Antibody mediated rejection associated with complement factor h-related protein 3/1 deficiency successfully treated with eculizumab.

PubMed

Noone, D; Al-Matrafi, J; Tinckam, K; Zipfel, P F; Herzenberg, A M; Thorner, P S; Pluthero, F G; Kahr, W H A; Filler, G; Hebert, D; Harvey, E; Licht, C

2012-09-01

Antibody mediated rejection (AMR) activates the classical complement pathway and can be detrimental to graft survival. AMR can be accompanied by thrombotic microangiopathy (TMA). Eculizumab, a monoclonal C5 antibody prevents induction of the terminal complement cascade (TCC) and has recently emerged as a therapeutic option for AMR. We present a highly sensitized 13-year-old female with end-stage kidney disease secondary to spina bifida-associated reflux nephropathy, who developed severe steroid-, ATG- and plasmapheresis-resistant AMR with TMA 1 week post second kidney transplant despite previous desensitization therapy with immunoglobulin infusions. Eculizumab rescue therapy resulted in a dramatic improvement in biochemical (C3; creatinine) and hematological (platelets) parameters within 6 days. The patient was proven to be deficient in complement Factor H-related protein 3/1 (CFHR3/1), a plasma protein that regulates the complement cascade at the level of C5 conversion and has been involved in the pathogenesis of atypical hemolytic uremic syndrome caused by CFH autoantibodies (DEAP-HUS). CFHR1 deficiency may have worsened the severe clinical progression of AMR and possibly contributed to the development of donor-specific antibodies. Thus, screening for CFHR3/1 deficiency should be considered in patients with severe AMR associated with TMA. © Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

PubMed Central

Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

2017-01-01

Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

Induction of passive Heymann nephritis in complement component 6-deficient PVG rats.

PubMed

Spicer, S Timothy; Tran, Giang T; Killingsworth, Murray C; Carter, Nicole; Power, David A; Paizis, Kathy; Boyd, Rochelle; Hodgkinson, Suzanne J; Hall, Bruce M

2007-07-01

Passive Heymann nephritis (PHN), a model of human membranous nephritis, is induced in susceptible rat strains by injection of heterologous antisera to rat renal tubular Ag extract. PHN is currently considered the archetypal complement-dependent form of nephritis, with the proteinuria resulting from sublytic glomerular epithelial cell injury induced by the complement membrane attack complex (MAC) of C5b-9. This study examined whether C6 and MAC are essential to the development of proteinuria in PHN by comparing the effect of injection of anti-Fx1A antisera into PVG rats deficient in C6 (PVG/C6(-)) and normal PVG rats (PVG/c). PVG/c and PVG/C6(-) rats developed similar levels of proteinuria at 3, 7, 14, and 28 days following injection of antisera. Isolated whole glomeruli showed similar deposition of rat Ig and C3 staining in PVG/c and PVG/C6(-) rats. C9 deposition was abundant in PVG/c but was not detected in PVG/C6(-) glomeruli, indicating C5b-9/MAC had not formed in PVG/C6(-) rats. There was also no difference in the glomerular cellular infiltrate of T cells and macrophages nor the size of glomerular basement membrane deposits measured on electron micrographs. To examine whether T cells effect injury, rats were depleted of CD8+ T cells which did not affect proteinuria in the early heterologous phase but prevented the increase in proteinuria associated with the later autologous phase. These studies showed proteinuria in PHN occurs without MAC and that other mechanisms, such as immune complex size, early complement components, CD4+ and CD8+ T cells, disrupt glomerular integrity and lead to proteinuria.

Sexual dimorphism of liver metastasis by murine pancreatic neuroendocrine tumors is affected by expression of complement C5.

PubMed

Contractor, Tanupriya; Kobayashi, Shinta; da Silva, Edaise; Clausen, Richard; Chan, Chang; Vosburgh, Evan; Tang, Laura H; Levine, Arnold J; Harris, Chris R

2016-05-24

In a mouse model for neuroendocrine tumors of the pancreas (PanNETs), liver metastasis occurred at a higher frequency in males. Male mice also had higher serum and intratumoral levels of the innate immunity protein complement C5. In mice that lost the ability to express complement C5, there was a lower frequency of metastasis, and males no longer had a higher frequency of metastasis than females. Treatment with PMX53, a small molecule antagonist of C5aR1/CD88, the receptor for complement C5a, also reduced metastasis. Mice lacking a functional gene for complement C5 had smaller primary tumors, which were less invasive and lacked the CD68+ macrophages that have previously been associated with metastasis in this type of tumor. This is the first report of a gene that causes sexual dimorphism of metastasis in a mouse model. In the human disease, which also shows sexual dimorphism for metastasis, clinically advanced tumors expressed more complement C5 than less advanced tumors.

Immunization with a Vaccine Combining Herpes Simplex Virus 2 (HSV-2) Glycoprotein C (gC) and gD Subunits Improves the Protection of Dorsal Root Ganglia in Mice and Reduces the Frequency of Recurrent Vaginal Shedding of HSV-2 DNA in Guinea Pigs Compared to Immunization with gD Alone ▿

PubMed Central

Awasthi, Sita; Lubinski, John M.; Shaw, Carolyn E.; Barrett, Shana M.; Cai, Michael; Wang, Fushan; Betts, Michael; Kingsley, Susan; DiStefano, Daniel J.; Balliet, John W.; Flynn, Jessica A.; Casimiro, Danilo R.; Bryan, Janine T.; Friedman, Harvey M.

2011-01-01

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation. PMID:21813597

Increased activity of the complement system in the liver of patients with alcoholic hepatitis.

PubMed

Shen, Hong; French, Barbara A; Liu, Hui; Tillman, Brittany C; French, Samuel W

2014-12-01

Inflammation has been suggested as a mechanism underlying the development of alcoholic hepatitis (AH). The activation of the complement system plays an important role in inflammation. Although it has been shown that ethanol-induced activation of the complement system contributes to the pathophysiology of ethanol-induced liver injury in mice, whether ethanol consumption activates the complement system in the human liver has not been investigated. Using antibodies against C1q, C3, and C5, the immunoreactivity of the complement system in patients with AH was examined by immunohistochemistry and quantified by morphometric image analysis. The immunoreactivity intensity of C1q, C3, and C5 in patients with AH was significantly higher than that seen in normal controls. Further, the gene expression of C1q, C3, and C5 was examined using real-time PCR. There were increases in the levels of C1q and C5, but not C3 mRNA in AH. Moreover, the immunoreactivity of C5a receptor (C5aR) also increased in AH. To explore the functional implication of the activation of the complement system in AH, we examined the colocalization of C5aR in Mallory-Denk bodies (MDBs) forming balloon hepatocytes. C5aR was focally overexpressed in the MDB forming cells. Collectively, our study suggests that alcohol consumption increases the activity of the complement system in the liver cells, which contributes to the inflammation-associated pathogenesis of AH. Copyright © 2014 Elsevier Inc. All rights reserved.

Polyanion-Induced Self Association of Complement Factor H1

PubMed Central

Pangburn, Michael K.; Rawal, Nenoo; Cortes, Claudio; Alam, M. Nurul; Ferreira, Viviana P.; Atkinson, Mark A. L.

2008-01-01

Factor H is the primary soluble regulator of activation of the alternative pathway of complement. It prevents activation of complement on host cells and tissues upon association with C3b and surface polyanions such as sialic acids, heparin and other glycosaminoglycans. Here we show that interaction with polyanions causes self-association forming tetramers of the 155,000 Da glycosylated protein. Monomeric human factor H is an extended flexible protein that exhibits an apparent size of 330,000 Da, relative to globular standards, during gel filtration chromatography in the absence of polyanions. In the presence of dextran sulfate (5,000 Da) or heparin an intermediate species of apparent m.w. 700,000 and a limit species of m.w. 1,400,000 were observed by gel filtration. Sedimentation equilibrium analysis by analytical ultracentrifugation indicated a monomer Mr of 163,000 in the absence of polyanions and a Mr of 607,000, corresponding to a tetramer, in the presence of less than a 2-fold molar excess of dextran sulfate. Increasing concentrations of dextran sulfate increased binding of factor H to zymosan-C3b 4.5-fold. This was accompanied by an increase in both the decay accelerating and cofactor activity of factor H on these cells. An expressed fragment encompassing the C-terminal polyanion binding site (complement control protein domains 18–20) also exhibited polyanion-induced self association, suggesting that the C-terminal ends of factor H mediate self-association. The results suggest that recognition of polyanionic markers on host cells and tissues by factor H, and the resulting regulation of complement activation, may involve formation of dimers and tetramers of factor H. PMID:19124749

Complementation of DNA repair defect in xeroderma pigmentosum cells of group C by the transfer of human chromosome 5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaur, G.P.; Athwal, R.S.

1993-01-01

Complementation of DNA excision repair defect in xeroderma pigmentosum cells of group C (XP-C) has been achieved by the transfer of human chromosome 5. Individual human chromosomes tagged with a selectable marker were transferred to XP-C cells by microcell fusion from mouse-human hybrid cell lines each bearing a single different human chromosome. Analysis of the chromosome transfer clones revealed that introduction of chromosome 5 into XP-C cells corrected the DNA repair defect as well as UV-sensitive phenotypes, while chromosomes 2, 6, 7, 9, 13, 15, 17, and 21 failed to complement. The introduced chromosome 5 in complemented UV[sup r] clonesmore » was distinguished from the parental XP-C chromosomes by polymorphism for dinucleotide (CA)[sub n] repeats at two loci, D5S117 and D5S209. In addition, an intact marked chromosome 5 was rescued into mouse cells from a complemented UV[sup r] clone by microcell fusion. Five subclones of a complemented clone that had lost the marked chromosome 5 exhibited UV-sensitive and repair-deficient phenotypes identical to parental XP-C cells. Concordant loss of the transferred chromosome and reappearance of XP-C phenotype further confirmed the presence of a DNA repair gene on human chromosome 5. 38 refs., 7 figs., 1 tab.« less

Production and Characterization of High-Affinity Human Monoclonal Antibodies to Human Immunodeficiency Virus Type 1 Envelope Glycoproteins in a Mouse Model Expressing Human Immunoglobulins▿ †

PubMed Central

Sheppard, Neil C.; Davies, Sarah L.; Jeffs, Simon A.; Vieira, Sueli M.; Sattentau, Quentin J.

2007-01-01

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-197CN54), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-197CN54 Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies. PMID:17167037

Production and characterization of high-affinity human monoclonal antibodies to human immunodeficiency virus type 1 envelope glycoproteins in a mouse model expressing human immunoglobulins.

PubMed

Sheppard, Neil C; Davies, Sarah L; Jeffs, Simon A; Vieira, Sueli M; Sattentau, Quentin J

2007-02-01

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1(97CN54)), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1(97CN54) Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.

Multi-functional mechanisms of immune evasion by the streptococcal complement inhibitor C5a peptidase

PubMed Central

Reglinski, Mark; Calay, Damien; Siggins, Matthew K.; Mason, Justin C.; Botto, Marina; Sriskandan, Shiranee

2017-01-01

The complement cascade is crucial for clearance and control of invading pathogens, and as such is a key target for pathogen mediated host modulation. C3 is the central molecule of the complement cascade, and plays a vital role in opsonization of bacteria and recruitment of neutrophils to the site of infection. Streptococcal species have evolved multiple mechanisms to disrupt complement-mediated innate immunity, among which ScpA (C5a peptidase), a C5a inactivating enzyme, is widely conserved. Here we demonstrate for the first time that pyogenic streptococcal species are capable of cleaving C3, and identify C3 and C3a as novel substrates for the streptococcal ScpA, which are functionally inactivated as a result of cleavage 7 amino acids upstream of the natural C3 convertase. Cleavage of C3a by ScpA resulted in disruption of human neutrophil activation, phagocytosis and chemotaxis, while cleavage of C3 generated abnormally-sized C3a and C3b moieties with impaired function, in particular reducing C3 deposition on the bacterial surface. Despite clear effects on human complement, expression of ScpA reduced clearance of group A streptococci in vivo in wildtype and C5 deficient mice, and promoted systemic bacterial dissemination in mice that lacked both C3 and C5, suggesting an additional complement-independent role for ScpA in streptococcal pathogenesis. ScpA was shown to mediate streptococcal adhesion to both human epithelial and endothelial cells, consistent with a role in promoting bacterial invasion within the host. Taken together, these data show that ScpA is a multi-functional virulence factor with both complement-dependent and independent roles in streptococcal pathogenesis. PMID:28806402

Acute and prolonged complement activation in the central nervous system during herpes simplex encephalitis.

PubMed

Eriksson, Charlotta E; Studahl, Marie; Bergström, Tomas

2016-06-15

Herpes simplex encephalitis (HSE) is characterized by a pronounced inflammatory activity in the central nervous system (CNS). Here, we investigated the acute and prolonged complement system activity in HSE patients, by using enzyme-linked immunosorbent assays (ELISAs) for numerous complement components (C). We found increased cerebrospinal fluid concentrations of C3a, C3b, C5 and C5a in HSE patients compared with healthy controls. C3a and C5a concentrations remained increased also compared with patient controls. Our results conclude that the complement system is activated in CNS during HSE in the acute phase, and interestingly also in later stages supporting previous reports of prolonged inflammation. Copyright © 2016 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 andmore » 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.« less

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

PubMed Central

Banadakoppa, M; Chauhan, M S; Havemann, D; Balakrishnan, M; Dominic, J S; Yallampalli, C

2014-01-01

Spontaneous abortion in early pregnancy due to unknown reasons is a common problem. The excess complement activation and consequent placental inflammation and anti-angiogenic milieu is emerging as an important associated factor in many pregnancy-related complications. In the present study we sought to examine the expression of complement inhibitory proteins at the feto–maternal interface and levels of complement split products in the circulation to understand their role in spontaneous abortion. Consenting pregnant women who either underwent elective abortion due to non-clinical reasons (n = 13) or suffered miscarriage (n = 14) were recruited for the study. Systemic levels of complement factors C3a and C5a were measured by enzyme-linked immunosorbent assay (ELISA). Plasma C5 and C3 protein levels were examined by Western blot. Expressions of complement regulatory proteins such as CD46 and CD55 in the decidua were investigated by quantitative polymerase chain reaction (PCR) and Western blot. The median of plasma C3a level was 82·83 ng/ml and 66·17 ng/ml in elective and spontaneous abortion patients, respectively. Medians of plasma C5a levels in elective and spontaneous abortion patients were 0·96 ng/ml and 1·14 ng/ml, respectively. Only plasma C5a levels but not C3a levels showed significant elevation in spontaneous abortion patients compared to elective abortion patients. Further, there was a threefold decrease in the mRNA expressions of complement inhibitory proteins CD46 and CD55 in the decidua obtained from spontaneous abortion patients compared to that of elective abortion patients. These data suggested that dysregulated complement cascade may be associated with spontaneous abortion. PMID:24802103

Molecular and expression analysis of complement component C5 in the nurse shark (Ginglymostoma cirratum) and its predicted functional role.

PubMed

Graham, Matthew; Shin, Dong-Ho; Smith, Sylvia L

2009-07-01

We present the complete cDNA sequence of shark (Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5' and 3' UTRs of 35 bp and 79 bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences shows that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved.

Molecular and expression analysis of complement component C5 in the nurse shark (Ginglymostoma cirratum) and its predicted functional role

PubMed Central

Graham, Matthew; Shin, Dong-Ho; Smith, Sylvia L.

2009-01-01

We present the complete cDNA sequence of shark (Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5′ and 3′ UTRs of 35bp and 79bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences show that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved. PMID:19410004

Complement anaphylatoxins as immune regulators in cancer.

PubMed

Sayegh, Eli T; Bloch, Orin; Parsa, Andrew T

2014-08-01

The role of the complement system in innate immunity is well characterized. However, a recent body of research implicates the complement anaphylatoxins C3a and C5a as insidious propagators of tumor growth and progression. It is now recognized that certain tumors elaborate C3a and C5a and that complement, as a mediator of chronic inflammation and regulator of immune function, may in fact foster rather than defend against tumor growth. A putative mechanism for this function is complement-mediated suppression of immune effector cells responsible for immunosurveillance within the tumor microenvironment. This paradigm accords with models of immune dysregulation, such as autoimmunity and infectious disease, which have defined a pathophysiological role for abnormal complement signaling. Several types of immune cells express the cognate receptors for the complement anaphylatoxins, C3aR and C5aR, and demonstrate functional modulation in response to complement stimulation. In turn, impairment of antitumor immunity has been intimately tied to tumor progression in animal models of cancer. In this article, the literature was systematically reviewed to identify studies that have characterized the effects of the complement anaphylatoxins on the composition and function of immune cells within the tumor microenvironment. The search identified six studies based upon models of lymphoma and ovarian, cervical, lung, breast, and mammary cancer, which collectively support the paradigm of complement as an immune regulator in the tumor microenvironment. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Complement activation promotes colitis-associated carcinogenesis through activating intestinal IL-1β/IL-17A axis.

PubMed

Ning, C; Li, Y-Y; Wang, Y; Han, G-C; Wang, R-X; Xiao, H; Li, X-Y; Hou, C-M; Ma, Y-F; Sheng, D-S; Shen, B-F; Feng, J-N; Guo, R-F; Li, Y; Chen, G-J

2015-11-01

Colitis-associated colorectal cancer (CAC) is the most serious complication of inflammatory bowel disease (IBD). Excessive complement activation has been shown to be involved in the pathogenesis of IBD. However, its role in the development of CAC is largely unknown. Here, using a CAC model induced by combined administration of azoxymethane (AOM) and dextran sulfate sodium (DSS), we demonstrated that complement activation was required for CAC pathogenesis. Deficiency in key components of complement (e.g., C3, C5, or C5a receptor) rendered tumor repression in mice subjected to AOM/DSS. Mechanistic investigation revealed that complement ablation dramatically reduced proinflammatory cytokine interleukin (IL)-1β levels in the colonic tissues that was mainly produced by infiltrating neutrophils. IL-1β promoted colon carcinogenesis by eliciting IL-17 response in intestinal myeloid cells. Furthermore, complement-activation product C5a represented a potent inducer for IL-1β in neutrophil, accounting for downregulation of IL-1β levels in the employed complement-deficient mice. Overall, our study proposes a protumorigenic role of complement in inflammation-related colorectal cancer and that the therapeutic strategies targeting complement may be beneficial for the treatment of CAC in clinic.

*C5a/CD88 signaling alters blood-brain barrier integrity in lupus through NFκb

PubMed Central

Jacob, Alexander; Hack, Bradley; Chen, Peili; Quigg, Richard J.; Alexander, Jessy J.

2011-01-01

Inflammation is a key factor in a number of neurodegenerative diseases including systemic lupus erythematosus (SLE). The complement system is an important mechanism in initiating and amplifying inflammation. Our recent studies demonstrate that C5a, a protein fragment generated during complement activation could alter the blood-brain barrier (BBB) integrity, and thereby disturb the brain microenvironment. To understand the mechanism by which this occurs, we examined the effects of C5a on apoptosis, translocation of nuclear factor-κB (NFκb) and the expression of Iκbα, MAPK, CREB and TJ protein, zona occludens (ZO-1) in mouse brain endothelial cells. Apoptosis was examined by DNA laddering and caspase-3 activity and the distribution of the ZO-1 and the p65 subunit of NFκB were determined by immunofluorescence. Inhibition of CD88 reduced translocation of NFκb into the nucleus, altered ZO-1 at the interfaces of neighboring cells, decreased caspase-3 activity and prevented apoptosis in these cells. Our results indicate that signaling through CD88 regulates the BBB in a NFκb dependent manner. These studies suggest that the C5a receptor, CD88 is a promising therapeutic target that will reduce NFκb signaling cascades in inflammatory settings. PMID:21929539

Blood SC5b-9 complement levels increase at parturition during term and preterm labor.

PubMed

Segura-Cervantes, Enrique; Mancilla-Ramirez, Javier; Zurita, Luis; Paredes, Yuriria; Arredondo, José Luis; Galindo-Sevilla, Norma

2015-06-01

We explored the hypothesis that complement, an innate and adaptive immune effector, is active in the plasma of parturient women and is deposited on fetal membranes collected after delivery. A cross-sectional study was designed to evaluate complement activity at parturition. Pregnant women (n = 97) between 15 and 41 years of age were enrolled in a hospital protocol during the perinatal period to assess both SC5b-9 complement activity in blood and complement deposition on fetal membranes during parturition. Soluble SC5b-9 complement activity in plasma fractions was measured using a standard enzyme-linked immunosorbent assay (ELISA) that included specific anti-complement antibodies. Complement deposition on membranes was analyzed using immuno-dot blots and immunohistochemistry. Soluble SC5b-9 complement complex levels were increased in the plasma of women during term labor (TL; median 3361; range 1726-5670 ng/mL), preterm labor (PL; median 2958; range 1552-7092 ng/mL), and preterm premature rupture of membranes (PPROM; median 2272; range 167-6540 ng/mL) compared with pregnant women who were not in labor (P; median 1384; range 174-4570 ng/mL; P < 0.001, Kruskal-Wallis test). Active complement, as assessed by the C9 neo-antigen in C5b-9 complexes, was deposited on fetal membranes, with no difference between term and preterm delivery. The deposition of active complement on fetal membranes was confirmed by immunohistochemistry. Women who underwent non-labor-indicated Cesarean sections did not exhibit complement deposition. Soluble SC5b-9 complement complex levels increased in the plasma of women during parturition, and complement C5b-9 complexes were deposited on fetal membranes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Complement C3 and C5 play critical roles in traumatic brain cryoinjury: blocking effects on neutrophil extravasation by C5a receptor antagonist☆

PubMed Central

Sewell, Diane L.; Nacewicz, Brendon; Liu, Frances; Macvilay, Sinarack; Erdei, Anna; Lambris, John D.; Sandor, Matyas; Fabry, Zsuzsa

2016-01-01

The role of complement components in traumatic brain injury is poorly understood. Here we show that secondary damage after acute cryoinjury is significantly reduced in C3−/− or C5−/− mice or in mice treated with C5a receptor antagonist peptides. Injury sizes and neutrophil extravasation were compared. While neutrophil density increased following traumatic brain injury in wild type (C57BL/6) mice, C3-deficient mice demonstrated lower neutrophil extravasation and injury sizes in the brain. RNase protection assay indicated that C3 contributes to the induction of brain inflammatory mediators, MIF, RANTES (CCL5) and MCP-1 (CCL2). Intracranial C3 injection induced neutrophil extravasation in injured brains of C3−/− mice suggesting locally produced C3 is important in brain inflammation. We show that neutrophil extravasation is significantly reduced in both C5−/− mice and C5a receptor antagonist treated cryoinjured mice suggesting that one of the possible mechanisms of C3 effect on neutrophil extravasation is mediated via downstream complement activation products such as C5a. Our data indicates that complement inhibitors may ameliorate traumatic brain injury. PMID:15342196

Role of Complement Activation in a Model of Adult Respiratory Distress Syndrome

PubMed Central

Hosea, Stephen; Brown, Eric; Hammer, Carl; Frank, Michael

1980-01-01

The adult respiratory distress syndrome is characterized by arterial hypoxemia as a result of increased alveolar capillary permeability to serum proteins in the setting of normal capillary hydrostatic pressures. Because bacterial sepsis is prominent among the various diverse conditions associated with altered alveolar capillary permeability, we studied the effect of bacteremia with attendant complement activation on the sequestration of microorganisms and the leakage of albumin in the lungs of guinea pigs. Pneumococci were injected intravenously into guinea pigs and their localization was studied. Unlike normal guinea pigs, complement-depleted guinea pigs did not localize injected bacteria to the lungs. Preopsonization of organisms did not correct this defect in pulmonary localization of bacteria in complement-depleted animals, suggesting that a fluid-phase component of complement activation was required. Genetically C5-deficient mice showed no pulmonary localization of bacteria. C5-sufficient mice demonstrated the usual pulmonary localization, thus further suggesting that the activation of C5 might be important in this localization. The infusion of activated C5 increased alveolar capillary permeability to serum proteins as assayed by the amount of radioactive albumin sequestered in the lung. Neutropenic animals did not develop altered capillary permeability after challenge with activated C5. Thus, complement activation through C5, in the presence of neutrophils, induces alterations in pulmonary alveolar capillary permeability and causes localization of bacteria to the pulmonary parenchyma. Complement activation in other disease states could potentially result in similar clinical manifestations. PMID:7400321

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

2017-05-01

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. Copyright © 2017 by The American Association of Immunologists, Inc.

Identification of C3b-binding Small Molecule Complement Inhibitors Using Cheminformatics

PubMed Central

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

2017-01-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of over two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology which include 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 which 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 which 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. PMID:28298523

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface

PubMed Central

Hovingh, Elise S.; Kuipers, Betsy; Pinelli, Elena; Rooijakkers, Suzan H. M.

2017-01-01

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis. PMID:28742139

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface.

PubMed

Hovingh, Elise S; van den Broek, Bryan; Kuipers, Betsy; Pinelli, Elena; Rooijakkers, Suzan H M; Jongerius, Ilse

2017-07-01

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.

Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid Correlate with Clinical Measures of Disease.

PubMed

Sass, Laura A; Hair, Pamela S; Perkins, Amy M; Shah, Tushar A; Krishna, Neel K; Cunnion, Kenji M

2015-01-01

In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, and inflammation. Here we explored complement inflammatory effectors in CF lung fluid. In this study soluble fractions (sols) from sputum samples of 15 CF patients were assayed for complement effectors and analyzed with clinical measurements. The pro-inflammatory peptide C5a was increased 4.8-fold (P = 0.04) in CF sols compared with controls. Incubation of CF sols with P. aeruginosa or S. aureus increased C5a concentration 2.3-fold (P = 0.02). A peptide inhibitor of complement C1 (PIC1) completely blocked the increase in C5a concentration from P. aeruginosa in CF sol in vitro (P = 0.001). C5a concentration in CF sol correlated inversely with body mass index (BMI) percentile in children (r = -0.77, P = 0.04). C3a, which has anti-inflammatory effects, correlated positively with FEV1% predicted (rs = 0.63, P = 0.02). These results suggest that complement effectors may significantly impact inflammation in CF lung fluid.

Relative Contribution of Cellular Complement Inhibitors CD59, CD46, and CD55 to Parainfluenza Virus 5 Inhibition of Complement-Mediated Neutralization

PubMed Central

Li, Yujia; Parks, Griffith D.

2018-01-01

The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA) to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5) incorporates CD55 and CD46 to delay complement-mediated neutralization. In this paper, we tested the role of a third individual RCA inhibitor CD59 in PIV5 interactions with complement pathways. Using a cell line engineered to express CD59, we show that small levels of functional CD59 are associated with progeny PIV5, which is capable of blocking assembly of the C5b-C9 membrane attack complex (MAC). PIV5 containing CD59 (PIV5-CD59) showed increased resistance to complement-mediated neutralization in vitro comparing to PIV5 lacking regulators. Infection of A549 cells with PIV5 and RSV upregulated CD59 expression. TGF-beta treatment of PIV5-infected cells also increased cell surface CD59 expression and progeny virions were more resistant to complement-mediated neutralization. A comparison of individual viruses containing only CD55, CD46, or CD59 showed a potency of inhibiting complement-mediated neutralization, which followed a pattern of CD55 > CD46 > CD59. PMID:29693588

21 CFR 866.5240 - Complement components immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

.... 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids, and tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these...

21 CFR 866.5240 - Complement components immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

.... 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids, and tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these...

21 CFR 866.5240 - Complement components immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

.... 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids, and tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these...

21 CFR 866.5240 - Complement components immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

.... 866.5240 Section 866.5240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... complement components C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, and C9, in serum, other body fluids, and tissues. Complement is a group of serum proteins which destroy infectious agents. Measurements of these...

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

PubMed

Banadakoppa, M; Chauhan, M S; Havemann, D; Balakrishnan, M; Dominic, J S; Yallampalli, C

2014-09-01

Spontaneous abortion in early pregnancy due to unknown reasons is a common problem. The excess complement activation and consequent placental inflammation and anti-angiogenic milieu is emerging as an important associated factor in many pregnancy-related complications. In the present study we sought to examine the expression of complement inhibitory proteins at the feto-maternal interface and levels of complement split products in the circulation to understand their role in spontaneous abortion. Consenting pregnant women who either underwent elective abortion due to non-clinical reasons (n = 13) or suffered miscarriage (n = 14) were recruited for the study. Systemic levels of complement factors C3a and C5a were measured by enzyme-linked immunosorbent assay (ELISA). Plasma C5 and C3 protein levels were examined by Western blot. Expressions of complement regulatory proteins such as CD46 and CD55 in the decidua were investigated by quantitative polymerase chain reaction (PCR) and Western blot. The median of plasma C3a level was 82·83 ng/ml and 66·17 ng/ml in elective and spontaneous abortion patients, respectively. Medians of plasma C5a levels in elective and spontaneous abortion patients were 0·96 ng/ml and 1·14 ng/ml, respectively. Only plasma C5a levels but not C3a levels showed significant elevation in spontaneous abortion patients compared to elective abortion patients. Further, there was a threefold decrease in the mRNA expressions of complement inhibitory proteins CD46 and CD55 in the decidua obtained from spontaneous abortion patients compared to that of elective abortion patients. These data suggested that dysregulated complement cascade may be associated with spontaneous abortion. © 2014 British Society for Immunology.

Differential Effects of Complement Activation Products C3a and C5a on Cardiovascular Function in Hypertensive Pregnant Rats

PubMed Central

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.

2014-01-01

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 (N2-[(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-inflammatory approach. PMID:25150279

Endotoxin-induced shock in the rat. A role for C5a.

PubMed Central

Smedegård, G.; Cui, L. X.; Hugli, T. E.

1989-01-01

Administration of endotoxin from gram-negative bacteria to rats results in systemic hypotension, an increased hematocrit, and decreased numbers of circulating leukocytes (polymorphonuclear), monocytes, and platelets. These potentially lethal physiologic changes may be partially attributed to complement activation and generation of anaphylatoxins by the endotoxin (LPS). We demonstrated an elevation in the plasma levels of both C3a and C5a in LPS-treated rats. Injection of 5 micrograms C5ades Arg (rat) into rats produced effects similar to those induced by LPS, including decreased mean arterial pressure (systemic hypotension) and decreased numbers of circulating polymorphonuclear leukocytes, monocytes, and platelets. Unlike the response to LPS, C5a did not increase the hematocrit, indicating little effect on vascular permeability at the doses used. When LPS-treated animals were pretreated with F(ab')2 fragments of rabbit anti-rat C5a, no changes were measured in the circulating cell counts compared with LPS alone; however a significant improvement in the mean arterial pressure and a decrease in hematocrit was observed. We conclude that LPS-induced (septic) shock in the rat may result, in part, from the effects of complement activation and particularly from the generation of C5a. The influence of C5a on the LPS effect in the rat appears to enhance both the hypotensive (mean arterial pressure) and vascular permeability (hematocrit) responses. These results appear to support and confirm earlier observations that anti-human C5a increased survival in a septic-shock monkey model by eliminating circulating C5a and presumably thereby reducing the effects of endotoxin on blood pressure. Our results demonstrate that C5a plays a significant role in the hemodynamic changes associated with endotoxin-induced shock. Neutralization of C5a with specific antibodies may reduce the hypotensive response to endotoxin sufficiently to prevent lethal septic shock both in animals and in man. PMID:2789475

Complement Inhibition Alleviates Paraquat-Induced Acute Lung Injury

PubMed Central

Sun, Shihui; Wang, Hanbin; Zhao, Guangyu; An, Yingbo; Guo, Yan; Du, Lanying; Song, Hongbin; Qiao, Fei; Yu, Hong; Wu, Xiaohong; Atkinson, Carl; Jiang, Shibo; Tomlinson, Stephen

2011-01-01

The widely used herbicide, paraquat (PQ), is highly toxic and claims thousands of lives from both accidental and voluntary ingestion. The pathological mechanisms of PQ poisoning–induced acute lung injury (ALI) are not well understood, and the role of complement in PQ-induced ALI has not been elucidated. We developed and characterized a mouse model of PQ-induced ALI and studied the role of complement in the pathogenesis of PQ poisoning. Intraperitoneal administration of PQ caused dose- and time-dependent lung damage and mortality, with associated inflammatory response. Within 24 hours of PQ-induced ALI, there was significantly increased expression of the complement proteins, C1q and C3, in the lung. Expression of the anaphylatoxin receptors, C3aR and C5aR, was also increased. Compared with wild-type mice, C3-deficient mice survived significantly longer and displayed significantly reduced lung inflammation and pathology after PQ treatment. Similar reductions in PQ-induced inflammation, pathology, and mortality were recorded in mice treated with the C3 inhibitors, CR2-Crry, and alternative pathway specific CR2-fH. A similar therapeutic effect was also observed by treatment with either C3a receptor antagonist or a blocking C5a receptor monoclonal antibody. Together, these studies indicate that PQ-induced ALI is mediated through receptor signaling by the C3a and C5a complement activation products that are generated via the alternative complement pathway, and that complement inhibition may be an effective clinical intervention for postexposure treatment of PQ-induced ALI. PMID:21421909

C4B gene influences intestinal microbiota through complement activation in patients with paediatric-onset inflammatory bowel disease.

PubMed

Nissilä, E; Korpela, K; Lokki, A I; Paakkanen, R; Jokiranta, S; de Vos, W M; Lokki, M-L; Kolho, K-L; Meri, S

2017-12-01

Complement C4 genes are linked to paediatric inflammatory bowel disease (PIBD), but the mechanisms have remained unclear. We examined the influence of C4B gene number on intestinal microbiota and in-vitro serum complement activation by intestinal microbes in PIBD patients. Complement C4A and C4B gene numbers were determined by genomic reverse transcription-polymerase chain reaction (RT-PCR) from 64 patients with PIBD (Crohn's disease or ulcerative colitis). The severity of the disease course was determined from faecal calprotectin levels. Intestinal microbiota was assessed using the HITChip microarray. Complement reactivity in patients was analysed by incubating their sera with Yersinia pseudotuberculosis and Akkermansia muciniphila and determining the levels of C3a and soluble terminal complement complex (SC5b-9) using enzyme immunoassays. The microbiota diversity was wider in patients with no C4B genes than in those with one or two C4B genes, irrespective of intestinal inflammation. C4B and total C4 gene numbers correlated positively with soluble terminal complement complex (TCC, SC5b-9) levels when patient serum samples were stimulated with bacteria. Our results suggest that the C4B gene number associates positively with inflammation in patients with PIBD. Multiple copies of the C4B gene may thus aggravate the IBD-associated dysbiosis through escalated complement reactivity towards the microbiota. © 2017 British Society for Immunology.

Defining the Complement Biomarker Profile of C3 Glomerulopathy

PubMed Central

Zhang, Yuzhou; Nester, Carla M.; Martin, Bertha; Skjoedt, Mikkel-Ole; Meyer, Nicole C.; Shao, Dingwu; Borsa, Nicolò; Palarasah, Yaseelan

2014-01-01

Background and objectives C3 glomerulopathy (C3G) applies to a group of renal diseases defined by a specific renal biopsy finding: a dominant pattern of C3 fragment deposition on immunofluorescence. The primary pathogenic mechanism involves abnormal control of the alternative complement pathway, although a full description of the disease spectrum remains to be determined. This study sought to validate and define the association of complement dysregulation with C3G and to determine whether specific complement pathway abnormalities could inform disease definition. Design, setting, participants, & measurements This study included 34 patients with C3G (17 with C3 glomerulonephritis [C3GN] and 17 with dense deposit disease [DDD]) diagnosed between 2008 and 2013 selected from the C3G Registry. Control samples (n=100) were recruited from regional blood drives. Nineteen complement biomarkers were assayed on all samples. Results were compared between C3G disease categories and with normal controls. Results Assessment of the alternative complement pathway showed that compared with controls, patients with C3G had lower levels of serum C3 (P<0.001 for both DDD and C3GN) and factor B (P<0.001 for both DDD and C3GN) as well as higher levels of complement breakdown products including C3d (P<0.001 for both DDD and C3GN) and Bb (P<0.001 for both DDD and C3GN). A comparison of terminal complement pathway proteins showed that although C5 levels were significantly suppressed (P<0.001 for both DDD and C3GN) its breakdown product C5a was significantly higher only in patients with C3GN (P<0.05). Of the other terminal pathway components (C6–C9), the only significant difference was in C7 levels between patients with C3GN and controls (P<0.01). Soluble C5b-9 was elevated in both diseases but only the difference between patients with C3GN and controls reached statistical significance (P<0.001). Levels of C3 nephritic factor activity were qualitatively higher in patients with DDD compared with patients with C3GN. Conclusions Complement biomarkers are significantly abnormal in patients with C3G compared with controls. These data substantiate the link between complement dysregulation and C3G and identify C3G interdisease differences. PMID:25341722

Alternative pathway regulation by factor H modulates Streptococcus pneumoniae induced proinflammatory cytokine responses by decreasing C5a receptor crosstalk.

PubMed

van der Maten, Erika; de Bont, Cynthia M; de Groot, Ronald; de Jonge, Marien I; Langereis, Jeroen D; van der Flier, Michiel

2016-12-01

Bacterial pathogens not only stimulate innate immune receptors, but also activate the complement system. Crosstalk between complement C5a receptor (C5aR) and other innate immune receptors is known to enhance the proinflammatory cytokine response. An important determinant of the magnitude of complement activation is the activity of the alternative pathway, which serves as an amplification mechanism for complement activation. Both alternative pathway activity as well as plasma levels of factor H, a key inhibitor of the alternative pathway, show large variation within the human population. Here, we studied the effect of factor H-mediated regulation of the alternative pathway on bacterial-induced proinflammatory cytokine responses. We used the human pathogen Streptococcus pneumoniae as a model stimulus to induce proinflammatory cytokine responses in human peripheral blood mononuclear cells. Serum containing active complement enhanced pneumococcal induced proinflammatory cytokine production through C5a release and C5aR crosstalk. We found that inhibition of the alternative pathway by factor H, with a concentration equivalent to a high physiological level, strongly reduced C5a levels and decreased proinflammatory cytokine production in human peripheral blood mononuclear cells. This suggests that variation in alternative pathway activity due to variation in factor H plasma levels affects individual cytokine responses during infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

Complement inhibition decreases early fibrogenic events in the lung of septic baboons.

PubMed

Silasi-Mansat, Robert; Zhu, Hua; Georgescu, Constantin; Popescu, Narcis; Keshari, Ravi S; Peer, Glenn; Lupu, Cristina; Taylor, Fletcher B; Pereira, Heloise Anne; Kinasewitz, Gary; Lambris, John D; Lupu, Florea

2015-11-01

Acute respiratory distress syndrome (ARDS) induced by severe sepsis can trigger persistent inflammation and fibrosis. We have shown that experimental sepsis in baboons recapitulates ARDS progression in humans, including chronic inflammation and long-lasting fibrosis in the lung. Complement activation products may contribute to the fibroproliferative response, suggesting that complement inhibitors are potential therapeutic agents. We have been suggested that treatment of septic baboons with compstatin, a C3 convertase inhibitor protects against ARDS-induced fibroproliferation. Baboons challenged with 10(9) cfu/kg (LD50) live E. coli by intravenous infusion were treated or not with compstatin at the time of challenge or 5 hrs thereafter. Changes in the fibroproliferative response at 24 hrs post-challenge were analysed at both transcript and protein levels. Gene expression analysis showed that sepsis induced fibrotic responses in the lung as early as 24 hrs post-bacterial challenge. Immunochemical and biochemical analysis revealed enhanced collagen synthesis, induction of profibrotic factors and increased cell recruitment and proliferation. Specific inhibition of complement with compstatin down-regulated sepsis-induced fibrosis genes, including transforming growth factor-beta (TGF-β), connective tissue growth factor (CTGF), tissue inhibitor of metalloproteinase 1 (TIMP1), various collagens and chemokines responsible for fibrocyte recruitment (e.g. chemokine (C-C motif) ligand 2 (CCL2) and 12 (CCL12)). Compstatin decreased the accumulation of myofibroblasts and proliferating cells, reduced the production of fibrosis mediators (TGF-β, phospho-Smad-2 and CTGF) and inhibited collagen deposition. Our data demonstrate that complement inhibition effectively attenuates collagen deposition and fibrotic responses in the lung after severe sepsis. Inhibiting complement could prove an attractive strategy for preventing sepsis-induced fibrosis of the lung. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

C3aR and C5aR1 act as key regulators of human and mouse β-cell function.

PubMed

Atanes, Patricio; Ruz-Maldonado, Inmaculada; Pingitore, Attilio; Hawkes, Ross; Liu, Bo; Zhao, Min; Huang, Guo Cai; Persaud, Shanta J; Amisten, Stefan

2018-02-01

Complement components 3 and 5 (C3 and C5) play essential roles in the complement system, generating C3a and C5a peptides that are best known as chemotactic and inflammatory factors. In this study we characterised islet expression of C3 and C5 complement components, and the impact of C3aR and C5aR1 activation on islet function and viability. Human and mouse islet mRNAs encoding key elements of the complement system were quantified by qPCR and distribution of C3 and C5 proteins was determined by immunohistochemistry. Activation of C3aR and C5aR1 was determined using DiscoverX beta-arrestin assays. Insulin secretion from human and mouse islets was measured by radioimmunoassay, and intracellular calcium ([Ca 2+ ]i), ATP generation and apoptosis were assessed by standard techniques. C3 and C5 proteins and C3aR and C5aR1 were expressed by human and mouse islets, and C3 and C5 were mainly localised to β- and α-cells. Conditioned media from islets exposed for 1 h to 5.5 and 20 mM glucose stimulated C3aR and C5aR1-driven beta-arrestin recruitment. Activation of C3aR and C5aR1 potentiated glucose-induced insulin secretion from human and mouse islets, increased [Ca 2+ ]i and ATP generation, and protected islets against apoptosis induced by a pro-apoptotic cytokine cocktail or palmitate. Our observations demonstrate a functional link between activation of components of the innate immune system and improved β-cell function, suggesting that low-level chronic inflammation may improve glucose homeostasis through direct effects on β-cells.

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.

Upregulation of CD59

PubMed Central

Griesemer, Adam D.; Okumi, Masayoshi; Shimizu, Akira; Moran, Shannon; Ishikawa, Yoshinori; Iorio, Justin; Arn, J. Scott; Yamada, Kazuhiko

2009-01-01

Background Survival of ABO-mismatched kidneys with stable renal function despite the persistence of anti-ABO antibodies is called accommodation. The mechanism of accommodation is unclear, but may involve complement regulatory proteins such as CD59. The development of alpha-1,3-Galactosyltransferase knock-out (GalT-KO) swine that produce anti-Gal antibodies provides a large animal model capable of determining the role of complement regulatory proteins in accommodation. Methods ELISA and antibody FACS were used to examine the rate of anti-Gal antibody expression as a function of age. MHC-matched kidneys were transplanted from Gal-positive MGH miniature swine to MGH GalT-KO swine with systemic immunosuppression. One recipient underwent adsorbtion of anti-Gal antibodies prior to transplantation. Graft survival, antibody and complement deposition patterns and CD59 expression were determined. Results Three animals rejected Gal-positive kidneys via humoral mechanisms. One animal with low titers of anti-Gal Ab displayed spontaneous accommodation and the animal that was treated with Ab adsorbtion also displayed accommodation. Rejected grafts had deposition of IgM, IgG, C3 and C5b-9 with low expression of CD59, while accommodated grafts had low deposition of C5b-9 and high expression of CD59. Re-transplantation of one accommodated graft to a naïve GalT-KO animal confirmed that changes in the graft were responsible for the lack of C5b-9 deposition. Conclusion GalT-KO miniature swine produce anti-Gal antibodies and titers increase with age. These anti-Gal antibodies can cause rejection of MHC matched kidneys unless accommodation occurs. CD59 upregulation appears to be involved in the mechanism of accommodation by preventing the formation of the MAC on the accommodated graft. PMID:19424030

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

Identification of a central role for complement in osteoarthritis

PubMed Central

Wang, Qian; Rozelle, Andrew L.; Lepus, Christin M.; Scanzello, Carla R.; Song, Jason J.; Larsen, D. Meegan; Crish, James F.; Bebek, Gurkan; Ritter, Susan Y.; Lindstrom, Tamsin M.; Hwang, Inyong; Wong, Heidi H.; Punzi, Leonardo; Encarnacion, Angelo; Shamloo, Mehrdad; Goodman, Stuart B.; Wyss-Coray, Tony; Goldring, Steven R.; Banda, Nirmal K.; Thurman, Joshua M.; Gobezie, Reuben; Crow, Mary K.; Holers, V. Michael; Lee, David M.; Robinson, William H.

2011-01-01

Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of “wear and tear”1. Although low-grade inflammation is detected in osteoarthritis, its role is unclear2–4. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in C5, C6, or CD59a, we show that complement, and specifically the membrane attack complex (MAC)-mediated arm of complement, is critical to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Furthermore, MAC co-localized with matrix metalloprotease (MMP)-13 and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis. PMID:22057346

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.

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

Prediction of inflammatory responses induced by biomaterials in contact with human blood using protein fingerprint from plasma.

PubMed

Engberg, Anna E; Nilsson, Per H; Huang, Shan; Fromell, Karin; Hamad, Osama A; Mollnes, Tom Eirik; Rosengren-Holmberg, Jenny P; Sandholm, Kerstin; Teramura, Yuji; Nicholls, Ian A; Nilsson, Bo; Ekdahl, Kristina N

2015-01-01

Inappropriate complement activation is often responsible for incompatibility reactions that occur when biomaterials are used. Complement activation is therefore a criterion included in legislation regarding biomaterials testing. However, no consensus is yet available regarding appropriate complement-activation-related test parameters. We examined protein adsorption in plasma and complement activation/cytokine release in whole blood incubated with well-characterized polymers. Strong correlations were found between the ratio of C4 to its inhibitor C4BP and generation of 10 (mainly pro-inflammatory) cytokines, including IL-17, IFN-γ, and IL-6. The levels of complement activation products correlated weakly (C3a) or not at all (C5a, sC5b-9), confirming their poor predictive values. We have demonstrated a direct correlation between downstream biological effects and the proteins initially adhering to an artificial surface after contact with blood. Consequently, we propose the C4/C4BP ratio as a robust, predictor of biocompatibility with superior specificity and sensitivity over the current gold standard. Copyright © 2014 Elsevier Ltd. All rights reserved.

Complement system biomarkers in epilepsy.

PubMed

Kopczynska, Maja; Zelek, Wioleta M; Vespa, Simone; Touchard, Samuel; Wardle, Mark; Loveless, Samantha; Thomas, Rhys H; Hamandi, Khalid; Morgan, B Paul

2018-05-24

To explore whether complement dysregulation occurs in a routinely recruited clinical cohort of epilepsy patients, and whether complement biomarkers have potential to be used as markers of disease severity and seizure control. Plasma samples from 157 epilepsy cases (106 with focal seizures, 46 generalised seizures, 5 unclassified) and 54 controls were analysed. Concentrations of 10 complement analytes (C1q, C3, C4, factor B [FB], terminal complement complex [TCC], iC3b, factor H [FH], Clusterin [Clu], Properdin, C1 Inhibitor [C1Inh] plus C-reactive protein [CRP]) were measured using enzyme linked immunosorbent assay (ELISA). Univariate and multivariate statistical analysis were used to test whether combinations of complement analytes were predictive of epilepsy diagnoses and seizure occurrence. Correlation between number and type of anti-epileptic drugs (AED) and complement analytes was also performed. We found: CONCLUSION: This study adds to evidence implicating complement in pathogenesis of epilepsy and may allow the development of better therapeutics and prognostic markers in the future. Replication in a larger sample set is needed to validate the findings of the study. Copyright © 2018. Published by Elsevier Ltd.

Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin

PubMed Central

Foley, Jonathan H.; Walton, Bethany L.; Aleman, Maria M.; O'Byrne, Alice M.; Lei, Victor; Harrasser, Micaela; Foley, Kimberley A.; Wolberg, Alisa S.; Conway, Edward M.

2016-01-01

Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution. PMID:27077125

Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin

PubMed Central

Alcorlo, Martín; Tortajada, Agustín; Rodríguez de Córdoba, Santiago; Llorca, Oscar

2013-01-01

Complement is an essential component of innate immunity. Its activation results in the assembly of unstable protease complexes, denominated C3/C5 convertases, leading to inflammation and lysis. Regulatory proteins inactivate C3/C5 convertases on host surfaces to avoid collateral tissue damage. On pathogen surfaces, properdin stabilizes C3/C5 convertases to efficiently fight infection. How properdin performs this function is, however, unclear. Using electron microscopy we show that the N- and C-terminal ends of adjacent monomers in properdin oligomers conform a curly vertex that holds together the AP convertase, interacting with both the C345C and vWA domains of C3b and Bb, respectively. Properdin also promotes a large displacement of the TED (thioester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains of C3b, which likely impairs C3-convertase inactivation by regulatory proteins. The combined effect of molecular cross-linking and structural reorganization increases stability of the C3 convertase and facilitates recruitment of fluid-phase C3 convertase to the cell surfaces. Our model explains how properdin mediates the assembly of stabilized C3/C5-convertase clusters, which helps to localize complement amplification to pathogen surfaces. PMID:23901101

Mesophilic Aeromonas sp. serogroup O:11 resistance to complement-mediated killing.

PubMed Central

Merino, S; Rubires, X; Aguilar, A; Albertí, S; Hernandez-Allés, S; Benedí, V J; Tomas, J M

1996-01-01

The complement activation by and resistance to complement-mediated killing of Aeromonas sp. strains from serogroup O:11 were investigated by using different wild-type strains (with an S-layer characteristic of this serogroup) and their isogenic mutants characterized for their surface components (S-layer and lipopolysaccharide [LPS]). All of the Aeromonas sp. serogroup O:11 wild-type strains are unable to activate complement, which suggested that the S-layer completely covered the LPS molecules. We found that the classical complement pathway is involved in serum killing of susceptible Aeromonas sp. mutant strains of serogroup O11, while the alternative complement pathway seems not to be involved, and that the complement activation seems to be independent of antibody. The smooth mutant strains devoid of the S-layer (S-layer isogenic mutants) or isogenic LPS mutant strains with a complete or rather complete LPS core (also without the S-layer) are able to activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b is rapidly degraded, and therefore the lytic membrane attack complex (C5b-9) is not formed. Isogenic LPS rough mutants with an incomplete LPS core are serum sensitive because they bind more C3b than the resistant strains, the C3b is not completely degraded, and therefore the lytic complex (C5b-9) is formed. PMID:8945581

Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

PubMed Central

Seow, Vernon; Lim, Junxian; Cotterell, Adam J.; Yau, Mei-Kwan; Xu, Weijun; Lohman, Rink-Jan; Kok, W. Mei; Stoermer, Martin J.; Sweet, Matthew J.; Reid, Robert C.; Suen, Jacky Y.; Fairlie, David P.

2016-01-01

Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1–3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents. PMID:27094554

Receptor residence time trumps drug-likeness and oral bioavailability in determining efficacy of complement C5a antagonists

NASA Astrophysics Data System (ADS)

Seow, Vernon; Lim, Junxian; Cotterell, Adam J.; Yau, Mei-Kwan; Xu, Weijun; Lohman, Rink-Jan; Kok, W. Mei; Stoermer, Martin J.; Sweet, Matthew J.; Reid, Robert C.; Suen, Jacky Y.; Fairlie, David P.

2016-04-01

Drug discovery and translation are normally based on optimizing efficacy by increasing receptor affinity, functional potency, drug-likeness (rule-of-five compliance) and oral bioavailability. Here we demonstrate that residence time of a compound on its receptor has an overriding influence on efficacy, exemplified for antagonists of inflammatory protein complement C5a that activates immune cells and promotes disease. Three equipotent antagonists (3D53, W54011, JJ47) of inflammatory responses to C5a (3nM) were compared for drug-likeness, receptor affinity and antagonist potency in human macrophages, and anti-inflammatory efficacy in rats. Only the least drug-like antagonist (3D53) maintained potency in cells against higher C5a concentrations and had a much longer duration of action (t1/2 ~ 20 h) than W54011 or JJ47 (t1/2 ~ 1-3 h) in inhibiting macrophage responses. The unusually long residence time of 3D53 on its receptor was mechanistically probed by molecular dynamics simulations, which revealed long-lasting interactions that trap the antagonist within the receptor. Despite negligible oral bioavailability, 3D53 was much more orally efficacious than W54011 or JJ47 in preventing repeated agonist insults to induce rat paw oedema over 24 h. Thus, residence time on a receptor can trump drug-likeness in determining efficacy, even oral efficacy, of pharmacological agents.

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

Novel Mutations Causing C5 Deficiency in Three North-African Families.

PubMed

Colobran, Roger; Franco-Jarava, Clara; Martín-Nalda, Andrea; Baena, Neus; Gabau, Elisabeth; Padilla, Natàlia; de la Cruz, Xavier; Pujol-Borrell, Ricardo; Comas, David; Soler-Palacín, Pere; Hernández-González, Manuel

2016-05-01

The complement system plays a central role in defense to encapsulated bacteria through opsonization and membrane attack complex (MAC) dependent lysis. The three activation pathways (classical, lectin, and alternative) converge in the cleavage of C5, which initiates MAC formation and target lysis. C5 deficiency is associated to recurrent infections by Neisseria spp. In the present study, complement deficiency was suspected in three families of North-African origin after one episode of invasive meningitis due to a non-groupable and two uncommon Meningococcal serotypes (E29, Y). Activity of alternative and classical pathways of complement were markedly reduced and the measurement of terminal complement components revealed total C5 absence. C5 gene analysis revealed two novel mutations as causative of the deficiency: Family A propositus carried a homozygous deletion of two adenines in the exon 21 of C5 gene, resulting in a frameshift and a truncated protein (c.2607_2608del/p.Ser870ProfsX3 mutation). Families B and C probands carried the same homozygous deletion of three consecutive nucleotides (CAA) in exon 9 of the C5 gene, leading to the deletion of asparagine 320 (c.960_962del/p.Asn320del mutation). Family studies confirmed an autosomal recessive inheritance pattern. Although sharing the same geographical origin, families B and C were unrelated. This prompted us to investigate this mutation prevalence in a cohort of 768 North-African healthy individuals. We identified one heterozygous carrier of the p.Asn320del mutation (allelic frequency = 0.065 %), indicating that this mutation is present at low frequency in North-African population.

Autocrine Complement Inhibits IL10-Dependent T-Cell Mediated Antitumor Immunity to Promote Tumor Progression

PubMed Central

Wang, Yu; Sun, Sheng-Nan; Liu, Qing; Yu, Yang-Yang; Guo, Jian; Wang, Kun; Xing, Bao-Cai; Zheng, Qing-Feng; Campa, Michael J.; Patz, Edward F.; Li, Shi-You; He, You-Wen

2016-01-01

In contrast to its inhibitory effects on many cells, IL-10 activates CD8+ tumor infiltrating lymphocytes (TILs) and enhances their antitumor activity. However, CD8+ TILs do not routinely express IL-10 as autocrine complement C3 inhibits IL-10 production through complement receptors C3aR and C5aR. CD8+ TILs from C3-deficient mice, however, express IL-10 and exhibit enhanced effector function. C3-deficient mice are resistant to tumor development in a T cell- and IL-10-dependent manner; human TILs expanded with IL-2 plus IL-10 increase the killing of primary tumors in vitro compared to IL-2 treated TILs. Complement-mediated inhibition of antitumor immunity is independent of the PD-1/PD-L1 immune checkpoint pathway. Our findings suggest that complement receptors C3aR and C5aR expressed on CD8+ TILs represent a novel class of immune checkpoints that could be targeted for tumor immunotherapy. Moreover, incorporation of IL-10 in the expansion of TILs and in gene-engineered T cells for adoptive cell therapy enhances their antitumor efficacy. PMID:27297552

Functional anatomy of complement factor H.

PubMed

Makou, Elisavet; Herbert, Andrew P; Barlow, Paul N

2013-06-11

Factor H (FH) is a soluble regulator of the proteolytic cascade at the core of the evolutionarily ancient vertebrate complement system. Although FH consists of a single chain of similar protein modules, it has a demanding job description. Its chief role is to prevent complement-mediated injury to healthy host cells and tissues. This entails recognition of molecular patterns on host surfaces combined with control of one of nature's most dangerous examples of a positive-feedback loop. In this way, FH modulates, where and when needed, an amplification process that otherwise exponentially escalates the production of the pro-inflammatory, pro-phagocytic, and pro-cytolytic cleavage products of complement proteins C3 and C5. Mutations and single-nucleotide polymorphisms in the FH gene and autoantibodies against FH predispose individuals to diseases, including age-related macular degeneration, dense-deposit disease, and atypical hemolytic uremic syndrome. Moreover, deletions or variations of genes for FH-related proteins also influence the risk of disease. Numerous pathogens hijack FH and use it for self-defense. As reviewed herein, a molecular understanding of FH function is emerging. While its functional oligomeric status remains uncertain, progress has been achieved in characterizing its three-dimensional architecture and, to a lesser extent, its intermodular flexibility. Models are proposed, based on the reconciliation of older data with a wealth of recent evidence, in which a latent circulating form of FH is activated by its principal target, C3b tethered to a self-surface. Such models suggest hypotheses linking sequence variations to pathophysiology, but improved, more quantitative, functional assays and rigorous data analysis are required to test these ideas.

Homologous species restriction of the complement-mediated killing of nucleated cells.

PubMed Central

Yamamoto, H; Blaas, P; Nicholson-Weller, A; Hänsch, G M

1990-01-01

The homologous restriction of complement (C) lysis is attributed to membrane proteins: decay-accelerating factor (DAF), C8 binding protein (C8bp) and P18/CD59. Since these proteins are also expressed on peripheral blood cells, species restriction was tested for in the complement-mediated killing of antibody-coated human leucocytes by human or rabbit complement. Killing was more efficient when rabbit complement was used. Preincubation of cells with an antibody to DAF abolished the difference. When C1-7 sites were first attached to the cells and either rabbit or human C8, C9 were added, the killing of monocytes and lymphocytes was equally efficient; only in polymorphonuclear neutrophils was a higher efficiency of rabbit C8, C9 seen. Thus, in contrast to haemolysis, restriction occurred predominantly at the C3 level and the action of the terminal complement components was not inhibited. Since C8bp isolated from peripheral blood cells showed essentially similar characteristics as the erythrocyte-derived C8bp, the failure of C8bp to inhibit the action of the terminal components on nucleated cells might reflect differences of the complement membrane interactions between erythrocytes or nucleated cells, respectively. Images Figure 5 PMID:1697561

Complement Activation: An Emerging Player in the Pathogenesis of Cardiovascular Disease

PubMed Central

Carter, Angela M.

2012-01-01

A wealth of evidence indicates a fundamental role for inflammation in the pathogenesis of cardiovascular disease (CVD), contributing to the development and progression of atherosclerotic lesion formation, plaque rupture, and thrombosis. An increasing body of evidence supports a functional role for complement activation in the pathogenesis of CVD through pleiotropic effects on endothelial and haematopoietic cell function and haemostasis. Prospective and case control studies have reported strong relationships between several complement components and cardiovascular outcomes, and in vitro studies and animal models support a functional effect. Complement activation, in particular, generation of C5a and C5b-9, influences many processes involved in the development and progression of atherosclerosis, including promotion of endothelial cell activation, leukocyte infiltration into the extracellular matrix, stimulation of cytokine release from vascular smooth muscle cells, and promotion of plaque rupture. Complement activation also influences thrombosis, involving components of the mannose-binding lectin pathway, and C5b-9 in particular, through activation of platelets, promotion of fibrin formation, and impairment of fibrinolysis. The participation of the complement system in inflammation and thrombosis is consistent with the physiological role of the complement system as a rapid effector system conferring protection following vessel injury. However, in the context of CVD, these same processes contribute to development of atherosclerosis, plaque rupture, and thrombosis. PMID:24278688

Short periods of high temperature during meiosis prevent normal meiotic progression and reduce grain number in hexaploid wheat (Triticum aestivum L.).

PubMed

Draeger, Tracie; Moore, Graham

2017-09-01

Exposure of wheat to high temperatures during male meiosis prevents normal meiotic progression and reduces grain number. We define a temperature-sensitive period and link heat tolerance to chromosome 5D. This study assesses the effects of heat on meiotic progression and grain number in hexaploid wheat (Triticum aestivum L. var. Chinese Spring), defines a heat-sensitive stage and evaluates the role of chromosome 5D in heat tolerance. Plants were exposed to high temperatures (30 or 35 °C) in a controlled environment room for 20-h periods during meiosis and the premeiotic interphase just prior to meiosis. Examination of pollen mother cells (PMCs) from immature anthers immediately before and after heat treatment enabled precise identification of the developmental phases being exposed to heat. A temperature-sensitive period was defined, lasting from premeiotic interphase to late leptotene, during which heat can prevent PMCs from progressing through meiosis. PMCs exposed to 35 °C were less likely to progress than those exposed to 30 °C. Grain number per spike was reduced at 30 °C, and reduced even further at 35 °C. Chinese Spring nullisomic 5D-tetrasomic 5B (N5DT5B) plants, which lack chromosome 5D, were more susceptible to heat during premeiosis-leptotene than Chinese Spring plants with the normal (euploid) chromosome complement. The proportion of plants with PMCs progressing through meiosis after heat treatment was lower for N5DT5B plants than for euploids, but the difference was not significant. However, following exposure to 30 °C, in euploid plants grain number was reduced (though not significantly), whereas in N5DT5B plants the reduction was highly significant. After exposure to 35 °C, the reduction in grain number was highly significant for both genotypes. Implications of these findings for the breeding of thermotolerant wheat are discussed.

Early Complementopathy after Multiple Injuries in Humans

PubMed Central

Burk, Anne-Maud; Martin, Myriam; Flierl, Michael A.; Rittirsch, Daniel; Helm, Matthias; Lampl, Lorenz; Bruckner, Uwe; Stahl, Gregory L.; Blom, Anna M.; Perl, Mario; Gebhard, Florian; Huber-Lang, Markus

2012-01-01

After severe tissue injury, innate immunity mounts a robust systemic inflammatory response. However, little is known about the immediate impact of multiple trauma on early complement function in humans. In the present study we hypothesized that multiple trauma results in immediate activation, consumption and dysfunction of the complement cascade and that the resulting severe “complementopathy” may be associated with morbidity and mortality. Therefore a prospective multicenter study with 25 healthy volunteers and 40 polytrauma patients (mean injury severity score [ISS] = 30.3 ± 2.9) was performed. After polytrauma serum was collected as early as possible at the scene, upon admission to the emergency room and 4, 12, 24, 120 and 240 hours post trauma and analysed for the complement profile. Complement hemolytic activity (CH-50) was massively reduced within the first 24 h after injury, recovered only 5 days after trauma and discriminated between lethal and non-lethal 28-day outcome. Serum levels of the complement activation products C3a and C5a were significantly elevated throughout the entire observation period and correlated with the severity of traumatic brain injury and survival. The soluble terminal complement complex SC5b-9 and mannose-binding lectin (MBL) showed a biphasic response after trauma. Key fluid phase inhibitors of complement, such as C4b-binding protein (C4BP) and factor I, were significantly diminished early after trauma. The present data indicate an almost synchronically rapid activation and dysfunction of complement suggesting a trauma-induced “complementopathy” early after injury. These events may participate to the impairment of the innate immune response observed after severe trauma. PMID:22258234

On the value of therapeutic interventions targeting the complement system in acute myocardial infarction.

PubMed

Emmens, Reindert W; Wouters, Diana; Zeerleder, Sacha; van Ham, S Marieke; Niessen, Hans W M; Krijnen, Paul A J

2017-04-01

The complement system plays an important role in the inflammatory response subsequent to acute myocardial infarction (AMI). The aim of this study is to create a systematic overview of studies that have investigated therapeutic administration of complement inhibitors in both AMI animal models and human clinical trials. To enable extrapolation of observations from included animal studies toward post-AMI clinical trials, ex vivo studies on isolated hearts and proof-of-principle studies on inhibitor administration before experimental AMI induction were excluded. Positive therapeutic effects in AMI animal models have been described for cobra venom factor, soluble complement receptor 1, C1-esterase inhibitor (C1-inh), FUT-175, C1s-inhibitor, anti-C5, ADC-1004, clusterin, and glycosaminoglycans. Two types of complement inhibitors have been tested in clinical trials, being C1-inh and anti-C5. Pexelizumab (anti-C5) did not result in reproducible beneficial effects for AMI patients. Beneficial effects were reported in AMI patients for C1-inhibitor, albeit in small patient groups. In general, despite the absence of consistent positive effects in clinical trials thus far, the complement system remains a potentially interesting target for therapy in AMI patients. Based on the study designs of previous animal studies and clinical trials, we discuss several issues which require attention in the design of future studies: adjustment of clinical trial design to precise mechanism of action of administered inhibitor, optimizing the duration of therapy, and optimization of time point(s) on which therapeutic effects will be evaluated. Copyright © 2016 Elsevier Inc. All rights reserved.

Activation of the complement system in patients with porphyrias after irradiation in vivo.

PubMed Central

Lim, H W; Poh-Fitzpatrick, M B; Gigli, I

1984-01-01

Irradiation of the forearms of two patients with erythropoietic protoporphyria and one patient with porphyria cutanea tarda resulted in an in vivo activation of the complement system, as assessed by diminution of the hemolytic titers of the third component of complement by 23-57%, and of the fifth component of complement (C5) by 19-47%. Such treatment also generated chemotactic activity for human polymorphonuclear cells; the chemotactic activity was stable at 56 degrees C and antigenically related to human C5. On Sephadex G-75 chromatography the chemotactic activity eluted with an apparent molecular weight of 15,000. These in vivo results extend our previous in vitro observation of photoactivation of complement in sera from patients with erythropoietic protoporphyria and porphyria cutanea tarda, and suggest that the complement system may participate in the pathogenesis of cutaneous phototoxicity in these patients. PMID:6392339

Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

PubMed Central

Khattab, Ayman; Barroso, Marta; Miettinen, Tiera; Meri, Seppo

2015-01-01

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood. PMID:25679788

Sex matters: Systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components.

PubMed

Kotimaa, Juha; Klar-Mohammad, Ngaisah; Gueler, Faikah; Schilders, Geurt; Jansen, Aswin; Rutjes, Helma; Daha, Mohamed R; van Kooten, Cees

2016-08-01

Experimental mouse models have been extensively used to elucidate the role of the complement system in different diseases and injuries. Contribution of gender has revealed an intriguing gender specific difference; female mice often show protection against most complement driven injuries such as ischemia/reperfusion injury, graft rejection and sepsis. Interestingly, early studies to the mouse complement system revealed that female mice have very low total complement activity (CH50), which is related to androgen regulation of hepatic complement synthesis. Here, our aim was to understand at which level the female specific differences in mouse complement resides. We have used recently developed complement assays to study the functional activities of female and male mice at the level of C3 and C9 activation, and furthermore assayed key complement factor levels in serum of age-matched female and male C57BL/6 mice. Our results show that the female mice have normal complement cascade functionality at the level of C3 activation, which was supported by determinations of early complement factors. However, all pathways are strongly reduced at the level of C9 activation, suggesting a terminal pathway specific difference. This was in line with C6 and C9 measurements, showing strongly decreased levels in females. Furthermore, similar gender differences were also found in BALB/cJ mice, but not in CD-1 mice. Our results clearly demonstrate that the complement system in females of frequently used mouse strains is restricted by the terminal pathway components and that the perceived female specific protection against experimental disease and injury might be in part explained by the inability promote inflammation through C5b-9. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Classical and alternative complement activation on photoreceptor outer segments drives monocyte-dependent retinal atrophy.

PubMed

Katschke, Kenneth J; Xi, Hongkang; Cox, Christian; Truong, Tom; Malato, Yann; Lee, Wyne P; McKenzie, Brent; Arceo, Rommel; Tao, Jianhua; Rangell, Linda; Reichelt, Mike; Diehl, Lauri; Elstrott, Justin; Weimer, Robby M; Campagne, Menno van Lookeren

2018-05-09

Geographic atrophy (GA), the advanced form of dry age-related macular degeneration (AMD), is characterized by progressive loss of retinal pigment epithelium cells and photoreceptors in the setting of characteristic extracellular deposits and remains a serious unmet medical need. While genetic predisposition to AMD is dominated by polymorphisms in complement genes, it remains unclear how complement activation contributes to retinal atrophy. Here we demonstrate that complement is activated on photoreceptor outer segments (POS) in the retina peripheral to atrophic lesions associated with GA. When exposed to human serum following outer blood-retinal barrier breakdown, POS act as potent activators of the classical and alternative complement pathway. In mouse models of retinal degeneration, classical and alternative pathway complement activation on photoreceptors contributed to the loss of photoreceptor function. This was dependent on C5a-mediated recruitment of peripheral blood monocytes but independent of resident microglia. Genetic or pharmacologic inhibition of both classical and alternative complement C3 and C5 convertases was required to reduce progressive degeneration of photoreceptor rods and cones. Our study implicates systemic classical and alternative complement proteins and peripheral blood monocytes as critical effectors of localized retinal degeneration with potential relevance for the contribution of complement activation to GA.

Oral treatment with complement factor C5a receptor (CD88) antagonists inhibits experimental periodontitis in rats.

PubMed

Breivik, T; Gundersen, Y; Gjermo, P; Taylor, S M; Woodruff, T M; Opstad, P K

2011-12-01

The complement activation product 5a (C5a) is a potent mediator of the innate immune response to infection, and may thus also importantly determine the development of periodontitis. The present study was designed to explore the effect of several novel, potent and orally active C5a receptor (CD88) antagonists (C5aRAs) on the development of ligature-induced periodontitis in an animal model. Three different cyclic peptide C5aRAs, termed PMX205, PMX218 and PMX273, were investigated. Four groups of Wistar rats (n = 10 in each group) were used. Starting 3 d before induction of experimental periodontitis, rats either received one of the C5aRas (1-2 mg/kg) in the drinking water or received drinking water only. Periodontitis was assessed when the ligatures had been in place for 14 d. Compared with control rats, PMX205- and PMX218-treated rats had significantly reduced periodontal bone loss. The findings suggest that complement activation, and particularly C5a generation, may play a significant role in the development and progression of periodontitis. Blockade of the major C5a receptor, CD88, with specific inhibitors such as PMX205, may offer novel treatment options for periodontitis. © 2011 John Wiley & Sons A/S.

Complement C5a Receptor 1 Exacerbates the Pathophysiology of N. meningitidis Sepsis and Is a Potential Target for Disease Treatment

PubMed Central

Herrmann, Johannes B.; Muenstermann, Marcel; Strobel, Lea; Schubert-Unkmeir, Alexandra; Woodruff, Trent M.; Klos, Andreas

2018-01-01

ABSTRACT Sepsis caused by Neisseria meningitidis (meningococcus) is a rapidly progressing, life-threatening disease. Because its initial symptoms are rather unspecific, medical attention is often sought too late, i.e., when the systemic inflammatory response is already unleashed. This in turn limits the success of antibiotic treatment. The complement system is generally accepted as the most important innate immune determinant against invasive meningococcal disease since it protects the host through the bactericidal membrane attack complex. However, complement activation concomitantly liberates the C5a peptide, and it remains unclear whether this potent anaphylatoxin contributes to protection and/or drives the rapidly progressing immunopathogenesis associated with meningococcal disease. Here, we dissected the specific contribution of C5a receptor 1 (C5aR1), the canonical receptor for C5a, using a mouse model of meningococcal sepsis. Mice lacking C3 or C5 displayed susceptibility that was enhanced by >1,000-fold or 100-fold, respectively, consistent with the contribution of these components to protection. In clear contrast, C5ar1−/− mice resisted invasive meningococcal infection and cleared N. meningitidis more rapidly than wild-type (WT) animals. This favorable outcome stemmed from an ameliorated inflammatory cytokine response to N. meningitidis in C5ar1−/− mice in both in vivo and ex vivo whole-blood infections. In addition, inhibition of C5aR1 signaling without interference with the complement bactericidal activity reduced the inflammatory response also in human whole blood. Enticingly, pharmacologic C5aR1 blockade enhanced mouse survival and lowered meningococcal burden even when the treatment was administered after sepsis induction. Together, our findings demonstrate that C5aR1 drives the pathophysiology associated with meningococcal sepsis and provides a promising target for adjunctive therapy. PMID:29362231

Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C51

PubMed Central

Fukuoka, Yoshihiro; Xia, Han-Zhang; Sanchez-Muñoz, Laura B.; Dellinger, Anthony L.; Escribano, Luis; Schwartz, Lawrence B.

2009-01-01

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether β-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of β-tryptase, and the β-tryptase-activating polyanion. The B12 mAb was used to stabilize β-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric β-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by β-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by β-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both β-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by β-tryptase in releasates of activated skin mast cells. Of further biologic interest, β-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest β-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of β-tryptase and complement anaphylatoxins are detected. PMID:18424754

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA.

PubMed

Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-08-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab')2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. © 2015 The Authors. American Journal of Transplantation Published by Wiley Periodicals, Inc.

An Anti-C1s Monoclonal, TNT003, Inhibits Complement Activation Induced by Antibodies Against HLA

PubMed Central

Thomas, K A; Valenzuela, N M; Gjertson, D; Mulder, A; Fishbein, M C; Parry, G C; Panicker, S; Reed, E F

2015-01-01

Antibody-mediated rejection (AMR) of solid organ transplants (SOT) is characterized by damage triggered by donor-specific antibodies (DSA) binding donor Class I and II HLA (HLA-I and HLA-II) expressed on endothelial cells. While F(ab′)2 portions of DSA cause cellular activation and proliferation, Fc regions activate the classical complement cascade, resulting in complement deposition and leukocyte recruitment, both hallmark features of AMR. We characterized the ability of an anti-C1s monoclonal antibody, TNT003, to inhibit HLA antibody (HLA-Ab)-induced complement activation. Complement deposition induced by HLA-Ab was evaluated using novel cell- and bead-based assays. Human aortic endothelial cells (HAEC) were cultured with HLA-Ab and human complement; production of activated complement proteins was measured by flow cytometry. Additionally, C3d deposition was measured on single antigen beads (SAB) mixed with HLA-Ab and human complement. TNT003 inhibited HLA-Ab mediated complement deposition on HAEC in a concentration-dependent manner; C3a, C4a and C5a anaphylatoxin production was also diminished by TNT003. Finally, TNT003 blocked C3d deposition induced by Class I (HLAI-Ab)- and Class II (HLAII-Ab)-specific antibodies on SAB. These data suggest TNT003 may be useful for modulating the effects of DSA, as TNT003 inhibits complement deposition and split product formation generated by HLA-I/II-Ab in vitro. PMID:25904443

Increased cerebrospinal fluid complement C5 levels in major depressive disorder and schizophrenia.

PubMed

Ishii, Takashi; Hattori, Kotaro; Miyakawa, Tomoko; Watanabe, Kentaro; Hidese, Shinsuke; Sasayama, Daimei; Ota, Miho; Teraishi, Toshiya; Hori, Hiroaki; Yoshida, Sumiko; Nunomura, Akihiko; Nakagome, Kazuyuki; Kunugi, Hiroshi

2018-03-04

Inflammation has been implicated in a variety of psychiatric disorders. We aimed to determine whether levels of complement C5 protein in the cerebrospinal fluid (CSF), which may reflect activation of the complement system in the brain, are altered in patients with major psychiatric disorders. Additionally, we examined possible associations of CSF C5 levels with clinical variables. Subjects comprised 89 patients with major depressive disorder (MDD), 66 patients with bipolar disorder (BPD), 96 patients with schizophrenia, and 117 healthy controls, matched for age, sex, and ethnicity (Japanese). Diagnosis was made according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, criteria. CSF C5 levels were measured by enzyme-linked immunosorbent assay. CSF C5 levels were significantly increased in the patients with MDD (p < 0.001) and in the patients with schizophrenia (p = 0.001), compared with the healthy controls. The rate of individuals with an "abnormally high C5 level" (i.e., above the 95th percentile value of the control subjects) was significantly increased in all psychiatric groups, relative to the control group (all p < 0.01). Older age, male sex, and greater body mass index tended to associate with higher C5 levels. There was a significantly positive correlation between C5 levels and chlorpromazine-equivalent dose in the patients with schizophrenia. Thus, we found, for the first time, elevated C5 levels in the CSF of patients with major psychiatric disorders. Our results suggest that the activated complement system may contribute to neurological pathogenesis in a portion of patients with major psychiatric disorders. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

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

Plasma-derived Extracellular Vesicles Contain Predictive Biomarkers and Potential Therapeutic Targets for Myocardial Ischemic (MI) Injury*

PubMed Central

Cheow, Esther Sok Hwee; Cheng, Woo Chin; Lee, Chuen Neng; de Kleijn, Dominique; Sorokin, Vitaly; Sze, Siu Kwan

2016-01-01

Myocardial infarction (MI) triggers a potent inflammatory response via the release of circulatory mediators, including extracellular vesicles (EVs) by damaged cardiac cells, necessary for myocardial healing. Timely repression of inflammatory response are critical to prevent and minimize cardiac tissue injuries, nonetheless, progression in this aspect remains challenging. The ability of EVs to trigger a functional response upon delivery of carried bioactive cargos, have made them clinically attractive diagnostic biomarkers and vectors for therapeutic interventions. Using label-free quantitative proteomics approach, we compared the protein cargo of plasma EVs between patients with MI and from patients with stable angina (NMI). We report, for the first time, the proteomics profiling on 252 EV proteins that were modulated with >1.2-fold after MI. We identified six up-regulated biomarkers with potential for clinical applications; these reflected post-infarct pathways of complement activation (Complement C1q subcomponent subunit A (C1QA), 3.23-fold change, p = 0.012; Complement C5 (C5), 1.27-fold change, p = 0.087), lipoprotein metabolism (Apoliporotein D (APOD), 1.86-fold change, p = 0.033; Apolipoprotein C-III (APOCC3), 2.63-fold change, p = 0.029) and platelet activation (Platelet glycoprotein Ib alpha chain (GP1BA), 9.18-fold change, p < 0.0001; Platelet basic protein (PPBP), 4.72-fold change, p = 0.027). The data have been deposited to the ProteomeXchange with identifier PXD002950. This novel biomarker panel was validated in 43 patients using antibody-based assays (C1QA (p = 0.005); C5 (p = 0.0047), APOD (p = 0.0267); APOC3 (p = 0.0064); GP1BA (p = 0.0031); PPBP (p = 0.0465)). We further present that EV-derived fibrinogen components were paradoxically down-regulated in MI, suggesting that a compensatory mechanism may suppress post-infarct coagulation pathways, indicating potential for therapeutic targeting of this mechanism in MI. Taken together, these data demonstrated that plasma EVs contain novel diagnostic biomarkers and therapeutic targets that can be further developed for clinical use to benefit patients with coronary artery diseases (CADs). PMID:27234505

Angiogenic factor imbalance precedes complement deposition in placentae of the BPH/5 model of preeclampsia.

PubMed

Sones, Jennifer L; Merriam, Audrey A; Seffens, Angelina; Brown-Grant, Dex-Ann; Butler, Scott D; Zhao, Anna M; Xu, Xinjing; Shawber, Carrie J; Grenier, Jennifer K; Douglas, Nataki C

2018-05-01

Preeclampsia (PE), a hypertensive disorder of pregnancy, is a leading cause of maternal and fetal morbidity and mortality. Although the etiology is unknown, PE is thought to be caused by defective implantation and decidualization in pregnancy. Pregnant blood pressure high (BPH)/5 mice spontaneously develop placentopathies and maternal features of human PE. We hypothesized that BPH/5 implantation sites have transcriptomic alterations. Next-generation RNA sequencing of implantation sites at peak decidualization, embryonic day (E)7.5, revealed complement gene up-regulation in BPH/5 vs. controls. In BPH/5, expression of complement factor 3 was increased around the decidual vasculature of E7.5 implantation sites and in the trophoblast giant cell layer of E10.5 placentae. Altered expression of VEGF pathway genes in E5.5 BPH/5 implantation sites preceded complement dysregulation, which correlated with abnormal vasculature and increased placental growth factor mRNA and VEGF 164 expression at E7.5. By E10.5, proangiogenic genes were down-regulated, whereas antiangiogenic sFlt-1 was up-regulated in BPH/5 placentae. We found that early local misexpression of VEGF genes and abnormal decidual vasculature preceded sFlt-1 overexpression and increased complement deposition in BPH/5 placentae. Our findings suggest that abnormal decidual angiogenesis precedes complement activation, which in turn contributes to the aberrant trophoblast invasion and poor placentation that underlie PE.-Sones, J. L., Merriam, A. A., Seffens, A., Brown-Grant, D.-A., Butler, S. D., Zhao, A. M., Xu, X., Shawber, C. J., Grenier, J. K., Douglas, N. C. Angiogenic factor imbalance precedes complement deposition in placentae of the BPH/5 model of preeclampsia.

Complement System in Dermatological Diseases – Fire Under the Skin

PubMed Central

Panelius, Jaana; Meri, Seppo

2015-01-01

The complement system plays a key role in several dermatological diseases. Overactivation, deficiency, or abnormality of the control proteins are often related to a skin disease. Autoimmune mechanisms with autoantibodies and a cytotoxic effect of the complement membrane attack complex on epidermal or vascular cells can cause direct tissue damage and inflammation, e.g., in systemic lupus erythematosus (SLE), phospholipid antibody syndrome, and bullous skin diseases like pemphigoid. By evading complement attack, some microbes like Borrelia spirochetes and staphylococci can persist in the skin and cause prolonged symptoms. In this review, we present the most important skin diseases connected to abnormalities in the function of the complement system. Drugs having an effect on the complement system are also briefly described. On one hand, drugs with free hydroxyl on amino groups (e.g., hydralazine, procainamide) could interact with C4A, C4B, or C3 and cause an SLE-like disease. On the other hand, progress in studies on complement has led to novel anti-complement drugs (recombinant C1-inhibitor and anti-C5 antibody, eculizumab) that could alleviate symptoms in diseases associated with excessive complement activation. The main theme of the manuscript is to show how relevant the complement system is as an immune effector system in contributing to tissue injury and inflammation in a broad range of skin disorders. PMID:25688346

Plasmin cleaves fibrinogen and the human complement proteins C3b and C5 in the presence of Leptospira interrogans proteins: A new role of LigA and LigB in invasion and complement immune evasion.

PubMed

Castiblanco-Valencia, Mónica Marcela; Fraga, Tatiana Rodrigues; Pagotto, Ana Helena; Serrano, Solange Maria de Toledo; Abreu, Patricia Antonia Estima; Barbosa, Angela Silva; Isaac, Lourdes

2016-05-01

Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host. Copyright © 2016. Published by Elsevier GmbH.

THE RELATIONSHIP OF THE CHEMOTACTIC BEHAVIOR OF THE COMPLEMENT-DERIVED FACTORS, C3a, C5a, AND C567, AND A BACTERIAL CHEMOTACTIC FACTOR TO THEIR ABILITY TO ACTIVATE THE PROESTERASE 1 OF RABBIT POLYMORPHONUCLEAR LEUKOCYTES

PubMed Central

Becker, Elmer L.

1972-01-01

The inhibition profiles obtained when a series of p-nitrophenyl ethyl alkylphosphonates and of p-nitrophenyl ethyl chloroalkylphosphonates were used to interfere with the chemotactic activity of polymorphonuclear leukocytes stimulated by C3a, C5a, and bacterial factor were the same as found previously when C567 was the chemotactic agent. This indicates that as in the chemotactic activity induced by C567, an obligatory step in the chemotaxis caused by C3a, C5a, and bacterial factor is the activation of proesterase 1 of the rabbit polymorphonuclear leukocyte. C5a and C3a activate proesterase 1 of peripheral blood polymophonuclear leukocytes as measured by the increase of acetyl DL-phenylalanine β-naphthyl esterase activity. Attempts to detect in a like manner the proesterase 1 of the same leukocytes using bacterial factor under varying circumstances have consistently failed. It is concluded that bacterial factor, for unknown reasons, is unable to activate proesterase 1 to the same extent as the complement-derived chemotactic factors. The hypothesis of there being a quantitative difference in the ability of bacterial factor to activate proesterase 1 compared with the complement-derived factors explains the previous observations that bacterial factor can not deactivate to itself or to the complement-derived factors, although these latter factors can deactivate to themselves, to each other, and to the bacterial factor. The quantitative difference in the ability of bacterial factor to activate proesterase 1 compared to the complement-derived factors is also associated with and explains the finding that the maximal chemotactic activity attainable when bacterial factor is the chemotactic agent is distinctly less than that obtained using either C3a, C5a, or C567. These results indicate that the activation of proesterase 1 is a general requirement for the chemotactic activity of rabbit polymorphonuclear leukocytes with known macromolecular chemotactic agents and suggest that under several different circumstances the level of chemotactic activity attained is related to the degree of such activation. PMID:4551218

Design, synthesis, and evaluation of A/C/D-ring analogs of the fungal metabolite K-76 as potential complement inhibitors.

PubMed

Kaufman, T S; Srivastava, R P; Sindelar, R D; Scesney, S M; Marsh, H C

1995-04-28

The terpenoid 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydrox y-2',5',5', 8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a; K-76), a natural product of fungal origin, and its monocarboxylate sodium salt 1c (R = COONa; K-76COONa) inhibit the classical and alternative pathways of complement, and 1c was shown to inhibit the classical pathway at the C5 activation step. In an attempt to elucidate the essential pharmacophore of 1a,c, the natural product was used as a "topographical model" for the design of partial analogs retaining the desired complement inhibiting potency. Therefore, A/C/D-ring analogs have been synthesized, as shown in Scheme 1 using 3-methoxyphenol (3) and limonene chloride (5) as starting materials, which contain functional groups similar to those found on the natural product. The use of (4R)-(+)- and (4S)(-)-limonene chloride (5a,b, respectively) provided two series of compounds differing in the stereochemistry of the C-4 chiral center (limonene moiety numbering). The in vitro assay results of the inhibition of anaphylatoxin production and classical complement-mediated hemolysis revealed that 7-carboxy-2-(R,S)-methyl-2-(1'-methylcyclohexen-(4'R)-yl)-4-met hoxybenzofuran (13a) and 7-carboxy-2-(R,S)-methyl-2-(1'-methylcyclohexen-(4'S)-yl)-4-met hoxybenzofuran (13b) were active in the same range of concentrations as the natural product.

Persistent complement activation on tumor cells in breast cancer.

PubMed Central

Niculescu, F.; Rus, H. G.; Retegan, M.; Vlaicu, R.

1992-01-01

The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer. Images Figure 1 PMID:1374587

Autocrine Complement Inhibits IL10-Dependent T-cell-Mediated Antitumor Immunity to Promote Tumor Progression.

PubMed

Wang, Yu; Sun, Sheng-Nan; Liu, Qing; Yu, Yang-Yang; Guo, Jian; Wang, Kun; Xing, Bao-Cai; Zheng, Qing-Feng; Campa, Michael J; Patz, Edward F; Li, Shi-You; He, You-Wen

2016-09-01

In contrast to its inhibitory effects on many cells, IL10 activates CD8(+) tumor-infiltrating lymphocytes (TIL) and enhances their antitumor activity. However, CD8(+) TILs do not routinely express IL10, as autocrine complement C3 inhibits IL10 production through complement receptors C3aR and C5aR. CD8(+) TILs from C3-deficient mice, however, express IL10 and exhibit enhanced effector function. C3-deficient mice are resistant to tumor development in a T-cell- and IL10-dependent manner; human TILs expanded with IL2 plus IL10 increase the killing of primary tumors in vitro compared with IL2-treated TILs. Complement-mediated inhibition of antitumor immunity is independent of the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) immune checkpoint pathway. Our findings suggest that complement receptors C3aR and C5aR expressed on CD8(+) TILs represent a novel class of immune checkpoints that could be targeted for tumor immunotherapy. Moreover, incorporation of IL10 in the expansion of TILs and in gene-engineered T cells for adoptive cell therapy enhances their antitumor efficacy. Our data suggest novel strategies to enhance immunotherapies: a combined blockade of complement signaling by antagonists to C3aR, C5aR, and anti-PD-1 to enhance anti-PD-1 efficacy; a targeted IL10 delivery to CD8(+) TILs using anti-PD-1-IL10 or anti-CTLA4-IL10 fusion proteins; and the addition of IL10 in TIL expansion for adoptive cellular therapy. Cancer Discov; 6(9); 1022-35. ©2016 AACR.See related commentary by Peng et al., p. 953This article is highlighted in the In This Issue feature, p. 932. ©2016 American Association for Cancer Research.

Evidence of a Pivotal Role for the Distal Part of the Complement Cascade in the Diurnal Release of Hematopoietic Stem Cells Into Peripheral Blood.

PubMed

Borkowska, Sylwia; Suszynska, Malwina; Ratajczak, Janina; Ratajczak, Mariusz Z

2016-01-01

We found that diurnal activation of the three evolutionarily ancient proteolytic cascades in peripheral blood (PB), namely, the complement, coagulation, and fibrinolytic cascades, late at night or in the early morning hours, precedes the diurnal release of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into PB in wild-type mice. Moreover, activation of the distal part of the complement cascade (ComC), involving cleavage of the fifth component (C5), seems to play a crucial role in pharmacological mobilization of HSPCs. In order to shed more light on the role of diurnal rhythms in the egress of HSPCs, we studied diurnal changes in the number of circulating HSPCs in C5-deficient mice and did not observe diurnal changes in the number of these cells circulating in PB in C5(-/-) animals. Based on this finding, we conclude that activation of the distal part of the ComC, C5 cleavage, and release of C5a and desArgC5a are required in executing the diurnal release of HSPCs from BM into PB. Moreover, the fact that C5(-/-) mice still displayed normal activation of the coagulation and fibrinolytic cascades indicates that, of all the proteolytic cascades, the ComC is the dominant player regulating diurnal egress of HSPCs.

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

Photodegradation of avobenzone: stabilization effect of antioxidants.

PubMed

Afonso, S; Horita, K; Sousa e Silva, J P; Almeida, I F; Amaral, M H; Lobão, P A; Costa, P C; Miranda, Margarida S; Esteves da Silva, Joaquim C G; Sousa Lobo, J M

2014-11-01

Avobenzone is one of the most common UVA-filters in sunscreens, and is known to be photounstable. Some of the strategies used to stabilize this filter present some drawbacks like photosensitization reactions. Antioxidants are widely used as cosmetic ingredients that prevent photoageing and complement the photoprotection offered by the UV-filters preventing or reducing photogenerated reactive species. The purpose of this work was to study the effect of antioxidants in the photostabilization of avobenzone. The filter dissolved in dimethyl sulfoxide or incorporated in a sunscreen formulation was irradiated with simulated solar radiation (750 W/m(2)). The tested antioxidants were vitamin C, vitamin E, and ubiquinone. The area under the curve of the absorption spectrum for UVA range and the sun protection factor (SPF) were calculated. Vitamin E (1:2), vitamin C (1:0.5) and ubiquinone (1:0.5) were the more effective concentrations increasing the photostability of avobenzone. In sunscreen formulations, the most effective photostabilizer was ubiquinone which also promoted an increase in SPF. This knowledge is important to improve effectiveness of sunscreen formulation. Antioxidants can be valuable ingredients for sunscreens with a triple activity of filter stabilization, SPF boosting and photoageing prevention. Copyright © 2014 Elsevier B.V. All rights reserved.

Human Tamm-Horsfall protein, a renal specific protein, serves as a cofactor in complement 3b degradation

PubMed Central

2017-01-01

Tamm-Horsfall protein (THP) is an abundant urinary protein of renal origin. We hypothesize that THP can act as an inhibitor of complement since THP binds complement 1q (C1q) of the classical complement pathway, inhibits activation of this pathway, and is important in decreasing renal ischemia-reperfusion injury (a complement-mediated condition). In this study, we began to investigate whether THP interacted with the alternate complement pathway via complement factor H (CFH). THP was shown to bind CFH using ligand blots and in an ELISA (KD of 1 × 10−6 M). Next, the ability of THP to alter CFH’s normal action as it functioned as a cofactor in complement factor I (CFI)–mediated complement 3b (C3b) degradation was investigated. Unexpectedly, control experiments in these in vitro assays suggested that THP, without added CFH, could act as a cofactor in CFI-mediated C3b degradation. This cofactor activity was present equally in THP isolated from 10 different individuals. While an ELISA demonstrated small amounts of CFH contaminating THP samples, these CFH amounts were insufficient to explain the degree of cofactor activity present in THP. An ELISA demonstrated that THP directly bound C3b (KD ~ 5 × 10−8 m), a prerequisite for a protein acting as a C3b degradation cofactor. The cofactor activity of THP likely resides in the protein portion of THP since partially deglycosylated THP still retained cofactor activity. In conclusion, THP appears to participate directly in complement inactivation by its ability to act as a cofactor for C3b degradation, thus adding support to the hypothesis that THP might act as an endogenous urinary tract inhibitor of complement. PMID:28742158

Effects of freezer storage time on levels of complement biomarkers.

PubMed

Morgan, Angharad R; O'Hagan, Caroline; Touchard, Samuel; Lovestone, Simon; Morgan, B Paul

2017-11-06

There is uncertainty regarding how stable complement analytes are during long-term storage at - 80 °C. As part of our work program we have measured 17 complement biomarkers (C1q, C1 inhibitor, C3, C3a, iC3b, C4, C5, C9, FB, FD, FH, FI, TCC, Bb, sCR1, sCR2, Clusterin) and the benchmark inflammatory marker C-reactive protein (CRP) in a large set of plasma samples (n = 720) that had been collected, processed and subsequently stored at - 80 °C over a period of 6.6-10.6 years, prior to laboratory analysis. The biomarkers were measured using solid-phase enzyme immunoassays with a combination of multiplex assays using the MesoScale Discovery Platform and single-plex enzyme-linked immunosorbent assays (ELISAs). As part of a post hoc analysis of extrinsic factors (co-variables) affecting the analyses we investigated the impact of freezer storage time on the values obtained for each complement analyte. With the exception of five analytes (C4, C9, sCR2, clusterin and CRP), storage time was significantly correlated with measured plasma concentrations. For ten analytes: C3, FI, FB, FD, C5, sCR1, C3a, iC3b, Bb and TCC, storage time was positively correlated with concentration and for three analytes: FH, C1q, and C1 inhibitor, storage time was negatively correlated with concentration. The results suggest that information on storage time should be regarded as an important co-variable and taken into consideration when analysing data to look for associations of complement biomarker levels and disease or other outcomes.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation.

PubMed

Kim, Sook Young; Son, Myoungsun; Lee, Sang Eun; Park, In Ho; Kwak, Man Sup; Han, Myeonggil; Lee, Hyun Sook; Kim, Eun Sook; Kim, Jae-Young; Lee, Jong Eun; Choi, Ji Eun; Diamond, Betty; Shin, Jeon-Soo

2018-01-01

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia-reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

PubMed Central

Kim, Sook Young; Son, Myoungsun; Lee, Sang Eun; Park, In Ho; Kwak, Man Sup; Han, Myeonggil; Lee, Hyun Sook; Kim, Eun Sook; Kim, Jae-Young; Lee, Jong Eun; Choi, Ji Eun; Diamond, Betty; Shin, Jeon-Soo

2018-01-01

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia–reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation. PMID:29696019

Functional basis for complement evasion by staphylococcal superantigen-like 7.

PubMed

Bestebroer, Jovanka; Aerts, Piet C; Rooijakkers, Suzan H M; Pandey, Manoj K; Köhl, Jörg; van Strijp, Jos A G; de Haas, Carla J C

2010-10-01

The human pathogen Staphylococcus aureus has a plethora of virulence factors that promote its colonization and survival in the host. Among such immune modulators are staphylococcal superantigen-like (SSL) proteins, comprising a family of 14 small, secreted molecules that seem to interfere with the host innate immune system. SSL7 has been described to bind immunoglobulin A (IgA) and complement C5, thereby inhibiting IgA-FcαRI binding and serum killing of Escherichia coli. As C5a generation, in contrast to C5b-9-mediated lysis, is crucial for immune defence against staphylococci, we investigated the impact of SSL7 on staphylococcal-induced C5a-mediated effects. Here, we show that SSL7 inhibits C5a generation induced by staphylococcal opsonization, slightly enhanced by its IgA-binding capacity. Moreover, we demonstrate a strong protective activity of SSL7 against staphylococcal clearance in human whole blood. SSL7 strongly inhibited the C5a-induced phagocytosis of S. aureus and oxidative burst in an in vitro whole-blood inflammation model. Furthermore, we found that SSL7 affects all three pathways of complement activation and inhibits the cleavage of C5 by interference of its binding to C5 convertases. Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a-driven influx of neutrophils in mouse peritoneum. © 2010 Blackwell Publishing Ltd.

Functional basis for complement evasion by staphylococcal superantigen-like 7

PubMed Central

Bestebroer, Jovanka; Aerts, Piet C.; Rooijakkers, Suzan H.M.; Pandey, Manoj K.; Köhl, Jörg; van Strijp, Jos A. G.; de Haas, Carla J. C.

2010-01-01

Summary The human pathogen Staphylococcus aureus has a plethora of virulence factors that promote its colonization and survival in the host. Among such immune modulators are staphylococcal superantigen-like (SSL) proteins, comprising a family of 14 small, secreted molecules that seem to interfere with the host innate immune system. SSL7 has been described to bind immunoglobulin A (IgA) and complement C5, thereby inhibiting IgA-FcαRI binding and serum killing of E. coli. As C5a generation, in contrast to C5b-9-mediated lysis, is crucial for immune defense against staphylococci, we investigated the impact of SSL7 on staphylococcal-induced C5a-mediated effects. Here, we show that SSL7 inhibits C5a generation induced by staphylococcal opsonization, slightly enhanced by its IgA-binding capacity. Moreover, we demonstrate a strong protective activity of SSL7 against staphylococcal clearance in human whole blood. SSL7 strongly inhibited the C5a-induced phagocytosis of S. aureus and oxidative burst in an in vitro whole blood inflammation model. Furthermore, we found that SSL7 affects all three pathways of complement activation and inhibits the cleavage of C5 by interference of its binding to C5 convertases. Finally, SSL7 effects were also demonstrated in vivo. In a murine model of immune complex peritonitis, SSL7 abrogated the C5a-driven influx of neutrophils in mouse peritoneum. PMID:20545943

Role of capsule and suilysin in mucosal infection of complement-deficient mice with Streptococcus suis.

PubMed

Seitz, Maren; Beineke, Andreas; Singpiel, Alena; Willenborg, Jörg; Dutow, Pavel; Goethe, Ralph; Valentin-Weigand, Peter; Klos, Andreas; Baums, Christoph G

2014-06-01

Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Experimental infection of wild-type (WT) and C3(-/-) mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3(-/-) mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3(-/-) mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3(-/-) blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR(-/-) mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.

Complement activation by carbon nanotubes and its influence on the phagocytosis and cytokine response by macrophages.

PubMed

Pondman, Kirsten M; Sobik, Martin; Nayak, Annapurna; Tsolaki, Anthony G; Jäkel, Anne; Flahaut, Emmanuel; Hampel, Silke; Ten Haken, Bennie; Sim, Robert B; Kishore, Uday

2014-08-01

Carbon nanotubes (CNTs) have promised a range of applications in biomedicine. Although influenced by the dispersants used, CNTs are recognized by the innate immune system, predominantly by the classical pathway of the complement system. Here, we confirm that complement activation by the CNT used continues up to C3 and C5, indicating that the entire complement system is activated including the formation of membrane-attack complexes. Using recombinant forms of the globular regions of human C1q (gC1q) as inhibitors of CNT-mediated classical pathway activation, we show that C1q, the first recognition subcomponent of the classical pathway, binds CNTs via the gC1q domain. Complement opsonisation of CNTs significantly enhances their uptake by U937 cells, with concomitant downregulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines in both U937 cells and human monocytes. We propose that CNT-mediated complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response. This study highlights the importance of the complement system in response to carbon nanontube administration, suggesting that the ensuing complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response. Copyright © 2014 Elsevier Inc. All rights reserved.

Host Defense against Opportunist Microorganisms Following Trauma.

DTIC Science & Technology

1979-06-01

patients were total hemolytic complement (CH5 0 ), C3 conversion by inulin and cobra venom factor (CoVF), and itmunochemical concentrations of Clq, C4, C2...were normal or elevated for the entire study period. C3 conversion by inulin and CoVF and the concentration of properdin were reduced in the sera of the...measured in all patients were total hemolytic complement (CH5 0), C3 conversion by inulin and cobra venom factor (CoVF), and inunochemical

Identity of the segment of human complement C8 recognized by complement regulatory protein CD59.

PubMed

Lockert, D H; Kaufman, K M; Chang, C P; Hüsler, T; Sodetz, J M; Sims, P J

1995-08-25

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development

PubMed Central

Jäckel, Sven; Saffarzadeh, Mona; Langer, Florian

2017-01-01

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)–dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3−/− mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5−/− mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells. PMID:28223279

Discontinuation of dialysis with eculizumab therapy in a pediatric patient with dense deposit disease.

PubMed

Tran, Cheryl L; Sethi, Sanjeev; Murray, David; Cramer, Carl H; Sas, David J; Willrich, Maria; Smith, Richard J; Fervenza, Fernando C

2016-04-01

Dense deposit disease (DDD) is a rare glomerular disease caused by an uncontrolled activation of the alternative complement pathway leading to end-stage renal disease in 50 % of patients. As such, DDD has been classified within the spectrum of complement component 3 (C3) glomerulopathies due to its pathogenesis from alternative pathway dysregulation. Conventional immunosuppressive therapies have no proven effectiveness. Eculizumab, a terminal complement inhibitor, has been reported to mitigate disease in some cases. We report on the efficacy of eculizumab in a pediatric patient who failed to respond to cyclophosphamide, corticosteroids, and plasma exchange. Complement biomarker profiling was remarkable for low serum C3, low properdin, and elevated soluble C5b-9. Consistent with these findings, the alternative pathway functional assay was abnormally low, indicative of alternative pathway activity, although neither C3-nephritic factors nor Factor H autoantibodies were detected. Eculizumab therapy was associated with significant improvement in proteinuria and renal function allowing discontinuation of hemodialysis (HD). Repeat C3 and soluble C5b-9 levels normalized, showing that terminal complement pathway activity was successfully blocked while the patient was receiving eculizumab therapy. Repeat testing for alternative pathway activation allowed for a successful decrease in eculizumab dosing. The case reported here demonstrates the successful recovery of renal function in a pediatric patient on HD following the use of eculizumab.

A scabies mite serpin interferes with complement-mediated neutrophil functions and promotes staphylococcal growth.

PubMed

Swe, Pearl M; Fischer, Katja

2014-06-01

Scabies is a contagious skin disease caused by the parasitic mite Sarcoptes scabiei. The disease is highly prevalent worldwide and known to predispose to secondary bacterial infections, in particular by Streptococcus pyogenes and Staphylococcus aureus. Reports of scabies patients co-infected with methicillin resistant S. aureus (MRSA) pose a major concern for serious down-stream complications. We previously reported that a range of complement inhibitors secreted by the mites promoted the growth of S. pyogenes. Here, we show that a recently characterized mite serine protease inhibitor (SMSB4) inhibits the complement-mediated blood killing of S. aureus. Blood killing of S. aureus was measured in whole blood bactericidal assays, counting viable bacteria recovered after treatment in fresh blood containing active complement and phagocytes, treated with recombinant SMSB4. SMSB4 inhibited the blood killing of various strains of S. aureus including methicillin-resistant and methicillin-sensitive isolates. Staphylococcal growth was promoted in a dose-dependent manner. We investigated the effect of SMSB4 on the complement-mediated neutrophil functions, namely phagocytosis, opsonization and anaphylatoxin release, by flow cytometry and in enzyme linked immuno sorbent assays (ELISA). SMSB4 reduced phagocytosis of S. aureus by neutrophils. It inhibited the deposition of C3b, C4b and properdin on the bacteria surface, but did not affect the depositions of C1q and MBL. SMSB4 also inhibited C5 cleavage as indicated by a reduced C5b-9 deposition. We postulate that SMSB4 interferes with the activation of all three complement pathways by reducing the amount of C3 convertase formed. We conclude that SMSB4 interferes with the complement-dependent killing function of neutrophils, thereby reducing opsonization, phagocytosis and further recruitment of neutrophils to the site of infection. As a consequence secreted scabies mites complement inhibitors, such as SMSB4, provide favorable conditions for the onset of S. aureus co-infection in the scabies-infected microenvironment by suppressing the immediate host immune response.

Relationship of Circulating C5a and Complement Factor H Levels With Disease Control in Pregnant Women With Asthma.

PubMed

Bohács, Anikó; Bikov, András; Ivancsó, István; Czaller, Ibolya; Böcskei, Renáta; Müller, Veronika; Rigó, János; Losonczy, György; Tamási, Lilla

2016-04-01

Asthma often complicates pregnancy and represents a risk of serious pregnancy complications. The complement system contributes to asthma pathogenesis and is up-regulated in healthy gestation as well. The anaphylatoxin C5a has a major pro-inflammatory role, and the complement factor H is a main soluble regulator protein both in asthma and during pregnancy; however, peripheral levels of these complement factors and their relationship to disease control have not yet been evaluated in pregnant subjects with asthma. The present study aimed to investigate circulating C5a and complement factor H levels in asthma (non-pregnant subjects with asthma; n = 19) and in pregnancy with asthma (pregnant subjects with asthma; n = 22), compared with healthy non-pregnant (n = 21) and healthy pregnant women (n = 13) and to test their relationship to clinical parameters of asthma (lung function, airway inflammation, and symptoms). Circulating C5a levels were higher in the pregnant asthma subject group compared with the healthy non-pregnant, healthy pregnant, and non-pregnant asthma groups: median 2.629 (interquartile range [IQR] 2.257-3.052) ng/mL versus 1.84 (IQR 1.576-2.563), 1.783 (IQR 0.6064-2.786), and 2.024 (IQR 1.232-2.615) ng/mL, respectively (P = .02 in all cases). C5a correlated negatively with FEV1 (r = -0.44, P = .039) and FVC values (r = -0.64, P = .001) in the pregnant asthma group and positively with fraction of exhaled nitric oxide levels in the non-pregnant asthma group (n = 12, r = 0.78, P = .004). Complement factor H levels were elevated in both the healthy pregnant and pregnant asthma subject groups compared with the healthy non-pregnant group (median 1,082 [IQR 734.9-1,224] and 910.7 [IQR 614.5-1076] μg/mL vs. 559.7 [IQR 388.7-783.1] μg/mL, P = .002 and P = .004, respectively) but not in the pregnant asthma group compared with the non-pregnant asthma group (median 687.4 [IQR 441.6-947.6] μg/mL, P = .10). Asthma during pregnancy increases the circulating level of pro-inflammatory C5a, which is accompanied by impaired lung function and partly counteracted by the gestation-specific elevation of regulatory complement factor H level (detected in pregnancy both in healthy and subjects with asthma). Copyright © 2016 by Daedalus Enterprises.

The role of complement in myasthenia gravis: serological evidence of complement consumption in vivo.

PubMed

Romi, Fredrik; Kristoffersen, Einar K; Aarli, Johan A; Gilhus, Nils Erik

2005-01-01

Antibodies to the acetylcholine receptor (AChR) titin and the ryanodine receptor (RyR) occur in myasthenia gravis (MG). These antibodies are capable of complement activation in vitro. The involvement of the complement system should cause consumption of complement components such as C3 and C4 in vivo. Complement components C3 and C4 were assayed in sera from 78 AChR antibody-positive MG patients and 52 healthy controls. Forty-eight of the patient sera contained titin antibodies as well, and 20 were also RyR antibody-positive. MG patients with AChR antibody concentrations above the median (11.2 nmol/l) had significantly lower mean C3 and C4 concentrations in serum compared to those with AChR antibody concentrations below the median. Titin antibody-positive MG patients, titin antibody-negative early-onset MG patients, titin antibody-negative late-onset MG patients, and controls had similar C3 and C4 concentrations. Nor did mean C3 and C4 concentrations differ in MG patients with RyR antibodies. Patients with severe MG (grades 4 and 5) had similar C3 and similar C4 levels compared to those with mild MG (grades 1 and 2). An increased in vivo complement consumption was detected in MG patients with high AChR antibody concentrations, unrelated to MG severity and non-AChR muscle antibodies.

CFH Y402H polymorphism and the complement activation product C5a: effects on NF-κB activation and inflammasome gene regulation.

PubMed

Cao, Sijia; Wang, Jay Ching Chieh; Gao, Jiangyuan; Wong, Matthew; To, Elliott; White, Valerie A; Cui, Jing Z; Matsubara, Joanne A

2016-05-01

The Y402H polymorphism in the complement factor H (CFH) gene is an important risk factor for age-related macular degeneration (AMD). Complement activation products and proinflammatory cytokines are associated with this polymorphism at the systemic level, but less is known of the associations in the outer retina of the genotyped eye. Here we investigate complement activation products and their role in nuclear factor (NF)-κB activation and gene expression of the NLRP3 inflammasome pathway. Postmortem donor eyes were genotyped for the CFH Y402H polymorphism and assessed for complement C3a, C5a, interleukin (IL)-18 and tumour necrosis factor (TNF)-α. ARPE19 cells were stimulated basolaterally with C5a or TNF-α in polarised cultures. NF-κB activation was assessed with a reporter cell line. Gene expression of inflammasome-related (NLRP3, caspase-1, IL-1β and IL-18) and classic inflammatory (IL-6 and IL-8) genes was studied. The distribution of inflammasome products, IL-1β and IL-18, was studied in postmortem donor eyes with AMD pathologies. Eyes with the homozygous at-risk variant demonstrated higher levels of C5a, IL-18 and TNF-α in Bruch's membrane and choroid. C5a promoted NF-κB activation and upregulation of IL-18 in polarised ARPE19. TNF-α promoted NF-κB activation and gene expression of caspase-1, IL-1β, IL-18, IL-6 and IL-8, but downregulated NLRP3. In eyes with geographic atrophy, strong immunoreactivity was observed for inflammasome products IL-1β and IL-18 compared with age-matched controls. The at-risk polymorphism of the CFH Y402H may contribute to AMD disease process through increased complement and NF-κB activation, and the upregulation of IL-18, a product of inflammasome activation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Neutrophil extracellular traps can activate alternative complement pathways.

PubMed

Wang, H; Wang, C; Zhao, M-H; Chen, M

2015-09-01

The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). ANCAs activate primed neutrophils to release neutrophil extracellular traps (NETs), which have recently gathered increasing attention in the development of AAV. The relationship between NETs and alternative complement pathway has not been elucidated. The current study aimed to investigate the relationship between NETs and alternative complement pathway. Detection of components of alternative complement pathway on NETs in vitro was assessed by immunostain and confocal microscopy. Complement deposition on NETs were detected after incubation with magnesium salt ethyleneglycol tetraacetic acid (Mg-EGTA)-treated human serum. After incubation of serum with supernatants enriched in ANCA-induced NETs, levels of complement components in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Complement factor B (Bb) and properdin deposited on NETs in vitro. The deposition of C3b and C5b-9 on NETs incubated with heat-inactivated normal human serum (Hi-NHS) or EGTA-treated Hi-NHS (Mg-EGTA-Hi-NHS) were significantly less than that on NETs incubated with NHS or EGTA-treated NHS (Mg-EGTA-NHS). NETs induced by ANCA could activate the alternative complement cascade in the serum. In the presence of EGTA, C3a, C5a and SC5b-9 concentration decreased from 800·42 ± 244·81 ng/ml, 7·68 ± 1·50 ng/ml, 382·15 ± 159·75 ng/ml in the supernatants enriched in ANCA induced NETs to 479·07 ± 156·2 ng/ml, 4·86 ± 1·26 ng/ml, 212·65 ± 44·40 ng/ml in the supernatants of DNase I-degraded NETs (P < 0·001, P = 0·008, P < 0·001, respectively). NETs could activate the alternative complement pathway, and might thus participate in the pathogenesis of AAV. © 2015 British Society for Immunology.

Synergy between the classical and alternative pathways of complement is essential for conferring effective protection against the pandemic influenza A(H1N1) 2009 virus infection

PubMed Central

Rattan, Ajitanuj; Pawar, Shailesh D.; Nawadkar, Renuka; Kulkarni, Neeraja

2017-01-01

The pandemic influenza A(H1N1) 2009 virus caused significant morbidity and mortality worldwide thus necessitating the need to understand the host factors that influence its control. Previously, the complement system has been shown to provide protection during the seasonal influenza virus infection, however, the role of individual complement pathways is not yet clear. Here, we have dissected the role of intact complement as well as of its individual activation pathways during the pandemic influenza virus infection using mouse strains deficient in various complement components. We show that the virus infection in C3-/- mice results in increased viral load and 100% mortality, which can be reversed by adoptive transfer of naïve wild-type (WT) splenocytes, purified splenic B cells, or passive transfer of immune sera from WT, but not C3-/- mice. Blocking of C3a and/or C5a receptor signaling in WT mice using receptor antagonists and use of C3aR-/- and C5aR-/- mice showed significant mortality after blocking/ablation of C3aR, with little or no effect after blocking/ablation of C5aR. Intriguingly, deficiency of C4 and FB in mice resulted in only partial mortality (24%-32%) suggesting a necessary cross-talk between the classical/lectin and alternative pathways for providing effective protection. In vitro virus neutralization experiments performed to probe the cross-talk between the various pathways indicated that activation of the classical and alternative pathways in concert, owing to coating of viral surface by antibodies, is needed for its efficient neutralization. Examination of the virus-specific complement-binding antibodies in virus positive subjects showed that their levels vary among individuals. Together these results indicate that cooperation between the classical and alternative pathways not only result in efficient direct neutralization of the pandemic influenza virus, but also lead to the optimum generation of C3a, which when sensed by the immune cells along with the antigen culminates in generation of effective protective immune responses. PMID:28301559

Modulation of post-stroke degenerative and regenerative processes and subacute protection by site-targeted inhibition of the alternative pathway of complement.

PubMed

Alawieh, Ali; Elvington, Andrew; Zhu, Hong; Yu, Jin; Kindy, Mark S; Atkinson, Carl; Tomlinson, Stephen

2015-12-30

Complement promotes neuroinflammation and injury in models of stroke. However, complement is also being increasingly implicated in repair and regeneration after central nervous system (CNS) injury, and some complement deficiencies have been shown to provide acute, but not subacute, protection after murine stroke. Here, we investigate the dual role of complement in injury and repair after cerebral ischemia and reperfusion. We used complement-deficient mice and different complement inhibitors in a model of transient middle cerebral artery occlusion to investigate complement-dependent cellular and molecular changes that occur through the subacute phase after stroke. C3 deficiency and site-targeted complement inhibition with either CR2-Crry (inhibits all pathways) or CR2-fH (inhibits alternative pathway) significantly reduced infarct size, reduced apoptotic cell death, and improved neurological deficit score in the acute phase after stroke. However, only in CR2-fH-treated mice was there sustained protection with no evolution of injury in the subacute phase. Whereas both inhibitors significantly reduced microglia/macrophage activation and astrogliosis in the subacute phase, only CR2-fH improved neurological deficit and locomotor function, maintained neurogenesis markers, enhanced neuronal migration, and increased VEGF expression. These findings in CR2-fH-treated mice correlated with improved performance in spatial learning and passive avoidance tasks. The complement anaphylatoxins have been implicated in repair and regenerative mechanisms after CNS injury, and in this context CR2-fH significantly reduced, but did not eliminate the generation of C5a within the brain, unlike CR2-Crry that completely blocked C5a generation. Gene expression profiling revealed that CR2-fH treatment downregulated genes associated with apoptosis, TGFβ signaling, and neutrophil activation, and decreased neutrophil infiltration was confirmed by immunohistochemistry. CR2-fH upregulated genes for neural growth factor and mediators of neurogenesis and neuronal migration. Live animal imaging demonstrated that following intravenous injection, CR2-fH targeted specifically to the post-ischemic brain, with a tissue half-life of 48.5 h. Finally, unlike C3 deficiency, targeted complement inhibition did not increase susceptibility to lethal post-stroke infection, an important consideration for stroke patients. Ischemic brain tissue-targeted and selective inhibition of alternative complement pathway provide self-limiting inhibition of complement activation and reduces acute injury while maintaining complement-dependent recovery mechanisms into the subacute phase after stroke.

Complement Evasion Strategies of Viruses: An Overview

PubMed Central

Agrawal, Palak; Nawadkar, Renuka; Ojha, Hina; Kumar, Jitendra; Sahu, Arvind

2017-01-01

Being a major first line of immune defense, the complement system keeps a constant vigil against viruses. Its ability to recognize large panoply of viruses and virus-infected cells, and trigger the effector pathways, results in neutralization of viruses and killing of the infected cells. This selection pressure exerted by complement on viruses has made them evolve a multitude of countermeasures. These include targeting the recognition molecules for the avoidance of detection, targeting key enzymes and complexes of the complement pathways like C3 convertases and C5b-9 formation – either by encoding complement regulators or by recruiting membrane-bound and soluble host complement regulators, cleaving complement proteins by encoding protease, and inhibiting the synthesis of complement proteins. Additionally, viruses also exploit the complement system for their own benefit. For example, they use complement receptors as well as membrane regulators for cellular entry as well as their spread. Here, we provide an overview on the complement subversion mechanisms adopted by the members of various viral families including Poxviridae, Herpesviridae, Adenoviridae, Flaviviridae, Retroviridae, Picornaviridae, Astroviridae, Togaviridae, Orthomyxoviridae and Paramyxoviridae. PMID:28670306

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. Copyright © 2013 Elsevier B.V. All rights reserved.

Tuning complement activation and pathway through controlled molecular architecture of dextran chains in nanoparticle corona.

PubMed

Coty, Jean-Baptiste; Eleamen Oliveira, Elquio; Vauthier, Christine

2017-11-05

The understanding of complement activation by nanomaterials is a key to a rational design of safe and efficient nanomedicines. This work proposed a systematic study investigating how molecular design of nanoparticle coronas made of dextran impacts on mechanisms that trigger complement activation. The nanoparticles used for this work consisted of dextran-coated poly(isobutylcyanoacrylate) (PIBCA) nanoparticles have already been thoroughly characterized. Their different capacity to trigger complement activation established on the cleavage of the protein C3 was also already described making these nanoparticles good models to investigate the relation between the molecular feature of their corona and the mechanism by which they triggered complement activation. Results of this new study show that complement activation pathways can be selected by distinct architectures formed by dextran chains composing the nanoparticle corona. Assumptions that explain the relation between complement activation mechanisms triggered by the nanoparticles and the nanoparticle corona molecular feature were proposed. These results are of interest to better understand how the design of dextran-coated nanomaterials will impact interactions with the complement system. It can open perspectives with regard to the selection of a preferential complement activation pathway or prevent the nanoparticles to activate the complement system, based on a rational choice of the corona configuration. Copyright © 2017 Elsevier B.V. All rights reserved.

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

PubMed Central

Wright, J K; Tschopp, J; Jaton, J C; Engel, J

1980-01-01

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability. Images Fig. 2. PMID:6985362

Smoke Exposure Causes Endoplasmic Reticulum Stress and Lipid Accumulation in Retinal Pigment Epithelium through Oxidative Stress and Complement Activation*

PubMed Central

Kunchithapautham, Kannan; Atkinson, Carl; Rohrer, Bärbel

2014-01-01

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. PMID:24711457

Role of Capsule and Suilysin in Mucosal Infection of Complement-Deficient Mice with Streptococcus suis

PubMed Central

Seitz, Maren; Beineke, Andreas; Singpiel, Alena; Willenborg, Jörg; Dutow, Pavel; Goethe, Ralph; Valentin-Weigand, Peter; Klos, Andreas

2014-01-01

Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Experimental infection of wild-type (WT) and C3−/− mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3−/− mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3−/− mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3−/− blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR−/− mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity. PMID:24686060

Cooperation between Hsp90 and mortalin/GRP75 in resistance to cell death induced by complement C5b-9.

PubMed

Rozenberg, Perri; Ziporen, Lea; Gancz, Dana; Saar-Ray, Moran; Fishelson, Zvi

2018-02-02

Cancer cells are commonly more resistant to cell death activated by the membranolytic protein complex C5b-9. Several surface-expressed and intracellular proteins that protect cells from complement-dependent cytotoxicity (CDC) have been identified. In this study, we investigated the function of heat shock protein 90 (Hsp90), an essential and ubiquitously expressed chaperone, overexpressed in cancer cells, in C5b-9-induced cell death. As shown, inhibition of Hsp90 with geldanamycin or radicicol is enhancing sensitivity of K562 erythroleukemia cells to CDC. Similarly, Hsp90 inhibition confers in Ramos B cell lymphoma cells elevated sensitivity to treatment with rituximab and complement. C5b-9 deposition is elevated on geldanamycin-treated cells. Purified Hsp90 binds directly to C9 and inhibits zinc-induced C9 polymerization, indicating that Hsp90 may act directly on the C5b-9 complex. Mortalin, also known as stress protein 70 or GRP75, is a mitochondrial chaperone that confers resistance to CDC. The postulated cooperation between Hsp90 and mortalin in protection from CDC was tested. Geldanamycin failed to sensitize toward CDC cells with knocked down mortalin. Direct binding of Hsp90 to mortalin was shown by co-immunoprecipitation in cell extracts after triggering with complement as well as by using purified recombinant proteins. These results provide an insight into the protective mechanisms utilized by cancer cells to evade CDC. They suggest that Hsp90 protects cells from CDC by inhibiting, together with mortalin, C5b-9 assembly and/or stability at the plasma membrane.

Complement C4a inhibits the secretion of hepatitis B virus screened by surface-enhanced laser desorption ionization time-flight mass spectrometry-based ProteinChip analysis.

PubMed

Song, Ya-Nan; Zhang, Gui-Biao; Hu, Xue-Qing; Lu, Yi-Yu; Zhao, Yu; Yang, Yang; Yang, Yi-Fu; Zhang, Yong-Yu; Hu, Yi-Yang; Su, Shi-Bing

2015-12-01

Chronic hepatitis B (CHB) is a kind of chronic liver disease caused by persistent hepatitis B virus (HBV) infection. The study aims to seek the factors of host resistance to HBV and investigate their roles. Protein profiles of 58 healthy controls and 121 CHB patients were obtained by SELDI-TOF/MS. Predicted protein was validated by ELISA. Protein expression was evaluated by Western blot in the persistently HBV expressing cell line HepG2.2.15 and non-HBV expressing cell line HepG2. The level of HBV DNA was subsequently detected by quantitative real-time PCR in HepG2.2.15 cells with complement C4a treatment. Significantly altered protein peaks were found through statistical analysis, and m/z 4300 was predicted by databases and successfully matched with the fragment of complement C4a. According to ELISA, serum complement C4a was found to be significantly lower in CHB patients compared with healthy controls (p < 0.001) and the area under receiver operating characteristics curve is 0.78. Furthermore, complement C4a showed lower expression in HepG2.2.5 cells and the secretion of HBV DNA was inhibited by complement C4a. The present study implied the important role of complement C4a in inhibiting the HBV DNA secretion in CHB. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibitor(s) of the classical complement pathway in mouse serum limit the utility of mice as experimental models of neuromyelitis optica.

PubMed

Ratelade, Julien; Verkman, A S

2014-11-01

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system in which anti-aquaporin-4 (AQP4) autoantibodies (AQP4-IgG) cause damage to astrocytes by complement-dependent cytotoxicity (CDC). Various approaches have been attempted to produce NMO lesions in rodents, some involving genetically modified mice with altered immune cell function. Here, we found that mouse serum strongly inhibits complement from multiple species, preventing AQP4-IgG-dependent CDC. Effects of mouse serum on complement activation were tested in CDC assays in which AQP4-expressing cells were incubated with AQP4-IgG and complement from different species. Biochemical assays and mass spectrometry were used to characterize complement inhibitor(s) in mouse serum. Sera from different strains of mice produced almost no AQP4-IgG-dependent CDC compared with human, rat and guinea pig sera. Remarkably, addition of mouse serum prevented AQP4-IgG-dependent CDC caused by human, rat or guinea pig serum, with 50% inhibition at <5% mouse serum. Hemolysis assays indicated that the inhibitor(s) in mouse serum target the classical and not the alternative complement pathway. We found that the complement inhibitor(s) in mouse serum were contained in a serum fraction purified with protein-A resin; however, the inhibitor was not IgG as determined using serum from IgG-deficient mice. Mass spectrometry on the protein A-purified fraction produced several inhibitor candidates. The low intrinsic complement activity of mouse serum and the presence of complement inhibitor(s) limit the utility of mouse models to study disorders, such as NMO, involving the classical complement pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structure of C3b reveals conformational changes that underlie complement activity.

PubMed

Janssen, Bert J C; Christodoulidou, Agni; McCarthy, Andrew; Lambris, John D; Gros, Piet

2006-11-09

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-A resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 A away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the alpha-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation.

Dynamics and reproductive effects of complement factors in the spontaneous abortion model of CBA/J×DBA/2 mice.

PubMed

Takeshita, Ai; Kusakabe, Ken Takeshi; Hiyama, Masato; Kuniyoshi, Nobue; Kondo, Tomohiro; Kano, Kiyoshi; Kiso, Yasuo; Okada, Toshiya

2014-05-01

The complement system is one component of innate immunity that could participate in fetal loss. We have already reported that adipsin, a complement activator in the alternative pathway, is stably expressed in the placenta and that an increase in this expression is related to spontaneous abortion. However, complement inhibitor Crry was concurrently expressed in the placenta, and the role of complement factors during pregnancy was not clear. In the present study, we examined the endogenous regulation of complement factors in placenta and serum by using another model mouse for spontaneous abortion and studied the effect of exogenous complement disruption on pregnancy. Compared to control mice, the CBA/J×DBA/2 model mice had higher expression levels of adipsin in the placenta and serum. Adipsin and complement C3 were localized in the metrial gland and labyrinth regions, and both positive reactive ranges were limited in the maternal blood current in normal implantation sites. These results suggest that extrauterine adipsin hematogenously reaches the placenta, activates complement C3, and promotes destruction of the feto-maternal barrier in aborted implantation sites. Crry was consistently expressed in the placenta and serum and reduced in the resorption sites of CBA/J×DBA/2 mice as compared to normal sites. Injection of recombinant adipsin increased the resorption rate and changed the expression of Th-type cytokines toward a Th1 bias. The present study indicates that adipsin could induce the fetal loss that accompanies the Th1 bias and may be a crucial cause of spontaneous abortion. In addition, the local expression of Crry prevents complement activation in placenta in response to a systemic increase of adipsin. Copyright © 2014 Elsevier GmbH. All rights reserved.

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

Nebulized C1-Esterase Inhibitor does not Reduce Pulmonary Complement Activation in Rats with Severe Streptococcus Pneumoniae Pneumonia.

PubMed

de Beer, Friso; Lagrand, Wim; Glas, Gerie J; Beurskens, Charlotte J P; van Mierlo, Gerard; Wouters, Diana; Zeerleder, Sacha; Roelofs, Joris J T H; Juffermans, Nicole P; Horn, Janneke; Schultz, Marcus J

2016-12-01

Complement activation plays an important role in the pathogenesis of pneumonia. We hypothesized that inhibition of the complement system in the lungs by repeated treatment with nebulized plasma-derived human C1-esterase inhibitor reduces pulmonary complement activation and subsequently attenuates lung injury and lung inflammation. This was investigated in a rat model of severe Streptococcus pneumoniae pneumonia. Rats were intra-tracheally challenged with S. pneumoniae to induce pneumonia. Nebulized C1-esterase inhibitor or saline (control animals) was repeatedly administered to rats, 30 min before induction of pneumonia and every 6 h thereafter. Rats were sacrificed 20 or 40 h after inoculation with bacteria. Brochoalveolar lavage fluid and lung tissue were obtained for measuring levels of complement activation (C4b/c), lung injury and inflammation. Induction of pneumonia was associated with pulmonary complement activation (C4b/c at 20 h 1.24 % [0.56-2.59] and at 40 h 2.08 % [0.98-5.12], compared to 0.50 % [0.07-0.59] and 0.03 % [0.03-0.03] in the healthy control animals). The functional fraction of C1-INH was detectable in BALF, but no effect was found on pulmonary complement activation (C4b/c at 20 h 0.73 % [0.16-1.93] and at 40 h 2.38 % [0.54-4.19]). Twenty hours after inoculation, nebulized C1-esterase inhibitor treatment reduced total histology score, but this effect was no longer seen at 40 h. Nebulized C1-esterase inhibitor did not affect other markers of lung injury or lung inflammation. In this negative experimental animal study, severe S. pneumoniae pneumonia in rats is associated with pulmonary complement activation. Repeated treatment with nebulized C1-esterase inhibitor, although successfully delivered to the lungs, does not affect pulmonary complement activation, lung inflammation or lung injury.

Complement C5a receptor antagonism by protamine and poly-L-Arg on human leukocytes.

PubMed

Olsen, U B; Selmer, J; Kahl, J U

1988-01-01

It is shown that protamine selectively and dose-dependently inhibits complement C5a-induced leukocyte responses such as histamine release from basophils, chemiluminescence and beta-glucuronidase release from neutrophils. Protamine produces parallel rightward displacements of the C5a dose-response curves. The inhibitory capacity of the polypeptide is reversible and disappears following repeated washing of exposed cells. In neutrophils poly-L-Arg similarly and specifically antagonizes C5a-induced chemiluminescence and enzyme release. This polymer alone, however, degranulates basophils and neutrophils, leading to histamine and enzyme release, respectively. It is concluded that on human neutrophils the arginine-rich polycations protamine and poly-L-Arg exhibit a competitive C5a receptor antagonism. In addition, protamine inhibits the C5a receptors on basophils. It is hypothesized that molecular conformations of the arginine-rich polycations might bind reversibly to, and block negatively charged groups at the C5a-receptor sites.

The C8-binding protein of human erythrocytes: interaction with the components of the complement-attack phase.

PubMed Central

Schönermark, S; Filsinger, S; Berger, B; Hänsch, G M

1988-01-01

C8-binding protein is an intrinsic membrane protein of the human erythrocyte. It inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. In the present study we incorporated C8bp, isolated from human erythrocytes, into sheep erythrocytes (SRBC). SRBC, normally sensitive to lysis by human C5b-9, became insensitive to lysis. Furthermore, we found that C8bp is incorporated into the membrane-attack complex C5b-9, most probably by interacting with C8, since C8bp has an affinity for C8, particularly for the C8 alpha-gamma-subunit. Antibodies to C8bp react with the C8 alpha-subunits and with C9, pointing to the possibility of a partial homology between these proteins. Images Figure 4 Figure 6 Figure 7 PMID:3366469

Loxoprofen sodium induces the production of complement C5a in human serum.

PubMed

Kumagai, Tomoaki; Yamaguchi, Nozomi; Hirai, Hiroyuki; Kojima, Shigeyuki; Kodani, Yoshiko; Hashiguchi, Akihiko; Haida, Michiko; Nakamura, Masataka

2016-04-01

Basophil activation test (BAT) is an in vitro allergy test that is useful to identify allergens that cause IgE-dependent allergies. The test has been used to detect not only food allergies and allergies caused by environmental factors but also to detect drug hypersensitivity, which has been known to include IgE-independent reactions. In our preliminary studies in which BAT was applied to detect hypersensitivity of loxoprofen, a non-steroidal anti-inflammatory drug (NSAID), conventional BAT with incubation for 30min did not show basophil activation by means of increased CD203c expression. In this study, we extended the incubation time to 24h on the basis of the hypothesis that loxoprofen indirectly activates basophils. Basophils from healthy control donors as well as allergic patients showed up-regulation of CD203c after incubation with loxoprofen for 24h. Activation was induced using loxoprofen-treated serum. Proteomic and pharmacologic analyses revealed that serum incubation with loxoprofen generated an active complement component C5a, which induced CD203c expression via binding to the C5a receptor on basophils. Because C3a production was also detected after incubation for 24h, loxoprofen is likely to stimulate the complement classical pathway. Our findings suggest that the complement activation is involved in drug hypersensitivity and the suppression of this activation may contribute to the elimination of false positive of BAT for drug allergies. Copyright © 2016 Elsevier B.V. All rights reserved.

NF-κB and enhancer-binding CREB protein scaffolded by CREB-binding protein (CBP)/p300 proteins regulate CD59 protein expression to protect cells from complement attack.

PubMed

Du, Yiqun; Teng, Xiaoyan; Wang, Na; Zhang, Xin; Chen, Jianfeng; Ding, Peipei; Qiao, Qian; Wang, Qingkai; Zhang, Long; Yang, Chaoqun; Yang, Zhangmin; Chu, Yiwei; Du, Xiang; Zhou, Xuhui; Hu, Weiguo

2014-01-31

The complement system can be activated spontaneously for immune surveillance or induced to clear invading pathogens, in which the membrane attack complex (MAC, C5b-9) plays a critical role. CD59 is the sole membrane complement regulatory protein (mCRP) that restricts MAC assembly. CD59, therefore, protects innocent host cells from attacks by the complement system, and host cells require the constitutive and inducible expression of CD59 to protect themselves from deleterious destruction by complement. However, the mechanisms that underlie CD59 regulation remain largely unknown. In this study we demonstrate that the widely expressed transcription factor Sp1 may regulate the constitutive expression of CD59, whereas CREB-binding protein (CBP)/p300 bridge NF-κB and CREB, which surprisingly functions as an enhancer-binding protein to induce the up-regulation of CD59 during in lipopolysaccharide (LPS)-triggered complement activation, thus conferring host defense against further MAC-mediated destruction. Moreover, individual treatment with LPS, TNF-α, and the complement activation products (sublytic MAC (SC5b-9) and C5a) could increase the expression of CD59 mainly by activating NF-κB and CREB signaling pathways. Together, our findings identify a novel gene regulation mechanism involving CBP/p300, NF-κB, and CREB; this mechanism suggests potential drug targets for controlling various complement-related human diseases.

Complement C5a-C5aR interaction enhances MAPK signaling pathway activities to mediate renal injury in trichloroethylene sensitized BALB/c mice.

PubMed

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

2016-02-01

We have previously shown complement activation as a possible mechanism for trichloroethylene (TCE) sensitization, leading to multi-organ damage including the kidneys. In particular, excessive deposition of C5 and C5b-9-the membrane attack complex, which can generate significant tissue damage, was observed in the kidney tissue after TCE sensitization. The present study tested the hypothesis that anaphylatoxin C5a binding to its receptor C5aR mediates renal injury in TCE-sensitized BALB/c mice. BALB/c mice were sensitized through skin challenge with TCE, with or without pretreatment by the C5aR antagonist W54011. Kidney histopathology and the renal functional test were performed to assess renal injury, and immunohistochemistry and fluorescent labeling were carried out to assess C5a and C5aR expressions. TCE sensitization up-regulated C5a and C5aR expressions in kidney tissue, generated inflammatory infiltration, renal tubule damage, glomerular hypercellularity and impaired renal function. Antagonist pretreatment blocked C5a binding to C5aR and attenuated TCE-induced tissue damage and renal dysfunction. TCE sensitization also caused the deposition of major pro-inflammatory cytokines IL-2, TNF-α and IFN-γ in the kidney tissue (P < 0.05); this was accompanied by increased expression of P-p38, P-ERK and P-JNK proteins (P < 0.05). Pretreatment with the C5aR antagonist attenuated the increase of expression of P-p38, P-ERK and P-JNK proteins (P < 0.05) and also consistently reduced the TCE sensitization-induced increase of IL-2, TNF-α and IFN-γ (P < 0.05). These data identify C5a binding to C5aR, MAP kinase activation, and inflammatory cytokine release as a novel mechanism for complement-mediated renal injury by sensitization with TCE or other environmental chemicals. Copyright © 2015 John Wiley & Sons, Ltd.

Phylogenetic aspects of the complement system.

PubMed

Zarkadis, I K; Mastellos, D; Lambris, J D

2001-01-01

During evolution two general systems of immunity have emerged: innate or, natural immunity and adaptive (acquired), or specific immunity. The innate system is phylogenetically older and is found in some form in all multicellular organisms, whereas the adaptive system appeared about 450 million years ago and is found in all vertebrates except jawless fish. The complement system in higher vertebrates plays an important role as an effector of both the innate and the acquired immune response, and also participates in various immunoregulatory processes. In lower vertebrates complement is activated by the alternative and lectin pathways and is primarily involved in the opsonization of foreign material. The Agnatha (the most primitive vertebrate species) possess the alternative and lectin pathways while cartilaginous fish are the first species in which the classical pathway appears following the emergence of immunoglobulins. The rest of the poikilothermic species, ranging from teleosts to reptilians, appear to contain a well-developed complement system resembling that of the homeothermic vertebrates. It seems that most of the complement components have appeared after the duplication of primordial genes encoding C3/C4/C5, fB/C2, C1s/C1r/MASP-1/MASP-2, and C6/C7/C8/C9 molecules, in a process that led to the formation of distinct activation pathways. However, unlike homeotherms, several species of poikilotherms (e.g. trout) have recently been shown to possess multiple forms of complement components (C3, factor B) that are structurally and functionally more diverse than those of higher vertebrates. We hypothesize that this remarkable diversity has allowed these animals to expand their innate capacity for immune recognition and response. Recent studies have also indicated the possible presence of complement receptors in protochordates and lower vertebrates. In conclusion, there is considerable evidence suggesting that the complement system is present in the entire lineage of deuterostomes, and regulatory complement components have been identified in all species beyond the protochordates, indicating that the mechanisms of complement activation and regulation have developed in parallel.

Site-targeted complement inhibition by a complement receptor 2-conjugated inhibitor (mTT30) ameliorates post-injury neuropathology in mouse brains.

PubMed

Rich, Megan C; Keene, Chesleigh N; Neher, Miriam D; Johnson, Krista; Yu, Zhao-Xue; Ganivet, Antoine; Holers, V Michael; Stahel, Philip F

2016-03-23

Intracerebral complement activation after severe traumatic brain injury (TBI) leads to a cascade of neuroinflammatory pathological sequelae that propagate host-mediated secondary brain injury and adverse outcomes. There are currently no specific pharmacological agents on the market to prevent or mitigate the development of secondary cerebral insults after TBI. A novel chimeric CR2-fH compound (mTT30) provides targeted inhibition of the alternative complement pathway at the site of tissue injury. This experimental study was designed to test the neuroprotective effects of mTT30 in a mouse model of closed head injury. The administration of 500 μg mTT30 i.v. at 1 h, 4 h and 24 h after head injury attenuated complement C3 deposition in injured brains, reduced the extent of neuronal cell death, and decreased post-injury microglial activation, compared to vehicle-injected placebo controls. These data imply that site-targeted alternative pathway complement inhibition may represent a new promising therapeutic avenue for the future management of severe TBI. Copyright © 2016. Published by Elsevier Ireland Ltd.

Anti-complement activities of human breast-milk.

PubMed

Ogundele, M O

1999-08-01

It has long been observed that the human milk possesses significant anti-inflammatory properties, while simultaneously protecting the infant against many intestinal and respiratory pathogens. There is, however, a paucity of information on the degree and extent of this anti-inflammatory activity. In the present study, the inhibitory effects of different fractions of human milk on serum complement activity were analysed. Colostrum and milk samples from healthy voluntary lactating donors at different postpartum ages were obtained and pooled normal human serum was used as source of complement in a modified CH50 assay. Inherent complement activity in human milk was also investigated by measuring the deposition of an activated C3 fragment on a serum-sensitive bacteria, and by haemolytic assays. Most whole- and defatted-milk samples consistently showed a dose-dependent inhibition of the serum complement activity. This inhibition was greater in mature milk compared to transitional milk samples. It was enhanced by inactivation of milk complement, and diminished by centrifugation of milk samples, which partly removed fat and larger protein components including casein micelles. Inherent complement activity in human milk was also demonstrated by haemolysis of sensitised sheep erythrocytes and deposition of C3 fragments on solid-phase bacteria. These activities were highest in the colostrum and gradually decreased as lactation proceeded. Several natural components abundant in the fluid phase of the human breast-milk have been shown to be inhibitors of complement activation in vitro. Their physiological significance probably reside in their ability to prevent inflammatory-induced tissue damage of the delicate immature gastrointestinal tract of the new-born as well as the mammary gland itself, which may arise from ongoing complement activation.

Chimeras of human complement C9 reveal the site recognized by complement regulatory protein CD59.

PubMed

Hüsler, T; Lockert, D H; Kaufman, K M; Sodetz, J M; Sims, P J

1995-02-24

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex, thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective and is most effective toward C9 derived from human or other primate plasma. By contrast, rabbit C9, which can substitute for human C9 in the membrane attack complex, mediates unrestricted lysis of human cells. To identify the peptide segment of human C9 that is recognized by CD59, rabbit C9 cDNA clones were isolated, characterized, and used to construct hybrid cDNAs for expression of full-length human/rabbit C9 chimeras in COS-7 cells. All resulting chimeras were hemolytically active, when tested against chicken erythrocytes bearing C5b-8 complexes. Assays performed in the presence or absence of CD59 revealed that this inhibitor reduced the hemolytic activity of those chimeras containing human C9 sequence between residues 334-415, irrespective of whether the remainder of the protein contained human or rabbit sequence. By contrast, when this segment of C9 contained rabbit sequence, lytic activity was unaffected by CD59. These data establish that human C9 residues 334-415 contain the site recognized by CD59, and they suggest that sequence variability within this segment of C9 is responsible for the observed species-selective inhibitory activity of CD59.

Role of Complement C5 in Experimental Blunt Chest Trauma-Induced Septic Acute Lung Injury (ALI)

PubMed Central

Karbach, Michael; Braumueller, Sonja; Kellermann, Philipp; Gebhard, Florian; Huber-Lang, Markus; Perl, Mario

2016-01-01

Background Severe blunt chest trauma is associated with high mortality. Sepsis represents a serious risk factor for mortality in acute respiratory distress syndrome (ARDS). In septic patients with ARDS complement activation products were found to be elevated in the plasma. In single models like LPS or trauma complement has been studied to some degree, however in clinically highly relevant double hit models such as the one used here little data is available. Here, we hypothesized that absence of C5 is correlated with a decreased inflammatory response in trauma induced septic acute lung injury. Methods 12 hrs after DH in mice the local and systemic cytokines and chemokines were quantified by multiplex bead array or ELISA, activated caspase-3 by western blot. Data were analyzed using one-way ANOVA followed by post-hoc Sidak’s multiple comparison test (significance, p≤ 0.05). Results In lung tissue interleukin (IL)-6, monocyte chemo attractant protein-1 (MCP-1) and granulocyte-colony stimulating factor (G-CSF) was elevated in both C5-/- mice and wildtype littermates (wt), whereas caspase-3 was reduced in lungs after DH in C5-/- mice. Systemically, reduced keratinocyte-derived chemokine (KC) levels were observed after DH in C5-/- compared to wt mice. Locally, lung myeloperoxidase (MPO), protein, IL-6, MCP-1 and G-CSF in brochoalveolar lavage fluid (BALF) were elevated after DH in C5-/- compared to wt. Conclusions In the complex but clinically relevant DH model the local and systemic inflammatory immune response features both, C5-dependent and C5-independent characteristics. Activation of caspase-3 in lung tissue after DH was C5-dependent whereas local inflammation in lung tissue was C5-independent. PMID:27437704

Role of Complement C5 in Experimental Blunt Chest Trauma-Induced Septic Acute Lung Injury (ALI).

PubMed

Kalbitz, Miriam; Karbach, Michael; Braumueller, Sonja; Kellermann, Philipp; Gebhard, Florian; Huber-Lang, Markus; Perl, Mario

2016-01-01

Severe blunt chest trauma is associated with high mortality. Sepsis represents a serious risk factor for mortality in acute respiratory distress syndrome (ARDS). In septic patients with ARDS complement activation products were found to be elevated in the plasma. In single models like LPS or trauma complement has been studied to some degree, however in clinically highly relevant double hit models such as the one used here little data is available. Here, we hypothesized that absence of C5 is correlated with a decreased inflammatory response in trauma induced septic acute lung injury. 12 hrs after DH in mice the local and systemic cytokines and chemokines were quantified by multiplex bead array or ELISA, activated caspase-3 by western blot. Data were analyzed using one-way ANOVA followed by post-hoc Sidak's multiple comparison test (significance, p≤ 0.05). In lung tissue interleukin (IL)-6, monocyte chemo attractant protein-1 (MCP-1) and granulocyte-colony stimulating factor (G-CSF) was elevated in both C5-/- mice and wildtype littermates (wt), whereas caspase-3 was reduced in lungs after DH in C5-/- mice. Systemically, reduced keratinocyte-derived chemokine (KC) levels were observed after DH in C5-/- compared to wt mice. Locally, lung myeloperoxidase (MPO), protein, IL-6, MCP-1 and G-CSF in brochoalveolar lavage fluid (BALF) were elevated after DH in C5-/- compared to wt. In the complex but clinically relevant DH model the local and systemic inflammatory immune response features both, C5-dependent and C5-independent characteristics. Activation of caspase-3 in lung tissue after DH was C5-dependent whereas local inflammation in lung tissue was C5-independent.

Translational Mini-Review Series on Complement Factor H: Structural and functional correlations for factor H

PubMed Central

Schmidt, C Q; Herbert, A P; Hocking, H G; Uhrín, D; Barlow, P N

2008-01-01

The 155-kDa glycoprotein, complement factor H (CFH), is a regulator of complement activation that is abundant in human plasma. Three-dimensional structures of over half the 20 complement control protein (CCP) modules in CFH have been solved in the context of single-, double- and triple-module segments. Proven binding sites for C3b occupy the N and C termini of this elongated molecule and may be brought together by a bend in CFH mediated by its central CCP modules. The C-terminal CCP 20 is key to the ability of the molecule to adhere to polyanionic markers on self-surfaces where CFH acts to regulate amplification of the alternative pathway of complement. The surface patch on CCP 20 that binds to model glycosaminoglycans has been mapped using nuclear magnetic resonance (NMR), as has a second glycosaminoglycan-binding patch on CCP 7. These patches include many of the residue positions at which sequence variations have been linked to three complement-mediated disorders: dense deposit disease, age-related macular degeneration and atypical haemolytic uraemic syndrome. In one plausible model, CCP 20 anchors CFH to self-surfaces via a C3b/polyanion composite binding site, CCP 7 acts as a ‘proof-reader’ to help discriminate self- from non-self patterns of sulphation, and CCPs 1–4 disrupt C3/C5 convertase formation and stability. PMID:18081691

Distinct polymer architecture mediates switching of complement activation pathways at the nanosphere-serum interface: implications for stealth nanoparticle engineering.

PubMed

Hamad, Islam; Al-Hanbali, Othman; Hunter, A Christy; Rutt, Kenneth J; Andresen, Thomas L; Moghimi, S Moein

2010-11-23

Nanoparticles with surface projected polyethyleneoxide (PEO) chains in "mushroom-brush" and "brush" configurations display stealth properties in systemic circulation and have numerous applications in site-specific targeting for controlled drug delivery and release as well as diagnostic imaging. We report on the "structure-activity" relationship pertaining to surface-immobilized PEO of various configurations on model nanoparticles, and the initiation of complement cascade, which is the most ancient component of innate human immunity, and its activation may induce clinically significant adverse reactions in some individuals. Conformational states of surface-projected PEO chains, arising from the block copolymer poloxamine 908 adsorption, on polystyrene nanoparticles trigger complement activation differently. Alteration of copolymer architecture on nanospheres from mushroom to brush configuration not only switches complement activation from C1q-dependent classical to lectin pathway but also reduces the level of generated complement activation products C4d, Bb, C5a, and SC5b-9. Also, changes in adsorbed polymer configuration trigger alternative pathway activation differently and through different initiators. Notably, the role for properdin-mediated activation of alternative pathway was only restricted to particles displaying PEO chains in a transition mushroom-brush configuration. Since nanoparticle-mediated complement activation is of clinical concern, our findings provide a rational basis for improved surface engineering and design of immunologically safer stealth and targetable nanosystems with polymers for use in clinical medicine.

Effects of partial replacement of fish meal by yeast hydrolysate on complement system and stress resistance in juvenile Jian carp (Cyprinus carpio var. Jian).

PubMed

Yuan, Xiang-Yang; Liu, Wen-Bin; Liang, Chao; Sun, Cun-Xin; Xue, Yun-Fei; Wan, Zu-De; Jiang, Guang-Zhen

2017-08-01

A 10-week feeding trial was carried out to investigate the effects of dietary fish meal replacement by yeast hydrolysate (YH) on growth performance, complement system and stress resistance of juvenile Jian carp (Cyprinus carpio var. Jian) (initial average weight 19.44 ± 0.06 g). In the study, there were five groups: one control group was fed with a basal diet (YH0), and four treatment groups were fed with dietary fish meal replaced by 1% YH (YH1), 3% (YH3), 5% (YH5) and 7% (YH7), respectively. Each group had four replicates. At the end of feeding trial, twelve fish from each group (three fish per replicate) were randomly selected for assessing the growth and immunity. Meanwhile, 20 fish per replicate were injected by Aeromonas hydrophila. The results showed that (1) Replacement levels of YH significantly affected the growth of the fish with the highest values of weight gain (WG) occurred in fish fed YH3 diet. However, no significant difference in feed conversion ratios (FCR) was observed among all groups. (2) Pre-stressed plasma lysozyme activity, total protein and albumin contents and complement component 3 (C3) and complement component 4 (C4) levels of fish fed YH3 diet were significantly higher than those of fish fed YH0 diet. However, post-stressed immune parameters of fish in all groups were significantly lower. (3) There was a trend that the expression levels of the complement-related genes (c1r/s-A, c4-1, c3-H1, c5-1, fb/c2-A, mbl-2 and masp) initially increased and then decreased except mbl-2 and masp, with the maximum values observed in fish fed YH3 diet. Before stress, the expression levels of the inflammation-related genes (alp, il-1β and tnf-α) in the hepatopancreas and spleen of fish fed YH1 diet and YH7 diet were significant higher than that of fish fed YH0 diet. After stress, no significant difference in the expression levels of those genes was observed among all groups. These results indicated that FM replacement by YH could improve growth performance, enhance innate immunity, and activate complement via the alternative complement pathway (ACP) and the classical complement pathway (CCP). Copyright © 2017 Elsevier Ltd. All rights reserved.

Alternative complement pathway activation during invasive coronary procedures in acute myocardial infarction and stable angina pectoris.

PubMed

Horváth, Zsófia; Csuka, Dorottya; Vargova, Katarina; Kovács, Andrea; Leé, Sarolta; Varga, Lilian; Préda, István; Tóth Zsámboki, Emese; Prohászka, Zoltán; Kiss, Róbert Gábor

2016-12-01

The effect of invasive percutaneous coronary procedures on complement activation has not been elucidated. We enrolled stable angina patients with elective percutaneous coronary intervention (SA-PCI, n=24), diagnostic coronary angiography (CA, n=52) and 23 patients with ST segment elevation myocardial infarction and primary PCI (STEMI-PCI). Complement activation products (C1rC1sC1inh, C3bBbP and SC5b-9) were measured on admission, 6 and 24h after coronary procedures. The alternative pathway product, C3bBbP significantly and reversibly increased 6h after elective PCI (baseline: 7.81AU/ml, 6h: 16.09AU/ml, 24h: 4.27AU/ml, p<0.01, n=23) and diagnostic angiography (baseline: 6.13AU/ml, 6h: 12.08AU/ml, 24h: 5.4AU/ml, p<0.01, n=52). Six hour C3bBbP values correlated with post-procedural CK, creatinine level and the applied contrast material volume (r=0.41, r=0.4, r=0.3, p<0.05, respectively). In STEMI-PCI, baseline C3bBbP level was higher, compared to SA-PCI or CA patients (11.33AU/ml vs. 7.81AU/ml or 6.13AU/ml, p<0.001). Similarly, the terminal complex (SC5b-9) level was already elevated at baseline compared to SA-PCI group (3.49AU/ml vs. 1.87AU/ml, p=0.011). Complement pathway products did not increase further after primary PCI. Elective coronary procedures induced transient alternative complement pathway activation, influenced by the applied contrast volume. In STEMI, the alternative complement pathway is promptly activated during the atherothrombotic event and PCI itself had no further detectable effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Role of Complement on Broken Surfaces After Trauma.

PubMed

Huber-Lang, Markus; Ignatius, Anita; Brenner, Rolf E

2015-01-01

Activation of both the complement and coagulation cascade after trauma and subsequent local and systemic inflammatory response represent a major scientific and clinical problem. After severe tissue injury and bone fracture, exposure of innate immunity to damaged cells and molecular debris is considered a main trigger of the posttraumatic danger response. However, the effects of cellular fragments (e.g., histones) on complement activation remain enigmatic. Furthermore, direct effects of "broken" bone and cartilage surfaces on the fluid phase response of complement and its interaction with key cells of connective tissues are still unknown. Here, we summarize data suggesting direct and indirect complement activation by extracellular and cellular danger associated molecular patterns. In addition, key complement components and the corresponding receptors (such as C3aR, C5aR) have been detected on "exposed surfaces" of the damaged regions. On a cellular level, multiple effects of complement activation products on osteoblasts, osteoclasts, chondrocytes and mesenchymal stem cells have been found.In conclusion, the complement system may be activated by trauma-altered surfaces and is crucially involved in connective tissue healing and posttraumatic systemic inflammatory response.

Eculizumab for dense deposit disease and C3 glomerulonephritis.

PubMed

Bomback, Andrew S; Smith, Richard J; Barile, Gaetano R; Zhang, Yuzhou; Heher, Eliot C; Herlitz, Leal; Stokes, M Barry; Markowitz, Glen S; D'Agati, Vivette D; Canetta, Pietro A; Radhakrishnan, Jai; Appel, Gerald B

2012-05-01

The principle defect in dense deposit disease and C3 glomerulonephritis is hyperactivity of the alternative complement pathway. Eculizumab, a monoclonal antibody that binds to C5 to prevent formation of the membrane attack complex, may prove beneficial. In this open-label, proof of concept efficacy and safety study, six subjects with dense deposit disease or C3 glomerulonephritis were treated with eculizumab every other week for 1 year. All had proteinuria >1 g/d and/or AKI at enrollment. Subjects underwent biopsy before enrollment and repeat biopsy at the 1-year mark. The subjects included three patients with dense deposit disease (including one patient with recurrent dense deposit disease in allograft) and three patients with C3 glomerulonephritis (including two patients with recurrent C3 glomerulonephritis in allograft). Genetic and complement function testing revealed a mutation in CFH and MCP in one subject each, C3 nephritic factor in three subjects, and elevated levels of serum membrane attack complex in three subjects. After 12 months, two subjects showed significantly reduced serum creatinine, one subject achieved marked reduction in proteinuria, and one subject had stable laboratory parameters but histopathologic improvements. Elevated serum membrane attack complex levels normalized on therapy and paralleled improvements in creatinine and proteinuria. Clinical and histopathologic data suggest a response to eculizumab in some but not all subjects with dense deposit disease and C3 glomerulonephritis. Elevation of serum membrane attack complex before treatment may predict response. Additional research is needed to define the subgroup of dense deposit disease/C3 glomerulonephritis patients in whom eculizumab therapy can be considered.

The profile of adsorbed plasma and serum proteins on methacrylic acid copolymer beads: Effect on complement activation.

PubMed

Wells, Laura A; Guo, Hongbo; Emili, Andrew; Sefton, Michael V

2017-02-01

Polymer beads made of 45% methacrylic acid co methyl methacrylate (MAA beads) promote vascular regenerative responses in contrast to control materials without methacrylic acid (here polymethyl methacrylate beads, PMMA). In vitro and in vivo studies suggest that MAA copolymers induce differences in macrophage phenotype and polarization and inflammatory responses, presumably due to protein adsorption differences between the beads. To explore differences in protein adsorption in an unbiased manner, we used high resolution shotgun mass spectrometry to identify and compare proteins that adsorb from human plasma or serum onto MAA and PMMA beads. From plasma, MAA beads adsorbed many complement proteins, such as C1q, C4-related proteins and the complement inhibitor factor H, while PMMA adsorbed proteins, such as albumin, C3 and apolipoproteins. Because of the differences in complement protein adsorption, follow-up studies focused on using ELISA to assess complement activation. When incubated in serum, MAA beads generated significantly lower levels of soluble C5b9 and C3a/C3a desarg in comparison to PMMA beads, indicating a decrease in complement activation with MAA beads. The differences in adsorbed protein on the two materials likely alter subsequent cell-material interactions that ultimately result in different host responses and local vascularization. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of C1q as a Binding Protein for Advanced Glycation End Products.

PubMed

Chikazawa, Miho; Shibata, Takahiro; Hatasa, Yukinori; Hirose, Sayumi; Otaki, Natsuki; Nakashima, Fumie; Ito, Mika; Machida, Sachiko; Maruyama, Shoichi; Uchida, Koji

2016-01-26

Advanced glycation end products (AGEs) make up a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum proteins that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGE-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including a collagen-like domain, as the AGE-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 to release C5a. These findings provide the first evidence of AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway.

Micrurus snake venoms activate human complement system and generate anaphylatoxins

PubMed Central

2012-01-01

Background The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. Results In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. Conclusion Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process. PMID:22248157

Micrurus snake venoms activate human complement system and generate anaphylatoxins.

PubMed

Tanaka, Gabriela D; Pidde-Queiroz, Giselle; de Fátima D Furtado, Maria; van den Berg, Carmen; Tambourgi, Denise V

2012-01-16

The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process.

Targeting the Human Complement Membrane Attack Complex to Selectively Kill Prostate Cancer Cells

DTIC Science & Technology

2013-10-01

prostate cancer cells in vitro . Evaluate CD59 expression in human prostate cancer microarrays. Aim 4: Evaluate toxicity and efficacy of the lead PAC5...fragment in vitro . Since PSA is the major chymotrypsin-like serine protease in the seminal plasma and prostatic fluid, we hypothesized that PSA was...that the evolution -related complement protein C5, but not C4, is a substrate of PSA as well. *Department of Pharmacology and Molecular Sciences, The

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. Copyright © 2010 Elsevier GmbH. All rights reserved.

Complement C5 controls liver lipid profile, promotes liver homeostasis and inflammation in C57BL/6 genetic background.

PubMed

Bavia, Lorena; de Castro, Íris Arantes; Cogliati, Bruno; Dettoni, Juliano Bertollo; Alves, Venancio Avancini Ferreira; Isaac, Lourdes

2016-07-01

Innate immunity contributes effectively to the development of alcoholic liver disease (ALD). In special, the activation of the complement system is involved in the pathogenesis of this disease. Here we investigated the contribution of complement C5 protein to the establishment and maintenance of ALD. Eight- to ten-week-old B6C5(+) and B6C5(-) male mice were fed with high fat diet (HFD) only or the same diet containing equicaloric supplements of ethanol (HFDE) or maltodextrin (HFDM) for 10 weeks. Serum parameters of liver function as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), albumin, glucose, triglycerides (TG) and cholesterol were evaluated. Liver tissue samples were collected for histopathological analysis, lipid extraction (TG and cholesterol), cytokines (TNF-α, IL-6, IL-1β, IL-10, IL-12, IL-17, IFN-γ, TGF-β) measurement and NO production. We observed that B6C5(-) mice HFDE-fed accumulated more liver cholesterol and TG, increased liver IL-17 and IL-10 levels and reduced liver TGF-β levels when compared to HFD-fed mice. We also observed that serum AST, AP and albumin were increased in B6C5(-) mice. Liver IL-1β, IL-6, IL-12 and IFN-γ were decreased in B6C5(-) mice independently of diet. We conclude that C5 acts in the control of serum TG and cholesterol, liver cholesterol deposition, liver homeostasis and C5 promotes a pro-inflammatory liver environment in our mouse model of ALD. Copyright © 2016 Elsevier GmbH. All rights reserved.

Function of Serum Complement in Drinking Water Arsenic Toxicity

PubMed Central

Islam, Laila N.; Zahid, M. Shamim Hasan; Nabi, A. H. M. Nurun; Hossain, Mahmud

2012-01-01

Serum complement function was evaluated in 125 affected subjects suffering from drinking water arsenic toxicity. Their mean duration of exposure was 7.4 ± 5.3 yrs, and the levels of arsenic in drinking water and urine samples were 216 ± 211 and 223 ± 302 μg/L, respectively. The mean bactericidal activity of complement from the arsenic patients was 92% and that in the unexposed controls was 99% (P < 0.01), but heat-inactivated serum showed slightly elevated activity than in controls. In patients, the mean complement C3 was 1.56 g/L, and C4 was 0.29 g/L compared to 1.68 g/L and 0.25 g/L, respectively, in the controls. The mean IgG in the arsenic patients was 24.3 g/L that was highly significantly elevated (P < 0.001). Arsenic patients showed a significant direct correlation between C3 and bactericidal activity (P = 0.014). Elevated levels of C4 indicated underutilization and possibly impaired activity of the classical complement pathway. We conclude reduced function of serum complement in drinking water arsenic toxicity. PMID:22545044

Immune functions of the garment workers.

PubMed

Sultana, R; Ferdous, K J; Hossain, M; Zahid, M S H; Islam, L N

2012-10-01

Occupational exposure to cotton dust, fibers, metal fumes and different chemicals used in the aparrel manufacturing industries cause a wide range of physical and psychological health problems in the garment workers that may also affect their immune function. To assess the immune system function in garment workers. A total of 45 workers of a garment factory, and 41 control subjects, not exposed to the garment working environment were enrolled in this study. In the study subjects, the complement system function was assessed as bactericidal activity on Escherichia coli DH5α cells using the standard plate count method. Serum complement components C3 and C4 were measured by immunoprecipitation, and IgG was measured by immunonephelometry. The bactericidal activity of serum complement in the garment workers (range: 93.5%-99.9%) was significantly (p<0.01) lower than that in the controls (range: 98.6%-100%). The heat-inactivated serum of the workers showed a significantly enhanced bactericidal activity. In the garment workers, the mean levels of complement C3, and C4 were 1.75 and 0.26 g/L, respectively that were close to those of the controls. The mean IgG level in the garment workers was 13.5 g/L that was significantly (p<0.001) higher than that in the controls. Working in a garment factory may affect the immune system.

The Lectin Complement Pathway Is Involved in Protection Against Enteroaggregative Escherichia coli Infection.

PubMed

Adler Sørensen, Camilla; Rosbjerg, Anne; Hebbelstrup Jensen, Betina; Krogfelt, Karen Angeliki; Garred, Peter

2018-01-01

Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea worldwide. Still, the involvement of host factors in EAEC infections is unresolved. Binding of recognition molecules from the lectin pathway of complement to EAEC strains have been observed, but the importance is not known. Our aim was to uncover the involvement of these molecules in innate complement dependent immune protection toward EAEC. Binding of mannose-binding lectin, ficolin-1, -2, and -3 to four prototypic EAEC strains, and ficolin-2 binding to 56 clinical EAEC isolates were screened by a consumption-based ELISA method. Flow cytometry was used to determine deposition of C4b, C3b, and the bactericidal C5b-9 membrane attack complex (MAC) on the bacteria in combination with different complement inhibitors. In addition, the direct serum bactericidal effect was assessed. Screening of the prototypic EAEC strains revealed that ficolin-2 was the major binder among the lectin pathway recognition molecules. However, among the clinical EAEC isolates only a restricted number ( n  = 5) of the isolates bound ficolin-2. Using the ficolin-2 binding isolate C322-17 as a model, we found that incubation with normal human serum led to deposition of C4b, C3b, and to MAC formation. No inhibition of complement deposition was observed when a C1q inhibitor was added, while partial inhibition was observed when ficolin-2 or factor D inhibitors were used separately. Combining the inhibitors against ficolin-2 and factor D led to virtually complete inhibition of complement deposition and protection against direct bacterial killing. These results demonstrate that ficolin-2 may play an important role in innate immune protection against EAEC when an appropriate ligand is exposed, but many EAEC strains evade lectin pathway recognition and may, therefore, circumvent this strategy of innate host immune protection.

Rituximab fails where eculizumab restores renal function in C3nef-related DDD.

PubMed

Rousset-Rouvière, Caroline; Cailliez, Mathilde; Garaix, Florentine; Bruno, Daniele; Laurent, Daniel; Tsimaratos, Michel

2014-06-01

Dense deposit disease (DDD), a C3 glomerulopathy (C3G), is a rare disease with unfavorable progression towards end-stage kidney disease. The pathogenesis of DDD is due to cytotoxic effects related to acquired or genetic dysregulation of the complement alternative pathway, which is at times accompanied by the production of C3 nephritic factor (C3NeF), an auto-antibody directed against the alternative C3 convertase. Available treatments include plasma exchange, CD20-targeted antibodies, and a terminal complement blockade via the anti-C5 monoclonal antibody eculizumab. We report here the case of an 8-year-old child with C3NeF and refractory DDD who presented with a nephritic syndrome. She tested positive for C3NeF activity; C3 was undetectable. Genetic analyses of the alternative complement pathway were normal. Methylprednisolone pulses and mycophenolate mofetil treatment resulted in complete recovery of renal function and a reduction in proteinuria. Corticosteroids were tapered and then withdrawn. Four months after corticosteroid discontinuation, hematuria and proteinuria recurred, and a renal biopsy confirmed an active DDD with a majority of extracapillary crescents. Despite an increase in immunosuppressive drugs, including methylprednisolone pulses and rituximab therapy, the patient suffered acute renal failure within 3 weeks, requiring dialysis. Eculizumab treatment resulted in a quick and impressive response. Hematuria very quickly resolved, kidney function improved, and no further dialysis was required. The patient received bimonthly eculizumab injections of 600 mg, allowing for normalization of renal function and reduction of proteinuria to <0.5 g per day. Since then, she continues to receive eculizumab. Complement regulation pathway-targeted therapy may be a specific and useful treatment for rapidly progressing DDD prior to the development of glomerulosclerosis. Our data provide evidence supporting the pivotal role of complement alternative pathway abnormalities in C3G with DDD.

Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

PubMed Central

Shpakovski, G V; Acker, J; Wintzerith, M; Lacroix, J F; Thuriaux, P; Vigneron, M

1995-01-01

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit. PMID:7651387

Stimulation of complement component C3 synthesis in macrophagelike cell lines by group B streptococci.

PubMed Central

Goodrum, K J

1987-01-01

Complement levels and complement activation are key determinants in streptococcus-induced inflammatory responses. Activation of macrophage functions, such as complement synthesis, by group B streptococci (GBS) was examined as a possible component of GBS-induced chronic inflammation. Using an enzyme-linked immunosorbent assay, secreted C3 from mouse macrophagelike cell lines (PU5-1.8 and J774A.1) was monitored after cultivation with GBS. Whole, heat-killed GBS (1 to 10 CFU per macrophage) of both type Ia and III strains induced 25 to 300% increases in secreted C3 in both cell lines after a 24-h cultivation. GBS-treated cell lines exhibited increases in secreted lysozyme (10%) and in cellular protein (25 to 50%). Inhibition of macrophage phagocytosis by cytochalasin B inhibited GBS stimulation of C3. Purified cell walls of GBS type III strain 603-79 (1 to 10 micrograms/ml) also enhanced C3 synthesis. Local enhancement of macrophage C3 production by ingested streptococci or by persistent cell wall antigens may serve to promote chronic inflammatory responses. PMID:3552987

C5a alters blood-brain barrier integrity in experimental lupus.

PubMed

Jacob, Alexander; Hack, Bradley; Chiang, Eddie; Garcia, Joe G N; Quigg, Richard J; Alexander, Jessy J

2010-06-01

The blood-brain barrier (BBB) is a crucial anatomic location in the brain. Its dysfunction complicates many neurodegenerative diseases, from acute conditions, such as sepsis, to chronic diseases, such as systemic lupus erythematosus (SLE). Several studies suggest an altered BBB in lupus, but the underlying mechanism remains unknown. In the current study, we observed a definite loss of BBB integrity in MRL/MpJ-Tnfrsf6(lpr) (MRL/lpr) lupus mice by IgG infiltration into brain parenchyma. In line with this result, we examined the role of complement activation, a key event in this setting, in maintenance of BBB integrity. Complement activation generates C5a, a molecule with multiple functions. Because the expression of the C5a receptor (C5aR) is significantly increased in brain endothelial cells treated with lupus serum, the study focused on the role of C5a signaling through its G-protein-coupled receptor C5aR in brain endothelial cells, in a lupus setting. Reactive oxygen species production increased significantly in endothelial cells, in both primary cells and the bEnd3 cell line treated with lupus serum from MRL/lpr mice, compared with those treated with control serum from MRL(+/+) mice. In addition, increased permeability monitored by changes in transendothelial electrical resistance, cytoskeletal remodeling caused by actin fiber rearrangement, and increased iNOS mRNA expression were observed in bEnd3 cells. These disruptive effects were alleviated by pretreating cells with a C5a receptor antagonist (C5aRant) or a C5a antibody. Furthermore, the structural integrity of the vasculature in MRL/lpr brain was maintained by C5aR inhibition. These results demonstrate the regulation of BBB integrity by the complement system in a neuroinflammatory setting. For the first time, a novel role of C5a in the maintenance of BBB integrity is identified and the potential of C5a/C5aR blockade highlighted as a promising therapeutic strategy in SLE and other neurodegenerative diseases.

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.

Binding of human and rat CD59 to the terminal complement complexes.

PubMed Central

Lehto, T; Morgan, B P; Meri, S

1997-01-01

CD59-antigen (protectin) is a widely distributed glycolipid-anchored inhibitor of complement lysis. CD59 interacts with complement components C8 and C9 during assembly of the membrane attack complex (MAC). To evaluate species specificity of these interactions we have in the present study examined cross-species binding of isolated human and rat CD59 to the terminal complement components C8 and C9. By using primarily soluble CD59 isolated from urine (CD59U) potentially non-specific binding interactions of the phospholipid portion of the membrane forms of CD59 could be avoided. Sucrose density gradient ultracentrifugation analysis showed that human CD59U bound to both human and rat C8 in the SC5b-8 complexes. Similar binding occurred when rat CD59U was used. The degree of binding did not significantly differ between the heterologous and homologous CD59-C8 combinations. C9 from both species inhibited the binding of CD59 to soluble SC5b-8. In ligand blotting analysis human and rat CD59U bound to human and rat C8 alpha gamma-subunit and C9. Binding of human and rat CD59U was stronger to human than rat C9. In plate binding assays the erythrocyte form of CD59 (CD59E) bound to both human and rat C8. Binding of CD59E to heterologous C9 was considerably weaker than to homologous C9. Our results imply that the reciprocal binding sites between C8 and CD59 and to a lesser degree between CD59 and C9 are conserved between human and rat. Interactions of CD59 with the terminal C components are thus species selective but not 'homologously restricted'. Images Figure 4 Figure 5 PMID:9038722

The Murine Factor H-Related Protein FHR-B Promotes Complement Activation.

PubMed

Cserhalmi, Marcell; Csincsi, Ádám I; Mezei, Zoltán; Kopp, Anne; Hebecker, Mario; Uzonyi, Barbara; Józsi, Mihály

2017-01-01

Factor H-related (FHR) proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH). While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3), and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM) extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

Complement mutations in diacylglycerol kinase-ε-associated atypical hemolytic uremic syndrome.

PubMed

Sánchez Chinchilla, Daniel; Pinto, Sheila; Hoppe, Bernd; Adragna, Marta; Lopez, Laura; Justa Roldan, Maria Luisa; Peña, Antonia; Lopez Trascasa, Margarita; Sánchez-Corral, Pilar; Rodríguez de Córdoba, Santiago

2014-09-05

Atypical hemolytic uremic syndrome is characterized by vascular endothelial damage caused by complement dysregulation. Consistently, complement inhibition therapies are highly effective in most patients with atypical hemolytic uremic syndrome. Recently, it was shown that a significant percentage of patients with early-onset atypical hemolytic uremic syndrome carry mutations in diacylglycerol kinase-ε, an intracellular protein with no obvious role in complement. These data support an alternative, complement-independent mechanism leading to thrombotic microangiopathy that has implications for treatment of early-onset atypical hemolytic uremic syndrome. To get additional insights into this new form of atypical hemolytic uremic syndrome, the diacylglycerol kinase-ε gene in a cohort with atypical hemolytic uremic syndrome was analyzed. Eighty-three patients with early-onset atypical hemolytic uremic syndrome (<2 years) enrolled in the Spanish atypical hemolytic uremic syndrome registry between 1999 and 2013 were screened for mutations in diacylglycerol kinase-ε. These patients were also fully characterized for mutations in the genes encoding factor H, membrane cofactor protein, factor I, C3, factor B, and thrombomodulin CFHRs copy number variations and rearrangements, and antifactor H antibodies. Four patients carried mutations in diacylglycerol kinase-ε, one p.H536Qfs*16 homozygote and three compound heterozygotes (p.W322*/p.P498R, two patients; p.Q248H/p.G484Gfs*10, one patient). Three patients also carried heterozygous mutations in thrombomodulin or C3. Extensive plasma infusions controlled atypical hemolytic uremic syndrome recurrences and prevented renal failure in the two patients with diacylglycerol kinase-ε and thrombomodulin mutations. A positive response to plasma infusions and complement inhibition treatment was also observed in the patient with concurrent diacylglycerol kinase-ε and C3 mutations. Data suggest that complement dysregulation influences the onset and disease severity in carriers of diacylglycerol kinase-ε mutations and that treatments on the basis of plasma infusions and complement inhibition are potentially useful in patients with combined diacylglycerol kinase-ε and complement mutations. A comprehensive understanding of the genetic component predisposing to atypical hemolytic uremic syndrome is, therefore, critical to guide an effective treatment. Copyright © 2014 by the American Society of Nephrology.

Complement Mutations in Diacylglycerol Kinase-ε–Associated Atypical Hemolytic Uremic Syndrome

PubMed Central

Sánchez Chinchilla, Daniel; Pinto, Sheila; Hoppe, Bernd; Adragna, Marta; Lopez, Laura; Justa Roldan, Maria Luisa; Peña, Antonia; Lopez Trascasa, Margarita; Sánchez-Corral, Pilar; Rodríguez de Córdoba, Santiago

2014-01-01

Background and objectives Atypical hemolytic uremic syndrome is characterized by vascular endothelial damage caused by complement dysregulation. Consistently, complement inhibition therapies are highly effective in most patients with atypical hemolytic uremic syndrome. Recently, it was shown that a significant percentage of patients with early-onset atypical hemolytic uremic syndrome carry mutations in diacylglycerol kinase-ε, an intracellular protein with no obvious role in complement. These data support an alternative, complement-independent mechanism leading to thrombotic microangiopathy that has implications for treatment of early-onset atypical hemolytic uremic syndrome. To get additional insights into this new form of atypical hemolytic uremic syndrome, the diacylglycerol kinase-ε gene in a cohort with atypical hemolytic uremic syndrome was analyzed. Design, setting, participants, & measurements Eighty-three patients with early-onset atypical hemolytic uremic syndrome (<2 years) enrolled in the Spanish atypical hemolytic uremic syndrome registry between 1999 and 2013 were screened for mutations in diacylglycerol kinase-ε. These patients were also fully characterized for mutations in the genes encoding factor H, membrane cofactor protein, factor I, C3, factor B, and thrombomodulin CFHRs copy number variations and rearrangements, and antifactor H antibodies. Results Four patients carried mutations in diacylglycerol kinase-ε, one p.H536Qfs*16 homozygote and three compound heterozygotes (p.W322*/p.P498R, two patients; p.Q248H/p.G484Gfs*10, one patient). Three patients also carried heterozygous mutations in thrombomodulin or C3. Extensive plasma infusions controlled atypical hemolytic uremic syndrome recurrences and prevented renal failure in the two patients with diacylglycerol kinase-ε and thrombomodulin mutations. A positive response to plasma infusions and complement inhibition treatment was also observed in the patient with concurrent diacylglycerol kinase-ε and C3 mutations. Conclusions Data suggest that complement dysregulation influences the onset and disease severity in carriers of diacylglycerol kinase-ε mutations and that treatments on the basis of plasma infusions and complement inhibition are potentially useful in patients with combined diacylglycerol kinase-ε and complement mutations. A comprehensive understanding of the genetic component predisposing to atypical hemolytic uremic syndrome is, therefore, critical to guide an effective treatment. PMID:25135762

The expression of Fc and complement receptors in young, adult and aged mice.

PubMed Central

Vĕtvicka, V; Fornůsek, L; Zídková, J

1985-01-01

Age-dependent changes in the expression of Fc receptors (FcR) for different isotypes of immunoglobulins and receptors for C3b, C5b and C3bi fragments of complement on the membranes of peritoneal macrophages were studied with mice of different ages. An age-related increase in expression of Fc receptors for IgM, IgE, IgA, IgG2b and IgG3, and a decrease in the expression of Fc receptors for IgG1 was observed. The expression of FcR on macrophages of donors of different ages corresponded with Fc-receptor mediated phagocytosis. The highest number of C3b-binding macrophages was found in aged mice, in contrast to low numbers of C3bi-binding macrophages at this age. The percentage of C5b-binding macrophages was lowest in adult animals. We also observed effective inhibition of binding of the C3b component of complement by preincubation of macrophages with aggregated IgG and vice versa. These observations suggest that fluctuation in expression of Fc but not C receptors may be important to the generalized changes that occur in macrophage function during development and ageing. PMID:2931351

Blocking herpes simplex virus 2 glycoprotein E immune evasion as an approach to enhance efficacy of a trivalent subunit antigen vaccine for genital herpes.

PubMed

Awasthi, Sita; Huang, Jialing; Shaw, Carolyn; Friedman, Harvey M

2014-08-01

Herpes simplex virus 2 (HSV-2) subunit antigen vaccines targeting virus entry molecules have failed to prevent genital herpes in human trials. Our approach is to include a virus entry molecule and add antigens that block HSV-2 immune evasion. HSV-2 glycoprotein C (gC2) is an immune evasion molecule that inhibits complement. We previously reported that adding gC2 to gD2 improved vaccine efficacy compared to the efficacy of either antigen alone in mice and guinea pigs. Here we demonstrate that HSV-2 glycoprotein E (gE2) functions as an immune evasion molecule by binding the IgG Fc domain. HSV-2 gE2 is synergistic with gC2 in protecting the virus from antibody and complement neutralization. Antibodies produced by immunization with gE2 blocked gE2-mediated IgG Fc binding and cell-to-cell spread. Mice immunized with gE2 were only partially protected against HSV-2 vaginal challenge in mice; however, when gE2 was added to gC2/gD2 to form a trivalent vaccine, neutralizing antibody titers with and without complement were significantly higher than those produced by gD2 alone. Importantly, the trivalent vaccine protected the dorsal root ganglia (DRG) of 32/33 (97%) mice between days 2 and 7 postchallenge, compared with 27/33 (82%) in the gD2 group. The HSV-2 DNA copy number was significantly lower in mice immunized with the trivalent vaccine than in those immunized with gD2 alone. The extent of DRG protection using the trivalent vaccine was better than what we previously reported for gC2/gD2 immunization. Therefore, gE2 is a candidate antigen for inclusion in a multivalent subunit vaccine that attempts to block HSV-2 immune evasion. Herpes simplex virus is the most common cause of genital ulcer disease worldwide. Infection results in emotional distress for infected individuals and their partners, is life threatening for infants exposed to herpes during childbirth, and greatly increases the risk of individuals acquiring and transmitting HIV infection. A vaccine that prevents genital herpes infection will have major public health benefits. Our vaccine approach includes strategies to prevent the virus from evading immune attack. Mice were immunized with a trivalent vaccine containing an antigen that induces antibodies to block virus entry and two antigens that induce antibodies that block immune evasion from antibody and complement. Immunized mice demonstrated no genital disease, and 32/33 (97%) animals had no evidence of infection of dorsal root ganglia, suggesting that the vaccine may prevent the establishment of latency and recurrent infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma.

PubMed

Manning, Michael L; Williams, Simon A; Jelinek, Christine A; Kostova, Maya B; Denmeade, Samuel R

2013-03-15

Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen1

PubMed Central

Clay, Corey D.; Soni, Shilpa; Gunn, John S.; Schlesinger, Larry S.

2009-01-01

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag. PMID:18832715

Eculizumab for Dense Deposit Disease and C3 Glomerulonephritis

PubMed Central

Smith, Richard J.; Barile, Gaetano R.; Zhang, Yuzhou; Heher, Eliot C.; Herlitz, Leal; Stokes, M. Barry; Markowitz, Glen S.; D’Agati, Vivette D.; Canetta, Pietro A.; Radhakrishnan, Jai; Appel, Gerald B.

2012-01-01

Summary Background and objectives The principle defect in dense deposit disease and C3 glomerulonephritis is hyperactivity of the alternative complement pathway. Eculizumab, a monoclonal antibody that binds to C5 to prevent formation of the membrane attack complex, may prove beneficial. Design, setting, participants, & measurements In this open-label, proof of concept efficacy and safety study, six subjects with dense deposit disease or C3 glomerulonephritis were treated with eculizumab every other week for 1 year. All had proteinuria >1 g/d and/or AKI at enrollment. Subjects underwent biopsy before enrollment and repeat biopsy at the 1-year mark. Results The subjects included three patients with dense deposit disease (including one patient with recurrent dense deposit disease in allograft) and three patients with C3 glomerulonephritis (including two patients with recurrent C3 glomerulonephritis in allograft). Genetic and complement function testing revealed a mutation in CFH and MCP in one subject each, C3 nephritic factor in three subjects, and elevated levels of serum membrane attack complex in three subjects. After 12 months, two subjects showed significantly reduced serum creatinine, one subject achieved marked reduction in proteinuria, and one subject had stable laboratory parameters but histopathologic improvements. Elevated serum membrane attack complex levels normalized on therapy and paralleled improvements in creatinine and proteinuria. Conclusions Clinical and histopathologic data suggest a response to eculizumab in some but not all subjects with dense deposit disease and C3 glomerulonephritis. Elevation of serum membrane attack complex before treatment may predict response. Additional research is needed to define the subgroup of dense deposit disease/C3 glomerulonephritis patients in whom eculizumab therapy can be considered. PMID:22403278

Insights into the mechanism of C5aR inhibition by PMX53 via implicit solvent molecular dynamics simulations and docking

PubMed Central

2014-01-01

Background The complement protein C5a acts by primarily binding and activating the G-protein coupled C5a receptor C5aR (CD88), and is implicated in many inflammatory diseases. The cyclic hexapeptide PMX53 (sequence Ace-Phe-[Orn-Pro-dCha-Trp-Arg]) is a full C5aR antagonist of nanomolar potency, and is widely used to study C5aR function in disease. Results We construct for the first time molecular models for the C5aR:PMX53 complex without the a priori use of experimental constraints, via a computational framework of molecular dynamics (MD) simulations, docking, conformational clustering and free energy filtering. The models agree with experimental data, and are used to propose important intermolecular interactions contributing to binding, and to develop a hypothesis for the mechanism of PMX53 antagonism. Conclusion This work forms the basis for the design of improved C5aR antagonists, as well as for atomic-detail mechanistic studies of complement activation and function. Our computational framework can be widely used to develop GPCR-ligand structural models in membrane environments, peptidomimetics and other chemical compounds with potential clinical use. PMID:25170421

C5a alters blood-brain barrier integrity in experimental lupus

PubMed Central

Jacob, Alexander; Hack, Bradley; Chiang, Eddie; Garcia, Joe G. N.; Quigg, Richard J.; Alexander, Jessy J.

2010-01-01

The blood-brain barrier (BBB) is a crucial anatomic location in the brain. Its dysfunction complicates many neurodegenerative diseases, from acute conditions, such as sepsis, to chronic diseases, such as systemic lupus erythematosus (SLE). Several studies suggest an altered BBB in lupus, but the underlying mechanism remains unknown. In the current study, we observed a definite loss of BBB integrity in MRL/MpJ-Tnfrsf6lpr (MRL/lpr) lupus mice by IgG infiltration into brain parenchyma. In line with this result, we examined the role of complement activation, a key event in this setting, in maintenance of BBB integrity. Complement activation generates C5a, a molecule with multiple functions. Because the expression of the C5a receptor (C5aR) is significantly increased in brain endothelial cells treated with lupus serum, the study focused on the role of C5a signaling through its G-protein-coupled receptor C5aR in brain endothelial cells, in a lupus setting. Reactive oxygen species production increased significantly in endothelial cells, in both primary cells and the bEnd3 cell line treated with lupus serum from MRL/lpr mice, compared with those treated with control serum from MRL+/+ mice. In addition, increased permeability monitored by changes in transendothelial electrical resistance, cytoskeletal remodeling caused by actin fiber rearrangement, and increased iNOS mRNA expression were observed in bEnd3 cells. These disruptive effects were alleviated by pretreating cells with a C5a receptor antagonist (C5aRant) or a C5a antibody. Furthermore, the structural integrity of the vasculature in MRL/lpr brain was maintained by C5aR inhibition. These results demonstrate the regulation of BBB integrity by the complement system in a neuroinflammatory setting. For the first time, a novel role of C5a in the maintenance of BBB integrity is identified and the potential of C5a/C5aR blockade highlighted as a promising therapeutic strategy in SLE and other neurodegenerative diseases.—Jacob, A., Hack, B., Chiang, E., Garcia, J. G. N., Quigg, R. J., Alexander, J. J. C5a alters blood-brain barrier integrity in experimental lupus. PMID:20065106

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. Copyright © 2017 Elsevier B.V. All rights reserved.

Update on hemolytic uremic syndrome: Diagnostic and therapeutic recommendations

PubMed Central

Salvadori, Maurizio; Bertoni, Elisabetta

2013-01-01

Hemolytic uremic syndrome (HUS) is a rare disease. In this work the authors review the recent findings on HUS, considering the different etiologic and pathogenetic classifications. New findings in genetics and, in particular, mutations of genes that encode the complement-regulatory proteins have improved our understanding of atypical HUS. Similarly, the complement proteins are clearly involved in all types of thrombotic microangiopathy: typical HUS, atypical HUS and thrombotic thrombocytopenic purpura (TTP). Furthermore, several secondary HUS appear to be related to abnormalities in complement genes in predisposed patients. The authors highlight the therapeutic aspects of this rare disease, examining both “traditional therapy” (including plasma therapy, kidney and kidney-liver transplantation) and “new therapies”. The latter include anti-Shiga-toxin antibodies and anti-C5 monoclonal antibody “eculizumab”. Eculizumab has been recently launched for the treatment of the atypical HUS, but it appears to be effective in the treatment of typical HUS and in TTP. Future therapies are in phases I and II. They include anti-C5 antibodies, which are more purified, less immunogenic and absorbed orally and, anti-C3 antibodies, which are more powerful, but potentially less safe. Additionally, infusions of recombinant complement-regulatory proteins are a potential future therapy. PMID:24255888

Structure analysis of a heteropolysaccharide from Taraxacum mongolicum Hand.-Mazz. and anticomplementary activity of its sulfated derivatives.

PubMed

Chen, MiaoMiao; Wu, Jianjun; Shi, Songshan; Chen, Yonglin; Wang, Huijun; Fan, Hongwei; Wang, Shunchun

2016-11-05

A homogenous water-soluble polysaccharide, DPSW-A, with a deduced chemical structure was extracted from the herb Taraxacum mongolicum Hand.-Mazz. Moreover, 80.813-kDa DPSW-A is composed of three types of monosaccharide, namely rhamnose, arabinose, and galactose, at a molar ratio of 1.0:10.7:11.9. The main chain of DPSW-A contains Terminal-Galp, 1,3-Galp, 1,6-Galp, 1,3,6-Galp, and 1,2,4-Rhap; the branched chain contains Terminal-Araf, 1,5-Araf, and 1,3,5-Araf. The sulfated derivatives prepared from DPSW-A showed inhibitory effects on complement activation through the classical pathway (CH50: Sul-DPSW-A, 3.94±0.43μg/mL; heparin, 104.40±3.82μg/mL) and alternative pathway (AP50: Sul-DPSW-A, 42.76±0.46μg/mL; heparin, 43.42±0.22μg/mL). Mechanism studies indicated that Sul-DPSW-A inhibited complement activation by blocking C1q, C1r, C1s, and C9, but not C2, C3, C4, and C5. In addition, Sul-DPSW-A displayed limited anticoagulant effects. These results suggest that Sul-DPSW-A prepared from DPSW-A is valuable for treating diseases caused by excessive complement system activation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quiescent complement in nonhuman primates during E coli Shiga toxin-induced hemolytic uremic syndrome and thrombotic microangiopathy.

PubMed

Lee, Benjamin C; Mayer, Chad L; Leibowitz, Caitlin S; Stearns-Kurosawa, D J; Kurosawa, Shinichiro

2013-08-01

Enterohemorrhagic Escherichia coli (EHEC) produce ribosome-inactivating Shiga toxins (Stx1, Stx2) responsible for development of hemolytic uremic syndrome (HUS) and acute kidney injury (AKI). Some patients show complement activation during EHEC infection, raising the possibility of therapeutic targeting of complement for relief. Our juvenile nonhuman primate (Papio baboons) models of endotoxin-free Stx challenge exhibit full spectrum HUS, including thrombocytopenia, hemolytic anemia, and AKI with glomerular thrombotic microangiopathy. There were no significant increases in soluble terminal complement complex (C5b-9) levels after challenge with lethal Stx1 (n = 6) or Stx2 (n = 5) in plasma samples from T0 to euthanasia at 49.5 to 128 hours post-challenge. d-dimer and cell injury markers (HMGB1, histones) confirmed coagulopathy and cell injury. Thus, complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these preclinical models, and benefits or risks of complement inhibition should be studied further for this infection.

An improved host-vector system for Candida maltosa using a gene isolated from its genome that complements the his5 mutation of Saccharomyces cerevisiae.

PubMed

Hikiji, T; Ohkuma, M; Takagi, M; Yano, K

1989-10-01

The host-vector system of an n-alkane-assimilating-yeast, Candida maltosa, which we previously constructed using an autonomously replicating sequence (ARS) region isolated from the genome of this yeast, utilizes C. maltosa J288 (leu2-) as a host. As this host had a serious growth defect on n-alkane, we developed an improved host-vector system using C. maltosa CH1 (his-) as host. The vectors were constructed with the Candida ARS region and a DNA fragment isolated from the genome of C. maltosa. Since this DNA fragment could complement histidine auxotrophy of both C. maltosa CH1 and S. cerevisiae (his5-), we termed the gene contained in this DNA fragment C-HIS5. The vectors were characterized in terms of transformation frequency and stability, and the nucleotide sequence of C-HIS5 was determined. The deduced amino acid sequence (389 residues) shared 51% homology with that of HIS5 of S. cerevisiae (384 residues; Nishiwaki et al. 1987).

A host-specific biological control of grape crown gall by Agrobacterium vitis strain F2/5: its regulation and population dynamics.

PubMed

Kaewnum, Supaporn; Zheng, Desen; Reid, Cheryl L; Johnson, Kameka L; Gee, Jodi C; Burr, Thomas J

2013-05-01

Nontumorigenic Agrobacterium vitis strain F2/5 is able to prevent crown gall caused by tumorigenic A. vitis on grape but not on other plant species such as tobacco. Mutations in a quorum-sensing transcription factor, aviR, and in caseinolytic protease (clp) component genes clpA and clpP1 resulted in reduced or loss of biological control. All mutants were complemented; however, restoration of biological control by complemented clpA and clpP1 mutants was dependent on the copy number of vector that was used as well as timing of application of the complemented mutants to grape wounds in relation to inoculation with pathogen. Mutations in other quorum-sensing and clp genes and in a gene associated with polyketide synthesis did not affect biological control. It was determined that, although F2/5 inhibits transformation by tumorigenic A. vitis strains on grape, it does not affect growth of the pathogen in wounded grape tissue over time.

Susceptibility of pathogenic and nonpathogenic Naegleria ssp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteman, L.Y.

1988-01-01

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with {sup 3}H-uridine and visual observation with a compound microscope were used as indices of lysis. Susceptibility or resistance to complement-mediated lysis in vitro correlated with the in vivo pathogenic potential. Nonpathogenic Naegleria amoebae were lysed at a faster rate and at higher cell concentrations than were pathogenic amoebae. Electrophoretic analysis of NHS incubated with pathogenicmore » or nonpathogenic Naegleria spp. demonstrated that amoebae activate the complement cascade resulting in the production of C3 and C5 complement cleavage products. Treatment with papain or trypsin for 1 h, but not with sialidase, increase the susceptibility of highly pathogenic, mouse-passaged N. fowleri to lysis. Treatment with actinomycin D, cycloheximide or various protease inhibitors for 4 h did not increase susceptibility to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appear to account for the increase resistance of N. fowleri amoebae to complement-mediated lysis. A binding study with {sup 125}I radiolabeled C9 indicated that the terminal complement component does not remain stably bound to the membrane of pathogenic amoebae.« less

Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses.

PubMed

Hueging, Kathrin; Weller, Romy; Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W R; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas

2015-01-01

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry.

Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses

PubMed Central

Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W. R.; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas

2015-01-01

Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to distinct characteristics of these apolipoproteins that influence HCV assembly and cell entry. This will guide future research to precisely pinpoint how apolipoproteins function during virus assembly and cell entry. PMID:26226615

Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy.

PubMed

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; Zhang, Hong

2015-05-01

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. Copyright © 2015 by the American Society of Nephrology.

In vitro C3 Deposition on Cryptococcus Capsule Occurs Via Multiple Complement Activation Pathways

PubMed Central

Mershon-Shier, Kileen L.; Vasuthasawat, Alex; Takahashi, Kazue; Morrison, Sherie L.; Beenhouwer, David O.

2011-01-01

Complement can be activated via three pathways: classical, alternative, and lectin. Cryptococcus gattii and C. neoformans are closely related fungal pathogens possessing a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM), which serves as a site for complement activation and deposition of complement components. We determined C3 deposition on Cryptococcus spp. by flow cytometry and confocal microscopy after incubation with serum from C57BL/6J mice as well as mice deficient in complement components C4, C3, factor B, and mannose binding lectin (MBL). C. gattii and C. neoformans activate complement in EGTA-treated serum indicating that they can activate the alternative pathway. However, complement activation was seen with factor B−/− serum suggesting activation could also take place in the absence of a functional alternative pathway. Furthermore, we uncovered a role for C4 in the alternative pathway activation by Cryptococcus spp. We also identified an unexpected and complex role for MBL in complement activation by Cryptococcus spp. No complement activation occurred in the absence of MBL-A and -C proteins although activation took place when the lectin binding activity of MBL was disrupted by calcium chelation. In addition, alternative pathway activation by C. neoformans required both MBL-A and -C, while either MBL-A or -C was sufficient for alternative pathway activation by C. gattii. Thus, complement activation by Cryptococcus spp. can take place through multiple pathways and complement activation via the alternative pathway requires the presence of C4 and MBL proteins. PMID:21723612

Mutations participating in interallelic complementation in propionic acidemia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gravel, R.A.; Akerman, B.R.; Lamhonwah, A.M.

1994-07-01

Deficiency of propionyl-CoA carboxylase (PCC; [alpha][sub 4][beta][sub 4]) results in the rare, autosomal recessive disease propionic acidemia. Cell fusion experiments have revealed two complementation groups, pccA and pccB, corresponding to defects of the PCCA ([alpha]-subunit) and PCCB ([beta]-subunit) genes, respectively. The pccBCC group includes subgroups, pccB and pccC, which are thought to reflect interallelic complementation between certain mutations of the PCCB gene. In this study, the authors have identified the mutations in two pccB, one pccC, and two pccBC cell lines and have deduced those alleles participating in interallelic complementation. One pccB line was a compound hetrozygote of Pro228Leu andmore » Asn536Asp. The latter mutation was also detected in a noncomplementing pccBC line. This leaves Pro228Leu responsible for complementation in the pccB cells. The second pccB line contained an insertional duplication, dupKICK140-143, and a splice mutation IVS+1 G[yields]T, located after Lys466. The authors suggest that the dupKICK mutation is the complementing allele, since the second allele is incompatible with normal splicing. The pccC line studied was homozygous for Arg410Trp, which is necessarily the complementing allele in that line. For a second pccC line, they previously had proposed that [Delta]Ile408 was the complementing allele. They now show that its second allele, [open quotes]Ins[center dot]Del[close quotes], a 14-bp deletion replaced by a 12-bp insertion beginning at codon 407, fails to complement in homozygous form. The authors conclude that the interallelic complementation results from mutations in domains that can interact between [beta]-subunits in the PCC heteromer to restore enzymatic function. On the basis of sequence homology with the Propionibacterium shermanii transcarboxylase 12S subunit, they suggest that the pccC domain, defined by Ile408 and Arg410, may involve the propionyl-CoA binding site. 37 refs., 5 figs., 2 tabs.« less

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

Multicentre physiological reference intervals for serum concentrations of immunoglobulins A, G and M, complement C3c and C4 measured with Tina-Quant reagents systems.

PubMed

Fuentes-Arderiu, Xavier; Alonso-Gregorio, Eduardo; Alvarez-Funes, Virtudes; Ambrós-Marigómez, Carmen; Coca-Fábregas, Lluís; Cruz-Placer, Marta; Díaz-Fernández, Julián; Pinel-Julián, María Pilar; Gutiérrez-Cecchini, Beatriz; Herrero-Bernal, Pilar; Sempere-Alcocer, Marcos; García-Caballero, Francisca; Del Mar Larrea-Ortiz-Quintana, María; La-Torre-Marcellán, Pedro; Del Señor López-Vélez, María; Mar-Medina, Carmen; Martín-Oncina, Javier; Rodríguez-Hernández, María Victoria; Romero-Sotomayor, María Victoria; Serrano-López, Cándido; Sicilia-Enríquez-de-Salamanca, Adolfo; Velasco-Romero, Ana María; Juvé-Cuxart, Santiago

2007-01-01

Clinical laboratories seeking accreditation for compliance with ISO 15189:2003 need to demonstrate that the physiological reference intervals communicated to all users of the laboratory service are appropriate for the patient population served and for the measurement systems used. In the case of immunological quantities, few articles have been published in peer-reviewed journals. A total of 21 clinical laboratories in different regions of Spain collaborated in identifying reference individuals and determining adult reference intervals for some immunological quantities measured using RD/Hitachi Modular Analytics analysers and Tina-Quant reagent systems. These immunological quantities are the mass concentrations of immunoglobulin A, immunoglobulin G, immunoglobulin M, complement C3c and complement C4 in serum. All the logistic work was carried out in co-operation with the supplier of the reagents and analysers (Roche Diagnostics España, S.L., Sant Cugat del Vallès, Catalonia, Spain). From the set of reference values obtained by each laboratory, multicentre reference limits were estimated non-parametrically. The reference intervals estimated in this study for concentrations of serum components under consideration are: complement C3c, 0.62-1.64 g/L for women and men; complement C4, 0.14-0.72 g/L for women and men; immunoglobulin A, 0.89-4.80 g/L for women and men; immunoglobulin G, 6.5-14.3 g/L for women and men; and immunoglobulin M, 0.48-3.38 g/L for women and 0.41-2.46 g/L for men.

The role of complement system in septic shock.

PubMed

Charchaflieh, Jean; Wei, Jiandong; Labaze, Georges; Hou, Yunfang Joan; Babarsh, Benjamin; Stutz, Helen; Lee, Haekyung; Worah, Samrat; Zhang, Ming

2012-01-01

Septic shock is a critical clinical condition with a high mortality rate. A better understanding of the underlying mechanisms is important to develop effective therapies. Basic and clinical studies suggest that activation of complements in the common cascade, for example, complement component 3 (C3) and C5, is involved in the development of septic shock. The involvement of three upstream complement pathways in septic shock is more complicated. Both the classical and alternative pathways appear to be activated in septic shock, but the alternative pathway may be activated earlier than the classical pathway. Activation of these two pathways is essential to clear endotoxin. Recent investigations have shed light on the role of lectin complement pathway in septic shock. Published reports suggest a protective role of mannose-binding lectin (MBL) against sepsis. Our preliminary study of MBL-associated serine protease-2 (MASP-2) in septic shock patients indicated that acute decrease of MASP-2 in the early phase of septic shock might correlate with in-hospital mortality. It is unknown whether excessive activation of these three upstream complement pathways may contribute to the detrimental effects in septic shock. This paper also discusses additional complement-related pathogenic mechanisms and intervention strategies for septic shock.

Complement activation on the surface of cell-derived microparticles during cardiac surgery with cardiopulmonary bypass - is retransfusion of pericardial blood harmful?

PubMed

Biró, E; van den Goor, J M; de Mol, B A; Schaap, M C; Ko, L-Y; Sturk, A; Hack, C E; Nieuwland, R

2011-01-01

To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.

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.

Variola virus immune evasion design: Expression of a highly efficient inhibitor of human complement

PubMed Central

Rosengard, Ariella M.; Liu, Yu; Nie, Zhiping; Jimenez, Robert

2002-01-01

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. PMID:12034872

Granulocyte and monocyte CD11b expression during plasma separation is dependent on complement factor 5 (C5) - an ex vivo study with blood from a C5-deficient individual.

PubMed

Hardersen, Randolf; Enebakk, Terje; Christiansen, Dorte; Bergseth, Grethe; Brekke, Ole-Lars; Mollnes, Tom Eirik; Lappegård, Knut Tore; Hovland, Anders

2018-04-01

The aim of the study was to investigate the role of complement factor 5 (C5) in reactions elicited by plasma separation using blood from a C5-deficient (C5D) individual, comparing it to C5-deficient blood reconstituted with C5 (C5DR) and blood from healthy donors. Blood was circulated through an ex vivo plasma separation model. Leukocyte CD11b expression and leukocyte-platelet conjugates were measured by flow cytometry during a 30-min period. Other markers were assessed during a 240-min period. Granulocyte and monocyte CD11b expression did not increase in C5D blood during plasma separation. In C5DR samples granulocytes CD11b expression, measured by mean fluorescence intensity (MFI), increased from 10481 ± 6022 (SD) to 62703 ± 4936, and monocytes CD11b expression changed from 13837 ± 7047 to 40063 ± 713. Granulocyte-platelet conjugates showed a 2.5-fold increase in the C5DR sample compared to the C5D sample. Monocyte-platelet conjugates increased independently of C5. In the C5D samples, platelet count decreased from 210 × 10 9 /L (201-219) (median and range) to 51 × 10 9 /L (50-51), and C3bc increased from 14 CAU/mL (21-7) to 198 CAU/mL (127-269), whereas TCC formation was blocked during plasma separation. In conclusion, up-regulation of granulocyte and monocyte CD11b during plasma separation was C5-dependent. The results also indicate C5 dependency in granulocyte-platelet conjugates formation. © 2018 APMIS. Published by John Wiley & Sons Ltd.

Complement fixation test to C burnetii

MedlinePlus

... complement fixation test; Coxiella burnetii - complement fixation test; C burnetii - complement fixation test ... a specific foreign substance ( antigen ), in this case, C burnetii . Antibodies defend the body against bacteria, viruses, ...

Conditional economic incentives for reducing HIV risk behaviors: integration of psychology and behavioral economics.

PubMed

Operario, Don; Kuo, Caroline; Sosa-Rubí, Sandra G; Gálarraga, Omar

2013-09-01

This article reviews psychology and behavioral economic approaches to HIV prevention, and examines the integration and application of these approaches in conditional economic incentive (CEI) programs for reducing HIV risk behavior. We discuss the history of HIV prevention approaches, highlighting the important insights and limitations of psychological theories. We provide an overview of the theoretical tenets of behavioral economics that are relevant to HIV prevention, and utilize CEIs as an illustrative example of how traditional psychological theories and behavioral economics can be combined into new approaches for HIV prevention. Behavioral economic interventions can complement psychological frameworks for reducing HIV risk by introducing unique theoretical understandings about the conditions under which risky decisions are amenable to intervention. Findings from illustrative CEI programs show mixed but generally promising effects of economic interventions on HIV and sexually transmitted infection (STI) prevalence, HIV testing, HIV medication adherence, and drug use. CEI programs can complement psychological interventions for HIV prevention and behavioral risk reduction. To maximize program effectiveness, CEI programs must be designed according to contextual and population-specific factors that may determine intervention applicability and success. PsycINFO Database Record (c) 2013 APA, all rights reserved.

Eculizumab treatment and impaired opsonophagocytic killing of meningococci by whole blood from immunized adults.

PubMed

Konar, Monica; Granoff, Dan M

2017-08-17

Eculizumab, a humanized anti-complement C5 monoclonal antibody (mAb) for treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome, blocks the terminal complement pathway required for serum bactericidal activity (SBA). Because treated patients are at >1000-fold increased risk of meningococcal disease, vaccination is recommended; whether vaccination can protect by opsonophagocytic activity in the absence of SBA is not known. Meningococci were added to anticoagulated blood from 12 healthy adults vaccinated with meningococcal serogroup B and serogroup A, C, W, Y vaccines. Bacterial survival was measured after 3-hour incubation in the presence of eculizumab or control complement factor D inhibitor ACH-4471, which blocks the complement alternative pathway (AP) and is in phase 2 development for treatment of PNH. In the absence of inhibitors, colony formation units (CFUs) per milliliter in blood from all 12 immunized subjects decreased from ∼4000 at time 0 to sterile cultures at 3 hours. In the presence of eculizumab, there was a >22-fold increase in geometric mean CFUs per milliliter (90 596 and 114 683 CFU/mL for serogroup B and C strains, respectively; P < .0001 compared with time 0). In the presence of ACH-4471, there was a >12-fold decrease (23 and 331 CFU/mL, respectively; P < .0001). The lack of meningococci killing by blood containing eculizumab resulted from inhibition of release of C5a, a C5 split product needed for upregulation of phagocytosis. The results provide an explanation for the large number of cases of meningococcal disease in immunized patients being treated with eculizumab and suggest that vaccination may provide better protection against meningococcal disease in patients treated with an AP-specific inhibitor. © 2017 by The American Society of Hematology.

All five host-range variants of Xanthomonas citri carry one pthA homolog with 17.5 repeats that determines pathogenicity on citrus, but none determine host-range variation.

PubMed

Al-Saadi, Abdulwahid; Reddy, Joseph D; Duan, Yong P; Brunings, Asha M; Yuan, Qiaoping; Gabriel, Dean W

2007-08-01

Citrus canker disease is caused by five groups of Xanthomonas citri strains that are distinguished primarily by host range: three from Asia (A, A*, and A(w)) and two that form a phylogenetically distinct clade and originated in South America (B and C). Every X. citri strain carries multiple DNA fragments that hybridize with pthA, which is essential for the pathogenicity of wide-host-range X. citri group A strain 3213. DNA fragments that hybridized with pthA were cloned from a representative strain from all five groups. Each strain carried one and only one pthA homolog that functionally complemented a knockout mutation of pthA in 3213. Every complementing homolog was of identical size to pthA and carried 17.5 nearly identical, direct tandem repeats, including three new genes from narrow-host-range groups C (pthC), A(w) (pthAW), and A* (pthA*). Every noncomplementing paralog was of a different size; one of these was sequenced from group A* (pthA*-2) and was found to have an intact promoter and full-length reading frame but with 15.5 repeats. None of the complementing homologs nor any of the noncomplementing paralogs conferred avirulence to 3213 on grapefruit or suppressed avirulence of a group A* strain on grapefruit. A knockout mutation of pthC in a group C strain resulted in loss of pathogenicity on lime, but the strain was unaffected in ability to elicit an HR on grapefruit. This pthC- mutant was fully complemented by pthA, pthB, or pthC. Analysis of the predicted amino-acid sequences of all functional pthA homologs and nonfunctional paralogs indicated that the specific sequence of the 17th repeat may be essential for pathogenicity of X. citri on citrus.

Targeted complement inhibition as a promising strategy for preventing inflammatory complications in hemodialysis

PubMed Central

DeAngelis, Robert A.; Reis, Edimara S.; Ricklin, Daniel; Lambris, John D.

2012-01-01

Hemodialysis is the most common method used to remove waste and hazardous products of metabolism in patients suffering from renal failure. Hundreds of thousands of people with end-stage renal disease undergo hemodialysis treatment in the United States each year. Strikingly, the 5-year survival rate for all dialysis patients is only 35%. Most of the patients succumb to cardiovascular disease that is exacerbated by the chronic induction of inflammation caused by contact of the blood with the dialysis membrane. The complement system, a strong mediator of pro-inflammatory networks, is a key contributor to such biomaterial-induced inflammation. Though only evaluated in experimental ex vivo settings, specific targeting of complement activation during hemodialysis has uncovered valuable information that points towards the therapeutic use of complement inhibitors as means to control the unwelcomed inflammatory responses and consequent pathologies in hemodialysis patients. PMID:22964235

Coagulation and complement system in critically ill patients.

PubMed

Helling, H; Stephan, B; Pindur, G

2015-01-01

Activation of coagulation and inflammatory response including the complement system play a major role in the pathogenesis of critical illness. However, only limited data are available addressing the relationship of both pathways and its assessment of a predictive value for the clinical outcome in intense care medicine. Therefore, parameters of the coagulation and complement system were studied in patients with septicaemia and multiple trauma regarded as being exemplary for critical illness. 34 patients (mean age: 51.38 years (±16.57), 15 females, 19 males) were investigated at day 1 of admittance to the intensive care unit (ICU). Leukocytes, complement factors C3a and C5a were significantly (p <  0.0500) higher in sepsis than in trauma, whereas platelet count and plasma fibrinogen were significantly lower in multiple trauma. Activation markers of coagulation were elevated in both groups, however, thrombin-antithrombin-complex was significantly higher in multiple trauma. DIC scores of 5 were not exceeded in any of the two groups. Analysing the influences on mortality (11/34; 32.35% ), which was not different in both groups, non-survivors were significantly older, had significantly higher multiple organ failure (MOF) scores, lactate, abnormal prothrombin times and lower C1-inhibitor activities, even more pronounced in early deaths, than survivors. In septic non-survivors protein C was significantly lower than in trauma. We conclude from these data that activation of the complement system as part of the inflammatory response is a significant mechanism in septicaemia, whereas loss and consumption of blood components including parts of the coagulation and complement system is more characteristic for multiple trauma. Protein C in case of severe reduction might be of special concern for surviving in sepsis. Activation of haemostasis was occurring in both diseases, however, overt DIC was not confirmed in this study to be a leading mechanism in critically ill patients. MOF score, lactate, C1-inhibitor and prothrombin time have been the only statistically significant predictors for lethal outcome suggesting that organ function, microcirculation, haemostasis and inflammatory response are essential elements of the pathomechanism and clinical course of diseases among critically ill patients.

Beneficial effects of Houttuynia cordata polysaccharides on "two-hit" acute lung injury and endotoxic fever in rats associated with anti-complementary activities.

PubMed

Lu, Yan; Jiang, Yun; Ling, Lijun; Zhang, Yunyi; Li, Hong; Chen, Daofeng

2018-03-01

Houttuynia cordata Thunb. is a traditional herb used for clearing heat and eliminating toxins, and has also been used for the treatment of severe acute respiratory syndrome (SARS). In vitro, the crude H. cordata polysaccharides (CHCP) exhibited potent anti-complementary activity through both the classical and alternative pathways by acting on components C3 and C4 of the complement system without interfering with the coagulation system. This study was to investigate the preventive effects of CHCP on acute lung injury (ALI) induced by hemorrhagic shock plus lipopolysaccharide (LPS) instillation (two-hit) and LPS-induced fever in rats. CHCP significantly attenuated pulmonary injury in the "two-hit" ALI model by reducing pulmonary edema and protein exudation in bronchoalveolar lavage fluid (BALF). In addition, it reduced the deposit of complement activation products in the lung and improved oxidant-antioxidant imbalance. Moreover, CHCP administration inhibited fever in rats, reduced the number of leukocytes and restored serum complement levels. The inhibition on the inappropriate activation of complement system by CHCP may play an important role in its beneficial effects on inflammatory diseases. The anti-complementary polysaccharides are likely to be among the key substances for the heat-clearing function of H. cordata .

Antibody-Mediated Rejection in Human Cardiac Allografts: Evaluation of Immunoglobulins and Complement Activation Products C4d and C3d as Markers

PubMed Central

Rodriguez, E. R.; Skojec, Diane V.; Tan, Carmela D.; Zachary, Andrea A.; Kasper, Edward K.; Conte, John V.; Baldwin, William M.

2005-01-01

Antibody-mediated rejection (AMR) in human heart transplantation is an immunopathologic process in which injury to the graft is in part the result of activation of complement and it is poorly responsive to conventional therapy. We evaluated by immunofluorescence (IF), 665 consecutive endomyocardial biopsies from 165 patients for deposits of immunoglobulins and complement. Diffuse IF deposits in a linear capillary pattern greater than 2+ were considered significant. Clinical evidence of graft dysfunction was correlated with complement deposits. IF 2+ or higher was positive for IgG, 66%; IgM, 12%; IgA, 0.6%; C1q, 1.8%; C4d, 9% and C3d, 10%. In 3% of patients, concomitant C4d and C3d correlated with graft dysfunction or heart failure. In these 5 patients AMR occurred 56–163 months after transplantation, and they responded well to therapy for AMR but not to treatment with steroids. Systematic evaluation of endomyocardial biopsies is not improved by the use of antibodies for immunoglobulins or C1q. Concomitant use of C4d and C3d is very useful to diagnose AMR, when correlated with clinical parameters of graft function. AMR in heart transplant patients can occur many months or years after transplant. PMID:16212640

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

The crystal structure of cobra venom factor, a cofactor for C3- and C5-convertase CVFBb.

PubMed

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

2009-04-15

Cobra venom factor (CVF) is a functional analog of human complement component C3b, the active fragment of C3. Similar to C3b, in human and mammalian serum, CVF binds factor B, which is then cleaved by factor D, giving rise to the CVFBb complex that targets the same scissile bond in C3 as the authentic complement convertases C4bC2a and C3bBb. Unlike the latter, CVFBb is a stable complex and an efficient C5 convertase. We solved the crystal structure of CVF, isolated from Naja naja kouthia venom, at 2.6 A resolution. The CVF crystal structure, an intermediate between C3b and C3c, lacks the TED domain and has the CUB domain in an identical position to that seen in C3b. The similarly positioned CUB and slightly displaced C345c domains of CVF could play a vital role in the formation of C3 convertases by providing important primary binding sites for factor B.

Germline Variation in Complement Genes and Event-Free Survival in Follicular and Diffuse Large B-Cell Lymphoma

PubMed Central

Charbonneau, Bridget; Maurer, Matthew J.; Fredericksen, Zachary S.; Zent, Clive S.; Link, Brian K.; Novak, Anne J.; Ansell, Stephen M.; Weiner, George J.; Wang, Alice H.; Witzig, Thomas E.; Dogan, Ahmet; Slager, Susan L.; Habermann, Thomas M.; Cerhan, James R.

2013-01-01

The complement pathway plays a central role in innate immunity, and also functions as a regulator of the overall immune response. We evaluated whether polymorphisms in complement genes are associated with event-free survival (EFS) in follicular (FL) and diffuse large B-cell (DLBCL) lymphoma. We genotyped 167 single nucleotide polymorphisms (SNPs) from 30 complement pathway genes in a prospective cohort study of newly diagnosed FL (N=107) and DLBCL (N=82) patients enrolled at the Mayo Clinic from 2002–2005. Cox regression was used to estimate Hazard Ratios (HRs) for individual SNPs with EFS, adjusting for FLIPI or IPI and treatment. For gene-level analyses, we used a principal components based gene-level test. In gene-level analyses for FL EFS, CFH (p=0.009), CD55 (p=0.006), CFHR5 (p=0.01), C9 (p=0.02), CFHR1 (p=0.03), and CD46 (p=0.03) were significant at p<0.05, and these genes remained noteworthy after accounting for multiple testing (q<0.15). SNPs in CFH, CFHR1, and CFHR5 showed stronger associations among patients receiving any rituximab, while SNPs from CD55 and CD46 showed stronger associations among patients who were observed. For DLBCL, only CLU (p=0.001) and C7 (p=0.03) were associated with EFS, but did not remain noteworthy after accounting for multiple testing (q>0.15). Genes from the Regulators of Complement Activation (CFH, CD55, CFHR1, CFHR5, CD46) at 1q32-q32.1, along with C9, were associated with FL EFS after adjusting for clinical variables, and if replicated, these findings add further support for the role of host innate immunity in FL prognosis. PMID:22718493

The Crystal Structure of Cobra Venom Factor, a Cofactor for C3- and C5-Convertase CVFBb

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krishnan, Vengadesan; Ponnuraj, Karthe; Xu, Yuanyuan

2009-05-26

Cobra venom factor (CVF) is a functional analog of human complement component C3b, the active fragment of C3. Similar to C3b, in human and mammalian serum, CVF binds factor B, which is then cleaved by factor D, giving rise to the CVFBb complex that targets the same scissile bond in C3 as the authentic complement convertases C4bC2a and C3bBb. Unlike the latter, CVFBb is a stable complex and an efficient C5 convertase. We solved the crystal structure of CVF, isolated from Naja naja kouthia venom, at 2.6 {angstrom} resolution. The CVF crystal structure, an intermediate between C3b and C3c, lacksmore » the TED domain and has the CUB domain in an identical position to that seen in C3b. The similarly positioned CUB and slightly displaced C345c domains of CVF could play a vital role in the formation of C3 convertases by providing important primary binding sites for factor B.« less

Complement inhibition prevents gut ischemia and endothelial cell dysfunction after hemorrhage/resuscitation.

PubMed

Fruchterman, T M; Spain, D A; Wilson, M A; Harris, P D; Garrison, R N

1998-10-01

Complement, a nonspecific immune response, is activated during hemorrhage/resuscitation (HEM/RES) and is involved in cellular damage. We hypothesized that activated complement injures endothelial cells (ETCs) and is responsible for intestinal microvascular hypoperfusion after HEM/RES. Four groups of rats were studied by in vivo videomicroscopy of the intestine: SHAM, HEM/RES, HEM/RES + sCR1 (complement inhibitor, 15 mg/kg intravenously given before resuscitation), and SHAM + sCR1. Hemorrhage was to 50% of mean arterial pressure for 60 minutes followed by resuscitation with shed blood plus an equal volume of saline. ETC function was assessed by response to acetylcholine. Resuscitation restored central hemodynamics to baseline after hemorrhage. After resuscitation, inflow A1 and premucosal A3 arterioles progressively constricted (-24% and -29% change from baseline, respectively), mucosal blood flow was reduced, and ETC function was impaired. Complement inhibition prevented postresuscitation vasoconstriction and gut ischemia. This protective effect appeared to involve preservation of ETC function in the A3 vessels (SHAM 76% of maximal dilation, HEM/RES 61%, HEM/RES + sCR1 74%, P < .05). Complement inhibition preserved ETC function after HEM/RES and maintained gut perfusion. Inhibition of complement activation before resuscitation may be a useful adjunct in patients experiencing major hemorrhage and might prevent the sequelae of gut ischemia.

Immunological properties of glycolipids from membranes of Acholeplasma laidlawii.

PubMed Central

Ryan, M D; Noker, P; Matz, L L

1975-01-01

Glycolipids, the predominant class of lipids in the membranes of Acholeplasma laidlawii, are the haptenic determinants that react with anti-A. Laidlawii serum to fix complement. The predominant complement-fixing activity of the membrane glycolipids was associated with the monoglucoysyl diglyceride, diglucosyl diglyceride, glycerlphosphoryl diglucosyl diglyceride (GPDD), and an unknown lipid B, which did not react with ninhydrin but release glucose and glycerol and traces of phosphorus upon hydrolysis. The glycolipids monoglucosyl diglyceride and diglucosyl diglyceride or GPDD and unknown lipid B were paired as a result of their cross-reactions with selective antisera prepared with the aid of reconstituted membrane complexes containing membrane lipids. Reconstituted membrane complexes assembled from [14C]monoglucosyl diglyceride and delipidated membrane proteins gave optimal complement fixation titers before saturation of the complexes with the ]14C]monoglucosyl diglyceride. The phosphoglycolipid of the membrane, GPDD, was anticomplementary as a pure lipid, a cholesterol liposome, and a reconstituted membrane complex. This anticomplementary activity, which was caused by 3 mug of pure GPDD, affected both human and guinea pig complement. Although human C1, C4, C3, and C5 were not inhibited by GPDD, C2 was inhibited 10-fold by reconstituted membrane complexes containing 150 mug of GPDD. A role for this phosphoglycolipid is discussed in the hypothetical mechanism of inhibition of C2 attachment to SAC1, 4 sites. PMID:1193716

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

Recent insights into C3 glomerulopathy

PubMed Central

Barbour, Thomas D.; Pickering, Matthew C.; Cook, H. Terence

2013-01-01

‘C3 glomerulopathy’ is a recent disease classification comprising several rare types of glomerulonephritis (GN), including dense deposit disease (DDD), C3 glomerulonephritis (C3GN) and CFHR5 nephropathy. These disorders share the key histological feature of isolated complement C3 deposits in the glomerulus. A common aetiology involving dysregulation of the alternative pathway (AP) of complement has been elucidated in the past decade, with genetic defects and/or autoantibodies able to be identified in a proportion of patients. We review the clinical and histological features of C3 glomerulopathy, relating these to underlying molecular mechanisms. The role of uncontrolled C3 activation in pathogenesis is emphasized, with important lessons from animal models. Methods, advantages and limitations of gene testing in the assessment of individuals or families with C3 glomerulopathy are discussed. While no therapy has yet been shown consistently effective, clinical evaluation of agents targeting specific components of the complement system is ongoing. However, limits to current knowledge regarding the natural history and the appropriate timing and duration of proposed therapies need to be addressed. PMID:23479095

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 the...

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 the...

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 the...

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 the...

Beneficial effects of omega-3 fatty acids in the proteome of high-density lipoprotein proteome

PubMed Central

2012-01-01

Background Omega-3 poly-unsaturated fatty acids (ω-3 PUFAs) have demonstrated to be beneficial in the prevention of cardiovascular disease, however, the mechanisms by which they perform their cardiovascular protection have not been clarified. Intriguingly, some of these protective effects have also been linked to HDL. The hypothesis of this study was that ω-3 PUFAs could modify the protein cargo of HDL particle in a triglyceride non-dependent mode. The objective of the study was to compare the proteome of HDL before and after ω-3 PUFAs supplemented diet. Methods A comparative proteomic analysis in 6 smoker subjects HDL before and after a 5 weeks ω-3 PUFAs enriched diet has been performed. Results Among the altered proteins, clusterin, paraoxonase, and apoAI were found to increase, while fibronectin, α-1-antitrypsin, complement C1r subcomponent and complement factor H decreased after diet supplementation with ω-3 PUFAs. Immunodetection assays confirmed these results. The up-regulated proteins are related to anti-oxidant, anti-inflammatory and anti-atherosclerotic properties of HDL, while the down-regulated proteins are related to regulation of complement activation and acute phase response. Conclusions Despite the low number of subjects included in the study, our findings demonstrate that ω-3 PUFAs supplementation modifies lipoprotein containing apoAI (LpAI) proteome and suggest that these protein changes improve the functionality of the particle. PMID:22978374

The complement factor 5a receptor 1 has a pathogenic role in chronic inflammation and renal fibrosis in a murine model of chronic pyelonephritis.

PubMed

Choudhry, Naheed; Li, Ke; Zhang, Ting; Wu, Kun-Yi; Song, Yun; Farrar, Conrad A; Wang, Na; Liu, Cheng-Fei; Peng, Qi; Wu, Weiju; Sacks, Steven H; Zhou, Wuding

2016-09-01

Complement factor 5a (C5a) interaction with its receptor (C5aR1) contributes to the pathogenesis of inflammatory diseases, including acute kidney injury. However, its role in chronic inflammation, particularly in pathogen-associated disorders, is largely unknown. Here we tested whether the development of chronic inflammation and renal fibrosis is dependent on C5aR1 in a murine model of chronic pyelonephritis. C5aR1-deficient (C5aR1-/-) mice showed a significant reduction in bacterial load, tubule injury and tubulointerstitial fibrosis in the kidneys following infection, compared with C5aR1-sufficient mice. This was associated with reduced renal leukocyte infiltration specifically for the population of Ly6Chi proinflammatory monocytes/macrophages and reduced intrarenal gene expression of key proinflammatory and profibrogenic factors in C5aR1-/- mice following infection. Antagonizing C5aR1 decreased renal bacterial load, tissue inflammation and tubulointerstitial fibrosis. Ex vivo and in vitro studies showed that under infection conditions, C5a/C5aR1 interaction upregulated the production of proinflammatory and profibrogenic factors by renal tubular epithelial cells and monocytes/macrophages, whereas the phagocytic function of monocytes/macrophages was down-regulated. Thus, C5aR1-dependent bacterial colonization of the tubular epithelium, C5a/C5aR1-mediated upregulation of local inflammatory responses to uropathogenic E. coli and impairment of phagocytic function of phagocytes contribute to persistent bacterial colonization of the kidney, chronic renal inflammation and subsequent tubulointerstitial fibrosis. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Activation of complement factor B contributes to murine and human myocardial ischemia/reperfusion injury

PubMed Central

Hou, Yunfang; Wong, Karen A.; Lee, Daniel; Rushbrook, Julie I.; Gulaya, Karan; Hines, Roberta; Hollis, Tamika; Nistal Nuno, Beatriz; Mangi, Abeel A.; Hashim, Sabet; Pekna, Marcela; Catalfamo, Amy; Chin, Hsiao-ying; Patel, Foramben; Rayala, Sravani; Shevde, Ketan; Heeger, Peter S.

2017-01-01

The pathophysiology of myocardial injury that results from cardiac ischemia and reperfusion (I/R) is incompletely understood. Experimental evidence from murine models indicates that innate immune mechanisms including complement activation via the classical and lectin pathways are crucial. Whether factor B (fB), a component of the alternative complement pathway required for amplification of complement cascade activation, participates in the pathophysiology of myocardial I/R injury has not been addressed. We induced regional myocardial I/R injury by transient coronary ligation in WT C57BL/6 mice, a manipulation that resulted in marked myocardial necrosis associated with activation of fB protein and myocardial deposition of C3 activation products. In contrast, in fB-/- mice, the same procedure resulted in significantly reduced myocardial necrosis (% ventricular tissue necrotic; fB-/- mice, 20 ± 4%; WT mice, 45 ± 3%; P < 0.05) and diminished deposition of C3 activation products in the myocardial tissue (fB-/- mice, 0 ± 0%; WT mice, 31 ± 6%; P<0.05). Reconstitution of fB-/- mice with WT serum followed by cardiac I/R restored the myocardial necrosis and activated C3 deposition in the myocardium. In translational human studies we measured levels of activated fB (Bb) in intracoronary blood samples obtained during cardio-pulmonary bypass surgery before and after aortic cross clamping (AXCL), during which global heart ischemia was induced. Intracoronary Bb increased immediately after AXCL, and the levels were directly correlated with peripheral blood levels of cardiac troponin I, an established biomarker of myocardial necrosis (Spearman coefficient = 0.465, P < 0.01). Taken together, our results support the conclusion that circulating fB is a crucial pathophysiological amplifier of I/R-induced, complement-dependent myocardial necrosis and identify fB as a potential therapeutic target for prevention of human myocardial I/R injury. PMID:28662037

Activation of complement factor B contributes to murine and human myocardial ischemia/reperfusion injury.

PubMed

Chun, Nicholas; Haddadin, Ala S; Liu, Junying; Hou, Yunfang; Wong, Karen A; Lee, Daniel; Rushbrook, Julie I; Gulaya, Karan; Hines, Roberta; Hollis, Tamika; Nistal Nuno, Beatriz; Mangi, Abeel A; Hashim, Sabet; Pekna, Marcela; Catalfamo, Amy; Chin, Hsiao-Ying; Patel, Foramben; Rayala, Sravani; Shevde, Ketan; Heeger, Peter S; Zhang, Ming

2017-01-01

The pathophysiology of myocardial injury that results from cardiac ischemia and reperfusion (I/R) is incompletely understood. Experimental evidence from murine models indicates that innate immune mechanisms including complement activation via the classical and lectin pathways are crucial. Whether factor B (fB), a component of the alternative complement pathway required for amplification of complement cascade activation, participates in the pathophysiology of myocardial I/R injury has not been addressed. We induced regional myocardial I/R injury by transient coronary ligation in WT C57BL/6 mice, a manipulation that resulted in marked myocardial necrosis associated with activation of fB protein and myocardial deposition of C3 activation products. In contrast, in fB-/- mice, the same procedure resulted in significantly reduced myocardial necrosis (% ventricular tissue necrotic; fB-/- mice, 20 ± 4%; WT mice, 45 ± 3%; P < 0.05) and diminished deposition of C3 activation products in the myocardial tissue (fB-/- mice, 0 ± 0%; WT mice, 31 ± 6%; P<0.05). Reconstitution of fB-/- mice with WT serum followed by cardiac I/R restored the myocardial necrosis and activated C3 deposition in the myocardium. In translational human studies we measured levels of activated fB (Bb) in intracoronary blood samples obtained during cardio-pulmonary bypass surgery before and after aortic cross clamping (AXCL), during which global heart ischemia was induced. Intracoronary Bb increased immediately after AXCL, and the levels were directly correlated with peripheral blood levels of cardiac troponin I, an established biomarker of myocardial necrosis (Spearman coefficient = 0.465, P < 0.01). Taken together, our results support the conclusion that circulating fB is a crucial pathophysiological amplifier of I/R-induced, complement-dependent myocardial necrosis and identify fB as a potential therapeutic target for prevention of human myocardial I/R injury.

A New Approach for the Treatment of Arthritis in Mice with a Novel Conjugate of an anti-C5aR1 antibody and C5 siRNA

PubMed Central

Mehta, Gaurav; Scheinman, Robert I.; Holers, V. Michael; Banda, Nirmal K.

2015-01-01

Rheumatoid Arthritis (RA) is an inflammatory autoimmune joint disease in which the complement system plays an important role. Of the several components of complement, current evidence points to C5 as the most important inducer of inflammation. Several groups generated antibodies or siRNAs or small molecule inhibitors against C5 and C5aR1 (CD88) which have showed some efficacy in RA in animal models. However, none of these candidate therapeutics has moved from bench to bedside. Here we test in CAIA a new therapeutic strategy using a novel anti-C5ab-C5 siRNA conjugate. We first demonstrate that while C5aR2 or C5L2 (GPR77) plays no role in collagen antibody induced arthritis (CAIA), C5aR1 contributes to pathogenesis. We demonstrate that injection of siRNAs blocking either C5, C5aR1 or the combination decreased clinical disease activity (CDA) in mice with CAIA by 45%, 51% and 58%, respectively. Anti-C5 antibody (BB5.1) has only limited efficacy nonetheless significantly reduced arthritis up to 66%. We then generated a novel anti-C5aR1ab-protamine-C5siRNA conjugate. Here we show for the first time that while unconjugated antibody plus siRNAs reduce arthritis by 19%, our an anti-C5aR1ab - protamine - C5 siRNA conjugate was effective in reducing arthritis by 83% along with a parallel decrease in histopathology, C3 deposition, neutrophils and macrophages in the joints of mice with CAIA. These data suggest that by targeting anti-C5 siRNAs to the receptor for its C5a activation fragment (C5aR1), a striking clinical effect can be realized. PMID:25917104

Complement C5-inhibiting therapy for the thrombotic microangiopathies: accumulating evidence, but not a panacea

PubMed Central

Brocklebank, Vicky

2017-01-01

Abstract Thrombotic microangiopathy (TMA), characterized by organ injury occurring consequent to severe endothelial damage, can manifest in a diverse range of diseases. In complement-mediated atypical haemolytic uraemic syndrome (aHUS) a primary defect in complement, such as a mutation or autoantibody leading to over activation of the alternative pathway, predisposes to the development of disease, usually following exposure to an environmental trigger. The elucidation of the pathogenesis of aHUS resulted in the successful introduction of the complement inhibitor eculizumab into clinical practice. In other TMAs, although complement activation may be seen, its role in the pathogenesis remains to be confirmed by an interventional trial. Although many case reports in TMAs other than complement-mediated aHUS hint at efficacy, publication bias, concurrent therapies and in some cases the self-limiting nature of disease make broader interpretation difficult. In this article, we will review the evidence for the role of complement inhibition in complement-mediated aHUS and other TMAs. PMID:28980670

Thrombomodulin Mutations in Atypical Hemolytic–Uremic Syndrome

PubMed Central

Delvaeye, Mieke; Noris, Marina; De Vriese, Astrid; Esmon, Charles T.; Esmon, Naomi L.; Ferrell, Gary; Del-Favero, Jurgen; Plaisance, Stephane; Claes, Bart; Lambrechts, Diether; Zoja, Carla; Remuzzi, Giuseppe; Conway, Edward M.

2012-01-01

BACKGROUND The hemolytic–uremic syndrome consists of the triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. The common form of the syndrome is triggered by infection with Shiga toxin–producing bacteria and has a favorable outcome. The less common form of the syndrome, called atypical hemolytic–uremic syndrome, accounts for about 10% of cases, and patients with this form of the syndrome have a poor prognosis. Approximately half of the patients with atypical hemolytic–uremic syndrome have mutations in genes that regulate the complement system. Genetic factors in the remaining cases are unknown. We studied the role of thrombomodulin, an endothelial glycoprotein with anticoagulant, antiinflammatory, and cytoprotective properties, in atypical hemolytic–uremic syndrome. METHODS We sequenced the entire thrombomodulin gene (THBD) in 152 patients with atypical hemolytic–uremic syndrome and in 380 controls. Using purified proteins and cell-expression systems, we investigated whether thrombomodulin regulates the complement system, and we characterized the mechanisms. We evaluated the effects of thrombomodulin missense mutations associated with atypical hemolytic–uremic syndrome on complement activation by expressing thrombomodulin variants in cultured cells. RESULTS Of 152 patients with atypical hemolytic–uremic syndrome, 7 unrelated patients had six different heterozygous missense THBD mutations. In vitro, thrombomodulin binds to C3b and factor H (CFH) and negatively regulates complement by accelerating factor I–mediated inactivation of C3b in the presence of cofactors, CFH or C4b binding protein. By promoting activation of the plasma procarboxypeptidase B, thrombomodulin also accelerates the inactivation of anaphylatoxins C3a and C5a. Cultured cells expressing thrombomodulin variants associated with atypical hemolytic–uremic syndrome had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and were thus less protected from activated complement. CONCLUSIONS Mutations that impair the function of thrombomodulin occur in about 5% of patients with atypical hemolytic–uremic syndrome. PMID:19625716

Anti-C1q Antibodies in Systemic Lupus Erythematosus

PubMed Central

ORBAI, ANA-MARIA; TRUEDSSON, LENNART; STURFELT, GUNNAR; NIVED, OLA; FANG, HONG; ALARCÓN, GRACIELA S.; GORDON, CAROLINE; MERRILL, JOAN T.; FORTIN, PAUL R.; BRUCE, IAN N.; ISENBERG, DAVID A.; WALLACE, DANIEL J.; RAMSEY-GOLDMAN, ROSALIND; BAE, SANG-CHEOL; HANLY, JOHN G.; SANCHEZ-GUERRERO, JORGE; CLARKE, ANN E.; ARANOW, CYNTHIA B.; MANZI, SUSAN; UROWITZ, MURRAY B.; GLADMAN, DAFNA D.; KALUNIAN, KENNETH C.; COSTNER, MELISSA I.; WERTH, VICTORIA P.; ZOMA, ASAD; BERNATSKY, SASHA; RUIZ-IRASTORZA, GUILLERMO; KHAMASHTA, MUNTHER A.; JACOBSEN, SOREN; BUYON, JILL P.; MADDISON, PETER; DOOLEY, MARY ANNE; VAN VOLLENHOVEN, RONALD F.; GINZLER, ELLEN; STOLL, THOMAS; PESCHKEN, CHRISTINE; JORIZZO, JOSEPH L.; CALLEN, JEFFREY P.; LIM, S. SAM; FESSLER, BARRI J.; INANC, MURAT; KAMEN, DIANE L.; RAHMAN, ANISUR; STEINSSON, KRISTJAN; FRANKS, ANDREW G.; SIGLER, LISA; HAMEED, SUHAIL; PHAM, NEENA; BREY, ROBIN; WEISMAN, MICHAEL H.; MCGWIN, GERALD; MAGDER, LAURENCE S.; PETRI, MICHELLE

2014-01-01

Objective Anti-C1q has been associated with systemic lupus erythematosus (SLE) and lupus nephritis in previous studies. We studied anti-C1q specificity for SLE (vs. rheumatic disease controls) and the association with SLE manifestations in an international multi-center study. Methods Information and blood samples were obtained in a cross-sectional study from patients with SLE (n=308) and other rheumatologic diseases (n=389) from 25 clinical sites (84% female, 68% Caucasian, 17% African descent, 8% Asian, 7% other). IgG anti-C1q against the collagen-like region was measured by ELISA. Results Prevalence of anti-C1q was 28% (86/308) in patients with SLE and 13% (49/389) in controls (OR=2.7, 95% CI: 1.8-4, p<0.001). Anti-C1q was associated with proteinuria (OR=3.0, 95% CI: 1.7-5.1, p<0.001), red cell casts (OR=2.6, 95% CI: 1.2-5.4, p=0.015), anti-dsDNA (OR=3.4, 95% CI: 1.9-6.1, p<0.001) and anti-Smith (OR=2.8, 95% CI: 1.5-5.0, p=0.01). Anti-C1q was independently associated with renal involvement after adjustment for demographics, ANA, anti-dsDNA and low complement (OR=2.3, 95% CI: 1.3-4.2, p<0.01). Simultaneously positive anti-C1q, anti-dsDNA and low complement was strongly associated with renal involvement (OR=14.9, 95% CI: 5.8-38.4, p<0.01). Conclusions Anti-C1q was more common in patients with SLE and those of Asian race/ethnicity. We confirmed a significant association of anti-C1q with renal involvement, independent of demographics and other serologies. Anti-C1q in combination with anti-dsDNA and low complement was the strongest serological association with renal involvement. These data support the usefulness of anti-C1q in SLE, especially in lupus nephritis. PMID:25124676

Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura.

PubMed

Cao, Wenjing; Pham, Huy P; Williams, Lance A; McDaniel, Jenny; Siniard, Rance C; Lorenz, Robin G; Marques, Marisa B; Zheng, X Long

2016-11-01

Acquired thrombotic thrombocytopenic purpura is primarily caused by the deficiency of plasma ADAMTS13 activity resulting from autoantibodies against ADAMTS13. However, ADAMTS13 deficiency alone is often not sufficient to cause acute thrombotic thrombocytopenic purpura. Infections or systemic inflammation may precede acute bursts of the disease, but the underlying mechanisms are not fully understood. Herein, 52 patients with acquired autoimmune thrombotic thrombocytopenic purpura and 30 blood donor controls were recruited for the study. The plasma levels of human neutrophil peptides 1-3 and complement activation fragments (i.e. Bb, iC3b, C4d, and sC5b-9) were determined by enzyme-linked immunosorbent assays. Univariate analyses were performed to determine the correlation between each biomarker and clinical outcomes. We found that the plasma levels of human neutrophil peptides 1-3 and Bb in patients with acute thrombotic thrombocytopenic purpura were significantly higher than those in the control (P<0.0001). The plasma levels of HNP1-3 correlated with the levels of plasma complement fragment Bb (rho=0.48, P=0.0004) and serum lactate dehydrogenase (rho=0.28, P=0.04); in addition, the plasma levels of Bb correlated with iC3b (rho=0.55, P<0.0001), sC5b-9 (rho=0.63, P<0.0001), serum creatinine (rho=0.42, p=0.0011), and lactate dehydrogenase (rho=0.40, P=0.0034), respectively. Moreover, the plasma levels of iC3b and sC5b-9 were correlated (rho=0.72, P<0.0001), despite no statistically significant difference of the two markers between thrombotic thrombocytopenic purpura patients and the control. We conclude that innate immunity, i.e. neutrophil and complement activation via the alternative pathway, may play a role in the pathogenesis of acute autoimmune thrombotic thrombocytopenic purpura, and a therapy targeted at these pathways may be considered in a subset of these patients. Copyright© Ferrata Storti Foundation.

Effects of CTLA4-Fc on glomerular injury in humorally-mediated glomerulonephritis in BALB/c mice

PubMed Central

Kitching, A R; Huang, X R; Ruth, A-J; Tipping, P G; Holdsworth, S R

2002-01-01

The effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury. PMID:12067297

Effects of CTLA4-Fc on glomerular injury in humorally-mediated glomerulonephritis in BALB/c mice.

PubMed

Kitching, A R; Huang, X R; Ruth, A-J; Tipping, P G; Holdsworth, S R

2002-06-01

The effect of cytotoxic T-lymphocyte-associated molecule 4-immunoglobulin fusion protein (CTLA4-Fc) on humorally-mediated glomerulonephritis was studied in accelerated anti-glomerular basement membrane (anti-GBM) glomerulonephritis induced in BALB/c mice. This strain of mice develops antibody and complement dependent glomerulonephritis under this protocol. Sensitized BALB/c mice developed high levels of circulating autologous antibody titres, intense glomerular deposition of mouse immunoglobulin and complement, significant proteinuria, renal impairment, significant glomerular necrosis and a minor component of crescent formation 10 days after challenge with a nephritogenic antigen (sheep anti-GBM globulin). Early treatment during the primary immune response, or continuous treatment throughout the disease with CTLA4-Fc, significantly suppressed mouse anti-sheep globulin antibody titres in serum, and immunoglobulin and complement deposition in glomeruli. The degree of glomerular necrosis was improved and proteinuria was reduced, particularly in the earlier stages of disease. Late treatment by CTLA4-Fc starting one day after challenge with sheep anti-mouse GBM did not affect antibody production and did not attenuate glomerulonephritis. The low level of crescent formation found in BALB/c mice developing glomerulonephritis was not prevented by the administration of CTLA4-Fc. These results demonstrate that CTLA4-Fc is of benefit in this model of glomerulonephritis by its capacity to attenuate antibody production, without affecting the minor degree of cell-mediated glomerular injury.

Asthma progression to airway remodeling and bone marrow eosinophil responses in genetically distinct strains of mice.

PubMed

Hogan, Mary Beth; Piktel, Debra; Hubbs, Ann F; McPherson, Leslie E; Landreth, Kenneth S

2008-12-01

Patient factors that cause long-term airway remodeling are largely unidentified. This suggests that genetic differences may determine which asthmatic patients develop airway remodeling. A murine model with repeated allergen exposure leading to peribronchial fibrosis in complement factor 5 (C5)-deficient A/J mice has been used to study asthma progression. No studies have addressed the systemic effects of allergen sensitization or chronic allergen exposure on bone marrow eosinophilopoiesis in this mouse strain. To investigate bone marrow eosinophil responses during acute sensitization and chronic allergen exposure using genetically distinct mouse strains differing in persistent airway reactivity and remodeling. The C5-sufficient BALB/c and C5-deficient A/J mice were repetitively exposed to intranasal ovalbumin for 12 weeks. Subsequently, the mice were evaluated for airway eosinophilia, mucus-containing goblet cells, and peribronchial fibrosis. Both strains of mice were also acutely sensitized to ovalbumin. Bone marrow eosinophil progenitor cells and mature eosinophils were enumerated. BALB/c and A/J mice have similar bone marrow responses after acute allergen exposure, with elevations in bone marrow eosinophil progenitor cell and eosinophil numbers. After chronic allergen exposure, only C5-deficient A/J mice that developed peribronchial fibrosis exhibited bone marrow eosinophilia. BALB/c mice lacked peribronchial fibrosis and extinguished accelerated eosinophil production after long-term allergen challenge. Chronic airway remodeling after repeated allergen exposure in genetically different mice correlated with differences in long-term bone marrow eosinophilopoiesis. Preventing asthma from progressing to chronic airway remodeling with fibrosis may involve identifying genetically determined influences on bone marrow responses to chronic allergen exposure.

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. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

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, ECLAM

Natural IgM mediates complement-dependent uptake of Francisella tularensis by human neutrophils via CR1 and CR3 in nonimmune serum

PubMed Central

Schwartz, Justin T.; Barker, Jason H.; Long, Matthew E.; Kaufman, Justin; McCracken, Jenna; Allen, Lee-Ann H.

2012-01-01

A fundamental step in the life cycle of F. tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum, nor the receptors that mediate infection of neutrophils has been defined. Herein human neutrophil uptake of GFP-expressing F. tularensis strains LVS and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis whereas C5 was not. Second, we used purification and immuno-depletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-antigen and capsule as prominent targets of these antibodies on the bacterial surface. Finally, we demonstrate using receptor-blocking antibodies that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-antigen polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3-opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner. PMID:22888138

Interallelic complementation of mutations in propionic acidemia by microinjection of mutant cDNAs into fibroblasts of affected patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loyer, M.; Leclerc, D.; Gravel, R.A.

1994-09-01

Propionic acidemia is a rare autosomal recessive disorder resulting from defects of the {alpha} or {beta} subunit of biotin-dependent propionyl-CoA carboxylase (PCC). Mutations are assigned to defects of the PCCA ({alpha} subunit) or PCCB ({beta} subunit) gene through complementation studies after somatic fusion of patient cell lines. About two-thirds of patients with {beta} subunit defects (complementation group pccBC) show interallelic complementation in cell fusion experiments (subgroups pccB and pccC), monitored by the PCC-dependent metabolisms of {sup 14}C-propionate. Most patient cell lines are heteroallelic for two different mutations, leaving ambiguous the identity of the mutation participating in interallelic complementation. To identifymore » the complementing mutations, we have expressed {beta}-subunit cDNAs containing individual mutations by microinjection of the cDNAs in recipient cells from patients with {beta} subunit defects. Correction of the PCC defect was monitored by autoradiography of {sup 14}C-propionate incorporation. In some experiments, cDNAs were co-injected with a plasmid expressing the E. coli lacZ gene as a positive control for successful injection. Two mutations from the pccB subgroup showed complementation when injected into pccC cells; dupKICK140-143 and Pro228Leu. Similarly, two mutations from the pccC subgroup complemented after injection into pccB cells; {Delta}Ile408 and Arg410Trp. No mutation complemented with mutation of the pccBC group which are classified as non-complementing in cell fusion experiments. The results show that the complementing pccB mutations are found in the N-terminal half of the {beta} subunit, while the complementing pccC mutations cluxter at a site in the C-terminal half. The latter site is a candidate for the propionyl-CoA binding site based on sequence identity with a region of transcarboxylase from Propionibacterium shermanii.« less

C5a and C5aR are elevated in joints of rheumatoid and psoriatic arthritis patients, and C5aR blockade attenuates leukocyte migration to synovial fluid.

PubMed

Hornum, Lars; Hansen, Anker Jon; Tornehave, Ditte; Fjording, Marianne Scheel; Colmenero, Paula; Wätjen, Inger Falbe; Søe Nielsen, Niels Henrik; Bliddal, Henning; Bartels, Else Marie

2017-01-01

Complement activation correlates to rheumatoid arthritis disease activity, and increased amounts of the complement split product C5a is observed in synovial fluids from rheumatoid arthritis patients. Blockade of C5a or its receptor (C5aR) is efficacious in several arthritis models. The aim of this study was to investigate the role of C5a and C5aR in human rheumatoid arthritis and psoriatic arthritis-both with respect to expression and function. Synovial fluid, blood and synovial samples were obtained from rheumatoid arthritis, psoriatic arthritis and osteoarthritis patients as a less inflammatory arthritis type, and blood from healthy subjects. Cells infiltrating synovial tissue were analysed by immunohistochemistry and flow cytometry. SF and blood were analysed for biomarkers by flow cytometry or ELISA. The effect of a blocking anti-human C5aR mAb on leukocyte migration was determined using a Boyden chamber. Appropriate statistical tests were applied for comparisons. C5aR+ cells were detected in most rheumatoid arthritis, in all psoriatic arthritis, but not in non-inflammatory control synovia. C5aR+ cells were primarily neutrophils and macrophages. C5aR+ macrophages were mainly found in lymphoid aggregates in close contact with T cells. C5a levels were increased in both rheumatoid arthritis and psoriatic arthritis synovial fluid compared to osteoarthritis, and in blood from rheumatoid arthritis compared to healthy subjects. Neutrophil and monocyte migration to rheumatoid arthritis synovial fluid was significantly inhibited by anti-C5aR. The data support that the C5a-C5aR axis may be driving the infiltration of inflammatory cells into the synovial fluid and synovium in both rheumatoid and psoriatic arthritis, and suggest that C5a or C5aR may be a promising treatment target in both diseases.

ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies: an infectious diseases perspective (Soluble immune effector molecules [II]: agents targeting interleukins, immunoglobulins and complement factors).

PubMed

Winthrop, K L; Mariette, X; Silva, J T; Benamu, E; Calabrese, L H; Dumusc, A; Smolen, J S; Aguado, J M; Fernández-Ruiz, M

2018-06-01

The present review is part of the ESCMID Study Group for Infections in Compromised Hosts (ESGICH) Consensus Document on the safety of targeted and biological therapies. To review, from an Infectious Diseases perspective, the safety profile of agents targeting interleukins, immunoglobulins and complement factors and to suggest preventive recommendations. Computer-based MEDLINE searches with MeSH terms pertaining to each agent or therapeutic family. Patients receiving interleukin-1 (IL-1) -targeted (anakinra, canakinumab or rilonacept) or IL-5-targeted (mepolizumab) agents have a moderate risk of infection and no specific prevention strategies are recommended. The use of IL-6/IL-6 receptor-targeted agents (tocilizumab and siltuximab) is associated with a risk increase similar to that observed with anti-tumour necrosis factor-α agents. IL-12/23-targeted agents (ustekinumab) do not seem to pose a meaningful risk of infection, although screening for latent tuberculosis infection may be considered and antiviral prophylaxis should be given to hepatitis B surface antigen-positive patients. Therapy with IL-17-targeted agents (secukinumab, brodalumab and ixekizumab) may result in the development of mild-to-moderate mucocutaneous candidiasis. Pre-treatment screening for Strongyloides stercoralis and other geohelminths should be considered in patients who come from areas where these are endemic who are receiving IgE-targeted agents (omalizumab). C5-targeted agents (eculizumab) are associated with a markedly increased risk of infection due to encapsulated bacteria, particularly Neisseria spp. Meningococcal vaccination and chemoprophylaxis must be administered 2-4 weeks before initiating eculizumab. Patients with high-risk behaviours and their partners should also be screened for gonococcal infection. Preventive strategies are particularly encouraged to minimize the occurrence of neisserial infection associated with eculizumab. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Cloning, expression, cellular distribution, and role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in a nonmammalian species

USGS Publications Warehouse

Boshra, Hani; Li, Jun; Peters, Rodney; Hansen, John; Matlapudi, Anjan; Sunyer, J. Oriol

2004-01-01

C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.

Pathogenesis of aortic dilatation in mucopolysaccharidosis VII mice may involve complement activation

PubMed Central

Baldo, Guilherme; Wu, Susan; Howe, Ruth A.; Ramamoothy, Meera; Knutsen, Russell H.; Fang, Jiali; Mecham, Robert P.; Liu, Yuli; Wu, Xiaobo; Atkinson, John P.; Ponder, Katherine P.

2012-01-01

Mucopolysaccharidosis VII (MPS VII) is due to mutations within the gene encoding the lysosomal enzyme β-glucuronidase, and results in the accumulation of glycosaminoglycans. MPS VII causes aortic dilatation and elastin fragmentation, which is associated with upregulation of the elastases cathepsin S (CtsS) and matrix metalloproteinase 12 (MMP12). To test the role of these enzymes, MPS VII mice were crossed with mice deficient in CtsS or MMP12, and the effect upon aortic dilatation was determined. CtsS deficiency did not protect against aortic dilatation in MPS VII mice, but also failed to prevent an upregulation of cathepsin enzyme activity. Further analysis with substrates and inhibitors specific for particular cathepsins suggests that this enzyme activity was due to CtsB, which could contribute to elastin fragmentation. Similarly, MMP12 deficiency and deficiency of both MMP12 and CtsS could not prevent aortic dilatation in MPS VII mice. Microarray and reverse-transcriptase real-time PCR were performed to look for upregulation of other elastases. This demonstrated that mRNA for complement component D was elevated in MPS VII mice, while immunostaining demonstrated high levels of complement component C3 on surfaces within the aortic media. Finally, we demonstrate that neonatal intravenous injection of a retroviral vector encoding β-glucuronidase reduced aortic dilatation. We conclude that neither CtsS nor MMP12 are necessary for elastin fragmentation in MPS VII mouse aorta, and propose that CtsB and/or complement component D may be involved. Complement may be activated by the GAGs that accumulate, and may play a role in signal transduction pathways that upregulate elastases. PMID:21944884

Correction of both spontaneous and DEB-induced chromosome instability in Fanconi anemia FA-C cells by FACC cDNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stavropoulos, D.J.; Tomkins, D.J.; Allingham-Hawkins, D.J.

1994-09-01

Cells from all four Fanconi anemia complementation groups show hypersensitivity to cell-killing by mitomycin C (MMC), diepoxybutane (DEB) and other DNA cross-linking agents, and increased spontaneous and DEB-induced chromosome aberrations (CA). The extent of these phenotypes varies between lymphoblastoid cell lines from different complementation groups. Our data showed that the difference in MMC hypersensitivity and DEB-CA was not always coupled. While 230N (FA-B) had higher DEB-induced CA/cell than 536N (FA-C) (7.42 vs. 4.46 respectively), that latter was much more sensitive to cell-killing by MMC (dose at 10% survival, D{sub 10}: 5.2 vs. 1.2 ng/ml respectively). Strathdes et al. (1992) clonedmore » a cDNA Fanconi anemia complementation group C (FACC) which complemented the hypersensitivity to MMC and DEB cell-killing of FA-C cells (536N) but not cells from the other three complementation groups. The present study was initiated to determine whether chromosome instability in 536N is also complemented by the FACC (FAC3) cDNA. The pREP4-FAC3 vector was transfected into 536N and transfectants selected with hygromycin B. The DEB D{sub 10} of 536N (1.0 {mu}M) was corrected to the control level (16.2 {mu}M for 3TO) by FACC (15.1 {mu}M for 536N-FACC), as previously demonstrated. Chromosome instability (cab, cse, ctb, cte) was determined without and with 0.1 {mu}g/ml DEB treatment. Spontaneous CA of 536N (0.30 aberrations/cell) was corrected to the control level (0.04 for 3TO) by FACC (0.06 for 536N-FACC). Similarly, the DEB-induced CA was corrected (2.74 for 536N vs. 0.06 and 0.02 for 3TO and 536N-FACC respectively). Thus, at least for FA complementation group C, hypersensitivity to cell-killing and chromosome instability are not dissociated and are most likely caused by the same gene defect.« less

A Novel Role for C5a in B-1 Cell Homeostasis

PubMed Central

Bröker, Katharina; Figge, Julia; Magnusen, Albert F.; Manz, Rudolf A.; Köhl, Jörg; Karsten, Christian M.

2018-01-01

B-1 cells constitute a unique subpopulation of lymphocytes residing mainly in body cavities like the peritoneal cavity (PerC) but are also found in spleen and bone marrow (BM). As innate-like B cells, they mediate first line immune defense through low-affinity natural IgM (nIgM) antibodies. PerC B-1 cells can egress to the spleen and differentiate into nIgM antibody-secreting plasma cells that recognize conserved exogenous and endogenous cellular structures. Homing to and homeostasis within the PerC are regulated by the chemokine CXCL13 released by PerC macrophages and stroma cells. However, the exact mechanisms underlying the regulation of CXCL13 and B-1 homeostasis are not fully explored. B-1 cells play important roles in the inflammatory response to infection, autoimmunity, ischemia/reperfusion injury, obesity, and atherosclerosis. Remarkably, this list of inflammatory entities has a strong overlap with diseases that are regulated by complement suggesting a link between B-1 cells and the complement system. Interestingly, up to now, no data exist regarding the role of complement in B-1 cell biology. Here, we demonstrate for the first time that C5a regulates B-1 cell steady-state dynamics within the peritoneum, the spleen, and the BM. We found decreased B-1a cell numbers in the peritoneum and the spleen of C5aR1−/− mice associated with increased B1-a and B1-b numbers in the spleen and high serum titers of nIgM antibodies directed against phosphorylcholine and several pneumococcal polysaccharides. Similarly, peritoneal B-1a cells were decreased in the peritoneum and splenic B-1a and B-1b cells were increased in C5aR2−/− mice. The decrease in peritoneal B-1 cell numbers was associated with decreased peritoneal CXCL13 levels in C5aR1−/− and C5aR2−/− mice. In search for mechanisms, we found that combined TLR2 and IL-10 receptor activation in PerC macrophages induced strong CXCL13 production, which was significantly reduced in cells from C5aR1- and C5aR2-deficient mice and after combined C5aR-targeting. Such stimulation also induced marked local C5 production by PerC macrophages and C5a generation. Importantly, peritoneal in vivo administration of C5a increased CXCL13 production. Taken together, our findings suggest that local non-canonical C5 activation in PerC macrophages fuels CXCL13 production as a novel mechanism to control B-1 cell homeostasis. PMID:29520270

Complement research in the 18th-21st centuries: Progress comes with new technology.

PubMed

Sim, R B; Schwaeble, W; Fujita, T

2016-10-01

The complement system has been studied for about 120 years. Progress in defining this large and complex system has been dependent on the research technologies available, but since the introduction of protein chromatography, electrophoresis, and antibody-based assay methods in the 1950s and 60s, and sequencing of proteins and DNA in the 70s and 80s, there has been very rapid accumulation of data. With more recent improvements in 3D structure determination (nmr and X-ray crystallography), the structures of most of the complement proteins have now been solved. Complement research since 1990 has been greatly stimulated by the discoveries of the multiple proteins in the lectin pathway, the strong association of Factor H, C3, Factor B allelic variants with adult macular degeneration and atypical haemolytic uremic syndrome, and the introduction of the anti-C5 monoclonal antibody as a therapy for paroxysmal nocturnal hemoglobinuria and atypical haemolytic uremic syndrome. Potential new roles for complement in tissue development and the search for novel therapeutics suggest a very active future for complement research. Copyright © 2016 Elsevier GmbH. All rights reserved.

Epididymal C4b-binding protein is processed and degraded during transit through the duct and is not essential for fertility.

PubMed

Nonaka, Mayumi I; Zsigmond, Eva; Kudo, Akihiko; Kawakami, Hayato; Yoshida, Kaoru; Yoshida, Manabu; Kawano, Natsuko; Miyado, Kenji; Nonaka, Masaru; Wetsel, Rick A

2015-04-01

C4b-binding protein (C4BP) is known as one of the circulating complement regulators that prevents excessive activation of the host-defense complement system. We have reported previously that C4BP is expressed abundantly in the rodent epididymis, one of the male reproductive organs connecting the testis and vas deferens, where immature spermatozoa acquire their motility and fertilizing ability during their transit through the duct. Epididymal C4BP (EpC4BP) is synthesized androgen-dependently by the epithelial cells, secreted into the lumen, and bound to the outer membrane of the passing spermatozoa. In this study, we found that EpC4BP is secreted as a large oligomer, similar to the serum C4BP, but is digested during the epididymal transit and is almost lost from both the luminal fluid and the sperm surface in the vas deferens. Such a processing pattern is not known in serum C4BP, suggesting that EpC4BP and serum C4BP might have different functional mechanisms, and that there is a novel function of EpC4BP in reproduction. In addition, the disappearance of EpC4BP from the sperm surface prior to ejaculation suggests that EpC4BP works only in the epididymis and would not work in the female reproductive tract to protect spermatozoa from complement attack. Next, we generated C4BP-deficient (C4BP-/-) mice to examine the possible role of EpC4BP in reproduction. However, the C4BP-/- mice were fertile and no significant differences were observed between the C4BP-/- and wild-type mouse spermatozoa in terms of morphology, motility, and rate of the spontaneous acrosome reaction. These results suggest that EpC4BP is involved in male reproduction, but not essential for sperm maturation. Copyright © 2014 Elsevier GmbH. All rights reserved.

Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

PubMed

Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

2016-01-01

Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

21 CFR 866.5260 - Complement C3b inactivator immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5260 Complement C3b inactivator immunological test system. (a) Identification. A complement... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Complement C3b inactivator immunological test...

Complement-mediated neutrophil activation in sepsis- and trauma-related adult respiratory distress syndrome. Clarification with radioaerosol lung scans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tennenberg, S.D.; Jacobs, M.P.; Solomkin, J.S.

Complement-mediated neutrophil activation (CMNA) has been proposed as an important pathogenic mechanism causing acute microvascular lung injury in the adult respiratory distress syndrome (ARDS). To clarify the relationship between CMNA and evolving lung injury, we studied 26 patients with multiple trauma and sepsis within 24 hours of risk establishment for ARDS. Pulmonary alveolar-capillary permeability (PACP) was quantified as the clearance rate of a particulate radioaerosol. Seventeen patients (65%) had increased PACP (six developed ARDS) while nine (35%) had normal PACP (none developed ARDS; clearance rates of 3.4%/min and 1.5%/min, respectively). These patients, regardless of evidence of early lung injury, hadmore » elevated plasma C3adesArg levels and neutrophil chemotactic desensitization to C5a/C5adesArg. Plasma C3adesArg levels correlated weakly, but significantly, with PACP. Thus, CMNA may be a necessary, but not a sufficient, pathogenic mechanism in the evolution of ARDS.« less

The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia

PubMed Central

Lynch, AM; Murphy, JR; Gibbs, RS; Levine, RJ; Giclas, PC; Salmon, JE; Holers, VM

2016-01-01

Objective To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. Design Prospective cohort study. Setting Denver complement study (June 2005–June 2008). Population A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. Methods Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. Main outcome measure Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. Results The mean (±SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (±0.26) µg/ml and 0.69 (±0.2) µg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 ± 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 ± 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4–3.1, P < 0.0003) and 0.2 (CI = 0.07–0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. Conclusions In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy. PMID:20074261

Steroids and triterpenes from the fruit bodies of Ganoderma lucidum and their anti-complement activity.

PubMed

Seo, Hyo Won; Hung, Tran Manh; Na, MinKyun; Jung, Hyun Ju; Kim, Jin Cheol; Choi, Jae Sue; Kim, Jung Hee; Lee, Hyeong-Kyu; Lee, IkSoo; Bae, KiHwan; Hattori, Masao; Min, Byung Sun

2009-11-01

To determine the anti-complement activity of natural triterpenes, chromatographic separation of the EtOAc-soluble fraction from the fruiting body of Ganoderma lucidum led to the isolation of three steroids and five triterpenoids. They were identified as ergosterol peroxide (1), ergosterol (2), genoderic acid Sz (3), stella sterol (4), ganoderic aic C1 (5), ganoderic acid A (6), methyl ganoderate A (7), and lucidenic acid A (8) based on spectroscopic evidence and physicochemical properties. These compounds were examined for their anti-complement activity against the classical pathway of the complement system. Compounds 2 and 3 showed potent anti-complement activity with IC50 values of 52.0 and 44.6 microM, respectively. Compound 1 exhibited significant inhibitory activity with an IC50 value of 126.8 microM, whereas compounds 4-8 were inactive. Our findings suggested that in addition to the ketone group at C-3, the delta7(8), delta9(11)-lanostadiene type triterpene also plays an important role in inhibiting the hemolytic activity of human serum against erythrocytes.

Streptococcus pneumoniae PspC Subgroup Prevalence in Invasive Disease and Differences in Contribution to Complement Evasion.

PubMed

van der Maten, Erika; van den Broek, Bryan; de Jonge, Marien I; Rensen, Kim J W; Eleveld, Marc J; Zomer, Aldert L; Cremers, Amelieke J H; Ferwerda, Gerben; de Groot, Ronald; Langereis, Jeroen D; van der Flier, Michiel

2018-04-01

The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition. Copyright © 2018 American Society for Microbiology.

RNA interference of GGTA1 physiological and immune functions in immortalized porcine aortic endothelial cells.

PubMed

Han, Wei; Zhou, Jingshi; Li, Xiao; Wang, Jianfeng; Li, Junjie; Zhang, Zhuochao; Yang, Zhaoxu; Wang, Desheng; Tao, Kaishan; Dou, Kefeng

2013-11-01

Pig organs are commonly used in xenotransplantation, and α-1,3-galactose has been shown to be the main cause of hyperacute rejection. The development of transgenic pigs that lack α-1,3-galactosyltransferase (GGTA1) has overcome this problem to a certain extent, but transgenic pigs are difficult to maintain, making their usefulness in basic research limited. For this reason, we propose to establish a cell model to study hyperacute rejection. Immortalized primary porcine aortic endothelial cells were transfected with a short hairpin RNA targeted to GGTA1. Cell proliferation, apoptosis, complement C3 activation, and the binding of human immunoglobulins and components of the complement system, including IgM, IgG, C3, and C5b-9, were examined. After RNA interference, GGTA1 was found to be reduced at both the transcript and protein level as assessed by quantitative polymerase chain reaction and flow cytometry, respectively. When cultured in the presence of human serum, the proliferation rate of the transfected cells was higher than that of untransfected cells, and the apoptosis rate was lower. Additionally, activation of C3 and the binding of human immunoglobulins IgM and IgG and complement component C3 and C5b-9 to the transfected cells were lower than in the immortalized group but higher than in untransfected cells. RNA interference of GGTA1 in cultured porcine endothelial cells reduces the reaction of immunoglobulin and complement system with the cells. Therefore, this in vitro cell model could be useful for further study of xenotransplantation. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular characterization of the alpha subunit of complement component C8 (GcC8alpha) in the nurse shark (Ginglymostoma cirratum).

PubMed

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

2009-09-01

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 alpha, beta, and gamma 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 C8alpha and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8gamma-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 C8alpha respectively. Southern blot analysis showed GcC8alpha 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 C8alpha orthologs and as a sister taxa to the Xenopus. 2009 Elsevier Ltd.

Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells

PubMed Central

Eadon, Michael T; Jacob, Alexander; Cunningham, Patrick N; Quigg, Richard J; Garcia, Joe G N; Alexander, Jessy J

2014-01-01

Blood–brain barrier (BBB) disturbance is a crucial occurrence in many neurological diseases, including systemic lupus erythematosus (SLE). Our previous studies showed that experimental lupus serum altered the integrity of the mouse brain endothelial layer, an important constituent of the BBB. Complement activation occurs in lupus with increased circulating complement components. Using a genomics approach, we identified the microRNA (miRNA) altered in mouse brain endothelial cells (bEnd3) by lupus serum and the complement protein, C5a. Of the 318 miRNA evaluated, 23 miRNAs were altered by lupus serum and 32 were altered by C5a alone compared with controls. Seven miRNAs (P < 0·05) were differentially expressed by both treatments: mmu-miR-133a*, mmu-miR-193*, mmu-miR-26b, mmu-miR-28*, mmu-miR-320a, mmu-miR-423-3p and mmu-miR-509-5p. The microarray results were validated by quantitative RT-PCR. In line with the in vitro results, expression of miR-26b and miR-28* were also significantly up-regulated in lupus mouse brain which was reduced by C5a receptor inhibition. Target prediction analysis revealed miR gene targets encoding components involved in inflammation, matrix arrangement, and apoptosis, pathways known to play important roles in central nervous system lupus. Our findings suggest that the miRNAs reported in this study may represent novel therapeutic targets in central nervous system lupus and other similar neuroinflammatory settings. PMID:24801999

Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells.

PubMed

Eadon, Michael T; Jacob, Alexander; Cunningham, Patrick N; Quigg, Richard J; Garcia, Joe G N; Alexander, Jessy J

2014-11-01

Blood-brain barrier (BBB) disturbance is a crucial occurrence in many neurological diseases, including systemic lupus erythematosus (SLE). Our previous studies showed that experimental lupus serum altered the integrity of the mouse brain endothelial layer, an important constituent of the BBB. Complement activation occurs in lupus with increased circulating complement components. Using a genomics approach, we identified the microRNA (miRNA) altered in mouse brain endothelial cells (bEnd3) by lupus serum and the complement protein, C5a. Of the 318 miRNA evaluated, 23 miRNAs were altered by lupus serum and 32 were altered by C5a alone compared with controls. Seven miRNAs (P < 0 · 05) were differentially expressed by both treatments: mmu-miR-133a*, mmu-miR-193*, mmu-miR-26b, mmu-miR-28*, mmu-miR-320a, mmu-miR-423-3p and mmu-miR-509-5p. The microarray results were validated by quantitative RT-PCR. In line with the in vitro results, expression of miR-26b and miR-28* were also significantly up-regulated in lupus mouse brain which was reduced by C5a receptor inhibition. Target prediction analysis revealed miR gene targets encoding components involved in inflammation, matrix arrangement, and apoptosis, pathways known to play important roles in central nervous system lupus. Our findings suggest that the miRNAs reported in this study may represent novel therapeutic targets in central nervous system lupus and other similar neuroinflammatory settings. © 2014 John Wiley & Sons Ltd.

Plasma-based proteomics reveals immune response, complement and coagulation cascades pathway shifts in heat-stressed lactating dairy cows.

PubMed

Min, Li; Cheng, Jianbo; Zhao, Shengguo; Tian, He; Zhang, Yangdong; Li, Songli; Yang, Hongjian; Zheng, Nan; Wang, Jiaqi

2016-09-02

Heat stress (HS) has an enormous economic impact on the dairy industry. In recent years, many researchers have investigated changes in the gene expression and metabolomics profiles in dairy cows caused by HS. However, the proteomics profiles of heat-stressed dairy cows have not yet been completely elucidated. We compared plasma proteomics from HS-free and heat-stressed dairy cows using an iTRAQ labeling approach. After the depletion of high abundant proteins in the plasma, 1472 proteins were identified. Of these, 85 proteins were differentially abundant in cows exposed to HS relative to HS-free. Database searches combined with GO and KEGG pathway enrichment analyses revealed that many components of the complement and coagulation cascades were altered in heat-stressed cows compared with HS-free cows. Of these, many factors in the complement system (including complement components C1, C3, C5, C6, C7, C8, and C9, complement factor B, and factor H) were down-regulated by HS, while components of the coagulation system (including coagulation factors, vitamin K-dependent proteins, and fibrinogens) were up-regulated by HS. In conclusion, our results indicate that HS decreases plasma levels of complement system proteins, suggesting that immune function is impaired in dairy cows exposed to HS. Though many aspects of heat stress (HS) have been extensively researched, relatively little is known about the proteomics profile changes that occur during heat exposure. In this work, we employed a proteomics approach to investigate differential abundance of plasma proteins in HS-free and heat-stressed dairy cows. Database searches combined with GO and KEGG pathway enrichment analyses revealed that HS resulted in a decrease in complement components, suggesting that heat-stressed dairy cows have impaired immune function. In addition, through integrative analyses of proteomics and previous metabolomics, we showed enhanced glycolysis, lipid metabolic pathway shifts, and nitrogen repartitioning in dairy cows exposed to HS. Our findings expand our current knowledge on the effects of HS on plasma proteomics in dairy cows and offer a new perspective for future research. Copyright © 2016 Elsevier B.V. All rights reserved.

C5b-9 Staining Correlates With Clinical and Tumor Stage in Gastric Adenocarcinoma.

PubMed

Chen, Jian; Yang, Wei-Jun; Sun, Hai-Jian; Yang, Xia; Wu, Yu-Zhang

2016-08-01

The complement system is a critical part of the immune response, acting in defense against viral infections, clearance of immune complexes, and maintenance of tissue homeostasis. Upregulated expression of the terminal complement complex, C5b-9, has been observed on various tumor cells, such as stomach carcinoma cells, and on cells in the necrotic regions of these tumors as well; however, whether and how C5b-9 is related to gastric cancer progression and severity remains unknown. In this study, human gastric adenocarcinoma (HGAC) tissues (n=47 cases) and patient-matched adjacent nontumoral parenchyma (n=20 cases) were evaluated by tissue microarray and immunohistochemistry. The HGAC tissues showed upregulated C5b-9 expression. Multinomial logistic regression and likelihood ratio testing showed that overexpression of C5b-9 in HGAC tissue was significantly correlated with clinical stage (P=0.007) and tumor stage (P=0.005), but not with tumor distant organ metastasis, lymphoid nodal status, sex, or age. Patients with late-stage gastric adenocarcinoma had a higher amount of tumor cells showing positive staining for C5b-9 than patients with early-stage disease. These results may help in diagnosis and assessment of disease severity of human gastric carcinoma.

Elevated serum complement C3 levels are related to the development of prediabetes in an adult population: the Tianjin Chronic Low-Grade Systematic Inflammation and Health Cohort Study.

PubMed

Bao, X; Xia, Y; Zhang, Q; Wu, H M; Du, H M; Liu, L; Wang, C J; Shi, H B; Guo, X Y; Liu, X; Li, C L; Su, Q; Meng, G; Yu, B; Sun, S M; Wang, X; Zhou, M; Jia, Q Y; Song, K; Niu, K J

2016-04-01

To investigate whether serum complement C3 is related to the prevalence and incidence of prediabetes in an adult population. A cross-sectional (n = 10 206) and prospective cohort study (n = 3333), with a mean (range; 95% CI) follow-up of 2.63 (1-6; 2.58-2.68) years, was conducted in people recruited from the Health Management Centre of Tianjin Medical University General Hospital in Tianjin, China. Measurement of serum C3 concentration, blood fasting glucose, oral glucose tolerance, HbA1c and other potential confounding factors was performed at baseline and each year during the follow-up. Prediabetes was defined according to the criteria of the American Diabetes Association. Adjusted logistic and Cox proportional hazards regression models were used to assess the relationships between C3 quintiles and prediabetes. The prevalence and incidence of prediabetes were 38.5% and 119 per 1000 person-years, respectively. In cross-sectional analysis, after adjustment for potential confounders, the odds ratios of prediabetes for increasing quintiles of C3 were 1.00 (reference), 1.32 (95% CI 1.14-1.53), 1.37 (95% CI 1.18-1.59), 1.75 (95% CI 1.51-2.03), 2.25 (95% CI 1.93-2.62; P for trend < 0.0001). In the cohort analysis, the multiple-adjusted hazard ratio of prediabetes in the highest quintile of baseline C3 was 1.43 (95% CI 1.15, 1.78; P for trend < 0.001), when compared with the lowest quintile. These findings indicate that elevated serum C3 levels are significantly related to an increased risk of developing prediabetes in an adult population, suggesting that C3 can be used as a biomarker in high-risk individuals to improve primary prevention of prediabetes and diabetes. © 2015 Diabetes UK.

21 CFR 866.5250 - Complement C2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

21 CFR 866.5250 - Complement C2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

21 CFR 866.5250 - Complement C2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

21 CFR 866.5250 - Complement C 2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

21 CFR 866.5250 - Complement C 2 inhibitor (inactivator) immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... the reagents used to measure by immunochemical techniques the complement C1 inhibitor (a plasma protein) in serum. Complement C1 inhibitor occurs normally in plasma and blocks the action of the C1...

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.

Identification of Hot Spots in the Variola Virus Complement Inhibitor (SPICE) for Human Complement Regulation▿

PubMed Central

Yadav, Viveka Nand; Pyaram, Kalyani; Mullick, Jayati; Sahu, Arvind

2008-01-01

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 ∼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. PMID:18216095

Regulation of endotoxin-induced inhibition of macrophage migration by fresh serum.

PubMed Central

Heilman, D H

1977-01-01

Purified endotoxin (LPS) caused macrophage migration inhibition (MMI) in capillary tube cultures of guinea pig peritoneal macrophages in medium prepared with 15% fresh-frozen guinea pig serum. The inactivation of serum by heating at 56 degrees C for 30 min or by zymosan absorption prevented LPS-induced MMI. LPS was fully inhibitory in fresh C4-deficient guinea pig serum. Heat treatment of normal serum at 50 to 52 degrees C for 30 min to inactivate the alternate complement (C) pathway prevented or significantly decreased LPS-induced MMI, but heating C4-deficient serum at 50 to 52 degrees C for 30 min prevented LPS-MMI in all instances. These results suggest that the reaction was effected via the alternate C pathway but that some inhibition of migration was permitted via the classical C pathway, presumably due to antibodies for LPS in some normal sera. Pretreatment of normal serum with cobra venom factor decreased or prevented LPS-MMI in most instances, but similar results were obtained with C4-deficient serum. Experiments with chelated sera were unsuccessful because of the immobilization of macrophages by 10 mM ethylenediamine-tetraacetic acid and by 10 mM Mg-ethyleneglycol-bis (beta-aminoethyl)-N,N-tetraacetic acid. Low doses of concanavalin A and staphylococcal enterotoxin B and large doses of pokeweed mitogen caused MMI in "inactivated serum" medium, but MMI was enhanced in fresh serum. PMID:330407

Abnormal Complement Activation and Inflammation in the Pathogenesis of Retinopathy of Prematurity

PubMed Central

Rathi, Sonika; Jalali, Subhadra; Patnaik, Satish; Shahulhameed, Shahna; Musada, Ganeswara R.; Balakrishnan, Divya; Rani, Padmaja K.; Kekunnaya, Ramesh; Chhablani, Preeti Patil; Swain, Sarpras; Giri, Lopamudra; Chakrabarti, Subhabrata; Kaur, Inderjeet

2017-01-01

Retinopathy of prematurity (ROP) is a neurovascular complication in preterm babies, leading to severe visual impairment, but the underlying mechanisms are yet unclear. The present study aimed at unraveling the molecular mechanisms underlying the pathogenesis of ROP. A comprehensive screening of candidate genes in preterms with ROP (n = 189) and no-ROP (n = 167) was undertaken to identify variants conferring disease susceptibility. Allele and genotype frequencies, linkage disequilibrium and haplotypes were analyzed to identify the ROP-associated variants. Variants in CFH (p = 2.94 × 10−7), CFB (p = 1.71 × 10−5), FBLN5 (p = 9.2 × 10−4), CETP (p = 2.99 × 10−5), and CXCR4 (p = 1.32 × 10−8) genes exhibited significant associations with ROP. Further, a quantitative assessment of 27 candidate proteins and cytokines in the vitreous and tear samples of babies with severe ROP (n = 30) and congenital cataract (n = 30) was undertaken by multiplex bead arrays and further validated by western blotting and zymography. Significant elevation and activation of MMP9 (p = 0.038), CFH (p = 2.24 × 10−5), C3 (p = 0.05), C4 (p = 0.001), IL-1ra (p = 0.0019), vascular endothelial growth factor (VEGF) (p = 0.0027), and G-CSF (p = 0.0099) proteins were observed in the vitreous of ROP babies suggesting an increased inflammation under hypoxic condition. Along with inflammatory markers, activated macrophage/microglia were also detected in the vitreous of ROP babies that secreted complement component C3, VEGF, IL-1ra, and MMP-9 under hypoxic stress in a cell culture model. Increased expression of the inflammatory markers like the IL-1ra (p = 0.014), MMP2 (p = 0.0085), and MMP-9 (p = 0.03) in the tears of babies at different stages of ROP further demonstrated their potential role in disease progression. Based on these findings, we conclude that increased complement activation in the retina/vitreous in turn activated microglia leading to increased inflammation. A quantitative assessment of inflammatory markers in tears could help in early prediction of ROP progression and facilitate effective management of the disease, thereby preventing visual impairment. PMID:29312345

A specific inactivator of mammalian C'4 isolated from nurse shark (Ginglymostoma cirratum) serum.

PubMed

Jensen, J A

1969-08-01

A material which specifically inactivates mammalian C'4 was isolated from low ionic strength precipitates of nurse shark serum. The C'4 inactivator was not detected in whole serum. The conditions of its generation and its immunoelectrophoretic behavior seem to indicate that it is an enzymatically formed cleavage product of a precursor contained in whole shark serum. The inactivator was partially purified and characterized. It had an S-value of 3.3 (sucrose gradient) which was in agreement with its retardation on gel filtration, was stable between pH 5.0 and 10.0, had a half-life of 5 min at 56 degrees C, pH 7.5, was inactivated by trypsin and was nontoxic. Its powerful anticomplementary activity in vitro and in vivo was solely due to the rapid inactivation of C'4; no other complement components were affected. No cofactor requirement was observed for the equally rapid inactivation of highly purified human and guinea pig C'4. The kinetics of C'4 inactivation and TAME hydrolysis, the greater anodic mobility of inactivated human C'4, and the influence of temperature on the rate of inactivation suggest that the inactivator is an enzyme and C'4 its substrate. This conclusion was supported by the more recent detection of a split product of C'4. Intravenous administration of the C'4 inactivator could prevent lethal Forssman shock and suppress the Arthus reaction in guinea pigs; it prolonged significantly the rejection time of renal xenografts but had no detectable effect on passive cutaneous anaphylaxis. Anaphylatoxin could be generated in C'4 depleted guinea pig serum with the cobra venom factor, but not with immune precipitates. The possible relationship between C'1 esterase and the C'4 inactivator is discussed on the basis of similarities and dissimilarities.

MHC class I–specific antibody binding to nonhematopoietic cells drives complement activation to induce transfusion-related acute lung injury in mice

PubMed Central

Strait, Richard T.; Hicks, Wyenona; Barasa, Nathaniel; Mahler, Ashley; Khodoun, Marat; Köhl, Jörg; Stringer, Keith; Witte, David; Van Rooijen, Nico; Susskind, Brian M.

2011-01-01

Transfusion-related acute lung injury (TRALI), a form of noncardiogenic pulmonary edema that develops during or within 6 h after a blood transfusion, is the most frequent cause of transfusion-associated death in the United States. Because development of TRALI is associated with donor antibodies (Abs) reactive with recipient major histocompatibility complex (MHC), a mouse model has been studied in which TRALI-like disease is caused by injecting mice with anti–MHC class I monoclonal Ab (mAb). Previous publications with this model have concluded that disease is caused by FcR-dependent activation of neutrophils and platelets, with production of reactive oxygen species that damage pulmonary vascular endothelium. In this study, we confirm the role of reactive oxygen species in the pathogenesis of this mouse model of TRALI and show ultrastructural evidence of pulmonary vascular injury within 5 min of anti–MHC class I mAb injection. However, we demonstrate that disease induction in this model involves macrophages rather than neutrophils or platelets, activation of complement and production of C5a rather than activation of FcγRI, FcγRIII, or FcγRIV, and binding of anti–MHC class I mAb to non-BM–derived cells such as pulmonary vascular endothelium. These observations have important implications for the prevention and treatment of TRALI. PMID:22025304

Hypocomplementemia is associated with worse renal survival in ANCA-positive granulomatosis with polyangiitis and microscopic polyangiitis

PubMed Central

Aouba, Achille; Khoy, Kathy; Mariotte, Delphine; Lobbedez, Thierry; Martin Silva, Nicolas

2018-01-01

Recent data suggest the existence of a complement alternative pathway activation in the pathogenesis of antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis (AAV), a condition that remains poorly understood. This study aims to assess the clinical characteristics and outcomes of granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) patients with regard to their plasma complement levels at diagnosis. A retrospective monocentric study carried out at Caen University Hospital led to the identification of proteinase-3- or myeloperoxidase-ANCA-positive GPA and MPA patients from January 2000 to June 2016 and from September 2011 to June 2016, respectively. All patients with available C3 and C4 levels at diagnosis were included. Patients were categorized in the hypocomplementemia group if their C3 and/or C4 levels at diagnosis were below the lower limit of the normal range. Among the 76 AAV patients (43 GPA, 33 MPA), 4 (5%) had hypocomplementemia, and the 72 remaining patients exhibited normal plasma complement levels. All 4 hypocomplementemia patients had renal involvement. Hypocomplementemia was followed in 1 patient whose post-treatment complement level normalized within 1 month. Among all clinical and ANCA specificity, including relapse-free survival (p = 0.093), only overall and renal survival rates were significantly lower in the hypocomplementemia group (p = 0.0011 and p<0.001, respectively). Hypocomplementemia with low C3 and/or C4 levels at GPA or MPA diagnosis may be responsible for worse survival and renal prognosis. These results argue for larger and prospective studies to better determine the epidemiology of the disease and to assess complement-targeting therapy in these patients. PMID:29621352

Capsule Influences the Deposition of Critical Complement C3 Levels Required for the Killing of Burkholderia pseudomallei via NADPH-Oxidase Induction by Human Neutrophils

PubMed Central

Woodman, Michael E.; Worth, Randall G.; Wooten, R. Mark

2012-01-01

Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD50 via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo. PMID:23251706

Changes of human serum proteome profile during 7-day “dry” immersion

NASA Astrophysics Data System (ADS)

Pakharukova, N. A.; Pastushkova, L. Kh.; Larina, I. M.; Grigoriev, A. I.

2011-05-01

The aim of this study was to characterize changes of serum proteome profile during 7-day "dry" immersion (DI). The experiment with DI consisted of three series: control group without countermeasures (10 men), with using mechanical stimulation (6 men) and low-frequency myostimulation (5 men) as preventive means. Serum samples were fractionated using ClinProt robot (Bruker Daltonics) on magnetic beads (weak cation exchange magnetic beads—MB WCX) prior to mass-spectral profiling. It was obtained 170 peaks after fractionation of serum samples in each group. On 7th immersion day peak areas of fibrinopeptide A ( m/ z=1206; 1464), angiotensin II ( m/ z=1051), high molecular mass kininogen fragment ( m/ z=2133 Da) and C3-fragment of the complement system ( m/ z=1350 Da) were significantly decreased comparing with pre-experimental values of all experimental series. Peak areas of apolipoprotein C III ( m/ z=9419) and C4a fragment of the complement system ( m/ z=3206 Da) were increased. On 7th day of the recovery peak areas of all changed peaks were not close to pre-experimental values. This fact provided evidence of incomplete recovery of an organism after DI. The depth of the alterations had considerable individual variability. Thereby the detected changes of serum proteome profile in the experiment. They indicated a reorganization of the hormonal, immune systems and lipid metabolism. The use of myostimulation and mechanical stimulation as countermeasures partly compensated adverse effects of 7-day dry immersion on the parameters of coagulation system (fibrinopeptide A) and lipid metabolism (apolipoprotein CIII).

Milk complement and the opsonophagocytosis and killing of Staphylococcus aureus mastitis isolates by bovine neutrophils.

PubMed

Barrio, Maria Belén; Rainard, Pascal; Poutrel, Bernard

2003-01-01

Phagocytosis of bacteria by bovine polymorphonuclear neutrophils (PMN) has long been regarded as essential for host defense against mastitis infection. Complement-mediated opsonisation by complement component 3 (C3) binding is an important component of the innate immune system. We investigated the role of milk complement as an opsonin and its involvement in the phagocytosis and killing of Staphylococcus aureus isolates from cases of bovine mastitis by bovine blood PMN. We show that deposition of milk C3 component occurred on six different isolates of S. aureus and that the alternative pathway was the sole complement pathway operating in milk of uninflamed mammary gland. This deposition was shown to occur at the same location as the capsule, but not on capsular antigen. Milk complement enhanced the chemiluminescence response of PMN induced by S. aureus. Nevertheless, the association of S. aureus to cells and the overall killing of bacteria by bovine PMN were not affected by the presence of milk complement. Therefore, as all milk samples contained antibodies to capsular polysaccharide type 5 and to other surface antigens, it is likely that milk antibodies were responsible for these two phagocytic events. Results of this study suggest that the deposition of milk complement components on the surface of S. aureus does not contribute to the defence of the mammary gland against S. aureus.

Granular C3 Dermatosis.

PubMed

Hashimoto, Takashi; Tsuruta, Daisuke; Yasukochi, Atsushi; Imanishi, Hisayoshi; Sekine, Hideharu; Fujita, Teizo; Wanibuchi, Hideki; Gi, Min; Kárpáti, Sarolta; Sitaru, Cassian; Zone, John J; Endo, Daisuke; Abe, Shinichi; Nishino, Tomoya; Koji, Takehiko; Ishii, Norito

2016-08-23

There has been no previous systematic study of bullous skin diseases with granular basement membrane zone deposition exclusively of C3. In this study we collected 20 such patients, none of whom showed cutaneous vasculitis histopathologically. Oral dapsone and topical steroids were effective. Various serological tests detected no autoantibodies or autoantigens. Direct immunofluorescence for various complement components revealed deposition only of C3 and C5-C9, indicating that no known complement pathways were involved. Studies of in situ hybridization and micro-dissection with quantitative RT-PCR revealed a slight reduction in expression of C3 in patient epidermis. These patients may represent a new disease entity, for which we propose the term "granular C3 dermatosis". The mechanism for granular C3 deposition in these patients is unknown, but it is possible that the condition is caused by autoantibodies to skin or aberrant C3 expression in epidermal keratinocytes.

Complement is a central mediator of radiotherapy-induced tumor-specific immunity and clinical response.

PubMed

Surace, Laura; Lysenko, Veronika; Fontana, Andrea Orlando; Cecconi, Virginia; Janssen, Hans; Bicvic, Antonela; Okoniewski, Michal; Pruschy, Martin; Dummer, Reinhard; Neefjes, Jacques; Knuth, Alexander; Gupta, Anurag; van den Broek, Maries

2015-04-21

Radiotherapy induces DNA damage and cell death, but recent data suggest that concomitant immune stimulation is an integral part of the therapeutic action of ionizing radiation. It is poorly understood how radiotherapy supports tumor-specific immunity. Here we report that radiotherapy induced tumor cell death and transiently activated complement both in murine and human tumors. The local production of pro-inflammatory anaphylatoxins C3a and C5a was crucial to the tumor response to radiotherapy and concomitant stimulation of tumor-specific immunity. Dexamethasone, a drug frequently given during radiotherapy, limited complement activation and the anti-tumor effects of the immune system. Overall, our findings indicate that anaphylatoxins are key players in radiotherapy-induced tumor-specific immunity and the ensuing clinical responses. Copyright © 2015 Elsevier Inc. All rights reserved.

Biocompatibility differences with respect to the dialyzer sterilization method.

PubMed

Müller, T F; Seitz, M; Eckle, I; Lange, H; Kolb, G

1998-01-01

The impact of the method of sterilization (steam vs. ethylene oxide, ETO) on indices of biocompatibility is investigated using polysulfone membranes. Eight patients were treated with a random choice of the high-flux membranes F60S (steam) and F60 (ETO) and the low-flux membrane F6 (ETO). Blood samples were taken prior to and 5, 15, 30, 60, and 180 min after the start of hemodialysis. White blood cell count, platelet count, and plasma concentrations of polymorphonuclear neutrophil elastase, complements C3a and C5a, and beta2-microglobulin were determined. The dialysis procedure was associated with a significant decrease in white blood cell count and beta2-microglobulin level and a significant increase in polymorphonuclear neutrophil elastase and complement C3a and C5a levels. However, the steam-sterilized F60S membrane had a significantly lower impact on the biocompatibility indices than the ETO-sterilized F60 and F6 membranes (p < 0.05 or p < 0.001 for the individual markers). We conclude that using steam instead of ETO for sterilization may improve the biocompatibility of membranes.

Preeclampsia in autologous and oocyte donation pregnancy: is there a different pathophysiology?

PubMed

Lashley, Lisa E E L O; Buurma, Aletta; Swings, Godelieve M J S; Eikmans, Michael; Anholts, Jacqueline D H; Bakker, Jaap A; Claas, Frans H J

2015-06-01

Oocyte donation (OD) is a specific method of artificial reproductive technology that is accompanied by a higher risk of preeclampsia during pregnancy. The pathophysiological mechanism underlying preeclampsia in OD pregnancies is thought to differ from preeclampsia in autologous pregnancies. As preeclampsia in autologous pregnancies is suggested to be associated with complement activation, we studied C4d deposition, circulating complement components and placental complement regulatory proteins in preeclamptic OD pregnancies. Women with uncomplicated and preeclamptic pregnancies after OD or spontaneous conception were selected. We stained the placentas for C4d, marker for complement activation, measured complement factors C1q, C3 and C4 in maternal sera and quantified the placental mRNA expression of complement regulatory proteins CD46, CD55 and CD59. A significantly (p < 0.03) higher incidence of C4d deposition was observed in placentas from women with preeclampsia compared with uncomplicated pregnancies, both OD and autologous. The level of complement factors in serum did not differ between the groups. Children born in the autologous preeclampsia group were significantly lower in birth weight (p < 10th percentile) compared with the preeclamptic OD group. In addition, the placental mRNA expression level of complement regulatory proteins was significantly lower in uncomplicated and preeclamptic OD compared with the autologous pregnancies. In line with autologous preeclampsia pregnancies, there is excessive activation of complement in preeclamptic OD pregnancies. However, in contrast to autologous pregnancies this is not associated with counterbalancing upregulation of complement regulatory proteins. Furthermore, C4d deposition in OD pregnancies is not related to the severity of preeclampsia, suggesting another trigger or regulatory mechanism of placental C4d deposition in preeclamptic OD pregnancies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

Mastellos, D C; Ricklin, D; Hajishengallis, E; Hajishengallis, G; Lambris, J D

2016-02-01

There is 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 as 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. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multimeric complement component C9 is necessary for killing of Escherichia coli J5 by terminal attack complex C5b-9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joiner, K.A.; Schmetz, M.A.; Sanders, M.E.

The authors studied the molecular composition of the complement C5b-9 complex required for optimal killing of Escherichia coli strain J5. J5 cells were incubated in 3.3%, 6.6%, or 10.0% C8-deficient serum previously absorbed to remove specific antibody and lysozyme. This resulted in the stable deposition after washing of 310, 560, and 890 C5b67 molecules per colony-forming unit, respectively, as determined by binding of /sup 125/I-labeled C7. Organisms were then incubated with excess C8 and various amounts of /sup 131/I-labeled C9. Plots of the logarithm (base 10) of E. coli J5 cells killed (log kill) vs. C9 input were sigmoidal, confirmingmore » the multihit nature of the lethal process. When C9 was supplied in excess, 3300, 5700, and 9600 molecules of C9 were bound per organism for cells bearing 310, 560, and 890 C5b-8 complexes, respectively, leading to C9-to-C7 ratios of 11.0:1, 10.8:1, and 11.4:1 and to log kill values of 1.3, 2.1, and 3.9. However, at low inputs of C9 that lead to C9-to-C7 ratios of less than 3.3:1, no killing occurred, and this was independent of the number of C5b-9 complexes bound. Formation of multimeric C9 at C9-to-C7 ratios permissive for killing was confirmed by electron microscopy and by binding of /sup 125/I-labeled antibody with specificity for multimeric but not monomeric C9. These experiments are the first to demonstrate a biological function for C9 polymerization and suggest that multimeric C9 is necessary for optimal killing of E. coli J5 cells by C5b-9.« less

C5a induces caspase-dependent apoptosis in brain vascular endothelial cells in experimental lupus.

PubMed

Mahajan, Supriya D; Tutino, Vincent M; Redae, Yonas; Meng, Hui; Siddiqui, Adnan; Woodruff, Trent M; Jarvis, James N; Hennon, Teresa; Schwartz, Stanley; Quigg, Richard J; Alexander, Jessy J

2016-08-01

Blood-brain barrier (BBB) dysfunction complicates central nervous system lupus, an important aspect of systemic lupus erythematosus. To gain insight into the underlying mechanism, vascular corrosion casts of brain were generated from the lupus mouse model, MRL/lpr mice and the MRL/MpJ congenic controls. Scanning electron microscopy of the casts showed loss of vascular endothelial cells in lupus mice compared with controls. Immunostaining revealed a significant increase in caspase 3 expression in the brain vascular endothelial cells, which suggests that apoptosis could be an important mechanism causing cell loss, and thereby loss of BBB integrity. Complement activation occurs in lupus resulting in increased generation of circulating C5a, which caused the endothelial layer to become 'leaky'. In this study, we show that C5a and lupus serum induced apoptosis in cultured human brain microvascular endothelial cells (HBMVECs), whereas selective C5a receptor 1 (C5aR1) antagonist reduced apoptosis in these cells, demonstrating C5a/C5aR1-dependence. Gene expression of initiator caspases, caspase 1 and caspase 8, and pro-apoptotic proteins death-associated protein kinase 1, Fas-associated protein (FADD), cell death-inducing DNA fragmentation factor 45 000 MW subunit A-like effector B (CIDEB) and BCL2-associated X protein were increased in HBMVECs treated with lupus serum or C5a, indicating that both the intrinsic and extrinsic apoptotic pathways could be critical mediators of brain endothelial cell apoptosis in this setting. Overall, our findings suggest that C5a/C5aR1 signalling induces apoptosis through activation of FADD, caspase 8/3 and CIDEB in brain endothelial cells in lupus. Further elucidation of the underlying apoptotic mechanisms mediating the reduced endothelial cell number is important in establishing the potential therapeutic effectiveness of C5aR1 inhibition that could prevent and/or reduce BBB alterations and preserve the physiological function of BBB in central nervous system lupus. © 2016 John Wiley & Sons Ltd.

O1.6. INCREASED COMPLEMENT FACTORS C3 AND C4 IN SCHIZOPHRENIA AND THE EARLY STAGES OF PSYCHOSIS: IMPLICATIONS FOR CLINICAL SYMPTOMATOLOGY AND CORTICAL THICKNESS

PubMed Central

Cropley, Vanessa; Laskaris, Liliana; Zalesky, Andrew; Weickert, Cynthia Shannon; Biase, Maria Di; Chana, Gursharan; Baune, Bernhard; Bousman, Chad; Nelson, Barnaby; McGorry, Patrick D; Everall, Ian; Pantelis, Christos

2018-01-01

Abstract Background The complement system - a key component of the innate immune system, has been proposed to contribute to the pathogenesis of schizophrenia. Recently, complement C4 was associated with increased risk of schizophrenia, and in a mouse model, developmentally-timed synaptic pruning. These observations have led to proposals that abnormal activation of the complement system might contribute to the development of schizophrenia by disrupting synaptic pruning during key developmental periods. However, despite renewed interest in the complement system in schizophrenia it remains unclear whether peripheral complement levels differ in cases compared to controls, change over the course of illness and whether they are associated with current symptomatology and brain cortical thickness. This study aimed to: i) investigate whether peripheral complement protein levels are altered at different stages of illness, and ii) identify patterns among complement protein levels that predict clinical symptoms and grey matter thickness across the cortex. Methods Complement factors C1q, C3 and C4 were quantified in 183 participants [n=83 Healthy Controls (HC), n=10 Ultra-High Risk (UHR) for psychosis, n=40 First Episode Psychosis (FEP), n=50 Chronic schizophrenia] using Multiplex ELISA. Permutation-based t-tests were used to assess between-group differences in complement protein levels at each of the three illness stages, relative to age- and gender-matched healthy controls. Canonical correlation analysis was used to identify patterns of complement protein levels that correlated with clinical symptoms and regional thickness across the cortex. Results C3 and C4 were significantly increased in FEP and UHR patients, whereas only C4 was significantly increased in chronic patients. A molecular pattern of increased C4 and decreased C3 was associated with positive and negative symptom severity in the pooled patient sample. Increased C4 levels alone, or decreased C3 levels alone, did not correlate with symptom severity as strongly as the pattern of increased C4 in combination with decreased C3. Preliminary canonical correlation analyses revealed that, in healthy controls, a molecular pattern characterised by increased C3 and decreased C4 was associated with relatively thinner paracentral, inferior parietal and inferior temporal cortices, but relatively thicker insular, in the left hemisphere. In the pooled patient group, a trend for increased C3 in combination with decreased C1q was associated with relatively thinner left lateral occipital cortex and pars orbitalis but relatively thicker pars opercularis and precuneus. Discussion Our findings indicate that peripheral complement concentration is particularly increased early and preceding psychosis and its imbalance may be associated with symptom severity and variation in regional grey matter thickness across the cortex.

Pneumococcal polysaccharides complexed with C3d bind to human B lymphocytes via complement receptor type 2.

PubMed Central

Griffioen, A W; Rijkers, G T; Janssens-Korpela, P; Zegers, B J

1991-01-01

The immunoregulatory function of the complement system has been the focus of many investigations. In particular, fragments of complement factor C3 have been shown to play a role in B-lymphocyte activation and proliferation, lymphokine production, and the generation of in vitro antibody production. Purified pneumococcal polysaccharides (PS) can induce direct activation of C3 via the alternative pathway. Using sera of C1q-deficient patients and healthy subjects, we demonstrated that C3d, a split product of C3 that is generated after degradation of iC3b, can be bound to PS antigens. The binding of C3d to PS can occur in the absence of specific antibodies. Subsequently, we showed that PS complexed with C3d can be recognized by complement receptor type 2 that is expressed on B cells. Treatment of B cells with a monoclonal antibody recognizing the C3d-binding site of complement receptor type 2 reduces the binding of PS-C3d to the cells. In addition, we showed that PS4 complexed with C3d exerted an increased immunogenicity compared with free PS4. Our results show that the complement system plays a role in the activation of PS-specific B cells, carrying membrane receptors for C3d. Consequently, the complement system plays a regulatory role in the antibody response to T-cell-independent type 2 antigens such as PS. PMID:1826897

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.

The Cytochrome b5 dependent C-5(6) sterol desaturase DES5A from the endoplasmic reticulum of Tetrahymena thermophila complements ergosterol biosynthesis mutants in Saccharomyces cerevisiae

PubMed Central

Poklepovich, Tomas J.; Rinaldi, Mauro A.; Tomazic, Mariela L.; Favale, Nicolas O.; Turkewitz, Aaron P.; Nudel, Clara B.; Nusblat, Alejandro D.

2012-01-01

Tetrahymena thermophila is a free-living ciliate with no exogenous sterol requirement. However, it can perform several modifications on externally added sterols including desaturation at C5(6), C7(8), and C22(23). Sterol desaturases in Tetrahymena are microsomal enzymes that require Cyt b5, Cyt b5 reductase, oxygen, and reduced NAD(P)H for their activity, and some of the genes encoding these functions have recently been identified. The DES5A gene encodes a C-5(6) sterol desaturase, as shown by gene knockout in Tetrahymena. To confirm and extend that result, and to develop new approaches to gene characterization in Tetrahymena, we have now, expressed DES5A in Saccharomyces cerevisiae. The DES5A gene was codon optimized and expressed in a yeast mutant, erg3Δ, which is disrupted for the gene encoding the S. cerevisiae C-5(6) sterol desaturase ERG3. The complemented strain was able to accumulate 74% of the wild type level of ergosterol, and also lost the hypersensitivity to cycloheximide associated with the lack of ERG3 function. C-5(6) sterol desaturases are expected to function at the endoplasmic reticulum. Consistent with this, a GFP-tagged copy of Des5Ap was localized to the endoplasmic reticulum in both Tetrahymena and yeast. This work shows for the first time that both function and localization are conserved for a microsomal enzyme between ciliates and fungi, notwithstanding the enormous evolutionary distance between these lineages. The results suggest that heterologous expression of ciliate genes in S. cerevisiae provides a useful tool for the characterization of genes in Tetrahymena, including genes encoding membrane protein complexes. PMID:22982564

The Complement C3a-C3aR Axis Promotes Development of Thoracic Aortic Dissection via Regulation of MMP2 Expression.

PubMed

Ren, Weihong; Liu, Yan; Wang, Xuerui; Piao, Chunmei; Ma, Youcai; Qiu, Shulan; Jia, Lixin; Chen, Boya; Wang, Yuan; Jiang, Wenjian; Zheng, Shuai; Liu, Chang; Dai, Nan; Lan, Feng; Zhang, Hongjia; Song, Wen-Chao; Du, Jie

2018-03-01

Thoracic aortic dissection (TAD), once ruptured, is devastating to patients, and no effective pharmaceutical therapy is available. Anaphylatoxins released by complement activation are involved in a variety of diseases. However, the role of the complement system in TAD is unknown. We found that plasma levels of C3a, C4a, and C5a were significantly increased in patients with TAD. Elevated circulating C3a levels were also detected in the developmental process of mouse TAD, which was induced by β-aminopropionitrile monofumarate (BAPN) treatment, with enhanced expression of C1q and properdin in mouse dissected aortas. These findings indicated activation of classical and alternative complement pathways. Further, expression of C3aR was obviously increased in smooth muscle cells of human and mouse dissected aortas, and knockout of C3aR notably inhibited BAPN-induced formation and rupture of TAD in mice. C3aR antagonist administered pre- and post-BAPN treatment attenuated the development of TAD. We found that C3aR knockout decreased matrix metalloproteinase 2 (MMP2) expression in BAPN-treated mice. Additionally, recombinant C3a stimulation enhanced MMP2 expression and activation in smooth muscle cells that were subjected to mechanical stretch. Finally, we generated MMP2-knockdown mice by in vivo MMP2 short hairpin RNA delivery using recombinant adeno-associated virus and found that MMP2 deficiency significantly reduced the formation of TAD. Therefore, our study suggests that the C3a - C3aR axis contributes to the development of TAD via regulation of MMP2 expression. Targeting the C3a-C3aR axis may represent a strategy for inhibiting the formation of TAD. Copyright © 2018 by The American Association of Immunologists, Inc.

Binding of Free and Immune Complex-Associated Hepatitis C Virus to Erythrocytes Is Mediated by the Complement System.

PubMed

Salam, Kazi Abdus; Wang, Richard Y; Grandinetti, Teresa; De Giorgi, Valeria; Alter, Harvey J; Allison, Robert D

2018-05-09

Erythrocytes bind circulating immune complexes (IC) and facilitate IC clearance from the circulation. Chronic hepatitis C virus (HCV) infection is associated with IC-related disorders. In this study we investigated the kinetics and mechanism of HCV and HCV-IC binding to and dissociation from erythrocytes. Cell culture-produced HCV was mixed with erythrocytes from healthy blood donors and erythrocyte-associated virus particles were quantified. Purified complement proteins, complement-depleted serum, and complement receptor antibodies were used to investigate complement-mediated HCV-erythrocyte binding. Purified HCV-specific immunoglobulin G from a chronic HCV-infected patient was used to study complement-mediated HCV-IC-erythrocyte binding. Binding of HCV to erythrocytes increased 200 to 1,000 fold after adding complement active human serum in the absence of antibody. Opsonization of free HCV occurred within 10 minutes and peak binding to erythrocytes was observed at 20-30 minutes. Complement protein C1 was required for binding, while C2, C3 and C4 significantly enhanced binding. Complement receptor 1 (CR1, CD35) antibodies blocked the binding of HCV to erythrocytes isolated from chronically infected HCV patients and healthy blood donors. HCV-ICs significantly enhanced complement-mediated binding to erythrocytes compared to unbound HCV. Dissociation of complement-opsonized HCV from erythrocytes depended on the presence of Factor I. HCV released by Factor I bound preferentially to CD19+ B cells compared to other leukocytes. These results demonstrate that complement mediates the binding of free and IC-associated HCV to CR1 on erythrocytes, and provide a mechanistic rationale for investigating the differential phenotypic expression of HCV-IC-related disease. This article is protected by copyright. All rights reserved. © 2018 by the American Association for the Study of Liver Diseases.

High Levels of Soluble C5b-9 Complex in Dialysis Fluid May Predict Poor Prognosis in Peritonitis in Peritoneal Dialysis Patients.

PubMed

Mizuno, Masashi; Suzuki, Yasuhiro; Higashide, Keiko; Sei, Yumi; Iguchi, Daiki; Sakata, Fumiko; Horie, Masanobu; Maruyama, Shoichi; Matsuo, Seiichi; Morgan, B Paul; Ito, Yasuhiko

2017-01-01

We searched for indicators to predict the prognosis of infectious peritonitis by measuring levels of complement proteins and activation products in peritoneal dialysis (PD) fluid (PDF) of patients at early stages of peritonitis. We retrospectively analyzed the relationship between the levels of sC5b-9, C3 and C4 in PDF and the subsequent clinical prognosis. We measured levels of sC5b-9, C3 and C4 in PDF on days 1, 2 and 5 post-onset of peritonitis in 104 episodes of infectious peritonitis in PD patients from 2008 and retrospectively compared levels with clinical outcomes. Further analysis for the presence of causative microorganisms or to demonstrate bacterial culture negative peritonitis was performed and correlated with change of levels of sC5b-9 in PDF. When PD patients with peritonitis were divided into groups that either failed to recover from peritonitis and were finally withdrawn from PD (group 1; n = 25) or recovered (group 2; n = 79), levels of sC5b-9, C3 and C4 in PDF were significantly higher in group 1 patients compared to those in group 2 on day5. Analysis of microorganisms showed significantly higher sC5b-9 levels in PDF of peritonitis cases caused by culture negative peritonitis in group 1 compared with group 2 when we analyzed for individual microorganisms. Of note, on day5, the sC5b-9 levels in PDF were similarly high in peritonitis caused by fungi or other organisms. Our results suggested that levels of complement markers in PDF, especially sC5b-9, have potential as surrogate markers to predict prognosis of PD-related peritonitis.

Complement Inhibition in the Immunotherapy of Breast Cancer

DTIC Science & Technology

2013-03-01

cancer. In both models the therapeutic efficacy of C5aR inhibitor was comparable to the efficacy of Listeria monocytogenes-delivered Her2 vaccine (Lm...C5aR) blockage synergizes with Listeria monocytogenes-based Her2 vaccine (Lm-LLO-Her2) in reducing growth of primary tumors in FVB/N wild-type and...results pertain. Aim 1 (TASK 1: months 1-6 and TASK 3: months 6-12) (i) C5aR blockage synergizes with Listeria monocytogenes-based Her2 vaccine

Preservation of renal function in atypical hemolytic uremic syndrome by eculizumab: a case report.

PubMed

Giordano, Mario; Castellano, Giuseppe; Messina, Giovanni; Divella, Claretta; Bellantuono, Rosa; Puteo, Flora; Colella, Vincenzo; Depalo, Tommaso; Gesualdo, Loreto

2012-11-01

Genetic mutations in complement components are associated with the development of atypical hemolytic uremic syndrome (aHUS), a rare disease with high morbidity rate triggered by infections or unidentified factors. The uncontrolled activation of the alternative pathway of complement results in systemic endothelial damage leading to progressive development of renal failure. A previously healthy 8-month-old boy was referred to our hospital because of onset of fever, vomiting, and a single episode of nonbloody diarrhea. Acute kidney injury with preserved diuresis, hemolytic anemia, and thrombocytopenia were detected, and common protocols for management of HUS were followed without considerable improvement. The persistent low levels of complement component C3 led us to hypothesize the occurrence of aHUS. In fact, the child carried a specific mutation in complement factor H (Cfh; nonsense mutation in 3514G>T, serum levels of Cfh 138 mg/L, normal range 350-750). Given the lack of response to therapy and the occurrence of kidney failure requiring dialysis, we used eculizumab as rescue therapy, a monoclonal humanized antibody against the complement component C5. One week from the first administration, we observed a significant improvement of all clinical and laboratory parameters with complete recovery from hemodialysis, even in the presence of systemic infections. Our case report shows that complement inhibiting treatment allows the preservation of renal function and avoids disease relapses during systemic infections.

Effect of radiographic contrast media on markers of complement activation and apoptosis in patients with chronic coronary artery disease undergoing coronary angiography.

PubMed

Deftereos, Spyridon; Giannopoulos, Georgios; Kossyvakis, Charalampos; Raisakis, Konstantinos; Kaoukis, Andreas; Driva, Metaxia; Ntzouvara, Olga; Panagopoulou, Vasiliki; Rentoukas, Ilias; Nikas, Dimitrios J; Pyrgakis, Vlasios; Alpert, Martin A

2009-09-01

The effects of radiographic contrast media on markers of complement activation and apoptosis in patients with chronic coronary artery disease (CAD) are unknown. The purpose of this study was to assess the comparative effects of ionic high-osmolar and non-ionic iso-osmolar radiographic contrast media on plasma markers of complement activation and apoptosis in patients with chronic CAD undergoing coronary angiography. Forty-four patients undergoing coronary angiography for chronic CAD were randomly assigned to receive the ionic high-osmolar radiographic contrast agent diatrizoate (Group A), or the non-ionic iso-osmolar contrast agent iodixanol (Group B) during angiography. Complement component 5 (C5a) and apoptotic markers sFas and sFasL were measured just prior to angiography and 1 hour after completion of angiography. Comparison of mean pre- and post-angiography plasma marker levels showed significantly greater increases in plasma levels in Group A than in Group B of C5a (29.30 +/- 5.45 ng/ml for Group A and 0.47 +/- 0.70 ng/ml for Group B (p < 0.00001), sFas (2.36 +/- 1.63 ng/ml for Group A and 0.23 +/- 0.90 ng/ml for Group B (p < 0.00001) and sFasL (14.00 +/- 5.41 pg/ml for Group A and 0.01 +/- 1.00 pg/ml for Group B (p < 0.00001). The results suggest that in patients with chronic CAD, the use of ionic high-osmolar radiographic contrast media during coronary angiography is associated with a more robust inflammatory and apoptotic milieu than that associated with the use of non-ionic iso-osmolar radiographic contrast media.

Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Haimou; Qin, Gangjian; Liang, Gang

Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanismmore » of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.« less

Regulation of macrophage migration by products of the complement system.

PubMed Central

Bianco, C; Götze, O; Cohn, Z A

1979-01-01

Agents formerly shown to induce rapid macrophage spreading were examined for their ability to modify the migration of macrophages in the capillary tube assay. Products of the activation of the contact phase of blood coagulation as well as the purified component Bb, the large cleavage fragment of factor B of the alternative complement pathway produced a dose-dependent inhibition of migration. In addition, inflammatory macrophages elicited with either a lipopolysaccharide endotoxin or thioglycollate medium exhibited rapid spreading and inhibited migration, whereas resident cells did not. A close correlation existed, therefore, between enhanced spreading and inhibited migration under both in vitro induced and in vivo situations. Cleavage products of component C5 of the classical complement pathway enhanced macrophage migration and did not alter spreading. In mixtures of C5 cleavage products and Bb, the predominant peptide determined the outcome of the reaction. Factor B, a normal secretory product of macrophages, may represent a common substrate for several of the proteases that induce spreading, inhibit migration, and lead to the generation of the enzymatically active fragment Bb. PMID:284412

Streptococcus pyogenes Endopeptidase O Contributes to Evasion from Complement-mediated Bacteriolysis via Binding to Human Complement Factor C1q*

PubMed Central

Honda-Ogawa, Mariko; Sumitomo, Tomoko; Mori, Yasushi; Hamd, Dalia Talat; Ogawa, Taiji; Yamaguchi, Masaya; Nakata, Masanobu; Kawabata, Shigetada

2017-01-01

Streptococcus pyogenes secretes various virulence factors for evasion from complement-mediated bacteriolysis. However, full understanding of the molecules possessed by this organism that interact with complement C1q, an initiator of the classical complement pathway, remains elusive. In this study, we identified an endopeptidase of S. pyogenes, PepO, as an interacting molecule, and investigated its effects on complement immunity and pathogenesis. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis findings revealed that S. pyogenes recombinant PepO bound to human C1q in a concentration-dependent manner under physiological conditions. Sites of inflammation are known to have decreased pH levels, thus the effects of PepO on bacterial evasion from complement immunity was analyzed in a low pH condition. Notably, under low pH conditions, PepO exhibited a higher affinity for C1q as compared with IgG, and PepO inhibited the binding of IgG to C1q. In addition, pepO deletion rendered S. pyogenes more susceptible to the bacteriocidal activity of human serum. Also, observations of the morphological features of the pepO mutant strain (ΔpepO) showed damaged irregular surfaces as compared with the wild-type strain (WT). WT-infected tissues exhibited greater severity and lower complement activity as compared with those infected by ΔpepO in a mouse skin infection model. Furthermore, WT infection resulted in a larger accumulation of C1q than that with ΔpepO. Our results suggest that interaction of S. pyogenes PepO with C1q interferes with the complement pathway, which enables S. pyogenes to evade complement-mediated bacteriolysis under acidic conditions, such as seen in inflammatory sites. PMID:28154192

Microvesicles released constitutively from prostate cancer cells differ biochemically and functionally to stimulated microvesicles released through sublytic C5b-9

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stratton, Dan; Moore, Colin; Antwi-Baffour, Samuel

2015-05-08

We have classified microvesicles into two subtypes: larger MVs released upon stimulation of prostate cancer cells, sMVs, and smaller cMVs, released constitutively. cMVs are released as part of cell metabolism and sMVs, released at 10-fold higher levels, produced upon activation, including sublytic C5b-9. From electron microscopy, nanosight tracking analysis, dynamic light scattering and flow cytometry, cMVs (194–210 nm in diameter) are smaller than sMVs (333–385 nm). Furthermore, using a Quartz Crystal Microbalance measuring changes in resonant frequency (Δf) that equate to mass deposited on a sensor, an sMV and a cMV are estimated at 0.267 and 0.241 pg, respectively. sMVs carry more calciummore » and protein, express higher levels of lipid rafts, GPI-anchored CD55 and phosphatidylserine including deposited C5b-9 compared to cMVs. This may allude to biological differences such as increased bound C4BP on sMVs inhibiting complement more effectively. - Highlights: • Prostate cells release microvesicles constitutively (cMVs) or upon stimulus (sMVs). • sMVs are larger than cMVs and carry more protein, lipid rafts and surface PstSer. • sMVs inhibit complement more effectively than cMVs.« less

Role of Non neuronal Cells in Tauopathies After Brain Injury

DTIC Science & Technology

2016-09-01

task is 100%. Complete. This has led to a delay in the ability to breed mice to obtain four Page 5 of 13 transgenes needed for the GFAP C5a Tg...the C1inh and C5GFAP transgenic mice from the respective institutions and breed them the obtain the crosses needed for the study and begin the TBI...understanding this elusive delay in onset of symptoms. This mouse is bred to mice with novel transgenes associated with complement activation: one

Do Archaea and bacteria co-infection have a role in the pathogenesis of chronic chagasic cardiopathy?

PubMed

Higuchi, Maria de Lourdes; Kawakami, Joyce; Ikegami, Renata; Clementino, Maysa Beatriz Mandetta; Kawamoto, Flavio M; Reis, Marcia M; Bocchi, Edimar

2009-07-01

Chronic cardiopathy (CC) in Chagas disease is a fibrotic myocarditis with C5b-9 complement deposition. Mycoplasma and Chlamydia may interfere with the complement response. Proteolytic enzymes and archaeal genes that have been described in Trypanosoma cruzi may increase its virulence. Here we tested the hypothesis that different ratios of Mycoplasma, Chlamydia and archaeal organisms, which are frequent symbionts, may be associated with chagasic clinical forms. eight indeterminate form (IF) and 20 CC chagasic endomyocardial biopsies were submitted to in situ hybridization, electron and immunoelectron microscopy and PCR techniques for detection of Mycoplasma pneumoniae (MP), Chlamydia pneumoniae(CP), C5b-9 and archaeal-like bodies. MP and CP-DNA were always present at lower levels in CC than in IF (p < 0.001) and were correlated with each other only in CC. Electron microscopy revealed Mycoplasma, Chlamydia and two types of archaeal-like bodies. One had electron dense lipid content (EDL) and was mainly present in IF. The other had electron lucent content (ELC) and was mainly present in CC. In this group, ELC correlated negatively with the other microbes and EDL and positively with C5b-9. The CC group was positive for Archaea and T. cruzi DNA. In conclusion, different amounts of Mycoplasma, Chlamydia and archaeal organisms may be implicated in complement activation and may have a role in Chagas disease outcome.

Protection of Nonself Surfaces from Complement Attack by Factor H-Binding Peptides: Implications for Therapeutic Medicine

PubMed Central

Wu, You-Qiang; Qu, Hongchang; Sfyroera, Georgia; Tzekou, Apostolia; Kay, Brian K.; Nilsson, Bo; Ekdahl, Kristina Nilsson; Ricklin, Daniel; Lambris, John D.

2011-01-01

Exposure of nonself surfaces such as those of biomaterials or transplanted cells and organs to host blood frequently triggers innate immune responses, thereby affecting both their functionality and tolerability. Activation of the alternative pathway of complement plays a decisive role in this unfavorable reaction. Whereas previous studies demonstrated that immobilization of physiological regulators of complement activation (RCA) can attenuate this foreign body-induced activation, simple and efficient approaches for coating artificial surfaces with intact RCA are still missing. The conjugation of small molecular entities that capture RCA with high affinity is an intriguing alternative, as this creates a surface with autoregulatory activity upon exposure to blood. We therefore screened two variable cysteine-constrained phage-displayed peptide libraries for factor H-binding peptides. We discovered three peptide classes that differed with respect to their main target binding areas. Peptides binding to the broad middle region of factor H (domains 5–18) were of particular interest, as they do not interfere with either regulatory or binding activities. One peptide in this group (5C6) was further characterized and showed high factor H-capturing activity while retaining its functional integrity. Most importantly, when 5C6 was coated to a model polystyrene surface and exposed to human lepirudin-anticoagulated plasma, the bound peptide captured factor H and substantially inhibited complement activation by the alternative pathway. Our study therefore provides a promising and novel approach to produce therapeutic materials with enhanced biocompatibility. PMID:21339361

Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.

PubMed

Mosqueira, V C; Legrand, P; Gulik, A; Bourdon, O; Gref, R; Labarre, D; Barratt, G

2001-11-01

The aim of our work was to examine the relationship between modifications of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the first time that such properties have been correlated with biological interactions for NC, a novel carrier system with a structure more complex than nanospheres. C3 crossed immunoelectrophoresis revealed the reduced activation for NC with longer PEG chain and higher density, although all formulations induced C3 cleavage to a lesser or greater extent. NC bearing PEG covalently bound to the surface were weaker activators of complement than plain PLA [poly(D,L-lactide)] NC or nanospheres (NS). Furthermore, the fluorescent/confocal microscopy of J774A1 cells in contact with NC reveal a dramatically reduced interaction with PEG-bearing NC. However, the way in which PEG was attached (covalent or adsorbed) seemed to affect the mechanism of uptake. Taken together, these results suggest that the low level of protein binding to NC covered with a high density of 20kDa PEG chains is likely to be due to the steric barriers surrounding these particles, which prevents protein adsorption and reduces their interaction with macrophages.

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.

Study on the immunological safety of universal plasma in the Chinese population in vitro.

PubMed

Chen, Guanyi; Zhu, Liguo; Wang, Shufang; Zhuang, Yuan; Yu, Yang; Wang, Deqing

2017-04-01

The prepared procedure for universal plasma in the Chinese population has been developed. However, the immunological safety with the level of antibodies, soluble immune complexes and complements is necessary to investigate. The universal plasma was pooled at the optimal ratio of A:B:AB=6:2.5:1.5 at 22°C for 1 hour. The titer of IgM antibodies was detected by saline agglutination, and the titer of IgG antibodies was detected by a Polybrene test after IgM destroyed by 2-mereaptoethanol. The hemolysis extent of RBC was investigated by an extracorporal hemolysis test, and the concentration of free-hemoglobin was determined by the ortho-tolidine method. The levels of CIC-C1q, C3b and TCC (C5-9) were tested using an enzyme linked immunosorbent assay (ELISA). The titer of IgM and IgG in universal plasma was lower than 2 and 4, respectively. The hemolysis of the universal plasma with A, B and AB group RBCs was negative with values of 5.5, 6.8 and 5.7, respectively. The level of CIC-C1q and TCC (C5-9) in universal plasma was comparable to that in A or B type pooled plasma, but CIC-C1q was lower than that and TCC (C5-9) was higher than that in AB type pooled plasma. The level of complement C3b was comparable to that in A type pooled plasma, but lower than that in B type pooled plasma and higher than that in AB type pooled plasma. The results of this study demonstrated that the immunological levels were within an acceptable range and confirmed the safety in vitro. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immunomodulating Activity of Aronia melanocarpa Polyphenols

PubMed Central

Ho, Giang T. T.; Bräunlich, Marie; Austarheim, Ingvild; Wangensteen, Helle; Malterud, Karl E.; Slimestad, Rune; Barsett, Hilde

2014-01-01

The immunomodulating effects of isolated proanthocyanidin-rich fractions, procyanidins C1, B5 and B2 and anthocyanins of Aronia melanocarpa were investigated. In this work, the complement-modulating activities, the inhibitory activities on nitric oxide (NO) production in LPS-induced RAW 264.7 macrophages and effects on cell viability of these polyphenols were studied. Several of the proanthocyanidin-rich fractions, the procyanidins C1, B5 and B2 and the cyanidin aglycone possessed strong complement-fixing activities. Cyanidin 3-glucoside possessed stronger activity than the other anthocyanins. Procyanidins C1, B5 and B2 and proanthocyanidin-rich fractions having an average degree of polymerization (PD) of 7 and 34 showed inhibitory activities on NO production in LPS-stimulated RAW 264.7 mouse macrophages. All, except for the fraction containing proanthocyanidins with PD 34, showed inhibitory effects without affecting cell viability. This study suggests that polyphenolic compounds of A. melanocarpa may have beneficial effects as immunomodulators and anti-inflammatory agents. PMID:24983479

Immunomodulating activity of Aronia melanocarpa polyphenols.

PubMed

Ho, Giang T T; Bräunlich, Marie; Austarheim, Ingvild; Wangensteen, Helle; Malterud, Karl E; Slimestad, Rune; Barsett, Hilde

2014-06-30

The immunomodulating effects of isolated proanthocyanidin-rich fractions, procyanidins C1, B5 and B2 and anthocyanins of Aronia melanocarpa were investigated. In this work, the complement-modulating activities, the inhibitory activities on nitric oxide (NO) production in LPS-induced RAW 264.7 macrophages and effects on cell viability of these polyphenols were studied. Several of the proanthocyanidin-rich fractions, the procyanidins C1, B5 and B2 and the cyanidin aglycone possessed strong complement-fixing activities. Cyanidin 3-glucoside possessed stronger activity than the other anthocyanins. Procyanidins C1, B5 and B2 and proanthocyanidin-rich fractions having an average degree of polymerization (PD) of 7 and 34 showed inhibitory activities on NO production in LPS-stimulated RAW 264.7 mouse macrophages. All, except for the fraction containing proanthocyanidins with PD 34, showed inhibitory effects without affecting cell viability. This study suggests that polyphenolic compounds of A. melanocarpa may have beneficial effects as immunomodulators and anti-inflammatory agents.

Role of a disulfide-bonded peptide loop within human complement C9 in the species-selectivity of complement inhibitor CD59.

PubMed

Husler, T; Lockert, D H; Sims, P J

1996-03-12

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective, and is most effective toward C9 derived from human or other primate plasma. The species-selective activity of CD59 was recently used to map the segment of human C9 that is recognized by this MAC inhibitor, using recombinant rabbit/human C9 chimeras that retain lytic function within the MAC [Husler, T., Lockert, D. H., Kaufman, K. M., Sodetz, J. M., & Sims, P. J. (1995) J. Biol. Chem. 270,3483-3486]. These experiments suggested that the CD59 recognition domain was contained between residues 334 and 415 in human C9. By analyzing the species-selective lytic activity of recombinant C9 with chimeric substitutions internal to this segment, we now demonstrate that the site in human C9 uniquely recognized by CD59 is centered on those residues contained between C9 Cys359/Cys384, with an additional contribution by residues C-terminal to this segment. Consistent with its role as a CD59 recognition domain, CD59 specifically bound a human C9-derived peptide corresponding to residues 359-384, and antibody (Fab) raised against this C9-derived peptide inhibited the lytic activity of human MAC. Mutant human C9 in which Ala was substituted for Cys359/384 was found to express normal lytic activity and to be fully inhibited by CD59. This suggests that the intrachain Cys359/Cys384 disulfide bond within C9 is not required to maintain the conformation of this segment of C9 for interaction with CD59.

Identification of a human non-interferon lymphokine activating monocyte complement biosynthesis.

PubMed Central

Drouet, C; Reboul, A; Colomb, M

1989-01-01

A monocyte-stimulating activity produced by mitogen-induced mononuclear cells has been defined by its ability to enhance the synthesis in vitro of complement C1 subcomponents, C2 and C3. A lymphokine responsible for this activity was purified from culture supernatants of peripheral blood mononuclear cells activated by staphylococcal enterotoxin A. From 0.5 litre of supernatant the purification procedure [(NH4)2SO4 precipitation, phenyl-Sepharose chromatography and preparative electrofocusing] yielded about 100 pmol of purified lymphokine. Its pI is 7.9 and its Mr, estimated by SDS/polyacrylamide-gel electrophoresis, is 14,600, 27,000 and 56,000, the high-Mr species representing oligomeric forms of the Mr-14,600 molecule. Its amino acid analysis reveals a high percentage of hydrophobic amino acids (34%); the absence of histidine residues suggests that it is a novel monocyte-activating lymphokine. It enhances C1r and C1s biosynthesis at a pretranslational level. From its structure and activity this lymphokine appears different from gamma-interferon. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:2481436

A novel factor H-Fc chimeric immunotherapeutic molecule against Neisseria gonorrhoeae

PubMed Central

Shaughnessy, Jutamas; Gulati, Sunita; Agarwal, Sarika; Unemo, Magnus; Ohnishi, Makoto; Su, Xia-Hong; Monks, Brian G.; Visintin, Alberto; Madico, Guillermo; Lewis, Lisa A.; Golenbock, Douglas T.; Reed, George W.; Rice, Peter A.; Ram, Sanjay

2015-01-01

Neisseria gonorrhoeae (Ng), the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including Ng, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18-20 (FH18-20). We explored the utility of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic. FH18-20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18-20 that eliminated complement activation on host cells, yet maintained binding to Ng, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18-20/Fc, but unlike FH18-20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical Ng isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10/15 (67%) strains and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant Ng. PMID:26773149

Crystal structures of the Erp protein family members ErpP and ErpC from Borrelia burgdorferi reveal the reason for different affinities for complement regulator factor H.

PubMed

Brangulis, Kalvis; Petrovskis, Ivars; Kazaks, Andris; Akopjana, Inara; Tars, Kaspars

2015-05-01

Borrelia burgdorferi is the causative agent of Lyme disease, which can be acquired after the bite of an infected Ixodes tick. As a strategy to resist the innate immunity and to successfully spread and proliferate, B. burgdorferi expresses a set of outer membrane proteins that are capable of binding complement regulator factor H (CFH), factor H-like protein 1 (CFHL-1) and factor H-related proteins (CFHR) to avoid complement-mediated killing. B. burgdorferi B31 contains three proteins that belong to the Erp (OspE/F-related) protein family and are capable of binding CFH and some CFHRs, namely ErpA, ErpC and ErpP. We have determined the crystal structure of ErpP at 2.53Å resolution and the crystal structure of ErpC at 2.15Å resolution. Recently, the crystal structure of the Erp family member OspE from B. burgdorferi N40 was determined in complex with CFH domains 19-20, revealing the residues involved in the complex formation. Despite the high sequence conservation between ErpA, ErpC, ErpP and the homologous protein OspE (78-80%), the affinity for CFH and CFHRs differs markedly among the Erp family members, suggesting that ErpC may bind only CFHRs but not CFH. A comparison of the binding site in OspE with those of ErpC and ErpP revealed that the extended loop region, which is only observed in the potential binding site of ErpC, plays an important role by preventing the binding of CFH. These results can explain the inability of ErpC to bind CFH, whereas ErpP and ErpA still possess the ability to bind CFH. Copyright © 2015 Elsevier B.V. All rights reserved.

Interactive Macroeconomics

NASA Astrophysics Data System (ADS)

Di Guilmi, Corrado; Gallegati, Mauro; Landini, Simone

2017-04-01

Preface; List of tables; List of figures, 1. Introduction; Part I. Methodological Notes and Tools: 2. The state space notion; 3. The master equation; Part II. Applications to HIA Based Models: 4. Financial fragility and macroeconomic dynamics I: heterogeneity and interaction; 5. Financial fragility and macroeconomic Dynamics II: learning; Part III. Conclusions: 6. Conclusive remarks; Part IV. Appendices and Complements: Appendix A: Complements to Chapter 3; Appendix B: Solving the ME to solve the ABM; Appendix C: Specifying transition rates; Index.

Role of CYP2E1 immunoglobulin G4 subclass antibodies and complement in pathogenesis of idiosyncratic drug-induced hepatitis.

PubMed

Njoku, Dolores B; Mellerson, Jenelle L; Talor, Monica V; Kerr, Douglas R; Faraday, Nauder R; Outschoorn, Ingrid; Rose, Noel R

2006-02-01

Idiosyncratic drug-induced hepatitis (IDDIH) is the third most common cause for acute liver failure in the United States. Previous studies have attempted to identify susceptible patients or early stages of disease with various degrees of success. To determine if total serum immunoglobulin subclasses, CYP2E1-specific subclass autoantibodies, complement components, or immune complexes could distinguish persons with IDDIH from others exposed to drugs, we studied persons exposed to halogenated volatile anesthetics, which have been associated with IDDIH and CYP2E1 autoantibodies. We found that patients with anesthetic-induced IDDIH had significantly elevated levels of CYP2E1-specific immunoglobulin G4 (IgG4) autoantibodies, while anesthetic-exposed healthy persons had significantly elevated levels of CYP2E1-specific IgG1 autoantibodies. Anesthetic IDDIH patients had significantly lower levels of C4a, C3a, and C5a compared to anesthetic-exposed healthy persons. C1q- and C3d-containing immune complexes were significantly elevated in anesthetic-exposed persons. In conclusion, our data suggest that anesthetic-exposed persons develop CYP2E1-specific IgG1 autoantibodies which may form detectable circulating immune complexes subsequently cleared by classical pathway activation of the complement system. Persons susceptible to anesthetic-induced IDDIH develop CYP2E1-specific IgG4 autoantibodies which form small, nonprecipitating immune complexes that escape clearance because of their size or by direct inhibition of complement activation.

Pre-transplant donor HLA-specific antibodies: characteristics causing detrimental effects on survival after lung transplantation.

PubMed

Smith, John D; Ibrahim, Mohamed W; Newell, Helen; Danskine, Anna J; Soresi, Simona; Burke, Margaret M; Rose, Marlene L; Carby, Martin

2014-10-01

The impact of Luminex-detected HLA antibodies on outcomes after lung transplantation is unclear. Herein we have undertaken a retrospective study of pre-transplant sera from 425 lung transplants performed between 1991 and 2003. Pre-transplant sera, originally screened by complement-dependent cytotoxicity (CDC) assays, were retrospectively tested for the presence of HLA-specific antibodies using HLA-coated Luminex beads and C4d deposition on Luminex beads. The results were correlated with graft survival at 1 year. Twenty-seven patients were retrospectively identified as having been transplanted against donor-specific HLA antibodies (DSA) and 36 patients against non-donor-specific HLA antibodies (NDSA). DSA-positive patients had 1-year survival of 51.9% compared with 77.8% for NDSA and 71.8% for antibody-negative patients (p = 0.029). One-year survival of patients with complement-fixing DSA was 12.5% compared with 62.5% for non-complement-fixing DSA, 75.8% for non-complement-fixing NDSA and 71.8% for antibody-negative patients (p < 0.0001). DSA-positive patients with mean fluorescence intensity (MFI) >5,000 had 1-year survival of 33.3% compared with 71.4% for MFI 2,000 to 5000 and 62.5% for MFI <2,000 (p = 0.0046). Multivariable analysis revealed DSA to be an independent predictor of poor patient survival within 1 year (p = 0.0010, hazard ratio [HR] = 3.569) as well as complement-fixing DSA (p < 0.0001, HR = 11.083) and DSA with MFI >5,000 (p = 0.0001, HR = 5.512). Pre-formed DSA, particularly complement-fixing DSA, and high MFI are associated with poor survival within the first year after lung transplantation. Risk stratification according to complement fixation or MFI levels may allow for increased transplantation in sensitized patients. Copyright © 2014 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.

Phenotypic characterization of ten methanol oxidation (Mox) mutant classes in methylobacterium AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nunn, D.N.; Lidstrom, M.E.

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium strain AM1 have been characterized by complementation analysis and assigned to ten complementation groups, Mox A1,A2,A3 and B-H. We have characterized each of the mutants belonging to the ten Mox complementation groups by PMS-DCPIP dye linked methanol dehydrogenase activity, by methanol-dependent whole cell oxygen consumption, by the presence or absence of methanol dehydrogenase protein by SDS-polyacrylamide gels and Western blotting, by the absorption spectra of purified mutant methanol dehydrogenase proteins and by the presence or absence of the soluble cytochrome c proteins of Methylobacterium AM1. We propose functions for each ofmore » the genes deficient in the mutants of the ten Mox complementation groups. These functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome c/sub L/, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome c/sub L/, three gene functions responsible for the proper association of the PQQ prosthetic group with the methanol dehydrogenase apoprotein and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol. 24 refs., 5 figs., 2 tabs.« less

Characterization of Pharmacologic and Pharmacokinetic Properties of CCX168, a Potent and Selective Orally Administered Complement 5a Receptor Inhibitor, Based on Preclinical Evaluation and Randomized Phase 1 Clinical Study.

PubMed

Bekker, Pirow; Dairaghi, Daniel; Seitz, Lisa; Leleti, Manmohan; Wang, Yu; Ertl, Linda; Baumgart, Trageen; Shugarts, Sarah; Lohr, Lisa; Dang, Ton; Miao, Shichang; Zeng, Yibin; Fan, Pingchen; Zhang, Penglie; Johnson, Daniel; Powers, Jay; Jaen, Juan; Charo, Israel; Schall, Thomas J

2016-01-01

The complement 5a receptor has been an attractive therapeutic target for many autoimmune and inflammatory disorders. However, development of a selective and potent C5aR antagonist has been challenging. Here we describe the characterization of CCX168 (avacopan), an orally administered selective and potent C5aR inhibitor. CCX168 blocked the C5a binding, C5a-mediated migration, calcium mobilization, and CD11b upregulation in U937 cells as well as in freshly isolated human neutrophils. CCX168 retains high potency when present in human blood. A transgenic human C5aR knock-in mouse model allowed comparison of the in vitro and in vivo efficacy of the molecule. CCX168 effectively blocked migration in in vitro and ex vivo chemotaxis assays, and it blocked the C5a-mediated neutrophil vascular endothelial margination. CCX168 was effective in migration and neutrophil margination assays in cynomolgus monkeys. This thorough in vitro and preclinical characterization enabled progression of CCX168 into the clinic and testing of its safety, tolerability, pharmacokinetic, and pharmacodynamic profiles in a Phase 1 clinical trial in 48 healthy volunteers. CCX168 was shown to be well tolerated across a broad dose range (1 to 100 mg) and it showed dose-dependent pharmacokinetics. An oral dose of 30 mg CCX168 given twice daily blocked the C5a-induced upregulation of CD11b in circulating neutrophils by 94% or greater throughout the entire day, demonstrating essentially complete target coverage. This dose regimen is being tested in clinical trials in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Trial Registration ISRCTN registry with trial ID ISRCTN13564773.

Characterization of Pharmacologic and Pharmacokinetic Properties of CCX168, a Potent and Selective Orally Administered Complement 5a Receptor Inhibitor, Based on Preclinical Evaluation and Randomized Phase 1 Clinical Study

PubMed Central

Bekker, Pirow; Dairaghi, Daniel; Seitz, Lisa; Leleti, Manmohan; Wang, Yu; Ertl, Linda; Baumgart, Trageen; Shugarts, Sarah; Lohr, Lisa; Dang, Ton; Miao, Shichang; Zeng, Yibin; Fan, Pingchen; Zhang, Penglie; Johnson, Daniel; Powers, Jay; Jaen, Juan; Charo, Israel; Schall, Thomas J.

2016-01-01

The complement 5a receptor has been an attractive therapeutic target for many autoimmune and inflammatory disorders. However, development of a selective and potent C5aR antagonist has been challenging. Here we describe the characterization of CCX168 (avacopan), an orally administered selective and potent C5aR inhibitor. CCX168 blocked the C5a binding, C5a-mediated migration, calcium mobilization, and CD11b upregulation in U937 cells as well as in freshly isolated human neutrophils. CCX168 retains high potency when present in human blood. A transgenic human C5aR knock-in mouse model allowed comparison of the in vitro and in vivo efficacy of the molecule. CCX168 effectively blocked migration in in vitro and ex vivo chemotaxis assays, and it blocked the C5a-mediated neutrophil vascular endothelial margination. CCX168 was effective in migration and neutrophil margination assays in cynomolgus monkeys. This thorough in vitro and preclinical characterization enabled progression of CCX168 into the clinic and testing of its safety, tolerability, pharmacokinetic, and pharmacodynamic profiles in a Phase 1 clinical trial in 48 healthy volunteers. CCX168 was shown to be well tolerated across a broad dose range (1 to 100 mg) and it showed dose-dependent pharmacokinetics. An oral dose of 30 mg CCX168 given twice daily blocked the C5a-induced upregulation of CD11b in circulating neutrophils by 94% or greater throughout the entire day, demonstrating essentially complete target coverage. This dose regimen is being tested in clinical trials in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Trial Registration ISRCTN registry with trial ID ISRCTN13564773. PMID:27768695

Complement-mediated bactericidal activity of anti-factor H binding protein monoclonal antibodies against the meningococcus relies upon blocking factor H binding.

PubMed

Giuntini, Serena; Reason, Donald C; Granoff, Dan M

2011-09-01

Binding of the complement-downregulating protein factor H (fH) to the surface of the meningococcus is important for survival of the organism in human serum. The meningococcal vaccine candidate factor H binding protein (fHbp) is an important ligand for human fH. While some fHbp-specific monoclonal antibodies (MAbs) block binding of fH to fHbp, the stoichiometry of blocking in the presence of high serum concentrations of fH and its effect on complement-mediated bactericidal activity are unknown. To investigate this question, we constructed chimeric antibodies in which the human IgG1 constant region was paired with three murine fHbp-specific binding domains designated JAR 3, JAR 5, and MAb502. By surface plasmon resonance, the association rates for binding of all three MAbs to immobilized fHbp were >50-fold higher than that for binding of fH to fHbp, and the MAb dissociation rates were >500-fold lower than that for fH. While all three MAbs elicited similar C1q-dependent C4b deposition on live bacteria (classical complement pathway), only those antibodies that inhibited binding of fH to fHbp (JAR 3 and JAR 5) had bactericidal activity with human complement. MAb502, which did not inhibit fH binding, had complement-mediated bactericidal activity only when tested with fH-depleted human complement. When an IgG1 anti-fHbp MAb binds to sparsely exposed fHbp on the bacterial surface, there appears to be insufficient complement activation for bacteriolysis unless fH binding also is inhibited. The ability of fHbp vaccines to elicit protective antibodies, therefore, is likely to be enhanced if the antibody repertoire is of high avidity and includes fH-blocking activity.

T Cell Lipid Rafts and Complement Ligands for Diagnosis and Monitoring of SLE

DTIC Science & Technology

2011-05-01

including complete blood count, erythrocyte sedimentation rate , serum levels of C3 and C4, antinuclear autoantibodies (ANA), and anti-dsDNA level. Tests... sedimentation rate , serum levels of C3 and C4, antinuclear autoantibody (ANA) and anti-dsDNA antibody levels. In addition, each patient underwent history...6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail: 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES

Human L-ficolin, a recognition molecule of the lectin activation pathway of complement, activates complement by binding to pneumolysin, the major toxin of Streptococcus pneumoniae.

PubMed

Ali, Youssif M; Kenawy, Hany I; Muhammad, Adnan; Sim, Robert B; Andrew, Peter W; Schwaeble, Wilhelm J

2013-01-01

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q(-/-) mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.

Human L-ficolin, a Recognition Molecule of the Lectin Activation Pathway of Complement, Activates Complement by Binding to Pneumolysin, the Major Toxin of Streptococcus pneumoniae

PubMed Central

Ali, Youssif M.; Kenawy, Hany I.; Muhammad, Adnan; Sim, Robert B.

2013-01-01

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q−/− mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum. PMID:24349316

The limits to global-warming mitigation by terrestrial carbon removal

NASA Astrophysics Data System (ADS)

Boysen, Lena R.; Lucht, Wolfgang; Gerten, Dieter; Heck, Vera; Lenton, Timothy M.; Schellnhuber, Hans Joachim

2017-05-01

Massive near-term greenhouse gas emissions reduction is a precondition for staying "well below 2°C" global warming as envisaged by the Paris Agreement. Furthermore, extensive terrestrial carbon dioxide removal (tCDR) through managed biomass growth and subsequent carbon capture and storage is required to avoid temperature "overshoot" in most pertinent scenarios. Here, we address two major issues: First, we calculate the extent of tCDR required to "repair" delayed or insufficient emissions reduction policies unable to prevent global mean temperature rise of 2.5°C or even 4.5°C above pre-industrial level. Our results show that those tCDR measures are unable to counteract "business-as-usual" emissions without eliminating virtually all natural ecosystems. Even if considerable (Representative Concentration Pathway 4.5 [RCP4.5]) emissions reductions are assumed, tCDR with 50% storage efficiency requires >1.1 Gha of the most productive agricultural areas or the elimination of >50% of natural forests. In addition, >100 MtN/yr fertilizers would be needed to remove the roughly 320 GtC foreseen in these scenarios. Such interventions would severely compromise food production and/or biosphere functioning. Second, we reanalyze the requirements for achieving the 160-190 GtC tCDR that would complement strong mitigation action (RCP2.6) in order to avoid 2°C overshoot anytime. We find that a combination of high irrigation water input and/or more efficient conversion to stored carbon is necessary. In the face of severe trade-offs with society and the biosphere, we conclude that large-scale tCDR is not a viable alternative to aggressive emissions reduction. However, we argue that tCDR might serve as a valuable "supporting actor" for strong mitigation if sustainable schemes are established immediately.

Distinct tissue site-specific requirements of mast cells and complement components C3/C5a receptor in IgG immune complex-induced injury of skin and lung.

PubMed

Baumann, U; Chouchakova, N; Gewecke, B; Köhl, J; Carroll, M C; Schmidt, R E; Gessner, J E

2001-07-15

We induced the passive reverse Arthus reaction to IgG immune complexes (IC) at different tissue sites in mice lacking C3 treated or not with a C5aR-specific antagonist, or in mice lacking mast cells (Kit(W)/Kit(W-v) mice), and compared the inflammatory responses with those in the corresponding wild-type mice. We confirmed that IC inflammation of skin can be mediated largely by mast cells expressing C5aR and FcgammaRIII. In addition, we provided evidence for C3-independent C5aR triggering, which may explain why the cutaneous Arthus reaction develops normally in C3(-/-) mice. Furthermore, some, but not all, of the acute changes associated with the Arthus response in the lung were significantly more intense in normal mice than in C3(-/-) or Kit(W)/Kit(W-v) mice, indicating for C3- and mast cell-dependent and -independent components. Finally, we demonstrated that C3 contributed to the elicitation of neutrophils to alveoli, which corresponded to an increased synthesis of TNF-alpha, macrophage-inflammatory protein-2, and cytokine-induced neutrophil chemoattractant. While mast cells similarly influenced alveolar polymorphonuclear leukocyte influx, the levels of these cytokines remained largely unaffected in mast cell deficiency. Together, the phenotypes of C3(-/-) mice and Kit(W)/Kit(W-v) mice suggest that complement and mast cells have distinct tissue site-specific requirements acting by apparently distinct mechanisms in the initiation of IC inflammation.

From orphan drugs to adopted therapies: Advancing C3-targeted intervention to the clinical stage

PubMed Central

Mastellos, Dimitrios C.; Reis, Edimara S.; Yancopoulou, Despina; Hajishengallis, George; Ricklin, Daniel; Lambris, John D.

2016-01-01

Complement dysregulation is increasingly recognized as an important pathogenic driver in a number of clinical disorders. Complement-triggered pathways intertwine with key inflammatory and tissue destructive processes that can either increase the risk of disease or exacerbate pathology in acute or chronic conditions. The launch of the first complement-targeted drugs in the clinic has undeniably stirred the field of complement therapeutic design, providing new insights into complement's contribution to disease pathogenesis and also helping to leverage a more personalized, comprehensive approach to patient management. In this regard, a rapidly expanding toolbox of complement therapeutics is being developed to address unmet clinical needs in several immune-mediated and inflammatory diseases. Elegant approaches employing both surface-directed and fluid-phase inhibitors have exploited diverse components of the complement cascade as putative points of therapeutic intervention. Targeting C3, the central hub of the system, has proven to be a promising strategy for developing biologics as well as small-molecule inhibitors with clinical potential. Complement modulation at the level of C3 has recently shown promise in preclinical primate models, opening up new avenues for therapeutic intervention in both acute and chronic indications fueled by uncontrolled C3 turnover. This review highlights recent developments in the field of complement therapeutics, focusing on C3-directed inhibitors and alternative pathway (AP) regulator-based approaches. Translational perspectives and considerations are discussed, particularly with regard to the structure-guided drug optimization and clinical advancement of a new generation of C3-targeted peptidic inhibitors. PMID:27353192

TolC is required for pathogenicity of Xylella fastidiosa in Vitis vinifera grapevines.

PubMed

Reddy, Joseph D; Reddy, Stephanie L; Hopkins, Don L; Gabriel, Dean W

2007-04-01

Xylella fastidiosa infects a wide range of hosts and causes serious diseases on some of them. The complete genomic sequences of both a citrus variegated chlorosis (CVC) and a Pierce's disease (PD) strain revealed two type I protein secretion plus two multidrug resistance efflux systems, and all evidently were dependent on a single tolC homolog. Marker exchange mutagenesis of the single tolC gene in PD strain Temecula resulted in a total loss of pathogenicity on grape. Importantly, the tolC- mutant strains were not recovered after inoculation into grape xylem, strongly indicating that multidrug efflux is critical to survival of this fastidious pathogen. Both survival and pathogenicity were restored by complementation using tolC cloned in shuttle vector pBBR1MCS-5, which was shown to replicate autonomously, without selection, for 60 days in Temecula growing in planta. These results also demonstrate the ability to complement mutations in X. fastidiosa.

Complement system studies in systemic lupus erythematosus (SLE)

PubMed

Teisberg, P

1975-01-01

Complement system involvement has been studied in 16 patients with systemic lupus erythematosus (SLE). Circulating conversion products of C3 were observed in 4 cases. Low mean values of C4 and C3 were found, while C3 proactivator (properdin factor B) levels were low in only a few of the patients. The levels of C4, C3 and C3 proactivator were not lower in the 4 patients in whom C3 conversion products could be demonstrated than in the others. It is concluded that the low complement values found in SLE may be caused mainly by deficient synthesis. Signs of complement activation are in this patient material demonstrated early in the disease, and chiefly in patients not receiving immunosuppressive therapy.

Novel Scabies Mite Serpins Inhibit the Three Pathways of the Human Complement System

PubMed Central

Mika, Angela; Reynolds, Simone L.; Mohlin, Frida C.; Willis, Charlene; Swe, Pearl M.; Pickering, Darren A.; Halilovic, Vanja; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Blom, Anna M.; Kemp, David J.; Fischer, Katja

2012-01-01

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage. PMID:22792350

Cpa, the outer membrane protease of Cronobacter sakazakii, activates plasminogen and mediates resistance to serum bactericidal activity.

PubMed

Franco, A A; Kothary, M H; Gopinath, G; Jarvis, K G; Grim, C J; Hu, L; Datta, A R; McCardell, B A; Tall, B D

2011-04-01

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ~131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii.

The Anticomplementary Activity of ’Fusobacterium polymorphum’ in Normal and C-4 Deficient Sources of Guinea Pig Complement.

DTIC Science & Technology

1977-01-12

A complement consumption assay was used to show that the anticomplementary activity of a cell wall preparation from F. polymorphum in guinea pig complement...tests with C𔃾-deficient guinea pig sera confirmed that F. polymorphum cell walls were capable of generating alternate complement pathway activity in guinea pig sera.

Loss of CD11b Exacerbates Murine Complement-Mediated Tubulointerstitial Nephritis

PubMed Central

Wang, Ying; Chang, Anthony; Haas, Mark; Quigg, Richard John

2014-01-01

Acute complement activation occurs in the tubulointerstitium (TI) of kidneys transplanted from Crry−/−C3−/− mice into complement-sufficient wildtype mice, followed by marked inflammatory cell infiltration, tubular damage and interstitial fibrosis. We postulated iC3b-CD11b interactions were critical in this TI nephritis model. We transplanted Crry−/−C3−/− mouse kidneys into CD11b−/− and wildtype C57BL/6 mice. Surprisingly, there was greater inflammation in Crry−/−C3−/− kidneys in CD11b−/− recipients compared to those in wildtype hosts. Kidneys in CD11b−/− recipients had large numbers of CD11b−Ly6ChiCCR2hiF4/80+ cells consistent with inflammatory (M1) macrophages recruited from circulating monocytes of the host CD11b−/− animal. There was also an expanded population of CD11b+CD11c+Ly6C−F4/80hi cells. Since these cells were CD11b+, they must have originated from the transplanted kidney; their surface protein expression and appearance within the kidney were consistent with the intrinsic renal mononuclear cellular population. These cells were markedly expanded relative to all relevant controls, including the contralateral donor kidney and Crry−/−C3−/− mouse kidneys in CD11b+/+ wildtype recipients. Direct evidence for their in situ proliferation was the presence of nuclear Ki67 and PCNA in CD11b+F4/80+ cells. Thus, in this experimental model in which there is unrestricted C3 activation, CD11b+ monocytes limit their own infiltration into the kidney and prevent proliferation of endogenous mononuclear cells. This suggests a role for outside-in iC3b-CD11b signals in limiting intrinsic organ inflammation. PMID:24632830

Small-molecule factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

PubMed

Yuan, Xuan; Gavriilaki, Eleni; Thanassi, Jane A; Yang, Guangwei; Baines, Andrea C; Podos, Steven D; Huang, Yongqing; Huang, Mingjun; Brodsky, Robert A

2017-03-01

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA -null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Copyright© Ferrata Storti Foundation.

An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q-mediated complement system in the basal chordate.

PubMed

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

2014-12-01

The origin of the classical complement pathway remains open during chordate evolution. A C1q-like member, BjC1q, was identified in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly upregulated following challenge with bacteria or lipoteichoic acid and LPS. Recombinant BjC1q and its globular head domain specifically interact with lipoteichoic acid and LPS, but BjC1q displays little lectin activity. Moreover, rBjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan-binding lectin-associated serine protease-2 as well as amphioxus serine proteases involved in the cleavage of C4/C2, and C3 activation. Importantly, rBjC1q binds with human IgG as well as an amphioxus Ig domain containing protein, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q-like protein in invertebrates is able to initiate classical pathway, raising the possibility that amphioxus possesses a C1q-mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of a Complement-Binding Protein, DRS, from Strains of Streptococcus pyogenes Containing the emm12 and emm55 Genes

PubMed Central

Binks, Michael; Sriprakash, K. S.

2004-01-01

An extracellular protein of Streptococcus pyogenes, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some S. pyogenes strains. SIC from type 1 (M1) isolates of S. pyogenes interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that S. pyogenes strains carrying emm12 and emm55 (the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity. PMID:15213143

Characterization of a complement-binding protein, DRS, from strains of Streptococcus pyogenes containing the emm12 and emm55 genes.

PubMed

Binks, Michael; Sriprakash, K S

2004-07-01

An extracellular protein of Streptococcus pyogenes, streptococcal inhibitor of complement (SIC), and its variant, called DRS (distantly related to SIC), are expressed by some S. pyogenes strains. SIC from type 1 (M1) isolates of S. pyogenes interferes with complement-mediated cell lysis, reportedly via its interaction with complement proteins. In this study we demonstrate that S. pyogenes strains carrying emm12 and emm55 (the genes for the M12 and M55 proteins, respectively) express and secrete DRS. This protein, like SIC, binds to the C6 and C7 complement proteins, and competition enzyme-linked immunosorbent assay experiments demonstrate that DRS competes with SIC for C6 and C7 binding. Similarly, SIC competes with DRS for binding to the complement proteins. Despite this, the recombinant DRS preparation showed no significant effect on complement function, as determined by lysis of sensitized sheep erythrocytes. Furthermore, the presence of DRS is not inhibitory to SIC activity.

Alternative complement pathway activation increases mortality in a model of burn injury in mice.

PubMed Central

Gelfand, J A; Donelan, M; Hawiger, A; Burke, J F

1982-01-01

We have studied the role of the complement system in burn injury in an experimental model in mice. A 25% body surface area, full-thickness scald wound was produced in anesthetized animals. Massive activation of the alternative complement pathway, but not the classical pathway, was seen. This activation was associated with the generation of neutrophil aggregating activity in the plasma, neutrophil aggregates in the lungs, increased pulmonary vascular permeability, and increased lung edema formation. Decomplementation with cobra venom factor (CVF) or genetic C5 deficiency diminished these pathologic changes, and CVF pretreatment substantially reduced burn mortality in the first 24 h. Preliminary data show that human burn patients have a similar pattern of complement activation involving predominantly the alternative pathway, indicating the possible relevance of the murine model to human disease. Images PMID:7174787

Streptococcus pyogenes Endopeptidase O Contributes to Evasion from Complement-mediated Bacteriolysis via Binding to Human Complement Factor C1q.

PubMed

Honda-Ogawa, Mariko; Sumitomo, Tomoko; Mori, Yasushi; Hamd, Dalia Talat; Ogawa, Taiji; Yamaguchi, Masaya; Nakata, Masanobu; Kawabata, Shigetada

2017-03-10

Streptococcus pyogenes secretes various virulence factors for evasion from complement-mediated bacteriolysis. However, full understanding of the molecules possessed by this organism that interact with complement C1q, an initiator of the classical complement pathway, remains elusive. In this study, we identified an endopeptidase of S. pyogenes , PepO, as an interacting molecule, and investigated its effects on complement immunity and pathogenesis. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis findings revealed that S. pyogenes recombinant PepO bound to human C1q in a concentration-dependent manner under physiological conditions. Sites of inflammation are known to have decreased pH levels, thus the effects of PepO on bacterial evasion from complement immunity was analyzed in a low pH condition. Notably, under low pH conditions, PepO exhibited a higher affinity for C1q as compared with IgG, and PepO inhibited the binding of IgG to C1q. In addition, pepO deletion rendered S. pyogenes more susceptible to the bacteriocidal activity of human serum. Also, observations of the morphological features of the pepO mutant strain (Δ pepO ) showed damaged irregular surfaces as compared with the wild-type strain (WT). WT-infected tissues exhibited greater severity and lower complement activity as compared with those infected by Δ pepO in a mouse skin infection model. Furthermore, WT infection resulted in a larger accumulation of C1q than that with Δ pepO. Our results suggest that interaction of S. pyogenes PepO with C1q interferes with the complement pathway, which enables S. pyogenes to evade complement-mediated bacteriolysis under acidic conditions, such as seen in inflammatory sites. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Lutein and atherosclerosis: Belfast versus Toulouse revisited.

PubMed

Howard, A N; Thurnham, D I

2017-01-01

In 1995 we reported that mean plasma lutein concentrations in salaried men and women from Toulouse in Southern France were double those in subjects recruited from general practitioner lists in Belfast, Northern Ireland. At the time incidence of coronary heart disease (CHD) in Southern France was among the lowest in Europe and was much higher in Northern Ireland. Plasma lutein is a biomarker of vegetable and fruit intake and evidence suggests that high concentrations are generally associated with better cardiometabolic health. At the time we speculated like others that role of the carotenoids may well have been to prevent oxidation of lipid in the lipoproteins and so reduce the uptake of oxidised lipid by macrophages and its deposition within the intimal layers of the major arteries as plaque. It is now widely accepted that CHD is an inflammatory disease and that macrophages within plaque together with tissue damage contribute to this inflammation. Stimulated macrophages release cytokines to activate the immune system both locally and systemically. Precursor complement proteins in the blood are activated to assist immune cells in phagocytosis and cell repair. Individuals with a history of arteriosclerosis display significantly higher concentrations of complement factors C3 and C3a than subjects without such a history. Metabolism of C3 via the alternate complement pathway can give rise to the membrane attack complex (MAC) which creates a hole or pore in pathogens or host cells, killing the cell. Recent studies in patients with early age related macular disease (AMD) who also exhibit similar elevated concentrations of complement proteins in their blood, showed supplementation with lutein progressively decreased the amount of the MAC and other complement factors in the blood. Lutein was used in the supplementation experiments because it is an important constituent of macular pigment. Thus the healthier cardiometabolic features displayed by the people in Toulouse may have been due to the effects of concurrent high concentrations of plasma lutein on the immune system and complement in particular. Other carotenoids may exert similar antioxidant effects but we and others found no differences in antioxidant nutrients between subjects in Toulouse and Belfast or between subjects with asymptomatic markers of atherosclerosis and controls. Copyright © 2016 Elsevier Ltd. All rights reserved.

Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria.

PubMed

Boyle, Michelle J; Reiling, Linda; Feng, Gaoqian; Langer, Christine; Osier, Faith H; Aspeling-Jones, Harvey; Cheng, Yik Sheng; Stubbs, Janine; Tetteh, Kevin K A; Conway, David J; McCarthy, James S; Muller, Ivo; Marsh, Kevin; Anders, Robin F; Beeson, James G

2015-03-17

Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C') inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement. Inhibitory activity was mediated predominately via C1q fixation, and merozoite surface proteins 1 and 2 were identified as major targets. Complement fixation by antibodies was very strongly associated with protection from both clinical malaria and high-density parasitemia in a prospective longitudinal study of children. Ab-C' inhibitory activity could be induced by human immunization with a candidate merozoite surface-protein vaccine. Our findings demonstrate that human anti-malarial antibodies have evolved to function by fixing complement for potent invasion-inhibitory activity and protective immunity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A Decision Criteria to Select an Associative-Memory Organization That Minimizes the Execution Time of a Mix of Associative-Search Operations

DTIC Science & Technology

1993-06-01

s )) = Op (C(s), db(s))" (26) Equation (27) defines OP. Brt(JxBR’T()-,BR as the complement of Op,. Op,(C(s), db(s)) = 0p,(C(s), db(s))’" (27) The...9.5 tf=13.0 t= 11.0 tf= 12.8 128 words tr-13.5 tf=16.9 t,=14.3 t(=17.3 t,=16.5 tf=22.0 Op,,(C(s), db( s )), Op .<(C(s), db( s )), Op ,(C(s), db( s )), Op >(C...OP.(C(s), db( s )), Op •(C(s), db( s )), Op <(C(s), db( s )), Op >(C(s), db( s )), Op .(C(s), db( s

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis.

PubMed

Tüzün, Erdem; Scott, Benjamin G; Goluszko, Elzbieta; Higgs, Stephen; Christadoss, Premkumar

2003-10-01

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.

Cell-derived microparticles and complement activation in preeclampsia versus normal pregnancy.

PubMed

Biró, E; Lok, C A R; Hack, C E; van der Post, J A M; Schaap, M C L; Sturk, A; Nieuwland, R

2007-01-01

Inflammation plays a major role in the vascular dysfunction seen in preeclampsia, and several studies suggest involvement of the complement system. To investigate whether complement activation on the surface of microparticles is increased in plasma of preeclamptic patients versus healthy pregnant controls. Microparticles from plasma of preeclamptic (n=10), healthy pregnant (n=10) and healthy nonpregnant (n=10) women were analyzed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C-reactive protein [CRP], serum amyloid P component [SAP], immunoglobulin [Ig]M, IgG). Fluid phase complement activation products and activator molecules were also determined. Levels of microparticles with bound complement components showed no increase in complement activation on the microparticle surface in preeclamptic women, in line with levels of fluid phase complement activation products. In healthy nonpregnant and pregnant women, bound CRP was associated with classical pathway activation on the microparticle surface, and in healthy pregnant women IgM and IgG molecules also contributed. In preeclamptic women, microparticles with bound SAP and those with IgG seemed to contribute to C1q binding without a clear association to further classical pathway activation. Furthermore, significantly increased levels of microparticles with bound CRP were present in preeclamptic compared with healthy pregnant women (median 178x10(6)/L versus 47x10(6)/L, P<0.01), but without concomitant increases in complement activation. We found no evidence of increased complement activation on the microparticle surface in preeclamptic women. Microparticles with bound CRP were significantly increased, but in contrast to healthy pregnant and nonpregnant women, this was not associated with increased classical pathway activation on the surface of the microparticles.

Death Is Associated with Complement C3 Depletion in Cerebrospinal Fluid of Patients with Pneumococcal Meningitis

PubMed Central

Goonetilleke, U. R.; Scarborough, M.; Ward, S. A.; Hussain, S.; Kadioglu, A.; Gordon, S. B.

2012-01-01

ABSTRACT Pneumococcal meningitis can lead to death or serious neurological sequelae as a result of the host inflammatory response. We investigated the association between host response protein expression and outcome in patients with pneumococcal meningitis. Cerebrospinal fluid (CSF) was obtained from 80 patients with pneumococcal meningitis (40 nonsurvivors and 40 survivors) and 10 normal controls. Candidate proteins were analyzed for an association with survival. Complement C3 levels were 5-fold lower in nonsurvivors than in survivors (P < 0.05). This C3 reduction was not associated with lower levels in serum, indicating a compartmentalized CSF response. Transferrin levels were significantly higher in CSF (but not serum) from nonsurvivors than in CSF from survivors, suggestive of blood-brain barrier damage. Classical apoptosis proteins caspase 3 and apoptosis-inducing factor were not present in CSF. Expression of creatine kinase BB in clinically infected CSF suggested neuronal necrosis, but there was no clear association between level of expression and clinical outcome. Increased blood-brain barrier permeability and complement C3 depletion may have a role in determining outcome from bacterial meningitis. Therapeutic use of citicoline or caspase inhibitors is unlikely to have beneficial effects in patients with meningitis. PMID:22415003

Enhanced susceptibility to acute pneumococcal otitis media in mice deficient in complement C1qa, factor B, and factor B/C2.

PubMed

Tong, Hua Hua; Li, Yong Xing; Stahl, Gregory L; Thurman, Joshua M

2010-03-01

To define the roles of specific complement activation pathways in host defense against Streptococcus pneumoniae in acute otitis media (AOM), we investigated the susceptibility to AOM in mice deficient in complement factor B and C2 (Bf/C2(-/)(-)), C1qa (C1qa(-/)(-)), and factor B (Bf(-)(/)(-)). Bacterial titers of both S. pneumoniae serotype 6A and 14 in the middle ear lavage fluid samples from Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice were significantly higher than in samples from wild-type mice 24 h after transtympanical infection (P < 0.05) and remained persistently higher in samples from Bf/C2(-/)(-) mice than in samples from wild-type mice. Bacteremia occurred in Bf/C2(-/)(-), Bf(-)(/)(-), and C1qa(-/)(-) mice infected with both strains, but not in wild-type mice. Recruitment of inflammatory cells was paralleled by enhanced production of inflammatory mediators in the middle ear lavage samples from Bf/C2(-/)(-) mice. C3b deposition on both strains was greatest for sera obtained from wild-type mice, followed by C1qa(-)(/)(-) and Bf(-)(/)(-) mice, and least for Bf/C2(-)(/)(-) mice. Opsonophagocytosis and whole-blood killing capacity of both strains were significantly decreased in the presence of sera or whole blood from complement-deficient mice compared to wild-type mice. These findings indicate that both the classical and alternative complement pathways are critical for middle ear immune defense against S. pneumoniae. The reduced capacity of complement-mediated opsonization and phagocytosis in the complement-deficient mice appears to be responsible for the impaired clearance of S. pneumoniae from the middle ear and dissemination to the bloodstream during AOM.

Complement Activation in Relation to Capillary Leakage in Children with Septic Shock and Purpura

PubMed Central

Hazelzet, Jan A.; de Groot, Ronald; van Mierlo, Gerard; Joosten, Koen F. M.; van der Voort, Edwin; Eerenberg, Anke; Suur, Marja H.; Hop, Wim C. J.; Hack, C. Erik

1998-01-01

To assess the relationship between capillary leakage and inflammatory mediators during sepsis, blood samples were taken on hospital admission, as well as 24 and 72 h later, from 52 children (median age, 3.3 years) with severe meningococcal sepsis, of whom 38 survived and 14 died. Parameters related to cytokines (interleukin 6 [IL-6] IL-8, plasma phospholipase A2, and C-reactive protein [CRP]), to neutrophil degranulation (elastase and lactoferrin), to complement activation (C3a, C3b/c, C4b/c, and C3- and C4-CRP complexes), and to complement regulation (functional and inactivated C1 inhibitor and C4BP) were determined. The degree of capillary leakage was derived from the amount of plasma infused and the severity of disease by assessing the pediatric risk of mortality (PRISM) score. Levels of IL-6, IL-8, C3b/c, C3-CRP complexes, and C4BP on admission, adjusted for the duration of skin lesions, were significantly different in survivors and nonsurvivors (C3b/c levels were on average 2.2 times higher in nonsurvivors, and C3-CRP levels were 1.9 times higher in survivors). Mortality was independently related to the levels of C3b/c and C3-CRP complexes. In agreement with this, levels of complement activation products correlated well with the PRISM score or capillary leakage. Thus, these data show that complement activation in patients with severe meningococcal sepsis is associated with a poor outcome and a more severe disease course. Further studies should reveal whether complement activation may be a target for therapeutical intervention in this disease. PMID:9784543

A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP

PubMed Central

1988-01-01

We report the organization of the human genes encoding the complement components C4-binding protein (C4BP), C3b/C4b receptor (CR1), decay accelerating factor (DAF), and C3dg receptor (CR2) within the regulator of complement activation (RCA) gene cluster. Using pulsed field gel electrophoresis analysis these genes have been physically linked and aligned as CR1-CR2-DAF-C4BP in an 800-kb DNA segment. The very tight linkage between the CR1 and the C4BP loci, contrasted with the relative long DNA distance between these genes, suggests the existence of mechanisms interfering with recombination within the RCA gene cluster. PMID:2450163

Assignment of C1q-binding HLA antibodies as unacceptable HLA antigens avoids positive CDC-crossmatches prior to transplantation of deceased donor organs.

PubMed

Juhl, David; Marget, Matthias; Hallensleben, Michael; Görg, Siegfried; Ziemann, Malte

2017-03-01

Soon, a virtual crossmatch shall replace the complement-dependent cytotoxicity (CDC) allocation crossmatch in the Eurotransplant region. To prevent positive CDC-crossmatches in the recipient centre, careful definition of unacceptable antigens is necessary. For highly sensitized patients, this is difficult by CDC alone. Assignment of all antibodies detected by sensitive assays, however, could prevent organ allocation. To assess the usefulness of the Luminex C1q-assay to prevent positive CDC-crossmatches, all CDC-crossmatches performed prior to deceased kidney transplantation in a 16-month-period were reviewed. Sera causing positive crossmatches were investigated by the C1q-assay. 31 out of 1432 crossmatches (2.2%) were positive. Sera involved in 26 positive crossmatches were available. C1q-binding donor-specific antibodies were detected in 19 sera (73.1%). The other sera were from recipients without any HLA antibodies detectable by CDC or common solid phase assays. Three patients had known Non-HLA antibodies causing positive CDC-results. Four crossmatches were only weak positive. Therefore, avoidance of donors with HLA antigens against whom C1q-binding antibodies were detected would have prevented all positive crossmatches due to HLA antibodies. Provided that all HLA specificities against which antibodies are detected by the Luminex C1q-assay are considered as unacceptable antigens, CDC-crossmatches prior to transplantation might safely be omitted in many patients. They should be maintained in highly immunized patients, however, for whom assignment of all C1q-positive antibodies as unacceptable antigens could lead to a significant delay or even prevention of transplantation. Copyright © 2017 Elsevier B.V. All rights reserved.

Bioactive peptides derived from natural proteins with respect to diversity of their receptors and physiological effects.

PubMed

Yoshikawa, Masaaki

2015-10-01

We have found various bioactive peptides derived from animal and plant proteins, which interact with receptors for endogenous bioactive peptides such as opioids, neurotensin, complements C3a and C5a, oxytocin, and formyl peptides etc. Among them, rubiscolin, a δ opioid peptide derived from plant RuBisCO, showed memory-consolidating, anxiolytic-like, and food intake-modulating effects. Soymorphin, a μ opioid peptide derived from β-conglycinin showed anxiolytic-like, anorexigenic, hypoglycemic, and hypotriglyceridemic effects. β-Lactotensin derived from β-lactoglobulin, the first natural ligand for the NTS2 receptor, showed memory-consolidating, anxiolytic-like, and hypocholesterolemic effects. Weak agonist peptides for the complements C3a and C5a receptors were released from many proteins and exerted various central effects. Peptides showing anxiolytic-like antihypertensive and anti-alopecia effects via different types of receptors such as OT, FPR and AT2 were also obtained. Based on these study, new functions and post-receptor mechanisms of receptor commom to endogenous and exogenous bioactive peptides have been clarified. Copyright © 2015 Elsevier Inc. All rights reserved.

Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1

PubMed Central

Abdelbaset-Ismail, A; Borkowska-Rzeszotek, S; Kubis, E; Bujko, K; Brzeźniakiewicz-Janus, K; Bolkun, L; Kloczko, J; Moniuszko, M; Basak, G W; Wiktor-Jedrzejczak, W; Ratajczak, M Z

2017-01-01

As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK–HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated. PMID:27451975

Activation of the complement cascade enhances motility of leukemic cells by downregulating expression of HO-1.

PubMed

Abdelbaset-Ismail, A; Borkowska-Rzeszotek, S; Kubis, E; Bujko, K; Brzeźniakiewicz-Janus, K; Bolkun, L; Kloczko, J; Moniuszko, M; Basak, G W; Wiktor-Jedrzejczak, W; Ratajczak, M Z

2017-02-01

As a crucial arm of innate immunity, the complement cascade (ComC) is involved both in mobilization of normal hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood and in their homing to BM. Despite the fact that ComC cleavage fragments alone do not chemoattract normal HSPCs, we found that leukemia cell lines as well as clonogenic blasts from chronic myeloid leukemia and acute myeloid leukemia patients respond robustly to C3 and C5 cleavage fragments by chemotaxis and increased adhesion. This finding was supported by the detection of C3a and C5a receptors in cells from human malignant hematopoietic cell lines and patient blasts at the mRNA (reverse transcriptase-polymerase chain reaction) and protein level (fluorescence-activated cell sorting), and by the demonstration that these receptors respond to stimulation by C3a and C5a by phosphorylation of p42/44 and p38 mitogen-activated protein kinases (MAPK), and protein kinase B (PKB/AKT). We also found that inducible heme oxygenase 1 (HO-1) is a negative regulator of ComC-mediated trafficking of leukemic cells, and that stimulation of leukemic cells by C3 or C5 cleavage fragments activates p38 MAPK, which downregulates HO-1 expression, rendering cells more mobile. We conclude that activation of the ComC in leukemia/lymphoma patients (for example, as a result of accompanying infections) enhances the motility of malignant cells and contributes to their spread in a p38 MAPK-HO-1-dependent manner. Therefore, inhibition of p38 MAPK or upregulation of HO-1 by small-molecule modulators would have a beneficial effect on ameliorating cell migration-mediated expansion of leukemia/lymphoma cells when the ComC becomes activated.

Exogenous C3 protein enhances the adaptive immune response to polymicrobial sepsis through down-regulation of regulatory T cells.

PubMed

Yuan, Yujie; Ren, Jianan; Cao, Shougen; Zhang, Weiwei; Li, Jieshou

2012-01-01

The role of complement system in bridging innate and adaptive immunity has been confirmed in various invasive pathogens. It is still obscure how complement proteins promote T cell-mediated immune response during sepsis. The aim of this study is to investigate the role of exogenous C3 protein in the T-cell responses to sepsis. Sepsis was induced by colon ascendens stent peritonitis (CASP) in wild-type C57BL/6 mice, sham-operated mice for control. Human purified C3 protein (HuC3, 1 mg) was intraperitoneally injected at 6 h post-surgery, with 200 μl phosphate-buffered saline as control. The levels of C3 and cytokines, the expression of FOXP3 and NF-κB, and the percentages of CD4(+) T-cell subsets were compared among the groups at given time points. The polymicrobial sepsis produced considerable release of TNF-α and IL-10, and caused complement C3 exhaustion. Exogenous C3 administration markedly improved the 48 h survival rate, as compared with nontreatment (40% vs. 5%, P<0.01). The expression of FOXP3 protein was increased during sepsis, but can be suppressed by HuC3 administration. A single injection of HuC3 postponed the decline of differentiated Th1 cells, and depressed the activation of Th2/Th17 cells. Besides, the Th1-Th2 shift in late stage of sepsis can be controlled under C3 supplementation. The suppression of NF-κB pathway might be related to the appearance of immunocompromise. The study confirmed the important role of exogenous C3 in up-regulation of adaptive immune response to sepsis. The complement pathway would be a pivotal target for severe sepsis management. Copyright © 2011 Elsevier B.V. All rights reserved.

Novel roles of complement in renal diseases and their therapeutic consequences.

PubMed

Wada, Takehiko; Nangaku, Masaomi

2013-09-01

The complement system functions as a part of the innate immune system. Inappropriate activation of the complement pathways has a deleterious effect on kidneys. Recent advances in complement research have provided new insights into the pathogenesis of glomerular and tubulointerstitial injury associated with complement activation. A new disease entity termed 'C3 glomerulopathy' has recently been proposed and is characterized by isolated C3 deposition in glomeruli without positive staining for immunoglobulins. Genetic and functional studies have demonstrated that several different mutations and disease variants, as well as the generation of autoantibodies, are potentially associated with its pathogenesis. The data from comprehensive analyses suggest that complement dysregulation can also be associated with hemolytic uremic syndrome and more common glomerular diseases, such as IgA nephropathy and diabetic kidney disease. In addition, animal studies utilizing genetically modified mice have begun to elucidate the molecular pathomechanisms associated with the complement system. From a diagnostic point of view, a noninvasive, MRI-based method for detecting C3 has recently been developed to serve as a novel tool for diagnosing complement-mediated kidney diseases. While novel therapeutic tools related to complement regulation are emerging, studies evaluating the precise roles of the complement system in kidney diseases will still be useful for developing new therapeutic approaches.

Low copy numbers of complement C4 and homozygous deficiency of C4A may predispose to severe disease and earlier disease onset in patients with systemic lupus erythematosus.

PubMed

Jüptner, M; Flachsbart, F; Caliebe, A; Lieb, W; Schreiber, S; Zeuner, R; Franke, A; Schröder, J O

2018-04-01

Objectives Low copy numbers and deletion of complement C4 genes are potent risk factors for systemic lupus erythematosus (SLE). However, it is not known whether this genetic association affects the clinical outcome. We investigated C4 copy number variation and its relationship to clinical and serological features in a Northern European lupus cohort. Methods We genotyped the C4 gene locus using polymerase chain reaction (PCR)-based TaqMan assays in 169 patients with SLE classified according to the 1997 revised American College of Rheumatology (ACR) criteria and in 520 matched controls. In the patient group the mean C4 serum protein concentrations nephelometrically measured during a 12-month period prior to genetic analysis were compared to C4 gene copy numbers. Severity of disease was classified according to the intensity of the immunosuppressive regimens applied and compared to C4 gene copy numbers, too. In addition, we performed a TaqMan based analysis of three lupus-associated single-nucleotide polymorphisms (SNPs) located inside the major histocompatibility complex (MHC) to investigate the independence of complement C4 in association with SLE. Results Homozygous deficiency of the C4A isotype was identified as the strongest risk factor for SLE (odds ratio (OR) = 5.329; p = 7.7 × 10 -3 ) in the case-control comparison. Moreover, two copies of total C4 were associated with SLE (OR = 3.699; p = 6.8 × 10 -3 ). C4 serum levels were strongly related to C4 gene copy numbers in patients, the mean concentration ranging from 0.110 g/l (two copies) to 0.256 g/l (five to six copies; p = 4.9 × 10 -6 ). Two copies of total C4 and homozygous deletion of C4A were associated with a disease course requiring cyclophosphamide therapy (OR = 4.044; p = 0.040 and OR = 5.798; p = 0.034, respectively). Homozygous deletion of C4A was associated with earlier onset of SLE (median 24 vs. 34 years; p = 0.019) but not significant after correction for multiple testing. SNP analysis revealed a significant association of HLA-DRB1*0301 with SLE (OR = 2.231; p = 1.33 × 10 -5 ). Conclusions Our findings confirm the important role of complement C4 genes in the development of SLE. Beyond the impact on the susceptibility for lupus, C4 copy numbers may be related to earlier onset and a more severe course of the disease. The association of homozygous deletion of C4A and SLE is accompanied by the presence of HLA-DRB1*0301 without a proven pathophysiological mechanism.

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation.

PubMed

Khandhadia, Samir; Hakobyan, Svetlana; Heng, Ling Z; Gibson, Jane; Adams, David H; Alexander, Graeme J; Gibson, Jonathan M; Martin, Keith R; Menon, Geeta; Nash, Kathryn; Sivaprasad, Sobha; Ennis, Sarah; Cree, Angela J; Morgan, B Paul; Lotery, Andrew J

2013-08-01

To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). Multicenter, cross-sectional study. We recruited 223 Western European patients ≥ 55 years old who had undergone LT ≥ 5 years previously. We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). We evaluated AMD status and recipient and donor CFH Y402H genotype. In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; P<0.001) and the CFH Y402H sequence variation (41.9% vs 36.2%; OR, 1.27; P = 0.014). Presence of AMD is not associated with modification of hepatic CFH production. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local intraocular complement activity is of greater importance in AMD pathogenesis. The high AMD prevalence observed in LT patients may be associated with the increased frequency of the CFH Y402H sequence variation. The authors have no proprietary or commercial interest in any materials discussed in this article. Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23.

PubMed Central

Masutani, C; Sugasawa, K; Yanagisawa, J; Sonoyama, T; Ui, M; Enomoto, T; Takio, K; Tanaka, K; van der Spek, P J; Bootsma, D

1994-01-01

Complementation group C of xeroderma pigmentosum (XP) represents one of the most common forms of this cancer-prone DNA repair syndrome. The primary defect is located in the subpathway of the nucleotide excision repair system, dealing with the removal of lesions from the non-transcribing sequences ('genome-overall' repair). Here we report the purification to homogeneity and subsequent cDNA cloning of a repair complex by in vitro complementation of the XP-C defect in a cell-free repair system containing UV-damaged SV40 minichromosomes. The complex has a high affinity for ssDNA and consists of two tightly associated proteins of 125 and 58 kDa. The 125 kDa subunit is an N-terminally extended version of previously reported XPCC gene product which is thought to represent the human homologue of the Saccharomyces cerevisiae repair gene RAD4. The 58 kDa species turned out to be a human homologue of yeast RAD23. Unexpectedly, a second human counterpart of RAD23 was identified. All RAD23 derivatives share a ubiquitin-like N-terminus. The nature of the XP-C defect implies that the complex exerts a unique function in the genome-overall repair pathway which is important for prevention of skin cancer. Images PMID:8168482

Complement component C3 plays a critical role in protecting the aging retina in a murine model of age-related macular degeneration.

PubMed

Hoh Kam, Jaimie; Lenassi, Eva; Malik, Talat H; Pickering, Matthew C; Jeffery, Glen

2013-08-01

Complement component C3 is the central complement component and a key inflammatory protein activated in age-related macular degeneration (AMD). AMD is associated with genetic variation in complement proteins that results in enhanced activation of C3 through the complement alternative pathway. These include complement factor H (CFH), a negative regulator of C3 activation. Both C3 inhibition and/or CFH augmentation are potential therapeutic strategies in AMD. Herein, we examined retinal integrity in aged (12 months) mice deficient in both factors H and C3 (CFH(-/-).C3(-/-)), CFH alone (CFH(-/-)), or C3 alone (C3(-/-)), and wild-type mice (C57BL/6). Retinal function was assessed by electroretinography, and retinal morphological features were analyzed at light and electron microscope levels. Retinas were also stained for amyloid β (Aβ) deposition, inflammation, and macrophage accumulation. Contrary to expectation, electroretinograms of CFH(-/-).C3(-/-) mice displayed more severely reduced responses than those of other mice. All mutant strains showed significant photoreceptor loss and thickening of Bruch's membrane compared with wild-type C57BL/6, but these changes were greater in CFH(-/-).C3(-/-) mice. CFH(-/-).C3(-/-) mice had significantly more Aβ on Bruch's membrane, fewer macrophages, and high levels of retinal inflammation than the other groups. Our data show that both uncontrolled C3 activation (CFH(-/-)) and complete absence of C3 (CFH(-/-).C3(-/-) and C3(-/-)) negatively affect aged retinas. These findings suggest that strategies that inhibit C3 in AMD may be deleterious. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

A Novel Role for the Receptor of the Complement Cleavage Fragment C5a, C5aR1, in CCR5-Mediated Entry of HIV into Macrophages.

PubMed

Moreno-Fernandez, Maria E; Aliberti, Julio; Groeneweg, Sander; Köhl, Jörg; Chougnet, Claire A

2016-04-01

The complement system is an ancient pattern recognition system that becomes activated during all stages of HIV infection. Previous studies have shown that C5a can enhance the infection of monocyte-derived macrophages and T cells indirectly through the production of interleukin (IL)-6 and tumor necrosis factor (TNF)-α and the attraction of dendritic cells. C5a exerts its multiple biologic functions mainly through activation of C5a receptor 1 (C5aR1). Here, we assessed the role of C5aR1 as an enhancer of CCR5-mediated HIV infection. We determined CCR5 and C5aR1 heterodimer formation in myeloid cells and the impact of C5aR1 blockade on HIV entry and genomic integration. C5aR1/CCR5 heterodimer formation was identified by immunoprecipitation and western blotting. THP-1 cells and monocyte-derived macrophages (MDM) were infected by R5 laboratory strains or HIV pseudotyped for the vesicular stomatitis virus (VSV) envelope. Levels of integrated HIV were measured by quantitative PCR after targeting of C5aR1 by a C5aR antagonist, neutralizing C5aR1 monoclonal antibody (mAb) or hC5a. C5aR1 was also silenced by specific siRNA prior to viral entry. We found that C5aR1 forms heterodimers with the HIV coreceptor CCR5 in myeloid cells. Targeting C5aR1 significantly decreased integration by R5 viruses but not by VSV-pseudotyped viruses, suggesting that C5aR1 is critical for viral entry. The level of inhibition achieved with C5aR1-blocking reagents was comparable to that of CCR5 antagonists. Mechanistically, C5aR1 targeting decreased CCR5 expression. MDM from CCR5Δ32 homozygous subjects expressed levels of C5aR1 similar to CCR5 WT individuals, suggesting that mere C5aR1 expression is not sufficient for HIV infection. HIV appeared to preferentially enter THP-1 cells expressing high levels of both C5aR1 and CCR5. Targeted reduction of C5aR1 expression in such cells reduced HIV infection by ~50%. Our data thus suggest that C5aR1 acts as an enhancer of CCR5-mediated HIV entry into macrophages, the targeting of which may prove useful to reduce HIV infection by R5 strains.

Complement factor H in host defense and immune evasion.

PubMed

Parente, Raffaella; Clark, Simon J; Inforzato, Antonio; Day, Anthony J

2017-05-01

Complement is the major humoral component of the innate immune system. It recognizes pathogen- and damage-associated molecular patterns, and initiates the immune response in coordination with innate and adaptive immunity. When activated, the complement system unleashes powerful cytotoxic and inflammatory mechanisms, and thus its tight control is crucial to prevent damage to host tissues and allow restoration of immune homeostasis. Factor H is the major soluble inhibitor of complement, where its binding to self markers (i.e., particular glycan structures) prevents complement activation and amplification on host surfaces. Not surprisingly, mutations and polymorphisms that affect recognition of self by factor H are associated with diseases of complement dysregulation, such as age-related macular degeneration and atypical haemolytic uremic syndrome. In addition, pathogens (i.e., non-self) and cancer cells (i.e., altered-self) can hijack factor H to evade the immune response. Here we review recent (and not so recent) literature on the structure and function of factor H, including the emerging roles of this protein in the pathophysiology of infectious diseases and cancer.

High Levels of Soluble C5b-9 Complex in Dialysis Fluid May Predict Poor Prognosis in Peritonitis in Peritoneal Dialysis Patients

PubMed Central

Mizuno, Masashi; Suzuki, Yasuhiro; Higashide, Keiko; Sei, Yumi; Iguchi, Daiki; Sakata, Fumiko; Horie, Masanobu; Maruyama, Shoichi; Matsuo, Seiichi; Morgan, B. Paul; Ito, Yasuhiko

2017-01-01

Background We searched for indicators to predict the prognosis of infectious peritonitis by measuring levels of complement proteins and activation products in peritoneal dialysis (PD) fluid (PDF) of patients at early stages of peritonitis. We retrospectively analyzed the relationship between the levels of sC5b-9, C3 and C4 in PDF and the subsequent clinical prognosis. Methods We measured levels of sC5b-9, C3 and C4 in PDF on days 1, 2 and 5 post-onset of peritonitis in 104 episodes of infectious peritonitis in PD patients from 2008 and retrospectively compared levels with clinical outcomes. Further analysis for the presence of causative microorganisms or to demonstrate bacterial culture negative peritonitis was performed and correlated with change of levels of sC5b-9 in PDF. Results When PD patients with peritonitis were divided into groups that either failed to recover from peritonitis and were finally withdrawn from PD (group 1; n = 25) or recovered (group 2; n = 79), levels of sC5b-9, C3 and C4 in PDF were significantly higher in group 1 patients compared to those in group 2 on day5. Analysis of microorganisms showed significantly higher sC5b-9 levels in PDF of peritonitis cases caused by culture negative peritonitis in group 1 compared with group 2 when we analyzed for individual microorganisms. Of note, on day5, the sC5b-9 levels in PDF were similarly high in peritonitis caused by fungi or other organisms. Conclusion Our results suggested that levels of complement markers in PDF, especially sC5b-9, have potential as surrogate markers to predict prognosis of PD-related peritonitis. PMID:28046064

Role of CYP2E1 Immunoglobulin G4 Subclass Antibodies and Complement in Pathogenesis of Idiosyncratic Drug-Induced Hepatitis

PubMed Central

Njoku, Dolores B.; Mellerson, Jenelle L.; Talor, Monica V.; Kerr, Douglas R.; Faraday, Nauder R.; Outschoorn, Ingrid; Rose, Noel R.

2006-01-01

Idiosyncratic drug-induced hepatitis (IDDIH) is the third most common cause for acute liver failure in the United States. Previous studies have attempted to identify susceptible patients or early stages of disease with various degrees of success. To determine if total serum immunoglobulin subclasses, CYP2E1-specific subclass autoantibodies, complement components, or immune complexes could distinguish persons with IDDIH from others exposed to drugs, we studied persons exposed to halogenated volatile anesthetics, which have been associated with IDDIH and CYP2E1 autoantibodies. We found that patients with anesthetic-induced IDDIH had significantly elevated levels of CYP2E1-specific immunoglobulin G4 (IgG4) autoantibodies, while anesthetic-exposed healthy persons had significantly elevated levels of CYP2E1-specific IgG1 autoantibodies. Anesthetic IDDIH patients had significantly lower levels of C4a, C3a, and C5a compared to anesthetic-exposed healthy persons. C1q- and C3d-containing immune complexes were significantly elevated in anesthetic-exposed persons. In conclusion, our data suggest that anesthetic-exposed persons develop CYP2E1-specific IgG1 autoantibodies which may form detectable circulating immune complexes subsequently cleared by classical pathway activation of the complement system. Persons susceptible to anesthetic-induced IDDIH develop CYP2E1-specific IgG4 autoantibodies which form small, nonprecipitating immune complexes that escape clearance because of their size or by direct inhibition of complement activation. PMID:16467335

Effects of a polyelectrolyte additive on the selective dialysis membrane permeability for low-molecular-weight proteins.

PubMed

Krieter, Detlef H; Morgenroth, Andreas; Barasinski, Artur; Lemke, Horst-Dieter; Schuster, Oliver; von Harten, Bodo; Wanner, Christoph

2007-02-01

Improving the sieving characteristics of dialysis membranes enhances the clearance of low-molecular-weight (LMW) proteins and may have an impact on outcome in patients receiving haemodialysis. To approach this goal, a novel polyelectrolyte additive process was applied to a polyethersulphone (PES) membrane. Polyelectrolyte-modified PES was characterized in vitro by measuring complement activation and sieving coefficients of cytochrome c and serum albumin. In a prospective, randomized, cross-over study, instantaneous plasma water clearances and reduction rates of LMW proteins [beta(2)-microglobulin (b2m), cystatin c, myoglobin, retinol binding protein] were determined in eight patients receiving dialysis treatment with PES in comparison with polysulphone (PSU). Biocompatibility was assessed by determination of transient leucopenia, plasma levels of complement C5a, thrombin-antithrombin III (TAT), myeloperoxidase (MPO) and elastase (ELT). PES showed a steeper sieving profile and lower complement activation in vitro compared with PSU. Instantaneous clearance (69 +/- 8 vs. 58 +/- 3 ml/min; P < 0.001) and reduction rate (72.3 +/- 1 5% vs 66.2 +/- 6.1%; P < 0.001) of b2m were significantly higher with PES as compared with PSU. With higher molecular weight of proteins, differences in the solute removal between PES and PSU further increased, whereas albumin loss remained low (PES, 0.53 +/- 0.17 vs PSU, <0.22 g/dialysis). MPO, ELT and TAT did not differ between the two membranes. In contrast, leucopenia was less pronounced and C5a generation was significantly lower during dialysis with PES. Polyelectrolyte modification of PES results in a higher LMW protein removal and in optimized biocompatibility. Whether these findings translate into better outcome of patients receiving haemodialysis requires further studies.

A novel model for studies of blood-mediated long-term responses to cellular transplants

PubMed Central

Lindblom, Susanne; Hong, Jaan; Nilsson, Bo; Korsgren, Olle; Ronquist, Gunnar

2015-01-01

Aims Interaction between blood and bio-surfaces is important in many medical fields. With the aim of studying blood-mediated reactions to cellular transplants, we developed a whole-blood model for incubation of small volumes for up to 48 h. Methods Heparinized polyvinyl chloride tubing was cut in suitable lengths and sealed to create small bags. Multiple bags, with fresh venous blood, were incubated attached to a rotating wheel at 37°C. Physiological variables in blood were monitored: glucose, blood gases, mono- and divalent cations and chloride ions, osmolality, coagulation (platelet consumption, thrombin-antithrombin complexes (TAT)), and complement activation (C3a and SC5b-9), haemolysis, and leukocyte viability. Results Basic glucose consumption was high. Glucose depletion resulted in successive elevation of extracellular potassium, while sodium and calcium ions decreased due to inhibition of energy-requiring ion pumps. Addition of glucose improved ion balance but led to metabolic acidosis. To maintain a balanced physiological environment beyond 6 h, glucose and sodium hydrogen carbonate were added regularly based on analyses of glucose, pH, ions, and osmotic pressure. With these additives haemolysis was prevented for up to 72 h and leukocyte viability better preserved. Despite using non-heparinized blood, coagulation and complement activation were lower during long-term incubations compared with addition of thromboplastin and collagen. Conclusion A novel whole-blood model for studies of blood-mediated responses to a cellular transplant is presented allowing extended observations for up to 48 h and highlights the importance of stringent evaluations and adjustment of physiological conditions. PMID:25322825

Solution structure of a small protein containing a fluorinated side chain in the core

PubMed Central

Cornilescu, Gabriel; Hadley, Erik B.; Woll, Matthew G.; Markley, John L.; Gellman, Samuel H.; Cornilescu, Claudia C.

2007-01-01

We report the first high-resolution structure for a protein containing a fluorinated side chain. Recently we carried out a systematic evaluation of phenylalanine to pentafluorophenylalanine (Phe → F5-Phe) mutants for the 35-residue chicken villin headpiece subdomain (c-VHP), the hydrophobic core of which features a cluster of three Phe side chains (residues 6, 10, and 17). Phe → F5-Phe mutations are interesting because aryl–perfluoroaryl interactions of optimal geometry are intrinsically more favorable than either aryl–aryl or perfluoroaryl–perfluoroaryl interactions, and because perfluoroaryl units are more hydrophobic than are analogous aryl units. Only one mutation, Phe10 → F5-Phe, was found to provide enhanced tertiary structural stability relative to the native core (by ∼1 kcal/mol, according to guanidinium chloride denaturation studies). The NMR structure of this mutant, described here, reveals very little variation in backbone conformation or side chain packing relative to the wild type. Thus, although Phe → F5-Phe mutations offer the possibility of greater tertiary structural stability from side chain–side chain attraction and/or side chain desolvation, the constraints associated with the native c-VHP fold apparently prevent the modified polypeptide from taking advantage of this possibility. Our findings are important because they complement several studies that have shown that fluorination of saturated side chain carbon atoms can provide enhanced conformational stability. PMID:17123960

Fanconi Anemia Complementation Group A (FANCA) Protein Has Intrinsic Affinity for Nucleic Acids with Preference for Single-stranded Forms*

PubMed Central

Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y.; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

2012-01-01

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5′-flap or 5′-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772–1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found. PMID:22194614

Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms.

PubMed

Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

2012-02-10

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.

Anticomplementary activity of horse IgG and F(ab')2 antivenoms.

PubMed

Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V

2014-03-01

Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs.

Anti-GM2 gangliosides IgM paraprotein induces neuromuscular block without neuromuscular damage.

PubMed

Santafé, Manel M; Sabaté, M Mar; Garcia, Neus; Ortiz, Nico; Lanuza, M Angel; Tomàs, Josep

2008-11-15

We analyzed the effect on the mouse neuromuscular synapses of a human monoclonal IgM, which binds specifically to gangliosides with the common epitope [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-]. We focused on the role of the complement. Evoked neurotransmission was partially blocked by IgM both acutely (1 h) and chronically (10 days). Transmission electron microscopy shows important nerve terminal growth and retraction remodelling though axonal injury can be ruled out. Synapses did not show mouse C5b-9 immunofluorescence and were only immunolabelled when human complement was added. Therefore, the IgM-induced synaptic changes occur without complement-mediated membrane attack.

Mycophenolic acid exposure and complement fraction C3 influence inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus.

PubMed

Mino, Yasuaki; Naito, Takafumi; Shimoyama, Kumiko; Ogawa, Noriyoshi; Kawakami, Junichi

2017-07-01

Background Mycophenolate mofetil has recently been reported to be effective against systemic lupus erythematosus. The influence of the pharmacokinetics of mycophenolic acid, the active form of mycophenolate mofetil and the major inactive mycophenolic acid phenolic glucuronide on the activity of the target enzyme inosine 5'-monophosphate dehydrogenase, is expected to be revealed. The aim of this study was to identify the factors associated with inosine 5'-monophosphate dehydrogenase activity in systemic lupus erythematosus patients. Methods Fifty systemic lupus erythematosus patients in remission maintenance phase (29 received mycophenolate mofetil [MMF+] and 21 did not [MMF-]) were enrolled. Median and interquartile range of dose of mycophenolate mofetil were 1500 and 1000-1500 mg/day, respectively. Stepwise multiple linear regression analysis was performed to assess the dependence between inosine 5'-monophosphate dehydrogenase activity and 25 predictor values including predose plasma concentrations of free mycophenolic acid and mycophenolic acid phenolic glucuronide. Results Median and interquartile range of predose total plasma concentrations of mycophenolic acid and mycophenolic acid phenolic glucuronide were 2.73 and 1.43-5.73 and 25.5 and 13.1-54.7  µg/mL, respectively. Predose inosine 5'-monophosphate dehydrogenase activity was significantly higher in MMF+ than MMF- patients (median 38.3 and 20.6 nmoL xanthosine 5'-monophosphate/g haemoglobin/h, P<0.01). The plasma concentration of free mycophenolic acid phenolic glucuronide, complement fraction C3 and body weight were significant predictors accounting for interindividual variability in the inosine 5'-monophosphate dehydrogenase activity (adjusted R 2  = 0.52, P < 0.01) in a multivariate analysis. Conclusions Predose inosine 5'-monophosphate dehydrogenase activity was higher in systemic lupus erythematosus patients receiving mycophenolate mofetil therapy. Inosine 5'-monophosphate dehydrogenase activity may be determined by mycophenolic acid exposure and complement fraction C3 in systemic lupus erythematosus patients.

Anti-GK1 antibodies damage Taenia crassiceps cysticerci through complement activation.

PubMed

Núñez, Guadalupe; Villalobos, Nelly; Herrera, Cinthia P; Navarrete-Perea, José; Méndez, Adriana; Martinez-Maya, José J; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Aguilar, Laura; Del Arenal, Irene P

2018-06-06

Taeniasis-cysticercosis, a zoonosis caused by Taenia solium, is prevalent in underdeveloped countries, where marginalization promotes its continued transmission. Pig cysticercosis, an essential stage for transmission, is preventable by vaccination. An efficient multiepitope vaccine against pig cysticercosis, S3Pvac, was developed. Previous studies showed that antibodies against one of the S3Pvac components, GK-1, are capable of damaging T. solium cysticerci, inhibiting their ability to transform into the adult stage in golden hamster gut. This study is aimed to evaluate one of the mechanisms that could mediate anti-GK-1 antibody-dependent protection. To this end, pig anti-GK-1 antibodies were produced and purified by using protein A. Proteomic analysis showed that the induced antibodies recognized the respective native cysticercal protein KE7 (Bobes et al. Infect Immun 85:e00395-17, 2017) and two additional T. solium proteins (endophilin B1 and Gp50). A new procedure to evaluate cysticercus viability, based on quantifying the cytochrome c released after parasite damage, was developed. Taenia crassiceps cysticerci were cultured in the presence of differing amounts of anti-GK-1 antibody and complement in a saturating concentration, along with the respective controls. Cysticercus viability was assessed by recording parasite motility, trypan blue exclusion, and cytochrome c levels in cysticercal soluble extract. Anti-GK-1 antibody significantly increased cysticercus damage as measured by all three methods. Parasite evaluation by electron microscopy after treatment with anti-GK-1 antibody plus complement demonstrated cysticercus damage as shorter, capsule-severed microtrichia; a decrease in glycocalyx length with respect to untreated cysts; and disaggregated desmosomes. These results demonstrate that anti-GK-1 antibodies damage cysticerci through classic complement activation.

Antibody persistence for up to 5 years after a fourth dose of Haemophilus influenzae type b and Neisseria meningitidis serogroups C and Y-tetanus toxoid conjugate vaccine (HibMenCY-TT) given at 12-15 months of age.

PubMed

Marshall, Gary S; Blatter, Mark; Marchant, Colin; Aris, Emmanuel; Mesaros, Narcisa; Miller, Jacqueline M

2013-06-01

A 4-dose series of recently licensed Haemophilus influenzae type b-meningococcal serogroups C and Y-tetanus toxoid conjugate vaccine (HibMenCY-TT) was immunogenic with a clinically acceptable safety profile in infants, with antibodies persisting in most participants for 1 year following dose 4. This study assessed antibody persistence up to 5 years after vaccination. Participants had received HibMenCY-TT or Hib-TT at 2, 4 and 6 months of age. At age 12-15 months, HibMenCY-TT vaccinees received a fourth HibMenCY-TT dose (HibMenCY x 4 group), whereas those who received Hib-TT received a fourth dose of either Hib-TT (Hib) or HibMenCY-TT (HibMenCY x 1). Blood samples were collected 1 month and 1, 3 and 5 years after the last dose for measurement of antipolyribosylribitol phosphate (the Hib capsular polysaccharide) antibodies and serum bactericidal activity (human complement source) against meningococcal serogroups C and Y. Five years after the fourth dose, the percentages of children with antipolyribosylribitol phosphate ≥0.15 μg/mL in HibMenCY x 4, HibMenCY x 1 and Hib groups were 98.8% (95% confidence interval: 93.5%-100%), 97.3% (85.8%-99.9%) and 92.3% (79.1%-98.4%), respectively. The percentages with human complement serum bactericidal activity ≥1:8 for meningococcal serogroup C were 82.9% (72.5%-90.6%), 73.5% (55.6%-87.1%) and 21.1% (9.6%-37.3%), respectively. The percentages with human complement serum bactericidal activity ≥1:8 for serogroup Y were 69.5% (58.4%-79.2%), 54.3% (36.6%-71.2%) and 18.4% (7.7%-34.3%), respectively. HibMenCY-TT given as a 4-dose series or as a single dose at 12-15 months of age induced immune responses for all 3 antigens that lasted for up to 5 years after vaccination in more than half of recipients.

Activated complement components and complement activator molecules on the surface of cell‐derived microparticles in patients with rheumatoid arthritis and healthy individuals

PubMed Central

Biró, Éva; Nieuwland, Rienk; Tak, Paul P; Pronk, Loes M; Schaap, Marianne C L; Sturk, Augueste; Hack, C Erik

2007-01-01

Objectives In vitro, microparticles can activate complement via the classical pathway. If demonstrable ex vivo, this mechanism may contribute to the pathogenesis of rheumatoid arthritis (RA). We therefore investigated the presence of activated complement components and complement activator molecules on the surface of cell‐derived microparticles of RA patients and healthy individuals. Methods Microparticles from synovial fluid (n = 8) and plasma (n = 9) of 10 RA patients and plasma of sex‐ and age‐matched healthy individuals (n = 10) were analysed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C‐reactive protein (CRP), serum amyloid P component (SAP), immunoglobulin (Ig) M, IgG). Results Microparticles with bound C1q, C4, and/or C3 were abundant in RA synovial fluid, while in RA and control plasma much lower levels were present. Microparticles with bound C1q correlated with those with bound C3 in synovial fluid (r = 0.961, p = 0.0001), and with those with bound C4 in plasma (RA: r = 0.908, p = 0.0007; control: r = 0.632, p = 0.0498), indicating classical pathway activation. In synovial fluid, microparticles with IgM and IgG correlated with those with C1q (r = 0.728, p = 0.0408; r = 0.952, p = 0.0003, respectively), and in plasma, microparticles with CRP correlated with those with C1q (RA: r = 0.903, p = 0.0021; control: r = 0.683, p = 0.0296), implicating IgG and IgM in the classical pathway activation in RA synovial fluid, and CRP in the low level classical pathway activation in plasma. Conclusions This study demonstrates the presence of bound complement components and activator molecules on microparticles ex vivo, and supports their role in low grade complement activation in plasma and increased complement activation in RA synovial fluid. PMID:17261534

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. Copyright © 2013 Elsevier Ltd. All rights reserved.

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

PubMed

Barata, Lidia; Miwa, Takashi; Sato, Sayaka; Kim, David; Mohammed, Imran; Song, Wen-Chao

2013-03-15

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Cpa, the Outer Membrane Protease of Cronobacter sakazakii, Activates Plasminogen and Mediates Resistance to Serum Bactericidal Activity▿

PubMed Central

Franco, A. A.; Kothary, M. H.; Gopinath, G.; Jarvis, K. G.; Grim, C. J.; Hu, L.; Datta, A. R.; McCardell, B. A.; Tall, B. D.

2011-01-01

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ∼131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii. PMID:21245266

Effect of Immobilized Antithrombin III on the Thromboresistance of Polycarbonate Urethane.

PubMed

Lukas, Karin; Stadtherr, Karin; Gessner, Andre; Wehner, Daniel; Schmid, Thomas; Wendel, Hans Peter; Schmid, Christof; Lehle, Karla

2017-03-24

The surface of foils and vascular grafts made from a thermoplastic polycarbonate urethanes (PCU) (Chronoflex AR) were chemically modified using gas plasma treatment, binding of hydrogels-(1) polyethylene glycol bisdiamine and carboxymethyl dextran (PEG-DEX) and (2) polyethyleneimine (PEI)-and immobilization of human antithrombin III (AT). Their biological impact was tested in vitro under static and dynamic conditions. Static test methods showed a significantly reduced adhesion of endothelial cells, platelets, and bacteria, compared to untreated PCU. Modified PCU grafts were circulated in a Chandler-Loop model for 90 min at 37 °C with human blood. Before and after circulation, parameters of the hemostatic system (coagulation, platelets, complement, and leukocyte activation) were analyzed. PEI-AT significantly inhibited the activation of both coagulation and platelets and prevented the activation of leukocytes and complement. In conclusion, both modifications significantly reduce coagulation activation, but only PEI-AT creates anti-bacterial and anti-thrombogenic functionality.

A randomized, first-in-human, healthy volunteer trial of BIVV009, a humanized antibody for the specific inhibition of the classical complement pathway.

PubMed

Bartko, Johann; Schoergenhofer, Christian; Schwameis, Michael; Firbas, Christa; Beliveau, Martin; Chang, Colin; Marier, Jean-Francois; Nix, Darrell; Gilbert, James C; Panicker, Sandip; Jilma, Bernd

2018-05-08

Aberrant activation of the classical complement pathway is the common underlying pathophysiology of orphan diseases such as bullous pemphigoid, antibody-mediated rejection of organ transplants, cold agglutinin disease and warm autoimmune haemolytic anaemia. Therapeutic options for these complement-mediated disorders are limited and BIVV009, a humanized monoclonal antibody directed against complement factor C1s, may be potentially useful for inhibition of the classical complement pathway. A phase-1, first-in-human, double-blind, randomized, placebo-controlled, dose-escalation trial of single and multiple doses of BIVV009 or placebo was conducted in 64 volunteers to evaluate safety, tolerability, pharmacokinetic, and pharmacodynamic profiles. Single and multiple infusions of BIVV009 were well tolerated without any safety concerns. BIVV009 exhibited a steep concentration-effect relationship with a Hill coefficient of 2.4, and an IC90 of 15.5 µg/mL. This study establishes the foundation for using BIVV009 as a highly selective inhibitor of the classical complement pathway in different diseases. This article is protected by copyright. All rights reserved. © 2018 American Society for Clinical Pharmacology and Therapeutics.

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 accelerate CVD, serve as a CVD biomarker, and represent a target for new therapies. PMID:28362874

Systemic hypoxia enhances exercise-mediated bactericidal and subsequent apoptotic responses in human neutrophils.

PubMed

Wang, Jong-Shyan; Chiu, Ya-Ting

2009-10-01

Phagocytosis and oxidative burst are critical host defense mechanisms in which neutrophils clear invading pathogens. Clearing phagocytic neutrophils by triggering apoptosis is an essential process for controlling inflammation. This study elucidates how various exercise bouts with/without hypoxia affected neutrophil bactericidal activity and subsequent apoptosis in humans. Fifteen sedentary males performed six distinct experimental tests in an air-conditioned normobaric hypoxia chamber: two normoxic exercises [strenuous exercise (SE; up to maximal O2 consumption) and moderate exercise (ME; 50% maximal O2 consumption for 30 min) while exposed to 21% O2], two hypoxic exercises (ME for 30 min while exposed to 12% and 15% O2), and two hypoxic exposures (resting for 30 min while exposed to 12% and 15% O2). The results showed that 1) plasma complement-C3a desArg/C4a desArg/C5a concentrations were increased, 2) expressions of L-selectin/lymphocyte functin-associated antigen-1/Mac-1/C5aR on neutrophils were enhanced, 3) phagocytosis of neutrophils to Esherichia coli and release of neutrophil oxidant products by E. coli were elevated, and 4) E. coli-induced phosphotidylserine exposure or caspase-3 activation of neutrophils were promoted immediately and 2 h after both 12% O2 exposure at rest and with ME as well as normoxic SE. Although neither normoxic ME nor breathing 15% O2 at rest influenced these complement- and neutrophil-related immune responses, ME at both 12% and 15% O2 resulted in enhanced complement activation in the blood, expressions of opsonic/complement receptors on neutrophils, or the bactericidal activity and apoptosis of neutrophils. Moreover, the increased neutrophil oxidant production and apoptosis by normoxic SE and hypoxic ME were ameliorated by treating neutrophils with diphenylene iodonium (a NADPH oxidase inhibitor). Therefore, we conclude that ME at 12-15% O2 enhances bactericidal capacity and facilitates the subsequent apoptosis of neutrophils.

Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP▿

PubMed Central

Barbosa, Angela S.; Monaris, Denize; Silva, Ludmila B.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Cianciarullo, Aurora M.; Isaac, Lourdes; Abreu, Patricia A. E.

2010-01-01

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis. PMID:20404075

Erythrocyte-bound C4d in combination with complement and autoantibody status for the monitoring of SLE.

PubMed

Merrill, Joan T; Petri, Michelle A; Buyon, Jill; Ramsey-Goldman, Rosalind; Kalunian, Kenneth; Putterman, Chaim; Conklin, John; Furie, Richard A; Dervieux, Thierry

2018-01-01

We examined the usefulness of erythrocyte-bound C4d (EC4d) to monitor disease activity in SLE. Data and blood samples were collected from three different studies, each of which included longitudinal evaluations using the Physicians Global Assessment (PGA) of disease activity and the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) SLE Disease Activity Index (SLEDAI), which was assessed without anti-double-stranded DNA (dsDNA) and low complement C3/C4 (clinical SELENA-SLEDAI). EC4d levels were determined using flow cytometry; other laboratory measures included antibodies to dsDNA, C3 and C4 proteins. Relationships between clinical SELENA-SLEDAI, PGA and the laboratory measures were analysed using linear mixed effect models. The three studies combined enrolled 124 patients with SLE (mean age 42 years, 97% women, 31% Caucasians and 34% African-Americans) followed for an average of 5 consecutive visits (range 2-13 visits). EC4d levels and low C3/C4 status were significantly associated the clinical SELENA-SLEDAI or PGA in each of the three study groups (p<0.05). Multivariate analysis revealed that EC4d levels (estimate=0.94±0.28) and low complement C3/C4 (estimate=1.24±0.43) were both independently and significantly associated with the clinical SELENA-SLEDAI (p<0.01) and PGA. EC4d levels were also associated with the clinical SELENA-SLEDAI (estimate: 1.20±0.29) and PGA (estimate=0.19±0.04) among patients with chronically low or normal C3/C4 (p<0.01). Anti-dsDNA titres were generally associated with disease activity. These data support the association of EC4d with disease activity regardless of complement C3/C4 status and its usefulness in monitoring SLE disease. Additional studies will be required to support these validation data.

In vitro C3 mRNA expression in Pemphigus vulgaris: complement activation is increased by IL-1alpha and TNF-alpha.

PubMed

Feliciani, C; Toto, P; Amerio, P

1999-01-01

Pemphigus vulgaris (PV) is a potentially life-threatening disease, characterized immunohistologically by IgG deposits and complement activation on the surface of keratinocytes. Complement activation has been implicated in the pathogenesis with C3 deposits in about 90% of patients. In order to further elucidate the role of complement in PV and to define which cytokines play a role in C3 mRNA expression, we performed an in vitro study in human keratinocytes. Normal human epidermal keratinocytes (NHuK) were incubated with PV serum and C3 mRNA was measured. We previously had shown that IL-1alpha and TNF-alpha are expressed in PV in vivo and in vitro. Since cytokines are able to modulate complement activation, mRNA expression was evaluated in a similar experiment after pretreatment using antibodies against IL-1alpha and TNF-alpha. Incubation of NHuK with PV sera caused their detachment from the plates after 20-30 minutes with a complete acantholysis within 12 hours. An early C3 mRNA expression was seen after 30 minutes with a peak level after 1 hour. Blocking studies, using antibodies against human IL-1alpha and TNF-alpha in NHuK together with PV-IgG, showed reduction of in vitro induced acantholysis and inhibition of C3 mRNA expression. This study supports the hypothesis that complement C3 is important in PV acantholysis and that complement activation is increased by IL-1alpha and TNF-alpha.

Genetic Variation in Complement Component 2 of the Classical Complement Pathway is Associated with Increased Mortality and Infection: A Study of 627 Trauma Patients

PubMed Central

Morris, John A.; Francois, Cedric; Olson, Paul K.; Cotton, Bryan A.; Summar, Marshall; Jenkins, Judith M.; Norris, Patrick R.; Moore, Jason H.; Williams, Anna E.; McNew, Brent S.; Canter, Jeffrey A.

2009-01-01

Trauma is a disease of inflammation. Complement Component 2 (C2) is a protease involved in activation of complement through the classical pathway and has been implicated in a variety of chronic inflammatory diseases. We hypothesized that genetic variation in C2 (E318D) identifies a high-risk subgroup of trauma patients reflecting increased mortality and infection (Ventilator associated pneumonia: VAP). Consequently, genetic variation in C2 may stratify patient risk and illuminate underlying mechanisms for therapeutic intervention. Methods DNA samples from 702 trauma patients were genotyped for C2 E318D and linked with covariates (age: mean 42.8 years, gender: 74% male, ethnicity: 80% Caucasian, mechanism: 84% blunt, ISS: mean 25.0, admission lactate: mean 3.13 mEq/L) and outcomes: mortality 9.9% and VAP: 18.5%. VAP was defined by quantitative bronchoalveolar lavage (>104). Multivariate regression determined the relationship of genotype and covariates to risk of death and VAP. However, patients with ISS ≥ 45 were excluded from the multivariate analysis, as magnitude of injury overwhelms genetics and covariates in determining outcome. Results 52 patients (8.3%) had the high-risk heterozygous genotype, associated with a significant increase in mortality and VAP. Conclusion In 702 trauma patients, 8.3% had a high-risk genetic variation in C2 associated with increased mortality (OR=2.65) and infection (OR=2.00). This variation: 1) Identifies a previously unknown high risk group for infection and mortality; 2) Can be determined on admission; 3) May provide opportunity for early therapeutic intervention; and 4) Requires validation in a distinct cohort of patients. PMID:19430225

Protective immune responses against West Nile virus are primed by distinct complement activation pathways.

PubMed

Mehlhop, Erin; Diamond, Michael S

2006-05-15

West Nile virus (WNV) causes a severe infection of the central nervous system in several vertebrate animals including humans. Prior studies have shown that complement plays a critical role in controlling WNV infection in complement (C) 3(-/-) and complement receptor 1/2(-/-) mice. Here, we dissect the contributions of the individual complement activation pathways to the protection from WNV disease. Genetic deficiencies in C1q, C4, factor B, or factor D all resulted in increased mortality in mice, suggesting that all activation pathways function together to limit WNV spread. In the absence of alternative pathway complement activation, WNV disseminated into the central nervous system at earlier times and was associated with reduced CD8+ T cell responses yet near normal anti-WNV antibody profiles. Animals lacking the classical and lectin pathways had deficits in both B and T cell responses to WNV. Finally, and somewhat surprisingly, C1q was required for productive infection in the spleen but not for development of adaptive immune responses after WNV infection. Our results suggest that individual pathways of complement activation control WNV infection by priming adaptive immune responses through distinct mechanisms.

Complement component 3 (C3)

MedlinePlus

... of a certain protein. This protein is part of the complement system. The complement system is a group of proteins ... system and play a role in the development of inflammation. The complement system protects the body from infections, dead cells and ...

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

PubMed Central

Ferreira, Viviana P.; Fazito Vale, Vladimir; Pangburn, Michael K.; Abdeladhim, Maha; Ferreira Mendes-Sousa, Antonio; Coutinho-Abreu, Iliano V.; Rasouli, Manoochehr; Brandt, Elizabeth A.; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Horácio Pereira, Marcos; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M. C.; Gontijo, Nelder F.; Collin, Nicolas; Valenzuela, Jesus G.

2016-01-01

Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

Marketplace Clinics Complementing Diabetes Care for Urban Residing American Indians

PubMed Central

Rick, Robert; Hoye, Robert E.; Thron, Raymond W.; Kumar, Vibha

2017-01-01

Introduction: For several decades, the Minneapolis American Indian population has experienced limited health care access and threefold diabetes health disparity. As part of an urban health initiative, the marketplace clinics located in nearby CVS, Target, and Supervalu stores committed financial support, providers, certified educators, and pharmacy staff for a community-based diabetes support group. Objectives: To measure the extent to which collaborating marketplace clinics and the community-based support group expanded diabetes care and provided self-management education for this largely urban Indian neighborhood. Methods: A controlled quasi-experimental study and 3-years retrospective analysis of secondary data were used to test whether the Minneapolis marketplace clinics and the community diabetes support group participants (n = 48) had improved diabetes health outcomes relative to the comparison group (n = 87). The marketplace complemented intervention group employed motivational interviewing and the patient activation measure (PAM®) in coaching diabetes self-care and behavioral modification. The federally funded comparison group received only basic self-management education. Results: T tests and effect sizes were used to quantify the difference between the study intervention and comparison groups. Statistical significance was determined for the following outcome variables: A1C (P < .01), body mass index (P < .04), and PAM® (P < .001). Discussion: Includes strengths, limitations, and future study recommendations. Conclusions: Positive effects of marketplace clinics and community health complementation were found with regard to improved blood glucose control, weight loss, and healthful lifestyle adaptation. Primary care and community health improvements could be realized by incorporating patient activation with diabetes prevention programs for the urban Indian two-thirds majority of the United States 5 million American Indian population. PMID:28707507

Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

PubMed

Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

2006-05-01

The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

Genotype-phenotype correlations of low frequency variants in the complement system in renal disease and age-related macular degeneration.

PubMed

Geerlings, M J; Volokhina, E B; de Jong, E K; van de Kar, N; Pauper, M; Hoyng, C B; van den Heuvel, L P; den Hollander, A I

2018-06-11

Genetic alterations in the complement system have been linked to a variety of diseases, including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy (C3G), and age-related macular degeneration (AMD). We performed sequence analysis of the complement genes CFH, CFI, and C3 in 866 aHUS/C3G and 697 AMD patients. In total we identified 505 low frequency alleles, representing 121 unique variants, of which 51 are novel. CFH contained the largest number of unique low frequency variants (n=64; 53%), followed by C3 (n=32; 26%) and CFI (n=25; 21%). A substantial number of variants were found in both patients groups (n=48; 40%), while 41 (34%) variants were found only in aHUS/C3G and 32 (26%) variants were AMD-specific. Genotype-phenotype correlations between the disease groups identified a higher frequency of protein-altering alleles in SCR20 of Factor H (FH), and in the serine protease domain of Factor I (FI) in aHUS/C3G patients. In AMD a higher frequency of protein-altering alleles was observed in SCR3, SCR5 and SCR7 of FH, the SRCR domain of FI, and in the MG3 domain of C3. In conclusion, we observed a substantial overlap of variants between aHUS/C3G and AMD, however, there is a distinct clustering of variants within specific domains. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Induction of protective and therapeutic antitumor immunity by a DNA vaccine with C3d as a molecular adjuvant.

PubMed

Xu, Gui-lian; Zhang, Ke-qin; Guo, Bo; Zhao, Ting-ting; Yang, Fei; Jiang, Man; Wang, Qing-hong; Shang, Yu-hang; Wu, Yu-zhang

2010-10-18

Although the critical role of complement component C3d as a molecular adjuvant in preventing virus infection is well established, its role in cancer therapies is unclear. In this study, we have engineered a DNA vaccine that expresses extracellular region of murine VEGFR-2 (FLK1(265-2493)) and 3 copies of C3d (C3d3), a component of complement as a molecular adjuvant, designed to increase antitumor immunity. VEGFR-2 has a more restricted expression on endothelial cells and is upregulated once these cells proliferate during angiogenesis in the tumor vasculature. Immunization of mice with vector encoding FLK1(265-2493) alone generated only background levels of anti-VEGFR-2 antibodies and slight inhibitory effect on tumor growth. However, the addition of C3d3 to the vaccine construct significantly augmented the anti-VEGFR-2 humoral immune response and inhibited the tumor growth. The antitumor activity induced by vaccination with vector encoding FLK1(265-2493)-C3d3 fusion protein was also demonstrated via growth inhibition of established tumors following passive transfer of immune serum from vaccinated mice. Our results suggest that vaccination with vector encoding FLK1(265-2493) with C3d3 as a molecular adjuvant induces adaptive humoral activity, which is directed against the murine VEGFR-2 and can significantly inhibit tumor growth, and that administration of C3d as a molecular adjuvant to increase antibodies levels to VEGFR-2 may provide an alternative treatment modality for cancer therapies. Copyright © 2010 Elsevier Ltd. All rights reserved.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

PubMed

Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin ( Ts -CRT), a Ca 2+ -binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts -CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts -CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts -CRT (r Ts -CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte-macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts -CRT on the surface of newborn larvae (NBL) of T. spiralis with anti- Ts -CRT antibody increased the C1q-mediated adherence of monocyte-macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis -expressed Ts -CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy

PubMed Central

Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin (Ts-CRT), a Ca2+-binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts-CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts-CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts-CRT (rTs-CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte–macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts-CRT on the surface of newborn larvae (NBL) of T. spiralis with anti-Ts-CRT antibody increased the C1q-mediated adherence of monocyte–macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis-expressed Ts-CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages. PMID:28620388

STUDIES ON THE ANTIGENIC PROPERTIES OF COMPLEMENT

PubMed Central

Klein, Paul G.; Burkholder, Peter M.

1960-01-01

Sheep erythrocytes sensitized with amboceptor and persensitized thereafter with guinea pig complement are agglutinated by rabbit anti-guinea pig globulin and by immune sera obtained by injection of rabbits with fixed complement. In this agglutination neither C'1 nor C'2 takes part. Fixed C'4 acts as an agglutinogen. An additional agglutinogen, distinct from C'4, was found on persensitized cells. This additional agglutinogen appears to be distinct from hemolytically active C'3. PMID:14409703

Predictive value of the complement system for sepsis-induced disseminated intravascular coagulation in septic patients in emergency department.

PubMed

Zhao, Xin; Chen, Yun-Xia; Li, Chun-Sheng

2015-04-01

To investigate changes in circulating complement component C3, membrane attack complex (MAC), and mannose-binding lectin (MBL) in patients with sepsis-induced disseminated intravascular coagulation (DIC). Adult septic patients admitted to the emergency department (ED) of Beijing Chao-Yang Hospital were enrolled. A DIC score of 5 or higher was considered sepsis-induced DIC. Circulating C3, MAC, and MBL levels were detected on ED arrival and compared between patients with and without DIC. The predictive value of C3, MAC, and MBL for sepsis-induced DIC at ED arrival and development of DIC after admission were assessed by receiver operating characteristic curve and logistic regression. We enrolled 267 septic patients between February and December 2013. Complement 3, MAC, and MBL were higher in the DIC group (P < .01). Membrane attack complex was the independent predictor of sepsis-induced DIC. The area under the curve of MAC in predicting sepsis-induced DIC was 0.793. During hospitalization, 25 patients without DIC at enrollment developed DIC. Membrane attack complex and Sequential Organ Failure Assessment independently predicted progress to DIC. The area under the curve of MAC was 0.741. Complement 3, MAC, and MBL were significantly increased in septic patients with DIC. Membrane attack complex independently predicted sepsis-induced DIC and development of DIC after ED admission. Copyright © 2014 Elsevier Inc. All rights reserved.

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

Protein A immunoadsorption therapy in HIV-related immune thrombocytopenia: a preliminary report.

PubMed

Bertram, J H; Snyder, H W; Gill, P S; Shulman, I; Henry, D H; Jenkins, D; Kiprov, D D

1988-12-01

Nine homosexual patients with immune thrombocytopenia were treated with autologous plasma that had been perfused over silica-immobilized Staphylococcus aureus protein A (SpA). Pretreatment platelet counts ranged from 10,000 to 98,000 cells/mm3 (mean: 54,000 cells/mm3). Six patients responded to therapy. Platelets increased by a mean of 95,000 cells/mm3 (p less than 0.007) and reached normal levels (greater than 150,000 cells/mm3) in four patients. Increased platelet counts are presently sustained in these four individuals after 5 months of follow-up. Increases in platelet counts significantly correlated with decreases in platelet-associated IgG (PAIgG), platelet-directed IgG (PDIgG), and immune complexes (CIC). PAIgG and PDIgG declined by a mean of 67% (p less than 0.003) and 58% (p less than 0.007), respectively. CIC decreased by a mean of 37% (p = 0.02). Complement was concomitantly activated in all four examined patients. C3a and C5a increased 23-fold and 2.6-fold, respectively, while total hemolytic complement decreased by 50%. Activated complement components and removal of CIC and IgG thus may contribute to the platelet-enhancing activity of SpA immunoadsorption therapy.

Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

PubMed Central

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M.

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases. PMID:22514678

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

PubMed

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Complement Protein C1q Modulates Neurite Outgrowth In Vitro and Spinal Cord Axon Regeneration In Vivo

PubMed Central

Peterson, Sheri L.; Nguyen, Hal X.; Mendez, Oscar A.

2015-01-01

Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI. PMID:25762679

On the intracellular trafficking of mouse S5 ribosomal protein from cytoplasm to nucleoli.

PubMed

Matragkou, Ch; Papachristou, H; Karetsou, Z; Papadopoulos, G; Papamarcaki, T; Vizirianakis, I S; Tsiftsoglou, A S; Choli-Papadopoulou, T

2009-10-09

The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus and then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase II and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase II, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its cellular functions.

Celecoxib restores angiogenic factor expression at the maternal-fetal interface in the BPH/5 mouse model of preeclampsia.

PubMed

Reijnders, Dorien; Liu, Chin-Chi; Xu, Xinjing; Zhao, Anna M; Olson, Kelsey N; Butler, Scott D; Douglas, Nataki C; Sones, Jenny L

2018-05-01

Preeclampsia (PE), a hypertensive disease of pregnancy, is a leading cause of fetal and maternal morbidity/mortality. Early angiogenic and inflammatory disturbances within the placenta are thought to underlie the development of the maternal PE syndrome and poor pregnancy outcomes. However, the exact etiology remains largely unknown. Here, we use the BPH/5 mouse model of PE to elucidate the way in which inflammation early in pregnancy contributes to abnormal expression of angiogenic factors at the maternal-fetal interface. We have previously described improvement in maternal hypertension and fetal growth restriction in this model after treatment with the anti-inflammatory cyclooxygenase-2 (Cox2) specific inhibitor celecoxib. To further characterize the mechanisms by which celecoxib improves poor pregnancy outcomes in BPH/5 mice, we determined expression of angiogenic factors and complement pathway components after celecoxib. In BPH/5 implantation sites there was increased hypoxia inducible factor-1α ( Hif1α), heme oxygenase-1 ( Ho-1), and stem cell factor ( Scf) mRNA concomitant with elevated prostaglandin synthase 2 ( Ptgs2), encoding Cox2, and elevated VEGF protein. Angiopoietin 1 ( Ang1), tunica interna endothelial cell kinase-2 receptor ( Tie2), complement factor 3 ( C3), and complement factor B ( CfB) were increased in midgestation BPH/5 placentae. Whereas BPH/5 expression levels of VEGF, Ang1, and Tie2 normalized after celecoxib, placental C3 and CfB mRNA remained unchanged. However, celecoxib did reduce the pregnancy-specific circulating soluble fms-like tyrosine kinase-1 (sFlt-1) rise in BPH/5 mice at midgestation. These data show that elevated Cox2 during implantation contributes to placental angiogenic factor imbalances in the BPH/5 mouse model of PE.

Structure of genes and an insertion element in the methane producing archaebacterium Methanobrevibacter smithii.

PubMed

Hamilton, P T; Reeve, J N

1985-01-01

DNA fragments cloned from the methanogenic archaebacterium Methanobrevibacter smithii which complement mutations in the purE and proC genes of E. coli have been sequenced. Sequence analyses, transposon mutagenesis and expression in E. coli minicells indicate that purE and proC complementations result from the synthesis of M. smithii polypeptides with molecular weights of 36,697 and 27,836 respectively. The encoding genes appear to be located in operons. The M. smithii genome contains 69% A/T basepairs (bp) which is reflected in unusual codon usages and intergenic regions containing approximately 85% A/T bp. An insertion element, designated ISM1, was found within the cloned M. smithii DNA located adjacent to the proC complementing region. ISM1 is 1381 bp in length, has 29 bp terminal inverted repeat sequences and contains one major ORF encoded in 87% of the ISM1 sequence. ISM1 is mobile, present in approximately 10 copies per genome and integration duplicates 8 bp at the site of insertion. The duplicated sequences show homology with sequences within the 29 bp terminal repeat sequence of ISM1. Comparison of our data with sequences from halophilic archaebacteria suggests that 5'GAANTTTCA and 5'TTTTAATATAAA may be consensus promoter sequences for archaebacteria. These sequences closely resemble the consensus sequences which precede Drosophila heat-shock genes (Pelham 1982; Davidson et al. 1983). Methanogens appear to employ the eubacterial system of mRNA: 16SrRNA hybridization to ensure initiation of translation; the consensus ribosome binding sequence is 5'AGGTGA.

Screening of the two-component-system histidine kinases of Listeria monocytogenes EGD-e. LiaS is needed for growth under heat, acid, alkali, osmotic, ethanol and oxidative stresses.

PubMed

Pöntinen, Anna; Lindström, Miia; Skurnik, Mikael; Korkeala, Hannu

2017-08-01

To study the role of each two-component system (TCS) histidine kinase (HK) in stress tolerance of Listeria monocytogenes EGD-e, we monitored the growth of individual HK deletion mutant strains under heat (42.5 °C), acid (pH 5.6), alkali (pH 9.4), osmotic (6% NaCl), ethanol (3.5 vol%), and oxidative (5 mM H 2 O 2 ) stresses. The growth of ΔliaS (Δlmo1021) strain was impaired under each stress, with the most notable decrease under heat and osmotic stresses. The ΔvirS (Δlmo1741) strain showed nearly completely restricted growth at high temperature and impaired growth in ethanol. The growth of ΔagrC (Δlmo0050) strain was impaired under osmotic stress and slightly under oxidative stress. We successfully complemented the HK mutations using a novel allelic exchange based approach. This approach avoided the copy-number problems associated with in trans complementation from a plasmid. The mutant phenotypes were restored to the wild-type level in the complemented strains. This study reveals novel knowledge on the HKs needed for growth of L. monocytogenes EGD-e under abovementioned stress conditions, with LiaS playing multiple roles in stress tolerance of L. monocytogenes EGD-e. Copyright © 2017 Elsevier Ltd. All rights reserved.

C1q-Mediated Complement Activation and C3 Opsonization Trigger Recognition of Stealth Poly(2-methyl-2-oxazoline)-Coated Silica Nanoparticles by Human Phagocytes.

PubMed

Tavano, Regina; Gabrielli, Luca; Lubian, Elisa; Fedeli, Chiara; Visentin, Silvia; Polverino De Laureto, Patrizia; Arrigoni, Giorgio; Geffner-Smith, Alessandra; Chen, Fangfang; Simberg, Dmitri; Morgese, Giulia; Benetti, Edmondo M; Wu, Linping; Moghimi, Seyed Moein; Mancin, Fabrizio; Papini, Emanuele

2018-05-23

Poly(2-methyl-2-oxazoline) (PMOXA) is an alternative promising polymer to poly(ethylene glycol) (PEG) for design and engineering of macrophage-evading nanoparticles (NPs). Although PMOXA-engineered NPs have shown comparable pharmacokinetics and in vivo performance to PEGylated stealth NPs in the murine model, its interaction with elements of the human innate immune system has not been studied. From a translational angle, we studied the interaction of fully characterized PMOXA-coated vinyltriethoxysilane-derived organically modified silica NPs (PMOXA-coated NPs) of approximately 100 nm in diameter with human complement system, blood leukocytes, and macrophages and compared their performance with PEGylated and uncoated NP counterparts. Through detailed immunological and proteomic profiling, we show that PMOXA-coated NPs extensively trigger complement activation in human sera exclusively through the classical pathway. Complement activation is initiated by the sensing molecule C1q, where C1q binds with high affinity ( K d = 11 ± 1 nM) to NP surfaces independent of immunoglobulin binding. C1q-mediated complement activation accelerates PMOXA opsonization with the third complement protein (C3) through the amplification loop of the alternative pathway. This promoted NP recognition by human blood leukocytes and monocyte-derived macrophages. The macrophage capture of PMOXA-coated NPs correlates with sera donor variability in complement activation and opsonization but not with other major corona proteins, including clusterin and a wide range of apolipoproteins. In contrast to these observations, PMOXA-coated NPs poorly activated the murine complement system and were marginally recognized by mouse macrophages. These studies provide important insights into compatibility of engineered NPs with elements of the human innate immune system for translational steps.

Pretranslational regulation of the synthesis of the third component of complement in human mononuclear phagocytes by the lipid A portion of lipopolysaccharide.

PubMed Central

Strunk, R C; Whitehead, A S; Cole, F S

1985-01-01

The third component of complement (C3) is a plasma glycoprotein with a variety of biologic functions in the initiation and maintenance of host response to infectious agents. While the hepatocyte is the primary source of plasma C3, mononuclear phagocytes contribute to the regulation of tissue availability of C3. Lipopolysaccharide (LPS), a constituent of cell walls of gram-negative bacteria, consists of a polysaccharide moiety (core polysaccharide and O antigen) covalently linked to a lipid portion (lipid A). Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis, we examined the effects of LPS on synthesis of C3 by human mononuclear phagocytes as well as synthesis of the second component of complement (C2), factor B, lysozyme, and total protein. LPS increased C3 synthesis 5-30-fold without affecting the kinetics of secretion of C3 or the synthesis of C2, lysozyme, or total protein. Factor B synthesis was consistently increased by LPS. Experiments with lipid A-inactivated LPS (alkaline treated), LPS from a polysaccharide mutant strain, and lipid X (a lipid A precursor) indicated that the lipid A portion is the structural element required for this effect. Northern blot analysis demonstrated at least a fivefold increase in C3 mRNA in LPS-treated monolayers, which suggests that the regulation of the increase in C3 synthesis is pretranslational. C2 mRNA and factor B mRNA were increased approximately twofold. The availability of specific gene products in human mononuclear phagocytes that respond to LPS should permit understanding of the molecular regulation of more complex functions of these cells elicited by LPS in which multiple gene products are coordinately expressed. Images PMID:3900137

Polyreactive Antibodies Plus Complement Enhance the Phagocytosis of Cells Made Apoptotic by UV-Light or HIV

PubMed Central

Zhou, Zhao-hua; Wild, Teresa; Xiong, Ying; Sylvers, Peter; Zhang, Yahong; Zhang, Luxia; Wahl, Larry; Wahl, Sharon M.; Kozlowski, Steven; Notkins, Abner L.

2013-01-01

Polyreactive antibodies are a major component of the natural antibody repertoire and are capable of binding a variety of structurally unrelated antigens. Many of the properties attributed to natural antibodies, in fact, are turning out to be due to polyreactive antibodies. In humans, each day, billions of cells undergo apoptosis. In the present experiments, we show by ImageStream technology that although polyreactive antibodies do not bind to live T cells they bind to both the plasma membrane and cytoplasm of late apoptotic cells, fix complement, generate the anaphylatoxin C5a and increase by as much as 5 fold complement-mediated phagocytosis by macrophages. Of particular importance, T cells undergoing apoptosis following infection with HIV also bind polyreactive antibodies and are phagocytosed. We conclude that the polyreactive antibodies in the natural antibody repertoire contribute in a major way to the clearance of cells made apoptotic by a variety of natural and infectious processes. PMID:23881356

Coagulation cascade and complement system in systemic lupus erythematosus

PubMed Central

Liang, Yan; Xie, Shang-Bo; Wu, Chang-Hao; Hu, Yuan; Zhang, Qin; Li, Si; Fan, Yin-Guang; Leng, Rui-Xue; Pan, Hai-Feng; Xiong, Hua-Bao; Ye, Dong-Qing

2018-01-01

This study was conducted to (1) characterize coagulation cascade and complement system in systemic lupus erythematosus (SLE); (2) evaluate the associations between coagulation cascade, complement system, inflammatory response and SLE disease severity; (3) test the diagnostic value of a combination of D-dimer and C4 for lupus activity. Transcriptomics, proteomics and metabolomics were performed in 24 SLE patients and 24 healthy controls. The levels of ten coagulations, seven complements and three cytokines were measured in 112 SLE patients. Clinical data were collected from 2025 SLE patients. The analysis of multi-omics data revealed the common links for the components of coagulation cascade and complement system. The results of ELISA showed coagulation cascade and complement system had an interaction effect on SLE disease severity, this effect was pronounced among patients with excess inflammation. The analysis of clinical data revealed a combination of D-dimer and C4 provided good diagnostic performance for lupus activity. This study suggested that coagulation cascade and complement system become ‘partners in crime’, contributing to SLE disease severity and identified the diagnostic value of D-dimer combined with C4for lupus activity. PMID:29599912

Clinical features of patients with homozygous complement C4A or C4B deficiency.

PubMed

Liesmaa, Inka; Paakkanen, Riitta; Järvinen, Asko; Valtonen, Ville; Lokki, Marja-Liisa

2018-01-01

Homozygous deficiencies of complement C4A or C4B are detected in 1-10% of populations. In genome-wide association studies C4 deficiencies are missed because the genetic variation of C4 is complex. There are no studies where the clinical presentation of these patients is analyzed. This study was aimed to characterize the clinical features of patients with homozygous C4A or C4B deficiency. Thirty-two patients with no functional C4A, 87 patients with no C4B and 120 with normal amount of C4 genes were included. C4A and C4B numbers were assessed with genomic quantitative real-time PCR. Medical history was studied retrospectively from patients' files. Novel associations between homozygous C4A deficiency and lymphoma, coeliac disease and sarcoidosis were detected. These conditions were present in 12.5%, (4/32 in patients vs. 0.8%, 1/120, in controls, OR = 17.00, 95%CI = 1.83-158.04, p = 0.007), 12.5% (4/32 in patients vs. 0%, 0/120 in controls, OR = 1.14, 95%CI = 1.00-1.30, p = 0.002) and 12.5%, respectively (4/32 in patients vs. 2.5%, 3/120 in controls, OR = 5.571, 95%CI = 1.79-2.32, p = 0.036). In addition, C4A and C4B deficiencies were both associated with adverse drug reactions leading to drug discontinuation (34.4%, 11/32 in C4A-deficient patients vs. 14.2%, 17/120 in controls, OR = 3.174, 95%CI = 1.30-7.74, p = 0.009 and 28.7%, 25/87 in C4B-deficient patients, OR = 2.44, 95%CI = 1.22-4.88, p = 0.010). This reported cohort of homozygous deficiencies of C4A or C4B suggests that C4 deficiencies may have various unrecorded disease associations. C4 gene should be considered as a candidate gene in studying these selected disease associations.

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

Complement proteins bind to nanoparticle protein corona and undergo dynamic exchange in vivo

NASA Astrophysics Data System (ADS)

Chen, Fangfang; Wang, Guankui; Griffin, James I.; Brenneman, Barbara; Banda, Nirmal K.; Holers, V. Michael; Backos, Donald S.; Wu, Linping; Moghimi, Seyed Moein; Simberg, Dmitri

2017-05-01

When nanoparticles are intravenously injected into the body, complement proteins deposit on the surface of nanoparticles in a process called opsonization. These proteins prime the particle for removal by immune cells and may contribute toward infusion-related adverse effects such as allergic responses. The ways complement proteins assemble on nanoparticles have remained unclear. Here, we show that dextran-coated superparamagnetic iron oxide core-shell nanoworms incubated in human serum and plasma are rapidly opsonized with the third complement component (C3) via the alternative pathway. Serum and plasma proteins bound to the nanoworms are mostly intercalated into the nanoworm shell. We show that C3 covalently binds to these absorbed proteins rather than the dextran shell and the protein-bound C3 undergoes dynamic exchange in vitro. Surface-bound proteins accelerate the assembly of the complement components of the alternative pathway on the nanoworm surface. When nanoworms pre-coated with human plasma were injected into mice, C3 and other adsorbed proteins undergo rapid loss. Our results provide important insight into dynamics of protein adsorption and complement opsonization of nanomedicines.

Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase*

PubMed Central

Bettoni, Serena; Bresin, Elena; Remuzzi, Giuseppe; Noris, Marina; Donadelli, Roberta

2016-01-01

The activated fragment of C3 (C3b) and factor B form the C3 proconvertase (C3bB), which is cleaved by factor D to C3 convertase (C3bBb). Older studies (Conrad, D. H., Carlo, J. R., and Ruddy, S. (1978) J. Exp. Med. 147, 1792–1805; Pangburn, M. K., and Müller-Eberhard, H. J. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2416–2420; Kazatchkine, M. D., Fearon, D. T., and Austen, K. F. (1979) J. Immunol. 122, 75–81) indicated that the complement alternative pathway regulator factor H (FH) competes with factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. Moreover, in the few published studies FH did not favor C3bB dissociation. Whether FH may affect C3bBb formation from C3bB is unknown. We set up user-friendly assays based on combined microplate/Western blotting techniques that specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on C3bB assembly and decay and C3bBb formation and decay. We document that FH does not affect C3bB assembly, indicating that FH does not efficiently compete with factor B for C3b binding. We also found that FH does not dissociate C3bB. FH showed a strong C3bBb decay-accelerating activity, as reported previously, and also exerted an apparent inhibitory effect on C3bBb formation. The latter effect was not fully attributable to a rapid FH-mediated dissociation of C3bBb complexes, because blocking decay with properdin and C3 nephritic factor did not restore C3bBb formation. FH almost completely prevented release of the smaller cleavage subunit of FB (Ba), without modifying the amount of C3bB complexes, suggesting that FH inhibits the conversion of C3bB to C3bBb. Thus, the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration. Further studies are required to confirm these findings in physiological cell-based settings. PMID:26903516

The lectin pathway in renal disease: old concept and new insights.

PubMed

Gaya da Costa, Mariana; Poppelaars, Felix; Berger, Stefan P; Daha, Mohamed R; Seelen, Marc A

2018-04-26

The complement system is composed of a network of at least 40 proteins, which significantly contributes to health and disease. The lectin pathway (LP) is one of three pathways that can activate the complement system. Next to protection of the host against pathogens, the LP has been shown to play a crucial role in multiple renal diseases as well as during renal replacement therapy. Therefore, several complement-targeted drugs are currently being explored in clinical trials. Among these complement inhibitors, specific LP inhibitors are also being tested in renal abnormalities such as in immunoglobulin A nephropathy and lupus nephritis. Using various in vitro models, Yaseen et al. (Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement component 3 (C3) in absence of C4 and/or C2. FASEB J 2017; 31: 2210-2219) showed that Mannan-associated serine protease2 can directly activate C3 thereby bypassing C2 and C4 in the activation of the LP. These new findings broaden our understanding of the mechanisms of complement activation and could potentially impact our strategies to inhibit the LP in renal diseases. In support of these findings, we present data of human renal biopsies, demonstrating the occurrence of the LP bypass mechanism in vivo. In conclusion, this review provides a detailed overview of the LP and clarifies the recently described bypass mechanism and its relevance. Finally, we speculate on the role of the C4 bypass mechanism in other renal diseases.

Complement is activated in progressive multiple sclerosis cortical grey matter lesions.

PubMed

Watkins, Lewis M; Neal, James W; Loveless, Sam; Michailidou, Iliana; Ramaglia, Valeria; Rees, Mark I; Reynolds, Richard; Robertson, Neil P; Morgan, B Paul; Howell, Owain W

2016-06-22

The symptoms of multiple sclerosis (MS) are caused by damage to myelin and nerve cells in the brain and spinal cord. Inflammation is tightly linked with neurodegeneration, and it is the accumulation of neurodegeneration that underlies increasing neurological disability in progressive MS. Determining pathological mechanisms at play in MS grey matter is therefore a key to our understanding of disease progression. We analysed complement expression and activation by immunocytochemistry and in situ hybridisation in frozen or formalin-fixed paraffin-embedded post-mortem tissue blocks from 22 progressive MS cases and made comparisons to inflammatory central nervous system disease and non-neurological disease controls. Expression of the transcript for C1qA was noted in neurons and the activation fragment and opsonin C3b-labelled neurons and glia in the MS cortical and deep grey matter. The density of immunostained cells positive for the classical complement pathway protein C1q and the alternative complement pathway activation fragment Bb was significantly increased in cortical grey matter lesions in comparison to control grey matter. The number of cells immunostained for the membrane attack complex was elevated in cortical lesions, indicating complement activation to completion. The numbers of classical (C1-inhibitor) and alternative (factor H) pathway regulator-positive cells were unchanged between MS and controls, whilst complement anaphylatoxin receptor-bearing microglia in the MS cortex were found closely apposed to cortical neurons. Complement immunopositive neurons displayed an altered nuclear morphology, indicative of cell stress/damage, supporting our finding of significant neurodegeneration in cortical grey matter lesions. Complement is activated in the MS cortical grey matter lesions in areas of elevated numbers of complement receptor-positive microglia and suggests that complement over-activation may contribute to the worsening pathology that underlies the irreversible progression of MS.

Future perspectives in target-specific immunotherapies of myasthenia gravis

PubMed Central

Dalakas, Marinos C.

2015-01-01

Myasthenia gravis (MG) is an autoimmune disease caused by complement-fixing antibodies against acetylcholine receptors (AChR); antigen-specific CD4+ T cells, regulatory T cells (Tregs) and T helper (Th) 17+ cells are essential in antibody production. Target-specific therapeutic interventions should therefore be directed against antibodies, B cells, complement and molecules associated with T cell signaling. Even though the progress in the immunopathogenesis of the disease probably exceeds any other autoimmune disorder, MG is still treated with traditional drugs or procedures that exert a non-antigen specific immunosuppression or immunomodulation. Novel biological agents currently on the market, directed against the following molecular pathways, are relevant and specific therapeutic targets that can be tested in MG: (a) T cell intracellular signaling molecules, such as anti-CD52, anti-interleukin (IL) 2 receptors, anti- costimulatory molecules, and anti-Janus tyrosine kinases (JAK1, JAK3) that block the intracellular cascade associated with T-cell activation; (b) B cells and their trophic factors, directed against key B-cell molecules; (c) complement C3 or C5, intercepting the destructive effect of complement-fixing antibodies; (d) cytokines and cytokine receptors, such as those targeting IL-6 which promotes antibody production and IL-17, or the p40 subunit of IL-12/1L-23 that affect regulatory T cells; and (e) T and B cell transmigration molecules associated with lymphocyte egress from the lymphoid organs. All drugs against these molecular pathways require testing in controlled trials, although some have already been tried in small case series. Construction of recombinant AChR antibodies that block binding of the pathogenic antibodies, thereby eliminating complement and antibody-depended-cell-mediated cytotoxicity, are additional novel molecular tools that require exploration in experimental MG. PMID:26600875

Complement deficiency predisposes for meningitis due to nongroupable meningococci and Neisseria-related bacteria.

PubMed

Fijen, C A; Kuijper, E J; Tjia, H G; Daha, M R; Dankert, J

1994-05-01

Nongroupable meningococci or bacteria related to the genus Neisseria rarely cause meningitis. Complement deficiency has been identified as a major predisposing factor for meningococcal disease. To assess whether patients with meningitis due to such strains have a complement deficiency, we studied 12 persons. Six patients had meningitis due to nongroupable strains of meningococci, and six patients had meningitis due to Moraxella species or Acinetobacter species. Inherited complement component C7 or C8 deficiency was found in two persons who had had meningitis due to nongroupable meningococci, and one C8-deficient person had had meningitis caused by Moraxella osloensis. Hypocomplementemia resulting from CSF drain-associated shunt nephritis was found in one person with meningitis due to Moraxella nonliquefaciens and in one person with meningitis due to Acinetobacter lwoffi. This rather high frequency of inherited or acquired complement deficiencies among patients with meningitis due to nongroupable meningococci, Moraxella species, and Acinetobacter species justifies the recommendation that such patients must be studied for complement deficiency.

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

PubMed Central

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

1974-01-01

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225

Calcineurin inhibitor-induced complement system activation via ERK1/2 signalling is inhibited by SOCS-3 in human renal tubule cells.

PubMed

Loeschenberger, Beatrix; Niess, Lea; Würzner, Reinhard; Schwelberger, Hubert; Eder, Iris E; Puhr, Martin; Guenther, Julia; Troppmair, Jakob; Rudnicki, Michael; Neuwirt, Hannes

2018-02-01

One factor that significantly contributes to renal allograft loss is chronic calcineurin inhibitor (CNI) nephrotoxicity (CIN). Among other factors, the complement (C-) system has been proposed to be involved CIN development. Hence, we investigated the impact of CNIs on intracellular signalling and the effects on the C-system in human renal tubule cells. In a qPCR array, CNI treatment upregulated C-factors and downregulated SOCS-3 and the complement inhibitors CD46 and CD55. Additionally, ERK1/-2 was required for these regulations. Following knock-down and overexpression of SOCS-3, we found that SOCS-3 inhibits ERK1/-2 signalling. Finally, we assessed terminal complement complex formation, cell viability and apoptosis. Terminal complement complex formation was induced by CNIs. Cell viability was significantly decreased, whereas apoptosis was increased. Both effects were reversed under complement component-depleted conditions. In vivo, increased ERK1/-2 phosphorylation and SOCS-3 downregulation were observed at the time of transplantation in renal allograft patients who developed a progressive decline of renal function in the follow-up compared to stable patients. The progressive cohort also had lower total C3 levels, suggesting higher complement activity at baseline. In conclusion, our data suggest that SOCS-3 inhibits CNI-induced ERK1/-2 signalling, thereby blunting the negative control of C-system activation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Conservation of nodulation genes between Rhizobium meliloti and a slow-growing Rhizobium strain that nodulates a nonlegume host

PubMed Central

Marvel, Deborah J.; Kuldau, Gretchen; Hirsch, Ann; Richards, Eric; Torrey, John G.; Ausubel, Frederick M.

1985-01-01

Parasponia, a woody member of the elm family, is the only nonlegume genus whose members are known to form an effective nitrogen-fixing symbiosis with a Rhizobium species. The bacterial strain RP501 is a slow-growing strain of Rhizobium isolated from Parasponia nodules. Strain RP501 also nodulates the legumes siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata). Using a cosmid clone bank of RP501 DNA, we isolated a 13.4-kilobase (kb) EcoRI fragment that complemented insertion and point mutations in three contiguous nodulation genes (nodABC) of Rhizobium meliloti, the endosymbiont of alfalfa (Medicago sativa). The complemented R. meliloti nod mutants induced effective nitrogen-fixing nodules on alfalfa seedlings but not on siratro, cowpeas, or Parasponia. The cloned RP501 nodulation locus hybridized to DNA fragments carrying the R. meliloti nodABC genes. A 3-kb cluster of Tn5 insertion mutations on the RP501 13.4-kb EcoRI fragment prevented complementation of R. meliloti nodABC mutations. Images PMID:16593600

Expression of recombinant CD59 with an N-terminal peptide epitope facilitates analysis of residues contributing to its complement-inhibitory function.

PubMed

Zhou, Q; Zhao, J; Hüsler, T; Sims, P J

1996-10-01

CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immunodominant epitopes of CD59 are known to be sensitive to disruption of native tertiary structure, complicating immunological measurement of expressed mutant constructs for structure function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of this complement inhibitor, independent of CD59 antigen, an 11-residue peptide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted before the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cell lines expressing 0 to > 10(5) CD59/cell. The TAG-CD59 fusion protein was confirmed to be GPI-anchored, N-glycosylated and showed identical complement-inhibitory function to wild-type CD59, lacking the TAG peptide sequence. Using this construct, the contribution of each of four surface-localized aromatic residues (4Y, 47F, 61Y, and 62Y) to CD59's complement-inhibitory function was examined. These assays revealed normal surface expression with complete loss of complement-inhibitory function in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 62Y --> S mutants retained approximately 40% of function of wild-type CD59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surface aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function.

Simple method to distinguish between primary and secondary C3 deficiencies.

PubMed

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

2003-03-01

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.

Trichinella spiralis Paramyosin Binds to C8 and C9 and Protects the Tissue-Dwelling Nematode from Being Attacked by Host Complement

PubMed Central

Zhang, Zhifei; Yang, Jing; Wei, Junfei; Yang, Yaping; Chen, Xiaoqin; Zhao, Xi; Gu, Yuan; Cui, Shijuan; Zhu, Xinping

2011-01-01

Background Paramyosin is a thick myofibrillar protein found exclusively in invertebrates. Evidence suggested that paramyosin from helminths serves not only as a structural protein but also as an immunomodulatory agent. We previously reported that recombinant Trichinella spiralis paramyosin (Ts-Pmy) elicited a partial protective immunity in mice. In this study, the ability of Ts-Pmy to bind host complement components and protect against host complement attack was investigated. Methods and Findings In this study, the transcriptional and protein expression levels of Ts-Pmy were determined in T. spiralis newborn larva (NBL), muscle larva (ML) and adult worm developmental stages by RT-PCR and western blot analysis. Expression of Ts-Pmy at the outer membrane was observed in NBL and adult worms using immunogold electron microscopy and immunofluorescence staining. Functional analysis revealed that recombinant Ts-Pmy(rTs-Pmy) strongly bound to complement components C8 and C9 and inhibited the polymerization of C9 during the formation of the membrane attack complex (MAC). rTs-Pmy also inhibited the lysis of rabbit erythrocytes (ER) elicited by an alternative pathway-activated complement from guinea pig serum. Inhibition of native Ts-Pmy on the surface of NBL with a specific antiserum reduced larvae viability when under the attack of complement in vitro. In vivo passive transfer of anti-Ts-Pmy antiserum and complement-treated larvae into mice also significantly reduced the number of larvae that developed to ML. Conclusion These studies suggest that the outer membrane form of T. spiralis paramyosin plays an important role in the evasion of the host complement attack. PMID:21750743

Interruption of the Sequential Release of Small and Large Molecules from Tumor Cells by Low Temperature During Cytolysis Mediated by Immune T-Cells or Complement

PubMed Central

Martz, Eric; Burakoff, Steven J.; Benacerraf, Baruj

1974-01-01

Specific lysis of tumor cells by thymus-derived lymphocytes from alloimmunized mice (T-effector specific lysis) was studied with target cells labeled with isotopes attached to both small (14C-labeled nicotinamide) and large (51Cr-labeled) molecules. The results confirm and extend previous reports that target cells release small molecules considerably earlier than large molecules during T-effector specific lysis. After interruption of T-effector specific lysis by specific antibody and complement directed against the killer cells, or by ethylenediaminetetraacetic acid, release of both isotopes continued, eventually reaching identical levels of specific release, the value of which represents the fraction of the target cell population which had been committed to die at the time these treatments were applied. On the other hand, release of both isotopes during T-effector specific lysis stops immediately when the cultures are cooled to 0°. Thus, while ethylenediaminetetraacetic acid or specific complement-mediated lysis of the killer cells merely prevents the initiation of any new damage to target cells, cooling to 0° also stops the lytic process in already-damaged target cells. The colloid osmotic phase of target cell lysis induced by specific antibody and complement was similarly stopped at 0° in tumor cells, but not in erythrocytes. Thus, in tumor target cells, both T-effector specific lysis and complement cause a sequential release of progressively larger molecules which can be immediately stopped at any point by cooling to 0°. PMID:4359327

An update for atypical haemolytic uraemic syndrome: diagnosis and treatment. A consensus document.

PubMed

Campistol, Josep M; Arias, Manuel; Ariceta, Gema; Blasco, Miguel; Espinosa, Laura; Espinosa, Mario; Grinyó, Josep M; Macía, Manuel; Mendizábal, Santiago; Praga, Manuel; Román, Elena; Torra, Roser; Valdés, Francisco; Vilalta, Ramón; Rodríguez de Córdoba, Santiago

2015-01-01

Haemolytic uraemic syndrome (HUS) is a clinical entity defined as the triad of nonimmune haemolytic anaemia, thrombocytopenia, and acute renal failure, in which the underlying lesions are mediated by systemic thrombotic microangiopathy (TMA). Different causes can induce the TMA process that characterizes HUS. In this document we consider atypical HUS (aHUS) a sub-type of HUS in which the TMA phenomena are the consequence of the endotelial damage in the microvasculature of the kidneys and other organs due to a disregulation of the activity of the complement system. In recent years, a variety of aHUs-related mutations have been identified in genes of the the complement system, which can explain approximately 60% of the aHUS cases, and a number of mutations and polymorphisms have been functionally characterized. These findings have stablished that aHUS is a consequence of the insufficient regulation of the activiation of the complement on cell surfaces, leading to endotelial damage mediated by C5 and the complement terminal pathway. Eculizumab is a monoclonal antibody that inhibits the activation of C5 and blocks the generation of the pro-inflammatory molecule C5a and the formation of the cell membrane attack complex. In prospective studies in patients with aHUS, the use of Eculizumab has shown a fast and sustained interruption of the TMA process and it has been associated with significative long-term improvements in renal function, the interruption of plasma therapy and important reductions in the need of dialysis. According to the existing literature and the accumulated clinical experience, the Spanish aHUS Group published a consensus document with recommendations for the treatment of aHUs (Nefrologia 2013;33[1]:27-45). In the current online version of this document, we update the aetiological classification of TMAs, the pathophysiology of aHUS, its differential diagnosis and its therapeutic management. Copyright © 2015 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

Serum complement C3 strongly correlates with whole-body insulin sensitivity in rheumatoid arthritis.

PubMed

Ursini, Francesco; D'Angelo, Salvatore; Russo, Emilio; Arturi, Franco; D'Antona, Lucia; Bruno, Caterina; Naty, Saverio; De Sarro, Giovambattista; Olivieri, Ignazio; Grembiale, Rosa Daniela

2017-01-01

Rheumatoid arthritis (RA) is characterised by an excess of cardiovascular diseases (CVD) risk, attributable to a synergy between under-diagnosed traditional risk factors (i.e. insulin resistance) and inflammatory disease activity. The aim of the present study was to evaluate the correlation between inflammatory measures and insulin sensitivity in RA patients. Forty non-diabetic RA patients (19 males) were recruited. All patients underwent anthropometric measurements, laboratory evaluation and oral glucose tolerance test (OGTT). Insulin sensitivity index (ISI) was calculated with the equation proposed by Matsuda et al., from dynamic values of glucose and insulin obtained during OGTT. In the univariate analysis, lnISI correlated inversely with age, BMI, waist circumference, sBP, ESR, lnCRP and complement C3, but not with disease duration, dBP or complement C4. In non-obese patients (BMI <30 kg/m2, n=28), only age, BMI, lnCRP and C3 maintained their correlation with lnISI. In a stepwise multiple regression using lnISI as the dependent variable and BMI, age, lnCRP and complement C3 as predictors, only BMI and C3 entered the equation and accounted for 38.2% of the variance in lnISI. In non-obese patients, only C3 entered the regression equation, accounting for 32.2% of the variance in lnISI. Using a ROC curve, we identified the best cut-off for complement C3 of 1.22 g/L that yielded a sensitivity of 67% and a specificity of 79% for classification of insulin resistant patients. In RA patients, complement C3 correlates strongly with insulin sensitivity, in both obese and non-obese individuals.

Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

PubMed Central

van den Bremer, Ewald TJ; Beurskens, Frank J; Voorhorst, Marleen; Engelberts, Patrick J; de Jong, Rob N; van der Boom, Burt G; Cook, Erika M; Lindorfer, Margaret A; Taylor, Ronald P; van Berkel, Patrick HC; Parren, Paul WHI

2015-01-01

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential. PMID:26037225

Cold activation of complement for monitoring the response to interferon in patients with chronic hepatitis C.

PubMed

Akahane, Y; Miyazaki, Y; Naitoh, S; Takeda, K; Tsuda, F; Okamoto, H; Itoh, K; Miyakawa, Y; Mayumi, M

1996-02-01

Because of its specific association with hepatitis C virus (HCV) infection, the cold activation of complement is an easy and inexpensive indicator of HCV viremia. It was evaluated for eligibility as a marker of response to interferon in patients with hepatitis C. The cold activation of complement was determined by the loss or decrease of hemolytic activity with the microtitration method in sera that had been stored at 4 degrees C overnight. We observed the loss of hemolytic activity by the cold activation of complement in 236 (72%) and a decrease in 56 (17%) of 327 sera from patients with HCV-associated chronic liver disease, which was much more (p < 0.001) that in 1 (1%) and 13 (14%), respectively, of 49 sera from patients with chronic liver disease associated with hepatitis B virus infection. Interferon-alpha (total dose 516 x 10(6) units) or interferon-alpha 2b (774 x 10(6) units) was given to 67 patients with chronic hepatitis C, of whom 56 had the cold activation of complement. The response to interferon was evaluated by the clearance of serum HCV RNA at 6 months after the completion of therapy. The cold activation of complement disappeared in 18 patients, of whom 15 (86%) responded. It persisted or fluctuated in the remaining 38 patients, only six (16%) of whom responded to interferon (p < 0.001). The cold activation of complement once disappeared at the completion of interferon and then reappeared in patients who relapsed after completing interferon therapy. These results indicate that the cold activation of complement may be associated with the presence of HCV in blood and a lower rate of durable response after completion of interferon therapy.

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

PubMed

Prechl, József; Papp, Krisztián; Hérincs, Zoltán; Péterfy, Hajna; Lóránd, Veronika; Szittner, Zoltán; Estonba, Andone; Rovero, Paolo; Paolini, Ilaria; Del Amo, Jokin; Uribarri, Maria; Alcaro, Maria Claudia; Ruiz-Larrañaga, Otsanda; Migliorini, Paola; Czirják, László

2016-01-01

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

Effect of the C3a-receptor antagonist SB 290157 on anti-OVA polyclonal antibody-induced arthritis.

PubMed

Hutamekalin, Pilaiwanwadee; Takeda, Kohei; Tani, Mitsuhiro; Tsuga, Yuko; Ogawa, Naoki; Mizutani, Nobuaki; Yoshino, Shin

2010-01-01

It was investigated whether the C3a-receptor antagonist (C3aRA) SB 290157 was involved in the suppression of anti-OVA pAb-induced arthritis because it is well known that anaphylatoxin C3a plays a crucial role in the development of an effective inflammatory response during complement activation. Anti-OVA pAb-induced arthritis was induced in DBA/1J mice by administration of anti-OVA pAb 0.5 h prior to intra-articular (i.a.) injection of OVA (0 h). Two peaks of joint swelling were observed at 0.5 and 3 h. The role of C3aRA in arthritis was investigated by injection of SB 290157 at concentrations of 10 and 30 mg/kg at 0 and 2 h. The antagonist was able to reduce joint swelling only at 3 h, and about 50% inhibition of joint swelling was observed with the concentration of 30 mg/kg. The C3 level was significantly decreased at 3 h compared with naïve mice showing complement consumption. Furthermore, the C3 activation was observed and increased corresponding to the graded concentration of anti-OVA pAb. The results also revealed that the C3aRA was able to reduce the expression of IL-1beta in synovial tissue. Taken together, the results suggested that C3aRA may be effective in the inhibition of arthritis.

Substitution of blood coagulation factor X-binding to Ad5 by position-specific PEGylation: Preventing vector clearance and preserving infectivity.

PubMed

Krutzke, L; Prill, J M; Engler, T; Schmidt, C Q; Xu, Z; Byrnes, A P; Simmet, T; Kreppel, F

2016-08-10

The biodistribution of adenovirus type 5 (Ad5) vector particles is heavily influenced by interaction of the particles with plasma proteins, including coagulation factor X (FX), which binds specifically to the major Ad5 capsid protein hexon. FX mediates hepatocyte transduction by intravenously-injected Ad5 vectors and shields vector particles from neutralization by natural antibodies and complement. In mice, mutant Ad5 vectors that are ablated for FX-binding become detargeted from hepatocytes, which is desirable for certain applications, but unfortunately such FX-nonbinding vectors also become sensitive to neutralization by mouse plasma proteins. To improve the properties of Ad5 vectors for systemic delivery, we developed a strategy to replace the natural FX shield by a site-specific chemical polyethylene glycol shield. Coupling of polyethylene glycol to a specific site in hexon hypervariable region 1 yielded vector particles that were protected from neutralization by natural antibodies and complement although they were unable to bind FX. These vector particles evaded macrophages in vitro and showed significantly improved pharmacokinetics and hepatocyte transduction in vivo. Thus, site-specific shielding of Ad5 vectors with polyethylene glycol rendered vectors FX-independent and greatly improved their properties for systemic gene therapy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Involvement of the TonB System in Tolerance to Solvents and Drugs in Pseudomonas putida DOT-T1E

PubMed Central

Godoy, Patricia; Ramos-González, María Isabel; Ramos, Juan L.

2001-01-01

Pseudomonas putida DOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM) p-hydroxybenzoate (4HBA) per liter. After random mini-Tn5′phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock). Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with 14C-labeled substrates. The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA). Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted the exbD gene, which forms part of the exbBD tonB operon. Complementation by the exbBD and tonB genes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain. PMID:11514511

Involvement of the TonB system in tolerance to solvents and drugs in Pseudomonas putida DOT-T1E.

PubMed

Godoy, P; Ramos-González, M I; Ramos, J L

2001-09-01

Pseudomonas putida DOT-T1E is able to grow with glucose as the carbon source in liquid medium with 1% (vol/vol) toluene or 17 g of (123 mM) p-hydroxybenzoate (4HBA) per liter. After random mini-Tn5'phoA-Km mutagenesis, we isolated the mutant DOT-T1E-PhoA5, which was more sensitive than the wild type to 4HBA (growth was prevented at 6 g/liter) and toluene (the mutant did not withstand sudden toluene shock). Susceptibility to toluene and 4HBA resulted from the reduced efflux of these compounds from the cell, as revealed by accumulation assays with (14)C-labeled substrates. The mutant was also more susceptible to a number of antibiotics, and its growth in iron-deficient minimal medium was inhibited in the presence of ethylenediamine-di(o-hydroxyphenylacetic acid (EDDHA). Cloning the mutation in the PhoA5 strain and sequencing the region adjacent showed that the mini-Tn5 transposor interrupted the exbD gene, which forms part of the exbBD tonB operon. Complementation by the exbBD and tonB genes cloned in pJB3-Tc restored the wild-type characteristics to the PhoA5 strain.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum

PubMed Central

Castiblanco-Valencia, Mónica M.; Fraga, Tatiana R.; Breda, Leandro C.D.; Vasconcellos, Sílvio A.; Figueira, Cláudio P.; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I.; Barbosa, Angela S.; Isaac, Lourdes

2017-01-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. PMID:26976804

Evaluation of serum levels of C3 and C4 complement factors in patients with beta thalassemia major in Khuzestan Province, Southwest Iran.

PubMed

Ghafourian, Mehri; Esmaeili, Mehrnosh; Dashti-Gerdabi, Nader; Sadeghi, Alireza; Malekei Naseri, Ali; Kazemi, Akhtar

2017-01-01

Thalassemia syndrome is the most common genetic disorder in the world and infection is the second cause of death in these patients. Measurement of serum C3 and C4 complement factors in serum was done in 60 patients with beta thalassemia major in comparison with 30 healthy subjects as control group. The serum level of C3 and C4 complement factors in 60 patients with beta thalassemia major who were randomly selected from among the patients referred to Shafa Hospital of Ahvaz was evaluated and compared with 30 samples from healthy individuals with no history of recent infectious or autoimmune diseases. It should be noted that single-radial-immunodiffusion assay was used in this study. This study has shown a significant reduction in serum levels of C3 and C4 in patients compared to controls (P value < 0.05). Decreased synthesis or increased consumption of complement factors in patients receiving multiple blood transfusions might lead to continuous contact between the immune system and various antigens, causing nonstop use of complement factors, recurrent infections, changes in parameters of the immune system due to iron overload as well as exposure to infectious factors such as HBV, HCV, HIV, and HTLV through blood transfusion.

Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy.

PubMed

Xu, Jijun; Zhang, Lingjun; Xie, Mian; Li, Yan; Huang, Ping; Saunders, Thomas L; Fox, David A; Rosenquist, Richard; Lin, Feng

2018-06-15

Chemotherapy-induced peripheral neuropathy (CIPN) is a painful and debilitating side effect of cancer chemotherapy with an unclear pathogenesis. Consequently, the available therapies for this neuropathic pain syndrome are inadequate, leading to a significantly reduced quality of life in many patients. Complement, a key component of the innate immune system, has been associated with neuroinflammation, a potentially important trigger of some types of neuropathic pain. However, the role of complement in CIPN remains unclear. To address this issue, we developed a C3 knockout (KO) rat model and induced CIPN in these KO rats and wild-type littermates via the i.p. administration of paclitaxel, a chemotherapeutic agent associated with CIPN. We then compared the severity of mechanical allodynia, complement activation, and intradermal nerve fiber loss between the groups. We found that 1) i.p. paclitaxel administration activated complement in wild-type rats, 2) paclitaxel-induced mechanical allodynia was significantly reduced in C3 KO rats, and 3) the paclitaxel-induced loss of intradermal nerve fibers was markedly attenuated in C3 KO rats. In in vitro studies, we found that paclitaxel-treated rat neuronal cells activated complement, leading to cellular injury. Our findings demonstrate a previously unknown but pivotal role of complement in CIPN and suggest that complement may be a new target for the development of novel therapeutics to manage this painful disease. Copyright © 2018 by The American Association of Immunologists, Inc.

Linkage specificity and role of properdin in activation of the alternative complement pathway by fungal glycans.

PubMed

Agarwal, Sarika; Specht, Charles A; Haibin, Huang; Ostroff, Gary R; Ram, Sanjay; Rice, Peter A; Levitz, Stuart M

2011-01-01

Fungal cell walls are predominantly composed of glucans, mannans, and chitin. Recognition of these glycans by the innate immune system is a critical component of host defenses against the mycoses. Complement, an important arm of innate immunity, plays a significant role in fungal pathogenesis, especially the alternative pathway (AP). Here we determine that the glycan monosaccharide composition and glycosidic linkages affect AP activation and C3 deposition. Furthermore, properdin, a positive regulator of the AP, contributes to these functions. AP activation by glycan particles that varied in composition and linkage was measured by C3a generation in serum treated with 10 mM EGTA and 10 mM Mg(2+) (Mg-EGTA-treated serum) (AP specific; properdin functional) or Mg-EGTA-treated serum that lacked functional properdin. Particles that contained either β1→3 or β1→6 glucans or both generated large and similar amounts of C3a when the AP was intact. Blocking properdin function resulted in 5- to 10-fold-less C3a production by particulate β1→3 glucans. However, particulate β1→6 glucans generated C3a via the AP only in the presence of intact properdin. Interestingly, zymosan and glucan-mannan particles (GMP), which contain both β-glucans and mannans, also required properdin to generate C3a. The β1→4 glycans chitin and chitosan minimally activated C3 even when properdin was functional. Finally, properdin binding to glucan particles (GP) and zymosan in serum required active C3. Properdin colocalized with bound C3, suggesting that in the presence of serum, properdin bound indirectly to glycans through C3 convertases. These findings provide a better understanding of how properdin facilitates AP activation by fungi through interaction with the cell wall components. Invasive fungal infections have increased in incidence with the widespread use of immunosuppressive therapy and invasive procedures. Activation of the complement system contributes to innate immunity against fungi by generating chemoattractants that recruit white blood cells and by coating the pathogen with complement fragments that "mark" them for phagocytosis. The fungal cell wall activates complement in an antibody-independent manner through the alternative pathway (AP). Properdin is a positive regulator of the AP. This study elucidates how the specificity of cell wall glycan linkages affects AP activation and the role properdin plays in this process. Particulate β1→3 glucans activated the AP even in the absence of properdin, while β1→6 glucans required properdin for AP activation. In contrast, the β1→4 glycans chitin and chitosan failed to activate the AP. These findings enhance our mechanistic understanding of how fungi activate complement and have implications for the use of glycans in biomedical applications.

Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

PubMed Central

Chen, Yan; Carrington-Lawrence, Stacy D.; Bai, Ping; Weller, Sandra K.

2005-01-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface. PMID:15994803

Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

PubMed

Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

2005-07-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

The two sides of complement C3d: evolution of electrostatics in a link between innate and adaptive immunity.

PubMed

Kieslich, Chris A; Morikis, Dimitrios

2012-01-01

The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of -1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic "hot-spots". Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic "hot-spots" at the two functional sites of C3d, while the surface of CR2 lacks electrostatic "hot-spots" despite its excessively positive nature. We propose that the electrostatic "hot-spots" of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the divergence of jawless fish.

The Two Sides of Complement C3d: Evolution of Electrostatics in a Link between Innate and Adaptive Immunity

PubMed Central

Kieslich, Chris A.; Morikis, Dimitrios

2012-01-01

The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of −1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic “hot-spots”. Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic “hot-spots” at the two functional sites of C3d, while the surface of CR2 lacks electrostatic “hot-spots” despite its excessively positive nature. We propose that the electrostatic “hot-spots” of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the divergence of jawless fish. PMID:23300422

Isolation and purification of C3 from human plasma.

PubMed

O'Rear, L D; Ross, G D

2001-05-01

The alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.

Complement activation on B lymphocytes opsonized with rituximab or ofatumumab produces substantial changes in membrane structure preceding cell lysis.

PubMed

Beum, Paul V; Lindorfer, Margaret A; Beurskens, Frank; Stukenberg, P Todd; Lokhorst, Henk M; Pawluczkowycz, Andrew W; Parren, Paul W H I; van de Winkel, Jan G J; Taylor, Ronald P

2008-07-01

Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.

C5a Receptor (CD88) Blockade Protects against MPO-ANCA GN

PubMed Central

Xiao, Hong; Dairaghi, Daniel J.; Powers, Jay P.; Ertl, Linda S.; Baumgart, Trageen; Wang, Yu; Seitz, Lisa C.; Penfold, Mark E.T.; Gan, Lin; Hu, Peiqi; Lu, Bao; Gerard, Norma P.; Gerard, Craig; Schall, Thomas J.; Jaen, Juan C.; Falk, Ronald J.

2014-01-01

Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO–induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO–induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN. PMID:24179165

C5a receptor (CD88) blockade protects against MPO-ANCA GN.

PubMed

Xiao, Hong; Dairaghi, Daniel J; Powers, Jay P; Ertl, Linda S; Baumgart, Trageen; Wang, Yu; Seitz, Lisa C; Penfold, Mark E T; Gan, Lin; Hu, Peiqi; Lu, Bao; Gerard, Norma P; Gerard, Craig; Schall, Thomas J; Jaen, Juan C; Falk, Ronald J; Jennette, J Charles

2014-02-01

Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO-induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO-induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.

Complement 3 activates the renal renin-angiotensin system by induction of epithelial-to-mesenchymal transition of the nephrotubulus in mice.

PubMed

Zhou, Xueli; Fukuda, Noboru; Matsuda, Hiroyuki; Endo, Morito; Wang, Xiaofei; Saito, Kosuke; Ueno, Takahiro; Matsumoto, Taro; Matsumoto, Koichi; Soma, Masayoshi; Kobayashi, Naohiko; Nishiyama, Akira

2013-10-01

We have demonstrated that mesenchymal cells from spontaneously hypertensive rats genetically express complement 3 (C3). Mature tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT) that is linked to the pathogenesis of renal fibrosis and injury. In this study, we investigated the contribution of C3 in EMT and in the renal renin-angiotensin (RA) systems associated with hypertension. C3a induced EMT in mouse TCMK-1 epithelial cells, which displayed increased expression of renin and Krüppel-like factor 5 (KLF5) and nuclear localization of liver X receptor α (LXRα). C3 and renin were strongly stained in the degenerated nephrotubulus and colocalized with LXRα and prorenin receptor in unilateral ureteral obstruction (UUO) kidneys from wild-type mice. In C3-deficient mice, hydronephrus and EMT were suppressed, with no expression of renin and C3. After UUO, systolic blood pressure was increased in wild-type but not C3-deficient mice. In wild-type mice, intrarenal angiotensin II (ANG II) levels were markedly higher in UUO kidneys than normal kidneys and decreased with aliskiren. There were no increases in intrarenal ANG II levels after UUO in C3-deficient mice. Thus C3 induces EMT and dedifferentiation of epithelial cells, which produce renin through induction of LXRα. These data indicate for the first time that C3 may be a primary factor to activate the renal RA systems to induce hypertension.

Assessment of Complement Activation by Nanoparticles: Development of a SPR Based Method and Comparison with Current High Throughput Methods.

PubMed

Coty, Jean-Baptiste; Noiray, Magali; Vauthier, Christine

2018-04-26

A Surface Plasmon Resonance chip (SPR) was developed to study the activation of complement system triggered by nanomaterials in contact with human serum, which is an important concern today to warrant safety of nanomedicines. The developed chip was tested for its specificity in complex medium and its longevity of use. It was then employed to assess the release of complement fragments upon incubation of nanoparticles in serum. A comparison was made with other current methods assessing complement activation (μC-IE, ELISA). The SPR chip was found to give a consistent response for C3a release upon activation by nanoparticles. Results were similar to those obtained by μC-IE. However, ELISA detection of iC3b fragments showed an explained high non-specific background. The impact of sample preparation preceding the analysis was assessed with the newly develop SPR method. The removal of nanoparticles before analysis showed an important modification in the obtained response, possibly leading to false negative results. The SPR chip developed in this work allows for an automated assessment of complement activation triggered by nanoparticles with possibility of multiplexed analysis. The design of the chip proved to give consistent results of complement activation by nanoparticles.

Distal radical migration strategy: an emerging synthetic means.

PubMed

Li, Weipeng; Xu, Wentao; Xie, Jin; Yu, Shouyun; Zhu, Chengjian

2018-02-05

The remote radical migration strategy has gained considerable momentum. During the past three years, we have witnessed the rapid development of sustainable and practical C-C and C-H bond functionalization by means of long-distance 1,n-radical migration (n = 4, 5, 6) events. Its advent brings our chemical community a new platform to deal with the challenging migration transformations and thus complements the existing ionic-type migration protocols. In this review, the recent achievements in distal radical migration triggered C-C and C-H bond functionalization are summarized.

[Cloning, expression and identification of functional fragment rC3B of human complement C3 in E. Coli].

PubMed

Gan, Hui; Zhou, Yong; Sun, Ping; Zhu, Xiao-Xia; Wang, Quan-Li; Zhan, Lin-Sheng

2007-08-01

This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.

[Complement deficiencies and meningococcal disease in The Netherlands].

PubMed

Swart, A G; Fijen, C A; te Bulte, M T; Daha, M R; Dankert, J; Kuijper, E J

1993-06-05

To determine the prevalence of complement system deficiencies in patients who have survived a Neisseria meningitidis infection. Retrospective. Reference laboratory for bacterial meningitis of the University of Amsterdam and the National Institute of Public Health and Environmental Protection. Out of the files of the laboratory 187 patients who had experienced a meningococcal infection in the Netherlands between 1959-1990 were selected in two groups according to the infecting bacterial strain: 97 patients with a serogroup X, Y, Z, W135, 29E, or non-groupable strains and 90 patients with an infection due to serogroup A or C. The patients were asked for their cooperation by their family doctor and one of us visited the patients at home to take blood samples. The complement activity was studied with a haemolysis in gel test and with an assay of haemolytic activity in free solution. Complement deficiency was present in 18% of the 187 patients who had experienced a meningococcal infection. The highest prevalence was found in patients older than 10 years who had developed infections due to serogroups X, Y, W135, or non-groupable strains (45%). Of the patients with a serogroup A or C infection, 3% had an complement deficiency. Of the complement deficiencies, 42% concerned a component of the alternative pathway, 12% a deficiency of C3, and 46% a component of the terminal route. The most commonly found deficiencies were properdin deficiency (39%) and C8 deficiency (18%). 30% of the complement deficient patients reported other family members having experienced meningitis. Recurrent meningitis was only observed in patients with terminal route deficiencies. We recommend that patients with a meningococcal infection due to serogroups X, Y, W135 or non-groupable strains should be screened for complement deficiency.

Cadmium-induced immune abnormality is a key pathogenic event in human and rat models of preeclampsia.

PubMed

Zhang, Qiong; Huang, Yinping; Zhang, Keke; Huang, Yanjun; Yan, Yan; Wang, Fan; Wu, Jie; Wang, Xiao; Xu, Zhangye; Chen, Yongtao; Cheng, Xue; Li, Yong; Jiao, Jinyu; Ye, Duyun

2016-11-01

With increased industrial development, cadmium is an increasingly important environmental pollutant. Studies have identified various adverse effects of cadmium on human beings. However, the relationships between cadmium pollution and the pathogenesis of preeclampsia remain elusive. The objective of this study is to explore the effects of cadmium on immune system among preeclamptic patients and rats. The results showed that the cadmium levels in the peripheral blood of preeclamptic patients were significantly higher than those observed in normal pregnancy. Based on it, a novel rat model of preeclampsia was established by the intraperitoneal administration of cadmium chloride (CdCl2) (0.125 mg of Cd/kg body weight) on gestational days 9-14. Key features of preeclampsia, including hypertension, proteinuria, placental abnormalities and small foetal size, appeared in pregnant rats after the administration of low-dose of CdCl2. Cadmium increased immunoglobulin production, mainly angiotensin II type 1-receptor-agonistic autoantibodies (AT1-AA), by increasing the expression of activation-induced cytosine deaminase (AID) in B cells. AID is critical for the maturation of antibody and autoantibody responses. In addition, angiotensin II type 1-receptor-agonistic autoantibody, which emerged recently as a potential pathogenic contributor to PE, was responsible for the deposition of complement component 5 (C5) in kidneys of pregnant rats via angiotensin II type 1 receptor (AT1R) activation. C5a is a fragment of C5 that is released during C5 activation. Selectively interfering with C5a signalling by a complement C5a receptor-specific antagonist significantly attenuated hypertension and proteinuria in Cd-injected pregnant rats. Our results suggest that cadmium induces immune abnormalities that may be a key pathogenic contributor to preeclampsia and provide new insights into treatment strategies of preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Low serum immunglobulin G (IgG) during nephrosis is a predictor of urinary tract infection (UTI) in children with nephrotic syndrome.

PubMed

Afroz, S; Roy, D K; Khan, A H

2013-04-01

Low serum level of IgG, complement C3 and C4 in nephrotic syndrome children may cause increased susceptibility to infection. Serum level of IgG and complements in nephrotic children (NS) with UTI has been analyzed in this cross sectional study. It was carried out in the department of Pediatric nephrology, National Institute of Kidney Diseases & Urology (NIKDU), Dhaka, Bangladesh. The study subjects were followed up prospectively for one year to see and compare the frequency of relapse of NS and UTI. Patients were selected in a nonrandom purposive technique. Nephrotic syndrome children with initial attack between 1-12 year of age were included over a period of one year. The patients were grouped into Group I - UTI positive and Group II - UTI negative depending on urine culture positivity and colony count >10⁵ CFU/ml. Serum IgG and complements C3, C4 levels were done in both groups during nephrosis and were compared. A total of 101 children M: F 1.7:1, mean age 5.96±3.2 years were included in this study. Group I, n=45 vs. Group II, n=56. The mean serum level of IgG was low in Group I (549.91±210.71 vs. 728.64±235.81mg/dl, p<0.001). Serum IgG level less than 700mg/dl was found in 37 vs. 23 children {x² (¹) 17.52 p<0.001, OR=6.63}. Mean serum complement C3 level was also low in Group I (123.09±40.52 vs. 143.38±37.06mg/dl, p<0.05). But complement C3 and C4 level do not carry any risk of developing UTI in nephrotic children. Higher number of children in Group II were at remission (n=24) during follow up, while frequent relapsers were high in Group I (n=22). Increased frequency of UTI attack (88 episodes) was found in Group I children compared to none in Group II during follow up. So low serum level of IgG in children with NS during nephrosis can predict UTI with an odds ratio of 6.63 as well as relapse. Serum level of C3, C4 do not associated with any risk of development of UTI in NS children.

Bone marrow transplantation across major histocompatibility barriers. V. Protection of mice from lethal graft-vs. -host disease by pretreatment of donor cells with monoclonal anti-Thy-1. 2 coupled to the toxin ricin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vallera, D.A.; Youle, R.J.; Neville, D.M. Jr.

1982-03-01

A new method has been devised to eliminate T cells from murine bone marrow grafts across major histocompatibility barriers and thus prevent graft-vs.-host disease (GVHD). The method utilizes a monoclonal antibody directed at the Thy-1.2 antigen but is complement independent. To make anti-Thy-1.2 toxic, the antibody is covalently linked to the toxin ricin. Ricin ordinarily binds, enters, and kills cells through receptors containing galactose. The hybrid protein, anti-Thy-1.2-ricin, can enter and kill cells via the Thy-1.2 receptor. In the presence of lactose the usual entry route for ricin is largely blocked and the hybrid is shown to be a highlymore » selective reagent that is T cell specific in its inhibition of mitogen-stimulated splenocytes. We have used a model of severe and fatal GVHD where BALB/c splenocytes and bone marrow cells are given to irradiated C57BL/6 recipients. Over 90% of these mice die by day 70, exhibiting signs of GVHD. When donor cells are pretreated with 0.5 microgram/ml of anti-Thy-1.2-ricin plus 200 mM lactose before injection, 10 of 11 animals survive through day 70 without signs of GVHD. These studies demonstrate that ricin linked to monoclonal antibodies may have utility related to the prevention of GVHD in human bone marrow transplantation.« less

[Toxoplasmosis in sick persons].

PubMed

Netrebko, I D

2002-01-01

A screening investigation was conducted designed to identify ill persons presenting with toxoplasmosis with the aid of an intracutaneous test with toxoplasmin and complement-fixing reaction with a dry toxoplasmic antigen. Toxoplasmosis was detected in those persons having lymphadenitis, therapeutical patients, women with aggravated obstetrical history having had spontaneous abortions and having given birth to sick children, patients with sluggish meningoencephalitis presenting with epileptoid and hypothalamic syndrome, polyneuritis, and patients with myopia, chorioretinitis and uveitis. Identification and treatment of toxoplasmic invasion constitutes a valid method of control of spontaneous abortions of toxoplasmic etiology. Detection in a timely fashion of toxoplasmic invasion in pregnant women and preventive treatment with two 5-day cycles of pirimetamin (0.025) twice a day and sulfodimezin (0.5) four times daily with a 10-day interval between the cycles has been found out to prevent toxoplasmosis affliction of the fetus.

Nonrenal and renal activity of systemic lupus erythematosus: a comparison of two anti-C1q and five anti-dsDNA assays and complement C3 and C4.

PubMed

Julkunen, Heikki; Ekblom-Kullberg, Susanne; Miettinen, Aaro

2012-08-01

Associations of different assays for antibodies to C1q (anti-C1q) and to dsDNA (anti-dsDNA) and of complements C3 and C4 with disease activity in patients with systemic lupus erythematosus (SLE) were studied. The clinical manifestations of 223 SLE patients were recorded, and the disease activity was assessed by the SLEDAI score. Anti-C1q were determined by two enzyme-linked immunosorbent assays (ELISA) and anti-dsDNA by a radioimmunoassay (RIA), a Crithidia immunofluorescence (IF) assay and three ELISA assays using human telomere DNA, plasmid DNA circles, or calf thymus DNA as antigens, respectively. Complement C3 and C4 were determined by nephelometry. Control sera were obtained from 98 blood donors. In patients with SLE, the prevalence of anti-C1q was 17-18% and that of anti-dsDNA was 36-69%. Anti-C1q, anti-dsDNA, and complement C3 and C4 correlated well with the overall activity of SLE (r = 0.323-0.351, 0.353-0.566, and -0.372-0.444, respectively; P < 0.001). Sensitivity, specificity, positive predictive value, and negative predictive value for active lupus nephritis among SLE patients were 40-44, 92, 29, and 91-92% for anti-C1q and 48-68, 29-66, 11-16, and 86-91% for anti-dsDNA, respectively. Patients with active nephritis had higher levels of anti-C1q and lower levels of C3 and C4 than patients with inactive nephritis (P = 0.003-0.018). The corresponding associations of anti-dsDNA were somewhat weaker (P = 0.023-0.198). Hematological parameters reflecting disease activity correlated clearly better with anti-dsDNA and complement C3 and C4 than with anti-C1q. Anti-C1q is inferior to anti-dsDNA as a diagnostic test in SLE and in the evaluation of overall clinical activity of the disease. Anti-C1q together with complement C3 and C4 may offer useful additional information to monitor lupus nephritis activity. There are no practical differences between different assays for anti-C1q and anti-dsDNA.

Xeroderma pigmentosum complementation group F: A rare cause of cerebellar ataxia with chorea.

PubMed

Carré, G; Marelli, C; Anheim, M; Geny, C; Renaud, M; Rezvani, H R; Koenig, M; Guissart, C; Tranchant, C

2017-05-15

The complementation group F of Xeroderma pigmentosum (XP-F) is rare in the Caucasian population, and usually devoid of neurological symptoms. We report two cases, both Caucasian, who exhibited progressive cerebellar ataxia, chorea, a mild subcortical frontal cognitive impairment, and in one case severe polyneuropathy. Brain MRI demonstrated cerebellar (2/2) and cortical (1/2) atrophy. Both patients had only mild sunburn sensitivity and no skin cancer. Mini-exome sequencing approach revealed in ERCC4, two heterozygous mutations, one of which was never described (c.580-584+1delCCAAGG, exon 3), in the first case, and an already reported homozygous mutation, in the second case. These cases emphasize that XP-F is a rare cause of recessive cerebellar ataxia and can in some cases clinically mimic Huntington's disease due to chorea and executive impairment. The association of ataxia, chorea, and sun hypersensitivity are major guidance for the diagnosis, which should not be missed, in order to prevent skin neoplastic complications. Copyright © 2017 Elsevier B.V. All rights reserved.

Complementation Studies of Bacteriophage λ O Amber Mutants by Allelic Forms of O Expressed from Plasmid, and O-P Interaction Phenotypes.

PubMed

Hayes, Sidney; Rajamanickam, Karthic; Hayes, Connie

2018-04-05

λ genes O and P are required for replication initiation from the bacteriophage λ origin site, ori λ, located within gene O . Questions have persisted for years about whether O-defects can indeed be complemented in trans . We show the effect of original null mutations in O and the influence of four origin mutations (three are in-frame deletions and one is a point mutation) on complementation. This is the first demonstration that O proteins with internal deletions can complement for O activity, and that expression of the N-terminal portion of gene P can completely prevent O complementation. We show that O-P co-expression can limit the lethal effect of P on cell growth. We explore the influence of the contiguous small RNA OOP on O complementation and P-lethality.

Shark complement: an assessment.

PubMed

Smith, S L

1998-12-01

The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA clone encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish (Triakis scyllia) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C8n and C9n, and is activated by immune complexes in the presence of Ca++ and Mg++ ions. The ACP is antibody independent, requiring Mg++ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C3 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q-like rather than like mannan-binding protein. N-terminal amino acid sequences of the alpha and beta chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4 gamma chain, shows little similarity to human C4 gamma chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and alpha 2-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum.

PubMed

Castiblanco-Valencia, Mónica M; Fraga, Tatiana R; Breda, Leandro C D; Vasconcellos, Sílvio A; Figueira, Cláudio P; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I; Barbosa, Angela S; Isaac, Lourdes

2016-05-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. Copyright © 2016 European Federation of Immunological Societies. All rights reserved.

Strains Responsible for Invasive Meningococcal Disease in Patients With Terminal Complement Pathway Deficiencies.

PubMed

Rosain, Jérémie; Hong, Eva; Fieschi, Claire; Martins, Paula Vieira; El Sissy, Carine; Deghmane, Ala-Eddine; Ouachée, Marie; Thomas, Caroline; Launay, David; de Pontual, Loïc; Suarez, Felipe; Moshous, Despina; Picard, Capucine; Taha, Muhamed-Kheir; Frémeaux-Bacchi, Véronique

2017-04-15

Patients with terminal complement pathway deficiency (TPD) are susceptible to recurrent invasive meningococcal disease (IMD). Neisseria meningitidis (Nm) strains infecting these patients are poorly documented in the literature. We identified patients with TPD and available Nm strains isolated during IMD. We investigated the genetic basis of the different TPDs and the characteristics of the Nm strains. We included 56 patients with C5 (n = 8), C6 (n = 20), C7 (n = 18), C8 (n = 9), or C9 (n = 1) deficiency. Genetic study was performed in 47 patients and 30 pathogenic variants were identified in the genes coding for C5 (n = 4), C6 (n = 5), C7 (n = 12), C8 (n = 7), and C9 (n = 2). We characterized 61 Nm strains responsible for IMD in the 56 patients with TPD. The most frequent strains belonged to groups Y (n = 27 [44%]), B (n = 18 [30%]), and W (n = 8 [13%]). Hyperinvasive clonal complexes (CC11, CC32, CC41/44, and CC269) were responsible for 21% of IMD cases. The CC23 predominates and represented 26% of all invasive isolates. Eleven of the 15 clonal complexes identified fit to 12 different clonal complexes belonging to carriage strains. Unusual meningococcal strains with low level of virulence similar to carriage strains are most frequently responsible for IMD in patients with TPD. © The Author 217. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.

PubMed

Siméone, Roxane; Constant, Patricia; Guilhot, Christophe; Daffé, Mamadou; Chalut, Christian

2007-07-01

Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.

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

PubMed Central

Hecker, Laura A.; Edwards, Albert O.; Ryu, Euijung; Tosakulwong, Nirubol; Baratz, Keith H.; Brown, William L.; Issa, Peter Charbel; 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. PMID:19825847

Isolation and preliminary characterization of temperature-sensitive mutants of influenza virus.

PubMed

Sugiura, A; Tobita, K; Kilbourne, E D

1972-10-01

Isolation of temperature-sensitive (ts) mutants was attempted from the WSN strain of influenza A virus which was grown and assayed in MDBK cells. After growth of wild-type virus in the presence of 5-fluorouracil, 15 ts mutants were selected for which the ratio of plaquing efficiency at 39.5 C to that at 33 C was 10(-3) or less. In pairwise crosses of ts mutants, recombination and complementation were either very efficient or undetectable. It is suggested, therefore, that the viral genome consists of physically discrete units and recombination occurs as an exchange of these units. All 15 mutants have been assigned with certainty into five recombination groups. Three mutants are suspected to be double mutants. Any two complementing mutants always recombined with each other, and noncomplementing mutants did not recombine. In physiological tests, mutants showed diverse patterns of functional defects at the nonpermissive temperature. However, it was not always possible to correlate these physiological defects with the results of genetic characterization.

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

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

2015-01-01

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. PMID:25802332

Complement activation and interleukin response in major abdominal surgery.

PubMed

Kvarnström, A L; Sarbinowski, R T; Bengtson, J-P; Jacobsson, L M; Bengtsson, A L

2012-05-01

The objective of this study was to evaluate whether major abdominal surgery leads to complement activation and interleukin response and whether the kind of anaesthesia influence complement activation and the release of inflammatory interleukins. The study design was prospective and randomised. Fifty patients undergoing open major colorectal surgery due to cancer disease or inflammatory bowel disease were studied. Twenty-five patients were given total intravenous anaesthesia (TIVA) with propofol and remifentanil, and 25 patients were given inhalational anaesthesia with sevoflurane and fentanyl. To determine complement activation (C3a and SC5b-9) and the release of pro- and anti-inflammatory interleukins (tumour necrosis factor-a (TNF-a)), interleukin-1b (IL-1b), IL-6, IL-8, IL-4 and IL-10), blood samples were drawn preoperatively, 60 minutes after start of surgery, 30 minutes after end of surgery and 24 hours postoperatively. Complement was activated and pro-inflammatory interleukins (IL-6 and IL-8) and anti-inflammatory interleukins (IL-10) were released during major colorectal surgery. There was no significant difference between TIVA and inhalational anaesthesia regarding complement activation and cytokine release. Major colorectal surgery leads to activation of the complement cascade and the release of both pro-inflammatory and anti-inflammatory cytokines. There are no significant differences between total intravenous anaesthesia (TIVA) with propofol and remifentanil and inhalational anaesthesia with sevoflurane and fentanyl regarding complement activation and the release of pro- and anti-inflammatory interleukins. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd. Scandinavian Journal of Immunology.

Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sjoewall, Christopher; Wetteroe, Jonas; Bengtsson, Torbjoern

2007-01-05

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fc{gamma} receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficientlymore » down-regulated at CRP levels >150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.« less

In vitro biosynthesis of a universal t6A tRNA modification in Archaea and Eukarya

PubMed Central

Perrochia, Ludovic; Crozat, Estelle; Hecker, Arnaud; Zhang, Wenhua; Bareille, Joseph; Collinet, Bruno; van Tilbeurgh, Herman; Forterre, Patrick

2013-01-01

N6-threonylcarbamoyladenosine (t6A) is a modified nucleotide found in all transfer RNAs (tRNAs) decoding codons starting with adenosine. Its role is to facilitate codon–anticodon pairing and to prevent frameshifting during protein synthesis. Genetic studies demonstrated that two universal proteins, Kae1/YgjD and Sua5/YrdC, are necessary for t6A synthesis in Saccharomyces cerevisiae and Escherichia coli. In Archaea and Eukarya, Kae1 is part of a conserved protein complex named kinase, endopeptidase and other proteins of small size (KEOPS), together with three proteins that have no bacterial homologues. Here, we reconstituted for the first time an in vitro system for t6A modification in Archaea and Eukarya, using purified KEOPS and Sua5. We demonstrated binding of tRNAs to archaeal KEOPS and detected two distinct adenosine triphosphate (ATP)-dependent steps occurring in the course of the synthesis. Our data, together with recent reconstitution of an in vitro bacterial system, indicated that t6A cannot be catalysed by Sua5/YrdC and Kae1/YgjD alone but requires accessory proteins that are not universal. Remarkably, we observed interdomain complementation when bacterial, archaeal and eukaryotic proteins were combined in vitro, suggesting a conserved catalytic mechanism for the biosynthesis of t6A in nature. These findings shed light on the reaction mechanism of t6A synthesis and evolution of molecular systems that promote translation fidelity in present-day cells. PMID:23258706

Preparation of Low Molecular Weight Chondroitin Sulfates, Screening of a High Anti-Complement Capacity of Low Molecular Weight Chondroitin Sulfate and Its Biological Activity Studies in Attenuating Osteoarthritis.

PubMed

Li, Lian; Li, Yan; Feng, Danyang; Xu, Linghua; Yin, Fengxin; Zang, Hengchang; Liu, Chunhui; Wang, Fengshan

2016-10-11

Chondroitin sulfate (CS) plays important roles in the complement system. However, the CS structure is complicated due to different sources and the number and positions of sulfate groups. The objective of this study was to prepare different low molecular weight chondroitin sulfates (LMWCSs) and to investigate the biological activity in anti-complement capacity. A series of LMWCSs was prepared from different sources and characterized by ultraviolet-visible (UV) spectroscopy, high-performance liquid chromatography (HPLC), size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) and nuclear magnetic resonance (NMR) spectroscopy. Hemolytic, anti-complement 3 deposition capacity and cell viability assays were carried out to investigate the biological activities in vitro. The results showed that LMWCS prepared from shark cartilage with the oxidative degradation method (LMWCS-S-O) had the best anti-complement capacity. LMWCS-S-O could inhibit the alternative pathway of the complement system and protect chondrocytes from cell death. The attenuating effect of LMWCS-S-O on Osteoarthritis (OA) was investigated by destabilization of the medial meniscus (DMM) model in vivo. Functional wind-up, histological and C5b-9 analyses were used to evaluate the treatment effect on the OA model. In vivo results showed that LMWCS-S-O could attenuate OA. LMWCS-S-O with a high content of ΔDi-2,6diS and ΔDi-6S could be used for attenuating OA through regulating the complement system.

Use of eculizumab in crescentic IgA nephropathy: proof of principle and conundrum?

PubMed Central

Ring, Troels; Pedersen, Birgitte Bang; Salkus, Giedrius; Goodship, Timothy H.J.

2015-01-01

IgA nephropathy (IgAN) is characterized by a variable clinical course and multifaceted pathophysiology. There is substantial evidence to suggest that complement activation plays a pivotal role in the pathogenesis of the disease. Therefore, complement inhibition using the humanized anti-C5 monoclonal antibody eculizumab could be a rational treatment. We report here a 16-year-old male with the vasculitic form of IgAN who failed to respond to aggressive conventional therapy including high-dose steroids, cyclophosphamide and plasma exchange and who was treated with four weekly doses of 900 mg eculizumab followed by a single dose of 1200 mg. He responded rapidly to this treatment and has had a stable creatinine around 150 µmol/L (1.67 mg/dL) for >6 months. However, proteinuria was unabated on maximal conventional anti-proteinuric treatment, and a repeat renal biopsy 11 months after presentation revealed severe chronic changes. We believe this case provides proof of principle that complement inhibition may be beneficial in IgAN but also that development of chronicity may be independent of complement. PMID:26413271

Complementizer Agreement in Modern Varieties of West Germanic: A Model of Reanalysis and Renewal

ERIC Educational Resources Information Center

Bousquette, Joshua

2013-01-01

This dissertation provides a comparative analysis of Complementizer Agreement (C-agr) in modern dialects of West Germanic from a diachronic perspective, attributing the rise and development of C-agr to the initiation and progression of a Linguistic Cycle specific to C-agr. Approached as a historical process of reanalysis and compensatory renewal…

Identification of Bacillus subtilis men mutants which lack O-succinylbenzoyl-coenzyme A synthetase and dihydroxynaphthoate synthase.

PubMed Central

Meganathan, R; Bentley, R; Taber, H

1981-01-01

Menaquinone (vitamin K2)-deficient mutants of Bacillus subtilis, whose growth requirement is satisfied by 1,4-dihydroxy-2-naphthoic acid but not by o-succinylbenzoic acid (OSB), have been analyzed for enzymatic defects. Complementation analysis of cell-free extracts of the mutants revealed that there are two groups, as already indicated by genetic analysis. The missing enzyme in each group was identified by complementation of the cell-free extracts with o-succinylbenzoyl-coenzyme A (CoA) synthetase and dihydroxynaphthoate synthase extracted from Mycobacterium phlei. Mutants found to lack dihydroxynaphthoate synthase, and which therefore complement with dihydroxynaphthoate synthase of M. phlei, were designated as menB; those lacking o-succinylbenzoyl-CoA synthetase, and therefore complementing with o-succinylbenzoyl-CoA synthetase, were designated as menE. The menB mutants RB413 (men-325) and RB415 (men-329), when incubated with [2,3-14C2]OSB, produced only the spirodilactone form of OSB in a reaction that was CoA and adenosine 5'-triphosphate dependent. PMID:6780515

New Insights into Disease-Specific Absence of Complement Factor H Related Protein C in Mouse Models of Spontaneous Autoimmune Diseases

PubMed Central

Mehta, Gaurav; Ferreira, Viviana P.; Pickering, Matthew C.; Skerka, Christine; Zipfel, Peter F.; Banda, Nirmal K.

2014-01-01

Complement factor H (CFH) protein is an inhibitor of the alternative pathway of complement (AP) both in the fluid phase and on the surface of host cells. Mouse and human complement factor H-related (CFHR) proteins also belong to the fH family of plasma glycoproteins. The main goal of the current study was to compare the presence of mRNA for two mCFHR proteins in spontaneously developing autoimmune diseases in mice such as dense deposit disease (DDD), diabetes mellitus (DM), basal laminar deposits (BLD), collagen antibody-induced arthrits (CAIA) and systemic lupus erythematosus (SLE). Here we report for the first time that the CFHR-C mRNA was universally absent in the liver from three strains of lupus-prone mice and in a diabetic-prone mouse strain. The mRNA levels (pg/ng) for CFH and CFHR-B in MRL-lpr/lpr, at 9 wks and 23 wks were 707.2 ± 44.4, 54.5 ± 5.75 and 729 ± 252.9, 74.04 ± 22.76 respectively. The mRNA levels for CFH and CFHR-B in NZB/NZW mice, at 9 wks and 54 wks were 579.9 ± 23.8, 58.8 ± 1.41 and 890.3 ± 135.2, 63.30 ± 9.2 respectively. CFHR-C protein was absent in the circulation of MRL-lpr/lpr and NZB/NZW mice before and after the development of lupus. Similarly, mRNA and protein for CFHR-C was universally absent in liver and other organs and in the circulation of NOD mice before and after the development of DM. In contrast, the mRNAs for CFH, CFHR-B and CFHR-C were universally present in the liver from mice with and without DDD, BLD and CAIA. The levels of mRNA for CFHR-B in mice with and without BLD were ~4 times higher than the mice with lupus. The complete absence of mRNA for CFHR-C in lupus and diabetic-prone strains indicates that polymorphic variation within the mouse CFHR family exists and raises the possibility that such variation contributes to lupus and diabetic phenotypes. PMID:25033230

Mutants of Yeast Defective in Sucrose Utilization

PubMed Central

Carlson, Marian; Osmond, Barbara C.; Botstein, David

1981-01-01

Utilization of sucrose as a source of carbon and energy in yeast (Saccharomyces) is controlled by the classical SUC genes, which confer the ability to produce the sucrose-degrading enzyme invertase (Mortimer and Hawthorne 1969). Mutants of S. cerevisiae strain S288C (SUC2+) unable to grow anaerobically on sucrose, but still able to use glucose, were isolated. Two major complementation groups were identified: twenty-four recessive mutations at the SUC2 locus (suc2-); and five recessive mutations defining a new locus, SNF1 (for sucrose nonfermenting), essential for sucrose utilization. Two minor complementation groups, each comprising a single member with a leaky sucrose-nonfermenting phenotype, were also identified. The suc2 mutations isolated include four suppressible amber mutations and five mutations apparently exhibiting intragenic complementation; complementation analysis and mitotic mapping studies indicated that all of the suc2 mutations are alleles of a single gene. These results suggest that SUC2 encodes a protein, probably a dimer or multimer. No invertase activity was detected in suc2 mutants.—The SNF1 locus is not tightly linked to SUC2. The snf1 mutations were found to be pleiotropic, preventing sucrose utilization by SUC2+ and SUC7+ strains, and also preventing utilization of galactose, maltose and several nonfermentable carbon sources. Although snf1 mutants thus display a petite phenotype, classic petite mutations do not interfere with utilization of sucrose, galactose or maltose. A common feature of all the carbon utilization systems affected by SNF1 is that all are regulated by glucose repression. The snf1 mutants were found to produce the constitutive nonglycosylated form of invertase, but failed to produce the glucose-repressible, glycosylated, secreted invertase. This failure cannot be attributed to a general defect in production of glycosylated and secreted proteins because synthesis of acid phosphatase, a glycosylated secreted protein not subject to glucose repression, was not affected by snf1 mutations. These findings suggest that the SNF1 locus is involved in the regulation of gene expression by glucose repression. PMID:7040163

Functional Study of the P32T ITPA Variant Associated with Drug Sensitivity in Humans

PubMed Central

Stepchenkova, Elena I.; Tarakhovskaya, Elena R.; Spitler, Kathryn; Frahm, Christin; Menezes, Miriam R.; Simone, Peter D.; Kolar, Carol; Marky, Luis A.; Borgstahl, Gloria E. O.; Pavlov, Youri I.

2009-01-01

Sanitization of the cellular nucleotide pools from mutagenic base analogs is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic and toxic incorporation of purine base analogs (i.e. ITP, dITP, XTP, dXTP or 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele 94C->A encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analog drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 degrees C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by western blot is severely diminished in a human fibroblast cell line with the 94C->A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability and availability. PMID:19631656

Aloe vera enhances the innate immune response of pacu (Piaractus mesopotamicus) after transport stress and combined heat killed Aeromonas hydrophila infection.

PubMed

Zanuzzo, Fábio S; Sabioni, Rafael E; Montoya, Luz Natalia F; Favero, Gisele; Urbinati, Elisabeth C

2017-06-01

In this study, pacu (Piaractus mesopotamicus) were fed with diets containing Aloe vera for 10 days prior to transport stress and infection with heat killed Aeromonas hydrophila. A. vera is popular around the world due to its medicinal properties, including immunostimulatory effects which was observed in this study. The results show that transport causes immunosuppression, an effect that was prevented by A. vera. Specifically, A. vera prevented reductions of both leukocyte respiratory burst and hemolytic activity of complement system caused by transport. Further, fish fed with A. vera also showed significantly higher leukocyte respiratory burst, serum lysozyme concentrations and activity of complement system 24 h after bacterial infection. Additionally, we observed that A. vera may modulate the innate response through activation of complement system during bacterial immune stimulation. In summary, A. vera extract enhanced innate immune parameters and consequently the ability of fish to cope with pathogens following transport stress. These findings show that A. vera has promise for use in aquaculture and add further evidence that medicinal herbs added to fish feed assist to prevent disease outbreaks. Copyright © 2017 Elsevier Ltd. All rights reserved.

Activation of the classical pathway of complement by binding of bovine lactoferrin to unencapsulated Streptococcus agalactiae.

PubMed Central

Rainard, P

1993-01-01

The ability of lactoferrin (Lf) bound to Streptococcus agalactiae to interfere with the deposition of complement components on the bacterial surface was investigated by enzyme-linked immunosorbent assay (ELISA). By using a strain of S. agalactiae which activates the alternative pathway of complement in the absence of antibodies, it was found that pretreatment of bacteria with Lf shortened the lag phase preceding the deposition of C3 on bacteria. The kinetics of C3 deposition was comparable to that obtained by adding antibodies against S. agalactiae to agammaglobulinaemic precolostral calf serum (PCS) heated at 56 degrees for 3 min to inactivate the alternative pathway. Accelerated C3 deposition did not occur in the absence of Ca2+ ions. Deposition of C4 on bacteria occurred only when either antibodies or Lf were added to PCS. These results demonstrate that the interaction of lactoferrin with bacteria activated the classical pathway of complement in the absence of antibodies. The binding of purified C1q to bacteria was promoted in a dose-dependent manner by Lf, suggesting that recruitment of classical pathway of complement resulted from the interaction of C1q with Lf adsorbed to the bacterial surface. Phagocytosis of bacteria opsonized with heated PCS (at 56 degrees for 3 min) and Lf was comparable to that occurring in the presence of heated PCS and antibodies. In conclusion, Lf was able to substitute for antibodies in order to activate the classical pathway of complement and to opsonize unencapsulated S. agalactiae efficiently. PMID:8406591

Improved ex vivo blood compatibility of central venous catheter with noble metal alloy coating.

PubMed

Vafa Homann, Manijeh; Johansson, Dorota; Wallen, Håkan; Sanchez, Javier

2016-10-01

Central line associated bloodstream infections (CLABSIs) are a serious cause of morbidity and mortality induced by the use of central venous catheters (CVCs). Nobel metal alloy (NMA) coating is an advanced surface modification that prevents microbial adhesion and growth on catheters and thereby reduces the risk of infection. In vitro microbiological analyses have shown up to 90% reduction in microbial adhesion on coated CVC compared to uncoated ones. This study aimed to assess the blood compatibility of NMA-coated CVC according to ISO 10993-4. Hemolysis, thrombin-antithrombin (TAT) complex, platelet counts, fibrin deposition, and C3a and SC5b-9 complement activation were analyzed in human blood exposed to the NMA-coated and control CVCs using a Chandler-loop model. NMA-coated CVC did not induce hemolysis and fell in the "nonhemolytic" category according to ASTM F756-00. Significantly lower amounts of TAT were generated and less fibrin was deposited on NMA-coated CVC than on uncoated ones. Slightly higher platelet counts and lower complement markers were observed for NMA-coated CVC compared to uncoated ones. These data suggest that the NMA-coated CVC has better ex vivo blood compatibility compared to uncoated CVC. © 2015 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1359-1365, 2016. © 2015 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc.

Kinetics of leukocyte sequestration in the lungs of acutely septic primates: A study using sup 111 In-labeled autologous leukocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hangen, D.H.; Segall, G.M.; Harney, E.W.

1990-03-01

To further clarify the role of leukocytes in the pathogenesis of ARDS, we studied the localization and kinetics of leukocyte migration using 111In-labeled autologous white cell scans ({sup 111}In wbc scans) in four primates made acutely septic with infusions of Escherichia coli. Whole body images were obtained with a gamma camera and were acquired on computer every 15 min beginning immediately after the E. coli infusion. Simultaneous measurements of C5a and peripheral blood leukocyte count were also obtained. Within 5 min of initiating sepsis, three major events occurred: complement activation as measured by the production of C5a, a profound fallmore » in peripheral leukocyte count, and a significant increase in the sequestration of leukocytes in the lungs. The pulmonary sequestration reached a peak at 15 min with a mean of 152% of baseline activity. This sequestration consisted of a population that was predominantly neutrophils. Damage to the pulmonary capillary endothelium was demonstrated by an increase in extravascular lung water. The results support a role for neutrophils and complement as mediators in the pathogenesis of ARDS.« less

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

PubMed

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

2014-01-01

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).

Staphylococcus aureus SdrE captures complement factor H's C-terminus via a novel ‘close, dock, lock and latch' mechanism for complement evasion

PubMed Central

Zhang, Yingjie; Wu, Minhao; Hang, Tianrong; Wang, Chengliang; Yang, Ye; Pan, Weimin; Zang, Jianye

2017-01-01

Complement factor H (CFH) is a soluble complement regulatory protein essential for the down-regulation of the alternative pathway on interaction with specific markers on the host cell surface. It recognizes the complement component 3b (C3b) and 3d (C3d) fragments in addition to self cell markers (i.e. glycosaminoglycans, sialic acid) to distinguish host cells that deserve protection from pathogens that should be eliminated. The Staphylococcus aureus surface protein serine–aspartate repeat protein E (SdrE) was previously reported to bind human CFH as an immune-evasion tactic. However, the molecular mechanism underlying SdrE–CFH-mediated immune evasion remains unknown. In the present study, we identified a novel region at CFH's C-terminus (CFH1206–1226), which binds SdrE N2 and N3 domains (SdrEN2N3) with high affinity, and determined the crystal structures of apo-SdrEN2N3 and the SdrEN2N3–CFH1206–1226 complex. Comparison of the structure of the CFH–SdrE complex with other CFH structures reveals that CFH's C-terminal tail flips from the main body to insert into the ligand-binding groove of SdrE. In addition, SdrEN2N3 adopts a ‘close’ state in the absence of CFH, which undergoes a large conformational change on CFH binding, suggesting a novel ‘close, dock, lock and latch' (CDLL) mechanism for SdrE to recognize its ligand. Our findings imply that SdrE functions as a ‘clamp' to capture CFH's C-terminal tail via a unique CDLL mechanism and sequesters CFH on the surface of S. aureus for complement evasion. PMID:28258151

Staphylococcus aureus SdrE captures complement factor H's C-terminus via a novel 'close, dock, lock and latch' mechanism for complement evasion.

PubMed

Zhang, Yingjie; Wu, Minhao; Hang, Tianrong; Wang, Chengliang; Yang, Ye; Pan, Weimin; Zang, Jianye; Zhang, Min; Zhang, Xuan

2017-05-04

Complement factor H (CFH) is a soluble complement regulatory protein essential for the down-regulation of the alternative pathway on interaction with specific markers on the host cell surface. It recognizes the complement component 3b (C3b) and 3d (C3d) fragments in addition to self cell markers (i.e. glycosaminoglycans, sialic acid) to distinguish host cells that deserve protection from pathogens that should be eliminated. The Staphylococcus aureus surface protein serine-aspartate repeat protein E (SdrE) was previously reported to bind human CFH as an immune-evasion tactic. However, the molecular mechanism underlying SdrE-CFH-mediated immune evasion remains unknown. In the present study, we identified a novel region at CFH's C-terminus (CFH 1206-1226 ), which binds SdrE N2 and N3 domains (SdrE N2N3 ) with high affinity, and determined the crystal structures of apo-SdrE N2N3 and the SdrE N2N3 -CFH 1206-1226 complex. Comparison of the structure of the CFH-SdrE complex with other CFH structures reveals that CFH's C-terminal tail flips from the main body to insert into the ligand-binding groove of SdrE. In addition, SdrE N2N3 adopts a 'close' state in the absence of CFH, which undergoes a large conformational change on CFH binding, suggesting a novel 'close, dock, lock and latch' (CDLL) mechanism for SdrE to recognize its ligand. Our findings imply that SdrE functions as a 'clamp' to capture CFH's C-terminal tail via a unique CDLL mechanism and sequesters CFH on the surface of S. aureus for complement evasion. © 2017 The Author(s).

Attenuation of S. aureus-induced bacteremia by human mini-antibodies targeting the complement inhibitory protein Efb

PubMed Central

Georgoutsou-Spyridonos, Maria; Ricklin, Daniel; Pratsinis, Haris; Perivolioti, Eustathia; Pirmettis, Ioannis; Garcia, Brandon L.; Geisbrecht, Brian V.; Foukas, Periklis G.; Lambris, John D.; Mastellos, Dimitrios C.; Sfyroera, Georgia

2015-01-01

Staphylococcus aureus (S. aureus) can cause a broad range of potentially fatal inflammatory complications (e.g. sepsis, endocarditis). Its emerging antibiotic resistance and formidable immune evasion arsenal have emphasized the need for more effective antimicrobial approaches. Complement is an innate immune sensor that rapidly responds to bacterial infection eliciting C3-mediated opsonophagocytic and immunomodulatory responses. Extracellular Fibrinogen-binding Protein (Efb) is a key immune evasion protein of S. aureus that intercepts complement at the level of C3. To date, Efb has not been explored as a target for monoclonal antibody (mAb)-based antimicrobial therapeutics. Herein we have isolated donor-derived anti-Efb IgGs that attenuate S. aureus survival through enhanced neutrophil killing. A phage library screen yielded mAbs (miniAbs) that selectively inhibit the interaction of Efb with C3 partly by disrupting contacts essential for complex formation. Surface Plasmon Resonance-based kinetic analysis enabled the selection of miniAbs with favorable Efb-binding profiles as therapeutic leads. MiniAb-mediated blockade of Efb attenuated S aureus survival in a whole blood model of bacteremia. This neutralizing effect was associated with enhanced neutrophil-mediated killing of S. aureus, increased C5a release and modulation of IL-6 secretion. Finally, these miniAbs afforded protection from S. aureus-induced bacteremia in a murine renal abscess model, attenuating bacterial inflammation in kidneys. Overall, these findings are anticipated to pave the way towards novel antibody-based therapeutics for S. aureus-related diseases. PMID:26342032

Diagnosis of eight groups of xeroderma pigmentosum by genetic complementation using recombinant adenovirus vectors.

PubMed

Yamashita, Toshiharu; Okura, Masae; Ishii-Osai, Yasue; Hida, Tokimasa

2016-10-01

Because patients with xeroderma pigmentosum (XP) must avoid ultraviolet (UV) light from an early age, an early diagnosis of this disorder is essential. XP is composed of seven genetic complementation groups, XP-A to -G, and a variant type (XP-V). To establish an easy and accurate diagnosis of the eight disease groups, we constructed recombinant adenoviruses that expressed one of the XP cDNA. When fibroblasts derived from patients with XP-A, -B, -C, -D, -F or -G were infected with the adenovirus expressing XPA, XPB, XPC, XPD, XPF or XPG, respectively, and UV-C at 5-20 J/m 2 was irradiated, cell viability was clearly recovered by the corresponding recombinant adenoviruses. In contrast, XP-E and XP-V cells were not significantly sensitive to UV irradiation and were barely complemented by the matched recombinant adenoviruses. However, co-infection of Ad-XPA with Ad-XPE increased survival rate of XP-E cells after UV-C exposure. When XP-V cell strains, including one derived from a Japanese patient, were infected with Ad-XPV, exposed to UV-B and cultured with 1 mmol/L of caffeine, flow cytometry detected a characteristic decrease in the S phase in all the XP-V cell strains. From these results, the eight groups of XP could be differentiated by utilizing a set of recombinant adenoviruses, indicating that our procedure provides a convenient and correct diagnostic method for all the XP groups including XP-E and XP-V. © 2016 Japanese Dermatological Association.

Hereditary deficiency of the sixth component of complement in man. II. Studies of hemostasis.

PubMed Central

Heusinkveld, R S; Leddy, J P; Klemperer, M R; Breckenridge, R T

1974-01-01

Prompted by previous observations of defective blood clotting in rabbits deficient in the sixth component of complement (C6), an evaluation was made of the hemostatic functions of the homozygous proband of a newly recognized human kindred with hereditary C6 deficiency. This human subject, who had no clinical evidence of a bleeding disorder, exhibited a total lack of C6 by functional and immunoprecipitin assays of serum or plasma. Standard tests of hemostatic function were normal; however, when the whole blood clotting time was measured at 25 degrees C in plastic tubes, it was at the upper range of our normal values. In confirmation of this observation, prothrombin consumption, when performed at 37 degrees C in plastic tubes, was at the lower range of normal. Inulin and endotoxin, in concentrations shown to cause activation of human complement, had little or no effect on clotting times or prothrombin consumption of normal or C6-deficient human blood. These observations indicate that absence of C6 does not have a significant effect on hemostatic function in man. In the light of other investigations, the observed differences in clotting function between C6-deficient human blood and C6-deficient rabbit blood could be due to species differences governing the susceptibility of platelets to complement activation. PMID:11344569

Treatment with the C5a receptor antagonist ADC-1004 reduces myocardial infarction in a porcine ischemia-reperfusion model

PubMed Central

2010-01-01

Background Polymorphonuclear neutrophils, stimulated by the activated complement factor C5a, have been implicated in cardiac ischemia/reperfusion injury. ADC-1004 is a competitive C5a receptor antagonist that has been shown to inhibit complement related neutrophil activation. ADC-1004 shields the neutrophils from C5a activation before they enter the reperfused area, which could be a mechanistic advantage compared to previous C5a directed reperfusion therapies. We investigated if treatment with ADC-1004, according to a clinically applicable protocol, would reduce infarct size and microvascular obstruction in a large animal myocardial infarct model. Methods In anesthetized pigs (42-53 kg), a percutaneous coronary intervention balloon was inflated in the left anterior descending artery for 40 minutes, followed by 4 hours of reperfusion. Twenty minutes after balloon inflation the pigs were randomized to an intravenous bolus administration of ADC-1004 (175 mg, n = 8) or saline (9 mg/ml, n = 8). Area at risk (AAR) was evaluated by ex vivo SPECT. Infarct size and microvascular obstruction were evaluated by ex vivo MRI. The observers were blinded to the treatment at randomization and analysis. Results ADC-1004 treatment reduced infarct size by 21% (ADC-1004: 58.3 ± 3.4 vs control: 74.1 ± 2.9%AAR, p = 0.007). Microvascular obstruction was similar between the groups (ADC-1004: 2.2 ± 1.2 vs control: 5.3 ± 2.5%AAR, p = 0.23). The mean plasma concentration of ADC-1004 was 83 ± 8 nM at sacrifice. There were no significant differences between the groups with respect to heart rate, mean arterial pressure, cardiac output and blood-gas data. Conclusions ADC-1004 treatment reduces myocardial ischemia-reperfusion injury and represents a novel treatment strategy of myocardial infarct with potential clinical applicability. PMID:20875134

Treatment with the C5a receptor antagonist ADC-1004 reduces myocardial infarction in a porcine ischemia-reperfusion model.

PubMed

van der Pals, Jesper; Koul, Sasha; Andersson, Patrik; Götberg, Matthias; Ubachs, Joey F A; Kanski, Mikael; Arheden, Håkan; Olivecrona, Göran K; Larsson, Bengt; Erlinge, David

2010-09-27

Polymorphonuclear neutrophils, stimulated by the activated complement factor C5a, have been implicated in cardiac ischemia/reperfusion injury. ADC-1004 is a competitive C5a receptor antagonist that has been shown to inhibit complement related neutrophil activation. ADC-1004 shields the neutrophils from C5a activation before they enter the reperfused area, which could be a mechanistic advantage compared to previous C5a directed reperfusion therapies. We investigated if treatment with ADC-1004, according to a clinically applicable protocol, would reduce infarct size and microvascular obstruction in a large animal myocardial infarct model. In anesthetized pigs (42-53 kg), a percutaneous coronary intervention balloon was inflated in the left anterior descending artery for 40 minutes, followed by 4 hours of reperfusion. Twenty minutes after balloon inflation the pigs were randomized to an intravenous bolus administration of ADC-1004 (175 mg, n = 8) or saline (9 mg/ml, n = 8). Area at risk (AAR) was evaluated by ex vivo SPECT. Infarct size and microvascular obstruction were evaluated by ex vivo MRI. The observers were blinded to the treatment at randomization and analysis. ADC-1004 treatment reduced infarct size by 21% (ADC-1004: 58.3 ± 3.4 vs control: 74.1 ± 2.9%AAR, p = 0.007). Microvascular obstruction was similar between the groups (ADC-1004: 2.2 ± 1.2 vs control: 5.3 ± 2.5%AAR, p = 0.23). The mean plasma concentration of ADC-1004 was 83 ± 8 nM at sacrifice. There were no significant differences between the groups with respect to heart rate, mean arterial pressure, cardiac output and blood-gas data. ADC-1004 treatment reduces myocardial ischemia-reperfusion injury and represents a novel treatment strategy of myocardial infarct with potential clinical applicability.

Complement and the control of HIV infection: an evolving story.

PubMed

Frank, Michael M; Hester, Christopher; Jiang, Haixiang

2014-05-01

Thirty years ago, investigators isolated and later determined the structure of HIV-1 and its envelope proteins. Using techniques that were effective with other viruses, they prepared vaccines designed to generate antibody or T-cell responses, but they were ineffective in clinical trials. In this article, we consider the role of complement in host defense against enveloped viruses, the role it might play in the antibody response and why complement has not controlled HIV-1 infection. Complement consists of a large group of cell-bound and plasma proteins that are an integral part of the innate immune system. They provide a first line of defense against microbes and also play a role in the immune response. Here we review the studies of complement-mediated HIV destruction and the role of complement in the HIV antibody response. HIV-1 has evolved a complex defense to prevent complement-mediated killing reviewed here. As part of these studies, we have discovered that HIV-1 envelope, on administration into animals, is rapidly broken down into small peptides that may prove to be very inefficient at provident the type of antigenic stimulation that leads to an effective immune response. Improving complement binding and stabilizing envelope may improve the vaccine response.

Influenza-associated thrombotic microangiopathies.

PubMed

Bitzan, Martin; Zieg, Jakub

2017-09-07

Thrombotic microangiopathy (TMA) refers to phenotypically similar disorders, including hemolytic uremic syndromes (HUS) and thrombotic thrombocytopenic purpura (TTP). This review explores the role of the influenza virus as trigger of HUS or TTP. We conducted a literature survey in PubMed and Google Scholar using HUS, TTP, TMA, and influenza as keywords, and extracted and analyzed reported epidemiological and clinical data. We identified 25 cases of influenza-associated TMA. Five additional cases were linked to influenza vaccination and analyzed separately. Influenza A was found in 83%, 10 out of 25 during the 2009 A(H1N1) pandemic. Two patients had bona fide TTP with ADAMTS13 activity <10%. Median age was 15 years (range 0.5-68 years), two thirds were male. Oligoanuria was documented in 81% and neurological involvement in 40% of patients. Serum C3 was reduced in 5 out of 14 patients (36%); Coombs test was negative in 7 out of 7 and elevated fibrin/fibrinogen degradation products were documented in 6 out of 8 patients. Pathogenic complement gene mutations were found in 7 out of 8 patients tested (C3, MCP, or MCP combined with CFB or clusterin). Twenty out of 24 patients recovered completely, but 3 died (12%). Ten of the surviving patients underwent plasma exchange (PLEX) therapy, 5 plasma infusions. Influenza-mediated HUS or TTP is rare. A sizable proportion of tested patients demonstrated mutations associated with alternative pathway of complement dysregulation that was uncovered by this infection. Further research is warranted targeting the roles of viral neuraminidase, enhanced virus-induced complement activation and/or ADAMTS13 antibodies, and rational treatment approaches.

Apigenin prevents ultraviolet-B radiation induced cyclobutane pyrimidine dimers formation in human dermal fibroblasts.

PubMed

Britto, S Mary; Shanthakumari, D; Agilan, B; Radhiga, T; Kanimozhi, G; Prasad, N Rajendra

2017-09-01

Exposure to solar ultraviolet-B (UVB) radiation leads to the formation of cyclobutane pyrimidine dimers (CPDs). We investigated the protective effect of apigenin against UVB-induced CPDs formation in human dermal fibroblasts cells (HDFa). For this purpose, HDFa cells were treated with apigenin (15μM) prior to UVB irradiation (20mJ/cm 2 ); DNA damage and subsequent molecular end points were observed. Exposure to UVB radiation increased significant CPDs formation in HDFa cells and the frequencies of CPDs were reduced by treatment with apigenin (15μM). UVB-induced CPDs downregulates the expression of nucleotide excision repair (NER) genes such as xeroderma pigmentosum complementation group C, B, G and F (XPC, XPB, XPG and XPF), transcription factor II human (TFIIH) and excision repair cross-complementation group 1 (ERCC1) in HDFa cells. Conversely, apigenin treatment restored UVB-induced loss of NER proteins in HDFa cells, which indicates its preventive effect against CPDs formation. Besides, single low dose UVB-exposure induced nuclear fragmentation, apoptotic frequency and apoptotic proteins expression (Bax and Caspase-3) have been prevented by the apigenin pretreatment. Furthermore, apigenin exhibits strong UV absorbance property and showed 10.08 SPF value. Thus, apigenin can protect skin cells against UVB-induced CPDs formation probably through its sunscreen effect. Hence, apigenin can be considered as an effective protective agent against UV induced skin damages. Copyright © 2017 Elsevier B.V. All rights reserved.

Exploiting a novel conformational switch to control innate immunity mediated by complement protein C3a.

PubMed

Lohman, Rink-Jan; Hamidon, Johan K; Reid, Robert C; Rowley, Jessica A; Yau, Mei-Kwan; Halili, Maria A; Nielsen, Daniel S; Lim, Junxian; Wu, Kai-Chen; Loh, Zhixuan; Do, Anh; Suen, Jacky Y; Iyer, Abishek; Fairlie, David P

2017-08-24

Complement C3a is an important protein in innate and adaptive immunity, but its specific roles in vivo remain uncertain because C3a degrades rapidly to form the C3a-desArg protein, which does not bind to the C3a receptor and is indistinguishable from C3a using antibodies. Here we develop the most potent, stable and highly selective small molecule modulators of C3a receptor, using a heterocyclic hinge to switch between agonist and antagonist ligand conformations. This enables characterization of C3 areceptor-selective pro- vs. anti-inflammatory actions in human mast cells and macrophages, and in rats. A C3a receptor-selective agonist induces acute rat paw inflammation by first degranulating mast cells before activating macrophages and neutrophils. An orally administered C3a receptor-selective antagonist inhibits mast cell degranulation, thereby blocking recruitment and activation of macrophages and neutrophils, expression of inflammatory mediators and inflammation in a rat paw edema model. These novel tools reveal the mechanism of C3a-induced inflammation and provide new insights to complement-based medicines.Complement C3a is an important protein in innate and adaptive immunity, but its roles in vivo are unclear. Here the authors develop novel chemical agonists and antagonists for the C3a receptor, and show that they modulate mast cell degranulation and inflammation in a rat paw edema model.

Innate immune humoral factors, C1q and factor H, with differential pattern recognition properties, alter macrophage response to carbon nanotubes.

PubMed

Pondman, Kirsten M; Pednekar, Lina; Paudyal, Basudev; Tsolaki, Anthony G; Kouser, Lubna; Khan, Haseeb A; Shamji, Mohamed H; Ten Haken, Bennie; Stenbeck, Gudrun; Sim, Robert B; Kishore, Uday

2015-11-01

Interaction between the complement system and carbon nanotubes (CNTs) can modify their intended biomedical applications. Pristine and derivatised CNTs can activate complement primarily via the classical pathway which enhances uptake of CNTs and suppresses pro-inflammatory response by immune cells. Here, we report that the interaction of C1q, the classical pathway recognition molecule, with CNTs involves charge pattern and classical pathway activation that is partly inhibited by factor H, a complement regulator. C1q and its globular modules, but not factor H, enhanced uptake of CNTs by macrophages and modulated the pro-inflammatory immune response. Thus, soluble complement factors can interact differentially with CNTs and alter the immune response even without complement activation. Coating CNTs with recombinant C1q globular heads offers a novel way of controlling classical pathway activation in nanotherapeutics. Surprisingly, the globular heads also enhance clearance by phagocytes and down-regulate inflammation, suggesting unexpected complexity in receptor interaction. Carbon nanotubes (CNTs) maybe useful in the clinical setting as targeting drug carriers. However, it is also well known that they can interact and activate the complement system, which may have a negative impact on the applicability of CNTs. In this study, the authors functionalized multi-walled CNT (MWNT), and investigated the interaction with the complement pathway. These studies are important so as to gain further understanding of the underlying mechanism in preparation for future use of CNTs in the clinical setting. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Complement activating properties of complexes containing rheumatoid factor in synovial fluids and sera from patients with rheumatoid arthritis.

PubMed Central

Elson, C J; Carter, S D; Cottrell, B J; Scott, D G; Bacon, P A; Wallington, T B

1985-01-01

The relationship between complexes containing rheumatoid factor and complexes activating complement was examined in synovial fluids and sera from patients with rheumatoid arthritis (RA). In each case this was performed by quantifying the amount of rheumatoid factor bound by solid phase Fab'2 anti-C3 and/or solid phase conglutinin. Both anti-C3 coated and conglutinin coated microtitre plates bound high levels of complexes containing rheumatoid factor from sera of RA patients with vasculitis. Unexpectedly, these complexes were detected in synovial fluids from only a minority of RA patients with synovitis. However, RA synovial fluids did contain other complexes as shown by the presence of complement consuming activity, C1q binding material and immunoglobulin attaching to conglutinin. It is considered that in RA synovial fluids the complexes containing RF and those activating complement are not necessarily the same whilst in vasculitic sera the complexes containing rheumatoid factor also activate complement. PMID:3978872

Characterization of Zea mays endosperm C-24 sterol methyltransferase: one of two types of sterol methyltransferase in higher plants.

PubMed

Grebenok, R J; Galbraith, D W; Penna, D D

1997-08-01

We report the characterization of a higher-plant C-24 sterol methyltransferase by yeast complementation. A Zea mays endosperm expressed sequence tag (EST) was identified which, upon complete sequencing, showed 46% identity to the yeast C-24 methyltransferase gene (ERG6) and 75% and 37% amino acid identity to recently isolated higher-plant sterol methyltransferases from soybean and Arabidopsis, respectively. When placed under GALA regulation, the Z. mays cDNA functionally complemented the erg6 mutation, restoring ergosterol production and conferring resistance to cycloheximide. Complementation was both plasmid-dependent and galactose-inducible. The Z. mays cDNA clone contains an open reading frame encoding a 40 kDa protein containing motifs common to a large number of S-adenosyl-L-methionine methyltransferases (SMTs). Sequence comparisons and functional studies of the maize, soybean and Arabidopsis cDNAs indicates two types of C-24 SMTs exist in higher plants.

Selective expression of a splice variant of decay-accelerating factor in c-erbB-2-positive mammary carcinoma cells showing increased transendothelial invasiveness

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brandt, Burkhard; Mikesch, Jan-Hendrik; Simon, Ronald

2005-04-01

By differential-display-PCR a subclone of the SK-BR-3 cell line with high in vitro transendothelial invasiveness was identified to express increased levels of a new alternative splice variant of decay-accelerating factor (DAF). DAF seems to play an important role in some malignant tumours since on the one hand the expression of complement inhibitors on the surface of tumour cells prevents the accumulation of complement factors and in consequence cell lysis. On the other hand, DAF has been identified as a ligand for the CD97 surface receptor which induces cell migration. Immunofluorescence procedures, Western blot analyses, and cDNA clone sequencing were employedmore » to confirm the expression of DAF restricted to invasive tumour cells. Using a radioactive RNA-in situ hybridisation on freshly frozen tissue microarrays and RT-PCR on native tumour tissue, the expression of alternative spliced DAF mRNA was demonstrated in invasive breast cancer. Due to the fact that it could thereby not be detected in normal mammary tissues, it has to be confirmed in larger studies that the DAF splice variant might be a specific tumour marker for invasive breast cancer.« less

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation.

PubMed

Lekowski, R; Collard, C D; Reenstra, W R; Stahl, G L

2001-02-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress.

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation

PubMed Central

Lekowski, Robert; Collard, Charles D.; Reenstra, Wende R.; Stahl, Gregory L.

2001-01-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O2, 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 ± 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (≤ 100 μmol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC50 = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC50 ≈ 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress. PMID:11266613

Interaction of extremophilic archaeal viruses with human and mouse complement system and viral biodistribution in mice

PubMed Central

Wu, Linping; Uldahl, Kristine Buch; Chen, Fangfang; Benasutti, Halli; Logvinski, Deborah; Vu, Vivian; Banda, Nirmal K.; Peng, Xu; Simberg, Dmitri; Moghimi, Seyed Moein

2017-01-01

Archaeal viruses offer exceptional biophysical properties for modification and exploration of their potential in bionanotechnology, bioengineering and nanotherapeutic developments. However, the interaction of archaeal viruses with elements of the innate immune system has not been explored, which is a necessary prerequisite if their potential for biomedical applications to be realized. Here we show complement activation through lectin (via direct binding of MBL/MASPs) and alternative pathways by two extremophilic archaeal viruses (Sulfolobus monocaudavirus 1 and Sulfolobus spindle-shaped virus 2) in human serum. We further show some differences in initiation of complement activation pathways between these viruses. Since, Sulfolobus monocaudavirus 1 was capable of directly triggering the alternative pathway, we also demonstrate that the complement regulator factor H has no affinity for the viral surface, but factor H deposition is purely C3-dependent. This suggests that unlike some virulent pathogens Sulfolobus monocaudavirus 1 does not acquire factor H for protection. Complement activation with Sulfolobus monocaudavirus 1 also proceeds in murine sera through MBL-A/C as well as factor D-dependent manner, but C3 deficiency has no overall effect on viral clearance by organs of the reticuloendothelial system on intravenous injection. However, splenic deposition was significantly higher in C3 knockout animals compared with the corresponding wild type mice. We discuss the potential application of these viruses in biomedicine in relation to their complement activating properties. PMID:28846925

C-Reactive Protein as a Therapeutic Target in Age-Related Macular Degeneration.

PubMed

Molins, Blanca; Romero-Vázquez, Sara; Fuentes-Prior, Pablo; Adan, Alfredo; Dick, Andrew D

2018-01-01

Age-related macular degeneration (AMD), a retinal degenerative disease, is the leading cause of central vision loss among the elderly population in developed countries and an increasing global burden. The major risk is aging, compounded by other environmental factors and association with genetic variants for risk of progression. Although the etiology of AMD is not yet clearly understood, several pathogenic pathways have been proposed, including dysfunction of the retinal pigment epithelium, inflammation, and oxidative stress. The identification of AMD susceptibility genes encoding complement factors and the presence of complement and other inflammatory mediators in drusen, the hallmark deposits of AMD, support the concept that local inflammation and immune-mediated processes play a key role in AMD pathogenesis that may be accelerated through systemic immune activation. In this regard, increased levels of circulating C-reactive protein (CRP) have been associated with higher risk of AMD. Besides being a risk marker for AMD, CRP may also play a role in the progression of the disease as it has been identified in drusen, and we have recently found that its monomeric form (mCRP) induces blood retinal barrier disruption in vitro . In this review, we will address recent evidence that links CRP and AMD pathogenesis, which may open new therapeutic opportunities to prevent the progression of AMD.

The relevance of coagulation factor X protection of adenoviruses in human sera

PubMed Central

Duffy, M R; Doszpoly, A; Turner, G; Nicklin, S A; Baker, A H

2016-01-01

Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulation factor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5–FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad–FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy. PMID:27014840

Pathogenetic mechanisms in dengue haemorrhagic fever: Report of an international collaborative study*

PubMed Central

1973-01-01

In a study of 55 persons with dengue haemorrhagic fever—36 of whom showed the dengue shock syndrome—clinical, haematological, virological, and serological changes were correlated with serial measurements of complement components and immunopathological studies. Viruses dengue-1 or dengue-2 were isolated from the sera of 9 patients. Serological responses indicative of secondary dengue virus infections were observed in 53 patients; 2 (infants) had primary infections. During the acute phase of the disease, dengue antibody titres rose logarithmically. Marked depression of complement components, especially C3, was observed. Activation of both the classical and alternative complement pathways was demonstrated, with depression of both C4 and C3 proactivator levels in most instances, although in some cases it appeared that one mechanism was involved to a greater extent than the other. The level of depression of C3 was correlated with the severity of the disease. Relatively stable transferrin levels indicated that depletion of complement proteins was not primarily due to extravasation. Fibrinogen levels were depressed and fibrinogen split products were found in the plasma. The accumulated data provide further evidence of the central role that activated complement components play in the pathogenesis of dengue haemorrhagic fever. PMID:4575523

Fanconi Anemia complementation group C protein in metabolic disorders.

PubMed

Nepal, Manoj; Ma, Chi; Xie, Guoxiang; Jia, Wei; Fei, Peiwen

2018-06-21

Given importance of 22-Fanconi Anemia (FA) proteins together to act in a signaling pathway in preventing deleterious clinical symptoms, e.g. severe bone marrow failure, congenital defects, an early onset of aging and cancer, studies on each FA protein become increasingly attractive. However, an unbiased and systematic investigation of cellular effects resulting from each FA protein is missing. Here, we report roles of FA complementation C group protein (FANCC) in the protection from metabolic disorders. This study was prompted by the diabetes-prone feature displayed in FANCC knockout mice, which is not typically shown in patients with FA. We found that in cells expressing FANCC at different levels, there are representative alterations in metabolites associated with aging (glycine, citrulline, ornithine, L-asparagine, L-tyrosine, L-arginine, L-glutamine, L-leucine, L-isoleucine, L-valine, L-proline and L-alanine), Diabetes Mellitus (DM) (carbon monoxide, collagens, fatty acids, D-glucose, fumaric acid, 2-oxoglutaric acid, C3), inflammation (inosine, L-arginine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, hypoxanthine, L-methionine), and cancer ( L-methionine, sphingomyelin, acetyl-L-carnitine, L-aspartic acid, L-glutamic acid, niacinamide, phospho-rylethanolamine). We also found that FANCC can act in an FA-pathway-independent manner in tumor suppression. Collectively, featured-metabolic alterations are readouts of functional mechanisms underlying reduced tumorigenicity driven by FANCC, demonstrating close links among cancer, aging, inflammation and DM.

Gene for ataxia-telangiectasia complementation group D (ATDC)

DOEpatents

Murnane, John P.; Painter, Robert B.; Kapp, Leon N.; Yu, Loh-Chung

1995-03-07

Disclosed herein is a new gene, an AT gene for complementation group D, the ATDC gene and fragments thereof. Nucleic acid probes for said gene are provided as well as proteins encoded by said gene, cDNA therefrom, preferably a 3 kilobase (kb) cDNA, and recombinant nucleic acid molecules for expression of said proteins. Further disclosed are methods to detect mutations in said gene, preferably methods employing the polymerase chain reaction (PCR). Also disclosed are methods to detect AT genes from other AT complementation groups.

A C3(H20) recycling pathway is a component of the intracellular complement system

PubMed Central

Elvington, Michelle; Bertram, Paula; Atkinson, John P.

2017-01-01

An intracellular complement system (ICS) has recently been described in immune and nonimmune human cells. This system can be activated in a convertase-independent manner from intracellular stores of the complement component C3. The source of these stores has not been rigorously investigated. In the present study, Western blotting identified a band corresponding to C3 in freshly isolated human peripheral blood cells that was absent in corresponding cell lines. One difference between native cells and cell lines was the time absent from a fluid-phase complement source; therefore, we hypothesized that loading C3 from plasma was a route of establishing intracellular C3 stores. We found that many types of human cells specifically internalized C3(H2O), the hydrolytic product of C3, and not native C3, from the extracellular milieu. Uptake was rapid, saturable, and sensitive to competition with unlabeled C3(H2O), indicating a specific mechanism of loading. Under steady-state conditions, approximately 80% of incorporated C3(H2O) was returned to the extracellular space. These studies identify an ICS recycling pathway for C3(H2O). The loaded C3(H2O) represents a source of C3a, and its uptake altered the cytokine profile of activated CD4+ T cells. Importantly, these results indicate that the impact of soluble plasma factors should be considered when performing in vitro studies assessing cellular immune function. PMID:28192370

RAN Translation as a Therapeutic in ALS

DTIC Science & Technology

2017-05-01

allow for HTS via CRISPR or drug screens to complement the in vitro screens using high-throughput microscopy or FACS. Figure 5: Mammalian G4C2...poly-GP in yeast (Figure 6A). [filler about RPS25 here?] This effect was further investigated in mammalian Hap1 cell lines with a CRISPR -mediated

A selection that reports on protein–protein interactions within a thermophilic bacterium

PubMed Central

Nguyen, Peter Q.; Silberg, Jonathan J.

2010-01-01

Many proteins can be split into fragments that exhibit enhanced function upon fusion to interacting proteins. While this strategy has been widely used to create protein-fragment complementation assays (PCAs) for discovering protein–protein interactions within mesophilic organisms, similar assays have not yet been developed for studying natural and engineered protein complexes at the temperatures where thermophilic microbes grow. We describe the development of a selection for protein–protein interactions within Thermus thermophilus that is based upon growth complementation by fragments of Thermotoga neapolitana adenylate kinase (AKTn). Complementation studies with an engineered thermophile (PQN1) that is not viable above 75°C because its adk gene has been replaced by a Geobacillus stearothermophilus ortholog revealed that growth could be restored at 78°C by a vector that coexpresses polypeptides corresponding to residues 1–79 and 80–220 of AKTn. In contrast, PQN1 growth was not complemented by AKTn fragments harboring a C156A mutation within the zinc-binding tetracysteine motif unless these fragments were fused to Thermotoga maritima chemotaxis proteins that heterodimerize (CheA and CheY) or homodimerize (CheX). This enhanced complementation is interpreted as arising from chemotaxis protein–protein interactions, since AKTn-C156A fragments having only one polypeptide fused to a chemotaxis protein did not complement PQN1 to the same extent. This selection increases the maximum temperature where a PCA can be used to engineer thermostable protein complexes and to map protein–protein interactions. PMID:20418388

A selection that reports on protein-protein interactions within a thermophilic bacterium.

PubMed

Nguyen, Peter Q; Silberg, Jonathan J

2010-07-01

Many proteins can be split into fragments that exhibit enhanced function upon fusion to interacting proteins. While this strategy has been widely used to create protein-fragment complementation assays (PCAs) for discovering protein-protein interactions within mesophilic organisms, similar assays have not yet been developed for studying natural and engineered protein complexes at the temperatures where thermophilic microbes grow. We describe the development of a selection for protein-protein interactions within Thermus thermophilus that is based upon growth complementation by fragments of Thermotoga neapolitana adenylate kinase (AK(Tn)). Complementation studies with an engineered thermophile (PQN1) that is not viable above 75 degrees C because its adk gene has been replaced by a Geobacillus stearothermophilus ortholog revealed that growth could be restored at 78 degrees C by a vector that coexpresses polypeptides corresponding to residues 1-79 and 80-220 of AK(Tn). In contrast, PQN1 growth was not complemented by AK(Tn) fragments harboring a C156A mutation within the zinc-binding tetracysteine motif unless these fragments were fused to Thermotoga maritima chemotaxis proteins that heterodimerize (CheA and CheY) or homodimerize (CheX). This enhanced complementation is interpreted as arising from chemotaxis protein-protein interactions, since AK(Tn)-C156A fragments having only one polypeptide fused to a chemotaxis protein did not complement PQN1 to the same extent. This selection increases the maximum temperature where a PCA can be used to engineer thermostable protein complexes and to map protein-protein interactions.

Polysialic acid blocks mononuclear phagocyte reactivity, inhibits complement activation, and protects from vascular damage in the retina.

PubMed

Karlstetter, Marcus; Kopatz, Jens; Aslanidis, Alexander; Shahraz, Anahita; Caramoy, Albert; Linnartz-Gerlach, Bettina; Lin, Yuchen; Lückoff, Anika; Fauser, Sascha; Düker, Katharina; Claude, Janine; Wang, Yiner; Ackermann, Johannes; Schmidt, Tobias; Hornung, Veit; Skerka, Christine; Langmann, Thomas; Neumann, Harald

2017-02-01

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly population. Its pathophysiology is linked to reactive oxygen species (ROS) and activation of the complement system. Sialic acid polymers prevent ROS production of human mononuclear phagocytes via the inhibitory sialic acid-binding immunoglobulin-like lectin-11 (SIGLEC11) receptor. Here, we show that low-dose intravitreal injection of low molecular weight polysialic acid with average degree of polymerization 20 (polySia avDP20) in humanized transgenic mice expressing SIGLEC11 on mononuclear phagocytes reduced their reactivity and vascular leakage induced by laser coagulation. Furthermore, polySia avDP20 prevented deposition of the membrane attack complex in both SIGLEC11 transgenic and wild-type animals. In vitro, polySia avDP20 showed two independent, but synergistic effects on the innate immune system. First, polySia avDP20 prevented tumor necrosis factor-α, vascular endothelial growth factor A, and superoxide production by SIGLEC11-positive phagocytes. Second, polySia avDP20 directly interfered with complement activation. Our data provide evidence that polySia avDP20 ameliorates laser-induced damage in the retina and thus is a promising candidate to prevent AMD-related inflammation and angiogenesis. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Modulation of risk of squamous cell carcinoma head and neck in North Indian population with polymorphisms in xeroderma pigmentosum complementation Group C gene.

PubMed

Yadav, Suresh Kumar; Singh, Sudhir; Gupta, Shalini; Brahma Bhatt, Madan Lal; Mishra, Durga P; Roy, D; Sanyal, Somali

2018-01-01

Genetic variations in nucleotide excision repair genes can alter the risk of squamous cell carcinoma of head and neck (SCCHN). The present study has genotyped 334 subjects from North Indian population for xeroderma pigmentosum complementation Group C (XPC) rs2228001A>C, XPC rs77907221 polyadenylate (PAT) deletion/insertion (D/I), xeroderma pigmentosum complementation Group D - rs13181A>C, and xeroderma pigmentosum complementation Type G rs17655 G>C polymorphisms with polymerase chain reaction (PCR)-restriction-fragment length polymorphism or allele-specific PCR methods. Compared to D allele, I allele for XPC PAT D/I polymorphism was associated with significantly decreased the risk of SCCHN (odds ratios = 0.67, 95% confidence interval [CI] =0.48-0.94, P = 0.03). Haplotype CI constituted from XPC polymorphisms was also associated with decreased risk of SCCHN (P = 0.004). In contrast, haplotype Crohn's disease significantly increased the risk for SCCHN (P < 0.00). A significant early onset of SCCHN was observed in individuals with CC genotype for XPC A>C polymorphism (P = 0.004). Our results suggest a possible risk modulation for SCCHN with XPC polymorphisms in North Indian population.

Antimicrobial Peptides and Complement in Neonatal Hypoxia-Ischemia Induced Brain Damage

PubMed Central

Rocha-Ferreira, Eridan; Hristova, Mariya

2015-01-01

Hypoxic-ischemic encephalopathy (HIE) is a clinical condition in the neonate, resulting from oxygen deprivation around the time of birth. HIE affects 1–5/1000 live births worldwide and is associated with the development of neurological deficits, including cerebral palsy, epilepsy, and cognitive disabilities. Even though the brain is considered as an immune-privileged site, it has innate and adaptive immune response and can produce complement (C) components and antimicrobial peptides (AMPs). Dysregulation of cerebral expression of AMPs and C can exacerbate or ameliorate the inflammatory response within the brain. Brain ischemia triggers a prolonged inflammatory response affecting the progression of injury and secondary energy failure and involves both innate and adaptive immune systems, including immune-competent and non-competent cells. Following injury to the central nervous system (CNS), including neonatal hypoxia-ischemia (HI), resident microglia, and astroglia are the main cells providing immune defense to the brain in a stimulus-dependent manner. They can express and secrete pro-inflammatory cytokines and therefore trigger prolonged inflammation, resulting in neurodegeneration. Microglial cells express and release a wide range of inflammation-associated molecules including several components of the complement system. Complement activation following neonatal HI injury has been reported to contribute to neurodegeneration. Astrocytes can significantly affect the immune response of the CNS under pathological conditions through production and release of pro-inflammatory cytokines and immunomodulatory AMPs. Astrocytes express β-defensins, which can chemoattract and promote maturation of dendritic cells (DC), and can also limit inflammation by controlling the viability of these same DC. This review will focus on the balance of complement components and AMPs within the CNS following neonatal HI injury and the effect of that balance on the subsequent brain damage. PMID:25729383