both trypsin and C'1a possess an anionic group in the active center of the respective enzyme, but the distance between the anionic and esteratic site in C'1a might be less than in trypsin. The pattern of inhibition of histamine relase by the alkyl, phenyl alkyl, and chloroalkyl phosphonates is similar to the inhibition of C'1a by these compounds, although distinct differences are apparent. The aminoalkyl phosphonates are distinctly less active inhibitors of histamine release than the corresponding alkyl phosphonates, whereas the reverse is true of the inhibition of C'1a. On the basis of these differences, it is tentatively concluded that the organophosphorus-inhibitable enzymes in the guinea pig systems studied here are similar but not identical. PMID:14212115

  4. In vitro cleavage by asbestos fibers of the fifth component of human complement through free-radical generation and kallikrein activation.

    PubMed

    Governa, M; Amati, M; Valentino, M; Visonà, I; Fubini, B; Botta, G C; Volpe, A R; Carmignani, M

    2000-04-14

    Chrysotile and crocidolite fibers incubated in normal human plasma (NHP) generated from the C5 component of complement C5a-type fragments that stimulated polymorphonuclear leukocyte (PMN) chemotaxis. Absorption of NHP with antiserum against C5a totally abolished neutrophil chemotactic activity. Asbestos fibers also produced C5a small peptides in the presence of ethylene glycol bis(beta-aminoethyl ether) N,N,N'N'-tetraacetic acid (EGTA) but not ethylene diamine tetraacetic acid (EDTA). Activation of C5 was significantly inhibited when asbestos fibers were pretreated with iron chelators such as sodium dithionite (DTN), deferoxamine (DFX), or ascorbate (AA). Concentration-related inhibition of C5 activation was also observed when asbestos fibers were added concurrently to plasma in the presence of DFX, 1,3-dimethyl-2-thiourea (DMTU), a strong hydroxyl scavenger, or aprotinin (APR), a specific protease inhibitor. Further, chrysotile and crocidolite significantly increased plasma kallikrein activity. Data demonstrate that asbestos-induced C5 activation plays a role in inflammatory reactions characteristic of asbestosis through mechanisms involving iron ions, hydroxyl radicals, and oxidized C5-ike fragments. The ferrous ions present at the asbestos fiber surface trigger this activation and catalyze, via Fenton reaction, the production of hydroxyl radicals, which in turn convert native C5 to an oxidized C5-like form. This product is then cleaved by kallikrein, activated by the same asbestos fibers, yielding an oxidized C5a with the same functional properties as C5a.

  5. NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways

    PubMed Central

    Yuen, Joshua; Pluthero, Fred G.; Douda, David N.; Riedl, Magdalena; Cherry, Ahmed; Ulanova, Marina; Kahr, Walter H. A.; Palaniyar, Nades; Licht, Christoph

    2016-01-01

    Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases. PMID:27148258

  6. Effect of ammonia-N and pathogen challenge on complement component 8α and 8β expression in the darkbarbel catfish Pelteobagrus vachellii.

    PubMed

    Qin, Chuanjie; Shao, Ting; Zhao, Daxian; Duan, Huiguo; Wen, Zhengyong; Yuan, Dengyue; Li, Huatao; Qi, Zemin

    2017-03-01

    The complement components C8α and C8β mediate the formation of the membrane attack complex (MAC) to resist pathogenic bacteria and play important roles in innate immunity. Full-length complement C8α (Pv-C8α) and C8β (Pv-C8β) cDNA were identified in the darkbarbel catfish Pelteobagrus vachellii, and their mRNA expression levels were analyzed after ammonia-N and pathogen treatment. The Pv-C8α gene contained 1983 bp, including a 1794-bp open reading frame (ORF) encoding 598 amino acids. The Pv-C8β gene contained 1952 bp, including a 1761-bp ORF encoding 587 amino acids. Pv-C8α and Pv-C8β had the highest amino acid identity with rainbow trout Oncorhynchus mykiss C8α (62%) and Japanese flounder Paralichthys olivaceus C8β (83%), respectively. Sequence analysis indicated that both Pv-C8α and Pv-C8β contained a thrombospondin type-1 (TSP1) domain, a low-density lipoprotein receptor class A (LDLR-A) domain, a membrane attack complex/perforin (MACPF) domain and an epidermal growth factor-like (EGF-like) domain. In addition, Pv-C8α and Pv-C8β were mainly distributed in the liver, head kidney, spleen, and eggs. Under ammonia-N stress, the Pv-C8α and Pv-C8β mRNA levels significantly decreased (P < 0.05), with minimum levels, respectively, attained at 24 and 48 h in the liver, 48 and 24 h in the head kidney, and 24 and 24 h in the spleen. After Aeromonas hydrophila challenge, the Pv-C8α and Pv-C8β mRNA levels significantly increased (P < 0.05), with maximum levels, respectively, attained at 48 and 24 h in the liver, 24 and 48 h in the head kidney, and 48 and 48 h in the spleen. The present study indicated that Pv-C8α and Pv-C8β exhibited important immune responses to infection and that ammonia-N in water decreased the immune responses of Pv-C8α and Pv-C8β.

  7. Complement Factor 3 Could Be an Independent Risk Factor for Mortality in Patients with HBV Related Acute-on-Chronic Liver Failure

    PubMed Central

    Zhang, Geng-lin; Zhang, Ting; Ye, Yi-nong; Liu, Jing; Zhang, Xiao-hong; Xie, Chan; Peng, Liang; Gao, Zhi-liang

    2016-01-01

    The complement is thought to be involved in the pathogenesis of multiple liver disorders. However, its role in patients with HBV related acute-on-chronic liver failure (HBV-ACLF) remains unclear. Serum levels of the third and fourth complement components (C3, C4) and complement function (CH50) were examined in this prospective, observational study. Associations between their expression and disease activity were analyzed. Survival was analyzed by Kaplan-Meier curves. Predictors of clinical outcome were determined by Cox regression analysis. C3, C4, and CH50 levels were significantly lower in HBV-ACLF patients compared to controls. C3, C4, and CH50 levels were negatively correlated with Tbil levels but positively associated with PTA levels. C3 levels were negatively associated with MELD-Na. C3 levels were significantly lower in HBV-ACLF patients who died compared to patients who survived. In a median hospital stay of 39 days, mortality occurred in 41 patients with a progressive increase based on C3 grade (P = 0.008). The actuarial probability of developing mortality was significantly higher in patients with low C3 grade compared to those with high C3 grade (P < 0.001). Multivariate Cox regression analysis showed that C3 levels were an independent predictor of mortality. Complement played a pathogenic role in HBV-ACLF patients and C3 was an independent predictor of mortality. PMID:27144164

  8. Smoke exposure causes endoplasmic reticulum stress and lipid accumulation in retinal pigment epithelium through oxidative stress and complement activation.

    PubMed

    Kunchithapautham, Kannan; Atkinson, Carl; Rohrer, Bärbel

    2014-05-23

    Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD.

  9. A local complement response by RPE causes early-stage macular degeneration

    PubMed Central

    Fernandez-Godino, Rosario; Garland, Donita L.; Pierce, Eric A.

    2015-01-01

    Inherited and age-related macular degenerations (AMDs) are important causes of vision loss. An early hallmark of these disorders is the formation of sub-retinal pigment epithelium (RPE) basal deposits. A role for the complement system in MDs was suggested by genetic association studies, but direct functional connections between alterations in the complement system and the pathogenesis of MD remain to be defined. We used primary RPE cells from a mouse model of inherited MD due to a p.R345W mutation in EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) to investigate the role of the RPE in early MD pathogenesis. Efemp1R345W RPE cells recapitulate the basal deposit formation observed in vivo by producing sub-RPE deposits in vitro. The deposits share features with basal deposits, and their formation was mediated by EFEMP1R345W or complement component 3a (C3a), but not by complement component 5a (C5a). Increased activation of complement appears to occur in response to an abnormal extracellular matrix (ECM), generated by the mutant EFEMP1R345W protein and reduced ECM turnover due to inhibition of matrix metalloproteinase 2 by EFEMP1R345W and C3a. Increased production of C3a also stimulated the release of cytokines such as interleukin (IL)-6 and IL-1B, which appear to have a role in deposit formation, albeit downstream of C3a. These studies provide the first direct indication that complement components produced locally by the RPE are involved in the formation of basal deposits. Furthermore, these results suggest that C3a generated by RPE is a potential therapeutic target for the treatment of EFEMP1-associated MD as well as AMD. PMID:26199322

  10. Interplay between invertebrate C3a with vertebrate macrophages: functional characterization of immune activities of amphioxus C3a.

    PubMed

    Gao, Zhan; Li, Mengyang; Wu, Jie; Zhang, Shicui

    2013-10-01

    Our current knowledge of the structure and function of C3a comes from the study of vertebrate C3a anaphylatoxins, virtually nothing is known about the structure and function of C3a molecules in invertebrates. Here we demonstrated that C3a from the invertebrate chordate Branchiostoma japonicum, BjC3a, was similar to vertebrate C3a possessing potential antibacterial activity, as revealed by sequence analysis and computational modeling. The antibacterial activity of BjC3a was definitely confirmed by both antibacterial assay and TEM observation showing that recombinant BjC3a was directly bactericidal. Additionally, recombinant BjC3a, like vertebrate C3a, was capable of inducing sea bass macrophage migration and enhancing macrophage phagocytosis and respiratory burst response. Moreover, recombinant BjC3a-desArg (generated by removal of the C-terminal arginine), like mammalian C3a-desArg, retained the immunological activities of BjC3a such as antibacterial and respiratory burst-stimulating activities, indicating that the immunological functions of C3a-desArg were conserved throughout chordate evolution. Altogether, our findings show that invertebrate (amphioxus) BjC3a is able to interact with vertebrate (sea bass) macrophages and mediate immune activities, suggesting the emergence of the inflammatory pathway of the complement system similar to that of vertebrates in the basal chordate amphioxus.

  11. The pivotal role of the complement system in aging and age-related macular degeneration: hypothesis re-visited.

    PubMed

    Anderson, Don H; Radeke, Monte J; Gallo, Natasha B; Chapin, Ethan A; Johnson, Patrick T; Curletti, Christy R; Hancox, Lisa S; Hu, Jane; Ebright, Jessica N; Malek, Goldis; Hauser, Michael A; Rickman, Catherine Bowes; Bok, Dean; Hageman, Gregory S; Johnson, Lincoln V

    2010-03-01

    During the past ten years, dramatic advances have been made in unraveling the biological bases of age-related macular degeneration (AMD), the most common cause of irreversible blindness in western populations. In that timeframe, two distinct lines of evidence emerged which implicated chronic local inflammation and activation of the complement cascade in AMD pathogenesis. First, a number of complement system proteins, complement activators, and complement regulatory proteins were identified as molecular constituents of drusen, the hallmark extracellular deposits associated with early AMD. Subsequently, genetic studies revealed highly significant statistical associations between AMD and variants of several complement pathway-associated genes including: Complement factor H (CFH), complement factor H-related 1 and 3 (CFHR1 and CFHR3), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3). In this article, we revisit our original hypothesis that chronic local inflammatory and immune-mediated events at the level of Bruch's membrane play critical roles in drusen biogenesis and, by extension, in the pathobiology of AMD. Secondly, we report the results of a new screening for additional AMD-associated polymorphisms in a battery of 63 complement-related genes. Third, we identify and characterize the local complement system in the RPE-choroid complex - thus adding a new dimension of biological complexity to the role of the complement system in ocular aging and AMD. Finally, we evaluate the most salient, recent evidence that bears directly on the role of complement in AMD pathogenesis and progression. Collectively, these recent findings strongly re-affirm the importance of the complement system in AMD. They lay the groundwork for further studies that may lead to the identification of a transcriptional disease signature of AMD, and hasten the development of new therapeutic approaches that will restore the complement-modulating activity that

  12. The chromosomal order of genes controlling the major histocompatibility complex, properdin factor B, and deficiency of the second component of complement.

    PubMed Central

    Raum, D; Glass, D; Carpenter, C B; Alper, C A; Schur, P H

    1976-01-01

    The relationship of the genes coding for HLA to those coding for properdin Factor B allotypes and for deficiency of the second component of complement (C2) was studied in families of patients with connective tissue disorders. Patients were selected because they were heterozygous or homozygous for C2 deficiency. 12 families with 15 matings informative for C2 deficiency were found. Of 57 informative meioses, two crossovers were noted between the C2 deficiency gene and the HLA-B gene, with a recombinant fraction of 0.035. A lod score of 13 was calculated for linkage between C2 deficiency and HLA-B at a maximum likelihood value of the recombinant fraction of 0.04. 18 families with 21 informative matings for both properdin Factor B allotype and HLA-B were found. Of 72 informative meioses, three recombinants were found, giving a recombinant fraction of 0.042. A lod score of 16 between HLA-B and Factor B allotypes was calculated at a maximum likelihood value of the recombinant fraction of 0.04. A crossover was shown to have occurred between genes for Factor B and HLA-D, in which HLA-D segregared with HLA-A and B. These studies suggest that the genes for Factor B and C2 deficiency are located outside those for HLA, that the order of genese is HLA-A, -B, -D, Factor B allotype, C2 deficiency, that the genes coding for C2 deficiency and Factor B allotypes are approximately 3--5 centimorgans from the HLA-A and HLA-B loci, and that the apparent lack of recombinants between the Factor B gene and C2 deficiency gene suggests that these two genes lie in close proximity to one another. PMID:993342

  13. Differential surface deposition of complement proteins on logarithmic and stationary phase Leishmania chagasi promastigotes.

    PubMed

    Ramer-Tait, Amanda E; Lei, Soi Meng; Bellaire, Bryan H; Beetham, Jeffrey K

    2012-12-01

    Previous works demonstrated that various species of Leishmania promastigotes exhibit differential sensitivity to complement-mediated lysis (CML) during development. Upon exposure to normal human serum (NHS), cultures of Leishmania chagasi promastigotes recently isolated from infected hamsters (fewer than 5 in vitro passages) are CML-sensitive when in the logarithmic growth phase but become CML-resistant upon transition to the stationary culture phase. Visualization by light and electron microscopy revealed dramatic morphological differences between promastigotes from the 2 culture phases following exposure to NHS. Flow cytometric analysis demonstrated that surface deposition of the complement components C3, C5, and C9 correlated inversely with promastigote CML-resistance. The highest levels of complement protein surface accumulation were observed for logarithmic phase promastigotes, while stationary phase promastigotes adsorbed the least amount of complement proteins. Additionally, fluorescence microscopy revealed that C3 and C5 localized in a fairly uniform pattern to the plasma membrane of promastigotes from logarithmic phase cultures, while the staining of promastigotes from stationary phase cultures was indistinguishable from background. By Western blot analysis, high levels of the complement proteins C3, C5, and C9 were detected in the total lysates of NHS-exposed logarithmic phase L. chagasi promastigotes, relative to NHS-exposed stationary phase promastigotes; this finding indicates that the low levels of C3 and C5 seen on the surface of stationary phase promastigotes were not due to protein uptake/internalization. Together, these data demonstrate the differential deposition of complement proteins on the surfaces of logarithmic and stationary phase L. chagasi promastigotes. The data support a model wherein stationary phase L. chagasi promastigotes resist CML by limiting the deposition of C3 and its derivatives, which, in turn, limit surface levels of

  14. Complement activation is critical for placental ischemia-induced hypertension in the rat.

    PubMed

    Lillegard, Kathryn E; Johnson, Alex C; Lojovich, Sarah J; Bauer, Ashley J; Marsh, Henry C; Gilbert, Jeffrey S; Regal, Jean F

    2013-11-01

    Preeclampsia is a major obstetric problem defined by new-onset hypertension and proteinuria associated with compromised placental perfusion. Although activation of the complement system is increased in preeclampsia compared to normal pregnancy, it remains unclear whether excess complement activation is a cause or consequence of placental ischemia. Therefore, we hypothesized that complement activation is critical for placental ischemia-induced hypertension. We employed the reduced utero-placental perfusion pressure (RUPP) model of placental ischemia in the rat to induce hypertension in the third trimester and evaluated the effect of inhibiting complement activation with a soluble recombinant form of an endogenous complement regulator, human complement receptor 1 (sCR1; CDX-1135). On day 14 of a 21-day gestation, rats received either RUPP or Sham surgery and 15 mg/kg/day sCR1 or saline intravenously on days 14-18. Circulating complement component 3 decreased and complement activation product C3a increased in RUPP vs. Sham (p<0.05), indicating complement activation had occurred. Mean arterial pressure (MAP) measured on day 19 increased in RUPP vs. Sham rats (109.8±2.8 mmHg vs. 93.6±1.6 mmHg). Treatment with sCR1 significantly reduced elevated MAP in RUPP rats (98.4±3.6 mmHg, p<0.05) and reduced C3a production. Vascular endothelial growth factor (VEGF) decreased in RUPP compared to Sham rats, and the decrease in VEGF was not affected by sCR1 treatment. Thus, these studies have identified a mechanistic link between complement activation and the pregnancy complication of hypertension apart from free plasma VEGF and have identified complement inhibition as a potential treatment strategy for placental ischemia-induced hypertension in preeclampsia.

  15. Immune competence of the Ciona intestinalis pharynx: complement system-mediated activity.

    PubMed

    Giacomelli, Stefano; Melillo, Daniela; Lambris, John D; Pinto, Maria Rosaria

    2012-10-01

    In the tunicate Ciona intestinalis, the ciliated pharynx, which connects the external environment to a highly developed and compartmentalized gastrointestinal system, represents the natural portal of entry for a vast and diverse, potentially pathogenic microbial community. To address the role of the pharynx in immune surveillance in Ciona, we asked whether C3, the key component of the complement system, was expressed in this organ and whether the encoded protein was functionally active. We found by real-time PCR that C3, constitutively expressed in the pharynx, is up-regulated by LPS injection. Using two specific anti-CiC3 and anti-CiC3a polyclonal antibodies in immunohistochemical staining of pharynx sections, we found that the gene product was localized to hemocytes of the pharyngeal bars (identified as granular amoebocytes) and in stigmata ciliated cells. Use of the same antibodies in Western blot analysis indicated that CiC3 and its activation products CiC3b and CiC3a are present in pharynx homogenates. Our observation that the amount of the bioactive fragment CiC3a increased in the pharynx of LPS-treated animals provides the first molecular and functional evidence for complement-mediated immunological activity in the tunicate pharynx.

  16. C3 deficiency ameliorates the negative effects of irradiation of the young brain on hippocampal development and learning

    PubMed Central

    Kalm, Marie; Andreasson, Ulf; Björk-Eriksson, Thomas; Zetterberg, Henrik; Pekny, Milos; Blennow, Kaj; Pekna, Marcela; Blomgren, Klas

    2016-01-01

    Radiotherapy in the treatment of pediatric brain tumors is often associated with debilitating late-appearing adverse effects, such as intellectual impairment. Areas in the brain harboring stem cells are particularly sensitive to irradiation (IR) and loss of these cells may contribute to cognitive deficits. It has been demonstrated that IR-induced inflammation negatively affects neural progenitor differentiation. In this study, we used mice lacking the third complement component (C3−/−) to investigate the role of complement in a mouse model of IR-induced injury to the granule cell layer (GCL) of the hippocampus. C3−/− and wild type (WT) mice received a single, moderate dose of 8 Gy to the brain on postnatal day 10. The C3−/− mice displayed 55 % more microglia (Iba-1+) and a trend towards increase in proliferating cells in the GCL compared to WT mice 7 days after IR. Importantly, months after IR C3−/− mice made fewer errors than WT mice in a reversal learning test indicating better learning capacity in C3−/− mice after IR. Notably, months after IR C3−/− and WT mice had similar GCL volumes, survival of newborn cells (BrdU), microglia (Iba-1) and astrocyte (S100β) numbers in the GCL. In summary, our data show that the complement system contributes to IR-induced loss of proliferating cells and maladaptive inflammatory responses in the acute phase after IR, leading to impaired learning capacity in adulthood. Targeting the complement system is hence promising for future strategies to reduce the long-term adverse consequences of IR in the young brain. PMID:27029069

  17. C3 deficiency ameliorates the negative effects of irradiation of the young brain on hippocampal development and learning.

    PubMed

    Kalm, Marie; Andreasson, Ulf; Björk-Eriksson, Thomas; Zetterberg, Henrik; Pekny, Milos; Blennow, Kaj; Pekna, Marcela; Blomgren, Klas

    2016-04-12

    Radiotherapy in the treatment of pediatric brain tumors is often associated with debilitating late-appearing adverse effects, such as intellectual impairment. Areas in the brain harboring stem cells are particularly sensitive to irradiation (IR) and loss of these cells may contribute to cognitive deficits. It has been demonstrated that IR-induced inflammation negatively affects neural progenitor differentiation. In this study, we used mice lacking the third complement component (C3-/-) to investigate the role of complement in a mouse model of IR-induced injury to the granule cell layer (GCL) of the hippocampus. C3-/- and wild type (WT) mice received a single, moderate dose of 8 Gy to the brain on postnatal day 10. The C3-/- mice displayed 55 % more microglia (Iba-1+) and a trend towards increase in proliferating cells in the GCL compared to WT mice 7 days after IR. Importantly, months after IR C3-/- mice made fewer errors than WT mice in a reversal learning test indicating better learning capacity in C3-/- mice after IR. Notably, months after IR C3-/- and WT mice had similar GCL volumes, survival of newborn cells (BrdU), microglia (Iba-1) and astrocyte (S100β) numbers in the GCL. In summary, our data show that the complement system contributes to IR-induced loss of proliferating cells and maladaptive inflammatory responses in the acute phase after IR, leading to impaired learning capacity in adulthood. Targeting the complement system is hence promising for future strategies to reduce the long-term adverse consequences of IR in the young brain.

  18. Insights into complement convertase formation based on the structure of the factor B-cobra venom factor complex.

    PubMed

    Janssen, Bert J C; Gomes, Lucio; Koning, Roman I; Svergun, Dmitri I; Koster, Abraham J; Fritzinger, David C; Vogel, Carl-Wilhelm; Gros, Piet

    2009-08-19

    Immune protection by the complement system critically depends on assembly of C3 convertases on the surface of pathogens and altered host cells. These short-lived protease complexes are formed through pro-convertases, which for the alternative pathway consist of the complement component C3b and the pro-enzyme factor B (FB). Here, we present the crystal structure at 2.2-A resolution, small-angle X-ray scattering and electron microscopy (EM) data of the pro-convertase formed by human FB and cobra venom factor (CVF), a potent homologue of C3b that generates more stable convertases. FB is loaded onto CVF through its pro-peptide Ba segment by specific contacts, which explain the specificity for the homologous C3b over the native C3 and inactive products iC3b and C3c. The protease segment Bb binds the carboxy terminus of CVF through the metal-ion dependent adhesion site of the Von Willebrand factor A-type domain. A possible dynamic equilibrium between a 'loading' and 'activation' state of the pro-convertase may explain the observed difference between the crystal structure of CVFB and the EM structure of C3bB. These insights into formation of convertases provide a basis for further development of complement therapeutics.

  19. Anti-complement activity of the Ixodes scapularis salivary protein Salp20

    PubMed Central

    Hourcade, Dennis E.; Akk, Antonina M.; Mitchell, Lynne M.; Zhou, Hui-fang; Hauhart, Richard; Pham, Christine T.N.

    2015-01-01

    Complement, a major component of innate immunity, presents a rapid and robust defense of the intravascular space. While regulatory proteins protect host cells from complement attack, when these measures fail, unrestrained complement activation may trigger self-tissue injury, leading to pathologic conditions. Of the three complement activation pathways, the alternative pathway (AP) in particular has been implicated in numerous disease and injury states. Consequently, the AP components represent attractive targets for therapeutic intervention. The common hard-bodied ticks from the family Ixodidae derive nourishment from the blood of their mammalian hosts. During its blood meal the tick is exposed to host immune effectors, including the complement system. In defense, the tick produces salivary proteins that can inhibit host immune functions. The Salp20 salivary protein of Ixodes scapularis inhibits the host AP pathway by binding properdin and dissociating C3bBbP, the active C3 convertase. In these studies we examined Salp20 activity in various complement-mediated pathologies. Our results indicate that Salp20 can inhibit AP-dependent pathogenesis in the mouse. Its efficacy may be part in due to synergic effects it provides with the endogenous AP regulator, factor H. While Salp20 itself would be expected to be highly immunogenic and therefore inappropriate for therapeutic use, its emergence speaks for the potential development of a non-immunogenic Salp20 mimic that replicates its anti-properdin activity. PMID:26675068

  20. Autocrine Effects of Tumor-Derived Complement

    PubMed Central

    Cho, Min Soon; Vasquez, Hernan G.; Rupaimoole, Rajesha; Pradeep, Sunila; Wu, Sherry; Zand, Behrouz; Han, Hee-Dong; Rodriguez-Aguayo, Cristian; Bottsford-Miller, Justin; Huang, Jie; Miyake, Takahito; Choi, Hyun-Jin; Dalton, Heather J.; Ivan, Cristina; Baggerly, Keith; Lopez-Berestein, Gabriel; Sood, Anil K.; Afshar-Kharghan, Vahid

    2014-01-01

    SUMMARY We describe a role for the complement system in enhancing cancer growth. Cancer cells secrete complement proteins that stimulate tumor growth upon activation. Complement promotes tumor growth via a direct autocrine effect that is partially independent of tumor-infiltrating cytotoxic T cells. Activated C5aR and C3aR signal through the PI3K/AKT pathway in cancer cells, and silencing the PI3K or AKT gene in cancer cells eliminates the progrowth effects of C5aR and C3aR stimulation. In patients with ovarian or lung cancer, higher tumoral C3 or C5aR mRNA levels were associated with decreased overall survival. These data identify a role for tumor-derived complement proteins in promoting tumor growth, and they therefore have substantial clinical and therapeutic implications. PMID:24613353

  1. Effects of complement inhibition with soluble complement receptor-1 on vascular injury and inflammation during renal allograft rejection in the rat.

    PubMed

    Pratt, J R; Hibbs, M J; Laver, A J; Smith, R A; Sacks, S H

    1996-12-01

    Complement is both an effector of the humoral immune response and a stimulator of leukocyte activation. To examine the influence of complement on the allograft response, we inhibited complement using recombinant human soluble complement receptor-1 (sCR1; TP10), in an unsensitized model of rat renal allograft rejection. Lewis to DA renal transplant recipients were treated daily with 25 mg/kg sCR1 or saline and sacrificed on days 1 to 5 after transplant. Transplanted organs were examined histologically and immunohistochemically for leukocyte subset markers and for the third component of complement, C3, and membrane attack complex deposition. A second set of recipients was followed from day 5 to day 9 to assess graft survival. sCR1-treated recipients displayed > 90% inhibition of plasma complement activity and a marked reduction in tissue C3 and membrane attack complex deposition. Inactivation of complement reduced the vascular injury such that there was almost complete sparing of vascular damage in day 5 sCR1-treated rats. There was a significant reduction in infiltrating leukocytes by day 5 after transplant, and complement inhibition delayed the time to reach a histologically defined end point of graft survival from 5 days in controls to 9 days in the sCR1-treated group. These results imply that the vascular and cell-mediated injury arises, in part, from complement activation. The partial inhibition of these injuries by sCR1 may have functional implications for strategies to inhibit allograft rejection.

  2. Skin Injuries Reduce Survival and Modulate Corticosterone, C-Reactive Protein, Complement Component 3, IgM, and Prostaglandin E2 after Whole-Body Reactor-Produced Mixed Field (n + γ-Photons) Irradiation

    PubMed Central

    Kiang, Juliann G.; Ledney, G. David

    2013-01-01

    Skin injuries such as wounds or burns following whole-body γ-irradiation (radiation combined injury (RCI)) increase mortality more than whole-body γ-irradiation alone. Wound-induced decreases in survival after irradiation are triggered by sustained activation of inducible nitric oxide synthase pathways, persistent alteration of cytokine homeostasis, and increased susceptibility to systemic bacterial infection. Among these factors, radiation-induced increases in interleukin-6 (IL-6) concentrations in serum were amplified by skin wound trauma. Herein, the IL-6-induced stress proteins including C-reactive protein (CRP), complement 3 (C3), immunoglobulin M (IgM), and prostaglandin E2 (PGE2) were evaluated after skin injuries given following a mixed radiation environment that might be found after a nuclear incident. In this report, mice received 3 Gy of reactor-produced mixed field (n + γ-photons) radiations at 0.38 Gy/min followed by nonlethal skin wounding or burning. Both wounds and burns reduced survival and increased CRP, C3, and PGE2 in serum after radiation. Decreased IgM production along with an early rise in corticosterone followed by a subsequent decrease was noted for each RCI situation. These results suggest that RCI-induced alterations of corticosterone, CRP, C3, IgM, and PGE2 cause homeostatic imbalance and may contribute to reduced survival. Agents inhibiting these responses may prove to be therapeutic for RCI and improve related survival. PMID:24175013

  3. Deletion of both the C3a and C5a receptors fails to protect against experimental autoimmune encephalomyelitis.

    PubMed

    Ramos, Theresa N; Wohler, Jillian E; Barnum, Scott R

    2009-12-31

    Multiple sclerosis (MS) is an autoimmune disease in which inflammation, leukocyte infiltration, and ultimately, demyelination occur as a result of innate and adaptive immune-mediated mechanisms. The pathophysiological role of the complement system, a major component of innate immunity, in the development and progression of experimental autoimmune encephalomyelitis (EAE), the animal model for MS has been extensively examined. Previous studies from our lab have shown that the complement receptor for the anaphylatoxin C3a, but not for C5a plays an important role in EAE. Based on the important contributions of the complement anaphylatoxin receptors to other inflammatory conditions in the CNS, we reasoned that deletion of both receptors may reveal underlying interactions between them that are important to EAE pathology. We performed EAE in C3aR/C5aR double knockout mice (C3aR/C5aR(-/-)) and observed delayed onset of disease but no attenuation of disease severity compared to wild type mice. Interestingly there was trend toward greater infiltration of CD4(+), but not CD8(+) T cells, in C3aR/C5aR(-/-) mice with EAE, suggesting altered trafficking of these cells. Antigen-specific T cells isolated from C3aR/C5aR(-/-) mice during acute EAE produced elevated levels of TNF-alpha, but markedly reduced levels of IFN-gamma and IL-12 compared to wild type mice. It remains unclear how the changes in these disease parameters contribute to the loss of the protective effect seen in C3aR(-/-) mice, however our data indicate a level of cross-modulation between the C3aR and C5aR during EAE.

  4. Deletion of Both the C3a and C5a Receptors Fails to Protect Against Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Ramos, Theresa N.; Wohler, Jillian E.; Barnum, Scott R.

    2009-01-01

    Multiple sclerosis (MS) is an autoimmune disease in which inflammation, leukocyte infiltration, and ultimately, demyelination occur as a result of innate and adaptive immune-mediated mechanisms. The pathophysiological role of the complement system, a major component of innate immunity, in the development and progression of experimental autoimmune encephalomyelitis (EAE), the animal model for MS has been extensively examined. Previous studies from our lab have shown that the complement receptor for the anaphylatoxin C3a, but not for C5a plays an important role in EAE. Based on the important contributions of the complement anaphylatoxin receptors to other inflammatory conditions in the CNS, we reasoned that deletion of both receptors may reveal underlying interactions between them that are important to EAE pathology. We performed EAE in C3aR/C5aR double knockout mice (C3aR/C5aR−/−) and observed delayed onset of disease but no attenuation of disease severity compared to wild type mice. Interestingly there was trend toward greater infiltration of CD4+, but not CD8+ T cells, in C3aR/C5aR−/− mice with EAE, suggesting altered trafficking of these cells. Antigen-specific T cells isolated from C3aR/C5aR−/− mice during acute EAE produced elevated levels of TNF-α, but markedly reduced levels of IFN-γ and IL-12 compared to wild type mice. It remains unclear how the changes in these disease parameters contribute to the loss of the protective effect seen in C3aR−/− mice, however our data indicate a level of cross modulation between the C3aR and C5aR during EAE. PMID:19850104

  5. Complement in health and disease.

    PubMed

    Carroll, Maria V; Sim, Robert B

    2011-09-16

    The complement system consists of about 35-40 proteins and glycoproteins present in blood plasma or on cell surfaces. Its main biological function is to recognise "foreign" particles and macromolecules, and to promote their elimination either by opsonisation or lysis. Although historically complement has been studied as a system for immune defence against bacteria, it has an important homeostatic role in which it recognises damaged or altered "self" components. Thus complement has major roles in both immune defence against microorganisms, and in clearance of damaged or "used" host components. Since complement proteins opsonise or lyse cells, complement can damage healthy host cells and tissues. The system is regulated by many endogenous regulatory proteins. Regulation is sometimes imperfect and both too much and too little complement activation is associated with many diseases. Excessive or inappropriate activation can cause tissue damage in diseases such as rheumatoid arthritis, age-related macular degeneration (AMD), multiple sclerosis, ischemia-reperfusion injury (e.g. ischemic stroke). Insufficient complement activity is associated with susceptibility to infection (mainly bacterial) and development of autoimmune disease, like SLE (systemic lupus erythematosus).

  6. A second case of human C3b inhibitor (KAF) deficiency.

    PubMed Central

    Thompson, R A; Lachmann, P J

    1977-01-01

    The second case of C3b inhibitor deficiency is described in an 11-year-old girl who presented with recurrent attacks of meningitis, in between which she was well. Her serum showed all of the complement component changes noted in the first described case, although showing only a relatively slight defect in its ability to opsonize bacteria for phagocytosis and killing by polymorphonuclear leucocytes. This correlated with the patient's freedom from other infections. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:849647

  7. Interaction between Host Complement and Mosquito-Midgut-Stage Plasmodium berghei

    PubMed Central

    Margos, Gabriele; Navarette, Sandra; Butcher, Geoff; Davies, Alex; Willers, Christine; Sinden, Robert E.; Lachmann, Peter J.

    2001-01-01

    After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement on Plasmodium developmental stages in the mosquito blood meal using the rodent malaria parasite Plasmodium berghei and rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion of P. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages of P. berghei survived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules. PMID:11447187

  8. Complement activation of electrogenic ion transport in isolated rat colon.

    PubMed

    McCole, D F; Otti, B; Newsholme, P; Baird, A W

    1997-11-15

    The complement cascade is an important component in many immune and inflammatory reactions and may contribute to both the diarrhoea and inflammation associated with inflammatory bowel disease. Isolated rat colonic mucosae were voltage clamped in Ussing chambers. Basolateral addition of zymosan-activated whole human serum (ZAS) induced a rapid onset, transient inward short circuit current (SCC). This response was concentration dependent and was significantly attenuated by pre-heating ZAS at 60 degrees C for 30 min. Depletion of complement from normal human serum with cobra venom factor (CVF) significantly lowered SCC responses. Chloride was the primary charge carrying ion as responses to ZAS were abolished in the presence of the loop diuretic bumetanide. The complement component C3a stimulated ion transport but not to the same extent as whole serum. Exogenous C5 was without effect. The cyclooxygenase inhibitor piroxicam significantly attenuated the response to ZAS. These findings support the possibility that complement activation may contribute to the pathophysiology of secretory diarrhoea since activation of electrogenic chloride secretion converts intestinal epithelia to a state of net fluid secretion.

  9. Differential mechanisms of complement-mediated neutralization of the closely related paramyxoviruses simian virus 5 and mumps virus

    SciTech Connect

    Johnson, John B.; Capraro, Gerald A.; Parks, Griffith D.

    2008-06-20

    The complement system is an important component of the innate immune response to virus infection. The role of human complement pathways in the in vitro neutralization of three closely related paramyxoviruses, Simian Virus 5 (SV5), Mumps virus (MuV) and Human Parainfluenza virus type 2 (HPIV2) was investigated. Sera from ten donors showed high levels of neutralization against HPIV2 that was largely complement-independent, whereas nine of ten donor sera were found to neutralize SV5 and MuV only in the presence of active complement pathways. SV5 and MuV neutralization proceeded through the alternative pathway of the complement cascade. Electron microscopy studies and biochemical analyses showed that treatment of purified SV5 with human serum resulted in C3 deposition on virions and the formation of massive aggregates, but there was relatively little evidence of virion lysis. Treatment of MuV with human serum also resulted in C3 deposition on virions, however in contrast to SV5, MuV particles were lysed by serum complement and there was relatively little aggregation. Assays using serum depleted of complement factors showed that SV5 and MuV neutralization in vitro was absolutely dependent on complement factor C3, but was not dependent on downstream complement factors C5 or C8. Our results indicate that even though antibodies exist that recognize both SV5 and MuV, they are mostly non-neutralizing and viral inactivation in vitro occurs through the alternative pathway of complement. The implications of our work for development of paramyxovirus vectors and vaccines are discussed.

  10. Deficiencies and excessive human complement system activation in disorders of multifarious etiology.

    PubMed

    Tichaczek-Goska, Dorota

    2012-01-01

    Complement is an integral part of the immune system protecting the host organism against invasion and proliferation of various microorganisms. It is also involved in the removal of the body's own damaged and altered cells. Activation of the complement system is a very precise process and it is strictly controlled by regulatory proteins present in both plasma and at host cells' surfaces. C3 protein plays a major role in the complement activation and generation of immune responses. Deficiencies of the C3 and other complement components, so-called early and late complement proteins, contribute to the emergence of recurrent bacterial, viral and fungal infections. The low level of mannose-binding lectin is also important. This protein plays a protective role in the early stages of infection and in the control of inflammation. Its deficit is one of the most common reasons for human immunodeficiency, observed in microbial infections as well as in autoimmune diseases such as rheumatoid arthritis. On the other hand, the excessive activation of complement proteins is often discovered to be the reason for many diseases. These include e.g. autoimmune diseases, Alzheimer's syndrome, schizophrenia, atypical hemolytic-uremic syndrome, angioedema, macular degeneration, and Crohn's disease.

  11. Phenotypic Heterogeneity Enables Uropathogenic Escherichia coli To Evade Killing by Antibiotics and Serum Complement

    PubMed Central

    Putrinš, Marta; Kogermann, Karin; Lukk, Eliisa; Lippus, Markus; Varik, Vallo

    2015-01-01

    Uropathogenic strains of Escherichia coli (UPEC) are the major cause of bacteremic urinary tract infections. Survival in the bloodstream is associated with different mechanisms that help to resist serum complement-mediated killing. While the phenotypic heterogeneity of bacteria has been shown to influence antibiotic tolerance, the possibility that it makes cells refractory to killing by the immune system has not been experimentally tested. In the present study we sought to determine whether the heterogeneity of bacterial cultures is relevant to bacterial targeting by the serum complement system. We monitored cell divisions in the UPEC strain CFT073 with fluorescent reporter protein. Stationary-phase cells were incubated in active or heat-inactivated human serum in the presence or absence of different antibiotics (ampicillin, norfloxacin, and amikacin), and cell division and complement protein C3 binding were measured by flow cytometry and immunofluorescence microscopy. Heterogeneity in the doubling times of CFT073 cells in serum enabled three phenotypically different subpopulations to be distinguished, all of them being recognized by the C3 component of the complement system. The population of rapidly growing cells resists serum complement-mediated lysis. The dominant subpopulation of cells with intermediate growth rate is susceptible to serum. The third population, which does not resume growth upon dilution from stationary phase, is simultaneously protected from serum complement and antibiotics. PMID:25561706

  12. Infections of People with Complement Deficiencies and Patients Who Have Undergone Splenectomy

    PubMed Central

    Ram, Sanjay; Lewis, Lisa A.; Rice, Peter A.

    2010-01-01

    Summary: The complement system comprises several fluid-phase and membrane-associated proteins. Under physiological conditions, activation of the fluid-phase components of complement is maintained under tight control and complement activation occurs primarily on surfaces recognized as “nonself” in an attempt to minimize damage to bystander host cells. Membrane complement components act to limit complement activation on host cells or to facilitate uptake of antigens or microbes “tagged” with complement fragments. While this review focuses on the role of complement in infectious diseases, work over the past couple of decades has defined several important functions of complement distinct from that of combating infections. Activation of complement in the fluid phase can occur through the classical, lectin, or alternative pathway. Deficiencies of components of the classical pathway lead to the development of autoimmune disorders and predispose individuals to recurrent respiratory infections and infections caused by encapsulated organisms, including Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. While no individual with complete mannan-binding lectin (MBL) deficiency has been identified, low MBL levels have been linked to predisposition to, or severity of, several diseases. It appears that MBL may play an important role in children, who have a relatively immature adaptive immune response. C3 is the point at which all complement pathways converge, and complete deficiency of C3 invariably leads to severe infections, including those caused by meningococci and pneumococci. Deficiencies of the alternative and terminal complement pathways result in an almost exclusive predisposition to invasive meningococcal disease. The spleen plays an important role in antigen processing and the production of antibodies. Splenic macrophages are critical in clearing opsonized encapsulated bacteria (such as pneumococci, meningococci, and Escherichia coli

  13. Molecular characterization of the alpha subunit of complement component C8 (GcC8α) in the nurse shark (Ginglymostoma cirratum)

    PubMed Central

    Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L.

    2009-01-01

    Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of α, β, and γ subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8α and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8γ-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4 % identity with human and Xenopus C8α respectively. Southern blot analysis showed GcC8α exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8α orthologs and as a sister taxa to the Xenopus. PMID:19524681

  14. Complement expression in retinal pigment epithelial cells is modulated by activated macrophages.

    PubMed

    Luo, Chang; Zhao, Jiawu; Madden, Angelina; Chen, Mei; Xu, Heping

    2013-07-01

    Complement activation is involved in a variety of retinal diseases. We have shown previously that a number of complement components and regulators can be produced locally in the eye, and that retinal pigment epithelial (RPE) cells are the major source of complement expression at the retina-choroidal interface. The expression of complement components by RPE cells is regulated by inflammatory cytokines. Under aging or inflammatory conditions, microglia and macrophages accumulate in the subretinal space, where they are in close contact with RPE cells. In this study, we investigated the effect of activated macrophages on complement expression by RPE cells. Mouse RPE cells were treated with the supernatants from un-activated bone marrow-derived macrophages (BM-DMs), the classically activated BM-DMs (M1) and different types of the alternatively activated BM-DMs (M2a by IL-4, M2b by immune complex and lipopolysaccharide (LPS), M2c by IL-10). The expression of inflammatory cytokines and complement genes by RPE cells were determined by real-time RT-PCR. The protein expression of CFB, C3, C1INH, and C1r was examined by Western blot. Our results show that un-stimulated RPE cells express a variety of complement-related genes, and that the expression levels of complement regulators, including C1r, factor H (CFH), DAF1, CD59, C1INH, Crry, and C4BP genes are significantly higher than those of complement component genes (C2, C4, CFB, C3, and C5). Macrophage supernatants increased inflammatory cytokine (IL-1β, IL-6, iNOS), chemokine (CCL2) and complement expression in RPE cells. The supernatants from M0, M2a and M2c macrophages mildly up-regulated (2-3.5-fold) CFB, CFH and C3 gene expression in RPE cells, whereas the supernatants from M1 and M2b macrophages massively increased (10-30-fold) CFB and C3 gene expression in RPE cells. The expression of other genes, including C1r, C2, C4, CFH, Masp1, C1INH, and C4BP in RPE cells was also increased by the supernatants of M1 and M2b

  15. Lamprey variable lymphocyte receptors mediate complement-dependent cytotoxicity.

    PubMed

    Wu, Fenfang; Chen, Liyong; Liu, Xin; Wang, Huaying; Su, Peng; Han, Yinglun; Feng, Bo; Qiao, Xu; Zhao, Jing; Ma, Ning; Liu, Huijie; Zheng, Zhen; Li, Qingwei

    2013-02-01

    An alternative adaptive-immune system is present in the most basal vertebrates--lampreys and hagfish--the only surviving jawless vertebrates. These eel-like fish use leucine-rich repeat-based receptors for Ag recognition instead of the Ig-based receptors used in jawed vertebrates. We report that in Japanese lamprey (Lampetra japonica), variable lymphocyte receptor (VLR)B interacts with C1q and C3 proteins to mediate complement-dependent cytotoxicity for bacteria and tumor cells. The immune-based lysis involves deposition of VLRB and C1q-like protein complex on the surface of target cells, activation of C3, and ultimate disruption of cell wall integrity. The demonstration of functional interaction between VLRB and complement components in lamprey provides evidence for the emergence of cooperative innate and adaptive-immune responses at a pivotal point in vertebrate evolution, before or in parallel with the evolution of Ig-based Abs and the classical complement-activation pathway.

  16. Anaphylatoxin-mediated regulation of the immune response. I. C3a- mediated suppression of human and murine humoral immune responses

    PubMed Central

    1982-01-01

    The C3a fragment of the third component of complement was found to have immunosuppressive properties. C3a is capable of suppressing both specific and polyclonal antibody responses. In contrast, C3a had no effect on antigen- or mitogen-induced B or T cell proliferative responses. The carboxy-terminal arginine is essential for C3a to exhibit its immunosuppressive properties. The serum carboxypeptidase inhibitor 2-mercaptomethyl-5-guanodinopentanoic acid, which prevents cleavage of the terminal arginine that would produce C3ades Arg-77, allowed us to assay the effects of C3a on in vitro immune response systems where serum is required. When the terminal arginine is removed from C3a, the resulting C3ades Arg-77 molecule is nonsuppressive. Helper T lymphocytes are the target of C3a-mediated suppression of the immune response. Substitution of T cells by soluble T cell factors was found to abrogate the C3a suppressive activity. PMID:6978374

  17. Meningococcal disease and the complement system

    PubMed Central

    Lewis, Lisa A; Ram, Sanjay

    2014-01-01

    Despite considerable advances in the understanding of the pathogenesis of meningococcal disease, this infection remains a major cause of morbidity and mortality globally. The role of the complement system in innate immune defenses against invasive meningococcal disease is well established. Individuals deficient in components of the alternative and terminal complement pathways are highly predisposed to invasive, often recurrent meningococcal infections. Genome-wide analysis studies also point to a central role for complement in disease pathogenesis. Here we review the pathophysiologic events pertinent to the complement system that accompany meningococcal sepsis in humans. Meningococci use several often redundant mechanisms to evade killing by human complement. Capsular polysaccharide and lipooligosaccharide glycan composition play critical roles in complement evasion. Some of the newly described protein vaccine antigens interact with complement components and have sparked considerable research interest. PMID:24104403

  18. Characterization of the complement inhibitory function of rhesus rhadinovirus complement control protein (RCP).

    PubMed

    Okroj, Marcin; Mark, Linda; Stokowska, Anna; Wong, Scott W; Rose, Nicola; Blackbourn, David J; Villoutreix, Bruno O; Spiller, O Brad; Blom, Anna M

    2009-01-02

    Rhesus rhadinovirus (RRV) is currently the closest known, fully sequenced homolog of human Kaposi sarcoma-associated herpesvirus. Both these viruses encode complement inhibitors as follows: Kaposi sarcoma-associated herpesvirus-complement control protein (KCP) and RRV-complement control protein (RCP). Previously we characterized in detail the functional properties of KCP as a complement inhibitor. Here, we performed comparative analyses for two variants of RCP protein, encoded by RRV strains H26-95 and 17577. Both RCP variants and KCP inhibited human and rhesus complement when tested in hemolytic assays measuring all steps of activation via the classical and the alternative pathway. RCP variants from both RRV strains supported C3b and C4b degradation by factor I and decay acceleration of the classical C3 convertase, similar to KCP. Additionally, the 17577 RCP variant accelerated decay of the alternative C3 convertase, which was not seen for KCP. In contrast to KCP, RCP showed no affinity to heparin and is the first described complement inhibitor in which the binding site for C3b/C4b does not interact with heparin. Molecular modeling shows a structural disruption in the region of RCP that corresponds to the KCP-heparin-binding site. This makes RRV a superior model for future in vivo investigations of complement evasion, as RCP does not play a supportive role in viral attachment as KCP does.

  19. Complement Activation and Inhibition in Wound Healing

    PubMed Central

    Cazander, Gwendolyn; Jukema, Gerrolt N.; Nibbering, Peter H.

    2012-01-01

    Complement activation is needed to restore tissue injury; however, inappropriate activation of complement, as seen in chronic wounds can cause cell death and enhance inflammation, thus contributing to further injury and impaired wound healing. Therefore, attenuation of complement activation by specific inhibitors is considered as an innovative wound care strategy. Currently, the effects of several complement inhibitors, for example, the C3 inhibitor compstatin and several C1 and C5 inhibitors, are under investigation in patients with complement-mediated diseases. Although (pre)clinical research into the effects of these complement inhibitors on wound healing is limited, available data indicate that reduction of complement activation can improve wound healing. Moreover, medicine may take advantage of safe and effective agents that are produced by various microorganisms, symbionts, for example, medicinal maggots, and plants to attenuate complement activation. To conclude, for the development of new wound care strategies, (pre)clinical studies into the roles of complement and the effects of application of complement inhibitors in wound healing are required. PMID:23346185

  20. The Complement System and Adverse Pregnancy Outcomes

    PubMed Central

    Regal, Jean F.; Gilbert, Jeffrey S.; Burwick, Richard M.

    2015-01-01

    Adverse pregnancy outcomes significantly contribute to morbidity and mortality for mother and child, with lifelong health consequences for both. The innate and adaptive immune system must be regulated to insure survival of the feta allograft, and the complement system is no exception. An intact complement system optimizes placental development and function and is essential to maintain host defense and fetal survival. Complement regulation is apparent at the placental interface from early pregnancy with some degree of complement activation occurring normally throughout gestation. However, a number of pregnancy complications including early pregnancy loss, fetal growth restriction, hypertensive disorders of pregnancy and preterm birth are associated with excessive or misdirected complement activation, and are more frequent in women with inherited or acquired complement system disorders or complement gene mutations. Clinical studies employing complement biomarkers in plasma and urine implicate dysregulated complement activation in components of each of the adverse pregnancy outcomes. In addition, mechanistic studies in rat and mouse models of adverse pregnancy outcomes address the complement pathways or activation products of importance and allow critical analysis of the pathophysiology. Targeted complement therapeutics are already in use to control adverse pregnancy outcomes in select situations. A clearer understanding of the role of the complement system in both normal pregnancy and complicated or failed pregnancy will allow a rational approach to future therapeutic strategies for manipulating complement with the goal of mitigating adverse pregnancy outcomes, preserving host defense, and improving long term outcomes for both mother and child. PMID:25802092

  1. Bioactive Lysophospholipids Generated by Hepatic Lipase Degradation of Lipoproteins Lead to Complement Activation via the Classical Pathway

    PubMed Central

    Ma, Wanchao; Paik, David C.; Barile, Gaetano R.

    2014-01-01

    Purpose. We determined bioactivity of lysophospholipids generated by degradation of the low-density (LDL), very low-density (VLDL), and high-density (HDL) lipoproteins with hepatic lipase (HL), cholesterol esterase (CE), and lipoprotein-associated phospholipase A2 (Lp-PLA2). Methods. The LDL, VLDL, and HDL were treated with HL, CE, and Lp-PLA2 after immobilization on plates, and complement activation studies were performed with diluted human serum. Complement component 3 (C3) fixation, a marker for complement activation, was determined with a monoclonal anti-human C3d antibody. Enzymatic properties of HL and CE were assayed with triglyceride and phosphatidylcholine substrates for triglyceride hydrolase and phospholipase A activities. The ARPE-19 cells were used for viability studies. Results. The HL degradation of human lipoproteins LDL, VLDL, or HDL results in the formation of modified lipoproteins that can activate the complement pathway. Complement activation is dose- and time-dependent upon HL and occurs via the classical pathway. Enzymatic studies suggest that the phospholipase A1 activity of HL generates complement-activating lysophospholipids. C-reactive protein (CRP), known to simultaneously interact with complement C1 and complement factor H (CFH), further enhances HL-induced complement activation. The lysophospholipids, 1-Palmitoyl-sn-glycero-3-phosphocholine and 1-Oleoyl-sn-glycero-3-phosphocholine, can be directly cytotoxic to ARPE-19 cells. Conclusions. The HL degradation of lipoproteins, known to accumulate in the outer retina and in drusen, can lead to the formation of bioactive lysophospholipids that can trigger complement activation and induce RPE cellular dysfunction. Given the known risk associations for age-related macular degeneration (AMD) with HL, CRP, and CFH, this study elucidates a possible damage pathway for age-related macular degeneration (AMD) in genetically predisposed individuals, that HL activity may lead to accumulation of

  2. Proteoform Profile Mapping of the Human Serum Complement Component C9 Revealing Unexpected New Features of N-, O-, and C-Glycosylation

    PubMed Central

    2017-01-01

    The human complement C9 protein (∼65 kDa) is a member of the complement pathway. It plays an essential role in the membrane attack complex (MAC), which forms a lethal pore on the cellular surface of pathogenic bacteria. Here, we charted in detail the structural microheterogeneity of C9 purified from human blood serum, using an integrative workflow combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The proteoform profile of C9 was acquired by high-resolution native mass spectrometry, which revealed the co-occurrence of ∼50 distinct mass spectrometry (MS) signals. Subsequent peptide-centric analysis, through proteolytic digestion of C9 and liquid chromatography (LC)-tandem mass spectrometry (MS/MS) measurements of the resulting peptide mixtures, provided site-specific quantitative profiles of three different types of C9 glycosylation and validation of the native MS data. Our study provides a detailed specification, validation, and quantification of 15 co-occurring C9 proteoforms and the first direct experimental evidence of O-linked glycans in the N-terminal region. Additionally, next to the two known glycosylation sites, a third novel, albeit low abundant, N-glycosylation site on C9 is identified, which surprisingly does not possess the canonical N-glycosylation sequence N-X-S/T. Our data also reveal a binding of up to two Ca2+ ions to C9. Mapping all detected and validated sites of modifications on a structural model of C9, as present in the MAC, hints at their putative roles in pore formation or receptor interactions. The applied methods herein represent a powerful tool for the unbiased in-depth analysis of plasma proteins and may advance biomarker discovery, as aberrant glycosylation profiles may be indicative of the pathophysiological state of the patients. PMID:28221766

  3. Genetic susceptibility to chronic wasting disease in free-ranging white-tailed deer: complement component C1q and Prnp polymorphisms

    USGS Publications Warehouse

    Blanchong, Julie A.; Heisey, Dennis M.; Scribner, Kim T.; Libants, Scot V.; Johnson, Chad; Aiken, Judd M.; Langenberg, Julia A.; Samuel, Michael D.

    2009-01-01

    The genetic basis of susceptibility to chronic wasting disease (CWD) in free-ranging cervids is of great interest. Association studies of disease susceptibility in free-ranging populations, however, face considerable challenges including: the need for large sample sizes when disease is rare, animals of unknown pedigree create a risk of spurious results due to population admixture, and the inability to control disease exposure or dose. We used an innovative matched case–control design and conditional logistic regression to evaluate associations between polymorphisms of complement C1q and prion protein (Prnp) genes and CWD infection in white-tailed deer from the CWD endemic area in south-central Wisconsin. To reduce problems due to admixture or disease-risk confounding, we used neutral genetic (microsatellite) data to identify closely related CWD-positive (n = 68) and CWD-negative (n = 91) female deer to serve as matched cases and controls. Cases and controls were also matched on factors (sex, location, age) previously demonstrated to affect CWD infection risk. For Prnp, deer with at least one Serine (S) at amino acid 96 were significantly less likely to be CWD-positive relative to deer homozygous for Glycine (G). This is the first characterization of genes associated with the complement system in white-tailed deer. No tests for association between any C1q polymorphism and CWD infection were significant at p < 0.05. After controlling for Prnp, we found weak support for an elevated risk of CWD infection in deer with at least one Glycine (G) at amino acid 56 of the C1qC gene. While we documented numerous amino acid polymorphisms in C1q genes none appear to be strongly associated with CWD susceptibility.

  4. Relationship of high CH50 level and interruption of cascade reaction of complement mRNA expression in acute venous thromboembolism patients

    PubMed Central

    Wen, Siwan; Yang, Fan; Wang, Lemin; Duan, Qianglin; Gong, Zhu; Lv, Wei

    2014-01-01

    In patients with pulmonary embolism (PE), forepart components of complements were activated. However there are interruption/decrease of cascade reaction and cytolytic effects in complement system. This study detected CRP, CH50, C3 and C4 levels in patients with venous thromboembolism (VTE) and compare with the imbalance of complement associated gene mRNA expression in PE patients. There was significant increase of CH50 in acute VTE patients. Even though CH50 increased significantly in acute VTE patients and had a relatively high sensitivity, cytolytic effects of complements might decrease, based on the genomics results of complement cascade reactions imbalance/interruption and increased total complements in VTE patients. PMID:25232435

  5. Plasmin(ogen) acquisition by group A Streptococcus protects against C3b-mediated neutrophil killing.

    PubMed

    Ly, Diane; Taylor, Jude M; Tsatsaronis, James A; Monteleone, Mercedes M; Skora, Amanda S; Donald, Cortny A; Maddocks, Tracy; Nizet, Victor; West, Nicholas P; Ranson, Marie; Walker, Mark J; McArthur, Jason D; Sanderson-Smith, Martina L

    2014-01-01

    The globally significant human pathogen group A Streptococcus (GAS) sequesters the host protease plasmin to the cell surface during invasive disease initiation. Recent evidence has shown that localized plasmin activity prevents opsonization of several bacterial species by key components of the innate immune system in vitro. Here we demonstrate that plasmin at the GAS cell surface resulted in degradation of complement factor C3b, and that plasminogen acquisition is associated with a decrease in C3b opsonization and neutrophil-mediated killing in vitro. Furthermore, the ability to acquire cell surface plasmin(ogen) correlates directly with a decrease in C3b opsonization, neutrophil phagocytosis, and increased bacterial survival in a humanized plasminogen mouse model of infection. These findings demonstrate that localized plasmin(ogen) plays an important role in facilitating GAS escape from the host innate immune response and increases bacterial virulence in the early stages of infection.

  6. Systemic complement activation in age-related macular degeneration.

    PubMed

    Scholl, Hendrik P N; Charbel Issa, Peter; Walier, Maja; Janzer, Stefanie; Pollok-Kopp, Beatrix; Börncke, Florian; Fritsche, Lars G; Chong, Ngaihang V; Fimmers, Rolf; Wienker, Thomas; Holz, Frank G; Weber, Bernhard H F; Oppermann, Martin

    2008-07-02

    Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH), factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001), were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes.This study is the first to show systemic complement activation in AMD patients. This suggests that AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were previously linked to AMD susceptibility.

  7. Development of a large scale human complement source for use in bacterial immunoassays.

    PubMed

    Brookes, Charlotte; Kuisma, Eeva; Alexander, Frances; Allen, Lauren; Tipton, Thomas; Ram, Sanjay; Gorringe, Andrew; Taylor, Stephen

    2013-05-31

    The serum bactericidal assay is the correlate of protection for meningococcal disease but the use and comparison of functional immunological assays for the assessment of meningococcal vaccines is complicated by the sourcing of human complement. This is due to high levels of immunity in the population acquired through natural meningococcal carriage and means that many individuals must be screened to find donors with suitably low bactericidal titres against the target strain. The use of different donors for each meningococcal strain means that comparisons of assay responses between strains and between laboratories is difficult. We have developed a method for IgG-depletion of 300 ml batches of pooled human lepirudin-derived plasma using Protein G sepharose affinity chromatography that retains complement activity. However, IgG-depletion also removed C1q. This was also eluted from the affinity matrix, concentrated and added to the complement source. The final complement source retained mean alternative pathway activity of 96.8% and total haemolytic activity of 84.2% in four batches. Complement components C3, C5, properdin and factor H were retained following the process and the IgG-depleted complement was shown to be suitable for use in antibody-mediated complement deposition and serum bactericidal activity assays against serogroup B meningococci. The generation of large IgG-depleted batches of pooled human plasma allows for the comparison of immunological responses to diverse meningococcal strain panels in large clinical trials.

  8. Systemic complement profiling in multiple sclerosis as a biomarker of disease state

    PubMed Central

    Ingram, G; Hakobyan, S; Hirst, CL; Harris, CL; Loveless, S; Mitchell, JP; Pickersgill, TP; Robertson, NP

    2012-01-01

    Background: There is increasing evidence of significant and dynamic systemic activation and upregulation of complement in multiple sclerosis (MS), which may contribute to disease pathogenesis. Objective: We aimed to investigate the pathological role of complement in MS and the potential role for complement profiling as a biomarker of MS disease state. Methods: Key components of the classical, alternative and terminal pathways of complement were measured in plasma and cerebrospinal fluid (CSF) of patients with MS in different clinical phases of disease and in matched controls. Results: Increased plasma levels of C3 (p<0.003), C4 (p<0.001), C4a (p<0.001), C1 inhibitor (p<0.001), and factor H (p<0.001), and reduced levels of C9 (p<0.001) were observed in MS patients compared with controls. Combined profiling of these analytes produced a statistical model with a predictive value of 97% for MS and 73% for clinical relapse when combined with selected demographic data. CSF-plasma correlations suggested that source of synthesis of these components was both systemic and central. Conclusion: These data provide further evidence of alterations in both local and systemic expression and activation of complement in MS and suggest that complement profiling may be informative as a biomarker of MS disease, although further work is needed to determine its use in distinguishing MS from its differential. PMID:22354735

  9. The Lectin Pathway of Complement and Rheumatic Heart Disease

    PubMed Central

    Beltrame, Marcia Holsbach; Catarino, Sandra Jeremias; Goeldner, Isabela; Boldt, Angelica Beate Winter; de Messias-Reason, Iara José

    2014-01-01

    The innate immune system is the first line of host defense against infection and is comprised of humoral and cellular mechanisms that recognize potential pathogens within minutes or hours of entry. The effector components of innate immunity include epithelial barriers, phagocytes, and natural killer cells, as well as cytokines and the complement system. Complement plays an important role in the immediate response against microorganisms, including Streptococcus sp. The lectin pathway is one of three pathways by which the complement system can be activated. This pathway is initiated by the binding of mannose-binding lectin (MBL), collectin 11 (CL-K1), and ficolins (Ficolin-1, Ficolin-2, and Ficolin-3) to microbial surface oligosaccharides and acetylated residues, respectively. Upon binding to target molecules, MBL, CL-K1, and ficolins form complexes with MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2), which cleave C4 and C2 forming the C3 convertase (C4b2a). Subsequent activation of complement cascade leads to opsonization, phagocytosis, and lysis of target microorganisms through the formation of the membrane-attack complex. In addition, activation of complement may induce several inflammatory effects, such as expression of adhesion molecules, chemotaxis and activation of leukocytes, release of reactive oxygen species, and secretion of cytokines and chemokines. In this chapter, we review the general aspects of the structure, function, and genetic polymorphism of lectin-pathway components and discuss most recent understanding on the role of the lectin pathway in the predisposition and clinical progression of Rheumatic Fever. PMID:25654073

  10. Complement System Part II: Role in Immunity

    PubMed Central

    Merle, Nicolas S.; Noe, Remi; Halbwachs-Mecarelli, Lise; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T.

    2015-01-01

    The complement system has been considered for a long time as a simple lytic cascade, aimed to kill bacteria infecting the host organism. Nowadays, this vision has changed and it is well accepted that complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. This review discusses recent advances in the understanding of the role of complement in physiology and pathology. It starts with a description of complement contribution to the normal physiology (homeostasis) of a healthy organism, including the silent clearance of apoptotic cells and maintenance of cell survival. In pathology, complement can be a friend or a foe. It acts as a friend in the defense against pathogens, by inducing opsonization and a direct killing by C5b–9 membrane attack complex and by triggering inflammatory responses with the anaphylatoxins C3a and C5a. Opsonization plays also a major role in the mounting of an adaptive immune response, involving antigen presenting cells, T-, and B-lymphocytes. Nevertheless, it can be also an enemy, when pathogens hijack complement regulators to protect themselves from the immune system. Inadequate complement activation becomes a disease cause, as in atypical hemolytic uremic syndrome, C3 glomerulopathies, and systemic lupus erythematosus. Age-related macular degeneration and cancer will be described as examples showing that complement contributes to a large variety of conditions, far exceeding the classical examples of diseases associated with complement deficiencies. Finally, we discuss complement as a therapeutic target. PMID:26074922

  11. Targeted complement inhibition and microvasculature in transplants: a therapeutic perspective.

    PubMed

    Khan, M A; Hsu, J L; Assiri, A M; Broering, D C

    2016-02-01

    Active complement mediators play a key role in graft-versus-host diseases, but little attention has been given to the angiogenic balance and complement modulation during allograft acceptance. The complement cascade releases the powerful proinflammatory mediators C3a and C5a anaphylatoxins, C3b, C5b opsonins and terminal membrane attack complex into tissues, which are deleterious if unchecked. Blocking complement mediators has been considered to be a promising approach in the modern drug discovery plan, and a significant number of therapeutic alternatives have been developed to dampen complement activation and protect host cells. Numerous immune cells, especially macrophages, develop both anaphylatoxin and opsonin receptors on their cell surface and their binding affects the macrophage phenotype and their angiogenic properties. This review discusses the mechanism that complement contributes to angiogenic injury, and the development of future therapeutic targets by antagonizing activated complement mediators to preserve microvasculature in rejecting the transplanted organ.

  12. Studies on immune adherence (C3b) receptor activity of human erythrocytes: relationship between receptor activity and presence of immune complexes in serum.

    PubMed Central

    Inada, Y; Kamiyama, M; Kanemitsu, T; Hyman, C L; Clark, W S

    1982-01-01

    Human erythrocytes (E) have surface receptors for the third component of complement (C3b-IA receptors) which mediate immune adherence haemagglutination (IAHA). We have observed that E from patients with systemic lupus erythematosus had imparied or defective C3b receptor (C3b-R) activity when circulating immune complexes (CIC) were found in serum. This phenomenon has been investigated by a newly developed method involving competitive inhibition of IAHA in patients with immune complex diseases. IAHA involving sheep E coated with antibody and complement (EAC), and indicator human E was inhibited by lysates of E with normal C3b-R activity from healthy donors and a monkey. In contrast, the lysates of E from 95% of patients bearing CIC did not inhibit IAHA, which indicated such E had defective or impaired C3b-R activity. This phenomenon was supported by control studies in which IAHA was not inhibited by lysates of E with absent, inactivated or occupied C3b-R. In those patients, in whom CIC disappeared during immunosuppressive therapy, C3b-R activity slowly returned to normal levels. Moreover, it was observed that C3b-R activity of patients' E decreased with the reappearance of CIC during exacerbations of disease. These observations suggest that CIC are carried in vivo by the C3b-R of E as well as those of the mononuclear phagocyte system, and that the E C3b-R may also contribute to the clearance of CIC. PMID:6216998

  13. Complement activation by a B cell superantigen.

    PubMed

    Kozlowski, L M; Soulika, A M; Silverman, G J; Lambris, J D; Levinson, A I

    1996-08-01

    Staphylococcal protein A (SpA), acting as a B cell superantigen, binds to the Fab region of human VH3+ Igs. Using SpA abrogated of its IgG Fc binding activity (Mod SpA) as a model B cell superantigen, we determined whether such an interaction causes complement activation. Addition of Mod SpA to human serum led to complement consumption and the generation of C3a. To determine whether this complement activation 1) was due to an interaction between VH3+ Igs and the Fab binding site of SpA and 2) proceeded via the classical complement pathway, we tested a panel of monoclonal IgM proteins for the ability to hind C1q following interaction with SpA. C1q binding was restricted to SpA-reactive, VH3+ IgM proteins. To formally determine whether the binding of SpA to the reactive VH3+ IgM proteins led to complement activation, we reconstituted the serum from a hypogammaglobulinemic patient with monoclonal IgM proteins and measured complement consumption and C3a generation following the addition of Mod SpA. We observed complement consumption and C3a production only in Mod SpA-treated serum reconstituted with a VH3+, SpA-binding, IgM protein. Taken together, these results provide compelling evidence that the interaction of the Fab binding site of SpA and VH3+ Igs can lead to complement activation via the classical pathway. This novel interaction may have significant implications for the in vivo properties of a B cell superantigen.

  14. Complement associated pathogenic mechanisms in myasthenia gravis.

    PubMed

    Tüzün, Erdem; Christadoss, Premkumar

    2013-07-01

    The complement system is profoundly involved in the pathogenesis of acetylcholine receptor (AChR) antibody (Ab) related myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). The most characteristic finding of muscle pathology in both MG and EAMG is the abundance of IgG and complement deposits at the nerve-muscle junction (NMJ), suggesting that AChR-Ab induces muscle weakness by complement pathway activation and consequent membrane attack complex (MAC) formation. This assumption has been supported with EAMG resistance of complement factor C3 knockout (KO), C4 KO and C5 deficient mice and amelioration of EAMG symptoms following treatment with complement inhibitors such as cobra venom factor, soluble complement receptor 1, anti-C1q, anti-C5 and anti-C6 Abs. Moreover, the complement inhibitor decay accelerating factor (DAF) KO mice exhibit increased susceptibility to EAMG. These findings have brought forward improvisation of novel therapy methods based on inhibition of classical and common complement pathways in MG treatment.

  15. Can Cell Bound Complement Activation Products Predict Inherited Complement Deficiency in Systemic Lupus Erythematosus?

    PubMed Central

    Waters, Barry

    2016-01-01

    Activation of the classical pathway complement system has long been implicated in stimulating immune complex mediated tissue destruction in systemic lupus erythematosus (SLE). C3 and C4 complement levels are utilized as part of SLE diagnosis and monitoring criteria. Recently, cell bound complement activation products (CBCAPs) have shown increased sensitivity in diagnosing and monitoring lupus activity, compared to traditional markers. CBCAPs are increasingly utilized in rheumatology practice as additional serological markers in evaluating SLE patients. We report a case of a patient diagnosed with SLE that had chronically low C3 and C4, along with negative CBCAPs. We surmise that the patient has an inherited complement deficiency as the etiology of her SLE and that CBCAPs could be used to predict such deficiency. PMID:28074166

  16. C3 dysregulation due to factor H deficiency is mannan-binding lectin-associated serine proteases (MASP)-1 and MASP-3 independent in vivo

    PubMed Central

    Ruseva, M M; Takahashi, M; Fujita, T; Pickering, M C

    2014-01-01

    Uncontrolled activation of the complement alternative pathway is associated with complement-mediated renal disease. Factor B and factor D are essential components of this pathway, while factor H (FH) is its major regulator. In complete FH deficiency, uncontrolled C3 activation through the alternative pathway results in plasma C3 depletion and complement-mediated renal disease. These are dependent on factor B. Mannan-binding lectin-associated serine proteases 1 and 3 (MASP-1, MASP-3) have been shown recently to contribute to alternative pathway activation by cleaving pro-factor D to its active form, factor D. We studied the contribution of MASP-1 and MASP-3 to uncontrolled alternative pathway activation in experimental complete FH deficiency. Co-deficiency of FH and MASP-1/MASP-3 did not ameliorate either the plasma C3 activation or glomerular C3 accumulation in FH-deficient mice. Our data indicate that MASP-1 and MASP-3 are not essential for alternative pathway activation in complete FH deficiency. PMID:24279761

  17. ROLE OF C/EBP-β, p38 MAPK and MKK6 IN IL-1β MEDIATED C3 GENE REGULATION IN ASTROCYTES

    PubMed Central

    Maranto, Jeffrey; Rappaport, Jay; Datta, Prasun K.

    2011-01-01

    Complement component C3, the central player in the complement cascade and the pro-inflammatory cytokine IL-1β is expressed by activated glial cells and may contribute to neurodegeneration. This study examines the regulation of the expression of C3 by IL-1β in astroglial cells focusing on the role of the upstream kinase MKK6, p38-α MAPK and C/EBP-β isoforms (LAP1, LAP2 or LIP) in astroglial cells. Activation of human astroglial cell line, U373 with IL-1β, led to the induction of C3 mRNA and protein expression as determined by real-time RT-PCR and western blot analysis, respectively. This induction was suppressed by the pharmacological inhibitor of p38 MAPK (i.e., SB202190-HCl), suggesting the involvement of p38 MAPK in C3 gene expression. IL-1β also induced C3 promoter activity in U373 cells in a MAP kinase- and C/EBP-β-dependent manner. Cotransfection of C3 luciferase reporter construct with constitutively active form of the upstream kinase in the MAP kinase cascade, i.e., MKK6 (the immediate upstream activator of p38 kinase) resulted in marked stimulation of the promoter activity, whereas, overexpression of a dominant negative forms of MKK6 and p38α MAPK inhibited C3 promoter activity. Furthermore, a mutant form of C/EBP-β, LAPT235A showed reduction in IL-1β mediated C3 promoter activation. These results suggest that the p38α MAPK and MKK6 play prominent roles in IL-1β and C/EBP-β mediated C3 gene expression in astrocytes. PMID:21308746

  18. Activation of C3a receptor is required in cigarette smoke-mediated emphysema

    PubMed Central

    Yuan, Xiaoyi; Shan, Ming; You, Ran; Frazier, Michael V.; Hong, Monica Jeongsoo; Wetsel, Rick A.; Drouin, Scott; Seryshev, Alexander; MD, Li-zhen Song; Cornwell, Lorraine; Rossen, Roger D; Corry, David B.; Kheradmand, Farrah

    2014-01-01

    Exposure to cigarette smoke can initiate sterile inflammatory responses in the lung and activate myeloid dendritic cells (mDCs) that induce differentiation of T helper type 1 (Th1) and Th17 cells in the emphysematous lungs. Consumption of complement proteins increases in acute inflammation, but the contribution of complement protein 3 (C3) to chronic cigarette smoke-induced immune responses in the lung is not clear. Here we show that following chronic exposure to cigarette smoke, C3 deficient (C3−/−) mice develop less emphysema and have fewer CD11b+CD11c+ mDCs infiltrating the lungs as compared to wild type mice. Proteolytic cleavage of C3 by neutrophil elastase releases C3a, which in turn increases expression of its receptor (C3aR) on lung mDCs. Mice deficient in the C3aR (C3ar−/−) partially phenocopy the attenuated responses to chronic smoke observed in C3−/− mice. Consistent with a role for C3 in emphysema C3 and its active fragments are deposited on the lung tissue of smokers with emphysema, and smoke exposed mice. Together, these findings suggest a critical role for C3a through autocrine/paracrine induction of C3aR in the pathogenesis of cigarette smoke induced sterile inflammation and provide new therapeutic targets for the treatment of emphysema. PMID:25465103

  19. The effects of soluble recombinant complement receptor 1 on complement-mediated experimental glomerulonephritis.

    PubMed

    Couser, W G; Johnson, R J; Young, B A; Yeh, C G; Toth, C A; Rudolph, A R

    1995-05-01

    Complement is a major mediator of tissue injury in several types of glomerulonephritis. However, no therapeutic agents that inhibit complement activation are available for human use. sCR1 (TP10, BRL 55736) is a recombinant, soluble human complement receptor 1 (CR1) molecule lacking transmembrane and cytoplasmic domains that inhibits C3 and C5 convertase activity by preferentially binding C4b and C3b. To test the efficacy of sCR1 on complement-mediated glomerulonephritis, rats were pretreated with sCR1 (60 mg/kg per day) before and during the induction of three models of complement-dependent glomerulonephritis (concanavalin A and antithymocyte serum models of proliferative glomerulonephritis, passive Heyman nephritis). Daily sCR1 and complement hemolytic activity levels were measured, and renal histology and urine protein excretion were examined. Mean serum sCR1 levels of 100 to 200 micrograms/mL were maintained with a reduction in complement hemolytic activity to less than 15% in most animals. In the antithymocyte serum model, sCR1-treated animals had significant reductions in mesangiolysis, glomerular platelet and macrophage infiltrates, and proteinuria at 48 h. In the concanavalin A model, sCR1 significantly reduced glomerular C3 and fibrin deposits, platelet infiltrates, and proteinuria at 48 h. In passive Heymann nephritis, proteinuria was also significantly reduced (199 +/- 8.5 versus 125 +/- 16 mg/day, P < 0.002) at 5 days. It was concluded that sCR1 significantly reduces both morphologic and functional consequences of several different types of complement-mediated glomerulonephritis and deserves evaluation as a potential therapeutic agent in complement-mediated immune glomerular disease in humans.

  20. Regulation of humoral immunity by complement.

    PubMed

    Carroll, Michael C; Isenman, David E

    2012-08-24

    The complement system of innate immunity is important in regulating humoral immunity largely through the complement receptor CR2, which forms a coreceptor on B cells during antigen-induced activation. However, CR2 also retains antigens on follicular dendritic cells (FDCs). Display of antigen on FDCs is critical for clonal selection and affinity maturation of activated B cells. This review will discuss the role of complement in adaptive immunity in general with a focus on the interplay between CR2-associated antigen on B cells with CR2 expressed on FDCs. This latter interaction provides an opportunity for memory B cells to sample antigen over prolonged periods. The cocrystal structure of CR2 with its ligand C3d provides insight into how the complement system regulates access of antigen by B cells with implications for therapeutic manipulations to modulate aberrant B cell responses in the case of autoimmunity.

  1. Biological effects of short-term, high-concentration exposure to methyl isocyanate. VI. In vitro and in vivo complement activation studies.

    PubMed Central

    Kolb, W P; Savary, J R; Troup, C M; Dodd, D E; Tamerius, J D

    1987-01-01

    The ability of MIC to induce complement activation in vitro and in vivo was investigated. For the in vitro studies, both human and guinea pig serum or EDTA-plasma samples were exposed to 1167 to 1260 ppm MIC vapor for 15 min at room temperature. The human serum samples exposed to MIC showed significant reductions in Factor B, C2, C4, C3, C5, and total hemolytic complement CH50 activity levels. C6 functional activity was unaffected. The C3, C5, and CH50 functional activities in guinea pig serum (the only functional tests conducted on these samples) were more sensitive to MIC-mediated reduction than the corresponding activity reductions observed in the human serum samples. The human and single guinea pig EDTA-plasma samples exposed to MIC vapor showed no evidence of C3 consumption but did show significant reductions in CH50 levels. Thus, MIC vapor was able to activate, and thereby reduce serum complement C3 activity in vitro by a complement-dependent process. However, the data suggest at least one complement component other than C3 was inactivated in EDTA-plasma by a complement-independent mechanism. For the in vivo studies, five pairs of guinea pigs were exposed to 644 to 702 ppm MIC vapor until one of the pair died (11-15 min). MIC exposure was then discontinued, the surviving guinea pig was sacrificed, and EDTA-plasma was obtained from both animals and analyzed for complement consumption.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3622434

  2. Loss of Properdin Exacerbates C3 Glomerulopathy Resulting from Factor H Deficiency

    PubMed Central

    Ruseva, Marieta M.; Vernon, Katherine A.; Lesher, Allison M.; Schwaeble, Wilhelm J.; Ali, Youssif M.; Botto, Marina; Cook, Terence; Song, Wenchao; Stover, Cordula M.

    2012-01-01

    Complement factor H (CFH) is a negative regulator of the alternative pathway of complement, and properdin is the sole positive regulator. CFH-deficient mice (CFH−/−) develop uncontrolled C3 activation and spontaneous renal disease characterized by accumulation of C3 along the glomerular basement membrane, but the role of properdin in the pathophysiology is unknown. Here, we studied mice deficient in both CFH and properdin (CFH−/−.P−/−). Although CFH−/− mice had plasma depleted of both C3 and C5, CFH−/−.P−/− animals exhibited depletion of C3 predominantly, recapitulating the plasma complement profile observed in humans with properdin-independent C3 nephritic factors. Glomerular inflammation, thickening of the capillary wall, and glomerular C3 staining were significantly increased in CFH−/−.P−/− compared with CFH−/− mice. We previously reported that exogenous CFH ameliorates C3 staining of the glomerular basement membrane and triggers the appearance of mesangial C3 deposits in CFH−/− mice; here, we show that these effects require properdin. In summary, during uncontrolled activation of C3 driven by complete CFH deficiency, properdin influences the intraglomerular localization of C3, suggesting that therapeutic inhibition of properdin would be detrimental in this setting. PMID:23184055

  3. Targeted Inhibition of Complement Using Complement Receptor 2-Conjugated Inhibitors Attenuates EAE

    PubMed Central

    Hu, Xianzhen; Tomlinson, Stephen; Barnum, Scott R.

    2012-01-01

    Multiple sclerosis (MS) is the most common autoimmune demyelinating disease, affecting millions of individuals worldwide. In the last two decades, many therapeutic options for the treatment of MS have become available, however they are limited in terms of effectiveness and some remain plagued by safety issues. The currently available treatment options target relapsing remitting forms of MS and are not effective against the more progressive forms of the disease. These limitations highlight a significant unmet treatment need for MS. In experimental autoimmune encephalomyelitis (EAE) studies from our laboratory, we have previously shown, using a number of complement mutant and transgenic mice, that inhibition of the alternative complement pathway and the C3 convertase confers significant protection from disease. We report here that targeted inhibition of complement activation using complement receptor 2 (CR2)-conjugated inhibitors significantly attenuates EAE. Administration of CR2-Crry (blocks all complement pathways at C3 activation) and CR2-fH (specifically blocks the alternative pathway) just prior to and during the onset of EAE blocks progression of both acute and chronic disease. These data indicate that inhibition of complement may offer an effective therapeutic approach to treating both acute and chronic forms of demyelinating disease through blocking the alternative pathway or complement convertases. PMID:23079547

  4. Targeted inhibition of complement using complement receptor 2-conjugated inhibitors attenuates EAE.

    PubMed

    Hu, Xianzhen; Tomlinson, Stephen; Barnum, Scott R

    2012-11-30

    Multiple sclerosis (MS) is the most common autoimmune demyelinating disease, affecting millions of individuals worldwide. In the last two decades, many therapeutic options for the treatment of MS have become available, however they are limited in terms of effectiveness and some remain plagued by safety issues. The currently available treatment options target relapsing remitting forms of MS and are not effective against the more progressive forms of the disease. These limitations highlight a significant unmet treatment need for MS. In experimental autoimmune encephalomyelitis (EAE) studies from our laboratory, we have previously shown, using a number of complement mutant and transgenic mice, that inhibition of the alternative complement pathway and the C3 convertase confers significant protection from disease. We report here that targeted inhibition of complement activation using complement receptor 2 (CR2)-conjugated inhibitors significantly attenuates EAE. Administration of CR2-Crry (blocks all complement pathways at C3 activation) and CR2-fH (specifically blocks the alternative pathway) just prior to and during the onset of EAE blocks progression of both acute and chronic disease. These data indicate that inhibition of complement may offer an effective therapeutic approach to treating both acute and chronic forms of demyelinating disease through blocking the alternative pathway or complement convertases.

  5. Therapeutic complement inhibition in complement-mediated hemolytic anemias: Past, present and future.

    PubMed

    Risitano, Antonio M; Marotta, Serena

    2016-06-01

    The introduction in the clinic of anti-complement agents represented a major achievement which gave to physicians a novel etiologic treatment for different human diseases. Indeed, the first anti-complement agent eculizumab has changed the treatment paradigm of paroxysmal nocturnal hemoglobinuria (PNH), dramatically impacting its severe clinical course. In addition, eculizumab is the first agent approved for atypical Hemolytic Uremic Syndrome (aHUS), a life-threatening inherited thrombotic microangiopathy. Nevertheless, such remarkable milestone in medicine has brought to the fore additional challenges for the scientific community. Indeed, the list of complement-mediated anemias is not limited to PNH and aHUS, and other human diseases can be considered for anti-complement treatment. They include other thrombotic microangiopathies, as well as some antibody-mediated hemolytic anemias. Furthermore, more than ten years of experience with eculizumab led to a better understanding of the individual steps of the complement cascade involved in the pathophysiology of different human diseases. Based on this, new unmet clinical needs are emerging; a number of different strategies are currently under development to improve current anti-complement treatment, trying to address these specific clinical needs. They include: (i) alternative anti-C5 agents, which may improve the heaviness of eculizumab treatment; (ii) broad-spectrum anti-C3 agents, which may improve the efficacy of anti-C5 treatment by intercepting the complement cascade upstream (i.e., preventing C3-mediated extravascular hemolysis in PNH); (iii) targeted inhibitors of selective complement activating pathways, which may prevent early pathogenic events of specific human diseases (e.g., anti-classical pathway for antibody-mediated anemias, or anti-alternative pathway for PNH and aHUS). Here we briefly summarize the status of art of current and future complement inhibition for different complement-mediated anemias

  6. Glycoproteins, antigens, and regulation of complement activation on the surface of the protozoan parasite Trypanosoma lewisi: implications for immune evasion

    SciTech Connect

    Sturtevant, J.E.

    1985-01-01

    The surface antigens and glycoproteins of the rat parasitic protozoan, Trypanosoma lewisi were characterized. Radioiodination with /sup 125/I identified 10 out of more 40 polypeptides separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis. All of these components were identified as glycoproteins by peroxidase-conjugated Conconavalin A (HR-Con A) lectin affinoblotting. This analysis detected that quantitative but not qualitative changes occurred during infection. Localization of most of the reactive determinants was indicated by immunoblotting extracts of radioiodinated T. lewisi. Changes in the antigenicity as related to survival in the host are discussed. The presence of IgG and IgM on the surface of T. lewisi isolated from intact and ..gamma..-irradiated rats (irr.) and that determinants bind Ig from uninfected rat sera (NRS) was indicated by flow cytometric analysis. Immunoblotting identified the major NRS IgG binding component as the 74 kd surface glycoprotein. Complement component C3 deposition during infection was indicated by flow cytometric analysis and immunoblotting. Incubation of intact T. lewisi with normal human sera indicated that C3, C5, and factor B deposition was Mg/sup 2 +/ dependent, Ca/sup 2 +/ independent and deposited C3 was rapidly processed to hemolytically inactive fragments. Radioiodination of intact and protease T. lewisi after cultivation identified three components which correlate with resistance to lysis. This suggests that surface moieties on intact T. lewisi modulate host complement activity by restricting C3/C5 convertase activity.

  7. [Incidence of factor VII and C3 in vascular anastomosis].

    PubMed

    Skóra, Jan; Janczak, Dariusz; Korta, Krzysztof; Pupka, Artur; Ruciński, Artur; Szyber, Piotr; Pawłowski, Stanisław; Milnerowicz, Artur; Stepiński, Piotr

    2004-06-01

    In order to study pathogenesis of vascular prosthesis healing process the following experiment was designed. 16 dogs underwent implantation of unilateral straight aorto-femoral teflon (PTFE, polytetrafluoroethylene) by-pass. After 6 months all dogs were killed, dissected and vascular prostheses with margin of adjacent aorta and femoral artery were collected for further study. Areas of proximal and distal anastomosis were examined immunohistochemically. Presence of coagulation factor VII, and C3 complement factor were studied. The obtained results were analyzed statistically by means of t-Student test. Factor VII as well C3 were found in areas of both proximal and distal anastomosis. Concentration of all two substances in proximal and distal anastomosis was compared. No statistically valid differences in factor VII concentration in proximal and distal anastomosis were found, whereas amounts of C3 factor as well as degree of extracellular matrix infiltration were markedly higher in distal anastomosis.

  8. Targeting complement in therapy.

    PubMed

    Kirschfink, M

    2001-04-01

    With increasing evidence that complement activation significantly contributes to the pathogenesis of a large number of inflammatory diseases, strategies that interfere with its deleterious action have become a major focus in pharmacological research. Endogenous soluble complement inhibitors (C1 inhibitor, recombinant soluble complement receptor 1, antibodies) blocking key proteins of the cascade reaction, neutralizing the action of the complement-derived anaphylatoxin C5a, or interfering with complement receptor 3 (CR3, CD18/11b)-mediated adhesion of inflammatory cells to the vascular endothelium have successfully been tested in various animal models over the past years. Promising results consequently led to clinical trials. Furthermore, incorporation of membrane-bound complement regulators (decay-accelerating factor (CD55), membrane co-factor protein (CD46), CD59) in transgenic animals has provided a major step forward in protecting xenografts from hyperacute rejection. At the same time, the poor contribution of complement to the antitumor response, which is caused by multiple resistance mechanisms that hamper the efficacy of antibody-based tumor therapy, is increasingly recognized and requires pharmacologic intervention. First attempts have now been made to interfere with the resistance mechanisms, thereby improving complement-mediated tumor cell destruction.

  9. Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes

    SciTech Connect

    Lutz, H.U.; Bussolino, F.; Flepp, R.; Fasler, S.; Stammler, P.; Kazatchkine, M.D.; Arese, P.

    1987-11-01

    Treatment of erythrocytes with the thiol-specific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3- antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which simulate senescent erythrocytes with respect to bound antibody and complement.

  10. Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement.

    PubMed

    Rosengard, Ariella M; Liu, Yu; Nie, Zhiping; Jimenez, Robert

    2002-06-25

    Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30-40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges.

  11. Role of complement systems in IVIG mediated attenuation of cognitive deterioration in Alzheimer's disease.

    PubMed

    Gong, Bing; Levine, Samara; Barnum, Scott R; Pasinetti, Giulio M

    2014-01-01

    Human intravenous immunoglobulin (IVIG) has been indicated as a potential therapy for autoimmune neurological disorders, as well as in many neurodegenerative diseases, with various underlying therapeutic mechanisms such as regulation of T-cell trafficking, cytokines, Fc receptor blocking, and interruption of complement activation cascade. In Alzheimer's disease (AD), IVIG presents naturally occurring antibodies against amyloid-beta (Aβ) aggregation, thus IVIG immunotherapy may increase the clearance of Aβ and protect brain function. Recently, we and others reported that besides Aβ clearance, IVIG specifically regulates the levels of complement-derived anaphylatoxins, such as C5a and C3, which play an important role in the regulation of AMPA and NMDA receptor expression in the brain and further upregulate the AMPA-PKA-CREB signaling pathway and synaptic function in AD mouse models. Since down-regulation of complement components has been linked with deficits of cognitive function in age-related dementia following the decline of innate immunity during aging, the IVIG immunotherapy could be an attractive novel AD therapeutic through its local regulation of C3, C5a component levels in brain.

  12. C5 Modulates Airway Hyperreactivity and Pulmonary Eosinophilia during Enhanced Respiratory Syncytial Virus Disease by Decreasing C3a Receptor Expression▿

    PubMed Central

    Melendi, Guillermina A.; Hoffman, Scott J.; Karron, Ruth A.; Irusta, Pablo M.; Laham, Federico R.; Humbles, Alison; Schofield, Brian; Pan, Chien-Hsiung; Rabold, Richard; Thumar, Bhagvanji; Thumar, Adeep; Gerard, Norma P.; Mitzner, Wayne; Barnum, Scott R.; Gerard, Craig; Kleeberger, Steven R.; Polack, Fernando P.

    2007-01-01

    Enhanced respiratory syncytial virus disease, a serious pulmonary disorder that affected recipients of an inactivated vaccine against respiratory syncytial virus in the 1960s, has delayed the development of vaccines against the virus. The enhanced disease was characterized by immune complex-mediated airway hyperreactivity and a severe pneumonia associated with pulmonary eosinophilia. In this paper, we show that complement factors contribute to enhanced-disease phenotypes. Mice with a targeted disruption of complement component C5 affected by the enhanced disease displayed enhanced airway reactivity, lung eosinophilia, and mucus production compared to wild-type mice and C5-deficient mice reconstituted with C5. C3aR expression in bronchial epithelial and smooth muscle cells in the lungs of C5-deficient mice was enhanced compared to that in wild-type and reconstituted rodents. Treatment of C5-deficient mice with a C3aR antagonist significantly attenuated airway reactivity, eosinophilia, and mucus production. These results indicate that C5 plays a crucial role in modulating the enhanced-disease phenotype, by affecting expression of C3aR in the lungs. These findings reveal a novel autoregulatory mechanism for the complement cascade that affects the innate and adaptive immune responses. PMID:17079327

  13. C3 photosynthesis in silico.

    PubMed

    Laisk, Agu; Eichelmann, Hillar; Oja, Vello

    2006-10-01

    A computer model comprising light reactions, electron-proton transport, enzymatic reactions, and regulatory functions of C3 photosynthesis has been developed as a system of differential budget equations for intermediate compounds. The emphasis is on electron transport through PSII and PSI and on the modeling of Chl fluorescence and 810 nm absorptance signals. Non-photochemical quenching of PSII excitation is controlled by lumenal pH. Alternative electron transport is modeled as the Mehler type O2 reduction plus the malate-oxaloacetate shuttle based on the chloroplast malate dehydrogenase. Carbon reduction enzymes are redox-controlled by the ferredoxin-thioredoxin system, sucrose synthesis is controlled by the fructose 2,6-bisphosphate inhibition of cytosolic FBPase, and starch synthesis is controlled by ADP-glucose pyrophosphorylase. Photorespiratory glycolate pathway is included in an integrated way, sufficient to reproduce steady-state rates of photorespiration. Rate-equations are designed on principles of multisubstrate-multiproduct enzyme kinetics. The parameters of the model were adopted from literature or were estimated from fitting the photosynthetic rate and pool sizes to experimental data. The model provided good simulations for steady-state photosynthesis, Chl fluorescence, and 810 nm transmittance signals under varying light, CO2 and O2 concentrations, as well as for the transients of post-illumination CO2 uptake, Chl fluorescence induction and the 810 nm signal. The modeling shows that the present understanding of photosynthesis incorporated in the model is basically correct, but still insufficient to reproduce the dark-light induction of photosynthesis, the time kinetics of non-photochemical quenching, 'photosynthetic control' of plastoquinone oxidation, cyclic electron flow around PSI, oscillations in photosynthesis. The model may find application for predicting the results of gene transformations, the analysis of kinetic experimental data, the

  14. Extinct I-129 in C3 chondrites

    NASA Astrophysics Data System (ADS)

    Crabb, J.; Lewis, R. S.; Anders, E.

    1982-12-01

    Eight C3 chondrites were examined by the I-129 to Xe-129 dating method to determine whether their initial I-129/I-127 ratios, or R(0), correlate with any other properties. The R(0)'s range from 1.60 x 10 to the -4th to 1.09 x 10 to the -4th, corresponding to I to Xe ages from 2.0 Myr before to 6.7 Myr after the Murchison magnetite. Three C30's have essentially indistinguishable R(0)'s, while a fourth is undatable. Four C3V's show a distinct spread, ranging from 1.60 + or 0.07 x 10 to the -4th to 1.09 + or - 0.10 x 10 to the -4th. These R(0)'s correlate inversely with four other properties: I, Br, and Cd content, olivine composition, both percent mean deviation, and proportion of iron-poor olivine grains. The simplest model that accounts for the correlations with R(0) involves mixing of two iodine components in the solar nebula, associated with gas and grains, respectively. The second, of lower I-129/I-127 ratio, predominated at later times and thus became enriched in late-formed meteorites.

  15. Complement in therapy and disease: Regulating the complement system with antibody-based therapeutics.

    PubMed

    Melis, Joost P M; Strumane, Kristin; Ruuls, Sigrid R; Beurskens, Frank J; Schuurman, Janine; Parren, Paul W H I

    2015-10-01

    Complement is recognized as a key player in a wide range of normal as well as disease-related immune, developmental and homeostatic processes. Knowledge of complement components, structures, interactions, and cross-talk with other biological systems continues to grow and this leads to novel treatments for cancer, infectious, autoimmune- or age-related diseases as well as for preventing transplantation rejection. Antibodies are superbly suited to be developed into therapeutics with appropriate complement stimulatory or inhibitory activity. Here we review the design, development and future of antibody-based drugs that enhance or dampen the complement system.

  16. Experimental Chagas' disease in complement-deficient mice and guinea pigs.

    PubMed Central

    Dalmasso, A P; Jarvinen, J A

    1980-01-01

    The course of infection with trypomastigotes of Trypanosoma cruzi (House 510 strain) in mice and guinea pigs with genetic complement deficiencies was compared with that in normocomplementemic animals. Parasitemias in a mouse strain (B10.D2/old) genetically deficient in C5 and therefore unable to sustain lysis were similar to or lower than in a congenic normocomplementemic strain (B10.D2/new). The levels of C3 measured immunochemically were generally unaffected. There were no significant differences in mortality rates. These results indicate that, in mice, complement-mediated lysis does not play a significant role in the control of T. cruzi (House 510) infections. Studies were also performed in normocomplementemic guinea pigs and in guinea pigs genetically deficient in the fourth component of complement and thus unable to support functions mediated by the classical pathway of complement activation. No significant differences were noted between the two strains in the course of infection, persistence of subpatent infection, or rate of mortality, indicating that if the classical complement pathway plays a role in resistance to T. cruzi (House 510) in guinea pigs, this role must be a small one. PMID:6772558

  17. The complement system and toll-like receptors as integrated players in the pathophysiology of atherosclerosis.

    PubMed

    Hovland, Anders; Jonasson, Lena; Garred, Peter; Yndestad, Arne; Aukrust, Pål; Lappegård, Knut T; Espevik, Terje; Mollnes, Tom E

    2015-08-01

    Despite recent medical advances, atherosclerosis is a global burden accounting for numerous deaths and hospital admissions. Immune-mediated inflammation is a major component of the atherosclerotic process, but earlier research focus on adaptive immunity has gradually switched towards the role of innate immunity. The complement system and toll-like receptors (TLRs), and the crosstalk between them, may be of particular interest both with respect to pathogenesis and as therapeutic targets in atherosclerosis. Animal studies indicate that inhibition of C3a and C5a reduces atherosclerosis. In humans modified LDL-cholesterol activate complement and TLRs leading to downstream inflammation, and histopathological studies indicate that the innate immune system is present in atherosclerotic lesions. Moreover, clinical studies have demonstrated that both complement and TLRs are upregulated in atherosclerotic diseases, although interventional trials have this far been disappointing. However, based on recent research showing an intimate interplay between complement and TLRs we propose a model in which combined inhibition of both complement and TLRs may represent a potent anti-inflammatory therapeutic approach to reduce atherosclerosis.

  18. Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies.

    PubMed

    Meulenbroek, Elisabeth M; de Haas, Masja; Brouwer, Conny; Folman, Claudia; Zeerleder, Sacha S; Wouters, Diana

    2015-11-01

    In autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal hemolytic anemia. Depending on whether IgG or IgM antibodies are involved, response to therapy is different. Proper identification of the isotype of the anti-erythrocyte autoantibodies is, therefore, crucial. However, detection of IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of complement C3 on a patient's erythrocytes could be used for indirect detection of anti-erythrocyte IgM. To this end, we fractionated patients' sera by size exclusion chromatography and tested which fractions yielded complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an IgM anti-erythrocyte component that could activate complement, even if no such autoantibody had been detected with any other test. This also included all tested patients with only IgG and C3 on their erythrocytes, who would previously have been classified as having an IgG-only mediated autoimmune hemolytic anemia. Depleting patients' sera of either IgG or IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in autoimmune hemolytic anemia is mostly IgM-mediated and the presence of covalent C3 on patients' erythrocytes can be taken as a footprint of the presence of anti-erythrocyte IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy.

  19. The reaction of iodine and thiol-blocking reagents with human complement components C2 and factor B. Purification and N-terminal amino acid sequence of a peptide from C2a containing a free thiol group.

    PubMed Central

    Parkes, C; Gagnon, J; Kerr, M A

    1983-01-01

    Human complement components C2 and Factor B each contain one free thiol group/molecule. Reaction with p-chloromercuribenzoate destroyed the haemolytic activity of C2 but had no effect on Factor B. Reaction of C2 with I2 gave a 16-fold enhancement of its haemolytic activity. The pH optimum for the reaction was 7.0. The I2 reacted at the thiol group in C2 with a stoicheiometry of 1 mol of I2/mol of C2. The product of the reaction was unaffected by millimolar concentrations of dithiothreitol; however, azide and cyanide were inhibitory. Reaction with azide did not result in re-expression of the thiol group. Mild oxidation of the thiol group with m-chloroperbenzoic acid did not enhance the haemolytic activity. The results suggest that reaction with I2 causes intramolecular covalent, but not disulphide, bond formation. I2 reacted with Factor B at the free thiol group without affecting the haemolytic activity. A CNBr-cleavage peptide from C2a (obtained by cleavage of C2 by subcomponent C1s) containing the free thiol group was isolated. Automated Edman degradation of the peptide showed that it was the N-terminal peptide of C2a. The free thiol group was identified at position 18. PMID:6555044

  20. Differential pathogenesis of lethal mousepox in congenic DBA/2 mice implicates natural killer cell receptor NKR-P1 in necrotizing hepatitis and the fifth component of complement in recruitment of circulating leukocytes to spleen.

    PubMed

    Brownstein, D G; Gras, L

    1997-04-01

    Innate resistance of C57BL/6 (B6) mice to lethal mousepox is controlled by multiple genes. Previously, four resistance genes were localized to specific subchromosomal regions and transferred onto a susceptible DBA/2 (D2) background by serial backcrossing and intercrossing to produce congenic strains. Intraperitoneally inoculated ectromelia virus was uniformly lethal and achieved similar titers in B6 and D2 mice but elicited differential responses in liver, spleen, and circulating blood leukocytes. The distribution of these response phenotypes in congenic strains linked control of phenotypes with specific subchromosomal regions. D2.R1 mice, which carried a differential segment of chromosome 6, exhibited a B6 liver response and intermediate spleen and circulating leukocyte responses. D2.R2 and D2.R4 mice, which carried differential segments of chromosomes 2 and 1, respectively, exhibited a D2 liver response, a B6 spleen response, and an intermediate circulating leukocyte response. The localization of control of liver response phenotypes to chromosome 6 implicates cells that express natural killer (NK) cell receptor NKR-P1 alloantigens. The localization of control of spleen and circulating leukocyte responses to chromosomes 1, 2, and 6 implicates NK cells, the fifth component of complement, and a gene near the selectin gene complex in recruitment of circulating leukocytes to spleen.

  1. Complement Interaction with Trypanosomatid Promastigotes in Normal Human Serum

    PubMed Central

    Domínguez, Mercedes; Moreno, Inmaculada; López-Trascasa, Margarita; Toraño, Alfredo

    2002-01-01

    In normal human serum (NHS), axenic promastigotes of Crithidia, Phytomonas, and Leishmania trigger complement activation, and from 1.2 to 1.8 × 105 C3 molecules are deposited per promastigote within 2.5 min. In Leishmania, promastigote C3 binding capacity remains constant during in vitro metacyclogenesis. C3 deposition on promastigotes activated through the classical complement pathway reaches a 50% maximum after ∼50 s, and represents >85% of total C3 bound. In C1q- and C2-deficient human sera, promastigotes cannot activate the classical pathway (CP) unless purified C1q or C2 factors, respectively, are supplemented, demonstrating a requirement for CP factor in promastigote C3 opsonization. NHS depleted of natural anti-Leishmania antibodies cannot trigger promastigote CP activation, but IgM addition restores C3 binding. Furthermore, Leishmania binds natural antibodies in ethylenediaminetetracetic acid (EDTA)-treated NHS; after EDTA removal, promastigote-bound IgM triggers C3 deposition in natural antibody-depleted NHS. Serum collectins and pentraxins thus do not participate significantly in NHS promastigote C3 opsonization. Real-time kinetic analysis of promastigote CP-mediated lysis indicates that between 85–95% of parasites are killed within 2.5 min of serum contact. These data indicate that successful Leishmania infection in man must immediately follow promastigote transmission, and that Leishmania evasion strategies are shaped by the selective pressure exerted by complement. PMID:11854358

  2. Inhibition of complement-mediated cytolysis by the terminal complement inhibitor of herpesvirus saimiri.

    PubMed

    Rother, R P; Rollins, S A; Fodor, W L; Albrecht, J C; Setter, E; Fleckenstein, B; Squinto, S P

    1994-02-01

    Herpesvirus saimiri (HVS) is a lymphotropic herpesvirus that induces T-cell transformation in vitro and causes lymphomas and leukemias in New World primates other than its natural host, the squirrel monkey. Nucleotide sequence analysis of the HVS genome revealed two open reading frames with significant homology to genes for human complement regulatory molecules. One of these genes encodes a predicted protein (designated HVSCD59) with 48% amino acid sequence identity to the human terminal complement regulatory protein CD59 (HuCD59). The CD59 homolog from squirrel monkey (SMCD59) was cloned, and the corresponding amino acid sequence showed 69% identity with HVSCD59. BALB/3T3 cells stably expressing HVSCD59, SMCD59, or HuCD59 were equally protected from complement-mediated lysis by human serum. However, only HVSCD59-expressing cells were effectively protected from complement-mediated lysis when challenged with rat serum, suggesting that HVSCD59 was less species restrictive. The complement regulatory activity of HVSCD59 and SMCD59 occurred after C3b deposition, indicating terminal complement inhibition. Treatment of BALB/3T3 stable transfectants with phosphatidylinositol-specific phospholipase C prior to complement attack decreased the complement regulatory function of HVSCD59, suggesting cell surface attachment via a glycosyl-phosphatidylinositol anchor. Cells expressing HVSCD59 effectively inhibited complement-mediated lysis by squirrel monkey serum in comparison with SMCD59-expressing cells. Finally HVSCD59-specific transcripts were detected in owl monkey cells permissive for lytic HVS replication but not in T cells transformed by HVS, which failed to produce virions. These data are the first to demonstrate a functional, virally encoded terminal complement inhibitor and suggest that HVSCD59 represents a humoral immune evasion mechanism supporting the lytic life cycle of HVS.

  3. Host Specificity of Ovine Bordetella parapertussis and the Role of Complement.

    PubMed

    Hester, Sara E; Goodfield, Laura L; Park, Jihye; Feaga, Heather A; Ivanov, Yury V; Bendor, Liron; Taylor, Dawn L; Harvill, Eric T

    2015-01-01

    The classical bordetellae are comprised of three subspecies that differ from broad to very limited host specificity. Although several lineages appear to have specialized to particular host species, most retain the ability to colonize and grow in mice, providing a powerful common experimental model to study their differences. One of the subspecies, Bordetella parapertussis, is composed of two distinct clades that have specialized to different hosts: one to humans (Bpphu), and the other to sheep (Bppov). While Bpphu and the other classical bordetellae can efficiently colonize mice, Bppov strains are severely defective in their ability to colonize the murine respiratory tract. Bppov genomic analysis did not reveal the loss of adherence genes, but substantial mutations and deletions of multiple genes involved in the production of O-antigen, which is required to prevent complement deposition on B. bronchiseptica and Bpphu strains. Bppov lacks O-antigen and, like O-antigen mutants of other bordetellae, is highly sensitive to murine complement-mediated killing in vitro. Based on these results, we hypothesized that Bppov failed to colonize mice because of its sensitivity to murine complement. Consistent with this, the Bppov defect in the colonization of wild type mice was not observed in mice lacking the central complement component C3. Furthermore, Bppov strains were highly susceptible to killing by murine complement, but not by sheep complement. These data demonstrate that the failure of Bppov to colonize mice is due to sensitivity to murine, but not sheep, complement, providing a mechanistic example of how specialization that accompanies expansion in one host can limit host range.

  4. Complement activation and liver impairment in trichloroethylene-sensitized BALB/c mice.

    PubMed

    Zhang, Jiaxiang; Zha, Wansheng; Wang, Feng; Jiang, Tao; Xu, Shuhai; Yu, Junfeng; Zhou, Chengfan; Shen, Tong; Wu, Changhao; Zhu, Qixing

    2013-01-01

    Our recent studies have shown that trichloroethylene (TCE) was able to induce multisystem injuries in the form of occupational medicamentosa-like dermatitis, including skin, kidney, and liver damages. However, the role of complement activation in the immune-mediated liver injury is not known. This study examined the role of complement activation in the liver injury in a mouse model of TCE-induced sensitization. Treatment of female BALB/c mice with TCE under specific dosing protocols resulted in skin inflammation and sensitization. Skin edema and erythema occurred in TCE-sensitized groups. Trichloroethylene sensitization produced liver histopathological lesions, increased serum alanine aminotransferase, aspartate transaminase activities, and the relative liver weight. The concentrations of serum complement components C3a-desArg, C5a-desArg, and C5b-9 were significantly increased in 24-hour, 48-hour, and 72-hour sensitization-positive groups treated with TCE and peaked in the 72-hour sensitization-positive group. Depositions of C3a, C5a, and C5b-9 into the liver tissue were also revealed by immunohistochemistry. Immunofluorescence further verified high C5b-9 expression in 24-hour, 48-hour, and 72-hour sensitization-positive groups in response to TCE treatment. Reverse transcription-polymerase chain reaction detected C3 messenger RNA expression in the liver, and this was significantly increased in 24-hour and 48-hour sensitization-positive groups with a transient reduction at 72 hours. These results provide the first experimental evidence that complement activation may play a key role in the generation and progression of immune-mediated hepatic injury by exposure to TCE.

  5. 42 CFR 52c.3 - Eligibility.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 42 Public Health 1 2010-10-01 2010-10-01 false Eligibility. 52c.3 Section 52c.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS MINORITY BIOMEDICAL RESEARCH SUPPORT PROGRAM § 52c.3 Eligibility. To be eligible for a grant under this Program, an applicant must...

  6. 42 CFR 52c.3 - Eligibility.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 42 Public Health 1 2014-10-01 2014-10-01 false Eligibility. 52c.3 Section 52c.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS MINORITY BIOMEDICAL RESEARCH SUPPORT PROGRAM § 52c.3 Eligibility. To be eligible for a grant under this Program, an applicant must...

  7. 42 CFR 52c.3 - Eligibility.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 42 Public Health 1 2013-10-01 2013-10-01 false Eligibility. 52c.3 Section 52c.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS MINORITY BIOMEDICAL RESEARCH SUPPORT PROGRAM § 52c.3 Eligibility. To be eligible for a grant under this Program, an applicant must...

  8. 42 CFR 52c.3 - Eligibility.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 42 Public Health 1 2012-10-01 2012-10-01 false Eligibility. 52c.3 Section 52c.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS MINORITY BIOMEDICAL RESEARCH SUPPORT PROGRAM § 52c.3 Eligibility. To be eligible for a grant under this Program, an applicant must...

  9. 42 CFR 52c.3 - Eligibility.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 42 Public Health 1 2011-10-01 2011-10-01 false Eligibility. 52c.3 Section 52c.3 Public Health PUBLIC HEALTH SERVICE, DEPARTMENT OF HEALTH AND HUMAN SERVICES GRANTS MINORITY BIOMEDICAL RESEARCH SUPPORT PROGRAM § 52c.3 Eligibility. To be eligible for a grant under this Program, an applicant must...

  10. The Importance of the C3 Framework

    ERIC Educational Resources Information Center

    Social Education, 2013

    2013-01-01

    "The C3 Framework for Social Studies State Standards will soon be released under the title "The College, Career, and Civic Life (C3) Framework for Social Studies State Standards: State Guidance for Enhancing the Rigor of K-12 Civics, Economics, Geography, and History." The C3 Project Director and Lead Writer was NCSS member Kathy…

  11. Soluble complement receptor 1 inhibits both complement and granulocyte activation during ex vivo hemodialysis.

    PubMed

    Himmelfarb, J; McMonagle, E; Holbrook, D; Toth, C

    1995-10-01

    Hemodialysis with cellulosic membranes results in both complement and granulocyte activation. We investigated the effects of soluble complement receptor 1 (sCR1), a potent complement inhibitor, on both complement and granulocyte activation in an ex vivo model of dialysis. Measurements were made of complement activation (radioimmunoassay for C3a desArg) as well as granulocyte activation (flow cytometric measurements of reactive oxygen species production, granulocyte CD11b/CD18 (MAC-1) expression and CD62L (L-selectin) expression). sCR1 completely abolished the generation of plasma C3a desArg during ex vivo hemodialysis. Without sCR1, C3a desArg levels rose from 968 +/- 373 ng/ml to 4961 +/- 40 ng/ml by the end of the ex vivo procedure (p < 0.001). sCR1 also completely inhibited MAC-1 upregulation and L-selectin shedding from granulocytes during ex vivo hemodialysis. With sCR1 there was still a statistically significant increase in granulocyte reactive oxygen species production (from 2.42 +/- 0.1 fluorescence channels to 6.47 +/- 0.7 fluorescence channels, p < 0.01) but a 50% inhibition when compared with experiments without sCR1 (3.15 +/- 0.5 to 11.2 +/- 1.9, p < 0.01). We conclude that sCR1 completely abolishes complement activation and changes in granulocyte cell adhesion molecules during ex vivo hemodialysis with cellulosic membranes. sCR1 partially inhibits granulocyte reactive oxygen species formation.

  12. A potential role for complement in immune evasion by Mycobacterium leprae.

    PubMed

    Callegaro-Filho, Donato; Shrestha, Niraj; Burdick, Anne E; Haslett, Patrick A J

    2010-11-01

    Lepromatous leprosy is a model of immune evasion wherein pathogen-specific IL-10-secreting T cells and concomitant failure of Th-1 immunity permit uncontrolled proliferation of the intracellular pathogen, Mycobacterium leprae (M. leprae). The mechanism of this immune escape is unknown. Here, the authors report that phenolic glycolipid-1 (PGL-1), a major and distinguishing feature of the M. leprae cell wall, is expressed in the cell membrane of M. leprae-infected human dendritic cells, where it can activate complement in human serum. The authors demonstrate that PGL-1 and the C3 component of complement colocalize in lipid rafts in the dendritic cell membrane, and enter the immune synapse upon co-culture of M. leprae-infected DCs and T cells. Hence, activated C3 is strategically located to costimulate naïve T cells via the complement regulatory protein, CD46, a process known to stimulate the differentiation of IL-10-secreting regulatory T cells. These observations suggest a potential novel mechanism of immune evasion, wherein M. leprae may subvert host natural immunity to provoke an adaptive response that favors bacillary survival.

  13. Effects of penicillinase on bactericidal and complement activities in normal human serum.

    PubMed Central

    Biggs, W H; Wunderlich, A C; Corbeil, L C; Davis, C E; Curd, J G

    1983-01-01

    During routine addition of penicillinase (beta-lactamase) to patients sera, we found that the capacity of some of these sera to kill serum-sensitive gram-negative organisms was significantly decreased. Further controlled studies showed that penicillinase decreased both the bactericidal activity of normal human sera and the total hemolytic activity (CH50) of complement in these sera. The decreased bactericidal activity correlated significantly (r = 0.57, P less than 0.05) with the reduction of CH50 in eight normal sera. These effects of penicillinase were time and temperature dependent. Measurement of individual complement component activities showed that penicillinase decreased the activity of C2, C4, and C3-C9, suggesting that the penicillinase preparation activated the classical pathway. These results cast doubts on the validity of bactericidal determinations when sera are pretreated with penicillinase. PMID:6603195

  14. Molecular Characterization and Expression Analyses of the Complement Component C8α, C8β and C9 Genes in Yellow Catfish (Pelteobagrus fulvidraco) after the Aeromonas hydrophila Challenge

    PubMed Central

    Zheng, Huan; Ji, Wei; Zhang, Gui-Rong; Zhang, Xiao-Ting; Shi, Ze-Chao; Wei, Kai-Jian; Yang, Rui-Bin; Gardner, Jonathan P. A.

    2016-01-01

    The complement components C8α, C8β and C9 have important roles in the innate immune system against invading microorganisms. Partial cDNA sequences of the Pf_C8α, Pf_C8β and Pf_C9 genes (Pf: abbreviation of Pelteobagrus fulvidraco) were cloned from yellow catfish. The Pf_C8α, Pf_C8β and Pf_C9 genes showed the greatest amino acid similarity to C8α (54%) and C8β (62%) of zebrafish and to C9 (52%) of grass carp, respectively. Ontogenetic expression analyses using real-time quantitative PCR suggested that the three genes may play crucial roles during embryonic and early larval development. The mRNA expressions of the three genes were all at the highest levels in liver tissue, and at lower or much lower levels in 16 other tissues, demonstrating that the liver is the primary site for the protein synthesis of Pf_C8α, Pf_C8β and Pf_C9. Injection of Aeromonas hydrophila led to up-regulation of the three genes in the spleen, head kidney, kidney, liver and blood tissues, indicating that the three genes may contribute to the host’s defense against invading pathogenic microbes. An increased understanding of the functions of the Pf_C8α, Pf_C8β and Pf_C9 genes in the innate immunity of yellow catfish will help enhance production of this valuable freshwater species. PMID:27005612

  15. Molecular Characterization and Expression Analyses of the Complement Component C8α, C8β and C9 Genes in Yellow Catfish (Pelteobagrus fulvidraco) after the Aeromonas hydrophila Challenge.

    PubMed

    Zheng, Huan; Ji, Wei; Zhang, Gui-Rong; Zhang, Xiao-Ting; Shi, Ze-Chao; Wei, Kai-Jian; Yang, Rui-Bin; Gardner, Jonathan P A

    2016-03-08

    The complement components C8α, C8β and C9 have important roles in the innate immune system against invading microorganisms. Partial cDNA sequences of the Pf_C8α, Pf_C8β and Pf_C9 genes (Pf: abbreviation of Pelteobagrus fulvidraco) were cloned from yellow catfish. The Pf_C8α, Pf_C8β and Pf_C9 genes showed the greatest amino acid similarity to C8α (54%) and C8β (62%) of zebrafish and to C9 (52%) of grass carp, respectively. Ontogenetic expression analyses using real-time quantitative PCR suggested that the three genes may play crucial roles during embryonic and early larval development. The mRNA expressions of the three genes were all at the highest levels in liver tissue, and at lower or much lower levels in 16 other tissues, demonstrating that the liver is the primary site for the protein synthesis of Pf_C8α, Pf_C8β and Pf_C9. Injection of Aeromonas hydrophila led to up-regulation of the three genes in the spleen, head kidney, kidney, liver and blood tissues, indicating that the three genes may contribute to the host's defense against invading pathogenic microbes. An increased understanding of the functions of the Pf_C8α, Pf_C8β and Pf_C9 genes in the innate immunity of yellow catfish will help enhance production of this valuable freshwater species.

  16. Inactivation of complement by Loxosceles reclusa spider venom.

    PubMed

    Gebel, H M; Finke, J H; Elgert, K D; Cambell, B J; Barrett, J T

    1979-07-01

    Zymosan depletion of serum complement in guinea pigs rendered them highly resistant to lesion by Loxosceles reclusa spider venom. Guinea pigs deficient in C4 of the complement system are as sensitive to the venom as normal guinea pigs. The injection of 35 micrograms of whole recluse venom intradermally into guinea pigs lowered their complement level by 35.7%. Brown recluse spider venom in concentrations as slight as 0.02 micrograms protein/ml can totally inactivate one CH50 of guinea pig complement in vitro. Bee, scorpion, and other spider venoms had no influence on the hemolytic titer of complement. Fractionation of recluse spider venom by Sephadex G-200 filtration separated the complement-inactivating property of the venom into three major regions which could be distinguished on the basis of heat stability as well as size. None was neutralized by antivenom. Polyacrylamide gel electrophoresis of venom resolved the complement inactivators into five fractions. Complement inactivated by whole venom or the Sephadex fractions could be restored to hemolytic activity by supplements of fresh serum but not by heat-inactivated serum, pure C3, pure C5, or C3 and C5 in combination.

  17. The effect of Tlr4 and/or C3 deficiency and of neonatal gene therapy on skeletal disease in mucopolysaccharidosis VII mice.

    PubMed

    Xing, Elizabeth M; Wu, Susan; Ponder, Katherine P

    2015-02-01

    Mucopolysaccharidosis (MPS) VII is a lysosomal storage disorder caused by the deficiency of the enzyme β-glucuronidase (Gusb(-/-)) and results in glycosaminoglycan (GAG) accumulation. Skeletal abnormalities include stunted long bones and bone degeneration. GAGs have been hypothesized to activate toll-like receptor 4 (Tlr4) signaling and the complement pathway, resulting in upregulation of inflammatory cytokines that suppress growth and cause degeneration of the bone. Gusb(-/-) mice were bred with Tlr4- and complement component 3 (C3)-deficient mice, and the skeletal manifestations of the doubly- and triply-deficient mice were compared to those of purebred Gusb(-/-) mice. Radiographs showed that purebred Gusb(-/-) mice had shorter tibias and femurs, and wider femurs, compared to normal mice. No improvement was seen in Tlr4, C3, or Tlr4/C3-deficient Gusb(-/-) mice. The glenoid cavity and humerus were scored on a scale from 0 (normal) to +3 (severely abnormal) for dysplasia and bone irregularities, and the joint space was measured. No improvement was seen in Tlr4, C3, or Tlr4/C3-deficient Gusb(-/-) mice, and their joint space remained abnormally wide. Gusb(-/-) mice treated neonatally with an intravenous retroviral vector (RV) had thinner femurs, longer legs, and a narrowed joint space compared with untreated purebred Gusb(-/-) mice, but no improvement in glenohumeral degeneration. We conclude that Tlr4- and/or C3-deficiency fail to ameliorate skeletal abnormalities, and other pathways may be involved. RV treatment improves some but not all aspects of bone disease. Radiographs may be an efficient method for future evaluation, as they readily show glenohumeral joint abnormalities.

  18. Outline of Hungarian Complementation.

    ERIC Educational Resources Information Center

    Szamosi, Michael

    This study presents a preliminary analysis of Hungarian complement constructions and the syntactic operations needed to account for them. The expository framework (and the implicit framework of the research itself) is based upon that of Rosenbaum (1967). The aim of the paper is to arrive at a rough picture of the kinds of structures and syntactic…

  19. Verbal Complementizers in Arabic

    ERIC Educational Resources Information Center

    Ahmed, Hossam Eldin Ibrahim

    2015-01-01

    A class of Modern Standard Arabic complementizers known as "'?inna' and its sisters" demonstrate unique case and word order restrictions. While CPs in Arabic allow both Subject-Verb (SV) and Verb-Subject (VS) word order and their subjects show nominative morphology, CPs introduced by "?inna" ban a verb from directly following…

  20. 670-nm light treatment reduces complement propagation following retinal degeneration

    PubMed Central

    2012-01-01

    Aim Complement activation is associated with the pathogenesis of age-related macular degeneration (AMD). We aimed to investigate whether 670-nm light treatment reduces the propagation of complement in a light-induced model of atrophic AMD. Methods Sprague–Dawley (SD) rats were pretreated with 9 J/cm2 670-nm light for 3 minutes daily over 5 days; other animals were sham treated. Animals were exposed to white light (1,000 lux) for 24 h, after which animals were kept in dim light (5 lux) for 7 days. Expression of complement genes was assessed by quantitative polymerase chain reaction (qPCR), and immunohistochemistry. Counts were made of C3-expressing monocytes/microglia using in situ hybridization. Photoreceptor death was also assessed using outer nuclear layer (ONL) thickness measurements, and oxidative stress using immunohistochemistry for 4-hydroxynonenal (4-HNE). Results Following light damage, retinas pretreated with 670-nm light had reduced immunoreactivity for the oxidative damage maker 4-HNE in the ONL and outer segments, compared to controls. In conjunction, there was significant reduction in retinal expression of complement genes C1s, C2, C3, C4b, C3aR1, and C5r1 following 670 nm treatment. In situ hybridization, coupled with immunoreactivity for the marker ionized calcium binding adaptor molecule 1 (IBA1), revealed that C3 is expressed by infiltrating microglia/monocytes in subretinal space following light damage, which were significantly reduced in number after 670 nm treatment. Additionally, immunohistochemistry for C3 revealed a decrease in C3 deposition in the ONL following 670 nm treatment. Conclusions Our data indicate that 670-nm light pretreatment reduces lipid peroxidation and complement propagation in the degenerating retina. These findings have relevance to the cellular events of complement activation underling the pathogenesis of AMD, and highlight the potential of 670-nm light as a non-invasive anti-inflammatory therapy. PMID:23181358

  1. Electron Interactions With C3F8

    NASA Astrophysics Data System (ADS)

    Christophorou, L. G.; Olthoff, J. K.

    1998-09-01

    To aid the many and diverse applications for which perfluoropropane (C3F8) is suited, we critically evaluate and synthesize existing knowledge on electron scattering and electron energy-loss processes for the C3F8 molecule, and provide recommendations for the most reliable data. We also draw attention to electron-interaction data that are not presently available, but are needed for modeling the behavior of C3F8 in practical uses, especially plasma processing.

  2. IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fcα/μ Receptor.

    PubMed

    Weinstein, Jonathan R; Quan, Yi; Hanson, Josiah F; Colonna, Lucrezia; Iorga, Michael; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira; Elkon, Keith B; Möller, Thomas

    2015-12-01

    Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR(-/-) mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR(-/-) mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18(-/-) and C3(-/-) mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of S. aureus by microglia.

  3. Complement related kidney diseases: Recurrence after transplantation.

    PubMed

    Salvadori, Maurizio; Bertoni, Elisabetta

    2016-12-24

    The recurrence of renal disease after renal transplantation is becoming one of the main causes of graft loss after kidney transplantation. This principally concerns some of the original diseases as the atypical hemolytic uremic syndrome (HUS), the membranoproliferative glomerulonephritis (MPGN), in particular the MPGN now called C3 glomerulopathy. Both this groups of renal diseases are characterized by congenital (genetic) or acquired (auto-antibodies) modifications of the alternative pathway of complement. These abnormalities often remain after transplantation because they are constitutional and poorly influenced by the immunosuppression. This fact justifies the high recurrence rate of these diseases. Early diagnosis of recurrence is essential for an optimal therapeutically approach, whenever possible. Patients affected by end stage renal disease due to C3 glomerulopathies or to atypical HUS, may be transplanted with extreme caution. Living donor donation from relatives is not recommended because members of the same family may be affected by the same gene mutation. Different therapeutically approaches have been attempted either for recurrence prevention and treatment. The most promising approach is represented by complement inhibitors. Eculizumab, a monoclonal antibody against C5 convertase is the most promising drug, even if to date is not known how long the therapy should be continued and which are the best dosing. These facts face the high costs of the treatment. Eculizumab resistant patients have been described. They could benefit by a C3 convertase inhibitor, but this class of drugs is by now the object of randomized controlled trials.

  4. Complement related kidney diseases: Recurrence after transplantation

    PubMed Central

    Salvadori, Maurizio; Bertoni, Elisabetta

    2016-01-01

    The recurrence of renal disease after renal transplantation is becoming one of the main causes of graft loss after kidney transplantation. This principally concerns some of the original diseases as the atypical hemolytic uremic syndrome (HUS), the membranoproliferative glomerulonephritis (MPGN), in particular the MPGN now called C3 glomerulopathy. Both this groups of renal diseases are characterized by congenital (genetic) or acquired (auto-antibodies) modifications of the alternative pathway of complement. These abnormalities often remain after transplantation because they are constitutional and poorly influenced by the immunosuppression. This fact justifies the high recurrence rate of these diseases. Early diagnosis of recurrence is essential for an optimal therapeutically approach, whenever possible. Patients affected by end stage renal disease due to C3 glomerulopathies or to atypical HUS, may be transplanted with extreme caution. Living donor donation from relatives is not recommended because members of the same family may be affected by the same gene mutation. Different therapeutically approaches have been attempted either for recurrence prevention and treatment. The most promising approach is represented by complement inhibitors. Eculizumab, a monoclonal antibody against C5 convertase is the most promising drug, even if to date is not known how long the therapy should be continued and which are the best dosing. These facts face the high costs of the treatment. Eculizumab resistant patients have been described. They could benefit by a C3 convertase inhibitor, but this class of drugs is by now the object of randomized controlled trials. PMID:28058212

  5. Renal expression of the C3a receptor and functional responses of primary human proximal tubular epithelial cells.

    PubMed

    Braun, Michael C; Reins, Rose Y; Li, Tong-Bin; Hollmann, Travis J; Dutta, Ranjan; Rick, Wetsel A; Teng, Ba-Bie; Ke, Baozhen

    2004-09-15

    Although complement activation and deposition have been associated with a variety of glomerulopathies, the pathogenic mechanisms by which complement directly mediates renal injury remain to be fully elucidated. Renal parenchymal tissues express a limited repertoire of receptors that directly bind activated complement proteins. We report the renal expression of the receptor for the C3 cleavage product C3a, a member of the anaphylatoxin family. C3aR is highly expressed in normal human and murine kidney, as demonstrated by immunohistochemistry and in situ hybridization. Its distribution is limited to epithelial cells only, as glomerular endothelial and mesangial cells showed no evidence of C3aR expression. The C3aR is also expressed by primary renal proximal tubular epithelial cells in vitro as demonstrated by FACS, Western blot, and RT-PCR. In vitro C3aR is functional in terms of its capacity to bind 125I-labeled C3a and generate inositol triphosphate. Finally, using microarray analysis, four novel genes were identified and confirmed as transcriptionally regulated by C3aR activation in proximal tubular cells. These studies define a new pathway by which complement activation may directly modulate the renal response to immunologic injury.

  6. A Serine Protease Isolated from the Bristles of the Amazonic Caterpillar, Premolis semirufa, Is a Potent Complement System Activator

    PubMed Central

    Villas Boas, Isadora Maria; Pidde-Queiroz, Giselle; Magnoli, Fabio Carlos; Gonçalves-de-Andrade, Rute M.; van den Berg, Carmen W.; Tambourgi, Denise V.

    2015-01-01

    Background The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called “Pararamose”, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa’s bristles extract could interfere with the human complement system. Results The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly

  7. Complements do not lie.

    PubMed

    Robert, Stefanie Christina; Forbes, Suzanne Helen; Soleimanian, Surusch; Hadley, Julia S

    2011-12-13

    A 74-year-old patient presented with constitutional symptoms and was found to have acute kidney injury. He was known to have a prosthetic aortic valve. He was febrile with splenomegaly and vasculitic lesions on both hands. Nephritic screen revealed strongly positive cytoplasmic-antineutrophil cytoplasmic antibodies (c-ANCA). Differential diagnosis thus included a small vessel vasculitis or infective endocarditis. Transoesophageal echocardiography demonstrated no vegetations and serial blood cultures were negative. Immunosuppression for presumed granulomatosis with polyangiitis (Wegeners granulomatosis) was therefore instituted. The patient deteriorated, requiring multi-organ support. Renal biopsy showed a proliferative glomerulopathy and complements were low. Atypical screen for culture negative endocarditis revealed a strongly positive IgG-antibody titre against Bartonella henselae. Immunosuppression was discontinued and treatment for chronic Bartonellosis commenced. The patient made a remarkable recovery. His renal function quickly returned to normal, and ANCA titres and complements normalised. He was discharged home after completing a 6 week course of antibiotic therapy.

  8. Complements do not lie

    PubMed Central

    Robert, Stefanie Christina; Forbes, Suzanne Helen; Soleimanian, Surusch; Hadley, Julia S

    2011-01-01

    A 74-year-old patient presented with constitutional symptoms and was found to have acute kidney injury. He was known to have a prosthetic aortic valve. He was febrile with splenomegaly and vasculitic lesions on both hands. Nephritic screen revealed strongly positive cytoplasmic-antineutrophil cytoplasmic antibodies (c-ANCA). Differential diagnosis thus included a small vessel vasculitis or infective endocarditis. Transoesophageal echocardiography demonstrated no vegetations and serial blood cultures were negative. Immunosuppression for presumed granulomatosis with polyangiitis (Wegeners granulomatosis) was therefore instituted. The patient deteriorated, requiring multi-organ support. Renal biopsy showed a proliferative glomerulopathy and complements were low. Atypical screen for culture negative endocarditis revealed a strongly positive IgG-antibody titre against Bartonella henselae. Immunosuppression was discontinued and treatment for chronic Bartonellosis commenced. The patient made a remarkable recovery. His renal function quickly returned to normal, and ANCA titres and complements normalised. He was discharged home after completing a 6 week course of antibiotic therapy. PMID:22674942

  9. Complements do not lie.

    PubMed

    Robert, Stefanie Christina; Forbes, Suzanne Helen; Soleimanian, Surusch; Hadley, Julia S

    2011-12-01

    A 74-year-old patient presented with constitutional symptoms and was found to have acute kidney injury. He was known to have a prosthetic aortic valve. He was febrile with splenomegaly and vasculitic lesions on both hands. Nephritic screen revealed strongly positive cytoplasmic-antineutrophil cytoplasmic antibodies (c-ANCA). Differential diagnosis thus included a small vessel vasculitis or infective endocarditis. Transoesophageal echocardiography demonstrated no vegetations and serial blood cultures were negative. Immunosuppression for presumed granulomatosis with polyangiitis (Wegeners granulomatosis) was therefore instituted. The patient deteriorated, requiring multi-organ support. Renal biopsy showed a proliferative glomerulopathy and complements were low. Atypical screen for culture negative endocarditis revealed a strongly positive IgG-antibody titre against Bartonella henselae. Immunosuppression was discontinued and treatment for chronic Bartonellosis commenced. The patient made a remarkable recovery. His renal function quickly returned to normal, and ANCA titres and complements normalised. He was discharged home after completing a 6 week course of antibiotic therapy.

  10. IXO: The Instrument Complement

    NASA Astrophysics Data System (ADS)

    Nousek, John A.; IWG, IXO

    2009-01-01

    The International X-ray Observatory (IXO) has recently been created as a mission concept by a joint team of NASA, ESA and JAXA scientists, based on the previous Constellation-X and XEUS concepts. Definition of the IXO instruments is still under evolution, but the core instrument complement will include a Wide Field X-ray Imager, an X-ray Calorimeter / Narrow Field X-ray Imager, and an X-ray Grating Spectrometer. Other, modest additional instruments (such as a hard X-ray capability, a polarimeter, and a high time resolution detector) will also be considered. We present the current status of the IXO instrument complement and offer the opportunity for discussion of ideas relevant to the IXO mission concept process.

  11. Plasma complement and vascular complement deposition in patients with coronary artery disease with and without inflammatory rheumatic diseases

    PubMed Central

    2017-01-01

    Purpose Inflammatory rheumatic diseases (IRD) are associated with accelerated coronary artery disease (CAD), which may result from both systemic and vascular wall inflammation. There are indications that complement may be involved in the pathogenesis of CAD in Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA). This study aimed to evaluate the associations between circulating complement and complement activation products with mononuclear cell infiltrates (MCI, surrogate marker of vascular inflammation) in the aortic media and adventitia in IRDCAD and non-IRDCAD patients undergoing coronary artery bypass grafting (CABG). Furthermore, we compared complement activation product deposition patterns in rare aorta adventitial and medial biopsies from SLE, RA and non-IRD patients. Methods We examined plasma C3 (p-C3) and terminal complement complexes (p-TCC) in 28 IRDCAD (SLE = 3; RA = 25), 52 non-IRDCAD patients, and 32 IRDNo CAD (RA = 32) from the Feiring Heart Biopsy Study. Aortic biopsies taken from the CAD only patients during CABG were previously evaluated for adventitial MCIs. The rare aortic biopsies from 3 SLE, 3 RA and 3 non-IRDCAD were assessed for the presence of C3 and C3d using immunohistochemistry. Results IRDCAD patients had higher p-TCC than non-IRDCAD or IRDNo CAD patients (p<0.0001), but a similar p-C3 level (p = 0.42). Circulating C3 was associated with IRD duration (ρ, p-value: 0.46, 0.03). In multiple logistic regression analysis, IRD remained significantly related to the presence and size of MCI (p<0.05). C3 was present in all tissue samples. C3d was detected in the media of all patients and only in the adventitia of IRD patients (diffuse in all SLE and focal in one RA). Conclusion The independent association of IRD status with MCI and the observed C3d deposition supports the unique relationship between rheumatic disease, and, in particular, SLE with the complement system. Exaggerated systemic and vascular complement activation may

  12. Bullous pemphigoid autoantibodies directly induce blister formation without complement activation.

    PubMed

    Ujiie, Hideyuki; Sasaoka, Tetsumasa; Izumi, Kentaro; Nishie, Wataru; Shinkuma, Satoru; Natsuga, Ken; Nakamura, Hideki; Shibaki, Akihiko; Shimizu, Hiroshi

    2014-11-01

    Complement activation and subsequent recruitment of inflammatory cells at the dermal/epidermal junction are thought to be essential for blister formation in bullous pemphigoid (BP), an autoimmune blistering disease induced by autoantibodies against type XVII collagen (COL17); however, this theory does not fully explain the pathological features of BP. Recently, the involvement of complement-independent pathways has been proposed. To directly address the question of the necessity of the complement activation in blister formation, we generated C3-deficient COL17-humanized mice. First, we show that passive transfer of autoantibodies from BP patients induced blister formation in neonatal C3-deficient COL17-humanized mice without complement activation. By using newly generated human and murine mAbs against the pathogenic noncollagenous 16A domain of COL17 with high (human IgG1, murine IgG2), low (murine IgG1), or no (human IgG4) complement activation abilities, we demonstrate that the deposition of Abs, and not complements, is relevant to the induction of blister formation in neonatal and adult mice. Notably, passive transfer of BP autoantibodies reduced the amount of COL17 in lesional mice skin, as observed in cultured normal human keratinocytes treated with the same Abs. Moreover, the COL17 depletion was associated with a ubiquitin/proteasome pathway. In conclusion, the COL17 depletion induced by BP autoantibodies, and not complement activation, is essential for the blister formation under our experimental system.

  13. Complement and thrombosis in the antiphospholipid syndrome.

    PubMed

    Oku, Kenji; Nakamura, Hiroyuki; Kono, Michihiro; Ohmura, Kazumasa; Kato, Masaru; Bohgaki, Toshiyuki; Horita, Tetsuya; Yasuda, Shinsuke; Amengual, Olga; Atsumi, Tatsuya

    2016-10-01

    The involvement of complement activation in the pathophysiology of antiphospholipid syndrome (APS) was first reported in murine models of antiphospholipid antibody (aPL)-related pregnancy morbidities. We previously reported that complement activation is prevalent and may function as a source of procoagulant cell activation in the sera of APS patients. Recently, autoantibodies against C1q, a component of complement 1, were reported to be correlated with complement activation in systemic lupus erythematosus. These antibodies target neoepitopes of deformed C1q bound to various molecules (i.e., anionic phospholipids) and induce accelerated complement activation. We found that anti-C1q antibodies are more frequently detected in primary APS patients than in control patients and in refractory APS patients with repeated thrombotic events. The titer of anti-C1q antibodies was significantly higher in refractory APS patients than in APS patients without flare. The binding of C1q to anionic phospholipids may be associated with the surge in complement activation in patients with anti-C1q antibodies when triggered by 'second-hit' biological stressors such as infection. Such stressors will induce overexpression of anionic phospholipids, with subsequent increases in deformed C1q that is targeted by anti-C1q antibodies.

  14. Remarkable Activation of the Complement System and Aberrant Neuronal Localization of the Membrane Attack Complex in the Brain Tissues of Scrapie-Infected Rodents.

    PubMed

    Lv, Yan; Chen, Cao; Zhang, Bao-Yun; Xiao, Kang; Wang, Jing; Chen, Li-Na; Sun, Jing; Gao, Chen; Shi, Qi; Dong, Xiao-Ping

    2015-12-01

    As an integral part of the innate immunity, the complement system has been reported to involve in the pathogenesis of prion diseases (PrD). However, the states of expression and activity of complement proteins in experimental models of scrapie infection are still not fully understood. Herein, the state of complement activation, the presence, and distribution as well as localization of C3 and membrane attack complex (MAC) in the brains of several scrapie-infected rodents were comparatively assessed through various methodologies. Our data illustrated a significant increase in the total complement activity (CH50, U/ml) in several scrapie-infected rodent brains at the terminal stage and a time-dependent upregulation of C1q in 263K-infected hamsters during the incubation period, intimating the sustained and progressive activation of the classical pathway during PrD progression. Confocal microscopy revealed robust activation of C3 and its localization to various central nervous system (CNS) cells with differential morphology in the brain tissues of both 263K-infected hamsters and 139A-infected C57BL/6 mice at disease end stages. Dynamic analyses of MAC in the brains of 263K-infected hamsters and 139A-infected C57BL/6 mice demonstrated remarkably time-dependent deposition during the incubation period, which may highlight a persistently activated terminal complement components. Moreover, immunofluorescent assays (IFAs) showed that MAC-specific signals appeared to overlap with morphologically abnormal neurons rather than proliferative astrocytes or activated microglia throughout the CNS of both 263K-infected hamsters and 139A-infected C57BL/6 mice. Overall, these results indicate that the activation of the complement system and the subsequent localization of the complement components to neurons may be a hallmark during prion infection, which ultimately contribute to the neurodegeneration in PrD.

  15. Analysis of C3b/C4b receptor (CR1) polymorphic variants by tryptic peptide mapping.

    PubMed

    Nickells, M W; Seya, T; Holers, V M; Atkinson, J P

    1986-06-01

    The human C3b/C4b receptor (CR1) binds the major activation and opsonic fragments of the third (C3) and fourth (C4) components of complement. CR1 is a single chain integral membrane glycoprotein widely distributed on peripheral blood cells. Four codominantly inherited allelic variants with Mrs of 160,000, 190,000, 220,000 and 250,000 have been described. To address the structural basis for this unusual polymorphism, CR1 from donors expressing three of the four allelic variants was purified from surface labeled (125I) erythrocytes by iC3-Sepharose affinity chromatography and the variants compared by tryptic peptide mapping (TPM). The TPMs of each variant contained the same major peaks and minor peak areas and were nearly identical to one another. Tryptic peptide mappings of the 190,000 Mr erythrocyte CR1, which was purified prior to iodination, were similar to those derived from surface iodinated CR1. The TPMs of erythrocyte and granulocyte CR1 from the same donor differed by a single peak of increased prominence in the granulocyte map. These results indicate a conservation in amino acid sequence for those peptides detected. In view of these data and those of other studies of the structure and genetics of CR1 and related proteins, it is suggested in this paper that the allelic variation relates to CR1, being composed of repeating amino acid sequences.

  16. Urinary C3dg and C5b-9 indicate active immune disease in human membranous nephropathy.

    PubMed

    Brenchley, P E; Coupes, B; Short, C D; O'Donoghue, D J; Ballardie, F W; Mallick, N P

    1992-04-01

    We have measured complement activation markers, C3dg and C5b-9 in plasma and urine from patients with idiopathic membranous nephropathy and IgA nephropathy. There was no significant difference in levels of plasma C5b-9 between the patient groups. However, high plasma concentrations of C3dg were associated significantly with IgA nephropathy with 45% of patients having levels over 25 U/ml (P less than 0.001). High concentrations of urinary C3dg and C5b-9 were associated significantly with membranous nephropathy (43% and 43% of the patient group, respectively) compared to patients with IgA nephropathy (10% and 0%, respectively, P less than 0.001). In a retrospective analysis of 31 patients with membranous nephropathy, 66% of patients with high initial urinary C5b-9 showed an unstable clinical course compared to 18% of patients with initially absent or low C5b-9 (P less than 0.001). We suggest that high urinary C5b-9 identifies those patients with a membranous lesion which retains an active immunological component contributing to the pathology of progressive glomerular damage.

  17. Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement.

    PubMed

    Choy, Henry A

    2012-01-01

    Microbial pathogens acquire the immediate imperative to avoid or counteract the formidable defense of innate immunity as soon as they overcome the initial physical barriers of the host. Many have adopted the strategy of directly disrupting the complement system through the capture of its components, using proteins on the pathogen's surface. In leptospirosis, pathogenic Leptospira spp. are resistant to complement-mediated killing, in contrast to the highly vulnerable non-pathogenic strains. Pathogenic L. interrogans uses LenA/LfhA and LcpA to respectively sequester and commandeer the function of two regulators, factor H and C4BP, which in turn bind C3b or C4b to interrupt the alternative or classical pathways of complement activation. LigB, another surface-proximal protein originally characterized as an adhesin binding multiple host proteins, has other activities suggesting its importance early in infection, including binding extracellular matrix, plasma, and cutaneous repair proteins and inhibiting hemostasis. In this study, we used a recent model of ectopic expression of LigB in the saprophyte, L. biflexa, to test the hypothesis that LigB also interacts with complement proteins C3b and C4b to promote the virulence of L. interrogans. The surface expression of LigB partially rescued the non-pathogen from killing by 5% normal human serum, showing 1.3- to 48-fold greater survival 4 to 6 d following exposure to complement than cultures of the non-expressing parental strain. Recombinant LigB7'-12 comprising the LigB-specific immunoglobulin repeats binds directly to human complement proteins, C3b and C4b, with respective K(d)s of 43±26 nM and 69±18 nM. Repeats 9 to 11, previously shown to contain the binding domain for fibronectin and fibrinogen, are also important in LigB-complement interactions, which interfere with the alternative and classical pathways measured by complement-mediated hemolysis of erythrocytes. Thus, LigB is an adaptable interface for L. interrogans

  18. COMPLEMENT REGULATION IN RENAL DISEASE MODELS

    PubMed Central

    Naik, Abhijit; Sharma, Shweta; Quigg, Richard J.

    2014-01-01

    Activation of the complement system is tightly regulated by plasma and cell-associated complement regulatory proteins (CRPs), such as factor H (fH), decay-accelerating factor (DAF), and membrane cofactor protein (MCP). Animal models of disease have provided considerable insights into the important roles for CRPs in the kidney. Mice deficient in fH have excessive fluid phase C3 activation and inactivation leading to deposition of iC3b in glomerular capillary walls (GCW), comparable to dense deposit disease. In contrast, when fH lacks C-terminal surface targeting regions, local activation on the GCW leads to a disease reminiscent of thrombotic microangiopathy. The uniquely rodent protein, CR1-related y (Crry), has features analogous to human MCP. Defective Crry leads to unrestricted alternative pathway activation in the tubulointerstitium (TI) resulting in pathological features ranging from TMA, acute kidney injury and TI nephritis. In the presence of initiators of the classical or lectin pathways, commonly in the form of immune complexes in human glomerular diseases, complement regulation on self is stressed, with the potential for recruitment of the spontaneously active alternative pathway. The threshold for this activation is set by CRPs; pathology is more likely when complement regulation is defective. Within the endocapillary region of the GCW, fH is key, while DAF and Crry are protective on mesangial cells and podocytes. Arguably, acquired alterations in these CRPs is a more common event, extending from pathological states of cellular injury or production of inhibitory antibodies, to physiological fine tuning of the adaptive immune response. PMID:24161042

  19. Structural basis for conserved complement factor-like function in the antimalarial protein TEP1

    PubMed Central

    Baxter, Richard H. G.; Chang, Chung-I; Chelliah, Yogarany; Blandin, Stéphanie; Levashina, Elena A.; Deisenhofer, Johann

    2007-01-01

    Thioester-containing proteins (TEPs) are a major component of the innate immune response of insects to invasion by bacteria and protozoa. TEPs form a distinct clade of a superfamily that includes the pan-protease inhibitors α2-macroglobulins and vertebrate complement factors. The essential feature of these proteins is a sequestered thioester bond that, after cleavage in a protease-sensitive region of the protein, is activated and covalently binds to its target. Recently, TEP1 from the malarial vector Anopheles gambiae was shown to mediate recognition and killing of ookinetes from the malarial parasite Plasmodium berghei, a model for the human malarial parasite Plasmodium falciparum. Here, we present the crystal structure of the TEP1 isoform TEP1r. Although the overall protein fold of TEP1r resembles that of complement factor C3, the TEP1r domains are repositioned to stabilize the inactive conformation of the molecule (containing an intact thioester) in the absence of the anaphylotoxin domain, a central component of complement factors. The structure of TEP1r provides a molecular basis for the differences between TEP1 alleles TEP1r and TEP1s, which correlate with resistance of A. gambiae to infection by P. berghei. PMID:17606907

  20. A complement-microglial axis drives synapse loss during virus-induced memory impairment.

    PubMed

    Vasek, Michael J; Garber, Charise; Dorsey, Denise; Durrant, Douglas M; Bollman, Bryan; Soung, Allison; Yu, Jinsheng; Perez-Torres, Carlos; Frouin, Arnaud; Wilton, Daniel K; Funk, Kristen; DeMasters, Bette K; Jiang, Xiaoping; Bowen, James R; Mennerick, Steven; Robinson, John K; Garbow, Joel R; Tyler, Kenneth L; Suthar, Mehul S; Schmidt, Robert E; Stevens, Beth; Klein, Robyn S

    2016-06-23

    Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34(-/-) mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease.

  1. Whole-exome sequencing of a patient with severe and complex hemostatic abnormalities reveals a possible contributing frameshift mutation in C3AR1

    PubMed Central

    Leinøe, Eva; Nielsen, Ove Juul; Jønson, Lars; Rossing, Maria

    2016-01-01

    The increasing availability of genome-wide analysis has made it possible to rapidly sequence the exome of patients with undiagnosed or unresolved medical conditions. Here, we present the case of a 64-yr-old male patient with schistocytes in the peripheral blood smear and a complex and life-threatening coagulation disorder causing recurrent venous thromboembolic events, severe thrombocytopenia, and subdural hematomas. Whole-exome sequencing revealed a frameshift mutation (C3AR1 c.355-356dup, p.Asp119Alafs*19) resulting in a premature stop codon in C3AR1 (Complement Component 3a Receptor 1). Based on this finding, atypical hemolytic uremic syndrome was suspected because of a genetic predisposition, and a targeted treatment regime with eculizumab was initiated. Life-threatening hemostatic abnormalities would most likely have persisted had it not been for the implementation of whole-exome sequencing in this particular clinical setting. PMID:27551680

  2. A previously unrecognized role of C3a in proteinuric progressive nephropathy

    PubMed Central

    Morigi, Marina; Locatelli, Monica; Rota, Cinzia; Buelli, Simona; Corna, Daniela; Rizzo, Paola; Abbate, Mauro; Conti, Debora; Perico, Luca; Longaretti, Lorena; Benigni, Ariela; Zoja, Carlamaria; Remuzzi, Giuseppe

    2016-01-01

    Podocyte loss is the initial event in the development of glomerulosclerosis, the structural hallmark of progressive proteinuric nephropathies. Understanding mechanisms underlying glomerular injury is the key challenge for identifying novel therapeutic targets. In mice with protein-overload induced by bovine serum albumin (BSA), we evaluated whether the alternative pathway (AP) of complement mediated podocyte depletion and podocyte-dependent parietal epithelial cell (PEC) activation causing glomerulosclerosis. Factor H (Cfh−/−) or factor B-deficient mice were studied in comparison with wild-type (WT) littermates. WT+BSA mice showed podocyte depletion accompanied by glomerular complement C3 and C3a deposits, PEC migration to capillary tuft, proliferation, and glomerulosclerosis. These changes were more prominent in Cfh−/− +BSA mice. The pathogenic role of AP was documented by data that factor B deficiency preserved glomerular integrity. In protein-overload mice, PEC dysregulation was associated with upregulation of CXCR4 and GDNF/c-Ret axis. In vitro studies provided additional evidence of a direct action of C3a on proliferation and CXCR4-related migration of PECs. These effects were enhanced by podocyte-derived GDNF. In patients with proteinuric nephropathy, glomerular C3/C3a paralleled PEC activation, CXCR4 and GDNF upregulation. These results indicate that mechanistically uncontrolled AP complement activation is not dispensable for podocyte-dependent PEC activation resulting in glomerulosclerosis. PMID:27345360

  3. Complotype affects the extent of down-regulation by Factor I of the C3b feedback cycle in vitro

    PubMed Central

    Lay, E; Nutland, S; Smith, J E; Hiles, I; Smith, R A G; Seilly, D J; Buchberger, A; Schwaeble, W; Lachmann, P J

    2015-01-01

    Sera from a large panel of normal subjects were typed for three common polymorphisms, one in C3 (R102G) and two in Factor H (V62I and Y402H), that influence predisposition to age-related macular degeneration and to some forms of kidney disease. Three groups of sera were tested; those that were homozygous for the three risk alleles; those that were heterozygous for all three; and those homozygous for the low-risk alleles. These groups vary in their response to the addition of exogenous Factor I when the alternative complement pathway is activated by zymosan. Both the reduction in the maximum amount of iC3b formed and the rate at which the iC3b is converted to C3dg are affected. For both reactions the at-risk complotype requires higher doses of Factor I to produce similar down-regulation. Because iC3b reacting with the complement receptor CR3 is a major mechanism by which complement activation gives rise to inflammation, the breakdown of iC3b to C3dg can be seen to have major significance for reducing complement-induced inflammation. These findings demonstrate for the first time that sera from subjects with different complement alleles behave as predicted in an in-vitro assay of the down-regulation of the alternative complement pathway by increasing the concentration of Factor I. These results support the hypothesis that exogenous Factor I may be a valuable therapeutic aid for down-regulating hyperactivity of the C3b feedback cycle, thereby providing a treatment for age-related macular degeneration and other inflammatory diseases of later life. PMID:25124117

  4. Cercarial glycocalyx of Schistosoma mansoni activates human complement.

    PubMed Central

    Samuelson, J C; Caulfield, J P

    1986-01-01

    Human complement activation by cercariae and schistosomula of the human parasite Schistosoma mansoni was studied in vitro. Cercariae are composed of tails which are shed after infection of the host and bodies which transform into the larvae or schistosomula after infection. After incubation in fresh normal human serum (NHS), cercarial tails bound more anti-C3 antibodies than did cercarial bodies (CB), and the tails were rapidly lysed, while the attached CB remained intact. Complement activation by cercariae was dependent on the alternative pathway but was independent of antibody, as shown by C3 deposition by hypogammaglobulinemic human sera. By transmission microscopy, the fibrillar glycocalyx on both CB and tails was stained by NHS but not by heat-inactivated serum (HI-NHS). The glycocalyx was labeled with periodate and tritiated borohydride, and parasites were incubated in NHS and HI-NHS. After solubilization, the labeled glycocalyx on organisms incubated in NHS but not HI-NHS bound anti-C3 antibodies. Of the CB incubated with eserine sulfate to prevent transformation, 78% +/- 10% were dead after culture for 24 h in NHS. In contrast, 21% +/- 12% of the CB were dead after culture in HI-NHS. Schistosomula incubated in NHS bound 37% of the amount of anti-C3 antibodies bound by cercariae but were not killed by NHS. In conclusion, the cercarial glycocalyx activated human complement, and schistosomula were less susceptible to killing than cercariae because they had less glycocalyx and activated less complement. Images PMID:3940995

  5. Identification of hot spots in the variola virus complement inhibitor (SPICE) for human complement regulation.

    PubMed

    Yadav, Viveka Nand; Pyaram, Kalyani; Mullick, Jayati; Sahu, Arvind

    2008-04-01

    Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is approximately 90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

  6. Complement and Antibody-Mediated Enhancement of Erythrocyte Invasion by Plasmodium Falciparum

    DTIC Science & Technology

    2016-04-01

    take place via antibody-dependent or independent mechanisms. Complement is activated during malaria infection (4), especially in the presence of...that complement activation during malaria infection enhances the invasion of RBCs by P. falciparum. In this research we tested our hypothesis by... infection have an enhancing effect on invasion and, if so, what is the mechanism. A - C3/C4 + C3/C4 % I n h ib it io n -10 -5 0 5 10 15 - C3/C4 + C3/C4

  7. C3-class Solar Flare Eruption

    NASA Video Gallery

    Just as sunspot 1105 was turning away from Earth on Sept. 8, the active region erupted, producing a C3-class solar flare (peak @ 2330 UT) and a fantastic prominence. This is a three color closeup o...

  8. 29 CFR 2575.502c-3 - Adjusted civil penalty under section 502(c)(3).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ACT OF 1974 ADJUSTMENT OF CIVIL PENALTIES UNDER ERISA TITLE I Adjustment of Civil Penalties Under ERISA Title I § 2575.502c-3 Adjusted civil penalty under section 502(c)(3). In accordance with the... section 502(c)(3) of the Employee Retirement Income Security Act of 1974, as amended (ERISA), is...

  9. 29 CFR 2575.502c-3 - Adjusted civil penalty under section 502(c)(3).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... ACT OF 1974 ADJUSTMENT OF CIVIL PENALTIES UNDER ERISA TITLE I Adjustment of Civil Penalties Under ERISA Title I § 2575.502c-3 Adjusted civil penalty under section 502(c)(3). In accordance with the... section 502(c)(3) of the Employee Retirement Income Security Act of 1974, as amended (ERISA), is...

  10. 29 CFR 2575.502c-3 - Adjusted civil penalty under section 502(c)(3).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... ACT OF 1974 ADJUSTMENT OF CIVIL PENALTIES UNDER ERISA TITLE I Adjustment of Civil Penalties Under ERISA Title I § 2575.502c-3 Adjusted civil penalty under section 502(c)(3). In accordance with the... section 502(c)(3) of the Employee Retirement Income Security Act of 1974, as amended (ERISA), is...

  11. 29 CFR 2575.502c-3 - Adjusted civil penalty under section 502(c)(3).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... ACT OF 1974 ADJUSTMENT OF CIVIL PENALTIES UNDER ERISA TITLE I Adjustment of Civil Penalties Under ERISA Title I § 2575.502c-3 Adjusted civil penalty under section 502(c)(3). In accordance with the... section 502(c)(3) of the Employee Retirement Income Security Act of 1974, as amended (ERISA), is...

  12. 29 CFR 2575.502c-3 - Adjusted civil penalty under section 502(c)(3).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... ACT OF 1974 ADJUSTMENT OF CIVIL PENALTIES UNDER ERISA TITLE I Adjustment of Civil Penalties Under ERISA Title I § 2575.502c-3 Adjusted civil penalty under section 502(c)(3). In accordance with the... section 502(c)(3) of the Employee Retirement Income Security Act of 1974, as amended (ERISA), is...

  13. C3 glomerulonephritis and autoimmune disease: more than a fortuitous association?

    PubMed

    Alexander, Mariam P; Fervenza, Fernando C; De Vriese, An S; Smith, Richard J H; Nasr, Samih H; Cornell, Lynn D; Herrera Hernandez, Loren P; Zhang, Yuzhou; Sethi, Sanjeev

    2016-04-01

    C3 glomerulonephritis (C3GN) results from genetic or acquired dysregulation of the alternative complement pathway. A subset of patients may have clinical and biochemical characteristics compatible with an autoimmune disorder. We studied a cohort of 85 patients with confirmed C3GN (2007-2014), of which ten patients (3 male, 7 female; mean age 38.5 years) had an associated autoimmune disorder. All patients had abnormal ANA titers, 6 also had positive ds-DNA titers. At the time of presentation with C3GN, all 7 female patients had autoimmune-related presentations. Of the 3 male patients, only 1 patient had autoimmune-related presentations. Kidney biopsy showed predominantly mesangial proliferative or membranoproliferative glomerulonephritis. In 5 patients, the alternative pathway was evaluated. All had allele variants/polymorphisms associated with C3GN. One patient was also positive for C3Nefs. Treatment varied form conservative management to the use of prednisone alone or with cytotoxic therapy. Mean serum creatinine decreased from 2.0 to 1.4 mg/dL while proteinuria decreased from 2300 to 994 mg/24 h in 8 patients with follow-up. The study highlights the association between C3GN and autoimmune disorders, particularly in female patients. The study suggests that an autoimmune milieu may act as a trigger for the development of C3GN in genetically susceptible patients. Short-term prognosis of C3GN associated with autoimmune disorders appears excellent.

  14. Applying Complement Therapeutics to Rare Diseases

    PubMed Central

    Reis, Edimara S.; Mastellos, Dimitrios C.; Yancopoulou, Despina; Risitano, Antonio M.; Ricklin, Daniel; Lambris, John D.

    2015-01-01

    Around 350 million people worldwide suffer from rare diseases. These may have a genetic, infectious, or autoimmune basis, and several include an inflammatory component. Launching of effective treatments can be very challenging when there is a low disease prevalence and limited scientific insights into the disease mechanisms. As a key trigger of inflammatory processes, complement has been associated with a variety of diseases and has become an attractive therapeutic target for conditions involving inflammation. In view of the clinical experience acquired with drugs licensed for the treatment of rare diseases such as hereditary angioedema and paroxysmal nocturnal hemoglobinuria, growing evidence supports the safety and efficacy of complement therapeutics in restoring immune balance and preventing aggravation of clinical outcomes. This review provides an overview of the candidates currently in the pharmaceutical pipeline with potential to treat orphan diseases and discusses the molecular mechanisms triggered by complement involved with the disease pathogenesis. PMID:26341313

  15. Applying complement therapeutics to rare diseases.

    PubMed

    Reis, Edimara S; Mastellos, Dimitrios C; Yancopoulou, Despina; Risitano, Antonio M; Ricklin, Daniel; Lambris, John D

    2015-12-01

    Around 350 million people worldwide suffer from rare diseases. These may have a genetic, infectious, or autoimmune basis, and several include an inflammatory component. Launching of effective treatments can be very challenging when there is a low disease prevalence and limited scientific insights into the disease mechanisms. As a key trigger of inflammatory processes, complement has been associated with a variety of diseases and has become an attractive therapeutic target for conditions involving inflammation. In view of the clinical experience acquired with drugs licensed for the treatment of rare diseases such as hereditary angioedema and paroxysmal nocturnal hemoglobinuria, growing evidence supports the safety and efficacy of complement therapeutics in restoring immune balance and preventing aggravation of clinical outcomes. This review provides an overview of the candidates currently in the pharmaceutical pipeline with potential to treat orphan diseases and discusses the molecular mechanisms triggered by complement involved with the disease pathogenesis.

  16. Increased sensitivity of early apoptotic cells to complement-mediated lysis.

    PubMed

    Attali, Gitit; Gancz, Dana; Fishelson, Zvi

    2004-11-01

    Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.

  17. Minor Role of Plasminogen in Complement Activation on Cell Surfaces

    PubMed Central

    Hyvärinen, Satu; Jokiranta, T. Sakari

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation. PMID:26637181

  18. Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy

    PubMed Central

    Zhu, Li; Zhai, Ya-Ling; Wang, Feng-Mei; Hou, Ping; Lv, Ji-Cheng; Xu, Da-Min; Shi, Su-Fang; Liu, Li-Jun; Yu, Feng; Zhao, Ming-Hui; Novak, Jan; Gharavi, Ali G.

    2015-01-01

    Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1–5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3–1 deletion (CFHR3–1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype–phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3–1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN. PMID:25205734

  19. Complementing Gender Analysis Methods.

    PubMed

    Kumar, Anant

    2016-01-01

    The existing gender analysis frameworks start with a premise that men and women are equal and should be treated equally. These frameworks give emphasis on equal distribution of resources between men and women and believe that this will bring equality which is not always true. Despite equal distribution of resources, women tend to suffer and experience discrimination in many areas of their lives such as the power to control resources within social relationships, and the need for emotional security and reproductive rights within interpersonal relationships. These frameworks believe that patriarchy as an institution plays an important role in women's oppression, exploitation, and it is a barrier in their empowerment and rights. Thus, some think that by ensuring equal distribution of resources and empowering women economically, institutions like patriarchy can be challenged. These frameworks are based on proposed equality principle which puts men and women in competing roles. Thus, the real equality will never be achieved. Contrary to the existing gender analysis frameworks, the Complementing Gender Analysis framework proposed by the author provides a new approach toward gender analysis which not only recognizes the role of economic empowerment and equal distribution of resources but suggests to incorporate the concept and role of social capital, equity, and doing gender in gender analysis which is based on perceived equity principle, putting men and women in complementing roles that may lead to equality. In this article the author reviews the mainstream gender theories in development from the viewpoint of the complementary roles of gender. This alternative view is argued based on existing literature and an anecdote of observations made by the author. While criticizing the equality theory, the author offers equity theory in resolving the gender conflict by using the concept of social and psychological capital.

  20. Reclassification of membranoproliferative glomerulonephritis: Identification of a new GN: C3GN

    PubMed Central

    Salvadori, Maurizio; Rosso, Giuseppina

    2016-01-01

    This review revises the reclassification of the membranoproliferative glomerulonephritis (MPGN) after the consensus conference that by 2015 reclassified all the glomerulonephritis basing on etiology and pathogenesis, instead of the histomorphological aspects. After reclassification, two types of MPGN are to date recognized: The immunocomplexes mediated MPGN and the complement mediated MPGN. The latter type is more extensively described in the review either because several of these entities are completely new or because the improved knowledge of the complement cascade allowed for new diagnostic and therapeutic approaches. Overall the complement mediated MPGN are related to acquired or genetic cause. The presence of circulating auto antibodies is the principal acquired cause. Genetic wide association studies and family studies allowed to recognize genetic mutations of different types as causes of the complement dysregulation. The complement cascade is a complex phenomenon and activating factors and regulating factors should be distinguished. Genetic mutations causing abnormalities either in activating or in regulating factors have been described. The diagnosis of the complement mediated MPGN requires a complete study of all these different complement factors. As a consequence, new therapeutic approaches are becoming available. Indeed, in addition to a nonspecific treatment and to the immunosuppression that has the aim to block the auto antibodies production, the specific inhibition of complement activation is relatively new and may act either blocking the C5 convertase or the C3 convertase. The drugs acting on C3 convertase are still in different phases of clinical development and might represent drugs for the future. Overall the authors consider that one of the principal problems in finding new types of drugs are both the rarity of the disease and the consequent poor interest in the marketing and the lack of large international cooperative studies. PMID:27458560

  1. Functional C3 Interoperability Architecture for Air Operations

    DTIC Science & Technology

    1991-08-20

    prevent effective joint operations. JTC3A developed this architecture after a review and analysis of joint and service documentation and discussions with...unified, component command, and service staff personnel. An interim report, referred to as the supporting analysis , was previously distributed for...service comment and provides extensive supplemental information on C3 for joint air operations. The supporting analysis will be available as soon as it is

  2. Complement inhibition in cancer therapy.

    PubMed

    Pio, Ruben; Ajona, Daniel; Lambris, John D

    2013-02-01

    For decades, complement has been recognized as an effector arm of the immune system that contributes to the destruction of tumor cells. In fact, many therapeutic strategies have been proposed that are based on the intensification of complement-mediated responses against tumors. However, recent studies have challenged this paradigm by demonstrating a tumor-promoting role for complement. Cancer cells seem to be able to establish a convenient balance between complement activation and inhibition, taking advantage of complement initiation without suffering its deleterious effects. Complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and activate cancer-related signaling pathways. In this context, inhibition of complement activation would be a therapeutic option for treating cancer. This concept is relatively new and deserves closer attention. In this article, we summarize the mechanisms of complement activation on cancer cells, the cancer-promoting effect of complement initiation, and the rationale behind the use of complement inhibition as a therapeutic strategy against cancer.

  3. Complement modulation of T cell immune responses during homeostasis and disease.

    PubMed

    Clarke, Elizabeth V; Tenner, Andrea J

    2014-11-01

    The complement system is an ancient and critical effector mechanism of the innate immune system as it senses, kills, and clears infectious and/or dangerous particles and alerts the immune system to the presence of the infection and/or danger. Interestingly, an increasing number of reports have demonstrated a clear role for complement in the adaptive immune system as well. Of note, a number of recent studies have identified previously unknown roles for complement proteins, receptors, and regulators in T cell function. Here, we will review recent data demonstrating the influence of complement proteins C1q, C3b/iC3b, C3a (and C3aR), and C5a (and C5aR) and complement regulators DAF (CD55) and CD46 (MCP) on T cell function during homeostasis and disease. Although new concepts are beginning to emerge in the field of complement regulation of T cell function, future experiments should focus on whether complement is interacting directly with the T cell or is having an indirect effect on T cell function via APCs, the cytokine milieu, or downstream complement activation products. Importantly, the identification of the pivotal molecular pathways in the human systems will be beneficial in the translation of concepts derived from model systems to therapeutic targeting for treatment of human disorders.

  4. Erythrocyte C3d and C4d for Monitoring Disease Activity in Systemic Lupus Erythematosus

    PubMed Central

    Kao, Amy H.; Navratil, Jeannine S.; Ruffing, Margie J.; Liu, Chau-Ching; Hawkins, Douglas; McKinnon, Kathleen M.; Danchenko, Natalya; Ahearn, Joseph M.; Manzi, Susan

    2010-01-01

    Objective Disease activity in systemic lupus erythematosus (SLE) is typically monitored by measuring serum C3 and C4. However, these proteins have limited utility as lupus biomarkers, because they are substrates rather than products of complement activation. The aim of this study was to evaluate the utility of measuring the erythrocyte-bound complement activation products, erythrocyte-bound C3d (E-C3d) and E-C4d, compared with that of serum C3 and C4 for monitoring disease activity in patients with SLE. Methods The levels of E-C3d and E-C4d were measured by flow cytometry in 157 patients with SLE, 290 patients with other diseases, and 256 healthy individuals. The patients with SLE were followed up longitudinally. Disease activity was measured at each visit, using the validated Systemic Lupus Activity Measure (SLAM) and the Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Results At baseline, patients with SLE had higher median levels of E-C3d and E-C4d (P < 0.0001) in addition to higher within-patient and between-patient variability in both E-C3d and E-C4d when compared with the 2 non-SLE groups. In a longitudinal analysis of patients with SLE, E-C3d, E-C4d, serum C3, and anti–double-stranded DNA (anti-dsDNA) antibodies were each significantly associated with the SLAM and SELENA–SLEDAI. In a multivariable analysis, E-C4d remained significantly associated with these SLE activity measures after adjusting for serum C3, C4, and anti-dsDNA antibodies; however, E-C3d was associated with the SLAM but not with the SELENA–SLEDAI. Conclusion Determining the levels of the erythrocyte-bound complement activation products, especially E-C4d, is an informative measure of SLE disease activity as compared with assessing serum C4 levels and should be considered for monitoring disease activity in patients with SLE. PMID:20187154

  5. The Carbohydrate-linked Phosphorylcholine of the Parasitic Nematode Product ES-62 Modulates Complement Activation*

    PubMed Central

    Ahmed, Umul Kulthum; Maller, N. Claire; Iqbal, Asif J.; Al-Riyami, Lamyaa; Harnett, William; Raynes, John G.

    2016-01-01

    Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications. PMID:27044740

  6. The Carbohydrate-linked Phosphorylcholine of the Parasitic Nematode Product ES-62 Modulates Complement Activation.

    PubMed

    Ahmed, Umul Kulthum; Maller, N Claire; Iqbal, Asif J; Al-Riyami, Lamyaa; Harnett, William; Raynes, John G

    2016-05-27

    Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications.

  7. A teleost complement factor Ba possesses antimicrobial activity and inhibits bacterial infection in fish.

    PubMed

    Li, Xue-Peng; Sun, Li

    2017-01-24

    Complement factor B (Bf) is a component of the complement system. Following activation of the alternative pathway of the complement system, factor B is cleaved into Ba and Bb fragments. In fish, the Bf of rainbow trout is known to act as a C3 convertase, but the function of the Ba fragment is essentially unknown. In this study, we examined the expression patterns of tongue sole Cynoglossus semilaevis Bf (named CsBf) and the biological activity of the Ba fragment of CsBf (named CsBa). CsBf possesses the conserved domains of Bf and shares 39.9%-56.4% sequence identities with other fish Bf. CsBf expression was high in liver, muscle, and heart, and low in intestine, blood, and kidney. Bacterial infection significantly induced CsBf expression in kidney, spleen, and liver in a time-dependent manner. Recombinant CsBa (rCsBa) exhibited apparent binding capacities to bacteria and tongue sole peripheral blood leukocytes, and binding of rCsBa to bacteria inhibited bacterial growth. When overexpressed in tongue sole, CsBa significantly reduced bacterial dissemination in fish tissues. Together these results indicate for the first time that a fish Ba possesses antibacterial effect as well as immune cell-binding capacity, and thus probably plays a role in host immune defense against bacterial infection.

  8. Complement activation in very early Alzheimer disease.

    PubMed

    Zanjani, H; Finch, C E; Kemper, C; Atkinson, J; McKeel, D; Morris, J C; Price, J L

    2005-01-01

    The activation of the classical complement (C)-system in early-stage Alzheimer disease (AD) and nondemented aging was examined with immunohistochemistry in subjects assessed by the Clinical Dementia Rating (CDR). Activation (staining for C3 and C4 fragments) was found in all brains with amyloid deposits, including all nondemented (CDR 0) cases, with either small numbers of diffuse plaques or with sufficient plaques and tangles to indicate preclinical AD. Staining for C3 and C4 increased in parallel with plaque density in very mild to severe clinical AD. A subset of very mild AD (CDR 0.5) cases also showed C1q (on plaques) and C5b-9 (on neuritic plaques and tangles), whereas these C-fragments were consistently found in severe AD (CDR 3). Mirror section (split-face) analysis showed that C1q, C3, and apoJ (clusterin) occurred on the same plaques. However, C-system regulators CD59, CR1, DAF, and MCP were not detected on plaques or tangles at any stage, indicating that C-activation related to AD is incompletely controlled.

  9. Recombinant Complement Receptor 2 Radiolabeled with [99mTc(CO)3]+ : A Potential New Radiopharmaceutical for Imaging Activated Complement

    PubMed Central

    McDonnell, James M.; Yahya, Norhakim; Thakor, David; Razavi, Reza; Smith, Richard; Sacks, Steven; Mullen, Gregory E. D.

    2011-01-01

    We describe the design and synthesis of a new Tc-99m labeled bioconjugate for imaging activated complement, based on Short Consensus Repeats 1 and 2 of Complement Receptor 2 (CR2), the binding domain for C3d. To avoid non specific modification of CR2 and the potential for modifying lysine residues critical to the CR2/C3d contact surface, we engineered a new protein, recombinant CR2 (rCR2), to include the C-terminal sequence VFPLECHHHHHH, a hexahistidine tag (for site-specific radiolabeling with [99mTc(CO)3(OH2)3]+). The protein was characterized by N-terminal sequencing, SDS-PAGE and size exclusion chromatography. To test the function of the recombinant CR2, binding to C3d was confirmed by enzyme-linked immunosorbent assay (ELISA). The function was further confirmed by binding of rCR2 to C3d+ red blood cells (RBC) which were generated by deposition of human or rat C3d and analyzed by fluorescence microscopy and flow cytometry. The affinity of rCR2 for C3d+, in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a KD≈500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [99mTc(CO)3(OH2)3]+ at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d+ RBC. Binding of radiolabeled rCR2 to C3d was inhibited by anti-C3d and the radiolabeled inactive mutant K41E CR2 and C2A did not bind to C3d+ RBCs. We conclude that rCR2-Tc99m has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent. PMID:21494666

  10. The Development of the C3 Framework

    ERIC Educational Resources Information Center

    Social Education, 2013

    2013-01-01

    Susan Griffin, executive director of NCSS, was chair of the Task Force of Professional Organizations that initiated and guided the development of "The College, Career, and Civic Life (C3) Framework for Social Studies State Standards", which will soon be released. In this interview with "Social Education", Susan explains how the…

  11. Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions.

    PubMed

    Guzman, Raul E; Miranda-Laferte, Erick; Franzen, Arne; Fahlke, Christoph

    2015-10-23

    ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl(-) currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.

  12. Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions*

    PubMed Central

    Guzman, Raul E.; Miranda-Laferte, Erick; Franzen, Arne; Fahlke, Christoph

    2015-01-01

    ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl− currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected. PMID:26342074

  13. Complement System in Lung Disease

    PubMed Central

    Pandya, Pankita H.

    2014-01-01

    In addition to its established contribution to innate immunity, recent studies have suggested novel roles for the complement system in the development of various lung diseases. Several studies have demonstrated that complement may serve as a key link between innate and adaptive immunity in a variety of pulmonary conditions. However, the specific contributions of complement to lung diseases based on innate and adaptive immunity are just beginning to emerge. Elucidating the role of complement-mediated immune regulation in these diseases will help to identify new targets for therapeutic interventions. PMID:24901241

  14. Detection of surface bound complement at increasing serum anticoagulant concentrations.

    PubMed

    Arvidsson, S; Askendal, A; Lindahl, T L; Tengvall, P

    2008-04-01

    Surface mediated immune complement activation can be detected by a variety of antibody utilizing methods such as ELISA, fluorescence- or radiolabelling techniques, QCM, and ellipsometry. In the present work we investigated how the common anticoagulants heparin, dalteparin, fondaparinux and sodium citrate affected the binding of anti-complement factor 3c (anti-C3c) on a model complement activator surface, immobilised IgG, after incubation in human blood serum. The results show, as expected, that different anticoagulants affect the antibody binding differently. Increasing amounts of heparin, dalteparin and sodium citrate in normal serum resulted in a decreasing anti-