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Sample records for complement virus opsonization

  1. Complement Opsonization Promotes Herpes Simplex Virus 2 Infection of Human Dendritic Cells

    PubMed Central

    Ellegård, Rada; Nyström, Sofia; Rondahl, Elin; Serrander, Lena; Bergström, Tomas; Sjöwall, Christopher; Eriksson, Kristina

    2016-01-01

    ABSTRACT Herpes simplex virus 2 (HSV-2) is one of the most common sexually transmitted infections globally, with a very high prevalence in many countries. During HSV-2 infection, viral particles become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of immune responses. In genital mucosa, the primary target cells for HSV-2 infection are epithelial cells, but resident immune cells, such as dendritic cells (DCs), are also infected. DCs are the activators of the ensuing immune responses directed against HSV-2, and the aim of this study was to examine the effects opsonization of HSV-2, either with complement alone or with complement and antibodies, had on the infection of immature DCs and their ability to mount inflammatory and antiviral responses. Complement opsonization of HSV-2 enhanced both the direct infection of immature DCs and their production of new infectious viral particles. The enhanced infection required activation of the complement cascade and functional complement receptor 3. Furthermore, HSV-2 infection of DCs required endocytosis of viral particles and their delivery into an acid endosomal compartment. The presence of complement in combination with HSV-1- or HSV-2-specific antibodies more or less abolished HSV-2 infection of DCs. Our results clearly demonstrate the importance of studying HSV-2 infection under conditions that ensue in vivo, i.e., conditions under which the virions are covered in complement fragments and complement fragments and antibodies, as these shape the infection and the subsequent immune response and need to be further elucidated. IMPORTANCE During HSV-2 infection, viral particles should become coated with complement proteins and antibodies, both present in genital fluids, which could influence the activation of the immune responses. The dendritic cells are activators of the immune responses directed against HSV-2, and the aim of this study was to examine the

  2. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1

    PubMed Central

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M.; Nyström, Sofia; Hinkula, Jorma

    2015-01-01

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection. PMID:26157174

  3. Impaired NK Cell Activation and Chemotaxis toward Dendritic Cells Exposed to Complement-Opsonized HIV-1.

    PubMed

    Ellegård, Rada; Crisci, Elisa; Andersson, Jonas; Shankar, Esaki M; Nyström, Sofia; Hinkula, Jorma; Larsson, Marie

    2015-08-15

    Mucosa resident dendritic cells (DCs) may represent one of the first immune cells that HIV-1 encounters during sexual transmission. The virions in body fluids can be opsonized with complement factors because of HIV-mediated triggering of the complement cascade, and this appears to influence numerous aspects of the immune defense targeting the virus. One key attribute of host defense is the ability to attract immune cells to the site of infection. In this study, we investigated whether the opsonization of HIV with complement (C-HIV) or a mixture of complement and Abs (CI-HIV) affected the cytokine and chemokine responses generated by DCs, as well as their ability to attract other immune cells. We found that the expression levels of CXCL8, CXCL10, CCL3, and CCL17 were lowered after exposure to either C-HIV or CI-HIV relative to free HIV (F-HIV). DCs exposed to F-HIV induced higher cell migration, consisting mainly of NK cells, compared with opsonized virus, and the chemotaxis of NK cells was dependent on CCL3 and CXCL10. NK cell exposure to supernatants derived from HIV-exposed DCs showed that F-HIV induced phenotypic activation (e.g., increased levels of TIM3, CD69, and CD25) and effector function (e.g., production of IFNγ and killing of target cells) in NK cells, whereas C-HIV and CI-HIV did not. The impairment of NK cell recruitment by DCs exposed to complement-opsonized HIV and the lack of NK activation may contribute to the failure of innate immune responses to control HIV at the site of initial mucosa infection. PMID:26157174

  4. Complement-Opsonized HIV-1 Overcomes Restriction in Dendritic Cells

    PubMed Central

    Posch, Wilfried; Steger, Marion; Knackmuss, Ulla; Blatzer, Michael; Baldauf, Hanna-Mari; Doppler, Wolfgang; White, Tommy E.; Hörtnagl, Paul; Diaz-Griffero, Felipe; Lass-Flörl, Cornelia; Hackl, Hubert; Moris, Arnaud; Keppler, Oliver T.; Wilflingseder, Doris

    2015-01-01

    DCs express intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. Thus, DCs are productively infected only at very low levels with HIV-1, and this non-permissiveness of DCs is suggested to go along with viral evasion. We now illustrate that complement-opsonized HIV-1 (HIV-C) efficiently bypasses SAMHD1 restriction and productively infects DCs including BDCA-1 DCs. Efficient DC infection by HIV-C was also observed using single-cycle HIV-C, and correlated with a remarkable elevated SAMHD1 T592 phosphorylation but not SAMHD1 degradation. If SAMHD1 phosphorylation was blocked using a CDK2-inhibitor HIV-C-induced DC infection was also significantly abrogated. Additionally, we found a higher maturation and co-stimulatory potential, aberrant type I interferon expression and signaling as well as a stronger induction of cellular immune responses in HIV-C-treated DCs. Collectively, our data highlight a novel protective mechanism mediated by complement opsonization of HIV to effectively promote DC immune functions, which might be in the future exploited to tackle HIV infection. PMID:26121641

  5. Complement receptor type 2 mediates infection of the human CD4-negative Raji B-cell line with opsonized HIV.

    PubMed

    Boyer, V; Delibrias, C; Noraz, N; Fischer, E; Kazatchkine, M D; Desgranges, C

    1992-12-01

    Opsonization of the HTLV-RF and HTLV-IIIB strains of HIV-1 with normal human HIV seronegative serum under conditions that allow complement activation resulted in the productive infection of cells of the Raji B lymphoblastoid cell line. Under the same experimental conditions, no infection of Raji cells was observed with unopsonized virus. Infection of Raji cells with complement-opsonized HIV-1 was totally suppressed by preblocking the function of CR2 (the C3dg receptor, CD21) on the cells with a monoclonal anti-CR2 antibody cross-linked with rabbit F(ab')2 anti-mouse immunoglobulin antibodies. Infection of Raji cells occurred independently of CD4 since the cells lacked the expression of CD4 antigen and of CD4 transcripts. Thus, Raji cells may be infected with complement-opsonized HIV independently of CD4 and in the absence of antibodies. By mediating and/or enhancing HIV infection, complement and complement receptors contribute to extend the range of target cells to the virus and may increase infection in patients with a low viral load. PMID:1281336

  6. Complement Opsonization of HIV-1 Results in Decreased Antiviral and Inflammatory Responses in Immature Dendritic Cells via CR3

    PubMed Central

    Ellegård, Rada; Crisci, Elisa; Burgener, Adam; Sjöwall, Christopher; Birse, Kenzie; Westmacott, Garrett; Hinkula, Jorma; Lifson, Jeffrey D.

    2014-01-01

    Immature dendritic cells (iDCs) in genital and rectal mucosa may be one of the first cells to come into contact with HIV-1 during sexual transmission of virus. HIV-1 activates the host complement system, which results in opsonization of virus by inactivated complement fragments, for example, iC3b. We investigated antiviral and inflammatory responses induced in human iDCs after exposure to free HIV-1 (F-HIV), complement-opsonized HIV-1 (C-HIV), and complement and Ab–opsonized HIV-1 (CI-HIV). F-HIV gave rise to a significantly higher expression of antiviral factors such as IFN-β, myxovirus resistance protein A, and IFN-stimulated genes, compared with C-HIV and CI-HIV. Additionally, F-HIV induced inflammatory factors such as IL-1β, IL-6, and TNF-α, whereas these responses were weakened or absent after C-HIV or CI-HIV exposure. The responses induced by F-HIV were TLR8-dependent with subsequent activation of IFN regulatory factor 1, p38, ERK, PI3K, and NF-κB pathways, whereas these responses were not induced by C-HIV, which instead induced activation of IFN regulatory factor 3 and Lyn. This modulation of TLR8 signaling was mediated by complement receptor 3 and led to enhanced infection. The impact that viral hijacking of the complement system has on iDC function could be an important immune evasion mechanism used by HIV-1 to establish infection in the host. PMID:25252956

  7. Modulatory Role of Surface Coating of Superparamagnetic Iron Oxide Nanoworms in Complement Opsonization and Leukocyte Uptake.

    PubMed

    Inturi, Swetha; Wang, Guankui; Chen, Fangfang; Banda, Nirmal K; Holers, V Michael; Wu, LinPing; Moghimi, Seyed Moein; Simberg, Dmitri

    2015-11-24

    Notwithstanding rapid advances of nanotechnology in diagnostic imaging and drug delivery, the engineered nanocarriers still exhibit substantial lack of hemocompatibility. Thus, when injected systemically, nanoparticles are avidly recognized by blood leukocytes and platelets, but the mechanisms of immune recognition are not well understood and strategies to mitigate these phenomena remain underexplored. Using superparamagnetic dextran iron oxide (SPIO) nanoworms (NWs) we demonstrate an efficient and predominantly complement-dependent uptake by mouse lymphocytes, neutrophils and monocytes from normal and tumor bearing mice in vitro. Following intravenous injection into wild type mice, blood leukocytes as well as platelets became magnetically labeled, while the labeling was decreased by 95% in complement C3-deficient mice. Using blood cells from healthy and cancer patient donors, we demonstrated that neutrophils, monocytes, lymphocytes and eosinophils took up SPIO NWs, and the uptake was prevented by EDTA (a general complement inhibitor) and by antiproperdin antibody (an inhibitor of the alternative pathway of the complement system). Cross-linking and hydrogelation of SPIO NWs surface by epichlorohydrin decreased C3 opsonization in mouse serum, and consequently reduced the uptake by mouse leukocytes by more than 70% in vivo. Remarkably, the cross-linked particles did not show a decrease in C3 opsonization in human serum, but showed a significant decrease (over 60%) of the uptake by human leukocytes. The residual uptake of cross-linked nanoparticles was completely blocked by EDTA. These findings demonstrate species differences in complement-mediated nanoparticle recognition and uptake by leukocytes, and further show that human hemocompatibility could be improved by inhibitors of complement alternative pathway and by nanoparticle surface coating. These results provide important insights into the mechanisms of hemocompatibility of nanomedicines.

  8. Modulatory Role of Surface Coating of Superparamagnetic Iron Oxide Nanoworms in Complement Opsonization and Leukocyte Uptake.

    PubMed

    Inturi, Swetha; Wang, Guankui; Chen, Fangfang; Banda, Nirmal K; Holers, V Michael; Wu, LinPing; Moghimi, Seyed Moein; Simberg, Dmitri

    2015-11-24

    Notwithstanding rapid advances of nanotechnology in diagnostic imaging and drug delivery, the engineered nanocarriers still exhibit substantial lack of hemocompatibility. Thus, when injected systemically, nanoparticles are avidly recognized by blood leukocytes and platelets, but the mechanisms of immune recognition are not well understood and strategies to mitigate these phenomena remain underexplored. Using superparamagnetic dextran iron oxide (SPIO) nanoworms (NWs) we demonstrate an efficient and predominantly complement-dependent uptake by mouse lymphocytes, neutrophils and monocytes from normal and tumor bearing mice in vitro. Following intravenous injection into wild type mice, blood leukocytes as well as platelets became magnetically labeled, while the labeling was decreased by 95% in complement C3-deficient mice. Using blood cells from healthy and cancer patient donors, we demonstrated that neutrophils, monocytes, lymphocytes and eosinophils took up SPIO NWs, and the uptake was prevented by EDTA (a general complement inhibitor) and by antiproperdin antibody (an inhibitor of the alternative pathway of the complement system). Cross-linking and hydrogelation of SPIO NWs surface by epichlorohydrin decreased C3 opsonization in mouse serum, and consequently reduced the uptake by mouse leukocytes by more than 70% in vivo. Remarkably, the cross-linked particles did not show a decrease in C3 opsonization in human serum, but showed a significant decrease (over 60%) of the uptake by human leukocytes. The residual uptake of cross-linked nanoparticles was completely blocked by EDTA. These findings demonstrate species differences in complement-mediated nanoparticle recognition and uptake by leukocytes, and further show that human hemocompatibility could be improved by inhibitors of complement alternative pathway and by nanoparticle surface coating. These results provide important insights into the mechanisms of hemocompatibility of nanomedicines. PMID:26488074

  9. Blocking of integrins inhibits HIV-1 infection of human cervical mucosa immune cells with free and complement-opsonized virions

    PubMed Central

    Tjomsland, Veronica; Ellegård, Rada; Kjölhede, Preben; Wodlin, Ninni Borendal; Hinkula, Jorma; Lifson, Jeffrey D; Larsson, Marie

    2013-01-01

    The initial interaction between HIV-1 and the host occurs at the mucosa during sexual intercourse. In cervical mucosa, HIV-1 exists both as free and opsonized virions and this might influence initial infection. We used cervical explants to study HIV-1 transmission, the effects of opsonization on infectivity, and how infection can be prevented. Complement opsonization enhanced HIV-1 infection of dendritic cells (DCs) compared with that by free HIV-1, but this increased infection was not observed with CD4+ T cells. Blockage of the α4-, β7-, and β1-integrins significantly inhibited HIV-1 infection of both DCs and CD4+ T cells. We found a greater impairment of HIV-1 infection in DCs for complement-opsonized virions compared with that of free virions when αM/β2- and α4-integrins were blocked. Blocking the C-type lectin receptor macrophage mannose receptor (MMR) inhibited infection of emigrating DCs but had no effect on CD4+ T-cell infection. We show that blocking of integrins decreases the HIV-1 infection of both mucosal DCs and CD4+ T cells emigrating from the cervical tissues. These findings may provide the basis of novel microbicidal strategies that may help limit or prevent initial infection of the cervical mucosa, thereby reducing or averting systemic HIV-1 infection. PMID:23686382

  10. Complement opsonization of HIV-1 results in a different intracellular processing pattern and enhanced MHC class I presentation by dendritic cells

    PubMed Central

    Tjomsland, Veronica; Ellegård, Rada; Burgener, Adam; Mogk, Kenzie; Che, Karlhans F; Westmacott, Garrett; Hinkula, Jorma; Lifson, Jeffrey D; Larsson, Marie

    2013-01-01

    Induction of optimal HIV-1-specific T-cell responses, which can contribute to controlling viral infection in vivo, depends on antigen processing and presentation processes occurring in DCs. Opsonization can influence the routing of antigen processing and pathways used for presentation. We studied antigen proteolysis and the role of endocytic receptors in MHC class I (MHCI) and II (MHCII) presentation of antigens derived from HIV-1 in human monocyte-derived immature DCs (IDCs) and mature DCs, comparing free and complement opsonized HIV-1 particles. Opsonization of virions promoted MHCI presentation by DCs, indicating that complement opsonization routes more virions toward the MHCI presentation pathway. Blockade of macrophage mannose receptor (MMR) and β7-integrin enhanced MHCI and MHCII presentation by IDCs and mature DCs, whereas the block of complement receptor 3 decreased MHCI and MHCII presentation. In addition, we found that IDC and MDC proteolytic activities were modulated by HIV-1 exposure; complement-opsonized HIV-1 induced an increased proteasome activity in IDCs. Taken together, these findings indicate that endocytic receptors such as MMR, complement receptor 3, and β7-integrin can promote or disfavor antigen presentation probably by routing HIV-1 into different endosomal compartments with distinct efficiencies for degradation of viral antigens and MHCI and MHCII presentation, and that HIV-1 affects the antigen-processing machinery. PMID:23526630

  11. Immunoglobulin isotype isolated from human placental extract does not interfere in complement-mediated bacterial opsonization within the wound milieu

    PubMed Central

    Sharma, Kanika; Bhattacharyya, Debasish

    2015-01-01

    The wound healing potency of an aqueous extract of placenta can be evaluated through the presence of numerous regulatory components. The presence of glycans was detected by thin layer chromatography and fluorophore-assisted carbohydrate electrophoresis. Mass spectrometric analysis revealed the existence of multiple fragments of immunoglobulin G (IgG). IgG was present in the extract at a concentration of 25.2 ± 3.97 μg/ml. IgG possesses anti-complementary activity by diverting the complement activation from target surface. Thus, effect of placental IgG on complement–bacteria interaction was investigated through classical and alternative pathway and the preparation was ascertained to be safe with respect to their interference in the process of bacterial opsonization. PMID:25984442

  12. Relative importance of complement-mediated bactericidal and opsonic activity for protection against meningococcal disease.

    PubMed

    Granoff, Dan M

    2009-06-24

    Killing of Neisseria meningitidis can result from complement-mediated serum bactericidal activity (SBA) or opsonophagocytosis (OPA), or a combination of the two mechanisms. While SBA titers > or =1:4 confer protection, recent evidence suggests that this threshold titer may not be required. For example, the incidence of meningococcal disease declines between ages 1 and 4 years without evidence of acquisition of SBA titers > or =1:4. Meningococcal polysaccharide vaccination also elicited OPA and lowered the risk of disease in patients with late complement component deficiencies whose sera did not support SBA. Sera from healthy adults immunized with an outer membrane vesicle vaccine showed OPA killing of N. meningitidis with C6-depleted complement, and whole blood from complement-sufficient non-immunized adults with SBA titers <1:4 also frequently had killing activity. Collectively the data indicate that SBA titers <1:4 and/or vaccine-induced OPA can confer protection against meningococcal disease.

  13. Human seminal plasma inhibition of antibody complement-mediated killing and opsonization of Neisseria gonorrhoeae and other gram-negative organisms.

    PubMed Central

    Brooks, G F; Lammel, C J; Petersen, B H; Stites, D P

    1981-01-01

    Seminal plasma diluted 1:5-1:1,000 gave marked inhibition of serum antibody complement-mediated bactericidal and opsonic effects against Neisseria gonorrhoeae and other gram-negative organisms. Serum that was bactericidal at a dilution of 1:5,120 was not bactericidal at a dilution of 1:10 when seminal plasma was added. Bactericidal action of immune human or rabbit sera, or purified immunoglobulin (Ig)G or IgM plus complement for six strains of N. gonorrhoeae, serogroups A, B, C, and Y of Neisseria meningitidis, Escherichia coli and other gram-negative rods was inhibited by seminal plasma. Using C8- or C7-deficient sera as antibody and complement sources, opsonization, phagocytosis, and killing of N. gonorrhoeae and E. coli 014-K7 were inhibited by seminal plasma. Opsonization, phagocytosis, and killing of Staphylococcus aureus 502A was not inhibited. For the gram-negative organisms, the early phase of the opsonization process, probably complement activation, appeared to be inhibited rather than the ingestion or polymorphonuclear leukocyte killing steps; addition of seminal plasma yielded a significant reduction in the percentage of polymorphonuclear cells with associated bacteria. Seminal plasma did not prevent attachment of IgG, IgM, or IgA antibodies to gonococci. It reduced serum hemolytic whole complement activity by 25%. The seminal plasma inhibitor was of low molecular weight and was stable at 56 degrees C for 30 min, but inhibitory activity was lost after heating to 100 degrees C for 10 min. It is likely that the inhibitory factor(s) is a low-molecular weight protease or protease inhibitor. Seminal plasma probably has an important role in inhibition of complement and antibody functions in the genital tract. It may enhance pathogenesis of agents of sexually transmitted diseases. PMID:6785314

  14. Complement regulator C4BP binds to Staphylococcus aureus surface proteins SdrE and Bbp inhibiting bacterial opsonization and killing☆

    PubMed Central

    Hair, Pamela S.; Foley, Caitlin K.; Krishna, Neel K.; Nyalwidhe, Julius O.; Geoghegan, Joan A.; Foster, Timothy J.; Cunnion, Kenji M.

    2013-01-01

    Staphylococcus aureus is a premier human pathogen and the most common cause of osteoarticular, wound, and implanted device infections. We recently demonstrated S. aureus efficiently binds the classical complement regulator C4b-binding protein (C4BP) inhibiting antibody-initiated complement-mediated opsonization. Here we identify S. aureus surface protein SdrE as a C4BP-binding protein. Recombinant SdrE and recombinant bone sialoprotein-binding protein (Bbp), an allelic variant of SdrE, both efficiently bound to C4BP in heat-inactivated human serum. We previously described SdrE as binding alternative pathway regulator factor H. Recombinant SdrE and Bbp efficiently bound C4BP and factor H in serum without apparent interference. Gain of function studies utilizing Lactococcus lactis clones expressing SdrE or Bbp increased serum C4BP and factor H binding, compared with empty-vector control (WT) approximately 2-fold. Correspondingly, classical pathway-mediated C3-fragment opsonization and bacterial killing by human neutrophils decreased by half for L. lactis clones expressing SdrE or Bbp compared with WT. In summary, we identify SdrE and allelic variant Bbp as S. aureus surface proteins that bind the complement regulator C4BP inhibiting classical pathway-mediated bacterial opsonization and killing. PMID:24600566

  15. Borrelia recurrentis Employs a Novel Multifunctional Surface Protein with Anti-Complement, Anti-Opsonic and Invasive Potential to Escape Innate Immunity

    PubMed Central

    Grosskinsky, Sonja; Schott, Melanie; Brenner, Christiane; Cutler, Sally J.; Kraiczy, Peter; Zipfel, Peter F.; Simon, Markus M.; Wallich, Reinhard

    2009-01-01

    Borrelia recurrentis, the etiologic agent of louse-borne relapsing fever in humans, has evolved strategies, including antigenic variation, to evade immune defence, thereby causing severe diseases with high mortality rates. Here we identify for the first time a multifunctional surface lipoprotein of B. recurrentis, termed HcpA, and demonstrate that it binds human complement regulators, Factor H, CFHR-1, and simultaneously, the host protease plasminogen. Cell surface bound factor H was found to retain its activity and to confer resistance to complement attack. Moreover, ectopic expression of HcpA in a B. burgdorferi B313 strain, deficient in Factor H binding proteins, protected the transformed spirochetes from complement-mediated killing. Furthermore, HcpA-bound plasminogen/plasmin endows B. recurrentis with the potential to resist opsonization and to degrade extracellular matrix components. Together, the present study underscores the high virulence potential of B. recurrentis. The elucidation of the molecular basis underlying the versatile strategies of B. recurrentis to escape innate immunity and to persist in human tissues, including the brain, may help to understand the pathological processes underlying louse-borne relapsing fever. PMID:19308255

  16. Complement mediates human immunodeficiency virus type 1 infection of a human T cell line in a CD4- and antibody-independent fashion.

    PubMed

    Boyer, V; Desgranges, C; Trabaud, M A; Fischer, E; Kazatchkine, M D

    1991-05-01

    Incubation of the human T cell lymphotropic virus (HTLV)-IIIB and HTLV-RF strains of human immunodeficiency virus type 1 (HIV-1) with normal seronegative human serum under conditions that allow complement activation resulted in enhancement of infection of the MT2 human T cell line cultured in the presence of low amounts of virus. Infection of MT2 cells was assessed by measuring reverse transcriptase activity in supernatants at day 9 of culture. Complement activation by viral suspensions occurred through the alternative pathway. Opsonization of HTLV-RF viral particles with complement was sufficient to allow a productive infection to occur in cells exposed to suboptimal amounts of virus. Infection of MT2 cells with suboptimal amounts of serum-opsonized HIV-1 was suppressed by blocking the C3dg receptor (CR2, CD21) on MT2 cells with monoclonal anti-CR2 antibody and rabbit F(ab')2 anti-mouse immunoglobulin antibodies. Blocking of the gp120-binding site on CD4 under similar experimental conditions had no inhibitory effect on infection of MT2 cells with opsonized virus. Opsonization of HIV-1 with seronegative serum also resulted in a CR2-mediated enhancement of the infection of normal peripheral blood mononuclear cells and T lymphocytes. These results indicate that complement in the absence of antibody may enhance infection of C3 receptor-bearing T cells with HIV-1, and that the interaction of opsonized virus with the CR2 receptor may result by itself in the infection of target T cells in a CD4- and antibody-independent fashion. PMID:1827139

  17. TRIM21 Promotes cGAS and RIG-I Sensing of Viral Genomes during Infection by Antibody-Opsonized Virus

    PubMed Central

    Watkinson, Ruth E.; McEwan, William A.; Tam, Jerry C. H.; Vaysburd, Marina; James, Leo C.

    2015-01-01

    Encapsidation is a strategy almost universally employed by viruses to protect their genomes from degradation and from innate immune sensors. We show that TRIM21, which targets antibody-opsonized virions for proteasomal destruction, circumvents this protection, enabling the rapid detection and degradation of viral genomes before their replication. TRIM21 triggers an initial wave of cytokine transcription that is antibody, rather than pathogen, driven. This early response is augmented by a second transcriptional program, determined by the nature of the infecting virus. In this second response, TRIM21-induced exposure of the viral genome promotes sensing of DNA and RNA viruses by cGAS and RIG-I. This mechanism allows early detection of an infection event and drives an inflammatory response in mice within hours of viral challenge. PMID:26506431

  18. Evidence for the role of CR1 (CD35), in addition to CR2 (CD21), in facilitating infection of human T cells with opsonized HIV.

    PubMed

    Delibrias, C C; Kazatchkine, M D; Fischer, E

    1993-08-01

    Complement activation by HIV results in the binding of C3 fragments to the gp160 complex and enhanced infection of C3 receptor-bearing target cells. We have studied complement-mediated enhancement of infection of the human CD4-positive T-cell line HPB-ALL which expresses the CR1 (CD35) and CR2 (CD21) receptors for C3. CR1 and CR2 are present on 15% and 40% of normal peripheral blood CD4-positive T lymphocytes respectively. Opsonization of the virus with complement resulted in a 3- to 10-fold enhancement of infection of HPB-ALL cells, as assessed by measuring the release of p24 antigen in culture supernatants throughout the culture period. Blockade of CR2 with cross-linked anti-CR2 monoclonal antibodies decreased infection to the level observed with unopsonized virus. Blocking CR1 reduced complement-mediated infection by 50-80%. Experiments using serum deficient in complement factor I demonstrated that CR1 mediates the interaction between opsonized virus and T cells in addition to its ability to serve as a cofactor for the cleavage of C3b into smaller fragments that interact with CR2. A requirement for CD4 in complement-mediated enhancement of infection was observed with HIV-1 Bru but not with HIV-1 RF. Thus, CR1 and CR2 contribute in an independent and complementary fashion to penetration of opsonized virus into complement receptor-expressing T cells. Involvement of CD4 in infection with opsonized virus depends on the viral strain. PMID:8346417

  19. CR1 on erythrocytes of Brazilian systemic lupus erythematosus patients: the influence of disease activity on expression and ability of this receptor to bind immune complexes opsonized with complement from normal human serum.

    PubMed

    Marzocchi-Machado, C M; Alves, C M O S; Azzolini, A E C S; Polizello, A C M; Carvalho, I F; Lucisano-Valim, Y M

    2005-12-01

    Hypocomplementaemia and low expression of CR1 on erythrocytes (E) of patients with systemic lupus erythematosus (SLE) are associated with defective clearance of circulating immune complexes (IC) and so they may have pathogenic significance. Here, we investigated whether the reduced CR1/E in SLE patients per se might affect the binding of IC to CR1/E. First, we analysed the expression of CR1 on E of active (n=30) and inactive (n=34) SLE patients using a FITC-conjugated mouse anti-CR1 monoclonal antibody E11 and flow cytometry. Both groups of patients had a significantly reduced CR1/E expression compared with healthy controls (n=40). It was also observed that the number of E bearing CR1 was reduced in both groups of SLE patients studied. Second, we determined the functional activity of CR1/E by measuring the binding to E of FITC-bovine serum albumin (BSA)/rabbit anti-BSA complexes, formed at equivalence, which were opsonized with complement from normal human serum (NHS). On the other hand, we did not find differences between the patient and control groups in the ability of E to bind IC/NHS. There was also a positive correlation between the CR1/E expression and the number of E bearing CR1 in control and inactive SLE groups, which was not observed in the group of active SLE patients. Considering the involvement of low levels of complement and CR1/E expression on complex processing, in this in vitro model the results show that an effective coating of the complexes with complement is sufficient to bind them preferentially to CR1 over normal levels of receptor expression.

  20. CD55 is a key complement regulatory protein that counteracts complement-mediated inactivation of Newcastle Disease Virus.

    PubMed

    Rangaswamy, Udaya S; Cotter, Christopher R; Cheng, Xing; Jin, Hong; Chen, Zhongying

    2016-08-01

    Newcastle disease virus (NDV) is being developed as an oncolytic virus for virotherapy. In this study we analysed the regulation of complement-mediated inactivation of a recombinant NDV in different host cells. NDV grown in human cells was less sensitive to complement-mediated virus inactivation than NDV grown in embryonated chicken eggs. Additionally, NDV produced from HeLa-S3 cells is more resistant to complement than NDV from 293F cells, which correlated with higher expression and incorporation of complement regulatory proteins (CD46, CD55 and CD59) into virions from HeLa-S3 cells. Further analysis of the recombinant NDVs individually expressing the three CD molecules showed that CD55 is the most potent in counteracting complement-mediated virus inactivation. The results provide important information on selecting NDV manufacture substrate to mitigate complement-mediated virus inactivation.

  1. Rapid complement fixation technique for estimating complement-fixing antigen elution profiles of viruses from gel filtration columns.

    PubMed

    Cornesky, R A; Hammon, W M; Sather, G E; Atchison, R

    1972-07-01

    The complement fixation elution profiles of dengue-2 virus-infected suckling mouse brain supernatant fluids from Sephadex G-200 columns were compared by the antigen end-point titration procedure and by a complement dilution technique. The latter technique was found to be a quick method for approximating the complement-fixing antigen in eluates.

  2. Activation of Complement by Cells Infected with Respiratory Syncytial Virus

    PubMed Central

    Smith, Thomas F.; Mcintosh, Kenneth; Fishaut, Mark; Henson, Peter M.

    1981-01-01

    The ability of respiratory syncytial virus (RSV)-infected HEp-2 cells in culture to activate complement was investigated. After incubation of cells with various complement sources and buffer, binding of C3b to surfaces of infected cells was demonstrated by immunofluorescence with a double-staining technique. Nonsyncytial and syncytial (i.e., fused, multinucleated) cells were separately enumerated. Also, lysis of RSV-infected cells was assessed by lactic dehydrogenase release. In this system only RSV-infected cells stained for C3b, and they did so only after incubation with functionally active complement. Blocking of classical pathway activation with ethylenediaminetetraacetic acid diminished the number of infected nonsyncytial cells positively stained for C3b, but had no effect on staining of syncytial cells. Blocking of alternative pathway activation with either zymosan incubation or heat treatment decreased the number of both syncytial and nonsyncytial cells stained for C3b. Decreasing immunoglobulin concentration of the serum used as the complement source also decreased numbers of both cell types stained for C3b. Eliminating specific anti-RSV antibody diminished numbers of both cell types stained for C3b, but staining was not eliminated. Lastly, incubation with functionally active complement markedly increased lactic dehydrogenase release from infected cells. This study demonstrated that RSV-infected nonsyncytial and syncytial cells are able to activate complement by both classical and alternative pathways. Activation of complement by syncytial cells appears to be less dependent on the classical pathway than is activation by nonsyncytial cells, and activation by syncytial cells may require immunoglobulin but not specific antibody. These experiments suggest the possibility of complement activation during respiratory tract infection by RSV. Implications of this are discussed. Images PMID:7263071

  3. 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.

  4. Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties.

    PubMed

    Moghimi, S M; Szebeni, J

    2003-11-01

    This article critically examines and evaluates the likely mechanisms that contribute to prolonged circulation times of sterically protected nanoparticles and liposomes. It is generally assumed that the macrophage-resistant property of sterically protected particles is due to suppression in surface opsonization and protein adsorption. However, recent evidence shows that sterically stabilized particles are prone to opsonization particularly by the opsonic components of the complement system. We have evaluated these phenomena and discussed theories that reconcile complement activation and opsonization with prolonged circulation times. With respect to particle longevity, the physiological state of macrophages also plays a critical role. For example, stimulated or newly recruited macrophages can recognize and rapidly internalize sterically protected nanoparticles by opsonic-independent mechanisms. These concepts are also examined.

  5. Complement

    MedlinePlus

    ... in: Cancer Certain infections Ulcerative colitis Decreased complement activity may be seen in: Cirrhosis Glomerulonephritis Hereditary angioedema Hepatitis Kidney transplant rejection Lupus nephritis Malnutrition Systemic lupus erythematosis

  6. Complementation of human immunodeficiency virus (HIV-1) gag particle formation.

    PubMed

    Zhoa, Y; Jones, I M; Hockley, D J; Nermut, M V; Roy, P

    1994-03-01

    The human immunodeficiency virus gag precursor protein Pr55Gag exhibits the ability of particle assembly when expressed using recombinant baculoviruses. In order to delineate the sequences required for particle formation, two mutants of Gag (D1 and D2) were constructed in which 10 amino acids within the CA domain were deleted. Both mutants yielded stable high levels of Gag antigen following expression in Spodoptera frugiperda insect cells. Electron microscopy of sections through infected cells revealed that neither mutant was able to assemble particles although targeting of the protein to the plasma membrane still occurred. The Gag antigen that accumulated beneath the plasma membrane exhibited distinctive morphologies when compared to each other and to parental (Pr46Gag) particles. Particle assembly was rescued when S. frugiperda cells were coinfected with both AcD1 and AcD2 viruses, or with AcD1 and a carboxyl-terminal deletion of Gag (Pr41.5) which was previously shown not to form particles (J.B.M., D.J. Hockley, M.V. Nermot, and I.M. Jones, 1992, J. Gen. Virol. 73, 3079-3086). The genetic complementation of Gag-driven assembly is discussed.

  7. Membrane-bound complement regulatory activity is decreased on vaccinia virus-infected cells.

    PubMed Central

    Baranyi, L; Okada, N; Baranji, K; Takizawa, H; Okada, H

    1994-01-01

    Decay accelerating factor (DAF), membrane cofactor protein (MCP), complement receptor 1 and mouse Crry are cell surface-bound complement regulatory proteins capable of inhibiting C3 convertase activity on cell membranes, and therefore provide a substantial protection from attack by homologous complement activated either by the classical or by the alternative pathway. Decrease in complement regulatory activity might lead to spontaneous complement deposition and subsequent cell injury. MoAb 5I2 can inhibit the complement regulatory activity of molecules on rat cells, resulting in deposition of homologous complement. The antigen recognized by 5I2 MoAb in rats is homologous to mouse Crry. Fifteen to 20 h after infection with vaccinia virus, in vitro cultured KDH-8 rat hepatoma cells show a strong decrease in expression of Crry-like antigen, and proved to be sensitive to complement deposition when 1:5 diluted normal rat serum was added to the culture medium as a source of complement. Addition of complement to the cultured KDH-8 cells infected with a very low dose of vaccinia virus (1 plaque-forming unit (PFU)/1000 cells) substantially reduced spreading of virus infection in the cell culture, while inactivation of complement by heat or zymosan treatment abrogated the protective effect. PMID:7923872

  8. Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

    PubMed Central

    Burge, Boyce W.; Pfefferkorn, E. R.

    1967-01-01

    Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes. PMID:5630228

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

    SciTech Connect

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

    2008-06-20

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

  10. Dissecting virus entry: replication-independent analysis of virus binding, internalization, and penetration using minimal complementation of β-galactosidase.

    PubMed

    Burkard, Christine; Bloyet, Louis-Marie; Wicht, Oliver; van Kuppeveld, Frank J; Rottier, Peter J M; de Haan, Cornelis A M; Bosch, Berend Jan

    2014-01-01

    Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).

  11. Glycosylated and nonglycosylated complement control protein of the lister strain of vaccinia virus.

    PubMed

    Meseda, Clement A; Kuhn, Jordan; Atukorale, Vajini; Campbell, Joseph; Weir, Jerry P

    2014-09-01

    The vaccinia virus complement control protein (VCP) is a secreted viral protein that binds the C3b and C4b complement components and inhibits the classic and alternative complement pathways. Previously, we reported that an attenuated smallpox vaccine, LC16m8, which was derived from the Lister strain of vaccinia virus (VV-Lister), expressed a glycosylated form of VCP, whereas published sequence data at that time indicated that the VV-Lister VCP has no motif for N-linked glycosylation. We were interested in determining whether the glycosylation of VCP impairs its biological activity, possibly contributing to the attenuation of LC16m8, and the likely origin of the glycosylated VCP. Expression analysis indicated that VV-Lister contains substrains expressing glycosylated VCP and substrains expressing nonglycosylated VCP. Other strains of smallpox vaccine, as well as laboratory strains of vaccinia virus, all expressed nonglycosylated VCP. Individual Lister virus clones expressing either the glycosylated VCP or the nonglycosylated species were isolated, and partially purified VCP from the isolates were found to be functional equivalents in binding human C3b and C4b complement proteins and inhibiting hemolysis and in immunogenicity. Recombinant vaccinia viruses expressing FLAG-tagged glycosylated VCP (FLAG-VCPg) and nonglycosylated VCP (FLAG-VCP) were constructed based on the Western Reserve strain. Purified FLAG-VCP and FLAG-VCPg bind human C3b and C4b and blocked complement-mediated hemolysis. Our data suggest that glycosylation did not affect the biological activity of VCP and thus may not have contributed to the attenuation of LC16m8. In addition, the LC16m8 virus likely originated from a substrain of VV-Lister that expresses glycosylated VCP.

  12. Complement-mediated opsonization of invasive group A Streptococcus pyogenes strain AP53 is regulated by the bacterial two-component cluster of virulence responder/sensor (CovRS) system.

    PubMed

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

    2013-09-20

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

  13. Bovine viral diarrhea virus structural protein E2 as a complement regulatory protein.

    PubMed

    Ostachuk, Agustín

    2016-07-01

    Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, family Flaviviridae, and is one of the most widely distributed viruses in cattle worldwide. Approximately 60 % of cattle in endemic areas without control measures are infected with BVDV during their lifetime. This wide prevalence of BVDV in cattle populations results in significant economic losses. BVDV is capable of establishing persistent infections in its host due to its ability to infect fetuses, causing immune tolerance. However, this cannot explain how the virus evades the innate immune system. The objective of the present work was to test the potential activity of E2 as a complement regulatory protein. E2 glycoprotein, produced both in soluble and transmembrane forms in stable CHO-K1 cell lines, was able to reduce complement-mediated cell lysis up to 40 % and complement-mediated DNA fragmentation by 50 %, in comparison with cell lines not expressing the glycoprotein. This work provides the first evidence of E2 as a complement regulatory protein and, thus, the finding of a mechanism of immune evasion by BVDV. Furthermore, it is postulated that E2 acts as a self-associated molecular pattern (SAMP), enabling the virus to avoid being targeted by the immune system and to be recognized as self. PMID:27038454

  14. 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

  15. 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.

  16. Complement-mediated neutralization of dengue virus requires mannose-binding lectin.

    PubMed

    Avirutnan, Panisadee; Hauhart, Richard E; Marovich, Mary A; Garred, Peter; Atkinson, John P; Diamond, Michael S

    2011-01-01

    Mannose-binding lectin (MBL) is a key soluble pathogen recognition protein of the innate immune system that binds specific mannose-containing glycans on the surfaces of microbial agents and initiates complement activation via the lectin pathway. Prior studies showed that MBL-dependent activation of the complement cascade neutralized insect cell-derived West Nile virus (WNV) in cell culture and restricted pathogenesis in mice. Here, we investigated the antiviral activity of MBL in infection by dengue virus (DENV), a related flavivirus. Using a panel of naïve sera from mouse strains deficient in different complement components, we showed that inhibition of infection by insect cell- and mammalian cell-derived DENV was primarily dependent on the lectin pathway. Human MBL also bound to DENV and neutralized infection of all four DENV serotypes through complement activation-dependent and -independent pathways. Experiments with human serum from naïve individuals with inherent variation in the levels of MBL in blood showed a direct correlation between the concentration of MBL and neutralization of DENV; samples with high levels of MBL in blood neutralized DENV more efficiently than those with lower levels. Our studies suggest that allelic variation of MBL in humans may impact complement-dependent control of DENV pathogenesis. IMPORTANCE Dengue virus (DENV) is a mosquito-transmitted virus that causes a spectrum of clinical disease in humans ranging from subclinical infection to dengue hemorrhagic fever and dengue shock syndrome. Four serotypes of DENV exist, and severe illness is usually associated with secondary infection by a different serotype. Here, we show that mannose-binding lectin (MBL), a pattern recognition molecule that initiates the lectin pathway of complement activation, neutralized infection of all four DENV serotypes through complement activation-dependent and -independent pathways. Moreover, we observed a direct correlation with the concentration of MBL in

  17. Complement-Fixation Analysis of Four Subtypes of Foot-and-Mouth Disease Virus Type A

    PubMed Central

    Lobo, C. A.; Cowan, K. M.; Hanson, R. P.

    1973-01-01

    Complement-fixation patterns were established for four subtypes of foot-and-mouth disease virus by block assays against homologous and heterologous antiserum. Inhibition of fixation by excess antigen was observed in most homologous systems but rarely in the heterologous systems. The heterologous antibody titers were, in all instances, considerably lower than those for the homologous systems. Although relatively high dilutions of antiserum may be desirable for subtyping, higher concentrations of antibody should be used for determining serological types. PMID:4356470

  18. ON THE NATURE OF THE OPSONIC SUBSTANCES OBNORMAL SERA.

    PubMed

    Zinsser, H; Cary, E G

    1914-04-01

    Our experiments show that the albumen fraction, or end-piece, obtained by the dialysis of normal guinea pig serum possesses definite opsonic action. This action is often almost equal to that residing in the unfractionated alexin. It is evident, however, only if the reaction maintained during the experiments approximates that of the original serum. By the addition of small quantities of a weak sodium hydrate solution to the dialyzed serum we have been able to bring back opsonic action which was not evident in the same end-piece if simply rendered isotonic. Although our attention was called to the question of reaction by the work of Bronfenbrenner and Noguchi, like Liefmann, we have been unable to reactivate the hemolytic function of end-piece by alteration of reaction. Our experiments suggest that the opsonic action of the albumen fraction is enhanced by preliminary sensitization of the bacteria with heated normal serum and by persensitization of such bacteria with the globulin fraction. However, we cannot be positive of this, since the slight differences of phagocytic counts upon which such an opinion can be based, fall within the limits of what we consider our experimental error. The fact that the albumen fraction can exert opsonic activity upon bacteria but cannot hemolyze blood cells seems to us particularly interesting in the light of the fact that alexin can be absorbed by unsensitized bacteria but not by similarly untreated blood cells. The literature upon the relation of the alexin fractions to bacteria and the bactericidal effect is confusing in that contradictory results have been obtained by other workers. We are studying this phase of the problem with particular attention to the alkalinity or acidity under which the reactions are carried out. We think that our experiments do not point to a differentiation of normal opsonin from alexin, but we believe they indicate that the so called end-piece can enter to a slight extent into non-specific relationship

  19. The Paramyxoviruses Simian Virus 5 and Mumps Virus Recruit Host Cell CD46 To Evade Complement-Mediated Neutralization ▿

    PubMed Central

    Johnson, John B.; Grant, Ken; Parks, Griffith D.

    2009-01-01

    The complement system is a critical component of the innate immune response that all animal viruses must face during natural infections. Our previous results have shown that treatment of the paramyxovirus simian virus 5 (SV5) with human serum results in deposition of complement C3-derived polypeptides on virion particles. Here, we show that the virion-associated C3 component includes the inactive form iC3b, suggesting that SV5 may have mechanisms to evade the host complement system. Electron microscopy, gradient centrifugation, and Western blot analysis indicated that purified SV5 virions derived from human A549 cells contained CD46, a plasma membrane-expressed regulator of complement that acts as a cofactor for cleavage and inactivation of C3b into iC3b. In vitro cleavage assays with purified complement components showed that SV5 virions had C3b cofactor activity, resulting in specific factor I-mediated cleavage of C3b into inactive iC3b. SV5 particles generated in CHO cells, which do not express CD46, did not have cofactor activity. Conversely, virions derived from a CHO cell line that was engineered to overexpress human CD46 contained elevated levels of virion-associated CD46 and displayed enhanced C3b cofactor activity. In comparison with C3b, purified SV5 virions had very low cofactor activity against C4b, consistent with the known preference of CD46 for C3b versus C4b. Similar results were obtained for the closely related mumps virus (MuV), except that MuV particles derived from CHO-CD46 cells had higher C4b cofactor activity than SV5 virions. In neutralization assays with human serum, SV5 and MuV containing CD46 showed slower kinetics and more resistance to neutralization than SV5 and MuV that lacked CD46. Our results support a model in which the rubulaviruses SV5 and MuV incorporate cell surface complement inhibitors into progeny virions as a mechanism to limit complement-mediated neutralization. PMID:19457998

  20. Opsonic requirements for phagocytosis of Legionella micdadei by polymorphonuclear leukocytes.

    PubMed Central

    Steffensen, D O; Weinbaum, D L; Dowling, J N

    1985-01-01

    The roles of the classical and alternative pathways of complement activation and of antibody in the phagocytosis of Legionella micdadei by polymorphonuclear leukocytes were studied. Normal serum was treated with the appropriate chelators or with heat to inactivate the classical, alternate, or both pathways of complement activation. Normal and complement-depleted sera with or without antibody were employed as opsonins for L. micdadei in phagocytosis assays. There was no difference in the phagocytosis of L. micdadei promoted by normal serum and either C4-deficient serum or serum in which the classical pathway had been inactivated. Both normal and classical pathway-deficient sera promoted significantly greater phagocytosis than did sera in which the alternate pathway or both the alternate and classical pathways had been inactivated. Thus, polymorphonuclear leukocyte phagocytosis of L. micdadei in the absence of antibody required an intact alternate pathway. Specific antibody partially restored opsonization to sera deficient in the alternate or both complement pathways, but phagocytosis was still significantly less than that with the alternate pathway intact. PMID:4030099

  1. Parainfluenza virus 5 upregulates CD55 expression to produce virions with enhanced resistance to complement-mediated neutralization.

    PubMed

    Li, Yujia; Johnson, John B; Parks, Griffith D

    2016-10-01

    Many enveloped RNA viruses recruit host cell proteins during assembly as a mechanism to limit antiviral effects of complement. Using viruses which incorporated CD46 alone, CD55 alone or both CD46 and CD55, we addressed the role of these two host cell regulators in limiting complement-mediated neutralization of Parainfluenza virus 5 (PIV5). PIV5 incorporated functional forms of both CD55 and CD46 into virions. PIV5 containing CD55 was highly resistant to complement-mediated neutralization, whereas CD46-containing PIV5 was as sensitive to neutralization as virus lacking both regulators. PIV5 infected cells had increased levels of cell surface CD55, which was further upregulated by exogenous treatment with tumor necrosis factor alpha. PIV5 derived from cells with higher CD55 levels was more resistant to complement-mediated neutralization in vitro than virus from control cells. We propose a role for virus induction of host cell complement inhibitors in defining virus growth and tissue tropism.

  2. Detachment of human immunodeficiency virus type 1 from germinal centers by blocking complement receptor type 2.

    PubMed

    Kacani, L; Prodinger, W M; Sprinzl, G M; Schwendinger, M G; Spruth, M; Stoiber, H; Döpper, S; Steinhuber, S; Steindl, F; Dierich, M P

    2000-09-01

    After the transition from the acute to the chronic phase of human immunodeficiency virus (HIV) infection, complement mediates long-term storage of virions in germinal centers (GC) of lymphoid tissue. The contribution of particular complement receptors (CRs) to virus trapping in GC was studied on tonsillar specimens from HIV-infected individuals. CR2 (CD21) was identified as the main binding site for HIV in GC. Monoclonal antibodies (MAb) blocking the CR2-C3d interaction were shown to detach 62 to 77% of HIV type 1 from tonsillar cells of an individual in the presymptomatic stage. Although they did so at a lower efficiency, these antibodies were able to remove HIV from tonsillar cells of patients under highly active antiretroviral therapy, suggesting that the C3d-CR2 interaction remains a primary entrapment mechanism in treated patients as well. In contrast, removal of HIV was not observed with MAb blocking CR1 or CR3. Thus, targeting CR2 may facilitate new approaches toward a reduction of residual virus in GC.

  3. Detachment of human immunodeficiency virus type 1 from germinal centers by blocking complement receptor type 2.

    PubMed

    Kacani, L; Prodinger, W M; Sprinzl, G M; Schwendinger, M G; Spruth, M; Stoiber, H; Döpper, S; Steinhuber, S; Steindl, F; Dierich, M P

    2000-09-01

    After the transition from the acute to the chronic phase of human immunodeficiency virus (HIV) infection, complement mediates long-term storage of virions in germinal centers (GC) of lymphoid tissue. The contribution of particular complement receptors (CRs) to virus trapping in GC was studied on tonsillar specimens from HIV-infected individuals. CR2 (CD21) was identified as the main binding site for HIV in GC. Monoclonal antibodies (MAb) blocking the CR2-C3d interaction were shown to detach 62 to 77% of HIV type 1 from tonsillar cells of an individual in the presymptomatic stage. Although they did so at a lower efficiency, these antibodies were able to remove HIV from tonsillar cells of patients under highly active antiretroviral therapy, suggesting that the C3d-CR2 interaction remains a primary entrapment mechanism in treated patients as well. In contrast, removal of HIV was not observed with MAb blocking CR1 or CR3. Thus, targeting CR2 may facilitate new approaches toward a reduction of residual virus in GC. PMID:10933708

  4. Exploration of binary virus-host interactions using an infectious protein complementation assay.

    PubMed

    Munier, Sandie; Rolland, Thomas; Diot, Cédric; Jacob, Yves; Naffakh, Nadia

    2013-10-01

    A precise mapping of pathogen-host interactions is essential for comprehensive understanding of the processes of infection and pathogenesis. The most frequently used techniques for interactomics are the yeast two-hybrid binary methodologies, which do not recapitulate the pathogen life cycle, and the tandem affinity purification mass spectrometry co-complex methodologies, which cannot distinguish direct from indirect interactions. New technologies are thus needed to improve the mapping of pathogen-host interactions. In the current study, we detected binary interactions between influenza A virus polymerase and host proteins during the course of an actual viral infection, using a new strategy based on trans-complementation of the Gluc1 and Gluc2 fragments of Gaussia princeps luciferase. Infectious recombinant influenza viruses that encode a Gluc1-tagged polymerase subunit were engineered to infect cultured cells transiently expressing a selected set of Gluc2-tagged cellular proteins involved in nucleocytoplasmic trafficking pathways. A random set and a literature-curated set of Gluc2-tagged cellular proteins were tested in parallel. Our assay allowed the sensitive and accurate recovery of previously described interactions, and it revealed 30% of positive, novel viral-host protein-protein interactions within the exploratory set. In addition to cellular proteins involved in the nuclear import pathway, components of the nuclear pore complex such as NUP62 and mRNA export factors such as NXF1, RMB15B, and DDX19B were identified for the first time as interactors of the viral polymerase. Gene silencing experiments further showed that NUP62 is required for efficient viral replication. Our findings give new insights regarding the subversion of host nucleocytoplasmic trafficking pathways by influenza A viruses. They also demonstrate the potential of our infectious protein complementation assay for high-throughput exploration of influenza virus interactomics in infected cells

  5. A complement-microglial axis drives synapse loss during virus-induced memory impairment.

    PubMed

    Vasek, Michael J; Garber, Charise; Dorsey, Denise; Durrant, Douglas M; Bollman, Bryan; Soung, Allison; Yu, Jinsheng; Perez-Torres, Carlos; Frouin, Arnaud; Wilton, Daniel K; Funk, Kristen; DeMasters, Bette K; Jiang, Xiaoping; Bowen, James R; Mennerick, Steven; Robinson, John K; Garbow, Joel R; Tyler, Kenneth L; Suthar, Mehul S; Schmidt, Robert E; Stevens, Beth; Klein, Robyn S

    2016-06-23

    Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34(-/-) mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease. PMID:27337340

  6. A complement-microglial axis drives synapse loss during virus-induced memory impairment.

    PubMed

    Vasek, Michael J; Garber, Charise; Dorsey, Denise; Durrant, Douglas M; Bollman, Bryan; Soung, Allison; Yu, Jinsheng; Perez-Torres, Carlos; Frouin, Arnaud; Wilton, Daniel K; Funk, Kristen; DeMasters, Bette K; Jiang, Xiaoping; Bowen, James R; Mennerick, Steven; Robinson, John K; Garbow, Joel R; Tyler, Kenneth L; Suthar, Mehul S; Schmidt, Robert E; Stevens, Beth; Klein, Robyn S

    2016-06-22

    Over 50% of patients who survive neuroinvasive infection with West Nile virus (WNV) exhibit chronic cognitive sequelae. Although thousands of cases of WNV-mediated memory dysfunction accrue annually, the mechanisms responsible for these impairments are unknown. The classical complement cascade, a key component of innate immune pathogen defence, mediates synaptic pruning by microglia during early postnatal development. Here we show that viral infection of adult hippocampal neurons induces complement-mediated elimination of presynaptic terminals in a murine WNV neuroinvasive disease model. Inoculation of WNV-NS5-E218A, a WNV with a mutant NS5(E218A) protein leads to survival rates and cognitive dysfunction that mirror human WNV neuroinvasive disease. WNV-NS5-E218A-recovered mice (recovery defined as survival after acute infection) display impaired spatial learning and persistence of phagocytic microglia without loss of hippocampal neurons or volume. Hippocampi from WNV-NS5-E218A-recovered mice with poor spatial learning show increased expression of genes that drive synaptic remodelling by microglia via complement. C1QA was upregulated and localized to microglia, infected neurons and presynaptic terminals during WNV neuroinvasive disease. Murine and human WNV neuroinvasive disease post-mortem samples exhibit loss of hippocampal CA3 presynaptic terminals, and murine studies revealed microglial engulfment of presynaptic terminals during acute infection and after recovery. Mice with fewer microglia (Il34(-/-) mice with a deficiency in IL-34 production) or deficiency in complement C3 or C3a receptor were protected from WNV-induced synaptic terminal loss. Our study provides a new murine model of WNV-induced spatial memory impairment, and identifies a potential mechanism underlying neurocognitive impairment in patients recovering from WNV neuroinvasive disease.

  7. Exploring the potential benefits of vaccinia virus complement control protein in controlling complement activation in pathogenesis of the central nervous system diseases.

    PubMed

    Kotwal, Girish J; Fernando, Nilisha; Zhou, Jianhua; Valter, Krisztina

    2014-10-01

    Aging is a major risk factor for the development of diseases related to the central nervous system (CNS), such as Alzheimer's disease (AD) and age-related macular degeneration (AMD). In both cases, linkage studies and genome-wide association studies found strong links with complement regulatory genes and disease risk. In AD, both CLU and CR1 genes were implicated in the late-onset form of the disease. In AMD, polymorphisms in CFH, CFB and C2 were similarly implicated. The cost of caring for patients with AD or AMD is approaching billions of dollars, and with the baby boomers reaching their 60's, this amount is likely to increase further. Intervention using complement inhibitors for individuals in their early 50s who are at a higher risk of disease development, (testing positive for genetic risk factors), could slow the progression of AD or AMD and possibly prevent the severity of late stage symptoms. Although we have used the vaccinia virus complement control protein (VCP) to elucidate the role of complement in CNS diseases, it has merely been an investigational tool but not the only possible potential therapeutic agent.

  8. Cofilin contributes to phagocytosis of IgG-opsonized particles but not non-opsonized particles in RAW264 macrophages.

    PubMed

    Lu, Yanmeng; Cao, Lei; Egami, Youhei; Kawai, Katsuhisa; Araki, Nobukazu

    2016-06-01

    Cofilin is an actin-binding protein that severs actin filaments. It plays a key role in regulating actin cytoskeletal remodeling, thereby contributing to diverse cellular functions. However, the involvement of cofilin in phagocytosis remains to be elucidated. We examined the spatiotemporal localization of cofilin during phagocytosis of IgG-opsonized erythrocytes, IgG-opsonized latex beads and non-opsonized latex beads. Live-cell imaging showed that GFP-cofilin accumulates in the sites of IgG-opsonized particle binding and in phagocytic cups. Moreover, immunofluorescence microscopy revealed that endogenous cofilin localizes to phagocytic cups engulfing IgG-opsonized particles, but not non-opsonized latex beads. Scanning electron microscopy demonstrated a notable difference in morphology between phagocytic structures in IgG-dependent and IgG-independent phagocytosis. In phagocytosis of IgG-opsonized particles, sheet-like pseudopodia extended along the surface of IgG-opsonized particles to form phagocytic cups. In contrast, in opsonin-independent phagocytosis, long finger-like filopodia captured non-opsonized latex beads. Importantly, non-opsonized beads sank into the cells without extending phagocytic cups. Our analysis of cofilin mutant expression demonstrates that phagocytosis of IgG-opsonized particles is enhanced in cells expressing wild-type cofilin or active mutant cofilin-S3A, whereas the uptake of non-opsonized latex beads is not. These data suggest that cofilin promotes actin cytoskeletal remodeling to form phagocytic cups by accelerating actin turnover and thereby facilitating phagosome formation. In contrast, cofilin is not involved in opsonin-independent phagocytosis of latex beads. PMID:26754560

  9. Administration of vaccinia virus complement control protein shows significant cognitive improvement in a mild injury model.

    PubMed

    Pillay, Nirvana S; Kellaway, Laurie A; Kotwal, Girish J

    2005-11-01

    Previous studies have shown that traumatic mild brain injury in a rat model is accompanied by breakdown of the blood brain barrier and the accumulation of inflammatory cells. A therapeutic agent, vaccinia virus complement control protein (VCP), inhibits both the classic and the alternative pathways of the complement system and, in so doing, prevents cell death and inflammation. With the use of a rat mild injury model, the effects of VCP on spatial learning and memory were tested. Training in a Morris water maze consisted of a total of 16 trials over a 2-day period before rats were anesthetized and subjected to mild (1.0-1.1 atm) lateral fluid percussion injury (FPI) 3.0 mm lateral to the sagittal suture and 4.5 mm posterior to bregma. Ten microl of VCP (1.7 mg/ml) was injected into the injury site immediately after FPI. Two weeks post-FPI the rats were assessed in the Morris water maze for spatial learning and memory. Neurologic motor function tests were carried out after FPI for 14 consecutive days and again after 28 days. The Morris water maze data show that FPI plus saline-injected rats spent a significantly (P <0.05) larger amount of time in one of the incorrect quadrants than did the FPI plus VCP-injected group. Neurologic evaluations 24 hours postinjury revealed differences in sensorimotor function between groups. The results suggest that in a mild injury model, VCP influences neurologic outcome and offers some enhancement in spatial memory and learning.

  10. Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.

    PubMed

    Song, Hongbin; He, Chun; Knaak, Christian; Guthridge, Joel M; Holers, V Michael; Tomlinson, Stephen

    2003-06-01

    In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the membrane complement inhibitors decay accelerating factor (DAF) or CD59. The targeted complement inhibitors bound to C3-opsonized cells, and all were significantly more effective (up to 20-fold) than corresponding untargeted inhibitors at protecting target cells from complement. CR2 fusion proteins also inhibited CR3-dependent adhesion of U937 cells to C3 opsonized erythrocytes, indicating a second potential anti-inflammatory mechanism of CR2 fusion proteins, since CR3 is involved in endothelial adhesion and diapedesis of leukocytes at inflammatory sites. Finally, the in vivo validity of the targeting strategy was confirmed by the demonstration that CR2-DAF, but not soluble DAF, targets to the kidney in mouse models of lupus nephritis that are associated with renal complement deposition.

  11. Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation.

    PubMed

    Song, Hongbin; He, Chun; Knaak, Christian; Guthridge, Joel M; Holers, V Michael; Tomlinson, Stephen

    2003-06-01

    In a strategy to specifically target complement inhibitors to sites of complement activation and disease, recombinant fusion proteins consisting of a complement inhibitor linked to a C3 binding region of complement receptor (CR) 2 were prepared and characterized. Natural ligands for CR2 are C3 breakdown products deposited at sites of complement activation. Fusion proteins were prepared consisting of a human CR2 fragment linked to either the N terminus or C terminus of soluble forms of the membrane complement inhibitors decay accelerating factor (DAF) or CD59. The targeted complement inhibitors bound to C3-opsonized cells, and all were significantly more effective (up to 20-fold) than corresponding untargeted inhibitors at protecting target cells from complement. CR2 fusion proteins also inhibited CR3-dependent adhesion of U937 cells to C3 opsonized erythrocytes, indicating a second potential anti-inflammatory mechanism of CR2 fusion proteins, since CR3 is involved in endothelial adhesion and diapedesis of leukocytes at inflammatory sites. Finally, the in vivo validity of the targeting strategy was confirmed by the demonstration that CR2-DAF, but not soluble DAF, targets to the kidney in mouse models of lupus nephritis that are associated with renal complement deposition. PMID:12813023

  12. Opsonic Fibronectin Deficiency and Sepsis: Cause or Effect?

    PubMed Central

    Lanser, Marc E.; Saba, Thomas M.

    1982-01-01

    Opsonic fibronectin is known to modulate macrophage (RE cell) and neutrophil Phagocytic function. Its depletion has been documented following trauma, burn, and operation in patients with rapid restoration of normal levels unless bacteremia and/or wound sepsis intervenes. Sepsis is associated with a secondary phase of opsonic fibronectin deficiency. We have observed in burn patients that this secondary phase of opsonic fibronectin depletion following trauma and burn is seen two to three days prior to the onset of clinical sepsis, raising the question of whether this deficiency sensitized the host to the subsequent development of sepsis or whether its deplection was merely an unsuspected sensitive indication of preclinical sepsis. To address the possibility that opsonic fibronectin deficiency might lower resistance to sepsis, Sprague-Dawley rats (200 gm) were partially depleted (35%) of their opsonic fibronectin prior to intraperitoneal inoculation with Staphylococcus aureus. Mortality to S. aureus peritonitis was significantly (p < 0.05) increased in animals with fibronectin deficiency. Furthermore, in control animals, nonsurvival was also associated with significantly (p < 0.05) lower initial fibronectin levels than survival. However, peritonitis itself also resulted in an early (within one hour) depletion of opsonic fibronectin followed by a marked “hyperopsonemia” within 12 hours in both groups. Thus, opsonic fibronectin depletion decreases resistance to sepsis, and the development of sepsis itself will initiate opsonic fibronectin deficiency. Host defense against infection may depend on early restoration and maintenance of normal opsonic fibronectin levels following trauma, burn, and operation, as well as the ability of the host to mount an appropriate hyperopsonemic elevation of fibronectin levels in response to infection. PMID:7059244

  13. Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cations.

    PubMed

    Bashford, C L; Alder, G M; Menestrina, G; Micklem, K J; Murphy, J J; Pasternak, C A

    1986-07-15

    Hemolytic viruses, bacterial and animal toxins, the components of activated complement, cationic proteins, and detergents induce a sequence of permeability changes at the plasma membrane that are in every case sensitive to changes in ionic strength and to divalent cations. Individually, each agent exhibits positive cooperativity; when two agents are present together, they show synergy. It is concluded that such cytotoxic agents damage membranes by a common mechanism. Hence permeability changes are unlikely to depend on the formation of specific, protein-lined channels, as previously envisaged in the case of activated complement or certain bacterial toxins.

  14. Virion-associated complement regulator CD55 is more potent than CD46 in mediating resistance of mumps virus and vesicular stomatitis virus to neutralization.

    PubMed

    Johnson, John B; Lyles, Douglas S; Alexander-Miller, Martha A; Parks, Griffith D

    2012-09-01

    Enveloped viruses can incorporate host cell membrane proteins during the budding process. Here we demonstrate that mumps virus (MuV) and vesicular stomatitis virus (VSV) assemble to include CD46 and CD55, two host cell regulators which inhibit propagation of complement pathways through distinct mechanisms. Using viruses which incorporated CD46 alone, CD55 alone, or both CD46 and CD55, we have tested the relative contribution of these regulators in resistance to complement-mediated neutralization. Virion-associated CD46 and CD55 were biologically active, with VSV showing higher levels of activity of both cofactors, which promoted factor I-mediated cleavage of C3b into iC3b as well as decay-accelerating factor (DAF) activity against the C3 convertase, than MuV. Time courses of in vitro neutralization with normal human serum (NHS) showed that both regulators could delay neutralization, but viruses containing CD46 alone were neutralized faster and more completely than viruses containing CD55 alone. A dominant inhibitory role for CD55 was most evident for VSV, where virus containing CD55 alone was not substantially different in neutralization kinetics from virus harboring both regulators. Electron microscopy showed that VSV neutralization proceeded through virion aggregation followed by lysis, with virion-associated CD55 providing a delay in both aggregation and lysis more substantial than that conferred by CD46. Our results demonstrate the functional significance of incorporation of host cell factors during virion envelope assembly. They also define pathways of virus complement-mediated neutralization and suggest the design of more effective viral vectors.

  15. Confirmation of herpes simplex virus type 2 infections in herpes-like genital lesions by a simple complement-fixation test.

    PubMed

    Arsenakis, M; May, J T

    1982-02-01

    The presence of complement-fixing antibody to an early herpes simplex virus type 2 (HSV-2) antigen (the AG-4 antigen) was correlated with HSV-2 infection in the sera of patients with genital herpes. Eighty-eight per cent of sera taken two weeks after clinical diagnosis of a primary or recurrent herpes infection in patients, confirmed to have HSV-2 by virus isolation and typing, contained the anti-AG-4 complement-fixing antibody. None of the patients with genital HSV-1 had the antibody, and only 9% of controls or patients with facial HSV-1 infection had positive results for the antibody. This correlation was used to identify genital HSV-2 infections when either no virus sample had been taken or when virus isolations had been unsuccessful. Thus, a simple complement-fixation test can confirm an HSV-2 virus infection without isolation of the virus from the herpetic lesion.

  16. In vitro inactivation of complement by a serum factor present in Junin-virus infected guinea-pigs.

    PubMed Central

    Rimoldi, M T; de Bracco, M M

    1980-01-01

    A serum factor(s) of guinea-pigs infected with Junin virus, the etiological agent of Argentine haemorrhagic fever, is endowed with a potent anticomplementary activity. It is resistant to heat (56 degrees, 30 min) and elutes from a Sephadex G-200 column between albumin and haemoglobin. It is ineffective in the presence of EDTA or EGTA and does not sediment at 82,000 g. It has no direct effect on C4 unless functional Cl is present. However, it induces Cl activation that consumes C4 haemolytic activity in normal human and guinea-pig sera. The evidence presented in this report demonstrates that the complement activation observed in experimental Argentine haemorrhagic fever is at least in part due to a direct effect of this serum factor on the classical complement pathway. PMID:6247264

  17. Human platelets efficiently kill IgG-opsonized E. coli.

    PubMed

    Riaz, Anum H; Tasma, Brian E; Woodman, Michael E; Wooten, R Mark; Worth, Randall G

    2012-06-01

    Platelets are known contributors of hemostasis but have recently been shown to be important in inflammation and infectious diseases. Moreover, thrombocytopenia is often observed in patients with sepsis. We previously reported that platelets actively phagocytosed IgG-coated latex beads. In this study, the capacity of human platelets to participate in host defense against bacterial infections was determined by assessing their ability to kill Escherichia coli. Washed human platelets were incubated with unopsonized or IgG-opsonized E. coli and evaluated for binding and killing of E. coli. We found that although both unopsonized and IgG-opsonized E. coli were associated with platelets, only IgG-opsonized E. coli were efficiently killed unless platelets were activated by a potent agonist. The bactericidal activity was dependent on FcγRIIA, was sensitive to cytochalasin D, but was not due to reactive oxygen metabolites. These data suggest that platelets may play an important role in protection against infection.

  18. Efficient, trans-complementing packaging systems for chimeric, pseudoinfectious dengue 2/yellow fever viruses

    SciTech Connect

    Shustov, Alexandr V.

    2010-04-25

    In our previous studies, we have stated to build a new strategy for developing defective, pseudoinfectious flaviviruses (PIVs) and applying them as a new type of vaccine candidates. PIVs combined the efficiency of live vaccines with the safety of inactivated or subunit vaccines. The results of the present work demonstrate further development of chimeric PIVs encoding dengue virus 2 (DEN2V) glycoproteins and yellow fever virus (YFV)-derived replicative machinery as potential vaccine candidates. The newly designed PIVs have synergistically functioning mutations in the prM and NS2A proteins, which abolish processing of the latter proteins and make the defective viruses capable of producing either only noninfectious, immature and/or subviral DEN2V particles. The PIV genomes can be packaged to high titers into infectious virions in vitro using the NS1-deficient YFV helper RNAs, and both PIVs and helpers can then be passaged as two-component genome viruses at an escalating scale.

  19. Complement-mediated, antibody-dependent enhancement of HIV-1 infection in vitro is characterized by increased protein and RNA syntheses and infectious virus release.

    PubMed

    Robinson, W E; Montefiori, D C; Gillespie, D H; Mitchell, W M

    1989-01-01

    Antibody-dependent enhancement (ADE) of human immunodeficiency virus type 1 (HIV-1) infection in vitro has been described recently and was shown to occur by two mechanisms: either participation of the alternative pathway of complement or to involve an Fc receptor-mediated, complement-independent mechanism. Complement-mediated ADE results in an accelerated cytopathic effect in target cells that can abrogate the protective properties of neutralizing antibodies. This study characterizes the surface antigens of MT-2 cells using flow cytometric analysis and shows that these cells express high levels of both CD4 and complement receptor type 2 (CR2) while several CD4+ cell lines that do not demonstrate complement-mediated ADE lack high levels of complement receptors. Further, utilizing MT-2 cell cultures, it is demonstrated that complement-mediated ADE of HIV-1 infection is conferred by the sera from more than 80% of HIV-1 antibody-positive individuals (N = 85). Complement-mediated ADE of HIV-1 infection causes an acceleration of several parameters indicative of HIV-1 infection in vitro including increased HIV-1 antigen synthesis as detected by indirect immunofluorescence, RNA accumulation as measured by a solution hybridization protocol, reverse transcriptase release, and progeny virus production. PMID:2465404

  20. Giant virus with a remarkable complement of genes infects marine zooplankton

    PubMed Central

    Fischer, Matthias G.; Allen, Michael J.; Wilson, William H.; Suttle, Curtis A.

    2010-01-01

    As major consumers of heterotrophic bacteria and phytoplankton, microzooplankton are a critical link in aquatic foodwebs. Here, we show that a major marine microflagellate grazer is infected by a giant virus, Cafeteria roenbergensis virus (CroV), which has the largest genome of any described marine virus (≈730 kb of double-stranded DNA). The central 618-kb coding part of this AT-rich genome contains 544 predicted protein-coding genes; putative early and late promoter motifs have been detected and assigned to 191 and 72 of them, respectively, and at least 274 genes were expressed during infection. The diverse coding potential of CroV includes predicted translation factors, DNA repair enzymes such as DNA mismatch repair protein MutS and two photolyases, multiple ubiquitin pathway components, four intein elements, and 22 tRNAs. Many genes including isoleucyl-tRNA synthetase, eIF-2γ, and an Elp3-like histone acetyltransferase are usually not found in viruses. We also discovered a 38-kb genomic region of putative bacterial origin, which encodes several predicted carbohydrate metabolizing enzymes, including an entire pathway for the biosynthesis of 3-deoxy-d-manno-octulosonate, a key component of the outer membrane in Gram-negative bacteria. Phylogenetic analysis indicates that CroV is a nucleocytoplasmic large DNA virus, with Acanthamoeba polyphaga mimivirus as its closest relative, although less than one-third of the genes of CroV have homologs in Mimivirus. CroV is a highly complex marine virus and the only virus studied in genetic detail that infects one of the major groups of predators in the oceans. PMID:20974979

  1. Influence of growth temperature of Escherichia coli on K1 capsular antigen production and resistance to opsonization.

    PubMed Central

    Bortolussi, R; Ferrieri, P; Quie, P G

    1983-01-01

    When Escherichia coli strains that produce K1 capsular polysaccharide antigen at 37 degrees C were grown at 22 degrees C, K1 antigen was not detected in the supernatant or washed-cell fraction of broth cultures. Significant amounts of K1 polysaccharide were detected only when the organism was grown at temperatures of 30 degrees C or higher. Rabbits immunized with an E. coli K1 strain (serotype O18ac:K1:H7) grown at 37 degrees C produced agglutinating antibody to somatic antigen and precipitating and agglutinating antibody to capsular K1 antigen; those immunized with this strain grown at 22 degrees C produced antibody to somatic antigen, but not to K1 antigen. Antibody to somatic antigen was markedly reduced by adsorption with the organism grown at 22 degrees C, while antibody to capsular antigen was not. E. coli K1 strains grown at 37 degrees C (K1 present) resisted phagocytosis and killing if they were opsonized solely by the alternative complement pathway (ACP) using magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid-chelated serum. When these strains were grown at 22 degrees C (K1 absent), they were opsonized efficiently by the ACP (28 versus 94% killing, respectively; P less than 0.001). In addition, a non-K1 mutant of an E. coli K1 strain was opsonized efficiently by the ACP although its encapsulated K1 parent was not. Sensitivity of E. coli strains to the bactericidal activity of serum was observed in strains with and without K1 capsular antigen. These studies demonstrated that production of K1 polysaccharide antigen was regulated by environmental temperature and that K1 capsule plays an essential role in rendering the organism resistant to opsonization by the ACP. PMID:6341228

  2. Protein engineering to target complement evasion in cancer.

    PubMed

    Carter, Darrick; Lieber, André

    2014-01-21

    The complement system is composed of soluble factors in plasma that enhance or "complement" immune-mediated killing through innate and adaptive mechanisms. Activation of complement causes recruitment of immune cells; opsonization of coated cells; and direct killing of affected cells through a membrane attack complex (MAC). Tumor cells up-regulate complement inhibitory factors - one of several strategies to evade the immune system. In many cases as the tumor progresses, dramatic increases in complement inhibitory factors are found on these cells. This review focuses on the classic complement pathway and the role of major complement inhibitory factors in cancer immune evasion as well as on how current protein engineering efforts are being employed to increase complement fixing or to reverse complement resistance leading to better therapeutic outcomes in oncology. Strategies discussed include engineering of antibodies to enhance complement fixation, antibodies that neutralize complement inhibitory proteins as well as engineered constructs that specifically target inhibition of the complement system.

  3. Viral complement regulatory proteins.

    PubMed

    Rosengard, A M; Ahearn, J M

    1999-05-01

    The inactivation of complement provides cells and tissues critical protection from complement-mediated attack and decreases the associated recruitment of other inflammatory mediators. In an attempt to evade the host immune response, viruses have evolved two mechanisms to acquire complement regulatory proteins. They can directly seize the host cell complement regulators onto their outer envelope and/or they can produce their own proteins which are either secreted into the neighboring intercellular space or expressed as membrane-bound proteins on the infected host cell. The following review will concentrate on the viral homologues of the mammalian complement regulatory proteins, specifically those containing complement control protein (CCP) repeats. PMID:10408371

  4. Temperature-sensitive mutants of herpes simplex virus type 2: description of three new complementation groups and studies on the inhibition of host cell DNA synthesis.

    PubMed

    Halliburton, I W; Timbury, M C

    1976-02-01

    Three new complementation groups of type 2 herpes simplex virus are described bringing the total number of complementation groups characterized to 13. Of the three new groups, ts 11 fails to make virus DNA at non-permissive temperature (38 degrees C) whereas ts 12 and ts 13 synthesize only very small amounts of virus or cellular DNA at 38 degrees C. ts 11, like ts 9 (Halliburton & Timbury, 1973) fails to switch off host cell DNA synthesis at 38 degrees C. That this is a failure to switch off cell DNA rather than a stimulation of cell DNA synthesis was confirmed in experiments using resting cells. Both the inability to make virus DNA and the inability to switch off cell DNA are reversed in temperature shift-down experiments with cells infected with ts 9 or ts 11. In temperature shift-up experiments, cellular DNA synthesis is inhibited after the shift but virus DNA is only made in very small amounts, probably due to the continuing functioning of a protein made at permissive temperature (31 degrees C) before the shift but which cannot be made at 38 degrees C. The shift-down experiments and the fact that ts 9 and ts 11 complement one another, suggest that the switch-off of host cell DNA synthesis may involve more than one virus specified function. U.v. irradiated virus fails to switch off host cell DNA synthesis.

  5. Complement-Dependent Lysis of Influenza A Virus-Infected Cells by Broadly Cross-Reactive Human Monoclonal Antibodies ▿

    PubMed Central

    Terajima, Masanori; Cruz, John; Co, Mary Dawn T.; Lee, Jane-Hwei; Kaur, Kaval; Wilson, Patrick C.; Ennis, Francis A.

    2011-01-01

    We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics. PMID:21994454

  6. Differential Complement Activation Pathways Promote C3b Deposition on Native and Acetylated LDL thereby Inducing Lipoprotein Binding to the Complement Receptor 1

    PubMed Central

    Klop, Boudewijn; van der Pol, Pieter; van Bruggen, Robin; Wang, Yanan; de Vries, Marijke A.; van Santen, Selvetta; O'Flynn, Joseph; van de Geijn, Gert-Jan M.; Njo, Tjin L.; Janssen, Hans W.; de Man, Peter; Jukema, J. Wouter; Rabelink, Ton J.; Rensen, Patrick C. N.; van Kooten, Cees; Cabezas, Manuel Castro

    2014-01-01

    Lipoproteins can induce complement activation resulting in opsonization and binding of these complexes to complement receptors. We investigated the binding of opsonized native LDL and acetylated LDL (acLDL) to the complement receptor 1 (CR1). Binding of complement factors C3b, IgM, C1q, mannose-binding lectin (MBL), and properdin to LDL and acLDL were investigated by ELISA. Subsequent binding of opsonized LDL and acLDL to CR1 on CR1-transfected Chinese Hamster Ovarian cells (CHO-CR1) was tested by flow cytometry. Both native LDL and acLDL induced complement activation with subsequent C3b opsonization upon incubation with normal human serum. Opsonized LDL and acLDL bound to CR1. Binding to CHO-CR1 was reduced by EDTA, whereas MgEGTA only reduced the binding of opsonized LDL, but not of acLDL suggesting involvement of the alternative pathway in the binding of acLDL to CR1. In vitro incubations showed that LDL bound C1q, whereas acLDL bound to C1q, IgM, and properdin. MBL did neither bind to LDL nor to acLDL. The relevance of these findings was demonstrated by the fact that ex vivo up-regulation of CR1 on leukocytes was accompanied by a concomitant increased binding of apolipoprotein B-containing lipoproteins to leukocytes without changes in LDL-receptor expression. In conclusion, CR1 is able to bind opsonized native LDL and acLDL. Binding of LDL to CR1 is mediated via the classical pathway, whereas binding of acLDL is mediated via both the classical and alternative pathways. Binding of lipoproteins to CR1 may be of clinical relevance due to the ubiquitous cellular distribution of CR1. PMID:25349208

  7. Evolution of the complement system.

    PubMed

    Nonaka, Masaru

    2014-01-01

    The mammalian complement system constitutes a highly sophisticated body defense machinery comprising more than 30 components. Research into the evolutionary origin of the complement system has identified a primitive version composed of the central component C3 and two activation proteases Bf and MASP in cnidaria. This suggests that the complement system was established in the common ancestor of eumetazoa more than 500 million years ago. The original activation mechanism of the original complement system is believed to be close to the mammalian lectin and alternative activation pathways, and its main role seems to be opsonization and induction of inflammation. This primitive complement system has been retained by most deuterostomes without major change until the appearance of jawed vertebrates. At this stage, duplication of the C3, Bf and MASP genes as well as recruitment of membrane attack components added the classical and lytic pathways to the primitive complement system, converting it to the modern complement system. In contrast, the complement system was lost multiple times independently in the protostome lineage.

  8. The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis.

    PubMed Central

    Stern, L B; Greenberg, M; Gershoni, J M; Rozenblatt, S

    1995-01-01

    Measles virus (MV) and canine distemper virus (CDV) are morbilliviruses that cause acute illnesses and several persistent central nervous system infections in humans and in dogs, respectively. Characteristically, the cytopathic effect of these viruses is the formation of syncytia in permissive cells. In this study, a vaccinia virus expression system was used to express MV and CDV hemagglutinin (HA) and fusion (F) envelope proteins. We found that cotransfecting F and HA genes of MV or F and HA genes of CDV resulted in extensive syncytium formation in permissive cells while transfecting either F or HA alone did not. Similar experiments with heterologous pairs of proteins, CDV-F with MV-HA or MV-F with CDV-HA, caused significant cell fusion in both cases. These results indicate that in this expression system, cell fusion requires both F and HA; however, the functions of these proteins are interchangeable between the two types of morbilliviruses. Human-mouse somatic hybrids were used to determine the human chromosome conferring susceptibility to either MV and CDV. Of the 12 hybrids screened, none were sensitive to MV. Two of the hybrids containing human chromosome 19 formed syncytia following CDV infection. In addition, these two hybrids underwent cell fusion when cotransfected with CDV-F and CDV-HA (but not MV-F and MV-HA) glycoproteins by using the vaccinia virus expression system. To discover the viral component responsible for cell specificity, complementation experiments coexpressing CDV-HA with MV-F or CDV-F with MV-HA in the CDV-sensitive hybrids were performed. We found that syncytia were formed only in the presence of CDV-HA. These results support the idea that the HA protein is responsible for cell tropism. Furthermore, while the F protein is necessary for the fusion process, it is interchangeable with the F protein from other morbilliviruses. PMID:7853502

  9. The NS3 protein of rice hoja blanca virus complements the RNAi suppressor function of HIV-1 Tat.

    PubMed

    Schnettler, Esther; de Vries, Walter; Hemmes, Hans; Haasnoot, Joost; Kormelink, Richard; Goldbach, Rob; Berkhout, Ben

    2009-03-01

    The question of whether RNA interference (RNAi) acts as an antiviral mechanism in mammalian cells remains controversial. The antiviral interferon (IFN) response cannot easily be distinguished from a possible antiviral RNAi pathway owing to the involvement of double-stranded RNA (dsRNA) as a common inducer molecule. The non-structural protein 3 (NS3) protein of rice hoja blanca virus (RHBV) is an RNA silencing suppressor (RSS) that exclusively binds to small dsRNA molecules. Here, we show that this plant viral RSS lacks IFN antagonistic activity, yet it is able to substitute the RSS function of the Tat protein of human immunodeficiency virus type 1. An NS3 mutant that is deficient in RNA binding and its associated RSS activity is inactive in this complementation assay. This cross-kingdom suppression of RNAi in mammalian cells by a plant viral RSS indicates the significance of the antiviral RNAi response in mammalian cells and the usefulness of well-defined RSS proteins. PMID:19218918

  10. Sensitivity of enzyme-linked immunosorbent assay, complement fixation, and hemagglutination inhibition serological tests for detection of Sendai virus antibody in laboratory mice.

    PubMed

    Parker, J C; O'Beirne, A J; Collins, M J

    1979-03-01

    The enzyme-linked immunosorbent assay technique for detection of Sendai virus antibody in mice was approximately 100- and 300-fold more sensitive than the complement fixation and hemagglutination inhibition tests, respectively. The assay also permitted direct quantitative measurement of the amount of antibody on a single serum dilution rather than by the more traditional serial titration.

  11. CD8+ T cells complement antibodies in protecting against yellow fever virus.

    PubMed

    Bassi, Maria R; Kongsgaard, Michael; Steffensen, Maria A; Fenger, Christina; Rasmussen, Michael; Skjødt, Karsten; Finsen, Bente; Stryhn, Anette; Buus, Søren; Christensen, Jan P; Thomsen, Allan R

    2015-02-01

    The attenuated yellow fever (YF) vaccine (YF-17D) was developed in the 1930s, yet little is known about the protective mechanisms underlying its efficiency. In this study, we analyzed the relative contribution of cell-mediated and humoral immunity to the vaccine-induced protection in a murine model of YF-17D infection. Using different strains of knockout mice, we found that CD4(+) T cells, B cells, and Abs are required for full clinical protection of vaccinated mice, whereas CD8(+) T cells are dispensable for long-term survival after intracerebral challenge. However, by analyzing the immune response inside the infected CNS, we observed an accelerated T cell influx into the brain after intracerebral challenge of vaccinated mice, and this T cell recruitment correlated with improved virus control in the brain. Using mice deficient in B cells we found that, in the absence of Abs, YF vaccination can still induce some antiviral protection, and in vivo depletion of CD8(+) T cells from these animals revealed a pivotal role for CD8(+) T cells in controlling virus replication in the absence of a humoral response. Finally, we demonstrated that effector CD8(+) T cells also contribute to viral control in the presence of circulating YF-specific Abs. To our knowledge, this is the first time that YF-specific CD8(+) T cells have been demonstrated to possess antiviral activity in vivo.

  12. Disulfide linked pyrazole derivatives inhibit phagocytosis of opsonized blood cells.

    PubMed

    Purohit, Meena K; Scovell, Iain; Neschadim, Anton; Katsman, Yulia; Branch, Donald R; Kotra, Lakshmi P

    2013-04-15

    Immune thrombocytopenia (ITP) is caused by production of an autoantibody to autologous platelets. ITP can be treated either by reducing platelet destruction or by increasing platelet production. Fcγ receptor mediated phagocytosis of the opsonized blood cells is a well-accepted mechanism for the underlying pathogenesis of ITP and inhibition of this phagocytosis process with small molecules is a potential strategy for the development of drugs against ITP. A broad screen indicated that 4-methyl-1-phenyl-pyrazole derivative (1) could inhibit the phagocytosis of opsonized blood cells with weak potency. We reveal here the discovery of the polysulfide products, synthesis of various 1-phenyl-pyrazole derivatives, and the biological evaluation of pyrazole derivatives as inhibitors of phagocytosis for potential use as therapeutics for ITP. Substitution at C4 of the pyrazole moiety in the disulfide-bridged dimers influenced the potency in the increasing order of 10 ~/= 11~/= 16 < 19 < 20. A novel scaffold, 20 with an IC(50) of 100 nM inhibiting opsonized blood cell phagocytosis was identified as a potential candidate for further studies. Confirmation of the disulfide bridge additionally provides clues for the non-thiol or non-disulfide bridge carrying ligands targeting ITP and other similar disorders.

  13. 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

  14. Immunologic injury of cultured cells infected with measles virus. I. role of IfG antibody and the alternative complement pathway

    PubMed Central

    1975-01-01

    In these studies, a number of human cell lines including epithelial, neural, glial, and lymphoid cells infected with several strains of measles virus were found to be lysed upon incubation with fresh sera from humans containing antibody measles virus. In all instances, the cytolytic event was mediated by alternative complement (C) pathway without a significant contribution from classical pathway. In contrast, isolated measles virus in conjunction with antibody was found to selectively activate the classical C pathway. Measles antibodies of the IgG class, but not the IgA class, possessed cytolytic potential against cells infected with measles virus. Human IgG antibodies could directly activate the alternative C pathway. No defect was found in cytolytic measles antibody in sera or cerebrospinal fluid from patients with subacute sclerosing panencephalitis, nor was the alternative C pathway impaired in sera from these patients. Sera from newborn humans exhibited a functional alternative C pathway. PMID:1092789

  15. Membrane-bound hemagglutinin mediates antibody and complement-dependent lysis of influenza virus-treated human platelets in autologous serum.

    PubMed Central

    Kazatchkine, M D; Lambré, C R; Kieffer, N; Maillet, F; Nurden, A T

    1984-01-01

    Influenza A virus-treated human platelets were lyzed in autologous serum. Lysis required the presence of antibody and occurred predominantly through activation of the classical complement pathway. Binding of the virus followed by its elution at 37 degrees C resulted in a dose-dependent desialation of the cells with a maximal release of 45% of total platelet sialic acid. In contrast, platelets that had been treated with Vibrio cholerae neuraminidase and from which 55% of total sialic acid had been removed were not lyzed in autologous serum and did not bind C3 as shown in binding assays using radiolabeled monoclonal anti-C3 antibody. Thus, the immune-mediated lysis of virus-treated platelets in autologous serum did not involve neoantigens expressed by desialated cells. To assess the effect of viruses on the platelet surface, treated platelets were incubated with galactose oxidase and sodium [3H]borohydride prior to separation and analysis of the labeled glycoproteins by SDS-PAGE. Viral treatment resulted in a desialation of each of the surface glycoproteins. At the same time, a labeled component of Mr 72,000 (nonreduced) and Mr 55,000 (reduced) was observed that was not present when V. cholerae-desialated platelets were examined in the same way. Immunoblotting experiments performed using antiwhole virus and anti-hemagglutinin antibodies demonstrated this component to be viral hemagglutinin. Involvement of membrane-bound hemagglutinin in antibody and in complement-mediated lysis of virus-treated platelets in autologous serum was supported by the increased lytic activity of a postvaccinal serum containing an elevated titer of complement fixing anti-hemagglutinin antibodies. Binding of a viral protein to the platelet surface provides a model for immune thrombocytopenias occurring during acute viral infections at the time of the specific immune response. Images PMID:6470149

  16. Adeno-Associated Virus Mediated Delivery of An Engineered Protein that Combines the Complement Inhibitory Properties of CD46, CD55 and CD59

    PubMed Central

    Leaderer, Derek; Cashman, Siobhan M.; Kumar-Singh, Rajendra

    2015-01-01

    Background A variety of disorders are associated with the activation of complement. CD46, CD55 and CD59 are the major membrane associated regulators of complement on human cells. Previously, we have found that independent expression of CD55, CD46 or CD59 through gene transfer protects murine tissues against human complement mediated attack. Herein we investigated the potential of combining the complement regulatory properties of CD46, CD55 and CD59 into single gene products expressed from an adeno-associated virus (AAV) vector in a soluble non-membrane anchored form. Methods Minigenes encoding the complement regulatory domains from CD46, CD55 and CD59 (SACT) or CD55 and CD59 (DTAC) were cloned into an AAV vector. The specific regulatory activity of each component of SACT and DTAC was measured in vitro. The recombinant AAV vectors were injected into the peritoneum of mice and the efficacy of the transgene products for being able to protect murine liver vasculature against human complement, specifically the membrane attack complex (MAC) was measured. Results SACT and DTAC exhibited properties similar to CD46, CD55 and CD59 or CD55 and CD59 respectively in vitro. AAV mediated delivery of SACT or DTAC protected murine liver vasculature from human MAC deposition by 63.2% and 56.7% respectively. Conclusions When delivered to mice in vivo via an AAV vector, SACT and DTAC are capable of limiting human complement mediated damage. SACT and DTAC merit further study as potential therapies for complement mediated disorders when delivered via a gene therapy approach. PMID:25917932

  17. Serum antibody responses in naturally occurring influenza A virus infection determined by enzyme-linked immunosorbent assay, hemagglutination inhibition, and complement fixation.

    PubMed

    Madore, H P; Reichman, R C; Dolin, R

    1983-12-01

    Serum antibody responses to influenza A virus infection were examined in 388 normal subjects during a trial of chemoprophylaxis in an outbreak of influenza A in 1980-1981 in which both A/H1N1 and A/H3N2 viruses circulated. Paired serum specimens obtained over a 6-week period were tested for antibodies to both A/H1N1 and A/H3N2 viruses by conventional hemagglutination inhibition, complement fixation, and an enzyme-linked immunosorbent assay (ELISA). Antibody responses detected by ELISA were determined by calculation of the area generated between titration curves of paired sera (area method), as well as by a conventional endpoint dilution method (endpoint method). Forty-two significant antibody rises were detected; 42 by ELISA (area method), 33 by ELISA (endpoint method), 32 by hemagglutination inhibition, and 13 by complement fixation. ELISA (area method) detected rises more frequently than either ELISA (endpoint method) (P less than 0.01), hemagglutination inhibition (P less than 0.005), or complement fixation (P less than 0.001). Another sensitive assay, the microneutralization test, detected significantly fewer rises (33, P less than 0.025) than the ELISA (area method). In the 42 subjects with ELISA (area method) rises, corroborating evidence of influenza A infection by other techniques (virus isolation, microneutralization, hemagglutination inhibition, or complement fixation tests) were available for 39 (93%). ELISA (area method) rises were subtype specific in all serum pairs in which other documentation of subtype-specific infection was available (38 of 38). Thus, ELISA (area method) was the single most sensitive assay for detection of serum antibody rises in this setting and possessed a high degree of subtype specificity.

  18. Human seminal plasma inhibition of complement.

    PubMed

    Petersen, B H; Lammel, C J; Stites, D P; Brooks, G F

    1980-10-01

    Recent studies have shown that human seminal plasma contains chemically and biologically distinct factors which inhibit lymphocyte functions and the serum bactericidal and opsonic activities associated with the killing of gram-negative organisms. Because of the direct association between complement action and serum bactericidal and opsonic activities, inhibition of complement may be one of the possible mechanisms of action of seminal plasma immunoinhibitory factors. Complement hemolytic activity was measured for C3 and C4 in serum Neisseria gonorrhoeae and Escherichia coli bactericidal reaction mixtures with and without addition of seminal plasma. In the presence of seminal plasma, where there was no bactericidal action, C3 and titers were reduced to approximately 50% of the titers in the reactions with complement donor serum. The C3 titers were lower than in the reaction mixtures with immune serum and complement donor serum, where N. gonorrhoeae bactericidal activity occurred. Individual human seminal plasma specimens depressed CH50 activity of pooled normal human sera up to 50% of normal levels. There were no differences in inhibition by seminal plasma specimens from normal or vasectomized men. Treatment with seminal plasma depressed the functional activity of complement components C1 and C3 by more than 50%. Seminal plasma also inhibited alternate pathway activity. Cleavage of factor B was demonstrated. The seminal plasma factor which inhibited complement was of low molecular weight. DPF blocked the seminal plasma complement-inhibitory factor. However, amidolytic activity for serine protease substrates could not be demonstrated. It is likely that the seminal plasma complement inhibitor is a protease inhibitor acting singly or in combination.

  19. Measles virus-induced down-regulation of CD46 is associated with enhanced sensitivity to complement-mediated lysis of infected cells.

    PubMed

    Schnorr, J J; Dunster, L M; Nanan, R; Schneider-Schaulies, J; Schneider-Schaulies, S; ter Meulen, V

    1995-04-01

    CD46, the major component of the measles virus (MV) receptor complex and a member of the regulators of complement activity (RCA) gene cluster, is down-regulated in MV-infected cells. We investigated whether the reduction of surface CD46 correlates with enhanced sensitivity of lymphoid and monocytic cells to lysis by activated complement. On human U937 cells, acutely or persistently infected with MV-Edmonston (ED) vaccine strain, infection-dependent down-regulation of CD46 confers sensitivity to activated complement, regardless of the pathway of activation and the specificity of the activating antibodies. Interestingly, down-regulation of CD46 alone is sufficient to confer susceptibility of cells to complement lysis despite the continued surface expression of other RCA proteins such as CD35 and CD55. In primary cultures, both peripheral blood lymphocytes and macrophages are efficiently lysed in the presence of complement activated via the alternative pathway after MV infection. In contrast to the MV-ED infection, infection of cells with the lymphotropic MV wild-type strain WTF does not down-regulate CD46. Cells infected with MV-WTF do not exhibit enhanced susceptibility to complement lysis. These data suggest that MV strains similar to WTF that do not down-regulate CD46 may have an enhanced potential for replication and dissemination within the human host, whereas complement-mediated elimination of cells infected with CD46-down-regulating strains of MV, such as ED, may limit the spread of MV infection, and could thus represent an attenuating factor for MV. PMID:7737301

  20. Sundanese Complementation

    ERIC Educational Resources Information Center

    Kurniawan, Eri

    2013-01-01

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

  1. Comparison of a newly developed enzyme-linked immunosorbent assay with complement fixation and neutralisation tests for serology of bovine respiratory syncytial virus infections.

    PubMed

    Westenbrink, F; Brinkhof, J M; Straver, P J; Quak, J; De Leeuw, P W

    1985-05-01

    An indirect double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the detection and titration of serum antibodies to bovine respiratory syncytial virus (BRSV). The ELISA was compared with a complement fixation (CF) test and a test for virus neutralising antibody in serum (virus neutralisation [VN] test). Testing sera collected in dairy herds revealed the closest correlation between the results of the ELISA and the CF test with respect to BRSV antibody titres. The VN test detected BRSV antibodies in a higher percentage of acute phase sera compared to the other two tests in field samples and in early bleedings of experimentally infected calves. However, the VN test was less effective in making a diagnosis of BRSV infections on the basis of a significant titre increase in paired sera. For this purpose the ELISA was found to be the most sensitive test.

  2. Mutations in the coat protein gene of plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana.

    PubMed

    Varrelmann, M; Maiss, E

    2000-03-01

    Two different motifs in the coat protein (CP) of Plum pox virus (PPV) (R(3015)Q(3016), D(3059)) were mutated by replacing the respective amino acids with others possessing different chemical properties. The mutated CP genes were introduced into an infectious full-length clone of PPV (p35PPV-NAT) to investigate their influence on systemic infection of transgenic wild-type PPV CP-expressing and non-transgenic plants of Nicotiana benthamiana. All mutants failed to establish systemic infections in non-transgenic N. benthamiana plants, but were complemented by intact CP in transgenic plants. Moreover, the CP-RQ-D mutant (carrying mutations in both the RQ and D motifs) was introduced into p35PPV-NAT engineered to express beta-glucuronidase (GUS) for direct observation of systemic movement and particle assembly in N. benthamiana leaves. GUS-staining revealed that the CP mutant (RQ-D) was restricted to initially infected cells without forming virions. Systemic movement and particle assembly were restored in CP-transgenic N. benthamiana plants. Finally, transgenic N. benthamiana plants were generated that expressed each of the three mutated CP genes. Homozygous T(2) lines were selected and tested for resistance to PPV. Immunogold labelling and electron microscopy revealed that heterologous encapsidation with challenging Chilli veinal mottle virus and Potato virus Y was suppressed in these lines. In addition, assembly mutants did not complement CP-defective p35PPV-NAT. The possible use of modified viral CP genes for the production of virus-resistant transgenic plants, thereby reducing the putative risks of heterologous encapsidation and complementation, is discussed. PMID:10675394

  3. Deletion of the complement C5a receptor alleviates the severity of acute pneumococcal otitis media following influenza A virus infection in mice.

    PubMed

    Tong, Hua Hua; Lambert, Garrett; Li, Yong Xing; Thurman, Joshua M; Stahl, Gregory L; Douthitt, Kelsey; Clancy, Caitlin; He, Yujuan; Bowman, Andrew S

    2014-01-01

    There is considerable evidence that influenza A virus (IAV) promotes adherence, colonization, and superinfection by S. pneumoniae (Spn) and contributes to the pathogenesis of otitis media (OM). The complement system is a critical innate immune defense against both pathogens. To assess the role of the complement system in the host defense and the pathogenesis of acute pneumococcal OM following IAV infection, we employed a well-established transtympanically-induced mouse model of acute pneumococcal OM. We found that antecedent IAV infection enhanced the severity of acute pneumococcal OM. Mice deficient in complement C1qa (C1qa-/-) or factor B (Bf -/-) exhibited delayed viral and bacterial clearance from the middle ear and developed significant mucosal damage in the eustachian tube and middle ear. This indicates that both the classical and alternative complement pathways are critical for the oto-immune defense against acute pneumococcal OM following influenza infection. We also found that Spn increased complement activation following IAV infection. This was characterized by sustained increased levels of anaphylatoxins C3a and C5a in serum and middle ear lavage samples. In contrast, mice deficient in the complement C5a receptor (C5aR) demonstrated enhanced bacterial clearance and reduced severity of OM. Our data support the concept that C5a-C5aR interactions play a significant role in the pathogenesis of acute pneumococcal OM following IAV infection. It is possible that targeting the C5a-C5aR axis might prove useful in attenuating acute pneumococcal OM in patients with influenza infection.

  4. [Biological roles of complement and recent topics in clinical medicine].

    PubMed

    Wakamiya, Nobutaka

    2012-08-01

    The complement has been identified as a complementation factor to compensate for the function of an antibody. The complement consists of C1-C9, a complement-related molecule, and its regulating molecules. Three major biological roles of the complement have been classified: First: opsonization following phagocytosis and the elimination of microbes; second: direct destruction of bacteria due to membrane attack complex (MAC); third: complement activation following the induction of anaphylactoid factors and local recruitment and activation of neutrophilic leukocytes. In this review, the basic findings and recent treatments of paroxysmal nocturnal hemoglobinuria (PNH) and hereditary angioedema (HAE) are summarized. Finally, there is a short review of a rare autosomal recessive disorder of 3MC syndrome and new biological functions of complement factors except for that of innate immunity are proposed.

  5. Members of a Novel Family of Mammalian Protein Kinases Complement the DNA-Negative Phenotype of a Vaccinia Virus ts Mutant Defective in the B1 Kinase

    PubMed Central

    Boyle, Kathleen A.; Traktman, Paula

    2004-01-01

    Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking sequence similarity between B1 and the VRK enzymes, we proposed that they might share overlapping substrate specificity. We therefore sought to determine whether the human and mouse VRK1 enzymes (hVRK1 and mVRK1, respectively) could complement a B1 deficiency in vivo. Recombinant ts2 viruses expressing hVRK1, mVRK1, or wild-type B1 were able to synthesize viral DNA at high temperature, but those expressing the more distantly related human casein kinase 1α2 could not. Complementation required the enzymatic activity of hVRK1, since a catalytically inactive allele of hVRK1 was unable to confer a temperature-insensitive phenotype. Interestingly, rescue of viral DNA synthesis was not coupled to the ability to phosphorylate H5, the only virus-encoded protein shown to be a B1 substrate in vivo. Expression of hVRK1 during nonpermissive ts2 infections restored virus production and plaque formation, whereas expression of mVRK1 resulted in an intermediate level of rescue. Taken together, these observations indicate that enzymatically active cellular VRK1 kinases can perform the function(s) of B1 required for genome replication, most likely due to overlapping specificity for cellular and/or viral substrates. PMID:14747564

  6. Antibody-mediated opsonization of red blood cells in parvovirus B19 infection.

    PubMed

    Chehadeh, Wassim; Halim, Medhat A; Al-Nakib, Widad

    2009-07-20

    Red blood cells (RBCs) express abundantly parvovirus B19 receptor, and their role in the dissemination or clearance of B19 infection is unknown. In this study, we report that in early, acute or persistent infection, B19 viremia is mostly associated with RBCs. The capacity of different patients' plasma or IgG to opsonize RBCs collected from patients with early B19 infection, was investigated. The highest opsonization activity was observed with plasma or IgG fractions from patients with past B19 infection. In contrast, IgG samples from patients with acute or persistent infection showed no or little opsonization activity. The depletion of antibodies specific to B19 VP1, but not VP2, from IgG samples, resulted in a significant suppression of opsonization. Furthermore, IgG samples preincubated with heated B19 particles exposing VP1-unique (VP1u) region were unable to opsonize RBCs. These observations clearly suggest a role for anti-VP1u IgG in the opsonization of RBC-bound B19 particles.

  7. Identification of a purified complement-fixing antigen as the Epstein-Barr-virus determined nuclear antigen (EBNA) by its binding to metaphase chromosomes.

    PubMed

    Ohno, S; Luka, J; Lindahl, T; Klein, G

    1977-04-01

    A soluble complement-fixing antigen carried by Epstein-Barr virus (EBV)-transformed human cells has been previously extracted from cell nuclei and purified by DNA-cellulose chromatography [Luka, J., Siegert, W. & Klein, G. (1977) J. Virol., in press]. On addition of this antigen to methanol/acetic acid-fixed metaphase chrmosomes, followed by exposure to human sera containing antibodies against the EBV-determined nuclear antigen (EBNA), brilliant positive staining was obtained by anti-complement immunofluorescence. There was no staining after exposure to EBV-negative sera. Moreover, a nuclear protein fraction, prepared from an EBV-negative cell line in an analogous fashion, failed to induce the staining reaction. These data identify the soluble purified antigen as the EBV-determined nuclear antigen. The purified antigen has a molecular weight of 174,000 +/- 15,000, as determined by sucrose gradient centrifugation and gel filtration experiments. In neutral buffers containing 0.5-1.0 M NaCl, the antigen dissociates into a form of approximately one-half the original molecular weight with retained complement-fixing activity. This "monomer" has a molecular weight of 98,000 +/- 8,000. PMID:67603

  8. Modulation of anxiety behavior by intranasally administered vaccinia virus complement control protein and curcumin in a mouse model of Alzheimer's disease.

    PubMed

    Kulkarni, A P; Govender, D A; Kotwal, G J; Kellaway, L A

    2011-02-01

    Widespread neuroinflammation in the central nervous system (CNS) of Alzheimer's disease (AD) patients, involving pro-inflammatory mediators such as complement components, might be responsible for AD associated behavioral symptoms such as anxiety. Vaccinia virus complement control protein (VCP) and curcumin (Cur) are the bioactive compounds of natural origin shown to inhibit the in-vitro complement activation. In order to develop complement regulatory compounds which could be delivered to the CNS by a non-invasive route, VCP, its truncated version (tVCP), and Cur were administered to Wistar rats intranasally. The distribution of these compounds in cerebrospinal fluid (CSF) was studied using an enzyme linked immunosorbent assay (ELISA), using VCP and tVCP as antigens and a modified fluorimetric method (Cur). VCP and tVCP were also detected in the olfactory lobes of the rat brain using immunohistochemical analysis. These compounds were then compared for their ability to attenuate the anxiety levels in APPswePS1δE9 mice using an elevated plus maze (EPM) apparatus. VCP treatment significantly improved the exploratory behavior and reduced the anxiety behavior in APPswePS1δE9 mice. tVCP however showed an opposite effect to VCP, whereas Cur showed no effect on the anxiety behavior of these mice. When these mice were subsequently tested for their cognitive performance in the Morris water maze (MWM), they showed tendencies to collide with the periphery of the walls of MWM. This unusual activity was termed "kissperi" behavior. This newly defined index of anxiety was comparable to the anxiety profile of the VCP and tVCP treated groups on EPM. VCP can thus be delivered to the CNS effectively via intranasal route of administration to attenuate anxiety associated with AD.

  9. Complement Test

    MedlinePlus

    ... helpful? Also known as: C1; C1q; C2; C3; C4; CH50; CH100 (among others) Formal name: Complement Activity; ... whether the system is functioning normally. C3 and C4 are the most frequently measured complement proteins. Total ...

  10. Protease-dependent mechanisms of complement evasion by bacterial pathogens.

    PubMed

    Potempa, Michal; Potempa, Jan

    2012-09-01

    The human immune system has evolved a variety of mechanisms for the primary task of neutralizing and eliminating microbial intruders. As the first line of defense, the complement system is responsible for rapid recognition and opsonization of bacteria, presentation to phagocytes and bacterial cell killing by direct lysis. All successful human pathogens have mechanisms of circumventing the antibacterial activity of the complement system and escaping this stage of the immune response. One of the ways in which pathogens achieve this is the deployment of proteases. Based on the increasing number of recent publications in this area, it appears that proteolytic inactivation of the antibacterial activities of the complement system is a common strategy of avoiding targeting by this arm of host innate immune defense. In this review, we focus on those bacteria that deploy proteases capable of degrading complement system components into non-functional fragments, thus impairing complement-dependent antibacterial activity and facilitating pathogen survival inside the host.

  11. The Role of Complement in Antibody Therapy for Infectious Diseases.

    PubMed

    Wibroe, Peter P; Helvig, Shen Y; Moein Moghimi, S

    2014-04-01

    The complement system is part of the innate immune system, eliciting central immunoregulatory functions. Detection of foreign surfaces is either achieved through complement-specific patternrecognition molecules or mediated by antigen recognition of antibodies. Immunoglobulin A (IgA), IgG, and IgM all have the potential to initiate a complement response, with the efficiency and response development closely related to the antibody isotype, multimeric state, and degree of glycosylation. A group of serum proteins constitutes the central effector functions of complement, thus allowing direct cell lysis, opsonization, and inflammation. These effector functions can be used in antibody therapies, especially against infectious diseases, as the target membranes lack complement regulatory proteins. The relative contribution of each function and the interplay with direct antibody-mediated clearance is not fully exploited, thus suggesting an option for further rational optimization of antibody therapies.

  12. Porcine complement regulatory protein CD46 and heparan sulfates are the major factors for classical swine fever virus attachment in vitro.

    PubMed

    Dräger, Carolin; Beer, Martin; Blome, Sandra

    2015-03-01

    Classical swine fever virus (CSFV) is the causative agent of a severe multi-systemic disease of pigs. While several aspects of virus-host-interaction are known, the early steps of infection remain unclear. For the closely related bovine viral diarrhea virus (BVDV), a cellular receptor is known: bovine complement regulatory protein CD46. Given that these two pestiviruses are closely related, porcine CD46 is also a candidate receptor for CSFV. In addition to CD46, cell-culture-adapted CSFV strains have been shown to use heparan sulfates as an additional cellular factor. In the present study, the interaction of field-type and cell-culture-adapted CSFV with a permanent porcine cell line or primary macrophages was assessed using anti-porcine CD46 monoclonal antibodies and a heparan-sulfate-blocking compound, DSTP-27. The influence of receptor blocking was assessed using virus titration and quantitative PCR. Treatment of cells with monoclonal antibodies against porcine CD46 led to a reduction of viral growth in both cell types. The effect was most pronounced with field-type CSFV. The blocking could be enhanced by addition of DSTP-27, especially for cell-culture-adapted CSFV. The combined use of both blocking agents led to a significant reduction of viral growth but was also not able to abolish infection completely. The results obtained in this study showed that both porcine CD46 and heparan sulfates play a major role in the initial steps of CSFV infection. Additional receptors might also play a role for attachment and entry; however, their impact is obviously limited in vitro in comparison to CD46 and heparan sulfates.

  13. Defective serum opsonization activity in children aged 6-48 months having acute purulent otitis media.

    PubMed

    Tezcan, I; Yilmaz, Y; Oner, F; Yel, L; Sanal, O; Ersoy, F; Onerci, M; Berkel, A I

    1997-01-01

    Serum opsonization of yeast (Saccharomyces) was investigated in 51 patients whose ages were between six and 48 months (median 15 months) with acute purulent otitis media and in an age-matched control group (median 13 months). Opsonization was assessed by measuring yeast particle uptake in an assay based on an electronic count of the unphagocytosed particles in serum by polymorphonuclear leukocytes. Despite normal levels of CH50 and serum immunoglobulins, a defective opsonization was determined in 13.7 percent of the patients (7 in 51). The corresponding figure was 2.9 percent in 103 healthy controls (p < 0.001). On the other hand, 218 percent (5 in 23) of the children having a history of recurrent purulent otitis media showed defective opsonization (p < 0.001). Previously, the presence of an opsonization defect has been linked to low levels of mannan binding lectin (MBL), a calcium dependent serum lectin that acts as an opsonin. Therefore, our findings indirectly support the idea that MBL has an important role as host defense, particularly in the earlier period of life when the antibody repertoire is restricted.

  14. Interaction between Epstein-Barr virus and a T cell line (HSB-2) via a receptor phenotypically distinct from complement receptor type 2.

    PubMed

    Hedrick, J A; Watry, D; Speiser, C; O'Donnell, P; Lambris, J D; Tsoukas, C D

    1992-05-01

    Epstein-Barr virus (EBV), the causative agent of mononucleosis and several human cancers, infects cells via complement receptor type 2 (CR2, CD21) which also serves as the receptor for the third complement component, C3. Expression of this receptor is restricted to B lymphocytes, immature thymocytes, and certain epithelial cells. In the present investigation; we describe the presence of a seemingly novel EBV receptor which is phenotypically distinct from CR2. Among various leukemic T cells studied, one, HSB-2, demonstrates no reactivity to several anti-CR2 antibodies, yet it reacts strongly with EBV as detected by incubation with biotin-conjugated virus and streptavidin-phycoerythrin. The virus binding is specific as demonstrated by blocking with anti-EBV antibodies and with non-conjugated virus. Aggregated C3 also binds HSB-2 and is capable of partially inhibiting EBV binding. The absence of CR2 on HSB-2 is further supported by the lack of expression of specific mRNA, assessed by Northern blotting analysis and polymerase chain reaction. Viral internalization and infection is demonstrated with electron microscopy, with detection of EBV-DNA by Southern blotting, and with detection of EBNA-1 transcripts by the polymerase chain reaction. Even though HSB-2 does not express CR2, it nevertheless displays transcripts which have some homology to a CR2 cDNA probe under low stringency hybridization conditions. This probe encompasses approximately the N-terminal half of CR2 which includes the EBV-binding epitope(s). The HSB-2 message is 5.2 kb, a size distinct from the 4.7-kb message of B cell CR2s. In contrast, the 5.2-kb message in not seen, under similar hybridization conditions, with a probe comprising the C-terminal half of CR2. Collectively, the data indicate that a receptor molecule having distinct phenotypic characteristics from the known CR2 protein on B cells is utilized by EBV to target human T lymphocytes. PMID:1315687

  15. Functional Interactions of the HHCC Domain of Moloney Murine Leukemia Virus Integrase Revealed by Nonoverlapping Complementation and Zinc-Dependent Dimerization

    PubMed Central

    Yang, Fan; Leon, Oscar; Greenfield, Norma J.; Roth, Monica J.

    1999-01-01

    The retroviral integrase (IN) is required for the integration of viral DNA into the host genome. The N terminus of IN contains an HHCC zinc finger-like motif, which is conserved among all retroviruses. To study the function of the HHCC domain of Moloney murine leukemia virus IN, the first N-terminal 105 residues were expressed independently. This HHCC domain protein is found to complement a completely nonoverlapping construct lacking the HHCC domain for strand transfer, 3′ processing and coordinated disintegration reactions, revealing trans interactions among IN domains. The HHCC domain protein binds zinc at a 1:1 ratio and changes its conformation upon binding to zinc. The presence of zinc within the HHCC domain stimulates selective integration processes. Zinc promotes the dimerization of the HHCC domain and protects it from N-ethylmaleimide modification. These studies dissect and define the requirement for the HHCC domain, the exact function of which remains unknown. PMID:9971758

  16. Isolating the Epstein-Barr virus gp350/220 binding site on complement receptor type 2 (CR2/CD21).

    PubMed

    Young, Kendra A; Chen, Xiaojiang S; Holers, V Michael; Hannan, Jonathan P

    2007-12-14

    Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lymphocytes in an interaction mediated by the viral envelope glycoprotein gp350. The heavily glycosylated structure of EBV gp350 has recently been elucidated by x-ray crystallography, and the CR2 binding site on this protein has been characterized. To identify the corresponding gp350 binding site on CR2, we have undertaken a site-directed mutagenesis study targeting regions of CR2 that have previously been implicated in the binding of CR2 to the C3d/C3dg fragments of complement component C3. Wild-type or mutant forms of CR2 were expressed on K562 cells, and the ability of these CR2-expressing cells to bind gp350 was measured using flow cytometry. Mutations directed toward the two N-terminal extracellular domains of CR2 (SCR1-2) reveal that a large contiguous surface of CR2 SCR1-2 is involved in gp350 binding, including a number of positively charged residues (Arg-13, (Arg-28, (Arg-36, Lys-41, Lys-57, Lys-67, and Arg-83). These data appear to complement the CR2 binding site on gp350, which is characterized by a preponderance of negative charge. In addition to identifying the importance of charge in the formation of a CR2-gp350 complex, we also provide evidence that both SCR1 and SCR2 make contact with gp350. Specifically, two anti-CR2 monoclonal antibodies, designated as monoclonal antibodies 171 and 1048 whose primary epitopes are located within SCR2, inhibit binding of wild-type CR2 to EBV gp350; with regard to SCR1, both K562 cells expressing an S15P mutation and recombinant S15P CR2 proteins exhibit diminished gp350 binding.

  17. Isolating the Epstein-Barr virus gp350/220 binding site on complement receptor type 2 (CR2/CD21).

    PubMed

    Young, Kendra A; Chen, Xiaojiang S; Holers, V Michael; Hannan, Jonathan P

    2007-12-14

    Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lymphocytes in an interaction mediated by the viral envelope glycoprotein gp350. The heavily glycosylated structure of EBV gp350 has recently been elucidated by x-ray crystallography, and the CR2 binding site on this protein has been characterized. To identify the corresponding gp350 binding site on CR2, we have undertaken a site-directed mutagenesis study targeting regions of CR2 that have previously been implicated in the binding of CR2 to the C3d/C3dg fragments of complement component C3. Wild-type or mutant forms of CR2 were expressed on K562 cells, and the ability of these CR2-expressing cells to bind gp350 was measured using flow cytometry. Mutations directed toward the two N-terminal extracellular domains of CR2 (SCR1-2) reveal that a large contiguous surface of CR2 SCR1-2 is involved in gp350 binding, including a number of positively charged residues (Arg-13, (Arg-28, (Arg-36, Lys-41, Lys-57, Lys-67, and Arg-83). These data appear to complement the CR2 binding site on gp350, which is characterized by a preponderance of negative charge. In addition to identifying the importance of charge in the formation of a CR2-gp350 complex, we also provide evidence that both SCR1 and SCR2 make contact with gp350. Specifically, two anti-CR2 monoclonal antibodies, designated as monoclonal antibodies 171 and 1048 whose primary epitopes are located within SCR2, inhibit binding of wild-type CR2 to EBV gp350; with regard to SCR1, both K562 cells expressing an S15P mutation and recombinant S15P CR2 proteins exhibit diminished gp350 binding. PMID:17925391

  18. Effect of sulfide ions on complement factor C3.

    PubMed Central

    Granlund-Edstedt, M; Johansson, E; Claesson, R; Carlsson, J

    1991-01-01

    In infected sites such as the gingival pockets of patients with periodontal disease, sulfide levels up to 1 mmol/liter may be reached. There is little information, however, on how sulfide may interact with the host defense. In a previous study (R. Claesson, M. Granlund-Edstedt, S. Persson, and J. Carlsson, Infect. Immun. 57:2776-2781, 1989), it was shown that polymorphonuclear leukocytes were able to kill bacteria in the presence of 1 mM sulfide. However, sulfide seemed to interfere with the opsonization of the bacteria. It has been claimed that sulfide may be toxic by splitting disulfide bonds of proteins. In the present study, serum was exposed to 2 mM sulfide under anaerobic conditions, and the capacity of sulfide to split disulfide bonds of 10 serum proteins involved in opsonization was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection of the proteins after blotting. Sulfide had a low capacity to split the disulfide bonds of most proteins. Sulfide had, however, a pronounced effect on the complement component C3 in the form of C3bi. Sulfide released the C-terminal region of the alpha chain from C3bi. When C3 opsonizes bacteria, it is this region of C3bi which binds to complement receptor 3 (CR3) of the polymorphonuclear leukocytes. If sulfide has the same effect on C3bi deposited on the bacterial surface as it has on C3bi in solution, it will annihilate the very important contribution of C3bi to opsonization. Images PMID:1987085

  19. Detection of opsonic antibodies against Enterococcus faecalis cell wall carbohydrates in immune globulin preparations.

    PubMed

    Hufnagel, M; Sixel, K; Hammer, F; Kropec, A; Sava, I G; Theilacker, C; Berner, R; Huebner, J

    2014-08-01

    Three different commercially available polyvalent immune globulins (IG) were investigated for the existence of antibodies against cell wall carbohydrates of four different E. faecalis serotypes (using a cell wall carbohydrate-enzyme-linked immunosorbent assay), and whether these antibodies mediated opsonic killing (using an opsonic-killing assay). All three IG preparations contained antibodies against all four serotypes (CPS-A to CPS-D). However, only one of the three IG preparations showed opsonic killing against all four serotypes. Average killing was higher against serotypes A and B (72 and 79 %, respectively) than against serotypes C and D (30 and 37 %, respectively). Such IG preparations could play a role as an adjuvant therapeutic option in life-threatening infections with E. faecalis, particularly when resistant strains are involved.

  20. Mechanisms of complement-mediated clearance of bacteria from the murine lung.

    PubMed

    Heidbrink, P J; Toews, G B; Gross, G N; Pierce, A K

    1982-05-01

    Complement factors enhance host defense against bacterial challenges by attracting phagocytic cells to the site of the inoculum and by opsonizing bacteria for phagocytic ingestion. The relative contribution of these 2 mechanisms to in vivo clearance of bacteria from the lung has not been described. Hypocomplementemic and normal animals were challenged with various bacteria. Clearance of bacteria was studied by quantitative lung culture. Phagocytic response was determined by bronchoalveolar lavage. Staphylococci were cleared by macrophages without regard to the complement status of the host. Hypocomplementemic animals cleared pneumococci less efficiently than did control animals. This defect correlated with decreased neutrophil recruitment. Pseudomonas was not cleared in hypocomplementemic animals, but there was no difference in the number or type of phagocytes. This implies that an opsonic rather thn a chemotactic defect was responsible. These data suggest that the mechanism of complement-mediated defense against bacterial challenge varies with the type of pathogen present.

  1. Encapsidation of poliovirus replicons encoding the complete human immunodeficiency virus type 1 gag gene by using a complementation system which provides the P1 capsid protein in trans.

    PubMed

    Porter, D C; Ansardi, D C; Morrow, C D

    1995-03-01

    Poliovirus genomes which contain small regions of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env genes substituted in frame for the P1 capsid region replicate and express HIV-1 proteins as fusion proteins with the P1 capsid precursor protein upon transfection into cells (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Since these genomes, referred to as replicons, do not express capsid proteins, a complementation system was developed to encapsidate the genomes by providing P1 capsid proteins in trans from a recombinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicons were generated after serial passage of the replicon genomes into cells previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this system, we have further defined the role of the P1 region in viral protein expression and RNA encapsidation. In the present study, we constructed poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investigate whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the complete gag gene was substituted for the VP2, VP3, and VP1 capsid sequences was constructed. Transfection of replicon RNA with and without the VP4 coding region into cells resulted in similar levels of expression of the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by immunoprecipitation using specific antibodies. Northern (RNA) blot analysis of RNA from transfected cells demonstrated comparable levels of RNA replication for each replicon. Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the genomes; serial passage in the presence of VV-P1 resulted in the generation of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated

  2. Complement in monoclonal antibody therapy of cancer.

    PubMed

    Rogers, Laura M; Veeramani, Suresh; Weiner, George J

    2014-08-01

    Monoclonal antibodies (mAb) have been used as targeted treatments against cancer for more than a decade, with mixed results. Research is needed to understand mAb mechanisms of action with the goal of improving the efficacy of currently used mAbs and guiding the design of novel mAbs. While some mAb-induced tumor cell killing is a result of direct effects on tumor cell signaling, mAb opsonization of tumor cells also triggers activation of immune responses due to complement activation and engagement of antibody receptors on immune effector cells. In fact, complement has been shown to play an important role in modulating the anti-tumor activity of many mAb through complement-dependent cytotoxicity, antibody-dependent cytotoxicity, and through indirect effects by modulating the tumor microenvironment. Complement activity can have both agonistic and antagonistic effects on these processes. How the balance of such effects impacts on the clinical efficacy of mAb therapy remains unclear. In this review, we discuss the mAbs currently approved for cancer treatment and examine how complement can impact their efficacy with a focus on how this information might be used to improve the clinical efficacy of mAb treatment.

  3. Low-dose autologous in vitro opsonized erythrocytes. Radioimmune method and autologous opsonized erythrocytes for refractory autoimmune thrombocytopenic purpura in adults

    SciTech Connect

    Ambriz, R.; Munoz, R.; Pizzuto, J.; Quintanar, E.; Morales, M.; Aviles, A.

    1987-01-01

    Adult patients with chronic autoimmune thrombocytopenic purpura (ATP), which proved refractory to various treatments, received a single dose of autologous in vitro opsonized erythrocytes with 100 micrograms of anti-D IgG. In 1983, 30 of these patients were treated with autologous erythrocytes that had been opsonized and labeled with 25 mCi (740 MBq) of technetium Tc 99m; this treatment was designated as the radioimmune method. Favorable responses were noted in 36% of patients so treated. In 1985, another group of 16 patients with refractory ATP received therapy with autologous opsonized erythrocytes (AOPE) and 55% of these patients showed favorable responses. Five (17%) of the patients treated using the radioimmune method attained a complete, long-term (greater than 35 months) remission of their ATP, and five (31%) of the patients treated using AOPE remained in complete remission over 270 days after cessation of therapy. Major complications were not seen. We concluded that the interaction of macrophages with low-dose AOPE is a successful therapeutic approach in ATP refractory to standard treatment.

  4. Hepatitis B virus X protein up-regulates C4b-binding protein α through activating transcription factor Sp1 in protection of hepatoma cells from complement attack

    PubMed Central

    Feng, Guoxing; Li, Jiong; Zheng, Minying; Yang, Zhe; Liu, Yunxia; Zhang, Shuqin; Ye, Lihong; Zhang, Weiying; Zhang, Xiaodong

    2016-01-01

    Hepatitis B virus X protein (HBx) plays crucial roles in the development of hepatocellular carcinoma (HCC). We previously showed that HBx protected hepatoma cells from complement attack by activation of CD59. Moreover, in this study we found that HBx protected hepatoma cells from complement attack by activation of C4b-binding protein α (C4BPα), a potent inhibitor of complement system. We observed that HBx were positively correlated with those of C4BPα in clinical HCC tissues. Mechanistically, HBx activated the promoter core region of C4BPα, located at −1199/−803nt, through binding to transcription factor Sp1. In addition, chromatin immunoprecipitation (ChIP) assays showed that HBx was able to bind to the promoter of C4BPα, which could be blocked by Sp1 silencing. Functionally, knockdown of C4BPα obviously increased the deposition of C5b-9, a complex of complement membrane attack, and remarkably abolished the HBx-induced resistance of hepatoma cells from complement attack in vitro and in vivo. Thus, we conclude that HBx up-regulates C4BPα through activating transcription factor Sp1 in protection of liver cancer cells from complement attack. Our finding provides new insights into the mechanism by which HBx enhances protection of hepatoma cells from complement attack. PMID:27050367

  5. Blue Native PAGE and Biomolecular Complementation Reveal a Tetrameric or Higher-Order Oligomer Organization of the Physiological Measles Virus Attachment Protein H▿

    PubMed Central

    Brindley, Melinda A.; Plemper, Richard K.

    2010-01-01

    Members of the Paramyxovirinae subfamily rely on the concerted action of two envelope glycoprotein complexes, attachment protein H and the fusion (F) protein oligomer, to achieve membrane fusion for viral entry. Despite advances in X-ray information, the organization of the physiological attachment (H) oligomer in functional fusion complexes and the molecular mechanism linking H receptor binding with F triggering remain unknown. Here, we have applied an integrated approach based on biochemical and functional assays to the problem. Blue native PAGE analysis indicates that native H complexes extract predominantly in the form of loosely assembled tetramers from purified measles virus (MeV) particles and cells transiently expressing the viral envelope glycoproteins. To gain functional insight, we have established a bimolecular complementation (BiC) assay for MeV H, on the basis of the hypothesis that physical interaction of H with F complexes, F triggering, and receptor binding constitute distinct events. Having experimentally confirmed three distinct H complementation groups, implementation of H BiC (H-BiC) reveals that a high-affinity receptor-to-paramyxovirus H monomer stoichiometry below parity is sufficient for fusion initiation, that F binding and fusion initiation are separable in H oligomers, and that a higher relative amount of F binding-competent than F fusion initiation- or receptor binding-competent H monomers per oligomer is required for optimal fusion. By capitalizing on these findings, H-BiC activity profiles confirm the organization of H into tetramers or higher-order multimers in functional fusion complexes. Results are interpreted in light of a model in which receptor binding may affect the oligomeric organization of the attachment protein complex. PMID:20861270

  6. An AGM model for changes in complement during pregnancy: neutralization of influenza virus by serum is diminished in late third trimester.

    PubMed

    Mayer, Anne E; Parks, Griffith D

    2014-01-01

    Pregnant women in the third trimester are at increased risk of severe influenza disease relative to the general population, though mechanisms behind this are not completely understood. The immune response to influenza infection employs both complement (C') and antibody (Ab). The relative contributions of these components to the anti-viral response are difficult to dissect because most humans have pre-existing influenza-specific Abs. We developed the African green monkey (AGM) as a tractable nonhuman primate model to study changes in systemic innate immunity to influenza during pregnancy. Because the AGMs were influenza-naïve, we were able to examine the role of C' in influenza virus neutralization using serum from non-pregnant animals before and after influenza infection. We determined that serum from naïve AGMs neutralized influenza via C', while post-infection neutralization did not require C', suggesting an Ab-mediated mechanism. The latter mimicked neutralization using human serum. Further, we found that ex vivo neutralization of influenza with both naïve and influenza-immune AGM serum occurred by virus particle aggregation and lysis, with immune serum lysing virus at a much higher rate than naïve serum. We hypothesized that the anti-influenza C' response would diminish late in AGM pregnancy, corresponding with the time when pregnant women suffer increased influenza severity. We found that influenza neutralization capacity is significantly diminished in serum collected late in the third trimester. Strikingly, we found that circulating levels of C3, C3a, and C4 are diminished late in gestation relative to nonpregnant animals, and while neutralization capacity and serum C3a return to normal shortly after parturition, C3 and C4 levels do not. This AGM model system will enable further studies of the role of physiologic and hormonal changes in downregulating C'-mediated anti-viral immunity during pregnancy, and it will permit the identification of therapeutic

  7. Hydrazinonicotinamide prolongs quantum dot circulation and reduces reticuloendothelial system clearance by suppressing opsonization and phagocyte engulfment

    NASA Astrophysics Data System (ADS)

    Jung, Kyung-Ho; Park, Jin Won; Paik, Jin-Young; Lee, Eun Jeong; Choe, Yearn Seong; Lee, Kyung-Han

    2012-12-01

    In this study, we investigated the effects of hydrazinonicotinamide (HYNIC)—a bifunctional crosslinker widely used to 99mTc radiolabel protein and nanoparticles for imaging studies—on quantum dot opsonization, macrophage engulfment and in vivo kinetics. In streptavidin-coated quantum dots (SA-QDots), conjugation with HYNIC increased the net negative charge without affecting the zeta potential. Confocal microscopy and fluorescence-activated cell sorting showed HYNIC attachment to suppress SA-QDot engulfment by macrophages. Furthermore, HYNIC conjugation suppressed surface opsonization by serum protein including IgG. When intravenously injected into mice, HYNIC conjugation significantly prolonged the circulation of SA-QDots and reduced their hepatosplenic uptake. Diminished reticuloendothelial system clearance of SA-QDots and aminoPEG-QDots by HYNIC conjugation was also demonstrated by in vivo and ex vivo optical imaging. The effects of HYNIC on the opsonization, phagocytosis and in vivo kinetics of quantum dots were reversed by removal of the hydrazine component from HYNIC. Thus, surface functionalization with HYNIC can improve the in vivo kinetics of quantum dots by reducing phagocytosis via suppression of surface opsonization.

  8. Chemiluminescence of neutrophiles stimulated by opsonized Zymosan in children with bronchial asthma and pneumonia

    NASA Astrophysics Data System (ADS)

    Lewandowicz-Uszynska, A.; Jankowski, A.

    2004-08-01

    Oxygen metabolism of neutrophils after stimulation with opsonized zymosan was examined using chemiluminescence test (in the presence of the patient serum or pooled serum). Into the study 37 children aged from 2 to 12 years were enrolled (20 girls and 17 boys). 10 healthy volunteers comprised the control group (group III). Two groups of patients were established: group I -- children with bronchial asthma (without infection), group II -- children with pneumonia. The examination in both groups was performed twice -- in acute phase and in remission period. The group I in acute phase comprised 16 children and in remission phase 9 children, group II - 21 children in acute phase and 9 children in remission phase, respectively. The following parameters of CL were estimated average value of so called spontaneous CL, maximal excitation of neutrophils after stimulation by zymogen (CLmax), time of zymosan opsonization. The following results were obtained: increased spontaneous CL and CLmax (at the presence of both sera) in acute phase of bronchial asthma and pneumonia in comparison to the control group. In the period of remission both these parameters were insignificantly decreased. The longest time of zymosan opsonization in acute period of disease was observed in children with pneumonia (18 min.). This time did not change during remission phase. Only slightly longer time of opsonization was observed in the patients from group I (in exacerbation) (15 min) than in the control group (13,1 min). This time was prolonged in the clinical remission (20 min).

  9. Construction, expression and immunoassay detection of recombinant plasmid encoding fusion protein of Roman chicken complement C3d and Newcastle disease virus F gene.

    PubMed

    Liu, D; Niu, Z-X

    2008-12-01

    The terminal degradation product (C3d) of mammalian complement component C3 plays an important role in modulation of the adaptive immune response through the interaction with complement receptor type 2 (CR2) on B cells. In this study, the gene fragment coding for the complement protein C3d (chC3d) from Roman chicken was cloned and expressed as a fusion protein for its application in the vaccine study of chicken, and for in vitro experiments. The chC3d fragment strengthened B-cell responses when complexed with antigen. Three potential vaccine construct units were engineered to contain two, four and six copies of chC3d coding gene linked to the F gene of Newcastle disease virus (NDV), an economically important pathogen of chicken that is classified as a list A contagious disease of poultry by the Office International des Epizooties. The cloned chC3d protein and different repeats of C3d proteins in addition to the F gene of NDV were generated separately in Escherichia coli and chicken embryo fibroblast cells with the help of expression vectors. All recombinant proteins were analysed by SDS-PAGE and Western blotting. Analysis of the immunogenicity of different repeats of C3d revealed that chC3d had an enhancing effect on the immunogenicity of antigens, and that six or more repeats of C3d may be necessary for efficient enhancement of antigen-specific immune responses. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice, rabbits and cattle. The adjuvant properties of C3d have not been assessed in poultry using homologous C3d in association with antigens relevant to the target species. The Roman chicken C3d fusion proteins described in this study is the first report and will provide a basis for immunization trials in chicken, studies of receptor binding and cell activation of chicken lymphocytes, and investigations of new types of vaccines, including recombinant vaccines and DNA vaccines for future use against other

  10. Mesenchymal Stromal Cells Engage Complement and Complement Receptor Bearing Innate Effector Cells to Modulate Immune Responses

    PubMed Central

    Moll, Guido; Jitschin, Regina; von Bahr, Lena; Rasmusson-Duprez, Ida; Sundberg, Berit; Lönnies, Lena; Elgue, Graciela; Nilsson-Ekdahl, Kristina; Mougiakakos, Dimitrios; Lambris, John D.; Ringdén, Olle; Le Blanc, Katarina; Nilsson, Bo

    2011-01-01

    Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the body's innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells. PMID:21747949

  11. Complement activation in the context of stem cells and tissue repair

    PubMed Central

    Schraufstatter, Ingrid U; Khaldoyanidi, Sophia K; DiScipio, Richard G

    2015-01-01

    The complement pathway is best known for its role in immune surveillance and inflammation. However, its ability of opsonizing and removing not only pathogens, but also necrotic and apoptotic cells, is a phylogenetically ancient means of initiating tissue repair. The means and mechanisms of complement-mediated tissue repair are discussed in this review. There is increasing evidence that complement activation contributes to tissue repair at several levels. These range from the chemo-attraction of stem and progenitor cells to areas of complement activation, to increased survival of various cell types in the presence of split products of complement, and to the production of trophic factors by cells activated by the anaphylatoxins C3a and C5a. This repair aspect of complement biology has not found sufficient appreciation until recently. The following will examine this aspect of complement biology with an emphasis on the anaphylatoxins C3a and C5a. PMID:26435769

  12. Studies on interaction of cucurbit aphid-borne yellow virus proteins using yeast two-hybrid system and bimolecular fluorescence complementation.

    PubMed

    Chen, X H; Xiang, H Y; Wang, Z; Zhang, Y J; Han, C G; Li, D W; Yu, J L; Cheng, Y Q

    2011-01-01

    In this article, yeast two-hybrid system (YTHS) and bimolecular fluorescence complementation (BiFC) were used to analyze the interactions of cucurbit aphid-borne yellows virus (CABYV)-encoded proteins. P0, P1, P1-2, P3, P4, and P5 were tested by YTHS in all possible pairwise combinations, and only P3/P3 interaction was detected. Results obtained by BiFC further confirmed the self-interaction of P3, and the subcellular localization of reconstituted YFP fluorescence was observed mainly in nuclei of Nicotiana benthamiana leaf epidermal cells. Domains involved in P3/P3 self-interaction were analyzed by YTHS and BiFC using deletion mutants. The results showed that R domain (residues 1-61) in the N-terminus could self-interact, and it also interacted with the S domain (residues 62-199) in the C-terminus of P3. The present work would serve as a molecular basis for further characterization of CABYV proteins, and the regions involved in P3/P3 self-interaction could provide the clue for understanding the capsid assembly pathway of CABYV. PMID:21978157

  13. Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: Implications for replication and genome packaging

    SciTech Connect

    Chaturvedi, Sonali; Rao, A.L.N.

    2014-09-15

    In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein–protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. - Highlights: • YFP fusion proteins of BMV p1a and p2a are biologically active. • Self-interaction was observed for p1a, p2a and CP. • CP interacts with p2a but not p1a. • Majority of reconstituted YFP resulting from bona fide fusion protein partners localized on ER.

  14. 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

  15. Rat C-reactive protein activates the autologous complement system.

    PubMed

    Diaz Padilla, Niubel; Bleeker, Wim K; Lubbers, Yvonne; Rigter, Gemma M M; Van Mierlo, Gerard J; Daha, Mohamed R; Hack, C Erik

    2003-08-01

    Activation of complement is a biological function of human C-reactive protein (hCRP), whereas rat CRP (rCRP) has been claimed to be unable to activate complement. As important biological functions of proteins are probably conserved among species, we re-evaluated, using various ligands, the capability of rCRP to activate complement. The activation of complement by hCRP and rCRP was investigated in solid- and fluid-phase systems. In the solid-phase system, purified CRP was fixed to enzyme-linked immunosorbent assay (ELISA) plates and incubated with human or rat recalcified plasma. Dose-dependent binding of human and rat C3 and C4 was observed to human and rat CRP, respectively. In the fluid-phase system, recalcified rat plasma, which contains about 500 mg/l of CRP, or human plasma supplemented with hCRP, were incubated with lyso-phosphatidylcholine. A dose-dependent activation of complement was observed upon incubation with this ligand, as reflected by the generation of activated C4 as well as of CRP-complement complexes. This activation was, in both cases, inhibited by preincubation of plasma with p-aminophosphorylcholine, a specific inhibitor of the interaction of CRP with its ligands, or by chelation of calcium ions. We conclude that rat CRP, similarly to human CRP, can activate autologous complement. These results support the notion that opsonization of ligands with complement is an important biological function of CRP.

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

    PubMed

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

    2016-04-01

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

  17. Pseudorabies virus mutants lacking the essential glycoprotein gII can be complemented by glycoprotein gI of bovine herpesvirus 1.

    PubMed Central

    Rauh, I; Weiland, F; Fehler, F; Keil, G M; Mettenleiter, T C

    1991-01-01

    The genome of pseudorabies virus (PrV) encodes at least seven glycoproteins. The glycoprotein complex gII consists of three related polypeptides, two of them derived by proteolytic cleavage from a common precursor and linked via disulfide bonds. It is homologous to herpes simplex virus (HSV) gB and is therefore thought to be essential for PrV replication, as is gB for HSV replication. To isolate PrV mutants deficient in gII expression, we established cell lines that stably carry the PrV gII gene. Line N7, of Vero cell origin, contains the gII gene under its own promoter and expresses gII after transactivation by herpesviral functions after infection. MDBK-derived line MT3 contains the gII gene under control of the mouse metallothionein promoter. However, it has essentially lost inducibility and constitutively produces high amounts of correctly processed glycoprotein gII. We used a beta-galactosidase expression cassette inserted into a partially deleted cloned copy of the gII gene for cotransfection with PrV DNA. gII- PrV mutants were isolated from viral progeny by taking advantage of their blue-plaque phenotype when incubated under an agarose overlay containing a chromogenic substrate. Analysis of these mutants proved that gII is indeed essential for PrV replication, since the gII- mutants grew normally on gII-complementing cells but were unable to produce plaques on noncomplementing cells. Surprisingly the PrV gII- mutants were also able to grow on a cell line constitutively expressing the gB-homologous glycoprotein gI from bovine herpesvirus 1 (BHV-1) to the same extent as on cells expressing PrV gII. gII- PrV propagated on cells expressing BHV-1 gI became susceptible to neutralization by anti-BHV-1 gI monoclonal antibodies. We also found that BHV-1 gI is present in the envelope of purified gII- pseudorabies virions grown on cells expressing BHV-1 gI, as judged by radioimmunoprecipitation and immunoelectron microscopy. These results prove that BHV-1 gI is

  18. Modulation of complement regulatory function and measles virus receptor function by the serine-threonine-rich domains of membrane cofactor protein (CD46).

    PubMed Central

    Iwata, K; Seya, T; Ueda, S; Ariga, H; Nagasawa, S

    1994-01-01

    Three major membrane cofactor protein (MCP) phenotypes with different serine-threonine (ST)-rich regions, namely STc (L-phenotype), STBC (H or U phenotype) and STABC, and the MCP without the ST domain (delta ST) were expressed in Chinese hamster ovary (CHO) cells by transfecting the respective cDNAs. The expressed molecules migrated with a larger molecular mass on SDS/PAGE than those expected from their amino acid sequences. O-Glycanase digestion showed that this was due to O-linked sugar chains. The apparent sugar contents in each ST segment were compatible with their serine and threonine contents in the ST regions. The functional properties of these phenotypes as inhibitors of human complement (C) and receptors of measles virus (MV) were compared. The classical pathway-dependent CHO cell lysis by human C was more effectively suppressed by the expressed delta ST and STC than by the STABC and STBC phenotypes, although the difference was not so prominent. In contrast, alternative C pathway-dependent CHO-cell lysis was most effectively suppressed by the STABC phenotype and was only slightly blocked by the ST-deleted mutant. MV infection occurred with all of the phenotypes, but the infectious dose required to cause the same level of syncytium formation was 100-times higher in large ST (STABC and STBC) than in small ST (STC and delta ST) phenotypes. Thus, the ST domain serves as a functional modulator in MCP: MCP with a large ST domain having high O-linked sugar contents is favourable to the effective suppression of both the alternative C pathway-mediated cytolysis and MV infection, whereas MCP with a small ST domain is favourable to the suppression of the classical C pathway. Images Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 PMID:7998929

  19. Recombinant form of human wild type mannan-binding lectin (MBL/A) but not its structural variant (MBL/C) promotes phagocytosis of zymosan by activating complement.

    PubMed

    Rajagopalan, Rema; Nyaundi, Takazvida; Salvi, Veena P; Rawal, Nenoo

    2010-09-01

    Mannan-binding lectin (MBL) mediates innate immune responses, such as activation of the complement lectin pathway and phagocytosis, to help fight infections. In the present study, employing recombinant forms of human MBL (rMBL), the role of wild type MBL (rMBL/A) and its structural variant rMBL/C in mediating THP-1 phagocytosis of fluorescent-labeled zymosan was examined and compared to MBL purified from human plasma (pMBL/A). Flow cytometric analyses revealed that opsonization of zymosan with rMBL/A and pMBL/A (0.5-30microg/ml) resulted in a 1.9- and 2.7-fold enhancement in its uptake by THP-1 cells in the presence of serum that was depleted of both MBL and the classical pathway component, C1q (MBL/C1q Dpl serum). In contrast, no enhancement in phagocytosis was observed when zymosan was opsonized with rMBL/C. Addition of MBL monoclonal antibody, EDTA, or mannan to the opsonization reaction mixture inhibited THP-1 phagocytosis of pMBL/A opsonized zymosan. Heat inactivation of MBL/C1q Dpl serum abolished the 2-fold increase in phagocytosis and in the absence of serum the direct opsonic activity of MBL did not contribute significantly to the uptake of zymosan into THP-1 cells. Activation products of complement components C3 and C4 were deposited on zymosan opsonized with pMBL/A and rMBL/A but not rMBL/C indicating that MBL-mediated phagocytosis of zymosan requires activation of the complement lectin pathway. The findings imply that impaired MBL-mediated phagocytosis may put individuals homozygous for the mutant allele MBL/C but not wild type MBL/A at increased risk to infections such as yeast. PMID:20579738

  20. The ancestral complement system in sea urchins.

    PubMed

    Smith, L C; Clow, L A; Terwilliger, D P

    2001-04-01

    The origin of adaptive immunity in the vertebrates can be traced to the appearance of the ancestral RAG genes in the ancestral jawed vertebrate; however, the innate immune system is more ancient. A central subsystem within innate immunity is the complement system, which has been identified throughout and seems to be restricted to the deuterostomes. The evolutionary history of complement can be traced from the sea urchins (members of the echinoderm phylum), which have a simplified system homologous to the alternative pathway, through the agnathans (hagfish and lamprey) and the elasmobranchs (sharks and rays) to the teleosts (bony fish) and tetrapods, with increases in the numbers of complement components and duplications in complement pathways. Increasing complexity in the complement system parallels increasing complexity in the deuterostome animals. This review focuses on the simplest of the complement systems that is present in the sea urchin. Two components have been identified that show significant homology to vertebrate C3 and factor B (Bf), called SpC3 and SpBf, respectively. Sequence analysis from both molecules reveals their ancestral characteristics. Immune challenge of sea urchins indicates that SpC3 is inducible and is present in coelomic fluid (the body fluids) in relatively high concentrations, while SpBf expression is constitutive and is present in much lower concentrations. Opsonization of foreign cells and particles followed by augmented uptake by phagocytic coelomocytes appears to be a central function for this simpler complement system and important for host defense in the sea urchin. These activities are similar to some of the functions of the homologous proteins in the vertebrate complement system. The selective advantage for the ancestral deuterostome may have been the amplification feedback loop that is still of central importance in the alternative pathway of complement in higher vertebrates. Feedback loop functions would quickly coat

  1. Malarial anemia: digestive vacuole of Plasmodium falciparum mediates complement deposition on bystander cells to provoke hemophagocytosis.

    PubMed

    Dasari, Prasad; Fries, Anja; Heber, Sophia D; Salama, Abdulgabar; Blau, Igor-Wolfgang; Lingelbach, Klaus; Bhakdi, Sebastian Chakrit; Udomsangpetch, Rachanee; Torzewski, Michael; Reiss, Karina; Bhakdi, Sucharit

    2014-12-01

    The digestive vacuole (DV) of Plasmodium falciparum, which is released into the bloodstream upon rupture of each parasitized red blood cell (RBC), was recently discovered to activate the alternative complement pathway. In the present work, we show that C3- and C5-convertases assembling on the parasitic organelle are able to provoke deposition of activated C3 and C5b-9 on non-infected bystander erythrocytes. Direct contact of DVs with cells is mandatory for the effect, and bystander complement deposition occurs focally, possibly at the sites of contact. Complement opsonization promotes protracted erythrophagocytosis by human macrophages, an effect that is magnified when ring-stage infected RBCs with reduced CD55 and CD59, or paroxysmal nocturnal hemoglobinuria (PNH)-RBCs lacking these complement inhibitors are employed as targets. Bystander attack can also directly induce lysis of PNH-RBCs. Direct evidence for complement activation and bystander attack mediated by DVs was obtained through immunohistochemical analyses of brain paraffin sections from autopsies of patients who had died of cerebral malaria. C3d and the assembled C5b-9 complex could be detected in all sections, colocalizing with and often extending locally beyond massive accumulations of DVs that were identified under polarized light. This is the first demonstration that a complement-activating particle can mediate opsonization of bystander cells to promote their antibody-independent phagocytosis. The phenomenon may act in concert with other pathomechanisms to promote the development of anemia in patients with severe malaria.

  2. Molecular basis of the interaction between complement receptor type 2 (CR2/CD21) and Epstein-Barr virus glycoprotein gp350.

    PubMed

    Young, Kendra A; Herbert, Andrew P; Barlow, Paul N; Holers, V Michael; Hannan, Jonathan P

    2008-11-01

    The binding of the Epstein-Barr virus glycoprotein gp350 by complement receptor type 2 (CR2) is critical for viral attachment to B lymphocytes. We set out to test hypotheses regarding the molecular nature of this interaction by developing an enzyme-linked immunosorbent assay (ELISA) for the efficient analysis of the gp350-CR2 interaction by utilizing wild-type and mutant forms of recombinant gp350 and also of the CR2 N-terminal domains SCR1 and SCR2 (designated CR2 SCR1-2). To delineate the CR2-binding site on gp350, we generated 17 gp350 single-site substitutions targeting an area of gp350 that has been broadly implicated in the binding of both CR2 and the major inhibitory anti-gp350 monoclonal antibody (MAb) 72A1. These site-directed mutations identified a novel negatively charged CR2-binding surface described by residues Glu-21, Asp-22, Glu-155, Asp-208, Glu-210, and Asp-296. We also identified gp350 amino acid residues involved in non-charge-dependent interactions with CR2, including Tyr-151, Ile-160, and Trp-162. These data were supported by experiments in which phycoerythrin-conjugated wild-type and mutant forms of gp350 were incubated with CR2-expressing K562 cells and binding was assessed by flow cytometry. The ELISA was further utilized to identify several positively charged residues (Arg-13, Arg-28, Arg-36, Lys-41, Lys-57, Lys-67, Arg-83, and Arg-89) within SCR1-2 of CR2 that are involved in the binding interaction with gp350. These experiments allowed a comparison of those CR2 residues that are important for binding gp350 to those that define the epitope for an effective inhibitory anti-CR2 MAb, 171 (Asn-11, Arg-13, Ser-32, Thr-34, Arg-36, and Tyr-64). The mutagenesis data were used to calculate a model of the CR2-gp350 complex using the soft-docking program HADDOCK.

  3. Molecular basis of the interaction between complement receptor type 2 (CR2/CD21) and Epstein-Barr virus glycoprotein gp350.

    PubMed

    Young, Kendra A; Herbert, Andrew P; Barlow, Paul N; Holers, V Michael; Hannan, Jonathan P

    2008-11-01

    The binding of the Epstein-Barr virus glycoprotein gp350 by complement receptor type 2 (CR2) is critical for viral attachment to B lymphocytes. We set out to test hypotheses regarding the molecular nature of this interaction by developing an enzyme-linked immunosorbent assay (ELISA) for the efficient analysis of the gp350-CR2 interaction by utilizing wild-type and mutant forms of recombinant gp350 and also of the CR2 N-terminal domains SCR1 and SCR2 (designated CR2 SCR1-2). To delineate the CR2-binding site on gp350, we generated 17 gp350 single-site substitutions targeting an area of gp350 that has been broadly implicated in the binding of both CR2 and the major inhibitory anti-gp350 monoclonal antibody (MAb) 72A1. These site-directed mutations identified a novel negatively charged CR2-binding surface described by residues Glu-21, Asp-22, Glu-155, Asp-208, Glu-210, and Asp-296. We also identified gp350 amino acid residues involved in non-charge-dependent interactions with CR2, including Tyr-151, Ile-160, and Trp-162. These data were supported by experiments in which phycoerythrin-conjugated wild-type and mutant forms of gp350 were incubated with CR2-expressing K562 cells and binding was assessed by flow cytometry. The ELISA was further utilized to identify several positively charged residues (Arg-13, Arg-28, Arg-36, Lys-41, Lys-57, Lys-67, Arg-83, and Arg-89) within SCR1-2 of CR2 that are involved in the binding interaction with gp350. These experiments allowed a comparison of those CR2 residues that are important for binding gp350 to those that define the epitope for an effective inhibitory anti-CR2 MAb, 171 (Asn-11, Arg-13, Ser-32, Thr-34, Arg-36, and Tyr-64). The mutagenesis data were used to calculate a model of the CR2-gp350 complex using the soft-docking program HADDOCK. PMID:18786993

  4. Secretion of a fungal protease represents a complement evasion mechanism in cerebral aspergillosis.

    PubMed

    Rambach, Günter; Dum, David; Mohsenipour, Iradj; Hagleitner, Magdalena; Würzner, Reinhard; Lass-Flörl, Cornelia; Speth, Cornelia

    2010-04-01

    Complement represents a central immune weapon in the brain, but the high lethality of cerebral aspergillosis indicates a low efficacy of the antifungal complement attack. Studies with cerebrospinal fluid (CSF) samples derived from a patient with cerebral aspergillosis showed a degradation of complement proteins, implying that Aspergillus might produce proteases to evade their antimicrobial potency. Further investigations of this hypothesis showed that Aspergillus, when cultured in CSF to simulate growth conditions in the brain, secreted a protease that can cleave various complement proteins. Aspergillus fumigatus, the most frequent cause of cerebral aspergillosis, destroyed complement activity more efficiently than other Aspergillus species. The degradation of complement in CSF resulted in a drastic reduction of the capacity to opsonize fungal hyphae. Furthermore, the Aspergillus-derived protease could diminish the amount of complement receptor CR3, a surface molecule to mediate eradication of opsonized pathogens, on granulocytes and microglia. The lack of these prerequisites caused a significant decrease in phagocytosis of primary microglia. Additional studies implied that the complement-degrading activity shares many characteristics with the previously described alkaline protease Alp1. To improve the current therapy for cerebral aspergillosis, we tried to regain the antifungal effects of complement by repressing the secretion of this degrading activity. Supplementation of CSF with nitrogen sources rescued the complement proteins and abolished any cleavage. Glutamine or arginine are of special interest for this purpose since they represent endogenous substances in the CNS and might be included in a future supportive therapy to reduce the high lethality of cerebral aspergillosis. PMID:20303595

  5. Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation

    PubMed Central

    Kumar, Shashi Kant; Singh, Padam; Sinha, Sudhir

    2015-01-01

    This study investigated the hypothesis that serum antibodies against Mycobacterium tuberculosis present in naturally infected healthy subjects of a tuberculosis (TB) endemic area could create and/or sustain the latent form of infection. All five apparently healthy Indian donors showed high titres of serum antibodies against M. tuberculosis cell membrane antigens, including lipoarabinomannan and alpha crystallin. Uptake and killing of bacilli by the donor macrophages was significantly enhanced following their opsonization with antibody-rich, heat-inactivated autologous sera. However, the capability to opsonize was apparent for antibodies against some and not other antigens. High-content cell imaging of infected macrophages revealed significantly enhanced colocalization of the phagosome maturation marker LAMP-1, though not of calmodulin, with antibody-opsonized compared with unopsonized M. tuberculosis. Key enablers of macrophage microbicidal action—proinflammatory cytokines (IFN-γ and IL-6), phagosome acidification, inducible NO synthase and nitric oxide—were also significantly enhanced following antibody opsonization. Interestingly, heat-killed M. tuberculosis also elevated these mediators to the levels comparable to, if not higher than, opsonized M. tuberculosis. Results of the study support the emerging view that an efficacious vaccine against TB should, apart from targeting cell-mediated immunity, also generate ‘protective’ antibodies. PMID:26674415

  6. Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation.

    PubMed

    Kumar, Shashi Kant; Singh, Padam; Sinha, Sudhir

    2015-12-01

    This study investigated the hypothesis that serum antibodies against Mycobacterium tuberculosis present in naturally infected healthy subjects of a tuberculosis (TB) endemic area could create and/or sustain the latent form of infection. All five apparently healthy Indian donors showed high titres of serum antibodies against M. tuberculosis cell membrane antigens, including lipoarabinomannan and alpha crystallin. Uptake and killing of bacilli by the donor macrophages was significantly enhanced following their opsonization with antibody-rich, heat-inactivated autologous sera. However, the capability to opsonize was apparent for antibodies against some and not other antigens. High-content cell imaging of infected macrophages revealed significantly enhanced colocalization of the phagosome maturation marker LAMP-1, though not of calmodulin, with antibody-opsonized compared with unopsonized M. tuberculosis. Key enablers of macrophage microbicidal action--proinflammatory cytokines (IFN-γ and IL-6), phagosome acidification, inducible NO synthase and nitric oxide--were also significantly enhanced following antibody opsonization. Interestingly, heat-killed M. tuberculosis also elevated these mediators to the levels comparable to, if not higher than, opsonized M. tuberculosis. Results of the study support the emerging view that an efficacious vaccine against TB should, apart from targeting cell-mediated immunity, also generate 'protective' antibodies.

  7. Dissimilar and similar functional properties of complement receptor-3 in microglia and macrophages in combating yeast pathogens by phagocytosis.

    PubMed

    Hadas, Smadar; Reichert, Fanny; Rotshenker, Shlomo

    2010-05-01

    Central nervous system (CNS) microglia (MG) and peripheral tissue macrophages (MO) remove pathogens by phagocytosis. Zymosan, a model yeast pathogen, is a beta-glucan rich particle that readily activates the complement system and then becomes C3bi-opsonized (op). Complement receptor-3 (CR3) has initially been implicated in mediating the phagocytosis of both C3bi-op and non-opsonized (nop) zymosan by MO through C3bi and beta-glucan binding sites, respectively. Later, the role of CR3 as a phagocytic beta-glucan receptor has been questioned and the supremacy of beta-glucan receptor Dectin-1 advocated. We compare here between primary mouse CNS MG and peripheral tissue MO with respect to CR3 and Dectin-1 mediated phagocytosis of C3bi-op and nop zymosan. We report that MG and MO display similar as well as dissimilar functional properties in this respect. Although CR3 and Dectin-1 function both as beta-glucan/non-opsonic receptors in MG during nop zymosan phagocytosis, Dectin-1, but not CR3, does so in MO. CR3 functions also as a C3bi/opsonic receptor in MG and MO during C3bi-op zymosan phagocytosis, leading to phagocytosis which is more efficient than that of nop zymosan. Dectin-1 contributes, albeit less than CR3, to phagocytosis of C3bi-op zymosan in MG and further less in MO, suggesting that C3bi-opsonization does not block all beta-glucan sites on zymosan from binding Dectin-1 on phagocytes. Thus, altogether CR3 and Dectin-1 contribute both to phagocytosis of nop and C3bi-op zymosan in MG, whereas MO switch from CR3-independent/Dectin-1-dependent phagocytosis of nop zymosan to phagocytosis of C3bi-op zymosan where CR3 dominates over Dectin-1.

  8. Investigation of the role of complement and complement receptors in the modulation of B cell activation by a Paracoccidioides brasiliensis cell wall fraction.

    PubMed

    de Agostino Biella, Carla; Uecker, Marilei; Fernandes da Silva, Marcelo; Barbosa, José Elpidio; Silva, Célio Lopes; Crott, Luciana Simon Pereira

    2006-01-01

    F1 fraction from Paracoccidioides brasiliensis is a potent activator of the complement system. Considering that complement receptors CR1 and CR2 are involved in the regulation of B cell response, we evaluated the in vitro effect of the F1 in the activation of B lymphocytes, as well as the participation of complement receptors in this process. Murine splenocytes were cultured in order to evaluate the expression of CD40, CD45RB and CD69 on B lymphocyte, and IgG and IgM were quantified in the culture supernatant. F1 participated in the activation of B cells, showing a positive modulation effect on all markers analyzed. An increase in the production of IgG was detected in the supernatants when the opsonized F1 fraction was present. Complement receptor blockade with monoclonal antibodies led to a partial reduction in immunoglobulin secretion, suggesting that these receptors, especially CR2, play a role in modulating the function of B lymphocyte stimulated with the opsonized F1 fraction. These results may contribute for a better understanding of the B cell activation and differentiation processes in response to the F1 fraction from P. brasiliensis.

  9. 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

  10. Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium

    PubMed Central

    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

  11. 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

  12. Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes

    SciTech Connect

    Lutz, H.U.; Bussolino, F.; Flepp, R.; Fasler, S.; Stammler, P.; Kazatchkine, M.D.; Arese, P.

    1987-11-01

    Treatment of erythrocytes with the thiol-specific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3- antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which simulate senescent erythrocytes with respect to bound antibody and complement.

  13. Neutrophils extracellular traps damage Naegleria fowleri trophozoites opsonized with human IgG.

    PubMed

    Contis-Montes de Oca, A; Carrasco-Yépez, M; Campos-Rodríguez, R; Pacheco-Yépez, J; Bonilla-Lemus, P; Pérez-López, J; Rojas-Hernández, S

    2016-08-01

    Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri.

  14. Neutrophils extracellular traps damage Naegleria fowleri trophozoites opsonized with human IgG.

    PubMed

    Contis-Montes de Oca, A; Carrasco-Yépez, M; Campos-Rodríguez, R; Pacheco-Yépez, J; Bonilla-Lemus, P; Pérez-López, J; Rojas-Hernández, S

    2016-08-01

    Naegleria fowleri infects humans through the nasal mucosa causing a disease in the central nervous system known as primary amoebic meningoencephalitis (PAM). Polymorphonuclear cells (PMNs) play a critical role in the early phase of N. fowleri infection. Recently, a new biological defence mechanism called neutrophil extracellular traps (NETs) has been attracting attention. NETs are composed of nuclear DNA combined with histones and antibacterial proteins, and these structures are released from the cell to direct its antimicrobial attack. In this work, we evaluate the capacity of N. fowleri to induce the liberation of NETs by human PMN cells. Neutrophils were cocultured with unopsonized or IgG-opsonized N. fowleri trophozoites. DNA, histone, myeloperoxidase (MPO) and neutrophil elastase (NE) were stained, and the formation of NETs was evaluated by confocal microscopy and by quantifying the levels of extracellular DNA. Our results showed N. fowleri induce the liberation of NETs including release of MPO and NE by human PMN cells as exposure interaction time is increased, but N. fowleri trophozoites evaded killing. However, when trophozoites were opsonized, they were susceptible to the neutrophils activity. Therefore, our study suggests that antibody-mediated PMNs activation through NET formation may be crucial for antimicrobial responses against N. fowleri. PMID:27189133

  15. Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies.

    PubMed

    Ricklin, Meret E; Vielle, Nathalie J; Python, Sylvie; Brechbühl, Daniel; Zumkehr, Beatrice; Posthaus, Horst; Zimmer, Gert; Summerfield, Artur

    2016-01-01

    This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4(+) T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value. PMID:27446083

  16. Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies

    PubMed Central

    Ricklin, Meret E.; Vielle, Nathalie J.; Python, Sylvie; Brechbühl, Daniel; Zumkehr, Beatrice; Posthaus, Horst; Zimmer, Gert; Summerfield, Artur

    2016-01-01

    This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4+ T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value. PMID:27446083

  17. The Vi capsular polysaccharide prevents complement receptor 3-mediated clearance of Salmonella enterica serotype Typhi.

    PubMed

    Wilson, R Paul; Winter, Sebastian E; Spees, Alanna M; Winter, Maria G; Nishimori, Jessalyn H; Sanchez, Jesus F; Nuccio, Sean-Paul; Crawford, Robert W; Tükel, Çagla; Bäumler, Andreas J

    2011-02-01

    Capsular polysaccharides are important virulence factors of invasive bacterial pathogens. Here we studied the role of the virulence (Vi) capsular polysaccharide of Salmonella enterica serotype Typhi (S. Typhi) in preventing innate immune recognition by complement. Comparison of capsulated S. Typhi with a noncapsulated mutant (ΔtviBCDE vexABCDE mutant) revealed that the Vi capsule interfered with complement component 3 (C3) deposition. Decreased complement fixation resulted in reduced bacterial binding to complement receptor 3 (CR3) on the surface of murine macrophages in vitro and decreased CR3-dependent clearance of Vi capsulated S. Typhi from the livers and spleens of mice. Opsonization of bacteria with immune serum prior to intraperitoneal infection increased clearance of capsulated S. Typhi from the liver. Our data suggest that the Vi capsule prevents CR3-dependent clearance, which can be overcome in part by a specific antibody response.

  18. 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). PMID:25646095

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

    PubMed Central

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

    2015-01-01

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

  20. Activation of Human Complement System by Dextran-Coated Iron Oxide Nanoparticles Is Not Affected by Dextran/Fe Ratio, Hydroxyl Modifications, and Crosslinking

    PubMed Central

    Wang, Guankui; Chen, Fangfang; Banda, Nirmal K.; Holers, V. Michael; Wu, LinPing; Moghimi, S. Moein; Simberg, Dmitri

    2016-01-01

    While having tremendous potential as therapeutic and imaging tools, the clinical use of engineered nanoparticles has been associated with serious safety concerns. Activation of the complement cascade and the release of proinflammatory factors C3a and C5a may contribute to infusion-related reactions, whereas opsonization with C3 fragments promotes rapid recognition and clearance of nanomaterials by mononuclear phagocytes. We used dextran-coated superparamagnetic iron oxide nanoparticles (SPIO), which are potent activators of the complement system, to study the role of nanoparticle surface chemistry in inciting complement in human serum. Using complement inhibitors and measuring levels of fluid phase markers (sC5b-9, C5a, and Bb), we found that the majority of human complement activation by SPIO is through the alternative pathways (AP). SPIO prepared with high dextran/iron ratio showed some complement activation via calcium-sensitive pathways, but the AP was responsible for the bulk of complement activation and amplification. Activation via the AP required properdin, the positive regulator of the alternative C3bBb convertase. Modification of sugar alcohols of dextran with alkylating, acylating, or crosslinking agents did not overcome complement activation and C3 opsonization. These data demonstrate that human complement activation is independent of dextran modification of SPIO and suggest a crucial role of the AP in immune recognition of nano-assemblies in human serum. PMID:27777575

  1. Real-time polymerase chain reaction detection of cauliflower mosaic virus to complement the 35S screening assay for genetically modified organisms.

    PubMed

    Cankar, Katarina; Ravnikar, Maja; Zel, Jana; Gruden, Kristina; Toplak, Natasa

    2005-01-01

    Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.

  2. Contribution of complement component C3 and complement receptor type 3 to carbohydrate-dependent uptake of oligomannose-coated liposomes by peritoneal macrophages.

    PubMed

    Abe, Yu; Kuroda, Yasuhiro; Kuboki, Noritaka; Matsushita, Misao; Yokoyama, Naoaki; Kojima, Naoya

    2008-11-01

    Peritoneal macrophages (PEMs) preferentially and rapidly take up oligomannose-coated liposomes (OMLs) and subsequently mature to induce a Th-1 immune response following administration of OMLs into the peritoneal cavity. Here, we examine the contributions of complement component C3 and complement receptor type 3 (CR3) to carbohydrate-dependent uptake of OMLs by PEMs. Effective uptake of OMLs into PEMs in vitro was observed only in the presence of peritoneal fluid (PF), and OMLs incubated with PF were incorporated by PEMs in vitro in the absence of PF. These phenomena were inhibited by methyl-alpha-mannoside, N-acetylglucosamine or EDTA, but not by galactose. Pull-down analysis followed by peptide mass fingerprinting of PF-treated OMLs indicated that the OMLs were opsonized with complement fragment iC3b. In vivo uptake of OMLs by PEMs was inhibited by intraperitoneal injection of an antibody against CR3, a receptor for iC3b, and OML uptake by PEMs in the peritoneal cavity was not observed in C3-deficient mice. Thus, our results indicate that OMLs are opsonized with iC3b in a mannose-dependent manner in the peritoneal cavity and then incorporated into PEMs via CR3. PMID:18694897

  3. Importance of antibody and complement for oxidative burst and killing of invasive nontyphoidal Salmonella by blood cells in Africans.

    PubMed

    Gondwe, Esther N; Molyneux, Malcolm E; Goodall, Margaret; Graham, Stephen M; Mastroeni, Pietro; Drayson, Mark T; MacLennan, Calman A

    2010-02-16

    Bacteremia caused by nontyphoidal strains of Salmonella is endemic among African children. Case-fatality rates are high and antibiotic resistance increasing, but no vaccine is currently available. T cells are important for clearance of Salmonella infection within macrophages, but in Africa, invasive Salmonella disease usually manifests in the blood and affects children between 4 months and 2 y of age, when anti-Salmonella antibody is absent. We have previously found a role for complement-fixing bactericidal antibody in protecting these children. Here we show that opsonic activity of antibody and complement is required for oxidative burst and killing of Salmonella by blood cells in Africans. Induction of neutrophil oxidative burst correlated with anti-Salmonella IgG and IgM titers and C3 deposition on bacteria and was significantly lower in African children younger than 2 y compared with older children. Preopsonizing Salmonella with immune serum overcame this deficit, indicating a requirement for antibody and/or complement. Using different opsonization procedures, both antibody and complement were found to be necessary for optimal oxidative burst, phagocytosis and killing of nontyphoidal Salmonella by peripheral blood cells in Africans. Although most strains of African nontyphoidal Salmonella can be killed with antibody and complement alone, phagocytes in the presence of specific antibody and complement can kill strains resistant to killing by immune serum. These findings increase the likelihood that an antibody-inducing vaccine will protect against invasive nontyphoidal Salmonella disease in African children.

  4. Binding and Phagocytosis by Opsonized and Nonopsonized Channel Catfish Macrophages of Viable DsRed-fluorescent-labeled Edwardsiella ictaluri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phagocyte-mediated killing of bacterial pathogens is one of the major defensive mechanisms in fish. The binding, uptake and destruction of recombinant fluorescent protein DsRed transformed Edwardsiella ictaluri by opsonized and nonopsonized channel catfish (Ictalurus punctatus) macrophages was chara...

  5. Antibodies That Efficiently Form Hexamers upon Antigen Binding Can Induce Complement-Dependent Cytotoxicity under Complement-Limiting Conditions

    PubMed Central

    Cook, Erika M.; Lindorfer, Margaret A.; van der Horst, Hilma; Oostindie, Simone; Beurskens, Frank J.; Schuurman, Janine; Zent, Clive S.; Burack, Richard; Parren, Paul W. H. I.

    2016-01-01

    Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation–enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (anti-CD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus providing additional insight into their enhanced efficacy in promoting CDC. PMID:27474078

  6. 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.

  7. The opsonic rejuvenation of tuberculin as portrayed in Bernard Shaw's Doctor's Dilemma.

    PubMed

    Breathnach, Caoimhghin

    2006-12-01

    In 1906, at a late evening tea party in Sir Almroth Wright's laboratory when the pathologist was working on his opsonic index as a guide to the therapeutic use of tuberculin, Bernard Shaw instigated a discussion on patient selection in the face of limited resources. In converting the various responses into a play Shaw turned the choice between a rogue artist and an honest doctor into a moral dilemma, and then thickened the plot by involving the artist's beguiling wife innocently in the decision. He added a blackly comedic denouement to answer a public challenge that he could not write a convincing death scene. With his penchant for irrepressible exaggeration, his doctors are amiable but inordinately opinionated, each convinced that he alone holds the secret of healing. The senior physician escapes censure and even measured praise is bestowed on the panel doctor who accepts a full-time public post. This article summarises the play and how it came to be written.

  8. Complement system in zebrafish.

    PubMed

    Zhang, Shicui; Cui, Pengfei

    2014-09-01

    Zebrafish is recently emerging as a model species for the study of immunology and human diseases. Complement system is the humoral backbone of the innate immune defense, and our knowledge as such in zebrafish has dramatically increased in the recent years. This review summarizes the current research progress of zebrafish complement system. The global searching for complement components in genome database, together with published data, has unveiled the existence of all the orthologues of mammalian complement components identified thus far, including the complement regulatory proteins and complement receptors, in zebrafish. Interestingly, zebrafish complement components also display some distinctive features, such as prominent levels of extrahepatic expression and isotypic diversity of the complement components. Future studies should focus on the following issues that would be of special importance for understanding the physiological role of complement components in zebrafish: conclusive identification of complement genes, especially those with isotypic diversity; analysis and elucidation of function and mechanism of complement components; modulation of innate and adaptive immune response by complement system; and unconventional roles of complement-triggered pathways.

  9. Analysis of Epstein-Barr virus-binding sites on complement receptor 2 (CR2/CD21) using human-mouse chimeras and peptides. At least two distinct sites are necessary for ligand-receptor interaction.

    PubMed

    Molina, H; Brenner, C; Jacobi, S; Gorka, J; Carel, J C; Kinoshita, T; Holers, V M

    1991-07-01

    The predicted amino acid sequence of human complement receptor 2 (CR2, CD21, C3d,g/Epstein-Barr virus receptor) and its genetic murine homologue are approximately 70% identical. The sequence of each consists of a linear array of 60-70 amino acid repeats designated short consensus repeats (SCRs). Although they share significant sequence identity, a major difference in the activities of these two proteins has been believed to be the ability of human, but not mouse, CR2 to mediate Epstein-Barr virus (EBV) infection of B lymphocytes. In order to formally address this question and to directly compare the activities of the CR2 protein of each species, we have expressed recombinant mouse CR2 (rMCR2) in a human K562 erythroleukemia cell line background. We have found that rMCR2 reacts with two previously described rat anti-MCR2 monoclonal antibodies (mAbs), 7G6 and 7E9, but not mAb 8C12, which recognizes only mouse complement receptor 1. rMCR2 rosettes with erythrocytes bearing mouse and human C3d,g and binds glutaraldehyde cross-linked human C3d,g with a similar Kd as human CR2 (HCR2). rMCR2 does not bind EBV. By using this observation and constructing chimeras bearing portions of MCR2 on a HCR2 background, we have been able to define unique sequences in HCR2 SCRs 1 and 2 important in the interaction with both mAb OKB7, which blocks EBV binding and infection, and with EBV. In addition, by using blocking peptides derived from HCR2 sequence, we have identified a second distinct region in SCR2 important in EBV binding. Therefore, within the first two SCRs of HCR2 are multiple distinct sites of interaction with EBV and with mAb OKB7.

  10. The split Renilla luciferase complementation assay is useful for identifying the interaction of Epstein-Barr virus protein kinase BGLF4 and a heat shock protein Hsp90.

    PubMed

    Wang, J; Guo, W; Long, C; Zhou, H; Wang, H; Sun, X

    2016-03-01

    Protein-protein interactions can regulate different cellular processes, such as transcription, translation, and oncogenic transformation. The split Renilla luciferase complementation assay (SRLCA) is one of the techniques that detect protein-protein interactions. The SRLCA is based on the complementation of the LN and LC non-functional halves of Renilla luciferase fused to possibly interacting proteins which after interaction form a functional enzyme and emit luminescence. The BGLF4 of Epstein-Barr virus (EBV) is a viral protein kinase that is expressed during the early and late stages of lytic cycles, which can regulate multiple cellular and viral substrates to optimize the DNA replication environment. The heat shock protein Hsp90 is a molecular chaperone that maintains the integrity of structure and function of various interacting proteins, which can form a complex with BGLF4 and stabilize its expression in cells. The interaction between BGLF4 and Hsp90 could be specifically detected through the SRLCA. The region of aa 250-295 of BGLF4 is essential for the BGLF4/Hsp90 interaction and the mutation of Phe-254, Leu-266, and Leu-267 can disrupt this interaction. These results suggest that the SRLCA can specifically detect the BGLF4/Hsp90 interaction and provide a reference to develop inhibitors that disrupt the BGLF4/Hsp90 interaction. PMID:26982469

  11. The split Renilla luciferase complementation assay is useful for identifying the interaction of Epstein-Barr virus protein kinase BGLF4 and a heat shock protein Hsp90.

    PubMed

    Wang, J; Guo, W; Long, C; Zhou, H; Wang, H; Sun, X

    2016-03-01

    Protein-protein interactions can regulate different cellular processes, such as transcription, translation, and oncogenic transformation. The split Renilla luciferase complementation assay (SRLCA) is one of the techniques that detect protein-protein interactions. The SRLCA is based on the complementation of the LN and LC non-functional halves of Renilla luciferase fused to possibly interacting proteins which after interaction form a functional enzyme and emit luminescence. The BGLF4 of Epstein-Barr virus (EBV) is a viral protein kinase that is expressed during the early and late stages of lytic cycles, which can regulate multiple cellular and viral substrates to optimize the DNA replication environment. The heat shock protein Hsp90 is a molecular chaperone that maintains the integrity of structure and function of various interacting proteins, which can form a complex with BGLF4 and stabilize its expression in cells. The interaction between BGLF4 and Hsp90 could be specifically detected through the SRLCA. The region of aa 250-295 of BGLF4 is essential for the BGLF4/Hsp90 interaction and the mutation of Phe-254, Leu-266, and Leu-267 can disrupt this interaction. These results suggest that the SRLCA can specifically detect the BGLF4/Hsp90 interaction and provide a reference to develop inhibitors that disrupt the BGLF4/Hsp90 interaction.

  12. Complement C3dg-mediated erythrophagocytosis: implications for paroxysmal nocturnal hemoglobinuria

    PubMed Central

    Lin, Zhuoer; Schmidt, Christoph Q.; Koutsogiannaki, Sophia; Ricci, Patrizia; Risitano, Antonio M.; Lambris, John D.

    2015-01-01

    The clinical management of paroxysmal nocturnal hemoglobinuria (PNH), a rare but life-threatening hematologic disease, has fundamentally improved with the introduction of a therapeutic that prevents complement-mediated intravascular hemolysis. However, a considerable fraction of PNH patients show insufficient treatment response and remain transfusion dependent. Because the current treatment only prevents C5-induced lysis but not upstream C3 activation, it has been speculated that ongoing opsonization with C3 fragments leads to recognition and phagocytosis of PNH erythrocytes by immune cells. Here, for the first time, we provide experimental evidence for such extravascular hemolysis and demonstrate that PNH erythrocytes from anti–C5-treated patients are phagocytosed by activated monocytes in vitro. Importantly, we show that this uptake can be mediated by the end-stage opsonin C3dg, which is not traditionally considered a phagocytic marker, via interaction with complement receptor 3 (CR3). Interaction studies confirmed that C3dg itself can act as a ligand for the binding domain of CR3. The degree of C3dg-mediated erythrophagocytosis in samples from different PNH patients correlated well with the individual level of C3dg opsonization. This finding may guide future treatment options for PNH but also has potential implications for the description and management of other complement-mediated diseases. PMID:26082452

  13. Complement activation cascade triggered by PEG-PL engineered nanomedicines and carbon nanotubes: the challenges ahead.

    PubMed

    Moghimi, S M; Andersen, A J; Hashemi, S H; Lettiero, B; Ahmadvand, D; Hunter, A C; Andresen, T L; Hamad, I; Szebeni, J

    2010-09-01

    Since their introduction, poly(ethylene glycol)-phospholipid (PEG-PL) conjugates have found many applications in design and engineering of nanosized delivery systems for controlled delivery of pharmaceuticals especially to non-macrophage targets. However, there are reports of idiosyncratic reactions to certain PEG-PL engineered nanomedicines in both experimental animals and man. These reactions are classified as pseudoallergy and may be associated with cardiopulmonary disturbance and other related symptoms of anaphylaxis. Recent studies suggest that complement activation may be a contributing, but not a rate limiting factor, in eliciting hypersensitivity reactions to such nanomedicines in sensitive individuals. This is rather surprising since PEGylated structures are generally assumed to suppress protein adsorption and blood opsonization events including complement. Here, we examine the molecular basis of complement activation by PEG-PL engineered nanomedicines and carbon nanotubes and discuss the challenges ahead.

  14. Complement and autoimmunity.

    PubMed

    Ballanti, Eleonora; Perricone, Carlo; Greco, Elisabetta; Ballanti, Marta; Di Muzio, Gioia; Chimenti, Maria Sole; Perricone, Roberto

    2013-07-01

    The complement system is a component of the innate immune system. Its main function was initially believed to be limited to the recognition and elimination of pathogens through direct killing or stimulation of phagocytosis. However, in recent years, the immunoregulatory functions of the complement system were demonstrated and it was determined that the complement proteins play an important role in modulating adaptive immunity and in bridging innate and adaptive responses. When the delicate mechanisms that regulate this sophisticated enzymatic system are unbalanced, the complement system may cause damage, mediating tissue inflammation. Dysregulation of the complement system has been involved in the pathogenesis and clinical manifestations of several autoimmune diseases, such as systemic lupus erythematosus, vasculitides, Sjögren's syndrome, antiphospholipid syndrome, systemic sclerosis, dermatomyositis, and rheumatoid arthritis. Complement deficiencies have been associated with an increased risk to develop autoimmune disorders. Because of its functions, the complement system is an attractive therapeutic target for a wide range of diseases. Up to date, several compounds interfering with the complement cascade have been studied in experimental models for autoimmune diseases. The main therapeutic strategies are inhibition of complement activation components, inhibition of complement receptors, and inhibition of membrane attack complex. At present, none of the available agents was proven to be both safe and effective for treatment of autoimmune diseases in humans. Nonetheless, data from preclinical studies and initial clinical trials suggest that the modulation of the complement system could constitute a viable strategy for the treatment of autoimmune conditions in the decades to come.

  15. Rapid platelet turnover in WASP(−) mice correlates with increased ex vivo phagocytosis of opsonized WASP(−) platelets

    PubMed Central

    Prislovsky, Amanda; Marathe, Bindumadhav; Hosni, Amira; Bolen, Alyssa L.; Nimmerjahn, Falk; Jackson, Carl W.; Weiman, Darryl; Strom, Ted S.

    2008-01-01

    Objective Our objective was to determine a mechanism for the thrombocytopenia of murine Wiskott-Aldrich syndrome (WAS). Materials and Methods Consumption rates of WAS protein (WASP)( −) and wild-type (WT) platelets were measured by injection of 5-chloromethylfluorescein diacetate (CMFDA)-labeled platelets into WT or WASP(−) recipients, and by in vivo biotinylation. Platelet and reticulated platelet counts were performed using quantitative flow cytometry. Bone marrow megakaryocyte number and ploidy was assessed by flow cytometry. Phagocytosis of CMFDA-labeled, opsonized platelets was assessed using bone marrow–derived macrophages. Serum antiplatelet antibodies were assayed via their binding to WT platelets. Results CMFDA-labeled WASP(−) platelets are consumed more rapidly than WT platelets in either WT or WASP(−) recipients. In vivo biotinylation studies corroborate these findings and show a normal consumption rate for WASP(−) reticulated platelets. The number of reticulated platelets is reduced in WASP(−) mice, but a significant number of the mice show an increased proportion of reticulated platelets and more severe thrombocytopenia. Sera from some of the latter group contain antiplatelet antibodies. Compared to WT platelets, WASP(−) platelets opsonized with anti-CD61 or 6A6 antibody are taken up more rapidly by bone marrow–derived macrophages. In vivo consumption rates of WASP(−) platelets are more accelerated by opsonization than are those of WT platelets. Conclusion Both rapid clearance and impaired production contribute to the thrombocytopenia of murine WAS. Increased susceptibility of opsonized WASP(−) platelets to phagocytosis leads to increased in vivo clearance. This correlates with a higher incidence of individuals with an elevated fraction of reticulated platelets, a more severe thrombocytopenia, and antiplatelet antibodies. PMID:18346836

  16. Source and role of diacylglycerol formed during phagocytosis of opsonized yeast particles and associated respiratory burst in human neutrophils

    SciTech Connect

    Della Bianca, V.; Grzeskowiak, M.; Lissandrini, D.; Rossi, F. )

    1991-06-28

    The results presented in this paper demonstrate that in human neutrophils phagocytosis of C3b/bi and IgG-opsonized yeast particles is associated with activation of phospholipase D and that this reaction is the main source of diglycerides. The demonstration is based upon the following findings: (1) the challenge of neutrophils with these opsonized particles was followed by a rapid formation of (3H)alkyl-phosphatidic acid (( 3H)alkyl-PA) and (3H)alkyl-diglyceride (( 3H)alkyl-DG) in cells labeled with (3H)alkyl-lyso-phosphatidylcholine; (2) in the presence of ethanol (3H)alkyl-phosphatidylethanol was formed, and accumulation of (3H)alkyl-PA and (3H)alkyl-DG was depressed; (3) propranolol, by inhibiting the dephosphorylation of (3H)alkyl-PA, completely inhibited the accumulation of (3H)alkyl-DG and depressed by about 75% the formation of diglyceride mass. Evidence is also presented that phagocytosis of C3b/bi and IgG-opsonized yeast particles and associated respiratory burst can take place independently of diglyceride formation and of the activity of this second messenger on protein kinase C. In fact: (a) propranolol while completely inhibited the formation of diglyceride mass did not modify either the phagocytosis or respiratory burst; (b) these two processes were insensitive to staurosporine.

  17. Stealth nanotubes: strategies of shielding carbon nanotubes to evade opsonization and improve biodistribution.

    PubMed

    Kotagiri, Nalinikanth; Kim, Jin-Woo

    2014-01-01

    Carbon nanotubes (CNTs) have recently been in the limelight for their potential role in disease diagnostics and therapeutics, as well as in tissue engineering. Before these medical applications can be realized, there is a need to address issues like opsonization, phagocytosis by macrophages, and sequestration to the liver and spleen for eventual elimination from the body; along with equally important issues such as aqueous solubility, dispersion, biocompatibility, and biofunctionalization. CNTs have not been shown to be able to evade such biological obstacles, which include their nonspecific attachments to cells and other biological components in the bloodstream, before reaching target tissues and cells in vivo. This will eventually determine their longevity in circulation and clearance rate from the body. This review article discusses the current status, challenges, practical strategies, and implementations of coating CNTs with biocompatible and opsonin-resistant moieties, rendering CNTs transparent to opsonins and deceiving the innate immune response to make believe that the CNTs are not foreign. A holistic approach to the development of such "stealth" CNTs is presented, which encompasses not only several biophysicochemical factors that are not limited to surface treatment of CNTs, but also extraneous biological factors such as the protein corona formation that inevitably controls the in vivo fate of the particles. This review also discusses the present and potential applications, along with the future directions, of CNTs and their hybrid-based nanotheranostic agents for multiplex, multimodal molecular imaging and therapy, as well as in other applications, such as drug delivery and tissue engineering.

  18. Stealth nanotubes: strategies of shielding carbon nanotubes to evade opsonization and improve biodistribution

    PubMed Central

    Kotagiri, Nalinikanth; Kim, Jin-Woo

    2014-01-01

    Carbon nanotubes (CNTs) have recently been in the limelight for their potential role in disease diagnostics and therapeutics, as well as in tissue engineering. Before these medical applications can be realized, there is a need to address issues like opsonization, phagocytosis by macrophages, and sequestration to the liver and spleen for eventual elimination from the body; along with equally important issues such as aqueous solubility, dispersion, biocompatibility, and biofunctionalization. CNTs have not been shown to be able to evade such biological obstacles, which include their nonspecific attachments to cells and other biological components in the bloodstream, before reaching target tissues and cells in vivo. This will eventually determine their longevity in circulation and clearance rate from the body. This review article discusses the current status, challenges, practical strategies, and implementations of coating CNTs with biocompatible and opsonin-resistant moieties, rendering CNTs transparent to opsonins and deceiving the innate immune response to make believe that the CNTs are not foreign. A holistic approach to the development of such “stealth” CNTs is presented, which encompasses not only several biophysicochemical factors that are not limited to surface treatment of CNTs, but also extraneous biological factors such as the protein corona formation that inevitably controls the in vivo fate of the particles. This review also discusses the present and potential applications, along with the future directions, of CNTs and their hybrid-based nanotheranostic agents for multiplex, multimodal molecular imaging and therapy, as well as in other applications, such as drug delivery and tissue engineering. PMID:24872705

  19. Opsonization modulates Rac-1 activation during cell entry by Leishmania amazonensis.

    PubMed

    Morehead, J; Coppens, I; Andrews, N W

    2002-08-01

    Lesions caused by Leishmania amazonensis normally heal, but relapses occur due to parasite persistence in host tissues. It has been proposed that infection of fibroblasts plays an important role in this process by providing the parasites with a safe haven in which to replicate. However, most previous studies have focused on the entry of Leishmania into macrophages, a process mediated by serum opsonins. To gain insight into a possible role of nonopsonic entry in the intracellular persistence of amastigotes, we examined the invasion of Chinese hamster ovary (CHO) cells. Amastigotes entered CHO cells by a cytochalasin D, genistein, wortmannin, and 2,3-butanedione monoxime-sensitive pathway and replicated within phagolysosomes. However, unlike most phagocytic processes described to date, amastigote internalization in CHO cells involved activation of the GTPases Rho and Cdc42 but not Rac-1. When uptake was mediated by fibronectin or when amastigotes were opsonized with immunoglobulin G and internalized by Fc receptor-expressing CHO cells, Rac-1 activation was restored and found to be required for parasite internalization. Given the essential role of Rac in assembly of the respiratory burst oxidase, invasion through this nonopsonic, Rac-1-independent pathway may play a central role in the intracellular survival of Leishmania in immune hosts.

  20. Opsonization Modulates Rac-1 Activation during Cell Entry by Leishmania amazonensis

    PubMed Central

    Morehead, J.; Coppens, I.; Andrews, N. W.

    2002-01-01

    Lesions caused by Leishmania amazonensis normally heal, but relapses occur due to parasite persistence in host tissues. It has been proposed that infection of fibroblasts plays an important role in this process by providing the parasites with a safe haven in which to replicate. However, most previous studies have focused on the entry of Leishmania into macrophages, a process mediated by serum opsonins. To gain insight into a possible role of nonopsonic entry in the intracellular persistence of amastigotes, we examined the invasion of Chinese hamster ovary (CHO) cells. Amastigotes entered CHO cells by a cytochalasin D, genistein, wortmannin, and 2,3-butanedione monoxime-sensitive pathway and replicated within phagolysosomes. However, unlike most phagocytic processes described to date, amastigote internalization in CHO cells involved activation of the GTPases Rho and Cdc42 but not Rac-1. When uptake was mediated by fibronectin or when amastigotes were opsonized with immunoglobulin G and internalized by Fc receptor-expressing CHO cells, Rac-1 activation was restored and found to be required for parasite internalization. Given the essential role of Rac in assembly of the respiratory burst oxidase, invasion through this nonopsonic, Rac-1-independent pathway may play a central role in the intracellular survival of Leishmania in immune hosts. PMID:12117970

  1. LL-37 opsonizes and inhibits biofilm formation of Aggregatibacter actinomycetemcomitans at subbactericidal concentrations.

    PubMed

    Sol, Asaf; Ginesin, Ofir; Chaushu, Stella; Karra, Laila; Coppenhagen-Glazer, Shunit; Ginsburg, Isaac; Bachrach, Gilad

    2013-10-01

    Host defense peptides are immediate responders of the innate immunity that express antimicrobial, immunoregulatory, and wound-healing activities. Neutrophils are a major source for oral host defense peptides, and phagocytosis by neutrophils is a major mechanism for bacterial clearance in the gingival tissue. Dysfunction of or reduction in the numbers of neutrophils or deficiency in the LL-37 host defense peptide was each previously linked with proliferation of oral Aggregatibacter actinomycetemcomitans which resulted in an aggressive periodontal disease. Surprisingly, A. actinomycetemcomitans shows resistance to high concentrations of LL-37. In this study, we demonstrated that submicrocidal concentrations of LL-37 inhibit biofilm formation by A. actinomycetemcomitans and act as opsonins and agglutinins that greatly enhance its clearance by neutrophils and macrophages. Improved uptake of A. actinomycetemcomitans by neutrophils was mediated by their opsonization with LL-37. Enhanced phagocytosis and killing of A. actinomycetemcomitans by murine macrophage-like RAW 264.7 cells were dependent on their preagglutination by LL-37. Although A. actinomycetemcomitans is resistant to the bactericidal effect of LL-37, our results offer a rationale for the epidemiological association between LL-37 deficiency and the expansion of oral A. actinomycetemcomitans and indicate a possible therapeutic use of cationic peptides for host defense.

  2. Stealth nanotubes: strategies of shielding carbon nanotubes to evade opsonization and improve biodistribution.

    PubMed

    Kotagiri, Nalinikanth; Kim, Jin-Woo

    2014-01-01

    Carbon nanotubes (CNTs) have recently been in the limelight for their potential role in disease diagnostics and therapeutics, as well as in tissue engineering. Before these medical applications can be realized, there is a need to address issues like opsonization, phagocytosis by macrophages, and sequestration to the liver and spleen for eventual elimination from the body; along with equally important issues such as aqueous solubility, dispersion, biocompatibility, and biofunctionalization. CNTs have not been shown to be able to evade such biological obstacles, which include their nonspecific attachments to cells and other biological components in the bloodstream, before reaching target tissues and cells in vivo. This will eventually determine their longevity in circulation and clearance rate from the body. This review article discusses the current status, challenges, practical strategies, and implementations of coating CNTs with biocompatible and opsonin-resistant moieties, rendering CNTs transparent to opsonins and deceiving the innate immune response to make believe that the CNTs are not foreign. A holistic approach to the development of such "stealth" CNTs is presented, which encompasses not only several biophysicochemical factors that are not limited to surface treatment of CNTs, but also extraneous biological factors such as the protein corona formation that inevitably controls the in vivo fate of the particles. This review also discusses the present and potential applications, along with the future directions, of CNTs and their hybrid-based nanotheranostic agents for multiplex, multimodal molecular imaging and therapy, as well as in other applications, such as drug delivery and tissue engineering. PMID:24872705

  3. Cloning and molecular characterization of complement component 1 inhibitor (C1INH) and complement component 8β (C8β) in Nile tilapia (Oreochromis niloticus).

    PubMed

    He, Anyuan; Yang, Jie; Tang, Shoujie; Wang, Chenghui

    2013-09-01

    Nile tilapia (Oreochromis niloticus), one of the most important groups of food fishes in the world, has frequently suffered from serious challenge from pathogens in recent years. Immune responses of Nile tilapia should be understood to protect the aquaculture industry of this fish. The complement system has an important function in recognizing bacteria, opsonizing these pathogens by phagocytes, or killing them by direct lysis. In this study, two Nile tilapia complement component genes, complement component 1 inhibitor (C1INH) and complement component 8β subunit (C8β), were cloned and their expression characteristics were analyzed. C1INH cDNA was found containing a 1791 bp open reading frame (ORF) encoding a putative protein with 597 amino acids, a 101 bp 5'-untranslated region (UTR) and a 236 bp 3'-UTR. The predicted protein structure for this gene consisted of two Ig-like domains and glycosyl hydrolase family-9 active site signature 2. The C8β cDNA consisted of a 1761 bp ORF encoding 587 amino acids, a 15 bp 5'-UTR and a 170 bp 3'-UTR. The predicted protein of C8β contained three motifs, thrombospondin type-1 repeat, membrane attack complex/perforin domain, and LDL-receptor class A. Expression analysis revealed that these two complement genes were highly expressed in the liver, however, were weakly expressed in the gill, heart, brain, kidney, intestine, spleen and dorsal muscle tissues. The present study provided insights into the complement system and immune functions of Nile tilapia.

  4. Complement and HIV-I infection/HIV-associated neurocognitive disorders.

    PubMed

    Liu, Fengming; Dai, Shen; Gordon, Jennifer; Qin, Xuebin

    2014-04-01

    The various neurological complications associated with HIV-1 infection, specifically HIV-associated neurocognitive disorders (HAND) persist as a major public health burden worldwide. Despite the widespread use of anti-retroviral therapy, the prevalence of HAND is significantly high. HAND results from the direct effects of an HIV-1 infection as well as secondary effects of HIV-1-induced immune reaction and inflammatory response. Complement, a critical mediator of innate and acquired immunity, plays important roles in defeating many viral infections by the formation of a lytic pore or indirectly by opsonization and recruitment of phagocytes. While the role of complement in the pathogenesis of HIV-1 infection and HAND has been previously recognized for over 15 years, it has been largely underestimated thus far. Complement can be activated through HIV-1 envelope proteins, mannose-binding lectins (MBL), and anti-HIV-1 antibodies. Complement not only fights against HIV-1 infection but also enhances HIV-1 infection. In addition, HIV-1 can hijack complement regulators such as CD59 and CD55 and can utilize these regulators and factor H to escape from complement attack. Normally, complement levels in brain are much lower than plasma levels and there is no or little complement deposition in brain cells. Interestingly, local production and deposition of complement are dramatically increased in HIV-1-infected brain, indicating that complement may contribute to the pathogenesis of HAND. Here, we review the current understanding of the role of complement in HIV-1 infection and HAND, as well as potential therapeutic approaches targeting the complement system for the treatment and eradications of HIV-1 infection.

  5. 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

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

    PubMed

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

    2008-12-01

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

  7. 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.

  8. The Lectin Pathway of Complement and Rheumatic Heart Disease

    PubMed Central

    Beltrame, Marcia Holsbach; Catarino, Sandra Jeremias; Goeldner, Isabela; Boldt, Angelica Beate Winter; de Messias-Reason, Iara José

    2014-01-01

    The innate immune system is the first line of host defense against infection and is comprised of humoral and cellular mechanisms that recognize potential pathogens within minutes or hours of entry. The effector components of innate immunity include epithelial barriers, phagocytes, and natural killer cells, as well as cytokines and the complement system. Complement plays an important role in the immediate response against microorganisms, including Streptococcus sp. The lectin pathway is one of three pathways by which the complement system can be activated. This pathway is initiated by the binding of mannose-binding lectin (MBL), collectin 11 (CL-K1), and ficolins (Ficolin-1, Ficolin-2, and Ficolin-3) to microbial surface oligosaccharides and acetylated residues, respectively. Upon binding to target molecules, MBL, CL-K1, and ficolins form complexes with MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2), which cleave C4 and C2 forming the C3 convertase (C4b2a). Subsequent activation of complement cascade leads to opsonization, phagocytosis, and lysis of target microorganisms through the formation of the membrane-attack complex. In addition, activation of complement may induce several inflammatory effects, such as expression of adhesion molecules, chemotaxis and activation of leukocytes, release of reactive oxygen species, and secretion of cytokines and chemokines. In this chapter, we review the general aspects of the structure, function, and genetic polymorphism of lectin-pathway components and discuss most recent understanding on the role of the lectin pathway in the predisposition and clinical progression of Rheumatic Fever. PMID:25654073

  9. Complement in hemolytic anemia.

    PubMed

    Brodsky, Robert A

    2015-11-26

    Complement is increasingly being recognized as an important driver of human disease, including many hemolytic anemias. Paroxysmal nocturnal hemoglobinuria (PNH) cells are susceptible to hemolysis because of a loss of the complement regulatory proteins CD59 and CD55. Patients with atypical hemolytic uremic syndrome (aHUS) develop a thrombotic microangiopathy (TMA) that in most cases is attributable to mutations that lead to activation of the alternative pathway of complement. For optimal therapy, it is critical, but often difficult, to distinguish aHUS from other TMAs, such as thrombotic thrombocytopenic purpura; however, novel bioassays are being developed. In cold agglutinin disease (CAD), immunoglobulin M autoantibodies fix complement on the surface of red cells, resulting in extravascular hemolysis by the reticuloendothelial system. Drugs that inhibit complement activation are increasingly being used to treat these diseases. This article discusses the pathophysiology, diagnosis, and therapy for PNH, aHUS, and CAD.

  10. Complement in hemolytic anemia.

    PubMed

    Brodsky, Robert A

    2015-01-01

    Complement is increasingly being recognized as an important driver of human disease, including many hemolytic anemias. Paroxysmal nocturnal hemoglobinuria (PNH) cells are susceptible to hemolysis because of a loss of the complement regulatory proteins CD59 and CD55. Patients with atypical hemolytic uremic syndrome (aHUS) develop a thrombotic microangiopathy (TMA) that in most cases is attributable to mutations that lead to activation of the alternative pathway of complement. For optimal therapy, it is critical, but often difficult, to distinguish aHUS from other TMAs, such as thrombotic thrombocytopenic purpura; however, novel bioassays are being developed. In cold agglutinin disease (CAD), immunoglobulin M autoantibodies fix complement on the surface of red cells, resulting in extravascular hemolysis by the reticuloendothelial system. Drugs that inhibit complement activation are increasingly being used to treat these diseases. This article discusses the pathophysiology, diagnosis, and therapy for PNH, aHUS, and CAD.

  11. Multi-Faceted Proteomic Characterization of Host Protein Complement of Rift Valley Fever Virus Virions and Identification of Specific Heat Shock Proteins, Including HSP90, as Important Viral Host Factors

    PubMed Central

    Nuss, Jonathan E.; Kehn-Hall, Kylene; Benedict, Ashwini; Costantino, Julie; Ward, Michael; Peyser, Brian D.; Retterer, Cary J.; Tressler, Lyal E.; Wanner, Laura M.; McGovern, Hugh F.; Zaidi, Anum; Anthony, Scott M.; Kota, Krishna P.; Bavari, Sina; Hakami, Ramin M.

    2014-01-01

    Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF. PMID:24809507

  12. Multi-faceted proteomic characterization of host protein complement of Rift Valley fever virus virions and identification of specific heat shock proteins, including HSP90, as important viral host factors.

    PubMed

    Nuss, Jonathan E; Kehn-Hall, Kylene; Benedict, Ashwini; Costantino, Julie; Ward, Michael; Peyser, Brian D; Retterer, Cary J; Tressler, Lyal E; Wanner, Laura M; McGovern, Hugh F; Zaidi, Anum; Anthony, Scott M; Kota, Krishna P; Bavari, Sina; Hakami, Ramin M

    2014-01-01

    Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.

  13. High-Sensitivity C-Reactive Protein Complements Plasma Epstein-Barr Virus Deoxyribonucleic Acid Prognostication in Nasopharyngeal Carcinoma: A Large-Scale Retrospective and Prospective Cohort Study

    SciTech Connect

    Tang, Lin-Quan; Li, Chao-Feng; Chen, Qiu-Yan; Zhang, Lu; Lai, Xiao-Ping; He, Yun; Xu, Yun-Xiu-Xiu; Hu, Dong-Peng; Wen, Shi-Hua; Peng, Yu-Tuan; Chen, Wen-Hui; Liu, Huai; Guo, Shan-Shan; Liu, Li-Ting; Li, Jing; Zhang, Jing-Ping; and others

    2015-02-01

    Purpose: To evaluate the effects of combining the assessment of circulating high-sensitivity C-reactive protein (hs-CRP) with that of Epstein-Barr virus DNA (EBV DNA) in the pretherapy prognostication of nasopharyngeal carcinoma (NPC). Patients and Methods: Three independent cohorts of NPC patients (training set of n=3113, internal validation set of n=1556, and prospective validation set of n=1668) were studied. Determinants of disease-free survival, distant metastasis–free survival, and overall survival were assessed by multivariate analysis. Hazard ratios and survival probabilities of the patient groups, segregated by clinical stage (T1-2N0-1M0, T3-4N0-1M0, T1-2N2-3M0, and T3-4N2-3M0) and EBV DNA load (low or high) alone, and also according to hs-CRP level (low or high), were compared. Results: Elevated hs-CRP and EBV DNA levels were significantly correlated with poor disease-free survival, distant metastasis–free survival, and overall survival in both the training and validation sets. Associations were similar and remained significant after excluding patients with cardiovascular disease, diabetes, and chronic hepatitis B. Patients with advanced-stage disease were segregated by high EBV DNA levels and high hs-CRP level into a poorest-risk group, and participants with either high EBV DNA but low hs-CRP level or high hs-CRP but low EBV DNA values had poorer survival compared with the bottom values for both biomarkers. These findings demonstrate a significant improvement in the prognostic ability of conventional advanced NPC staging. Conclusion: Baseline plasma EBV DNA and serum hs-CRP levels were significantly correlated with survival in NPC patients. The combined interpretation of EBV DNA with hs-CRP levels led to refinement of the risks for the patient subsets, with improved risk discrimination in patients with advanced-stage disease.

  14. CSF coccidioides complement fixation

    MedlinePlus

    ... eds. Henry's Clinical Diagnosis and Management by Laboratory Methods . 22nd ed. Philadelphia, PA: Elsevier Saunders; 2011:chap 61. Read More Complement Update Date 5/1/2015 Updated by: Jatin M. Vyas, MD, ...

  15. Hereditary deficiency of the seventh component of complement.

    PubMed Central

    Boyer, J T; Gall, E P; Norman, M E; Nilsson, U R; Zimmerman, T S

    1975-01-01

    Deficiency of the seventh component of complement has been found in the serum of a 42-yr-old Caucasian woman who has Raynaud's phenomenon, sclerodactyly, and telangiectasia. Partial deficiency was found in the serum of the patient's parents and children, indicating a pattern of inheritance of autosomal codominance. Transfusion experiments indicated that exogenous C7 had a 91-h halk-life in the patient. There was no evidence for C7 synthesis after transfusion. No C7 inhibitors were detected in the patient's serum. The patient's serum was found to support the activation of complement by both the classical and properdin pathways to the C7 stage. The addition of C7 to the patient's serum permitted it to support hemolytic reactions initiated by either pathway. No defects could be detected in plasma or whole blood coagulation. The patient's serum was deficient in opsonizing unsensitized yeast particles in serum and in the generation of chemotactic factor by antigen-antibody complexes and endotoxin. Both deficiencies were corrected by the addition of C7. These observations suggest a key role for C7 for in vitro yeast phagocytosis and chemotaxis generation. However, the patient's lack of infections indicates a relatively minor role for C7 in human resistance to infection. PMID:1099121

  16. Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity.

    PubMed

    Colonna, Lucrezia; Parry, Graham C; Panicker, Sandip; Elkon, Keith B

    2016-02-01

    Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.

  17. Laboratory tests for disorders of complement and complement regulatory proteins.

    PubMed

    Shih, Angela R; Murali, Mandakolathur R

    2015-12-01

    The complement pathway is a cascade of proteases that is involved in immune surveillance and innate immunity, as well as adaptive immunity. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, complement regulatory proteins, or complement deficiencies, and has been linked to a number of hematologic disorders, including paroxysmal noctural hemoglobinuria (PNH), hereditary angioedema (HAE), and atypical hemolytic-uremic syndrome (aHUS). Here, current laboratory tests for disorders of the complement pathway are reviewed, and their utility and limitations in hematologic disorders and systemic diseases are discussed. Current therapeutic advances targeting the complement pathway in treatment of complement-mediated hematologic disorders are also reviewed.

  18. Anti-Glycoprotein G Antibodies of Herpes Simplex Virus 2 Contribute to Complete Protection after Vaccination in Mice and Induce Antibody-Dependent Cellular Cytotoxicity and Complement-Mediated Cytolysis

    PubMed Central

    Görander, Staffan; Ekblad, Maria; Bergström, Tomas; Liljeqvist, Jan-Åke

    2014-01-01

    We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2) of herpes simplex virus 2 (HSV-2) in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1) and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (ACMC) activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials. PMID:25398047

  19. 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

  20. Complement component 3 (C3)

    MedlinePlus

    C3 and C4 are the most commonly measured complement components. A complement test may be used to monitor people with an ... normal levels of the complement proteins C3 and C4 . Complement activity varies throughout the body. For example, ...

  1. Verbal Complementizers in Arabic

    ERIC Educational Resources Information Center

    Ahmed, Hossam Eldin Ibrahim

    2015-01-01

    A class of Modern Standard Arabic complementizers known as "'?inna' and its sisters" demonstrate unique case and word order restrictions. While CPs in Arabic allow both Subject-Verb (SV) and Verb-Subject (VS) word order and their subjects show nominative morphology, CPs introduced by "?inna" ban a verb from directly following…

  2. Inhibition of biomaterial-induced complement activation attenuates the inflammatory host response to implantation

    PubMed Central

    Kourtzelis, Ioannis; Rafail, Stavros; DeAngelis, Robert A.; Foukas, Periklis G.; Ricklin, Daniel; Lambris, John D.

    2013-01-01

    Although complement is a known contributor to biomaterial-induced complications, pathological implications and therapeutic options remain to be explored. Here we investigated the involvement of complement in the inflammatory response to polypropylene meshes commonly used for hernia repair. In vitro assays revealed deposition of complement activation fragments on the mesh after incubation in plasma. Moreover, significant mesh-induced complement and granulocyte activation was observed in plasma and leukocyte preparations, respectively. Pretreatment of plasma with the complement inhibitor compstatin reduced opsonization >2-fold, and compstatin and a C5a receptor antagonist (C5aRa) impaired granulocyte activation by 50 and 67%, respectively. We established a clinically relevant mouse model of implantation and could confirm deposition of C3 activation fragments on mesh implants in vivo using immunofluorescence. In meshes extracted after subcutaneous or peritoneal implantation, the amount of immune cell infiltrate in mice deficient in key complement components (C3, C5aR), or treated with C5aRa, was approximately half of that observed in wild-type littermates or mice treated with inactive C5aRa, respectively. Our data suggest that implantation of a widely used surgical mesh triggers the formation of an inflammatory cell microenvironment at the implant site through complement activation, and indicates a path for the therapeutic modulation of implant-related complications.—Kourtzelis, I., Rafail, S., DeAngelis, R. A., Foukas, P. G., Ricklin, D., Lambris, J. D. Inhibition of biomaterial-induced complement activation attenuates the inflammatory host response to implantation. PMID:23558338

  3. Genetic Association of the Porcine C9 Complement Component with Hemolytic Complement Activity

    PubMed Central

    Khoa, D. V. A.; Wimmers, K.

    2015-01-01

    The complement system is a part of the natural immune regulation mechanism against invading pathogens. Complement activation from three different pathways (classical, lectin, and alternative) leads to the formation of C5-convertase, an enzyme for cleavage of C5 into C5a and C5b, followed by C6, C7, C8, and C9 in membrane attack complex. The C9 is the last complement component of the terminal lytic pathway, which plays an important role in lysis of the target cells depending on its self-polymerization to form transmembrane channels. To address the association of C9 with traits related to disease resistance, the complete porcine C9 cDNA was comparatively sequenced to detect single nucleotide polymorphisms (SNPs) in pigs of the breeds Hampshire (HS), Duroc (DU), Berlin miniature pig (BMP), German Landrace (LR), Pietrain (PIE), and Muong Khuong (Vietnamese potbelly pig). Genotyping was performed in 417 F2 animals of a resource population (DUMI: DU×BMP) that were vaccinated with Mycoplasma hyopneumoniae, Aujeszky diseases virus and porcine respiratory and reproductive syndrome virus at 6, 14 and 16 weeks of age, respectively. Two SNPs were detected within the third exon. One of them has an amino acid substitution. The European porcine breeds (LR and PIE) show higher allele frequency of these SNPs than Vietnamese porcine breed (MK). Association of the substitution SNP with hemolytic complement activity indicated statistically significant differences between genotypes in the classical pathway but not in the alternative pathway. The interactions between eight time points of measurement of complement activity before and after vaccinations and genotypes were significantly different. The difference in hemolytic complement activity in the both pathways depends on genotype, kind of vaccine, age and the interaction to the other complement components. These results promote the porcine C9 (pC9) as a candidate gene to improve general animal health in the future. PMID:26194222

  4. IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fcα/μ Receptor.

    PubMed

    Weinstein, Jonathan R; Quan, Yi; Hanson, Josiah F; Colonna, Lucrezia; Iorga, Michael; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira; Elkon, Keith B; Möller, Thomas

    2015-12-01

    Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR(-/-) mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR(-/-) mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18(-/-) and C3(-/-) mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of S. aureus by microglia.

  5. A co-operative interaction between Neisseria gonorrhoeae and complement receptor 3 mediates infection of primary cervical epithelial cells.

    PubMed

    Edwards, Jennifer L; Brown, Eric J; Uk-Nham, Sang; Cannon, Janne G; Blake, Milan S; Apicella, Michael A

    2002-09-01

    Little is known about the pathogenesis of gonococcal infection within the lower female genital tract. We recently described the distribution of complement receptor 3 (CR3) on epithelia of the female genital tract. Our studies further indicate that CR3-mediated endocytosis serves as a primary mechanism by which N. gonorrhoeae elicits membrane ruffling and cellular invasion of primary, human, cervical epithelial cells. We have extended these studies to describe the nature of the gonococcus-CR3 interaction. Western Blot analysis demonstrated production of alternative pathway complement components by ecto- and endocervical cells which allows C3b deposition on gonococci and its rapid conversion to iC3b. Anti-iC3b and -factor I antibodies significantly inhibited adherence and invasion of primary cervical cells, suggesting that iC3b covalently bound to the gonococcus serves as a primary ligand for CR3 adherence. However, gonococcal porin and pili also bound to the I-domain of CR3 in a non-opsonic manner. Binding of porin and pili to CR3 were required for adherence to and invasion of cervical epithelia. Collectively, these data suggest that gonococcal adherence to CR3 occurs in a co-operative manner, which requires gonococcal iC3b-opsonization, porin and pilus. In conjunction, these molecules facilitate targeting to and successful infection of the cervical epithelium. PMID:12390350

  6. Role of complement receptor 1 (CR1; CD35) on epithelial cells: A model for understanding complement-mediated damage in the kidney.

    PubMed

    Java, Anuja; Liszewski, M Kathryn; Hourcade, Dennis E; Zhang, Fan; Atkinson, John P

    2015-10-01

    The regulators of complement activation gene cluster encodes a group of proteins that have evolved to control the amplification of complement at the critical step of C3 activation. Complement receptor 1 (CR1) is the most versatile of these inhibitors with both receptor and regulatory functions. While expressed on most peripheral blood cells, the only epithelial site of expression in the kidney is by the podocyte. Its expression by this cell population has aroused considerable speculation as to its biologic function in view of many complement-mediated renal diseases. The goal of this investigation was to assess the role of CR1 on epithelial cells. To this end, we utilized a Chinese hamster ovary cell model system. Among our findings, CR1 reduced C3b deposition by ∼ 80% during classical pathway activation; however, it was an even more potent regulator (>95% reduction in C3b deposition) of the alternative pathway. This inhibition was primarily mediated by decay accelerating activity. The deposited C4b and C3b were progressively cleaved with a t½ of ∼ 30 min to C4d and C3d, respectively, by CR1-dependent cofactor activity. CR1 functioned intrinsically (i.e, worked only on the cell on which it was expressed). Moreover, CR1 efficiently and stably bound but didn't internalize C4b/C3b opsonized immune complexes. Our studies underscore the potential importance of CR1 on an epithelial cell population as both an intrinsic complement regulator and an immune adherence receptor. These results provide a framework for understanding how loss of CR1 expression on podocytes may contribute to complement-mediated damage in the kidney.

  7. Type IV pilus glycosylation mediates resistance of Pseudomonas aeruginosa to opsonic activities of the pulmonary surfactant protein A.

    PubMed

    Tan, Rommel M; Kuang, Zhizhou; Hao, Yonghua; Lee, Francis; Lee, Timothy; Lee, Ryan J; Lau, Gee W

    2015-04-01

    Pseudomonas aeruginosa is a major bacterial pathogen commonly associated with chronic lung infections in cystic fibrosis (CF). Previously, we have demonstrated that the type IV pilus (Tfp) of P. aeruginosa mediates resistance to antibacterial effects of pulmonary surfactant protein A (SP-A). Interestingly, P. aeruginosa strains with group I pilins are O-glycosylated through the TfpO glycosyltransferase with a single subunit of O-antigen (O-ag). Importantly, TfpO-mediated O-glycosylation is important for virulence in mouse lungs, exemplified by more frequent lung infection in CF with TfpO-expressing P. aeruginosa strains. However, the mechanism underlying the importance of Tfp glycosylation in P. aeruginosa pathogenesis is not fully understood. Here, we demonstrated one mechanism of increased fitness mediated by O-glycosylation of group 1 pilins on Tfp in the P. aeruginosa clinical isolate 1244. Using an acute pneumonia model in SP-A+/+ versus SP-A-/- mice, the O-glycosylation-deficient ΔtfpO mutant was found to be attenuated in lung infection. Both 1244 and ΔtfpO strains showed equal levels of susceptibility to SP-A-mediated membrane permeability. In contrast, the ΔtfpO mutant was more susceptible to opsonization by SP-A and by other pulmonary and circulating opsonins, SP-D and mannose binding lectin 2, respectively. Importantly, the increased susceptibility to phagocytosis was abrogated in the absence of opsonins. These results indicate that O-glycosylation of Tfp with O-ag specifically confers resistance to opsonization during host-mediated phagocytosis. PMID:25605768

  8. Complement receptor activity of recombinant porcine CR1-like protein expressed in a eukaryotic system.

    PubMed

    Yin, Wei; Wei, Xiaoming; Jiang, Junbing; Fan, Kuohai; Zhao, Junxing; Sun, Na; Wang, Zhiwei; Sun, Yaogui; Ma, Haili; Zhao, Xin; Li, Hongquan

    2016-08-01

    Primate complement receptor type 1 (CR1) protein, a single-chain transmembrane glycoprotein, plays an important role in immune adherence and clearing complement-opsonized immune complexes. Here, the mRNA of the porcine primate-like complement receptor (CR1-like) gene was analyzed, and two domain sequences with potential functions were cloned into the pwPICZalpha vector for expression in Pichia pastoris. The recombinant proteins were purified with both Protein Pure Ni-NTA resin and strong anion exchange resin. The activities of the purified recombinant proteins were evaluated by SDS-PAGE, western blotting, and complement receptor assays. The results indicated that two domains of the CR1-like protein, CCP36 and CCP811 with molecular weights of 29.8 kDa and 30 kDa, respectively, were successfully expressed in P. pastoris. These two recombinant proteins possess some of the functions of the primate CR1 protein. Using these two proteins coupled with an antibody blocking technique, we also showed that CR1-like is expressed on natural porcine erythrocytes. PMID:26903010

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

    PubMed Central

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

    2015-01-01

    There is a growing awareness that complement plays an integral role in human physiology and disease, transcending its traditional perception as an accessory system for pathogen clearance and opsonic cell killing. As the list of pathologies linked to dysregulated complement activation grows longer, it has become clear that targeted modulation of this innate immune system opens new windows of therapeutic opportunity for anti-inflammatory drug design. Indeed, the introduction of the first complement-targeting drugs has reignited a vibrant interest in the clinical translation of complement-based inhibitors. Compstatin was discovered as a cyclic peptide that inhibits complement activation by binding C3 and interfering with convertase formation and C3 cleavage. As the convergence point of all activation pathways and a molecular hub for crosstalk with multiple pathogenic pathways, C3 represents an attractive target for therapeutic modulation of the complement cascade. A multidisciplinary drug optimization effort encompassing rational “wet” and in silico synthetic approaches and an array of biophysical, structural, and analytical tools has culminated in an impressive structure-function refinement of compstatin, yielding a series of analogs that show promise for a wide spectrum of clinical applications. These new derivatives have improved inhibitory potency and pharmacokinetic profiles and show efficacy in clinically relevant primate models of disease. This review provides an up-to-date survey of the drug design effort placed on the compstatin family of C3 inhibitors, highlighting the most promising drug candidates. It also discusses translational challenges in complement drug discovery and peptide drug development and reviews concerns related to systemic C3 interception. PMID:25678219

  10. Defect of a complement receptor 3 epitope in a patient with systemic lupus erythematosus.

    PubMed Central

    Witte, T; Dumoulin, F L; Gessner, J E; Schubert, J; Götze, O; Neumann, C; Todd, R F; Deicher, H; Schmidt, R E

    1993-01-01

    Complement receptor 3 (CR3) is expressed on cells of the reticuloendothelial system and involved in the clearance of immune complexes. In this article a patient with a deficiency of the C3bi binding site of this receptor is described. Clinically this patient exhibited predominantly cutaneous manifestations of a systemic lupus erythematosus with an immune vasculitis and panniculitis. The deficiency of the CR3 epitope was demonstrated using flow cytometry. The functional relevance of this defect was demonstrated in a rosetting assay with C3bi-loaded erythrocytes. C3bi binding was found to be significantly decreased. Furthermore, there was an impairment of phagocytosis of opsonized Escherichia coli. The CR3 defect is not due to an autoantibody but is assumed to have a genetic basis. These data suggest that the defect of the CR3 may be involved in the pathogenesis of the immune vasculitis in this patient. Images PMID:7690773

  11. Complementing Gender Analysis Methods.

    PubMed

    Kumar, Anant

    2016-01-01

    The existing gender analysis frameworks start with a premise that men and women are equal and should be treated equally. These frameworks give emphasis on equal distribution of resources between men and women and believe that this will bring equality which is not always true. Despite equal distribution of resources, women tend to suffer and experience discrimination in many areas of their lives such as the power to control resources within social relationships, and the need for emotional security and reproductive rights within interpersonal relationships. These frameworks believe that patriarchy as an institution plays an important role in women's oppression, exploitation, and it is a barrier in their empowerment and rights. Thus, some think that by ensuring equal distribution of resources and empowering women economically, institutions like patriarchy can be challenged. These frameworks are based on proposed equality principle which puts men and women in competing roles. Thus, the real equality will never be achieved. Contrary to the existing gender analysis frameworks, the Complementing Gender Analysis framework proposed by the author provides a new approach toward gender analysis which not only recognizes the role of economic empowerment and equal distribution of resources but suggests to incorporate the concept and role of social capital, equity, and doing gender in gender analysis which is based on perceived equity principle, putting men and women in complementing roles that may lead to equality. In this article the author reviews the mainstream gender theories in development from the viewpoint of the complementary roles of gender. This alternative view is argued based on existing literature and an anecdote of observations made by the author. While criticizing the equality theory, the author offers equity theory in resolving the gender conflict by using the concept of social and psychological capital. PMID:25941756

  12. Complementing Gender Analysis Methods.

    PubMed

    Kumar, Anant

    2016-01-01

    The existing gender analysis frameworks start with a premise that men and women are equal and should be treated equally. These frameworks give emphasis on equal distribution of resources between men and women and believe that this will bring equality which is not always true. Despite equal distribution of resources, women tend to suffer and experience discrimination in many areas of their lives such as the power to control resources within social relationships, and the need for emotional security and reproductive rights within interpersonal relationships. These frameworks believe that patriarchy as an institution plays an important role in women's oppression, exploitation, and it is a barrier in their empowerment and rights. Thus, some think that by ensuring equal distribution of resources and empowering women economically, institutions like patriarchy can be challenged. These frameworks are based on proposed equality principle which puts men and women in competing roles. Thus, the real equality will never be achieved. Contrary to the existing gender analysis frameworks, the Complementing Gender Analysis framework proposed by the author provides a new approach toward gender analysis which not only recognizes the role of economic empowerment and equal distribution of resources but suggests to incorporate the concept and role of social capital, equity, and doing gender in gender analysis which is based on perceived equity principle, putting men and women in complementing roles that may lead to equality. In this article the author reviews the mainstream gender theories in development from the viewpoint of the complementary roles of gender. This alternative view is argued based on existing literature and an anecdote of observations made by the author. While criticizing the equality theory, the author offers equity theory in resolving the gender conflict by using the concept of social and psychological capital.

  13. How antibodies alter the cell entry pathway of dengue virus particles in macrophages

    PubMed Central

    Ayala-Nunez, Nilda V.; Hoornweg, Tabitha E.; van de Pol, Denise P.I.; Sjollema, Klaas A.; Flipse, Jacky; van der Schaar, Hilde M.; Smit, Jolanda M.

    2016-01-01

    Antibody-dependent enhancement of dengue virus (DENV) infection plays an important role in the exacerbation of DENV-induced disease. To understand how antibodies influence the fate of DENV particles, we explored the cell entry pathway of DENV in the absence and presence of antibodies in macrophage-like P388D1 cells. Recent studies unraveled that both mature and immature DENV particles contribute to ADE, hence, both particles were studied. We observed that antibody-opsonized DENV enters P388D1 cells through a different pathway than non-opsonized DENV. Antibody-mediated DENV entry was dependent on FcγRs, pH, Eps15, dynamin, actin, PI3K, Rab5, and Rab7. In the absence of antibodies, DENV cell entry was FcγR, PI3K, and Rab5-independent. Live-cell imaging of fluorescently-labeled particles revealed that actin-mediated membrane protrusions facilitate virus uptake. In fact, actin protrusions were found to actively search and capture antibody-bound virus particles distantly located from the cell body, a phenomenon that is not observed in the absence of antibodies. Overall, similar results were seen for antibody-opsonized standard and antibody-bound immature DENV preparations, indicating that the maturation status of the virus does not control the entry pathway. Collectively, our findings suggest that antibodies alter the cell entry pathway of DENV and trigger a novel mechanism of initial virus-cell contact. PMID:27385443

  14. Complement: the Iceman of immunology?

    PubMed

    Würzner, R

    1997-12-01

    Complement provides an important host defence system involved in a multitude of immune reactions, including opsonisation of micro-organisms, enhancement of inflammatory response, immunomodulation, clearance of immune complexes and cell lysis. The 6th European Meeting on Complement in Human Disease, 12-15 March 1997 in Innsbruck, Austria, the preservation site of the neolithic Iceman, addressed the functional role of complement and its regulators in human disease. The scientific presentations clearly demonstrated that complement is not a redundant fossil, evolving since the dawn of vertebrate existence on the earth, but remains continuously important for mankind.

  15. Complement system in lung disease.

    PubMed

    Pandya, Pankita H; Wilkes, David S

    2014-10-01

    In addition to its established contribution to innate immunity, recent studies have suggested novel roles for the complement system in the development of various lung diseases. Several studies have demonstrated that complement may serve as a key link between innate and adaptive immunity in a variety of pulmonary conditions. However, the specific contributions of complement to lung diseases based on innate and adaptive immunity are just beginning to emerge. Elucidating the role of complement-mediated immune regulation in these diseases will help to identify new targets for therapeutic interventions.

  16. Complement fixation by rheumatoid factor.

    PubMed Central

    Tanimoto, K; Cooper, N R; Johnson, J S; Vaughan, J H

    1975-01-01

    The capacity for fixation and activation of hemolytic complement by polyclonal IgM rheumatoid factors (RF) isolated from sera of patients with rheumatoid arthritis and monoclonal IgM-RF isolated from the cryoprecipitates of patients with IgM-IgG mixed cryoglobulinemia was examined. RF mixed with aggregated, reduced, and alkylated human IgG (Agg-R/A-IgG) in the fluid phase failed to significantly reduce the level of total hemolytic complement, CH50, or of individual complement components, C1, C2, C3, and C5. However, sheep erythrocytes (SRC) coated with Agg-R/A-IgG or with reduced and alkylated rabbit IgG anti-SRC antibody were hemolyzed by complement in the presence of polyclonal IgM-RF. Human and guinea pig complement worked equally well. The degree of hemolysis was in direct proportion to the hemagglutination titer of the RF against the same coated cells. Monoclonal IgM-RF, normal human IgM, and purified Waldenström macroglobulins without antiglobulin activity were all inert. Hemolysis of coated SRC by RF and complement was inhibited by prior treatment of the complement source with chelating agents, hydrazine, cobra venom factor, specific antisera to C1q, CR, C5, C6, or C8, or by heating at 56 degrees C for 30 min. Purified radiolabeled C4, C3, and C8 included in the complement source were bound to hemolysed SRC in direct proportion to the degree of hemolysis. These data indicate that polyclonal IgM-RF fix and activate complement via the classic pathway. The system described for assessing complement fixation by isolated RF is readily adaptable to use with whole human serum. PMID:1078825

  17. Bimolecular fluorescence complementation.

    PubMed

    Wong, Katy A; O'Bryan, John P

    2011-01-01

    Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1). A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and Förster resonance energy transfer (FRET). For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET

  18. Selectivity of C3-opsonin targeted complement inhibitors: A distinct advantage in the protection of erythrocytes from paroxysmal nocturnal hemoglobinuria patients.

    PubMed

    Schmidt, Christoph Q; Harder, Markus J; Nichols, Eva-Maria; Hebecker, Mario; Anliker, Markus; Höchsmann, Britta; Simmet, Thomas; Csincsi, Ádám I; Uzonyi, Barbara; Pappworth, Isabel Y; Ricklin, Daniel; Lambris, John D; Schrezenmeier, Hubert; Józsi, Mihály; Marchbank, Kevin J

    2016-04-01

    Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated cell lysis due to deficiency of GPI-anchored complement regulators. Blockage of the lytic pathway by eculizumab is the only available therapy for PNH patients and shows remarkable benefits, but regularly yields PNH erythrocytes opsonized with fragments of complement protein C3, rendering such erythrocytes prone to extravascular hemolysis. This effect is associated with insufficient responsiveness seen in a subgroup of PNH patients. Novel C3-opsonin targeted complement inhibitors act earlier in the cascade, at the level of activated C3 and are engineered from parts of the natural complement regulator Factor H (FH) or complement receptor 2 (CR2). This inhibitor class comprises three variants of "miniFH" and the clinically developed "FH-CR2" fusion-protein (TT30). We show that the approach of FH-CR2 to target C3-opsonins was more efficient in preventing complement activation induced by foreign surfaces, whereas the miniFH variants were substantially more active in controlling complement on PNH erythrocytes. Subtle differences were noted in the ability of each version of miniFH to protect human PNH cells. Importantly, miniFH and FH-CR2 interfered only minimally with complement-mediated serum killing of bacteria when compared to untargeted inhibition of all complement pathways by eculizumab. Thus, the molecular design of each C3-opsonin targeted complement inhibitor determines its potency in respect to the nature of the activator/surface providing potential functionality in PNH. PMID:26792457

  19. N-truncation and pyroglutaminylation enhances the opsonizing capacity of Aβ-peptides and facilitates phagocytosis by macrophages and microglia.

    PubMed

    Condic, Mateja; Oberstein, Timo Jan; Herrmann, Martin; Reimann, Mareike Carola; Kornhuber, Johannes; Maler, Juan Manuel; Spitzer, Philipp

    2014-10-01

    Abnormal accumulations of amyloid-β (Aβ)-peptides are one of the pathological hallmarks of Alzheimer's disease (AD). The precursor of the Aβ-peptides, the amyloid precursor protein (APP), is also found in peripheral blood cells, but its function in these cells remains elusive. We previously observed that mononuclear phagocytes release Aβ-peptides during activation and phagocytosis, suggesting a physiologic role in inflammatory processes. Here, we show that supplementing the media with soluble N-terminally truncated Aβ(2-40) and Aβ(2-42) as well as Aβ(1-42) induced the phagocytosis of polystyrene particles (PSPs) by primary human monocytes. If the PSPs were pre-incubated with Aβ-peptides, phagocytosis was induced by all tested Aβ-peptide species. N-terminally truncated Aβ(x-42) induced the phagocytosis of PSPs significantly more effectively than did Aβ(x-40). Similarly, the phagocytosis of Escherichia coli by GM-CSF- and M-CSF-elicited macrophages as well as microglia was particularly facilitated by pre-incubation with N-terminally truncated Aβ(x-42). The proinflammatory polarization of monocytes was indicated by the reduced MSRI expression and IL-10 secretion after phagocytosis of PSPs coated with Aβ(1-42), Aβ(2-42) and Aβ(3p-42). Polarization of the macrophages by GM-CSF reduced the phagocytic activity, but it did not affect the capabilities of Aβ-peptides to opsonize prey. Taken together, Aβ-peptides support phagocytosis as soluble factors and act as opsonins. Differential effects among the Aβ-peptide variants point to distinct mechanisms of interaction among monocytes/macrophages, prey and Aβ-peptides. A proinflammatory polarization induced by the phagocytosis of Aβ-peptide coated particles may provide a model for the chronic inflammatory reaction and sustained plaque deposition in AD.

  20. Complement-dependent cytotoxicity crossmatch.

    PubMed

    Peña, Jeremy Ryan; Fitzpatrick, Donna; Saidman, Susan L

    2013-01-01

    The complement-dependent cytotoxic crossmatch is an informative test that detects alloantibodies in pre- and post-transplant patients, which may dictate clinical management of transplant patients. While challenging to perform, the cytotoxic crossmatch represents the only assay that provides direct evidence for the presence of potentially pathologic (i.e., cytotoxic) alloantibodies. The cytotoxic crossmatch combines patient (recipient) serum and donor cells. If donor-reactive alloantibodies are present in patient serum, these antibodies can bind donor cells. Antibody-antigen complexes, in turn, can activate the complement cascade, leading to complement-mediated cytotoxicity. Two commonly performed cytotoxic crossmatches, using donor lymphocytes as target cells, are described.

  1. Heat differentiated complement factor profiling.

    PubMed

    Hamsten, Carl; Skattum, Lillemor; Truedsson, Lennart; von Döbeln, Ulrika; Uhlén, Mathias; Schwenk, Jochen M; Hammarström, Lennart; Nilsson, Peter; Neiman, Maja

    2015-08-01

    Complement components and their cascade of reactions are important defense mechanisms within both innate and adaptive immunity. Many complement deficient patients still remain undiagnosed because of a lack of high throughput screening tools. Aiming towards neonatal proteome screening for immunodeficiencies, we used a multiplex profiling approach with antibody bead arrays to measure 9 complement proteins in serum and dried blood spots. Several complement components have been described as heat sensitive, thus their heat-dependent detectability was investigated. Using sera from 16 patients with complement deficiencies and 23 controls, we confirmed that the proteins C1q, C2, C3, C6, C9 and factor H were positively affected by heating, thus the identification of deficient patients was improved when preheating samples. Measurements of C7, C8 and factor I were negatively affected by heating and non-heated samples should be used in analysis of these components. In addition, a proof of concept study demonstrated the feasibility of labeling eluates from dried blood spots to perform a subsequent correct classification of C2-deficiencies. Our study demonstrates the potential of using multiplexed single binder assays for screening of complement components that open possibilities to expand such analysis to other forms of deficiencies.

  2. Two African viruses serologically and morphologically related to rabies virus.

    PubMed

    Shope, R E; Murphy, F A; Harrison, A K; Causey, O R; Kemp, G E; Simpson, D I; Moore, D L

    1970-11-01

    Lagos bat virus and an isolate from shrews (IbAn 27377), both from Nigeria, were found to be bullet-shaped and to mature intracytoplasmically in association with a distinct matrix. They were related to, but readily distinguishable from, rabies virus and each other by complement fixation and neutralization tests. The three viruses, including rabies, form a subgrouping within the rhabdoviruses. PMID:5530013

  3. The Tyrosine Kinase Pyk2 Contributes to Complement-Mediated Phagocytosis in Murine Macrophages.

    PubMed

    Paone, Christoph; Rodrigues, Natalie; Ittner, Ella; Santos, Carina; Buntru, Alexander; Hauck, Christof R

    2016-01-01

    Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family and is mainly expressed in neuronal and hematopoietic cells. As FAK family members are involved in signaling connections downstream of integrins, we studied the role of Pyk2 in complement-receptor 3 (CR3, also known as Mac-1, integrin αMβ2, CD11b/CD18)-mediated phagocytosis, a key process in innate immunity. Using 3 independent approaches, we observed that Pyk2 contributes to CR3-dependent phagocytosis by RAW 264.7 macrophages, but is dispensable for Fcγ receptor (FcγR)-mediated uptake. Reduction of Pyk2 expression levels via siRNA, the pharmacological inhibition of Pyk2 kinase activity as well as macrophage treatment with a cell permeable TAT fusion protein containing the C-terminus of Pyk2 (TAT-PRNK) significantly impaired CR3-mediated phagocytosis without affecting FcγR-mediated uptake. In addition, Pyk2 was strongly recruited to complement opsonized Escherichia coli and the pharmacological inhibition of Pyk2 significantly decreased uptake of the bacteria. Finally, CRISPR/Cas-mediated disruption of the pyk2 gene in RAW 264.7 macrophages confirmed the role of this protein tyrosine kinase in CR3-mediated phagocytosis. Together, our data demonstrate that Pyk2 selectively contributes to the coordination of phagocytosis-promoting signals downstream of CR3, but is dispensable for FcγR-mediated phagocytosis.

  4. Distinct localization of the complement C5b-9 complex on Gram-positive bacteria.

    PubMed

    Berends, Evelien T M; Dekkers, Johanna F; Nijland, Reindert; Kuipers, Annemarie; Soppe, Jasper A; van Strijp, Jos A G; Rooijakkers, Suzan H M

    2013-12-01

    The plasma proteins of the complement system fulfil important immune defence functions, including opsonization of bacteria for phagocytosis, generation of chemo-attractants and direct bacterial killing via the Membrane Attack Complex (MAC or C5b-9). The MAC is comprised of C5b, C6, C7, C8, and multiple copies of C9 that generate lytic pores in cellular membranes. Gram-positive bacteria are protected from MAC-dependent lysis by their thick peptidoglycan layer. Paradoxically, several Gram-positive pathogens secrete small proteins that inhibit C5b-9 formation. In this study, we found that complement activation on Gram-positive bacteria in serum results in specific surface deposition of C5b-9 complexes. Immunoblotting revealed that C9 occurs in both monomeric and polymeric (SDS-stable) forms, indicating the presence of ring-structured C5b-9. Surprisingly, confocal microscopy demonstrated that C5b-9 deposition occurs at specialized regions on the bacterial cell. On Streptococcus pyogenes, C5b-9 deposits near the division septum whereas on Bacillus subtilis the complex is located at the poles. This is in contrast to C3b deposition, which occurs randomly on the bacterial surface. Altogether, these results show a previously unrecognized interaction between the C5b-9 complex and Gram-positive bacteria, which might ultimately lead to a new model of MAC assembly and functioning.

  5. Complement component 3 binding to Haemophilus influenzae type b in the presence of anticapsular and anti-outer membrane antibodies.

    PubMed Central

    Hetherington, S V; Patrick, C C

    1992-01-01

    Antibodies directed against the capsular polysaccharide (polyribosyl ribitol phosphate [PRP]) or the outer membrane proteins (OMP) of Haemophilus influenzae type b (Hib) promote bactericidal activity, complement 3 (C3) binding, and ingestion by phagocytic cells. To assess the relative contribution of anti-OMP to host defense against Hib, we compared the opsonic activities of anti-PRP and anti-OMP as reflected by the amounts of C3 bound to the bacterial surface. Immunoglobulin G (IgG) fractions containing either anti-PRP or anti-OMP were incubated with Hib in the presence of a C5-deficient complement source. C3, total IgG, and IgG subclasses bound to the bacteria were quantified by enzyme-linked immunosorbent assay. The maximum amount of C3 which could be bound to Hib was greater in the presence of anti-PRP than in the presence of anti-OMP. Also, except at low IgG concentrations, the rate of increase in bound C3 as a function of increasing IgG concentration was greater for anti-PRP than for anti-OMP. Hib-bound anti-OMP consisted primarily of IgG1 and IgG3, whereas bound anti-PRP was primarily IgG1 and IgG2. Thus, the potential for C3 binding to Hib is greater in the presence of anti-PRP than in the presence of anti-OMP, probably because of the larger number of binding sites available to the former. Nonetheless, OMP appear to provide important targets for opsonic antibody and would be logical components of a PRP-conjugate vaccine or may be efficacious as vaccines against nontypeable H. influenzae. Images PMID:1729183

  6. Intracellular sensing of complement C3 activates cell autonomous immunity

    PubMed Central

    Tam, Jerry C.H.; Bidgood, Susanna R.; McEwan, William A.; James, Leo C.

    2014-01-01

    Pathogens traverse multiple barriers during infection including cell membranes. Here we show that during this transition pathogens carry covalently attached complement C3 into the cell, triggering immediate signalling and effector responses. Sensing of C3 in the cytosol activates MAVS-dependent signalling cascades and induces proinflammatory cytokine secretion. C3 also flags viruses for rapid proteasomal degradation, thereby preventing their replication. This system can detect both viral and bacterial pathogens but is antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral Rupintrivir inhibits 3C protease and prevents C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol. PMID:25190799

  7. Complement activation on platelets correlates with a decrease in circulating immature platelets in patients with immune thrombocytopenic purpura.

    PubMed

    Peerschke, Ellinor I B; Andemariam, Biree; Yin, Wei; Bussel, James B

    2010-02-01

    The role of the complement system in immune thrombocytopenic purpura (ITP) is not well defined. We examined plasma from 79 patients with ITP, 50 healthy volunteers, and 25 patients with non-immune mediated thrombocytopenia, to investigate their complement activation/fixation capacity (CAC) on immobilized heterologous platelets. Enhanced CAC was found in 46 plasma samples (59%) from patients with ITP, but no samples from patients with non-immune mediated thrombocytopenia. Plasma from healthy volunteers was used for comparison. In patients with ITP, an enhanced plasma CAC was associated with a decreased circulating absolute immature platelet fraction (A-IPF) (<15 x 10(9)/l) (P = 0.027) and thrombocytopenia (platelet count < 100 x 10(9)/l) (P = 0.024). The positive predictive value of an enhanced CAC for a low A-IPF was 93%, with a specificity of 77%. The specificity and positive predictive values increased to 100% when plasma CAC was defined strictly by enhanced C1q and/or C4d deposition on test platelets. Although no statistically significant correlation emerged between CAC and response to different pharmacological therapies, an enhanced response to splenectomy was noted (P < 0.063). Thus, complement fixation may contribute to the thrombocytopenia of ITP by enhancing clearance of opsonized platelets from the circulation, and/or directly damaging platelets and megakaryocytes. PMID:19925495

  8. Improvisation: A Complement to Curriculum

    ERIC Educational Resources Information Center

    Ronald, Green A.

    2006-01-01

    With the growth of standardized assessment benchmarks in both the public and private paradigms, testing performance matters to institutions more than ever. In an attempt to take as many hindering variables out of this process, such as test anxiety, socioeconomic influences, and latency in cognition, Improvisation: A Complement to Curriculum seeks…

  9. Allele mining in the pepper gene pool provided new complementation effects between pvr2-eIF4E and pvr6-eIF(iso)4E alleles for resistance to pepper veinal mottle virus.

    PubMed

    Rubio, Manuel; Nicolaï, Maryse; Caranta, Carole; Palloix, Alain

    2009-11-01

    Molecular cloning of recessive resistance genes to potyviruses in a large range of host species identified the eukaryotic translation initiation factor 4E (eIF4E) as an essential determinant in the outcome of potyvirus infection. Resistance results from a few amino acid changes in the eIF4E protein encoded by the recessive resistance allele that disrupt the direct interaction with the potyviral protein VPg. In plants, several loci encode two protein subfamilies, eIF4E and eIF(iso)4E. While most eIF4E-mediated resistance to potyviruses depends on mutations in a single eIF4E protein, simultaneous mutations in eIF4E (corresponding to the pvr2 locus) and eIF(iso)4E (corresponding to the pvr6 locus) are required to prevent pepper veinal mottle virus (PVMV) infection in pepper. We used this model to look for additional alleles at the pvr2-eIF4E locus that result in resistance when combined with the pvr6-eIF(iso)4E resistant allele. Among the 12 pvr2-eIF4E resistance alleles sequenced in the pepper gene pool, three were shown to have a complementary effect with pvr6-eIF(iso)4E for resistance. Two amino acid changes were exclusively shared by these three alleles and were systematically associated with a second amino acid change, suggesting that these substitutions are associated with resistance expression. The availability of new resistant allele combinations increases the possibility for the durable deployment of resistance against this pepper virus which is prevalent in Africa.

  10. Microparticles Provide a Novel Biomarker To Predict Severe Clinical Outcomes of Dengue Virus Infection

    PubMed Central

    Punyadee, Nuntaya; Mairiang, Dumrong; Thiemmeca, Somchai; Komoltri, Chulaluk; Pan-ngum, Wirichada; Chomanee, Nusara; Charngkaew, Komgrid; Tangthawornchaikul, Nattaya; Limpitikul, Wannee; Vasanawathana, Sirijitt; Malasit, Prida

    2014-01-01

    ABSTRACT Shedding of microparticles (MPs) is a consequence of apoptotic cell death and cellular activation. Low levels of circulating MPs in blood help maintain homeostasis, whereas increased MP generation is linked to many pathological conditions. Herein, we investigated the role of MPs in dengue virus (DENV) infection. Infection of various susceptible cells by DENV led to apoptotic death and MP release. These MPs harbored a viral envelope protein and a nonstructural protein 1 (NS1) on their surfaces. Ex vivo analysis of clinical specimens from patients with infections of different degrees of severity at multiple time points revealed that MPs generated from erythrocytes and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of red blood cell-derived MPs (RMPs) directly correlated with DENV disease severity, whereas a significant decrease in platelet-derived MPs was associated with a bleeding tendency. Removal by mononuclear cells of complement-opsonized NS1–anti-NS1 immune complexes bound to erythrocytes via complement receptor type 1 triggered MP shedding in vitro, a process that could explain the increased levels of RMPs in severe dengue. These findings point to the multiple roles of MPs in dengue pathogenesis. They offer a potential novel biomarker candidate capable of differentiating dengue fever from the more serious dengue hemorrhagic fever. IMPORTANCE Dengue is the most important mosquito-transmitted viral disease in the world. No vaccines or specific treatments are available. Rapid diagnosis and immediate treatment are the keys to achieve a positive outcome. Dengue virus (DENV) infection, like some other medical conditions, changes the level and composition of microparticles (MPs), tiny bag-like structures which are normally present at low levels in the blood of healthy individuals. This study investigated how MPs in culture and patients' blood are changed in response to DENV infection. Infection of cells

  11. Influence of minor thermal injury on expression of complement receptor CR3 on human neutrophils.

    PubMed Central

    Nelson, R. D.; Hasslen, S. R.; Ahrenholz, D. H.; Haus, E.; Solem, L. D.

    1986-01-01

    Thermal injury is well known to inhibit functions of the circulating neutrophil related to its role in host defense against infection, but the mechanism(s) of this phenomenon are not fully understood. To gain further clues to these mechanisms, the authors have studied patients with thermal injury in terms of altered expression of neutrophil cell membrane receptors for the opsonic complement-derived ligand C3bi--complement receptor Type 3, or CR3. CR3 expression was selected for study because an increase in the number of receptors on the cell surface can be stimulated by products of complement activation known to accumulate after thermal injury and because of the role of CR3 in phagocytic and adherence functions of the neutrophil. Expression of CR3 was monitored semiquantitatively by flow cytometry with the use of a murine monoclonal antibody (OKM1) specific for an antigen (CD11) associated with this receptor. Patients evaluated were limited in this study to those with minor degrees of thermal injury (second-degree burn involving less than 20% of total body surface area) so that possible confounding effects of major injury and its complications could be eliminated. It was observed that patient neutrophil CR3 becomes significantly up-regulated during the first week, as early as 1 day after injury. The maximum level of expression of CR3 averaged greater than 150% (range, 70-314%) of the respective minimum level observed for each patient. The minimum levels of expression of CR3 on patient neutrophils, reached 11-37 days after injury for 7 of 8 patients, were comparable to the level of expression of CR3 on unstimulated control neutrophils. Such temporal up-regulation of patient neutrophil CR3 suggests the early generation of stimuli of CR3 mobilization in response to thermal injury. Increased numbers of CR3 on patient neutrophils may augment microbicidal function and enhance or inhibit delivery of cells to the burn site. PMID:3541642

  12. Identification of OprF as a Complement Component C3 Binding Acceptor Molecule on the Surface of Pseudomonas aeruginosa

    PubMed Central

    Mishra, Meenu; Ressler, Adam; Schlesinger, Larry S.

    2015-01-01

    Pseudomonas aeruginosa is a versatile opportunistic pathogen that can cause devastating persistent infections. Complement is a highly conserved pathway of the innate immune system, and its role in the first line of defense against pathogens is widely appreciated. One of the earliest events in the complement cascade is the conversion of C3 to C3a and C3b, the latter typically binds to one or more acceptor molecules on the pathogen surface. We previously demonstrated that complement C3b binding acceptors exist on the P. aeruginosa surface. In the current study, we utilized either C3 polyclonal or C3b monoclonal antibodies in a far-Western technique followed by mass spectroscopy to identify the C3b acceptor molecule(s) on the P. aeruginosa surface. Our data provide evidence that OprF (an outer membrane porin, highly conserved in the Pseudomonadaceae) binds C3b. An oprF-deficient P. aeruginosa strain exhibits reduced C3 deposition compared to the wild type. We observed reduced internalization of oprF-deficient bacteria by neutrophils after opsonization compared with wild-type P. aeruginosa. Heterologous expression of OprF significantly enhanced C3b binding and increased serum-mediated bactericidal effects in complement-susceptible Escherichia coli. Furthermore, the predicted secondary structure of the C-terminal, surface-exposed region of OprF has high structural identity to the OmpA domain of several other Gram-negative bacteria, one of which is known to bind C3b. Therefore, these findings provide new insights into the biology of complement interactions with P. aeruginosa and other Gram-negative bacteria. PMID:25964476

  13. Emerging concepts in dengue pathogenesis: interplay between plasmablasts, platelets, and complement in triggering vasculopathy.

    PubMed

    Nascimento, Eduardo J M; Hottz, Eugenio D; Garcia-Bates, Tatiana M; Bozza, Fernando; Marques, Ernesto T A; Barratt-Boyes, Simon M

    2014-01-01

    Dengue is a mosquito-borne disease caused by infection with dengue virus (DENV) that represents a serious and expanding global health threat. Most DENV infections are inapparent or produce mild and self-limiting illness; however a significant proportion results in severe disease characterized by vasculopathy and plasma leakage that may culminate in shock and death. The cause of dengue-associated vasculopathy is likely to be multifactorial but remains essentially unknown. Severe disease is manifest during a critical phase from 4 to 7 days after onset of symptoms, once the virus has disappeared from the circulation but before the peak of T-cell activation, suggesting that other factors mediate vasculopathy. Here, we present evidence for a combined role of plasmablasts, complement, and platelets in driving severe disease in DENV infection. Massive expansion of virus-specific plasmablasts peaks during the critical phase of infection, coincident with activation of complement and activation and depletion of platelets. We propose a step-wise model in which virus-specific antibodies produced by plasmablasts form immune complexes, leading to activation of complement and release of vasoactive anaphylatoxins. Platelets become activated through binding of complement- and antibody-coated virus, as well as direct binding of virus to DC-SIGN, leading to the release of inflammatory microparticles and cytokines and sequestration of platelets in the microvasculature. We suggest that the combined effects of anaphylatoxins, inflammatory microparticles, and platelet sequestration serve as triggers of vasculopathy in severe dengue.

  14. Cigarette smoking complements the prognostic value of baseline plasma Epstein-Barr virus deoxyribonucleic acid in patients with nasopharyngeal carcinoma undergoing intensity-modulated radiation therapy: a large-scale retrospective cohort study.

    PubMed

    Lv, Jia-Wei; Chen, Yu-Pei; Zhou, Guan-Qun; Tang, Ling-Long; Mao, Yan-Ping; Li, Wen-Fei; Guo, Rui; Lin, Ai-Hua; Ma, Jun; Sun, Ying

    2016-03-29

    We evaluated the combined prognostic value of cigarette smoking and baseline plasma Epstein-Barr virus deoxyribonucleic acid (EBV DNA) in patients with nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiation therapy (IMRT). Of consecutive patients, 1501 with complete data were eligible for retrospective analysis. Smoking index (SI; cigarette packs per day times smoking duration [years]), was used to evaluate the cumulative effect of smoking. Primary end-point was overall survival (OS); progression-free survival (PFS), distant metastasis-free survival (DMFS) and locoregional relapse-free survival (LRFS) were secondary end-points. Both cigarette smoking and baseline plasma EBV DNA load were associated with poorer survival (P <0.001). Patients were divided into four groups: low EBV DNA and light smoker (LL), low EBV DNA and heavy smoker (LH), high EBV DNA and light smoker (HL), and high EBV DNA and heavy smoker (HH). The respective 5-year survival rates were: OS (93.1%, 87.2%, 82.9%, and 76.3%, P<0.001), PFS (87.0%, 84.0%, 73.9%, and 64.6%, P<0.001), DMFS (94.1%, 92.1%, 82.4%, and72.5%, P<0.001), and LRFS (92.8%, 92.4%, 88.7%, and 84.0%, P=0.012).OS and PFS were significantly different between the LH and HL groups and HL and HH groups, but not LL and LH groups (pairwise comparisons). The combined risk stratification remained an independent prognostic factor for all endpoints (all Ptrend<0.001; multivariate analysis). Both cigarette smoking and baseline plasma EBV DNA were independent prognostic factors for survival outcomes. Combined interpretation of EBV DNA with smoking led to the refinement of the risks stratification for patient subsets, especially with improved risk discrimination in patients with high baseline plasma EBV DNA. PMID:26919237

  15. CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

    PubMed

    Amash, Alaa; Wang, Lin; Wang, Yawen; Bhakta, Varsha; Fairn, Gregory D; Hou, Ming; Peng, Jun; Sheffield, William P; Lazarus, Alan H

    2016-04-15

    Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions.

  16. CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

    PubMed

    Amash, Alaa; Wang, Lin; Wang, Yawen; Bhakta, Varsha; Fairn, Gregory D; Hou, Ming; Peng, Jun; Sheffield, William P; Lazarus, Alan H

    2016-04-15

    Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions. PMID:26944929

  17. The Complement System and Adverse Pregnancy Outcomes

    PubMed Central

    Regal, Jean F.; Gilbert, Jeffrey S.; Burwick, Richard M.

    2015-01-01

    Adverse pregnancy outcomes significantly contribute to morbidity and mortality for mother and child, with lifelong health consequences for both. The innate and adaptive immune system must be regulated to insure survival of the feta allograft, and the complement system is no exception. An intact complement system optimizes placental development and function and is essential to maintain host defense and fetal survival. Complement regulation is apparent at the placental interface from early pregnancy with some degree of complement activation occurring normally throughout gestation. However, a number of pregnancy complications including early pregnancy loss, fetal growth restriction, hypertensive disorders of pregnancy and preterm birth are associated with excessive or misdirected complement activation, and are more frequent in women with inherited or acquired complement system disorders or complement gene mutations. Clinical studies employing complement biomarkers in plasma and urine implicate dysregulated complement activation in components of each of the adverse pregnancy outcomes. In addition, mechanistic studies in rat and mouse models of adverse pregnancy outcomes address the complement pathways or activation products of importance and allow critical analysis of the pathophysiology. Targeted complement therapeutics are already in use to control adverse pregnancy outcomes in select situations. A clearer understanding of the role of the complement system in both normal pregnancy and complicated or failed pregnancy will allow a rational approach to future therapeutic strategies for manipulating complement with the goal of mitigating adverse pregnancy outcomes, preserving host defense, and improving long term outcomes for both mother and child. PMID:25802092

  18. The complement system and adverse pregnancy outcomes.

    PubMed

    Regal, Jean F; Gilbert, Jeffrey S; Burwick, Richard M

    2015-09-01

    Adverse pregnancy outcomes significantly contribute to morbidity and mortality for mother and child, with lifelong health consequences for both. The innate and adaptive immune system must be regulated to insure survival of the fetal allograft, and the complement system is no exception. An intact complement system optimizes placental development and function and is essential to maintain host defense and fetal survival. Complement regulation is apparent at the placental interface from early pregnancy with some degree of complement activation occurring normally throughout gestation. However, a number of pregnancy complications including early pregnancy loss, fetal growth restriction, hypertensive disorders of pregnancy and preterm birth are associated with excessive or misdirected complement activation, and are more frequent in women with inherited or acquired complement system disorders or complement gene mutations. Clinical studies employing complement biomarkers in plasma and urine implicate dysregulated complement activation in components of each of the adverse pregnancy outcomes. In addition, mechanistic studies in rat and mouse models of adverse pregnancy outcomes address the complement pathways or activation products of importance and allow critical analysis of the pathophysiology. Targeted complement therapeutics are already in use to control adverse pregnancy outcomes in select situations. A clearer understanding of the role of the complement system in both normal pregnancy and complicated or failed pregnancy will allow a rational approach to future therapeutic strategies for manipulating complement with the goal of mitigating adverse pregnancy outcomes, preserving host defense, and improving long term outcomes for both mother and child.

  19. Complement deposition in glomerular diseases.

    PubMed

    di Belgiojoso, G B; Tarantino, A; Durante, A; Guerra, L

    1975-01-01

    Biopsies from 400 patients affected by glomerular diseases, both "primary" and secondary to systemic diseases, have been studied by immunofluorescence. Staining was performed for immunoglobulins fibrogen and C1q, C4, C3 and C3A. C1q, C4 and C3 were positive in a high percentage of cases in focal glomerulosclerosis, membranoproliferative glomerulonephritis, lupus nephritis and essential cryoglobulinaemia glomerulonephritis. C1q and C4 were very rarely present in focal proliferative glomerulonephritis and rheumatoid purpura glomerulonephritis. C3A was found frequently only in acute glomerulonephritis. Results are discussed with reference to their diagnostic value and to information about mechanisms of complement activation.

  20. Complement in the Homeostatic and Ischemic Brain

    PubMed Central

    Alawieh, Ali; Elvington, Andrew; Tomlinson, Stephen

    2015-01-01

    The complement system is a component of the immune system involved in both recognition and response to pathogens, and it is implicated in an increasing number of homeostatic and disease processes. It is well documented that reperfusion of ischemic tissue results in complement activation and an inflammatory response that causes post-reperfusion injury. This occurs following cerebral ischemia and reperfusion and triggers secondary damage that extends beyond the initial infarcted area, an outcome that has rationalized the use of complement inhibitors as candidate therapeutics after stroke. In the central nervous system, however, recent studies have revealed that complement also has essential roles in synaptic pruning, neurogenesis, and neuronal migration. In the context of recovery after stroke, these apparent divergent functions of complement may account for findings that the protective effect of complement inhibition in the acute phase after stroke is not always maintained in the subacute and chronic phases. The development of effective stroke therapies based on modulation of the complement system will require a detailed understanding of complement-dependent processes in both early neurodegenerative events and delayed neuro-reparatory processes. Here, we review the role of complement in normal brain physiology, the events initiating complement activation after cerebral ischemia-reperfusion injury, and the contribution of complement to both injury and recovery. We also discuss how the design of future experiments may better characterize the dual role of complement in recovery after ischemic stroke. PMID:26322048

  1. Complement in Lupus Nephritis: New Perspectives

    PubMed Central

    Bao, Lihua; Cunningham, Patrick N.; Quigg, Richard J.

    2015-01-01

    Background Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to self-antigens, the production of autoantibodies and deposition of complement-fixing immune complexes (ICs) in injured tissues. SLE is characterized by a wide range of clinical manifestations and targeted organs, with lupus nephritis being one of the most serious complications. The complement system consists of three pathways and is tightly controlled by a set of regulatory proteins to prevent injudicious complement activation on host tissue. The involvement of the complement system in the pathogenesis of SLE is well accepted; yet, its exact role is still not clear. Summary Complement plays dual roles in the pathogenesis of SLE. On the one hand, the complement system appears to have protective features in that hereditary homozygous deficiencies of classical pathway components, such as C1q and C4, are associated with an increased risk for SLE. On the other hand, IC-mediated activation of complement in affected tissues is clearly evident in both experimental and human SLE along with pathological features that are logical consequences of complement activation. Studies in genetically altered mice have shown that lack of complement inhibitors, such as complement factor H (CFH) or decay-accelerating factor (DAF) accelerates the development of experimental lupus nephritis, while treatment with recombinant protein inhibitors, such as Crry-Ig, CR2-Crry, CR2-DAF and CR2-CFH, ameliorates the disease development. Complement-targeted drugs, including soluble complement receptor 1 (TP10), C1 esterase inhibitor and a monoclonal anti-C5 antibody (eculizumab), have been shown to inhibit complement safely, and are now being investigated in a variety of clinical conditions. Key Messages SLE is an autoimmune disorder which targets multiple systems. Complement is centrally involved and plays dual roles in the pathogenesis of SLE. Studies from experimental lupus models and clinical

  2. Dynamics of interaction between complement-fixing antibody/dsDNA immune complexes and erythrocytes. In vitro studies and potential general applications to clinical immune complex testing

    SciTech Connect

    Taylor, R.P.; Horgan, C.; Hooper, M.; Burge, J.

    1985-01-01

    Soluble antibody//sub 3/H-double-stranded PM2 DNA (dsDNA) immune complexes were briefly opsonized with complement and then allowed to bind to human erythrocytes (via complement receptors). The cells were washed and subsequently a volume of autologous blood in a variety of media was added, and the release of the bound immune complexes from the erythrocytes was studied as a function of temperature and time. After 1-2 h, the majority of the bound immune complexes were not released into the serum during blood clotting at either 37 degrees C or room temperature, but there was a considerably greater release of the immune complexes into the plasma of blood that was anticoagulated with EDTA. Similar results were obtained using various conditions of opsonization and also using complexes that contained lower molecular weight dsDNA. Thus, the kinetics of release of these antibody/dsDNA immune complexes differed substantially from the kinetics of release of antibody/bovine serum albumin complexes that was reported by others. Studies using the solution phase C1q immune complex binding assay confirmed that in approximately half of the SLE samples that were positive for immune complexes, there was a significantly higher level of detectable immune complexes in plasma vs. serum. Freshly drawn erythrocytes from some SLE patients exhibiting this plasma/serum discrepancy had IgG antigen on their surface that was released by incubation in EDTA plasma. Thus, the higher levels of immune complexes observed in EDTA plasma vs. serum using the C1q assay may often reflect the existence of immune complexes circulating in vivo bound to erythrocytes.

  3. Interaction of non-human primate complement and antibodies with hypermucoviscous Klebsiella pneumoniae.

    PubMed

    Soto, Esteban; Marchi, Sylvia; Beierschmitt, Amy; Kearney, Michael; Francis, Stewart; VanNess, Kimberly; Vandenplas, Michel; Thrall, MaryAnna; Palmour, Roberta

    2016-01-01

    Emergent hypermucoviscosity (HMV) phenotypes of Klebsiella pneumoniae have been associated with increased invasiveness and pathogenicity in primates. In this study, we investigated the interaction of African green monkeys (AGM) (Chlorocebus aethiops sabaeus) complement and antibody with HMV and non-HMV isolates as in vitro models of primate infection. Significantly greater survival of HMV isolates was evident after incubation in normal serum or whole blood (p < 0.05) of AGM donors when compared to non-HMV strains. Greater survival of HMV strains (p < 0.05) was found after incubation in whole blood and serum from seropositive donors when compared to seronegative donor samples. Additionally, significantly greater amounts of K. pneumoniae were phagocytozed by AGM leukocytes when complement was active (p < 0.05), but no difference in uptake was observed when serum from seropositive or seronegative animals was used in challenged cells utilizing flow cytometry. Results demonstrate that interaction of cellular and humoral immune elements play a role in the in vitro killing of K. pneumoniae, particularly HMV isolates. Neither AGM serum, nor washed whole blood effectively killed HMV isolates; however, assays using heparinized whole blood of seronegative donors significantly reduced viability of HMV and non-HMV strains. The lack of bacterial killing observed in seropositive donors treatments could be at least partially associated with low IgG2 present in these animals. A better understanding of the pathogenesis of klebsiellosis in primates and host immune response is necessary to identify surface molecules that can induce both opsonizing and bactericidal antibody facilitating killing of Klebsiella, and the development of vaccines in human and animals. PMID:26951091

  4. Interaction of non-human primate complement and antibodies with hypermucoviscous Klebsiella pneumoniae.

    PubMed

    Soto, Esteban; Marchi, Sylvia; Beierschmitt, Amy; Kearney, Michael; Francis, Stewart; VanNess, Kimberly; Vandenplas, Michel; Thrall, MaryAnna; Palmour, Roberta

    2016-03-08

    Emergent hypermucoviscosity (HMV) phenotypes of Klebsiella pneumoniae have been associated with increased invasiveness and pathogenicity in primates. In this study, we investigated the interaction of African green monkeys (AGM) (Chlorocebus aethiops sabaeus) complement and antibody with HMV and non-HMV isolates as in vitro models of primate infection. Significantly greater survival of HMV isolates was evident after incubation in normal serum or whole blood (p < 0.05) of AGM donors when compared to non-HMV strains. Greater survival of HMV strains (p < 0.05) was found after incubation in whole blood and serum from seropositive donors when compared to seronegative donor samples. Additionally, significantly greater amounts of K. pneumoniae were phagocytozed by AGM leukocytes when complement was active (p < 0.05), but no difference in uptake was observed when serum from seropositive or seronegative animals was used in challenged cells utilizing flow cytometry. Results demonstrate that interaction of cellular and humoral immune elements play a role in the in vitro killing of K. pneumoniae, particularly HMV isolates. Neither AGM serum, nor washed whole blood effectively killed HMV isolates; however, assays using heparinized whole blood of seronegative donors significantly reduced viability of HMV and non-HMV strains. The lack of bacterial killing observed in seropositive donors treatments could be at least partially associated with low IgG2 present in these animals. A better understanding of the pathogenesis of klebsiellosis in primates and host immune response is necessary to identify surface molecules that can induce both opsonizing and bactericidal antibody facilitating killing of Klebsiella, and the development of vaccines in human and animals.

  5. Complement the hemostatic system: an intimate relationship.

    PubMed

    Weitz, Ilene Ceil

    2014-05-01

    The complement system is important part of our innate immune system and interacts directly with the hemostatic system. Disorders of complement activation or dysregulation resulting in excess complement generation, such as Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical Hemolytic uremic Syndrome (aHUS) and antiphospholipid syndrome (APLS) have been associated with significant thrombophilia. Terminal Complement (C5b-9) deposition on endothelial and tumor cell membranes has also been reported in a variety of cancer. Recent developments in complement inhibition have given us new insights into the mechanism of thrombosis in these disorders.

  6. The Semantics of Complementation in English: A Cognitive Semantic Account of Two English Complement Constructions

    ERIC Educational Resources Information Center

    Smith, Michael B.

    2009-01-01

    Studies on complementation in English and other languages have traditionally focused on syntactic issues, most notably on the constituent structures of different complement types. As a result, they have neglected the role of meaning in the choice of different complements. This paper investigates the semantics of complementation within the…

  7. Role of complement in experiment silicosis

    SciTech Connect

    Callis, A.H.; Sohnle, P.G.; Mandel, G.S.; Mandel, N.S.

    1986-08-01

    The role of the complement system in the pathogenesis of crystal-induced pulmonary inflammation and fibrosis was evaluated using a mouse model of silicosis and congenitally complement-deficient mice. Mice lacking the fifth component of complement (B10.D2/o) were compared to C5-sufficient animals (B10.D2/n) for pulmonary changes following intratracheal instillation of silica crystals. Complement-deficient mice demonstrated a significant reduction compared to complement-sufficient mice in both cell number and protein content of lung lavage fluid throughout the 12 weeks following silica exposure. Lung hydroxyproline content (indicative of collagen deposition) was equivalent for both strains and significantly higher than controls at all times points following silica instillation. Moreover, studies in vitro have shown that silica crystals are capable of activating complement via the alternative pathway. These studies indicate that the complement system may be responsible for some of the pulmonary inflammation, but not fibrosis elicited by silica exposure.

  8. Meningococcal disease and the complement system

    PubMed Central

    Lewis, Lisa A; Ram, Sanjay

    2014-01-01

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

  9. The complement system in inflammatory bowel disease.

    PubMed

    Jain, Umang; Otley, Anthony R; Van Limbergen, Johan; Stadnyk, Andrew W

    2014-09-01

    Complement is well appreciated to be a potent innate immune defense against microbes and is important in the housekeeping act of removal of apoptotic and effete cells. It is also understood that hyperactivation of complement, or the lack of regulators, may underlie chronic inflammatory diseases. A pipeline of products to intervene in complement activation, some already in clinical use, is being studied in various chronic inflammatory diseases. To date, the role of complement in inflammatory bowel disease has not received a lot of research interest. Novel genetically modified laboratory animals and experiments using antagonists to complement effector molecules have kindled important research observations implicating the complement system in inflammatory bowel disease pathogenesis. We review the evidence base for the role and potential therapeutic manipulation of the complement cascade in inflammatory bowel disease.

  10. Overview of Complement Activation and Regulation

    PubMed Central

    Noris, Marina; Remuzzi, Giuseppe

    2013-01-01

    Summary Complement is an important component of the innate immune system that is crucial for defense from microbial infections and for clearance of immune complexes and injured cells. In normal conditions complement is tightly controlled by a number of fluid-phase and cell surface proteins to avoid injury to autologous tissues. When complement is hyperactivated, as occurs in autoimmune diseases or in subjects with dysfunctional regulatory proteins, it drives a severe inflammatory response in numerous organs. The kidney appears to be particularly vulnerable to complement-mediated inflammatory injury. Injury may derive from deposition of circulating active complement fragments in glomeruli, but complement locally produced and activated in the kidney also may have a role. Many kidney disorders have been linked to abnormal complement activation, including immune-complex–mediated glomerulonephritis and rare genetic kidney diseases, but also tubulointerstitial injury associated with progressive proteinuric diseases or ischemia-reperfusion. PMID:24161035

  11. Herbal complement inhibitors in the treatment of neuroinflammation: future strategy for neuroprotection.

    PubMed

    Kulkarni, Amod P; Kellaway, Laurie A; Kotwal, Girish J

    2005-11-01

    The upregulated complement system plays a damaging role in disorders of the central nervous system (CNS). The classical and alternate pathways are two major pathways activated in neuroinflammatory disorders such as Alzheimer's disease, multiple sclerosis, traumatic brain injury, spinal cord injury, HIV-associated dementia, Parkinson's disease, and mad cow disease. Failure of currently available anti-inflammatory agents, especially cyclooxygenase inhibitors, in offering significant neuroprotection in large epidemiologic clinical trials of CNS disorders suggests an urgent need for the development of new neuroprotective agents. The positive preclinical outcomes in treating CNS disorders by complement regulatory molecules, such as vaccinia virus complement control protein, suggest the possibility of using complement-inhibitory molecules as neuroprotective agents. Several active ingredients of herbal origin are found to have complement-inhibitory activity. These herbal ingredients along with other anti-inflammatory roles might be useful in treating neuroinflammation associated with CNS disorders. Active ingredients of herbal origin with complement inhibitory ingredients are summarized and classified according to their chemical nature and specificity towards the major pathways activating the complement system. The structure activity relationship of some specific examples is also discussed in this report. This information might be helpful in formulating a natural panacea against complement-mediated neuroinflammation. PMID:16387706

  12. Effects of complement activation on allograft injury

    PubMed Central

    Sheen, Joong Hyuk; Heeger, Peter S.

    2015-01-01

    Purpose of review To summarize the current knowledge regarding mechanisms linking the complement system to transplant injury, highlighting findings reported since 2013. Recent findings Building upon the documentation that complement activation is a pathogenic mediator of post-transplant ischemia-reperfusion (IR) injury, emerging evidence indicates blocking either the classical or lectin pathways attenuates IR injury in animal models. Immune cell-derived and locally activated complement, including intracellular C3 positively modulates allo-reactive T cell activation and expansion, while simultaneously inhibiting regulatory T cell induction and function, together promoting transplant rejection. While alloantibody-initiated complement activation directly injures target cells, complement-dependent signals activate endothelial cells to facilitate T cell dependent inflammation. Complement activation within allografts contributes to progressive chronic injury and fibrosis. Summary The complement cascade, traditionally considered relevant to transplantation only as an effector mechanism of antibody-initiated allograft injury, is now understood to damage the allograft through multiple mechanisms. Complement activation promotes post-transplant IR injury, formation and function of allo-antibody, differentiation and function of alloreactive T cells, and contributes to chronic progressive allograft failure. The recognition that complement impacts transplant injury at many levels provides a foundation for targeting complement as a therapy to prolong transplant survival and improve patient health. PMID:26132735

  13. Complement Activation and Inhibition in Wound Healing

    PubMed Central

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

    2012-01-01

    Complement activation is needed to restore tissue injury; however, inappropriate activation of complement, as seen in chronic wounds can cause cell death and enhance inflammation, thus contributing to further injury and impaired wound healing. Therefore, attenuation of complement activation by specific inhibitors is considered as an innovative wound care strategy. Currently, the effects of several complement inhibitors, for example, the C3 inhibitor compstatin and several C1 and C5 inhibitors, are under investigation in patients with complement-mediated diseases. Although (pre)clinical research into the effects of these complement inhibitors on wound healing is limited, available data indicate that reduction of complement activation can improve wound healing. Moreover, medicine may take advantage of safe and effective agents that are produced by various microorganisms, symbionts, for example, medicinal maggots, and plants to attenuate complement activation. To conclude, for the development of new wound care strategies, (pre)clinical studies into the roles of complement and the effects of application of complement inhibitors in wound healing are required. PMID:23346185

  14. The complement system in human cardiometabolic disease.

    PubMed

    Hertle, E; Stehouwer, C D A; van Greevenbroek, M M J

    2014-10-01

    The complement system has been implicated in obesity, fatty liver, diabetes and cardiovascular disease (CVD). Complement factors are produced in adipose tissue and appear to be involved in adipose tissue metabolism and local inflammation. Thereby complement links adipose tissue inflammation to systemic metabolic derangements, such as low-grade inflammation, insulin resistance and dyslipidaemia. Furthermore, complement has been implicated in pathophysiological mechanisms of diet- and alcohol induced liver damage, hyperglycaemia, endothelial dysfunction, atherosclerosis and fibrinolysis. In this review, we summarize current evidence on the role of the complement system in several processes of human cardiometabolic disease. C3 is the central component in complement activation, and has most widely been studied in humans. C3 concentrations are associated with insulin resistance, liver dysfunction, risk of the metabolic syndrome, type 2 diabetes and CVD. C3 can be activated by the classical, the lectin and the alternative pathway of complement activation; and downstream activation of C3 activates the terminal pathway. Complement may also be activated via extrinsic proteases of the coagulation, fibrinolysis and the kinin systems. Studies on the different complement activation pathways in human cardiometabolic disease are limited, but available evidence suggests that they may have distinct roles in processes underlying cardiometabolic disease. The lectin pathway appeared beneficial in some studies on type 2 diabetes and CVD, while factors of the classical and the alternative pathway were related to unfavourable cardiometabolic traits. The terminal complement pathway was also implicated in insulin resistance and liver disease, and appears to have a prominent role in acute and advanced CVD. The available human data suggest a complex and potentially causal role for the complement system in human cardiometabolic disease. Further, preferably longitudinal studies are needed to

  15. A Homology Model Reveals Novel Structural Features and an Immunodominant Surface Loop/Opsonic Target in the Treponema pallidum BamA Ortholog TP_0326

    PubMed Central

    Luthra, Amit; Anand, Arvind; Hawley, Kelly L.; LeDoyt, Morgan; La Vake, Carson J.; Caimano, Melissa J.; Cruz, Adriana R.; Salazar, Juan C.

    2015-01-01

    ABSTRACT We recently demonstrated that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses characteristic BamA bipartite topology. Herein, we used immunofluorescence analysis (IFA) to show that only the β-barrel domain of TP_0326 contains surface-exposed epitopes in intact T. pallidum. Using the solved structure of Neisseria gonorrhoeae BamA, we generated a homology model of full-length TP_0326. Although the model predicts a typical BamA fold, the β-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops, loop 4 (L4), L6, and L7, covers the barrel's extracellular opening. L4, the dome's major surface-accessible loop, contains mainly charged residues, while L7 is largely neutral and contains a polyserine tract in a two-tiered conformation. L6 projects into the β-barrel but lacks the VRGF/Y motif that anchors L6 within other BamAs. IFA and opsonophagocytosis assay revealed that L4 is surface exposed and an opsonic target. Consistent with B cell epitope predictions, immunoblotting and enzyme-linked immunosorbent assay (ELISA) confirmed that L4 is an immunodominant loop in T. pallidum-infected rabbits and humans with secondary syphilis. Antibody capture experiments using Escherichia coli expressing OM-localized TP_0326 as a T. pallidum surrogate further established the surface accessibility of L4. Lastly, we found that a naturally occurring substitution (Leu593 → Gln593) in the L4 sequences of T. pallidum strains affects antibody binding in sera from syphilitic patients. Ours is the first study to employ a “structure-to-pathogenesis” approach to map the surface topology of a T. pallidum OMP within the context of syphilitic infection. IMPORTANCE Previously, we reported that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses the bipartite topology characteristic of a BamA ortholog

  16. Antibody-directed complement-mediated cytotoxicity to hepatocytes from patients with chronic hepatitis B.

    PubMed Central

    Michalak, T I; Lau, J Y; McFarlane, B M; Alexander, G J; Eddleston, A L; Williams, R

    1995-01-01

    The susceptibility of hepatocytes from patients with chronic hepatitis B to complement-dependent cytotoxicity mediated by heterologous antibodies to hepatitis B virus core (anti-HBc) and surface (anti-HBs) antigens and to hepatic asialoglycoprotein receptor was examined using a microcytotoxicity assay. The anti-HBc-induced cytotoxicity was found to be markedly enhanced against hepatocytes isolated from patients with chronic active hepatitis (72.6 +/- 9.5% (mean +/- s.e.m.); n = 6) over that against hepatocytes from individuals with chronic persistent hepatitis or inactive liver cirrhosis (40.6 +/- 18.6%; n = 4) (P = 0.019). Overall, values of the anti-HBc-directed cytotoxicity were higher in patients positive for HBcAg in hepatocytes and seropositive for hepatitis B virus e antigen (HBeAg). Hepatocytotoxicity was also exerted by anti-HBs and anti-asialoglycoprotein receptor antibodies in the presence of complement, but it was not seemingly related to disease activity. These results indicate that hepatitis B virus core and surface antigens and asialoglycoprotein receptor at the hepatocyte surface can be recognized by antibodies, and raise the possibility that complement-dependent cytolysis may contribute to the injury of hepatitis B virus-infected hepatocytes. The data also suggest that liver cells of patients with severe chronic hepatitis might be more susceptible to anti-HBc antibody-directed complement-mediated cytotoxicity than those with inactive liver histology. PMID:7743660

  17. Complement diagnostics: concepts, indications, and practical guidelines.

    PubMed

    Nilsson, Bo; Ekdahl, Kristina Nilsson

    2012-01-01

    Aberrations in the complement system have been shown to be direct or indirect pathophysiological mechanisms in a number of diseases and pathological conditions such as autoimmune disease, infections, cancer, allogeneic and xenogeneic transplantation, and inflammation. Complement analyses have been performed on these conditions in both prospective and retrospective studies and significant differences have been found between groups of patients, but in many diseases, it has not been possible to make predictions for individual patients because of the lack of sensitivity and specificity of many of the assays used. The basic indications for serological diagnostic complement analysis today may be divided into three major categories: (a) acquired and inherited complement deficiencies; (b) disorders with complement activation; (c) inherited and acquired C1INH deficiencies. Here, we summarize indications, techniques, and interpretations for basic complement analyses and present an algorithm, which we follow in our routine laboratory.

  18. Role of Complement in Autoimmune Hemolytic Anemia.

    PubMed

    Berentsen, Sigbjørn

    2015-09-01

    The classification of autoimmune hemolytic anemias and the complement system are reviewed. In autoimmune hemolytic anemia of the warm antibody type, complement-mediated cell lysis is clinically relevant in a proportion of the patients but is hardly essential for hemolysis in most patients. Cold antibody-mediated autoimmune hemolytic anemias (primary cold agglutinin disease, secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria) are entirely complement-mediated disorders. In cold agglutinin disease, efficient therapies have been developed in order to target the pathogenic B-cell clone, but complement modulation remains promising in some clinical situations. No established therapy exists for secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria, and the possibility of therapeutic complement inhibition is interesting. Currently, complement modulation is not clinically documented in any autoimmune hemolytic anemia. The most relevant candidate drugs and possible target levels of action are discussed.

  19. 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

  20. Immune Protection of Retroviral Vectors Upon Molecular Painting with the Complement Regulatory Protein CD59.

    PubMed

    Heider, Susanne; Kleinberger, Sandra; Kochan, Feliks; Dangerfield, John A; Metzner, Christoph

    2016-07-01

    Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications. PMID:27170144

  1. Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent.

    PubMed Central

    Cywes, C; Hoppe, H C; Daffé, M; Ehlers, M R

    1997-01-01

    The choice of host cell receptor and the mechanism of binding (opsonic versus nonopsonic) may influence the intracellular fate of Mycobacterium tuberculosis. We have identified two substrains of M. tuberculosis H37Rv, designated H37Rv-CC and -HH, that differed in their modes of binding to complement receptor type 3 (CR3) expressed in transfected Chinese hamster ovary (CHO-Mac-1) cells: H37Rv-CC bound nonopsonically, whereas H37Rv-HH bound only after opsonization in fresh serum. H37Rv-CC also bound nonopsonically to untransfected CHO cells, whereas H37Rv-HH binding was enhanced by serum and was mediated by the 1D1 antigen, a bacterial adhesin previously identified as a polar phosphatidylinositol mannoside. H37Rv-CC and -HH had identical IS6110 DNA fingerprint patterns. Of five M. tuberculosis clinical isolates examined, four displayed the same binding phenotype as H37Rv-CC, as did the Erdman strain, whereas one isolate, as well as Mycobacterium smegmatis, behaved like H37Rv-HH. Nonopsonic binding of H37Rv-CC to CHO cell-expressed CR3 was apparently to the beta-glucan lectin site, as it was cation independent and inhibited by laminarin (seaweed beta-glucan) and N-acetylglucosamine; laminarin also inhibited the binding of H37Rv-CC to monocyte-derived macrophages. Further, binding of H37Rv-CC to CHO-Mac-1 cells was inhibited by prior agitation of bacteria with glass beads (which strips outer capsular polysaccharides) and by preincubation with amyloglucosidase, as well as by the presence of capsular D-glucan and D-mannan from M. tuberculosis Erdman, but not by Erdman D-arabino-D-mannan, yeast mannan, or capsular components from H37Rv-HH. Analysis of capsular carbohydrates revealed that H37Rv-CC expressed 5-fold more glucose and 2.5-fold more arabinose and mannose than H37Rv-HH. Flow cytometric detection of surface epitopes indicated that H37Rv-CC displayed twofold less surface-exposed phosphatidylinositol mannoside and bound complement C3 less efficiently than H37Rv

  2. Complement: an overview for the clinician.

    PubMed

    Varela, Juan Carlos; Tomlinson, Stephen

    2015-06-01

    The complement system is an essential component of the immune system. It is a highly integrative system and has a number of functions, including host defense, removal of injured cells and debris, modulation of metabolic and regenerative processes, and regulation of adaptive immunity. Complement is activated via different pathways and it is regulated tightly by several mechanisms to prevent host injury. Imbalance between complement activation and regulation can manifest in disease and injury to self. This article provides an outline of complement activation pathways, regulatory mechanisms, and normal physiologic functions of the system.

  3. Efficient internalization of mesoporous silica particles of different sizes by primary human macrophages without impairment of macrophage clearance of apoptotic or antibody-opsonized target cells

    SciTech Connect

    Witasp, Erika; Kupferschmidt, Natalia; Bengtsson, Linnea; Hultenby, Kjell; Smedman, Christian; Paulie, Staffan; Garcia-Bennett, Alfonso E.; Fadeel, Bengt

    2009-09-15

    Macrophage recognition and ingestion of apoptotic cell corpses, a process referred to as programmed cell clearance, is of considerable importance for the maintenance of tissue homeostasis and in the resolution of inflammation. Moreover, macrophages are the first line of defense against microorganisms and other foreign materials including particles. However, there is sparse information on the mode of uptake of engineered nanomaterials by primary macrophages. In this study, mesoporous silica particles with cubic pore geometries and covalently fluorescein-grafted particles were synthesized through a novel route, and their interactions with primary human monocyte-derived macrophages were assessed. Efficient and active internalization of mesoporous silica particles of different sizes was observed by transmission electron microscopic and flow cytometric analysis and studies using pharmacological inhibitors suggested that uptake occurred through a process of endocytosis. Moreover, uptake of silica particles was independent of serum factors. The silica particles with very high surface areas due to their porous structure did not impair cell viability or function of macrophages, including the ingestion of different classes of apoptotic or opsonized target cells. The current findings are relevant to the development of mesoporous materials for drug delivery and other biomedical applications.

  4. Controlling the complement system in inflammation.

    PubMed

    Kirschfink, M

    1997-12-01

    Inappropriate or excessive activation of the complement system can lead to harmful, potentially life-threatening consequences due to severe inflammatory tissue destruction. These consequences are clinically manifested in various disorders, including septic shock, multiple organ failure and hyperacute graft rejection. Genetic complement deficiencies or complement depletion have been proven to be beneficial in reducing tissue injury in a number of animal models of severe complement-dependent inflammation. It is therefore believed that therapeutic inhibition of complement is likely to arrest the process of certain diseases. Attempts to efficiently inhibit complement include the application of endogenous soluble complement inhibitors (C1-inhibitor, recombinant soluble complement receptor 1- rsCR1), the administration of antibodies, either blocking key proteins of the cascade reaction (e.g. C3, C5), neutralizing the action of the complement-derived anaphylatoxin C5a, or interfering with complement receptor 3 (CR3, CD18/11b)-mediated adhesion of inflammatory cells to the vascular endothelium. In addition, incorporation of membrane-bound complement regulators (DAF-CD55, MCP-CD46, CD59) has become possible by transfection of the correspondent cDNA into xenogeneic cells. Thereby, protection against complement-mediated inflammatory tissue damage could be achieved in various animal models of sepsis, myocardial as well as intestinal ischemia/reperfusion injury, adult respiratory distress syndrome, nephritis and graft rejection. Supported by results from first clinical trials, complement inhibition appears to be a suitable therapeutic approach to control inflammation. Current strategies to specifically inhibit complement in inflammation have been discussed at a recent meeting on the 'Immune Consequences of Trauma, Shock and Sepsis', held from March 4-8, 1997, in Munich, Germany. The Congress (chairman: E. Faist, Munich, Germany), which was held in close cooperation with various

  5. Infectious diseases associated with complement deficiencies.

    PubMed

    Figueroa, J E; Densen, P

    1991-07-01

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

  6. Infectious diseases associated with complement deficiencies.

    PubMed Central

    Figueroa, J E; Densen, P

    1991-01-01

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

  7. Review on complement analysis method and the roles of glycosaminoglycans in the complement system.

    PubMed

    Li, Lian; Li, Yan; Ijaz, Muhammad; Shahbaz, Muhammad; Lian, Qianqian; Wang, Fengshan

    2015-12-10

    Complement system is composed of over 30 proteins and it plays important roles in self-defence and inflammation. There are three activation pathways, including classical pathway, alternative pathway and lectin pathway, in complement system, and they are associated with many diseases such as osteoarthritis and age-related macular degeneration. Modulation of the complement system may be a promising strategy in the treatment of related diseases. Glycosaminoglycans are anionic linear polysaccharides without branches. They are one kind of multi-functional macromolecules which have great potential in regulating complement system. This review is organized around two aspects between the introduction of complement system and the interaction of glycosaminoglycans with complement system. Three complement activation pathways and the biological significance were introduced first. Then functional analysis methods were compared to provide a strategy for potential glycosaminoglycans screen. Finally, the roles of glycosaminoglycans played in the complement system were summed up.

  8. Progress and Trends in Complement Therapeutics

    PubMed Central

    Ricklin, Daniel; Lambris, John D.

    2012-01-01

    The past few years have proven to be a highly successful and exciting period for the field of complement-directed drug discovery and development. Driven by promising experiences with the first marketed complement drugs, increased knowledge about the involvement of complement in health and disease, and improvements in structural and analytical techniques as well as animal models of disease, the field has seen a surge in creative approaches to therapeutically intervene at various stages of the cascade. An impressive panel of compounds that show promise in clinical trials is meanwhile being lined up in the pipelines of both small biotechnology and big pharmaceutical companies. Yet with this new focus on complement-targeted therapeutics, important questions concerning target selection, point and length of intervention, safety, and drug delivery emerge. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases and affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This chapter highlights the key changes in the field that shape our current perception of complement-targeted drugs and provides a brief overview of recent strategies and emerging trends. Selected examples of complement-related diseases and inhibitor classes are highlighted to illustrate the diversity and creativity in field. PMID:22990692

  9. Commitment and Evidence in Arabic Complementation.

    ERIC Educational Resources Information Center

    Awad, Maher

    The study examines one component of the system of complementation in Palestinian Arabic. It is argued that the complementizer in question has an inherent semantics capable of influencing the meaning of sentences in which it is embedded. Specifically, its presence in a complex sentence communicates modal meanings distinct from those communicated by…

  10. Complement associated pathogenic mechanisms in myasthenia gravis.

    PubMed

    Tüzün, Erdem; Christadoss, Premkumar

    2013-07-01

    The complement system is profoundly involved in the pathogenesis of acetylcholine receptor (AChR) antibody (Ab) related myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). The most characteristic finding of muscle pathology in both MG and EAMG is the abundance of IgG and complement deposits at the nerve-muscle junction (NMJ), suggesting that AChR-Ab induces muscle weakness by complement pathway activation and consequent membrane attack complex (MAC) formation. This assumption has been supported with EAMG resistance of complement factor C3 knockout (KO), C4 KO and C5 deficient mice and amelioration of EAMG symptoms following treatment with complement inhibitors such as cobra venom factor, soluble complement receptor 1, anti-C1q, anti-C5 and anti-C6 Abs. Moreover, the complement inhibitor decay accelerating factor (DAF) KO mice exhibit increased susceptibility to EAMG. These findings have brought forward improvisation of novel therapy methods based on inhibition of classical and common complement pathways in MG treatment.

  11. The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H

    PubMed Central

    Riva, Rauna; Korhonen, Timo K.; Meri, Seppo

    2015-01-01

    The virulence factor PgtE is an outer membrane protease (omptin) of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e., rough LPS, as observed e.g., in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B) and H (H), key proteins controlling formation and inactivation of the complement protein C3b and thereby the activity of the complement system. S. enterica serovar Typhimurium or omptin-expressing recombinant E. coli bacteria were incubated with purified human complement proteins or recombinant H fragments. PgtE cleaved both B and H, whereas its close homolog Pla of Yersinia pestis cleaved only H. H was cleaved at both N- and C-termini, while the central region resisted proteolysis. Because of multiple effects of PgtE on complement components (cleavage of C3, C3b, B, and H) we assessed its effect on the opsonophagocytosis of Salmonella. In human serum, C3 cleavage was dependent on proteolytically active PgtE. Human neutrophils interacted less with serum-opsonized FITC-stained S. enterica 14028R than with the isogenic ΔpgtE strain, as analyzed by flow cytometry. In conclusion, cleavage of B and H by PgtE, together with C3 cleavage, affects the C3-mediated recognition of S. enterica by human neutrophils, thus thwarting the immune protection against Salmonella. PMID:25705210

  12. Complement activation in progressive renal disease

    PubMed Central

    Fearn, Amy; Sheerin, Neil Stephen

    2015-01-01

    Chronic kidney disease (CKD) is common and the cause of significant morbidity and mortality. The replacement of functioning nephrons by fibrosis is characteristic of progressive disease. The pathways that lead to fibrosis are not fully understood, although chronic non-resolving inflammation in the kidney is likely to drive the fibrotic response that occurs. In patients with progressive CKD there is histological evidence of inflammation in the interstitium and strategies that reduce inflammation reduce renal injury in pre-clinical models of CKD. The complement system is an integral part of the innate immune system but also augments adaptive immune responses. Complement activation is known to occur in many diverse renal diseases, including glomerulonephritis, thrombotic microangiopathies and transplant rejection. In this review we discuss current evidence that complement activation contributes to progression of CKD, how complement could cause renal inflammation and whether complement inhibition would slow progression of renal disease. PMID:25664245

  13. Molecules Great and Small: The Complement System

    PubMed Central

    Mathern, Douglas R.

    2015-01-01

    The complement cascade, traditionally considered an effector arm of innate immunity required for host defense against pathogens, is now recognized as a crucial pathogenic mediator of various kidney diseases. Complement components produced by the liver and circulating in the plasma undergo activation through the classical and/or mannose-binding lectin pathways to mediate anti-HLA antibody-initiated kidney transplant rejection and autoantibody-initiated GN, the latter including membranous glomerulopathy, antiglomerular basement membrane disease, and lupus nephritis. Inherited and/or acquired abnormalities of complement regulators, which requisitely limit restraint on alternative pathway complement activation, contribute to the pathogenesis of the C3 nephropathies and atypical hemolytic uremic syndrome. Increasing evidence links complement produced by endothelial cells and/or tubular cells to the pathogenesis of kidney ischemia-reperfusion injury and progressive kidney fibrosis. Data emerging since the mid-2000s additionally show that immune cells, including T cells and antigen-presenting cells, produce alternative pathway complement components during cognate interactions. The subsequent local complement activation yields production of the anaphylatoxins C3a and C5a, which bind to their respective receptors (C3aR and C5aR) on both partners to augment effector T-cell proliferation and survival, while simultaneously inhibiting regulatory T-cell induction and function. This immune cell–derived complement enhances pathogenic alloreactive T-cell immunity that results in transplant rejection and likely contributes to the pathogenesis of other T cell–mediated kidney diseases. C5a/C5aR ligations on neutrophils have additionally been shown to contribute to vascular inflammation in models of ANCA-mediated renal vasculitis. New translational immunology efforts along with the development of pharmacologic agents that block human complement components and receptors now permit

  14. Molecules Great and Small: The Complement System.

    PubMed

    Mathern, Douglas R; Heeger, Peter S

    2015-09-01

    The complement cascade, traditionally considered an effector arm of innate immunity required for host defense against pathogens, is now recognized as a crucial pathogenic mediator of various kidney diseases. Complement components produced by the liver and circulating in the plasma undergo activation through the classical and/or mannose-binding lectin pathways to mediate anti-HLA antibody-initiated kidney transplant rejection and autoantibody-initiated GN, the latter including membranous glomerulopathy, antiglomerular basement membrane disease, and lupus nephritis. Inherited and/or acquired abnormalities of complement regulators, which requisitely limit restraint on alternative pathway complement activation, contribute to the pathogenesis of the C3 nephropathies and atypical hemolytic uremic syndrome. Increasing evidence links complement produced by endothelial cells and/or tubular cells to the pathogenesis of kidney ischemia-reperfusion injury and progressive kidney fibrosis. Data emerging since the mid-2000s additionally show that immune cells, including T cells and antigen-presenting cells, produce alternative pathway complement components during cognate interactions. The subsequent local complement activation yields production of the anaphylatoxins C3a and C5a, which bind to their respective receptors (C3aR and C5aR) on both partners to augment effector T-cell proliferation and survival, while simultaneously inhibiting regulatory T-cell induction and function. This immune cell-derived complement enhances pathogenic alloreactive T-cell immunity that results in transplant rejection and likely contributes to the pathogenesis of other T cell-mediated kidney diseases. C5a/C5aR ligations on neutrophils have additionally been shown to contribute to vascular inflammation in models of ANCA-mediated renal vasculitis. New translational immunology efforts along with the development of pharmacologic agents that block human complement components and receptors now permit

  15. Antagonism of the complement component C4 by flavivirus nonstructural protein NS1

    PubMed Central

    Avirutnan, Panisadee; Fuchs, Anja; Hauhart, Richard E.; Somnuke, Pawit; Youn, Soonjeon

    2010-01-01

    The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection. PMID:20308361

  16. Properdin in complement activation and tissue injury.

    PubMed

    Lesher, Allison M; Nilsson, Bo; Song, Wen-Chao

    2013-12-15

    The plasma protein properdin is the only known positive regulator of complement activation. Although regarded as an initiator of the alternative pathway of complement activation at the time of its discovery more than a half century ago, the role and mechanism of action of properdin in the complement cascade has undergone significant conceptual evolution since then. Despite the long history of research on properdin, however, new insight and unexpected findings on the role of properdin in complement activation, pathogen infection and host tissue injury are still being revealed by ongoing investigations. In this article, we provide a brief review on recent studies that shed new light on properdin biology, focusing on the following three topics: (1) its role as a pattern recognition molecule to direct and trigger complement activation, (2) its context-dependent requirement in complement activation on foreign and host cell surfaces, and (3) its involvement in alternative pathway complement-mediated immune disorders and considerations of properdin as a potential therapeutic target in human diseases.

  17. The role of complement in membranous nephropathy

    PubMed Central

    Ma, Hong; Sandor, Dana G.; Beck, Laurence H.

    2013-01-01

    Membranous nephropathy (MN) describes a histopathological pattern of injury marked by glomerular subepithelial immune deposits and collectively represents one of the most common causes of adult nephrotic syndrome. Studies in Heymann nephritis, an experimental model of MN, have established a paradigm in which these deposits locally activate complement to cause podocyte injury, culminating in cytoskeletal reorganization, loss of slit diaphragms, and proteinuria. There is much circumstantial evidence for a prominent role of complement in human MN, as C3 and C5b-9 are consistently found within immune deposits. Secondary MN often exhibits the additional presence of C1q, implicating the classical pathway of complement activation. Primary MN, however, is IgG4-predominant and IgG4 is considered incapable of binding C1q and activating the complement pathway. Recent studies have identified the M-type phospholipase A2 receptor (PLA2R) as the major target antigen in primary MN. Early evidence hints that IgG4 anti-PLA2R autoantibodies can bind mannan-binding lectin and activate the lectin complement pathway. The identification of anti-PLA2R antibodies as likely participants in the pathogenesis of disease will allow focused investigation into the role of complement in MN. Definitive therapy for MN is immunosuppression, although future therapeutic agents that specifically target complement activation may represent an effective temporizing measure to forestall further glomerular injury. PMID:24161038

  18. Complement regulation: physiology and disease relevance

    PubMed Central

    2015-01-01

    The complement system is part of the innate immune response and as such defends against invading pathogens, removes immune complexes and damaged self-cells, aids organ regeneration, confers neuroprotection, and engages with the adaptive immune response via T and B cells. Complement activation can either benefit or harm the host organism; thus, the complement system must maintain a balance between activation on foreign or modified self surfaces and inhibition on intact host cells. Complement regulators are essential for maintaining this balance and are classified as soluble regulators, such as factor H, and membrane-bound regulators. Defective complement regulators can damage the host cell and result in the accumulation of immunological debris. Moreover, defective regulators are associated with several autoimmune diseases such as atypical hemolytic uremic syndrome, dense deposit disease, age-related macular degeneration, and systemic lupus erythematosus. Therefore, understanding the molecular mechanisms by which the complement system is regulated is important for the development of novel therapies for complement-associated diseases. PMID:26300937

  19. Oral vaccination with heat inactivated Mycobacterium bovis activates the complement system to protect against tuberculosis.

    PubMed

    Beltrán-Beck, Beatriz; de la Fuente, José; Garrido, Joseba M; Aranaz, Alicia; Sevilla, Iker; Villar, Margarita; Boadella, Mariana; Galindo, Ruth C; Pérez de la Lastra, José M; Moreno-Cid, Juan A; Fernández de Mera, Isabel G; Alberdi, Pilar; Santos, Gracia; Ballesteros, Cristina; Lyashchenko, Konstantin P; Minguijón, Esmeralda; Romero, Beatriz; de Juan, Lucía; Domínguez, Lucas; Juste, Ramón; Gortazar, Christian

    2014-01-01

    Tuberculosis (TB) remains a pandemic affecting billions of people worldwide, thus stressing the need for new vaccines. Defining the correlates of vaccine protection is essential to achieve this goal. In this study, we used the wild boar model for mycobacterial infection and TB to characterize the protective mechanisms elicited by a new heat inactivated Mycobacterium bovis vaccine (IV). Oral vaccination with the IV resulted in significantly lower culture and lesion scores, particularly in the thorax, suggesting that the IV might provide a novel vaccine for TB control with special impact on the prevention of pulmonary disease, which is one of the limitations of current vaccines. Oral vaccination with the IV induced an adaptive antibody response and activation of the innate immune response including the complement component C3 and inflammasome. Mycobacterial DNA/RNA was not involved in inflammasome activation but increased C3 production by a still unknown mechanism. The results also suggested a protective mechanism mediated by the activation of IFN-γ producing CD8+ T cells by MHC I antigen presenting dendritic cells (DCs) in response to vaccination with the IV, without a clear role for Th1 CD4+ T cells. These results support a role for DCs in triggering the immune response to the IV through a mechanism similar to the phagocyte response to PAMPs with a central role for C3 in protection against mycobacterial infection. Higher C3 levels may allow increased opsonophagocytosis and effective bacterial clearance, while interfering with CR3-mediated opsonic and nonopsonic phagocytosis of mycobacteria, a process that could be enhanced by specific antibodies against mycobacterial proteins induced by vaccination with the IV. These results suggest that the IV acts through novel mechanisms to protect against TB in wild boar.

  20. Oral Vaccination with Heat Inactivated Mycobacterium bovis Activates the Complement System to Protect against Tuberculosis

    PubMed Central

    Garrido, Joseba M.; Aranaz, Alicia; Sevilla, Iker; Villar, Margarita; Boadella, Mariana; Galindo, Ruth C.; Pérez de la Lastra, José M.; Moreno-Cid, Juan A.; Fernández de Mera, Isabel G.; Alberdi, Pilar; Santos, Gracia; Ballesteros, Cristina; Lyashchenko, Konstantin P.; Minguijón, Esmeralda; Romero, Beatriz; de Juan, Lucía; Domínguez, Lucas; Juste, Ramón; Gortazar, Christian

    2014-01-01

    Tuberculosis (TB) remains a pandemic affecting billions of people worldwide, thus stressing the need for new vaccines. Defining the correlates of vaccine protection is essential to achieve this goal. In this study, we used the wild boar model for mycobacterial infection and TB to characterize the protective mechanisms elicited by a new heat inactivated Mycobacterium bovis vaccine (IV). Oral vaccination with the IV resulted in significantly lower culture and lesion scores, particularly in the thorax, suggesting that the IV might provide a novel vaccine for TB control with special impact on the prevention of pulmonary disease, which is one of the limitations of current vaccines. Oral vaccination with the IV induced an adaptive antibody response and activation of the innate immune response including the complement component C3 and inflammasome. Mycobacterial DNA/RNA was not involved in inflammasome activation but increased C3 production by a still unknown mechanism. The results also suggested a protective mechanism mediated by the activation of IFN-γ producing CD8+ T cells by MHC I antigen presenting dendritic cells (DCs) in response to vaccination with the IV, without a clear role for Th1 CD4+ T cells. These results support a role for DCs in triggering the immune response to the IV through a mechanism similar to the phagocyte response to PAMPs with a central role for C3 in protection against mycobacterial infection. Higher C3 levels may allow increased opsonophagocytosis and effective bacterial clearance, while interfering with CR3-mediated opsonic and nonopsonic phagocytosis of mycobacteria, a process that could be enhanced by specific antibodies against mycobacterial proteins induced by vaccination with the IV. These results suggest that the IV acts through novel mechanisms to protect against TB in wild boar. PMID:24842853

  1. Infections Revealing Complement Deficiency in Adults

    PubMed Central

    Audemard-Verger, A.; Descloux, E.; Ponard, D.; Deroux, A.; Fantin, B.; Fieschi, C.; John, M.; Bouldouyre, A.; Karkowsi, L.; Moulis, G.; Auvinet, H.; Valla, F.; Lechiche, C.; Davido, B.; Martinot, M.; Biron, C.; Lucht, F.; Asseray, N.; Froissart, A.; Buzelé, R.; Perlat, A.; Boutboul, D.; Fremeaux-Bacchi, V.; Isnard, S.; Bienvenu, B.

    2016-01-01

    Abstract Complement system is a part of innate immunity, its main function is to protect human from bacterial infection. As genetic disorders, complement deficiencies are often diagnosed in pediatric population. However, complement deficiencies can also be revealed in adults but have been poorly investigated. Herein, we describe a case series of infections revealing complement deficiency in adults to study clinical spectrum and management of complement deficiencies. A nationwide retrospective study was conducted in French university and general hospitals in departments of internal medicine, infectious diseases enrolling patients older than 15 years old who had presented at least one infection leading to a complement deficiency diagnosis. Forty-one patients included between 2002 and 2015 in 19 different departments were enrolled in this study. The male-to-female ratio was 1.3 and the mean age at diagnosis was 28 ± 14 (15–67) years. The main clinical feature was Neisseria meningitidis meningitis 75% (n = 31/41) often involving rare serotype: Y (n = 9) and W 135 (n = 7). The main complement deficiency observed was the common final pathway deficiency 83% (n = 34/41). Half of the cohort displayed severe sepsis or septic shock at diagnosis (n = 22/41) but no patient died. No patient had family history of complement deficiency. The mean follow-up was 1.15 ± 1.95 (0.1–10) years. Half of the patients had already suffered from at least one infection before diagnosis of complement deficiency: meningitis (n = 13), pneumonia (n = 4), fulminans purpura (n = 1), or recurrent otitis (n = 1). Near one-third (n = 10/39) had received prophylactic antibiotics (cotrimoxazole or penicillin) after diagnosis of complement deficiency. The vaccination coverage rate, at the end of the follow-up, for N meningitidis, Streptococcus pneumonia, and Haemophilius influenzae were, respectively, 90% (n = 33/37), 47% (n = 17/36), and 35

  2. On complements of coradicals of finite groups

    NASA Astrophysics Data System (ADS)

    Vedernikov, V. A.; Sorokina, M. M.

    2016-06-01

    Let F be an ω-local Fitting formation, and G a finite group that can be represented in the form of a product of n subnormal subgroups whose F-coradicals are ω-soluble, and whose Sylow p-subgroups are abelian for any p\\inω. It is established that there exist ω-complements of the F-coradical of G. New theorems on the existence of complements of coradicals of a group are obtained as corollaries. For an ω-local formation F, conditions are established for the existence of complements and ω-complements of the F-coradical of a group in any of its extensions. Bibliography: 21 titles.

  3. Regulation of humoral immunity by complement.

    PubMed

    Carroll, Michael C; Isenman, David E

    2012-08-24

    The complement system of innate immunity is important in regulating humoral immunity largely through the complement receptor CR2, which forms a coreceptor on B cells during antigen-induced activation. However, CR2 also retains antigens on follicular dendritic cells (FDCs). Display of antigen on FDCs is critical for clonal selection and affinity maturation of activated B cells. This review will discuss the role of complement in adaptive immunity in general with a focus on the interplay between CR2-associated antigen on B cells with CR2 expressed on FDCs. This latter interaction provides an opportunity for memory B cells to sample antigen over prolonged periods. The cocrystal structure of CR2 with its ligand C3d provides insight into how the complement system regulates access of antigen by B cells with implications for therapeutic manipulations to modulate aberrant B cell responses in the case of autoimmunity.

  4. Autocrine effects of tumor-derived complement.

    PubMed

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

    2014-03-27

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

  5. [Complement system regulation and C3 glomerulopathy].

    PubMed

    Xiao, Hui-jie; He, Rui-juan

    2013-04-18

    Complement system is a key system for immune surveillance and homeostasis. Excessive activation of complement system,especially the activation of alternative pathway may play a very important role in the pathogenesis of primary and secondary glomerulonephritis. C3 glomerulopathy is a newly named disease characterized by evident C3 deposition in the glomeruli with little or no immunoglobulin under immunofluorescence (IF). Its clinical and pathological manifestations vary a lot. The decreased plasma C3 and Factor H(FH)suggest that abnormal regulation of complement system plays an importment role in its pathogenesis. C3 glomerulopathy varies a lot as to its clinical manifestation, treatment and prognosis. The inhibition of excessive complement activation might be the key to treating C3 glomerulopathy.

  6. The complement system in systemic autoimmune disease.

    PubMed

    Chen, Min; Daha, Mohamed R; Kallenberg, Cees G M

    2010-05-01

    Complement is part of the innate immune system. Its major function is recognition and elimination of pathogens via direct killing and/or stimulation of phagocytosis. Activation of the complement system is, however, also involved in the pathogenesis of the systemic autoimmune diseases. Activation via the classical pathway has long been recognized in immune complex-mediated diseases such as cryoglobulinemic vasculitis and systemic lupus erythematosus (SLE). In SLE, the role of complement is somewhat paradoxical. It is involved in autoantibody-initiated tissue damage on the one hand, but, on the other hand, it appears to have protective features as hereditary deficiencies of classical pathway components are associated with an increased risk for SLE. There is increasing evidence that the alternative pathway of complement, even more than the classical pathway, is involved in many systemic autoimmune diseases. This is true for IgA-dominant Henoch Schönlein Purpura, in which additional activation of the lectin pathway contributes to more severe disease. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis the complement system was considered not to be involved since immunoglobulin deposition is generally absent in the lesions. However, recent studies, both in human and animal models, demonstrated complement activation via the alternative pathway as a major pathogenic mechanism. Insight into the role of the various pathways of complement in the systemic autoimmune diseases including the vasculitides opens up new ways of treatment by blocking effector pathways of complement. This has been demonstrated for monoclonal antibodies to C5 or C5a in experimental anti-phospholipid antibody syndrome and ANCA-associated vasculitis.

  7. Polyphosphate suppresses complement via the terminal pathway

    PubMed Central

    Wat, Jovian M.; Foley, Jonathan H.; Krisinger, Michael J.; Ocariza, Linnette Mae; Lei, Victor; Wasney, Gregory A.; Lameignere, Emilie; Strynadka, Natalie C.; Smith, Stephanie A.; Morrissey, James H.

    2014-01-01

    Polyphosphate, synthesized by all cells, is a linear polymer of inorganic phosphate. When released into the circulation, it exerts prothrombotic and proinflammatory activities by modulating steps in the coagulation cascade. We examined the role of polyphosphate in regulating the evolutionarily related proteolytic cascade complement. In erythrocyte lysis assays, polyphosphate comprising more than 1000 phosphate units suppressed total hemolytic activity with a concentration to reduce maximal lysis to 50% that was 10-fold lower than with monophosphate. In the ion- and enzyme-independent terminal pathway complement assay, polyphosphate suppressed complement in a concentration- and size-dependent manner. Phosphatase-treated polyphosphate lost its ability to suppress complement, confirming that polymer integrity is required. Sequential addition of polyphosphate to the terminal pathway assay showed that polyphosphate interferes with complement only when added before formation of the C5b-7 complex. Physicochemical analyses using native gels, gel filtration, and differential scanning fluorimetry revealed that polyphosphate binds to and destabilizes C5b,6, thereby reducing the capacity of the membrane attack complex to bind to and lyse the target cell. In summary, we have added another function to polyphosphate in blood, demonstrating that it dampens the innate immune response by suppressing complement. These findings further establish the complex relationship between coagulation and innate immunity. PMID:24335501

  8. Complement and thrombosis in the antiphospholipid syndrome.

    PubMed

    Oku, Kenji; Nakamura, Hiroyuki; Kono, Michihiro; Ohmura, Kazumasa; Kato, Masaru; Bohgaki, Toshiyuki; Horita, Tetsuya; Yasuda, Shinsuke; Amengual, Olga; Atsumi, Tatsuya

    2016-10-01

    The involvement of complement activation in the pathophysiology of antiphospholipid syndrome (APS) was first reported in murine models of antiphospholipid antibody (aPL)-related pregnancy morbidities. We previously reported that complement activation is prevalent and may function as a source of procoagulant cell activation in the sera of APS patients. Recently, autoantibodies against C1q, a component of complement 1, were reported to be correlated with complement activation in systemic lupus erythematosus. These antibodies target neoepitopes of deformed C1q bound to various molecules (i.e., anionic phospholipids) and induce accelerated complement activation. We found that anti-C1q antibodies are more frequently detected in primary APS patients than in control patients and in refractory APS patients with repeated thrombotic events. The titer of anti-C1q antibodies was significantly higher in refractory APS patients than in APS patients without flare. The binding of C1q to anionic phospholipids may be associated with the surge in complement activation in patients with anti-C1q antibodies when triggered by 'second-hit' biological stressors such as infection. Such stressors will induce overexpression of anionic phospholipids, with subsequent increases in deformed C1q that is targeted by anti-C1q antibodies.

  9. Humoral pattern recognition and the complement system.

    PubMed

    Degn, S E; Thiel, S

    2013-08-01

    In the context of immunity, pattern recognition is the art of discriminating friend from foe and innocuous from noxious. The basis of discrimination is the existence of evolutionarily conserved patterns on microorganisms, which are intrinsic to these microorganisms and necessary for their function and existence. Such immutable or slowly evolving patterns are ideal handles for recognition and have been targeted by early cellular immune defence mechanisms such as Toll-like receptors, NOD-like receptors, RIG-I-like receptors, C-type lectin receptors and by humoral defence mechanisms such as the complement system. Complement is a proteolytic cascade system comprising around 35 different soluble and membrane-bound proteins. It constitutes a central part of the innate immune system, mediating several major innate effector functions and modulating adaptive immune responses. The complement cascade proceeds via controlled, limited proteolysis and conformational changes of constituent proteins through three activation pathways: the classical pathway, the alternative pathway and the lectin pathway, which converge in common effector functions. Here, we review the nature of the pattern recognition molecules involved in complement activation, as well as their close relatives with no or unknown capacity for activating complement. We proceed to examine the composition of the pattern recognition complexes involved in complement activation, focusing on those of the lectin pathway, and arrive at a new model for their mechanism of operation, supported by recently emerging evidence.

  10. Chromatography of arthropod-borne viruses on calcium phosphate columns*

    PubMed Central

    Smith, C. E. Gordon; Holt, Dolores

    1961-01-01

    This is an interim report on the fractionation of arthropod-borne viruses of groups A and B by chromatography on calcium phosphate. The method used provides an excellent, cheap and simple tool for the preparation of stable haemagglutinating and complement-fixing antigens for routine diagnostic and other purposes and for the concentration of such products. In the results reported, viruses of groups A and B have been shown to have two haemagglutinins, one of which is the virus particle. The haemagglutinins are antigenically similar but differ in sedimentation characteristics and in reaction with protamine sulfate. Group B viruses have also been shown to have two complement-fixing antigens with different sedimentation properties; one of these antigens is the virus particle. So far no complement-fixing antigen other than the virus particle has been found with group A viruses. ImagesFIG. 1 PMID:20604091

  11. Complement-fixing antibody response to rotavirus infection.

    PubMed Central

    Gust, I D; Pringle, R C; Barnes, G L; Davidson, G P; Bishop, R F

    1977-01-01

    A human rotavirus complement-fixing (CF) antigen, prepared by purification of large volumes of fluid feces collected from children with winter diarrhea, was used to study the development and persistence of antibody in children with diarrhea and the prevalence of rotavirus antibody in Melbourne. In children with diarrhea, antibody rises were detectable within 4 to 6 weeks of the onset of illness, and the titers usually remained elevated for the next 1 to 2 years. CF antibody did not develop in two children with proven rotavirus infection aged less than 6 months, an age at which poor CF responses to other viruses have also been observed. A study of CF antibody levels in the general community showed that in Melbourne, most children have been infected with human rotavirus by the age of 3 years. PMID:403196

  12. Binding of complement inhibitor C4b-binding protein to a highly virulent Streptococcus pyogenes M1 strain is mediated by protein H and enhances adhesion to and invasion of endothelial cells.

    PubMed

    Ermert, David; Weckel, Antonin; Agarwal, Vaibhav; Frick, Inga-Maria; Björck, Lars; Blom, Anna M

    2013-11-01

    Streptococcus pyogenes AP1, a strain of the highly virulent M1 serotype, uses exclusively protein H to bind the complement inhibitor C4b-binding protein (C4BP). We found a strong correlation between the ability of AP1 and its isogenic mutants lacking protein H to inhibit opsonization with complement C3b and binding of C4BP. C4BP bound to immobilized protein H or AP1 bacteria retained its cofactor activity for degradation of (125)I-C4b. Furthermore, C4b deposited from serum onto AP1 bacterial surfaces was processed into C4c/C4d fragments, which did not occur on strains unable to bind C4BP. Recombinant C4BP mutants, which (i) lack certain CCP domains or (ii) have mutations in single aa as well as (iii) mutants with additional aa between different CCP domains were used to determine that the binding is mainly mediated by a patch of positively charged amino acid residues at the interface of domains CCP1 and CCP2. Using recombinant protein H fragments, we narrowed down the binding site to the N-terminal domain A. With a peptide microarray, we identified one single 18-amino acid-long peptide comprising residues 92-109, which specifically bound C4BP. Biacore was used to determine KD = 6 × 10(-7) M between protein H and a single subunit of C4BP. C4BP binding also correlated with elevated levels of adhesion and invasion to endothelial cells. Taken together, we identified the molecular basis of C4BP-protein H interaction and found that it is not only important for decreased opsonization but also for invasion of endothelial cells by S. pyogenes.

  13. Dimerization of complement factor H-related proteins modulates complement activation in vivo.

    PubMed

    Goicoechea de Jorge, Elena; Caesar, Joseph J E; Malik, Talat H; Patel, Mitali; Colledge, Matthew; Johnson, Steven; Hakobyan, Svetlana; Morgan, B Paul; Harris, Claire L; Pickering, Matthew C; Lea, Susan M

    2013-03-19

    The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.

  14. Complement in therapy and disease: Regulating the complement system with antibody-based therapeutics.

    PubMed

    Melis, Joost P M; Strumane, Kristin; Ruuls, Sigrid R; Beurskens, Frank J; Schuurman, Janine; Parren, Paul W H I

    2015-10-01

    Complement is recognized as a key player in a wide range of normal as well as disease-related immune, developmental and homeostatic processes. Knowledge of complement components, structures, interactions, and cross-talk with other biological systems continues to grow and this leads to novel treatments for cancer, infectious, autoimmune- or age-related diseases as well as for preventing transplantation rejection. Antibodies are superbly suited to be developed into therapeutics with appropriate complement stimulatory or inhibitory activity. Here we review the design, development and future of antibody-based drugs that enhance or dampen the complement system.

  15. Serological relationship of the Tacaribe complex of viruses to lymphocytic choriomeningitis virus.

    PubMed

    Rowe, W P; Pugh, W E; Webb, P A; Peters, C J

    1970-03-01

    By means of the indirect fluorescent-antibody test, cross serological reactivity was demonstrated between lymphocytic choriomeningitis (LCM) virus and the viruses of the Tacaribe complex. Antisera to all members of the Tacaribe complex reacted with LCM virus; LCM antisera gave significant staining of Amapari virus, but minimal or inconsistent reactions with Tacaribe virus, and no reaction with two other viruses of the Tacaribe complex. A low level cross-reaction was observed in complement fixation tests of Machupo and Pichinde antisera against LCM antigen. Immunization with Tacaribe and Amapari viruses did not protect mice against challenge with LCM virus. Because of the identical appearance of the virions, the sharing of antigens, and the many biological similarities between LCM and the Tacaribe complex viruses, it is proposed that they be considered as constituting a new taxonomic group of viruses.

  16. Methods and procedures for use of complement-fixation technique in type- and strain-specific diagnosis of influenza*†

    PubMed Central

    Lief, Florence S.; Henle, Werner

    1959-01-01

    It is now well established that there are two kinds of complement-fixing antigens of influenza virus: the soluble, type-specific, antigens, found mainly in infected tissue but also within the virus particle; and the virus, strain-specific, antigens, closely linked with the haemagglutinating component. For true results in complement-fixation testing in influenza, antigens or antisera of the one kind must be free of the other kind. This paper describes in detail the preparation of reagents for such testing and a variety of basic aspects of test procedures. PMID:13651922

  17. Inhibition of Complement Retards Ankylosing Spondylitis Progression

    PubMed Central

    Yang, Chaoqun; Ding, Peipei; Wang, Qingkai; Zhang, Long; Zhang, Xin; Zhao, Jianquan; Xu, Enjie; Wang, Na; Chen, Jianfeng; Yang, Guang; Hu, Weiguo; Zhou, Xuhui

    2016-01-01

    Ankylosing spondylitis (AS) is a chronic axial spondyloarthritis (SpA) resulting in back pain and progressive spinal ankyloses. Currently, there are no effective therapeutics targeting AS largely due to elusive pathogenesis mechanisms, even as potential candidates such as HLA-B27 autoantigen have been identified. Herein, we employed a proteoglycan (PG)-induced AS mouse model together with clinical specimens, and found that the complement system was substantially activated in the spinal bone marrow, accompanied by a remarkable proportion alteration of neutrophils and macrophage in bone marrow and spleen, and by the significant increase of TGF-β1 in serum. The combined treatment with a bacteria-derived complement inhibitor Efb-C (C-terminal of extracellular fibrinogen-binding protein of Staphylococcus aureus) remarkably retarded the progression of mouse AS by reducing osteoblast differentiation. Furthermore, we demonstrated that two important modulators involved in AS disease, TGF-β1 and RANKL, were elevated upon in vitro complement attack in osteoblast and/or osteoclast cells. These findings further unravel that complement activation is closely related with the pathogenesis of AS, and suggest that complement inhibition may hold great potential for AS therapy. PMID:27698377

  18. Maggot excretions affect the human complement system.

    PubMed

    Cazander, Gwendolyn; Schreurs, Marco W J; Renwarin, Lennaert; Dorresteijn, Corry; Hamann, Dörte; Jukema, Gerrolt N

    2012-01-01

    The complement system plays an important role in the activation of the inflammatory response to injury, although inappropriate complement activation (CA) can lead to severe tissue damage. Maggot therapy is successfully used to treat infected wounds. In this study, we hypothesized that maggot excretions/secretions influence CA in order to modulate the host's inflammatory response. Therefore, the effect of maggot excretions on CA was investigated in preoperatively and postoperatively obtained sera from patients. Our results show that maggot excretions reduce CA in healthy and postoperatively immune-activated human sera up to 99.9%, via all pathways. Maggot excretions do not specifically initiate or inhibit CA, but break down complement proteins C3 and C4 in a cation-independent manner and this effect proves to be temperature tolerant. This study indicates a CA-reducing substrate that is already successfully used in clinical practice and may explain part of the improved wound healing caused by maggot therapy. Furthermore, the complement activation-reducing substance present in maggot excretions could provide a novel treatment modality for several diseases, resulting from an (over)active complement system.

  19. Interallelic complementation at the mouse Mitf locus.

    PubMed Central

    Steingrímsson, Eiríkur; Arnheiter, Heinz; Hallsson, Jón Hallsteinn; Lamoreux, M Lynn; Copeland, Neal G; Jenkins, Nancy A

    2003-01-01

    Mutations at the mouse microphthalmia locus (Mitf) affect the development of different cell types, including melanocytes, retinal pigment epithelial cells of the eye, and osteoclasts. The MITF protein is a member of the MYC supergene family of basic-helix-loop-helix-leucine-zipper (bHLHZip) transcription factors and is known to regulate the expression of cell-specific target genes by binding DNA as homodimer or as heterodimer with related proteins. The many mutations isolated at the locus have different effects on the phenotype and can be arranged in an allelic series in which the phenotypes range from near normal to white microphthalmic animals with osteopetrosis. Previous investigations have shown that certain combinations of Mitf alleles complement each other, resulting in a phenotype more normal than that of each homozygote alone. Here we analyze this interallelic complementation in detail and show that it is limited to one particular allele, Mitf(Mi-white) (Mitf(Mi-wh)), a mutation affecting the DNA-binding domain. Both loss- and gain-of-function mutations are complemented, as are other Mitf mutations affecting the DNA-binding domain. Furthermore, this behavior is not restricted to particular cell types: Both eye development and coat color phenotypes are complemented. Our analysis suggests that Mitf(Mi-wh)-associated interallelic complementation is due to the unique biochemical nature of this mutation. PMID:12586714

  20. Complement activation in chronic liver disease.

    PubMed Central

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

    1982-01-01

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

  1. Complement activation in discordant hepatic xenotransplantation.

    PubMed

    Tector, A J; Chen, X; Soderland, C; Tchervenkov, J I

    1998-11-01

    Little is known about hyperacute rejection in hepatic xenotransplantation. Information from clinical xenoperfusions suggests that the liver may be rejected by a mechanism less vigorous than either kidney or heart xenografts. We used the in vitro model of porcine hepatic sinusoidal endothelial cells (PHEC) incubated with either complement replete or deficient human serum to determine the relative roles of the classical and alternate pathways of complement in the immediate response to hepatic xenotransplantation. Our results suggest that either the classical or alternate pathways are capable of independently activating the complement cascade upon exposure to the porcine hepatic sinusoidal endothelium. Our results also imply that either pathway alone is capable of initiating similar degrees of injury as the entire cascade. PMID:9915253

  2. Supramolecular Control over Split-Luciferase Complementation.

    PubMed

    Bosmans, Ralph P G; Briels, Jeroen M; Milroy, Lech-Gustav; de Greef, Tom F A; Merkx, Maarten; Brunsveld, Luc

    2016-07-25

    Supramolecular split-enzyme complementation restores enzymatic activity and allows for on-off switching. Split-luciferase fragment pairs were provided with an N-terminal FGG sequence and screened for complementation through host-guest binding to cucurbit[8]uril (Q8). Split-luciferase heterocomplex formation was induced in a Q8 concentration dependent manner, resulting in a 20-fold upregulation of luciferase activity. Supramolecular split-luciferase complementation was fully reversible, as revealed by using two types of Q8 inhibitors. Competition studies with the weak-binding FGG peptide revealed a 300-fold enhanced stability for the formation of the ternary heterocomplex compared to binding of two of the same fragments to Q8. Stochiometric binding by the potent inhibitor memantine could be used for repeated cycling of luciferase activation and deactivation in conjunction with Q8, providing a versatile module for in vitro supramolecular signaling networks.

  3. The Complement System in Lupus Nephritis.

    PubMed

    Birmingham, Daniel J; Hebert, Lee A

    2015-09-01

    The complement system is composed of a family of soluble and membrane-bound proteins that historically has been viewed as a key component of the innate immune system, with a primary role of providing a first-line defense against microorganisms. Although this role indeed is important, complement has many other physiological roles, including the following: (1) influencing appropriate immune responses, (2) disposing of waste in the circulation (immune complexes, cellular debris), and (3) contributing to damage of self-tissue through inflammatory pathways. These three roles are believed to be significant factors in the pathogenesis of systemic lupus erythematosus, particularly its renal manifestation (lupus nephritis), contributing both protective and damaging effects. In this review, we provide an overview of the human complement system and its functions, and discuss its intricate and seemingly contradictory roles in the pathogenesis of lupus nephritis.

  4. Complement inhibition in C3 glomerulopathy.

    PubMed

    Nester, Carla M; Smith, Richard J H

    2016-06-01

    C3 glomerulopathy (C3G) describes a spectrum of glomerular diseases defined by shared renal biopsy pathology: a predominance of C3 deposition on immunofluorescence with electron microscopy permitting disease sub-classification. Complement dysregulation underlies the observed pathology, a causal relationship that is supported by well described studies of genetic and acquired drivers of disease. In this article, we provide an overview of the features of C3G, including a discussion of disease definition and a review of the causal role of complement. We discuss molecular markers of disease and how biomarkers are informing our evolving understanding of underlying pathology. Research advances are laying the foundation for complement inhibition as a targeted approach to treatment of C3G. PMID:27402056

  5. Applying complement therapeutics to rare diseases.

    PubMed

    Reis, Edimara S; Mastellos, Dimitrios C; Yancopoulou, Despina; Risitano, Antonio M; Ricklin, Daniel; Lambris, John D

    2015-12-01

    Around 350 million people worldwide suffer from rare diseases. These may have a genetic, infectious, or autoimmune basis, and several include an inflammatory component. Launching of effective treatments can be very challenging when there is a low disease prevalence and limited scientific insights into the disease mechanisms. As a key trigger of inflammatory processes, complement has been associated with a variety of diseases and has become an attractive therapeutic target for conditions involving inflammation. In view of the clinical experience acquired with drugs licensed for the treatment of rare diseases such as hereditary angioedema and paroxysmal nocturnal hemoglobinuria, growing evidence supports the safety and efficacy of complement therapeutics in restoring immune balance and preventing aggravation of clinical outcomes. This review provides an overview of the candidates currently in the pharmaceutical pipeline with potential to treat orphan diseases and discusses the molecular mechanisms triggered by complement involved with the disease pathogenesis.

  6. The extracellular RNA complement of Escherichia coli

    PubMed Central

    Ghosal, Anubrata; Upadhyaya, Bimal Babu; Fritz, Joëlle V; Heintz-Buschart, Anna; Desai, Mahesh S; Yusuf, Dilmurat; Huang, David; Baumuratov, Aidos; Wang, Kai; Galas, David; Wilmes, Paul

    2015-01-01

    The secretion of biomolecules into the extracellular milieu is a common and well-conserved phenomenon in biology. In bacteria, secreted biomolecules are not only involved in intra-species communication but they also play roles in inter-kingdom exchanges and pathogenicity. To date, released products, such as small molecules, DNA, peptides, and proteins, have been well studied in bacteria. However, the bacterial extracellular RNA complement has so far not been comprehensively characterized. Here, we have analyzed, using a combination of physical characterization and high-throughput sequencing, the extracellular RNA complement of both outer membrane vesicle (OMV)-associated and OMV-free RNA of the enteric Gram-negative model bacterium Escherichia coli K-12 substrain MG1655 and have compared it to its intracellular RNA complement. Our results demonstrate that a large part of the extracellular RNA complement is in the size range between 15 and 40 nucleotides and is derived from specific intracellular RNAs. Furthermore, RNA is associated with OMVs and the relative abundances of RNA biotypes in the intracellular, OMV and OMV-free fractions are distinct. Apart from rRNA fragments, a significant portion of the extracellular RNA complement is composed of specific cleavage products of functionally important structural noncoding RNAs, including tRNAs, 4.5S RNA, 6S RNA, and tmRNA. In addition, the extracellular RNA pool includes RNA biotypes from cryptic prophages, intergenic, and coding regions, of which some are so far uncharacterised, for example, transcripts mapping to the fimA-fimL and ves-spy intergenic regions. Our study provides the first detailed characterization of the extracellular RNA complement of the enteric model bacterium E. coli. Analogous to findings in eukaryotes, our results suggest the selective export of specific RNA biotypes by E. coli, which in turn indicates a potential role for extracellular bacterial RNAs in intercellular communication. PMID:25611733

  7. Activation of Complement Following Total Hip Replacement.

    PubMed

    Thordardottir, S; Vikingsdottir, T; Bjarnadottir, H; Jonsson, H; Gudbjornsson, B

    2016-03-01

    The aim of this study was to investigate whether complement activation, via the classical and alternative pathways, occurs following a cemented total hip replacement (THR) surgery due to osteoarthritis. Blood samples were collected systematically from 12 patients - six male and six women, with a median age of 75 (range: 59-90 years) - preoperatively, 6 h post-operatively and on the first, second and third post-operative day. Total function of classical (CH50) and alternative pathways (AH50) was evaluated, along with the determination of serum concentrations of the complement proteins C3, C4, C3d, the soluble terminal complement complex (sTCC) sC5b-9, as well as C-reactive protein (CRP) and albumin. Measurements of CRP and albumin levels elucidated a marked inflammatory response following the operation. The CH50, AH50 and C3 and C4 levels were significantly lower 6 h after the surgery compared with the preoperative levels, but elevated above the preoperative levels during the following 3 days. The complement activation product C3d levels increased continually during the whole observation period, from 13.5 AU/ml (range: 8-19 AU/ml) preoperative to 20 AU/ml (range: 12-34 AU/ml) on the third post-operative day. Furthermore, we observed an increase in the sC5b-9 levels between the preoperative and the third post-operative day. These results demonstrate a significant activation of the complement system following cemented THR. Further studies are needed to elucidate the time frame and the pathogenic role of this observed complement activation.

  8. Ixodes dammini: salivary anti-complement activity.

    PubMed

    Ribeiro, J M

    1987-12-01

    Saliva of the tick Ixodes dammini prevents hemolysis of rabbit erythrocytes by the human alternative pathway of complement. Deposition of C3b to activating surfaces and concomitant C3a release are inhibited. C3b deposition to activating surfaces is inhibited regardless the origin (humans, rat, mouse, guinea pig, and hamster) of the serum. The inhibitor elutes as a single peak upon gel filtration, with an apparent molecular weight of 49,000. Salivary anti-complement may contribute to successful feeding of I. dammini in their natural hosts. PMID:3119364

  9. HUS and the case for complement.

    PubMed

    Conway, Edward M

    2015-10-29

    Hemolytic-uremic syndrome (HUS) is a thrombotic microangiopathy that is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Excess complement activation underlies atypical HUS and is evident in Shiga toxin-induced HUS (STEC-HUS). This Spotlight focuses on new knowledge of the role of Escherichia coli-derived toxins and polyphosphate in modulating complement and coagulation, and how they affect disease progression and response to treatment. Such new insights may impact on current and future choices of therapies for STEC-HUS.

  10. Therapeutic complement inhibition in complement-mediated hemolytic anemias: Past, present and future.

    PubMed

    Risitano, Antonio M; Marotta, Serena

    2016-06-01

    The introduction in the clinic of anti-complement agents represented a major achievement which gave to physicians a novel etiologic treatment for different human diseases. Indeed, the first anti-complement agent eculizumab has changed the treatment paradigm of paroxysmal nocturnal hemoglobinuria (PNH), dramatically impacting its severe clinical course. In addition, eculizumab is the first agent approved for atypical Hemolytic Uremic Syndrome (aHUS), a life-threatening inherited thrombotic microangiopathy. Nevertheless, such remarkable milestone in medicine has brought to the fore additional challenges for the scientific community. Indeed, the list of complement-mediated anemias is not limited to PNH and aHUS, and other human diseases can be considered for anti-complement treatment. They include other thrombotic microangiopathies, as well as some antibody-mediated hemolytic anemias. Furthermore, more than ten years of experience with eculizumab led to a better understanding of the individual steps of the complement cascade involved in the pathophysiology of different human diseases. Based on this, new unmet clinical needs are emerging; a number of different strategies are currently under development to improve current anti-complement treatment, trying to address these specific clinical needs. They include: (i) alternative anti-C5 agents, which may improve the heaviness of eculizumab treatment; (ii) broad-spectrum anti-C3 agents, which may improve the efficacy of anti-C5 treatment by intercepting the complement cascade upstream (i.e., preventing C3-mediated extravascular hemolysis in PNH); (iii) targeted inhibitors of selective complement activating pathways, which may prevent early pathogenic events of specific human diseases (e.g., anti-classical pathway for antibody-mediated anemias, or anti-alternative pathway for PNH and aHUS). Here we briefly summarize the status of art of current and future complement inhibition for different complement-mediated anemias

  11. The contribution of both oxygen and nitrogen intermediates to the intracellular killing mechanisms of C1q-opsonized Listeria monocytogenes by the macrophage-like IC-21 cell line.

    PubMed

    Alvarez-Domínguez, C; Carrasco-Marín, E; López-Mato, P; Leyva-Cobián, F

    2000-09-01

    Listeria monocytogenes is a facultative intracellular pathogen which is internalized by host mammalian cells upon binding to their surface. Further listerial growth occurs in the cytosol after escape from the phagosomal-endosomal compartment. We have previously reported that C1q is able to potentiate L. monocytogenes phagocytosis upon bacterial opsonization by ingestion through C1q-binding structures. In this report, we analysed the post-phagocytic events upon internalization of C1q-opsonized L. monocytogenes and found an induction of macrophage (Mphi)-like IC-21 cell bactericidal mechanisms displayed by the production of oxygen and nitrogen metabolites. Both types of molecules are effective in L. monocytogenes killing. Further analysis of the cellular responses promoted by interaction of C1q with its surface binding structures, leads us to consider C1q as a collaborative molecule involved in Mphi activation. Upon interaction with surface binding structures, C1q was able to trigger and/or amplify the production of reactive oxygen and nitrogen intermediates induced by stimuli such as interferon-gamma and L. monocytogenes phagocytosis. PMID:11012757

  12. The contribution of both oxygen and nitrogen intermediates to the intracellular killing mechanisms of C1q-opsonized Listeria monocytogenes by the macrophage-like IC-21 cell line

    PubMed Central

    Álvarez-Domínguez, C; Carrasco-Marín, E; López-Mato, P; Leyva-Cobián, F

    2000-01-01

    Listeria monocytogenes is a facultative intracellular pathogen which is internalized by host mammalian cells upon binding to their surface. Further listerial growth occurs in the cytosol after escape from the phagosomal–endosomal compartment. We have previously reported that C1q is able to potentiate L. monocytogenes phagocytosis upon bacterial opsonization by ingestion through C1q-binding structures. In this report, we analysed the post-phagocytic events upon internalization of C1q-opsonized L. monocytogenes and found an induction of macrophage (Mφ)-like IC-21 cell bactericidal mechanisms displayed by the production of oxygen and nitrogen metabolites. Both types of molecules are effective in L. monocytogenes killing. Further analysis of the cellular responses promoted by interaction of C1q with its surface binding structures, leads us to consider C1q as a collaborative molecule involved in Mφ activation. Upon interaction with surface binding structures, C1q was able to trigger and/or amplify the production of reactive oxygen and nitrogen intermediates induced by stimuli such as interferon-γ and L. monocytogenes phagocytosis. PMID:11012757

  13. Association of complement receptor 2 polymorphisms with innate resistance to HIV-1 infection.

    PubMed

    Herrero, R; Real, L M; Rivero-Juárez, A; Pineda, J A; Camacho, Á; Macías, J; Laplana, M; Konieczny, P; Márquez, F J; Souto, J C; Soria, J M; Saulle, I; Lo Caputo, S; Biasin, M; Rivero, A; Fibla, J; Caruz, A

    2015-03-01

    HIV-1 induces activation of complement through the classical and lectin pathways. However, the virus incorporates several membrane-bound or soluble regulators of complement activation (RCA) that inactivate complement. HIV-1 can also use the complement receptors (CRs) for complement-mediated antibody-dependent enhancement of infection (Ć-ADE). We hypothesize that hypofunctional polymorphisms in RCA or CRs may protect from HIV-1 infection. For this purpose, 139 SNPs located in 19 RCA and CRs genes were genotyped in a population of 201 Spanish HIV-1-exposed seronegative individuals (HESN) and 250 HIV-1-infected patients. Two SNPs were associated with infection susceptibility, rs1567190 in CR2 (odds ratio (OR) = 2.27, P = 1 × 10(-4)) and rs2842704 in C4BPA (OR = 2.11, P = 2 × 10(-4)). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. Although not significant (P = 0.25, OR = 1.57), similar genotypic proportions were obtained for the CR2 marker rs1567190. The results of the two association analyses were combined through a random effect meta-analysis, with a significant P-value of 2.6 x 10(-5) (OR = 2.07). Furthermore, we found that the protective CR2 genotype is correlated with lower levels CR2 mRNA as well as differences in the ratio of the long and short CR2 isoforms.

  14. Complement Constructions in English: Fairly Difficult for EFL Language Learners

    ERIC Educational Resources Information Center

    Fazeli, Fatemeh; Shokrpour, Nasrin

    2012-01-01

    Complement constructions vary significantly in English and Persian. There are more complementation structures in English than in Persian and a complement structure in Persian might have more than one equivalent in English. Producing complement structures (CSs) in English is very difficult for native speakers of Persian, especially in an EFL…

  15. Emai Sentence Complements in Typological Perspective.

    ERIC Educational Resources Information Center

    Schaefer, Ronald P.; Egbokhare, Francis O.

    This paper explores the syntactic and semantic character of previously undescribed sentence complements (SCs) in Emai, a Benue-Congo language of Nigeria's Edoid group. Data come from ongoing documentation incorporating oral narrative texts as well as dictionary and grammar descriptions. To delineate the grammatical properties of SCs, the paper…

  16. Spacelab carrier complement thermal design and performance

    NASA Astrophysics Data System (ADS)

    Bancroft, S.; Key, R.; Kittredge, S.

    1992-07-01

    The present discussion of the Spacelab carrier complement, which encompasses a Module Carrier, a Module-Pallet Carrier, and a Multiplexer/Demultiplexer Pallet, gives attention to both active and passive thermal performance capabilities, and presents ground testing and analytical results obtained to date. An account is given of the prospective use of a Spacelab Multipurpose Experiment Support Structure.

  17. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  18. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  19. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  20. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  1. 21 CFR 866.4100 - Complement reagent.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Complement reagent. 866.4100 Section 866.4100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents §...

  2. Genetics Home Reference: complement component 2 deficiency

    MedlinePlus

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

  3. Spacelab carrier complement thermal design and performance

    NASA Technical Reports Server (NTRS)

    Bancroft, S.; Key, R.; Kittredge, S.

    1992-01-01

    The present discussion of the Spacelab carrier complement, which encompasses a Module Carrier, a Module-Pallet Carrier, and a Multiplexer/Demultiplexer Pallet, gives attention to both active and passive thermal performance capabilities, and presents ground testing and analytical results obtained to date. An account is given of the prospective use of a Spacelab Multipurpose Experiment Support Structure.

  4. Role of Complement and Complement Regulatory Proteins in the Complications of Diabetes

    PubMed Central

    Ghosh, Pamela; Sahoo, Rupam; Vaidya, Anand; Chorev, Michael

    2015-01-01

    It is well established that the organ damage that complicates human diabetes is caused by prolonged hyperglycemia, but the cellular and molecular mechanisms by which high levels of glucose cause tissue damage in humans are still not fully understood. The prevalent hypothesis explaining the mechanisms that may underlie the pathogenesis of diabetes complications includes overproduction of reactive oxygen species, increased flux through the polyol pathway, overactivity of the hexosamine pathway causing intracellular formation of advanced glycation end products, and activation of protein kinase C isoforms. In addition, experimental and clinical evidence reported in past decades supports a strong link between the complement system, complement regulatory proteins, and the pathogenesis of diabetes complications. In this article, we summarize the body of evidence that supports a role for the complement system and complement regulatory proteins in the pathogenesis of diabetic vascular complications, with specific emphasis on the role of the membrane attack complex (MAC) and of CD59, an extracellular cell membrane-anchored inhibitor of MAC formation that is inactivated by nonenzymatic glycation. We discuss a pathogenic model of human diabetic complications in which a combination of CD59 inactivation by glycation and hyperglycemia-induced complement activation increases MAC deposition, activates pathways of intracellular signaling, and induces the release of proinflammatory, prothrombotic cytokines and growth factors. Combined, complement-dependent and complement-independent mechanisms induced by high glucose promote inflammation, proliferation, and thrombosis as characteristically seen in the target organs of diabetes complications. PMID:25859860

  5. Engineering of human complement component C3 for catalytic inhibition of complement.

    PubMed

    Kölln, Johanna; Bredehorst, Reinhard; Spillner, Edzard

    2005-04-15

    As a novel therapeutic approach in complement-mediated pathologies, we recently developed a human C3 derivative capable of obliterating functional complement by a catalytic, non-inhibitory mechanism. In this derivative, the C-terminal region of hC3 was substituted by a 275 amino acid sequence derived from the corresponding sequence of cobra venom factor (CVF), a complement-activating C3b homologue from snake venom. In this study, we replaced shorter C-terminal sequences of hC3 by corresponding CVF sequences to further reduce potential immunogenicity and to identify domains essential for the formation of functionally stable C3 convertases. In one of these derivatives that is still capable of obliterating functional complement in vitro, the non-human portion could be reduced to a small domain located in the C-terminus of different complement proteins. This conserved NTR/C345C motif is known to be involved in assembly of different convertases of the complement system. These results suggest a major role of the C345C domain in the regulation of the half-life of the C3 convertase. Moreover, its overall identity of 96% to human C3 renders this derivative a promising candidate for therapeutic intervention in complement-mediated pathologies. PMID:15790508

  6. Targeted inhibition of complement using complement receptor 2-conjugated inhibitors attenuates EAE.

    PubMed

    Hu, Xianzhen; Tomlinson, Stephen; Barnum, Scott R

    2012-11-30

    Multiple sclerosis (MS) is the most common autoimmune demyelinating disease, affecting millions of individuals worldwide. In the last two decades, many therapeutic options for the treatment of MS have become available, however they are limited in terms of effectiveness and some remain plagued by safety issues. The currently available treatment options target relapsing remitting forms of MS and are not effective against the more progressive forms of the disease. These limitations highlight a significant unmet treatment need for MS. In experimental autoimmune encephalomyelitis (EAE) studies from our laboratory, we have previously shown, using a number of complement mutant and transgenic mice, that inhibition of the alternative complement pathway and the C3 convertase confers significant protection from disease. We report here that targeted inhibition of complement activation using complement receptor 2 (CR2)-conjugated inhibitors significantly attenuates EAE. Administration of CR2-Crry (blocks all complement pathways at C3 activation) and CR2-fH (specifically blocks the alternative pathway) just prior to and during the onset of EAE blocks progression of both acute and chronic disease. These data indicate that inhibition of complement may offer an effective therapeutic approach to treating both acute and chronic forms of demyelinating disease through blocking the alternative pathway or complement convertases. PMID:23079547

  7. Role of complement and complement regulatory proteins in the complications of diabetes.

    PubMed

    Ghosh, Pamela; Sahoo, Rupam; Vaidya, Anand; Chorev, Michael; Halperin, Jose A

    2015-06-01

    It is well established that the organ damage that complicates human diabetes is caused by prolonged hyperglycemia, but the cellular and molecular mechanisms by which high levels of glucose cause tissue damage in humans are still not fully understood. The prevalent hypothesis explaining the mechanisms that may underlie the pathogenesis of diabetes complications includes overproduction of reactive oxygen species, increased flux through the polyol pathway, overactivity of the hexosamine pathway causing intracellular formation of advanced glycation end products, and activation of protein kinase C isoforms. In addition, experimental and clinical evidence reported in past decades supports a strong link between the complement system, complement regulatory proteins, and the pathogenesis of diabetes complications. In this article, we summarize the body of evidence that supports a role for the complement system and complement regulatory proteins in the pathogenesis of diabetic vascular complications, with specific emphasis on the role of the membrane attack complex (MAC) and of CD59, an extracellular cell membrane-anchored inhibitor of MAC formation that is inactivated by nonenzymatic glycation. We discuss a pathogenic model of human diabetic complications in which a combination of CD59 inactivation by glycation and hyperglycemia-induced complement activation increases MAC deposition, activates pathways of intracellular signaling, and induces the release of proinflammatory, prothrombotic cytokines and growth factors. Combined, complement-dependent and complement-independent mechanisms induced by high glucose promote inflammation, proliferation, and thrombosis as characteristically seen in the target organs of diabetes complications.

  8. Utilizing complement evasion strategies to design complement-based antibacterial immunotherapeutics: Lessons from the pathogenic Neisseriae.

    PubMed

    Ram, Sanjay; Shaughnessy, Jutamas; DeOliveira, Rosane B; Lewis, Lisa A; Gulati, Sunita; Rice, Peter A

    2016-10-01

    Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac and resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms. PMID:27297292

  9. Utilizing complement evasion strategies to design complement-based antibacterial immunotherapeutics: Lessons from the pathogenic Neisseriae.

    PubMed

    Ram, Sanjay; Shaughnessy, Jutamas; DeOliveira, Rosane B; Lewis, Lisa A; Gulati, Sunita; Rice, Peter A

    2016-10-01

    Novel therapies are urgently needed to combat the global threat of multidrug-resistant pathogens. Complement forms an important arm of innate defenses against infections. In physiological conditions, complement activation is tightly controlled by soluble and membrane-associated complement inhibitors, but must be selectively activated on invading pathogens to facilitate microbial clearance. Many pathogens, including Neisseria gonorrhoeae and N. meningitidis, express glycans, including N-acetylneuraminic acid (Neu5Ac), that mimic host structures to evade host immunity. Neu5Ac is a negatively charged 9-cabon sugar that inhibits complement, in part by enhancing binding of the complement inhibitor factor H (FH) through C-terminal domains (19 and 20) on FH. Other microbes also bind FH, in most instances through FH domains 6 and 7 or 18-20. Here we describe two strategies to target complement activation on Neisseriae. First, microbial binding domains of FH were fused to IgG Fc to create FH18-20/Fc (binds gonococci) and FH6,7/Fc (binds meningococci). A point mutation in FH domain 19 eliminated hemolysis caused by unmodified FH18-20, but retained binding to gonococci. FH18-20/Fc and FH6,7/Fc mediated complement-dependent killing in vitro and showed efficacy in animal models of gonorrhea and meningococcal bacteremia, respectively. The second strategy utilized CMP-nonulosonate (CMP-NulO) analogs of sialic acid that were incorporated into LOS and prevented complement inhibition by physiologic CMP-Neu5Ac and resulted in attenuated gonococcal infection in mice. While studies to establish the safety of these agents are needed, enhancing complement activation on microbes may represent a promising strategy to treat antimicrobial resistant organisms.

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

    PubMed

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

    2012-01-01

    Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner. PMID:22611464

  11. Decoration of Histophilus somni lipooligosaccharide with N-acetyl-5-neuraminic acid enhances bacterial binding of complement factor H and resistance to killing by serum and polymorphonuclear leukocytes.

    PubMed

    Inzana, Thomas J; Balyan, Rajiv; Howard, Michael D

    2012-12-28

    The incorporation of N-acetyl-5-neuraminic acid (Neu5Ac), or sialic acid, onto surface components of some bacterial species may enhance their virulence. We have previously shown that Neu5Ac can be incorporated onto the lipooligosaccharide (LOS) of the bovine pathogen Histophilus somni, resulting in diminished antibody binding and enhanced serum resistance (Inzana et al., 2002. Infect. Immun. 70, 4870). In the present study, we assessed the effect of sialylation of H. somni LOS on the interaction with bovine innate host defenses. Incubation of non-sialylated H. somni with pre-colostral calf serum (PCS) resulted in dose-dependent, complement-mediated killing of the bacteria by the alternative pathway. However, sialylated H. somni was significantly more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated and consumed less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized red blood cells, and by Western blotting of C(3) activation products. Sialylated H. somni bound more factor H and iC(3)b and less C(3) than non-sialylated bacteria, as determined by enzyme-linked immunosorbent assay, supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni LOS inhibited both polymorphonuclear leukocyte phagocytosis of (3)H-thymidine-labeled bacteria and intracellular killing of the bacteria, compared to non-sialylated bacteria. Furthermore, sialylated H. somni bound less non-specific antibodies in normal bovine sera than non-sialylated bacteria. Therefore, sialylation of H. somni LOS had profound effects on resistance of the bacteria to innate bovine host defenses, which should be taken into consideration during in vitro studies of H. somni.

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

    PubMed

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

    2012-01-01

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

  13. Early complement components in Alzheimer's disease brains.

    PubMed

    Veerhuis, R; Janssen, I; Hack, C E; Eikelenboom, P

    1996-01-01

    Activation products of the early complement components C1, C4 and C3 can be found colocalized with diffuse and fibrillar beta-amyloid (beta/A4) deposits in Alzheimer's disease (AD) brains. Immunohistochemically, C1-esterase inhibitor (C1-Inh) and the C1 subcomponents C1s and C1r can not, or only occasionally, be detected in plaques or in astrocytes. The present finding that C1q, C1s and C1-Inh mRNA are present in both AD and control brains suggests that the variable immunohistochemical staining results for C1r, C1s and C1-Inh are due to a rapid consumption, and that the inability to detect C1s, C1r or C1-Inh is probably due to the dissociation of C1s-C1-Inh and C1r-C1-Inh complexes from the activator-bound C1q into the fluid phase. Employing monoclonal antibodies specific for different forms of C1-Inh, no complexed C1-Inh could be found, whereas inactivated C1-Inh seems to be present in astrocytes surrounding beta/A4 plaques in AD brains. These findings, together with our finding (using reverse transcriptase-polymerase chain reaction) that C1-Inh is locally produced in the brain, suggest that in the brain complement activation at the C1 level is regulated by C1-Inh. Immunohistochemically, no evidence for the presence of the late complement components C5, C7 and C9, or of the membrane attack complex (MAC), was found in beta/A4 plaques. In contrast to the mRNA encoding the early components, that of the late complement components appears to be hardly detectable (C7) or absent (C9). Thus, without blood-brain-barrier impairment, the late complement components are probably present at too low a concentration to allow the formation of the MAC, which is generally believed to be responsible for at least some of the neurodegenerative effects observed in AD. Therefore, the present findings support the idea that in AD, complement does not function as an inflammatory mediator through MAC formation, but through the action of early component activation products.

  14. Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation

    PubMed Central

    Sun, Ran; Zhao, Xi; Wang, Zixia; Yang, Jing; Zhao, Limei; Zhan, Bin; Zhu, Xinping

    2015-01-01

    Background Trichinella spiralis expresses paramyosin (Ts-Pmy) as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host’s immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated. Methods and Findings The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy) to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs) elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration. Conclusion Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9. PMID:26720603

  15. Tanker avionics and aircrew complement evaluation.

    PubMed

    Moss, R W; Barbato, G J

    1982-11-01

    This paper describes an effort to determine control and display criteria for operating SAC's KC-135 tanker with a reduced crew complement. The Tanker Avionics and Aircrew Complement Evaluation (TAACE) Program was a four-phase effort addressing the control and display design issues associated with operating the tanker without the navigator position. Discussed are: the mission analysis phase, during which the tanker's operational responsibilities were defined and documented; the design phase, during which alternative crew station design concepts were developed; the mockup evaluation phase, which accomplished initial SAC crew member assessment of cockpit designs; and the simulation phase, which validated the useability of the crew system redesign. The paper also describes a recommended crew station configuration and discusses some of the philosophy underlying the selection of cockpit hardware and systems.

  16. Elucidating the role of the complement control protein in monkeypox pathogenicity.

    PubMed

    Hudson, Paul N; Self, Joshua; Weiss, Sonja; Braden, Zachary; Xiao, Yuhong; Girgis, Natasha M; Emerson, Ginny; Hughes, Christine; Sammons, Scott A; Isaacs, Stuart N; Damon, Inger K; Olson, Victoria A

    2012-01-01

    Monkeypox virus (MPXV) causes a smallpox-like disease in humans. Clinical and epidemiological studies provide evidence of pathogenicity differences between two geographically distinct monkeypox virus clades: the West African and Congo Basin. Genomic analysis of strains from both clades identified a ∼10 kbp deletion in the less virulent West African isolates sequenced to date. One absent open reading frame encodes the monkeypox virus homologue of the complement control protein (CCP). This modulatory protein prevents the initiation of both the classical and alternative pathways of complement activation. In monkeypox virus, CCP, also known as MOPICE, is a ∼24 kDa secretory protein with sequence homology to this superfamily of proteins. Here we investigate CCP expression and its role in monkeypox virulence and pathogenesis. CCP was incorporated into the West African strain and removed from the Congo Basin strain by homologous recombination. CCP expression phenotypes were confirmed for both wild type and recombinant monkeypox viruses and CCP activity was confirmed using a C4b binding assay. To characterize the disease, prairie dogs were intranasally infected and disease progression was monitored for 30 days. Removal of CCP from the Congo Basin strain reduced monkeypox disease morbidity and mortality, but did not significantly decrease viral load. The inclusion of CCP in the West African strain produced changes in disease manifestation, but had no apparent effect on disease-associated mortality. This study identifies CCP as an important immuno-modulatory protein in monkeypox pathogenesis but not solely responsible for the increased virulence seen within the Congo Basin clade of monkeypox virus. PMID:22496894

  17. Complement and phagocytes - A complicated interaction.

    PubMed

    Roos, Dirk

    2015-11-01

    Mohamed Daha and I share a common interest in innate immunity. Working in institutes only 25 miles away from each other, that meant ample opportunity and relevance for collaboration. And so we did. Moreover, we have both been members of boards and councils of Dutch national organizations, and we have also become good friends. In this short recollection, I look back on 40 years of common activities in complement research and friendship.

  18. Anti-complement sesquiterpenes from Viola yedoensis.

    PubMed

    Du, Dongsheng; Cheng, Zhihong; Chen, Daofeng

    2015-03-01

    Two new germacrane sesquiterpenes, yedoensins A (1) and B (2), together with 8 known ones (3-10) were isolated from the herb of Viola yedoensis. The structures of the new compounds were established by extensive spectroscopic means including 1D ((1)H and (13)C) and 2D NMR experiments (HSQC, HMBC, and NOESY) as well as HR-ESI-MS analysis. The absolute configurations of the known sesquiterpenes versicolactone B (3) and madolin W (6) were determined by a modified Mosher's method for the first time. The sesquiterpenes 1-3, and 5-9 exhibited anti-complement activity against the classical pathway (CP) and the alternative pathway (AP) with the CH50 and AP50 values ranging from 0.14 to 0.37mg/mL and 0.32 to 0.54mg/mL, respectively. Preliminary mechanism study using complement-depleted sera showed that yedoensin A (1) and versicolactone B (3) acted on C1q, C3 and C9, while madolin W (6), aristoyunnolin E (7) and madolin Y (9) interacted with C1q, C3, C5 and C9 components in the complement activation cascade.

  19. Bacteria under stress by complement and coagulation.

    PubMed

    Berends, Evelien T M; Kuipers, Annemarie; Ravesloot, Marietta M; Urbanus, Rolf T; Rooijakkers, Suzan H M

    2014-11-01

    The complement and coagulation systems are two related protein cascades in plasma that serve important roles in host defense and hemostasis, respectively. Complement activation on bacteria supports cellular immune responses and leads to direct killing of bacteria via assembly of the Membrane Attack Complex (MAC). Recent studies have indicated that the coagulation system also contributes to mammalian innate defense since coagulation factors can entrap bacteria inside clots and generate small antibacterial peptides. In this review, we will provide detailed insights into the molecular interplay between these protein cascades and bacteria. We take a closer look at how these pathways are activated on bacterial surfaces and discuss the mechanisms by which they directly cause stress to bacterial cells. The poorly understood mechanism for bacterial killing by the MAC will be reevaluated in light of recent structural insights. Finally, we highlight the strategies used by pathogenic bacteria to modulate these protein networks. Overall, these insights will contribute to a better understanding of the host defense roles of complement and coagulation against bacteria.

  20. Complement Activation and Inhibition in Retinal Diseases.

    PubMed

    Kleinman, Mark E; Ambati, Jayakrishna

    2016-01-01

    Within the past several decades, a brigade of dedicated researchers from around the world has provided essential insights into the critical niche of immune-mediated inflammation in the pathogenesis of age-related macular degeneration (AMD). Yet, the question has lingered as to whether disease-initiating events are more or less dependent on isolated immune-related responses, unimpeded inflammation, endogenous pathways of age-related cell senescence and oxidative stress, or any of the other numerous molecular derangements that have been identified in the natural history of AMD. There is now an abundant cache of data signifying immune system activation as an impetus in the pathogenesis of this devastating condition. Furthermore, recent rigorous investigations have revealed multiple inciting factors, including several important complement-activating components, thus creating a new array of disease-modulating targets for the research and development of molecular therapeutic interventions. While the precise in vivo effects of complement activation and inhibition in the progression and treatment of AMD remain to be determined, ongoing clinical trials of the first generation of complement-targeted therapeutics are hoped to yield critical data on the contribution of this pathway to the disease process. PMID:26501209

  1. Complement activation in amyloid plaques in Alzheimer's dementia.

    PubMed

    Eikelenboom, P; Hack, C E; Rozemuller, J M; Stam, F C

    1989-01-01

    Amyloid plaques in Alzheimer's dementia contain complement factors C1q, C4 and C3. In the present study we demonstrate complement activation in amyloid plaques using immunoenzymatical techniques and specific antibodies against subunits of individual complement components and activated complement products. Amyloid plaques contain C1q and activated C3 fragments (C3c and C3d, g) but no C1s and C3a. These findings demonstrate that the complement components are not passively bound to the amyloid plaque structures but are the result of an activation process. The role of complement activation in the genesis of senile plaques is discussed.

  2. Role of complement in graft rejection after organ transplantation.

    PubMed

    Bos, Ineke G A; Ten Berge, Ineke J M; Hack, C Erik

    2002-07-01

    Activation of the complement system may significantly contribute to the inflammatory reaction after solid organ transplantation. In allotransplantation, the complement system may be activated by ischemia/reperfusion and, possibly, by antibodies directed against the graft. In xenotransplantation from nonprimates to primates, the major activators for complement are preexisting antibodies. Studies in animal models have shown that the use of complement inhibitors may significantly prolong graft survival. This review describes the role of the complement system in organ injury after organ transplantation and the use of complement inhibitors to prevent damage to the graft after allo- or xenotransplantation.

  3. Paroxysmal nocturnal hemoglobinuria: a complement-mediated hemolytic anemia.

    PubMed

    DeZern, Amy E; Brodsky, Robert A

    2015-06-01

    Paroxysmal nocturnal hemoglobinuria is manifests with a chronic hemolytic anemia from uncontrolled complement activation, a propensity for thrombosis and marrow failure. The hemolysis is largely mediated by the alternative pathway of complement. Clinical manifestations result from the lack of specific cell surface proteins, CD55 and CD59, on PNH cells. Complement inhibition by eculizumab leads to dramatic clinical improvement. While this therapeutic approach is effective, there is residual complement activity resulting from specific clinical scenarios as well as from upstream complement components that can account for suboptimal responses in some patients. Complement inhibition strategies are an area of active research.

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

    PubMed

    Arroyave, C M; Tan, E M

    1976-01-01

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

  5. Complement component C3b and immunoglobulin Fc receptors on neutrophils from calves with leukocyte adhesion deficiency.

    PubMed

    Worku, M; Paape, M J; Di Carlo, A; Kehrli, M E; Marquardt, W W

    1995-04-01

    Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence-related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (LAD) on ligand binding and receptor expression. We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from LAD, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (CL) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of CL was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1. A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from LAD-affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and LAD-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between LAD-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from LAD-affected calves.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7785817

  6. Another really, really big virus.

    PubMed

    Van Etten, James L

    2011-01-01

    Viruses with genomes larger than 300 kb and up to 1.2 Mb, which encode hundreds of proteins, are being discovered and characterized with increasing frequency. Most, but not all, of these large viruses (often referred to as giruses) infect protists that live in aqueous environments. Bioinformatic analyses of metagenomes of aqueous samples indicate that large DNA viruses are quite common in nature and await discovery. One issue that is perhaps not appreciated by the virology community is that large viruses, even those classified in the same family, can differ significantly in morphology, lifestyle, and gene complement. This brief commentary, which will mention some of these unique properties, was stimulated by the characterization of the newest member of this club, virus CroV (Fischer, M.G.; Allen, M.J.; Wilson, W.H.; Suttle, C.A. Giant virus with a remarkable complement of genes infects marine zooplankton. Proc. Natl. Acad. Sci. USA 2010, 107, 19508-19513). CroV has a 730 kb genome (with ∼544 protein-encoding genes) and infects the marine microzooplankton Cafeteria roenbergensis producing a lytic infection.

  7. C-reactive protein activates complement in infarcted human myocardium.

    PubMed

    Nijmeijer, Remco; Lagrand, Wim K; Lubbers, Yvonne T P; Visser, Cees A; Meijer, Chris J L M; Niessen, Hans W M; Hack, C Erik

    2003-07-01

    Circulating levels of C-reactive protein (CRP) constitute a cardiovascular risk marker. Immunohistochemical studies have revealed co-localization of CRP and activated complement in human infarcted myocardium suggesting CRP to enhance inflammation in ischemic myocardium by inducing local complement activation. The aim was to establish whether CRP activates complement in infarcted human myocardium and to assess the relationship between this activation and the duration of infarction. Myocardial tissue samples from 56 patients that had died from acute myocardial infarction were evaluated. Specimens were taken from infarcted as well as noninfarcted sites of the heart. CRP-mediated complement activation was assessed by immunohistochemistry and by measuring levels of complement, CRP, and CRP-complement complexes, specific markers for CRP-mediated activation, in homogenates of the heart. Infarctions of 12 hours to 5 days had significantly more extensive depositions of complement and CRP and contained significantly more CRP, activated complement, and CRP-complement complexes than infarctions that were less than 12 hours old. Levels of CRP complexes correlated significantly with CRP and complement concentrations in the infarctions, as well as with the extent of complement and CRP depositions as measured via immunohistochemistry. Specific activation products of CRP-mediated activation of complement are increased in infarcts of more than 12 hours in duration and correlate with the extent of complement depositions. Hence, CRP seems to enhance local inflammatory reactions ensuing in human myocardial infarcts of more than 12 hours duration.

  8. Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction.

    PubMed Central

    Oh, Y K; Straubinger, R M

    1996-01-01

    Mycobacterium avium is a facultative intracellular pathogen that can survive and replicate within macrophages. We tested the hypotheses that survival mechanisms may include alteration of phagosomal pH or inhibition of phagosome-lysosome fusion. M. avium was surface labeled with N-hydroxysuccinimidyl esters of carboxyfluorescein (CF) and rhodamine (Rho) to enable measurement of the pH of individual M. avium-containing phagosomes and the interactions of bacterium-containing phagosomes with labeled secondary lysosomes. CF fluorescence is pH sensitive, whereas Rho is pH insensitive; pH can be calculated from their fluorescence ratios. Surface labeling of M. avium did not affect viability in broth cultures or within J774, a murine macrophage-like cell line. By fluorescence spectroscopy, live M. avium was exposed to an environmental pH of approximately 5.7 at 6 h after phagocytosis, whereas similarly labeled Salmonella typhimurium, zymosan A, or heat-killed M. avium encountered an environmental pH of < 5.0. Video fluorescence and laser scanning confocal microscopy gave consistent pH results and demonstrated the heterogeneity of intracellular fate early in infection. pH became more homogeneous 6 h after infection. M. avium cells were coated with immunoglobulin G (IgG) or opsonized to investigate whether phagocytosis by the corresponding receptors would alter intracellular fate. Opsonized, unopsonized, and IgG-coated M. avium cells entered compartments of similar pH. Finally, the spatial distribution of intracellular bacteria and secondary lysosomes was compared. Only 18% of live fluorescent M. avium cells colocalized with fluorescent lysosomes, while 98% of heat-killed bacteria colocalized. Thus, both inhibition of phagosome-lysosome fusion and alteration of phagosomal pH may contribute to the intracellular survival of M. avium. PMID:8557358

  9. Complement receptor type two (CR2,CR21): a target for influencing the humoral immune response and antigen-trapping.

    PubMed

    Prodinger, W M

    1999-01-01

    Cellular receptors for complement C3 fragments deposited on antigens are important bricks in the wall defending against microbial pathogens. The part of complement receptor type 2 (CR2; CD21) deals with enhancing humoral immune responses and with long-term trapping of C3d-coated antigen by follicular dendritic cells. CR2 is also pivotal for Epstein-Barr virus (EBV) infection. Here, the current understanding, how CR2 interacts with its ligands C3d, EBV, and CD23 is summarized. The potential to target CR2 for clinical therapy or immunization purposes are discussed. PMID:10741859

  10. Factor H-related proteins determine complement-activating surfaces.

    PubMed

    Józsi, Mihály; Tortajada, Agustin; Uzonyi, Barbara; Goicoechea de Jorge, Elena; Rodríguez de Córdoba, Santiago

    2015-06-01

    Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

  11. Age-related macular degeneration and the complement system.

    PubMed

    Khandhadia, S; Cipriani, V; Yates, J R W; Lotery, A J

    2012-02-01

    Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world. It is a complex multifactorial disease, and despite new advances in treatment, many patients still succumb to visual impairment. The complement pathway has been implicated in the pathogenesis of many diseases, and recently variants in several genes encoding complement pathway proteins have been associated with AMD. Complement proteins have been found in histological specimens of eyes with AMD. Altered levels of both intrinsic complement proteins and activated products have been found in the circulation of patients with AMD. Complement activation may be triggered by oxidative stress, resulting from retinal exposure to incoming light; indeed an inter-play between these two pathological processes seems to exist. Finally, complement inhibitors are currently being evaluated in clinical trials. This article reviews the role of the complement system in AMD, and the potential of complement inhibition in preventing the devastating blindness resulting from this disease.

  12. Versatile microfluidic complement fixation test for disease biomarker detection.

    PubMed

    Li, Man; Shi, ZhuanZhuan; Fang, Can; Gao, AnXiu; Li, Chang Ming; Yu, Ling

    2016-04-15

    The complement fixation test (CFT) is a serological test that can be used to detect the presence of specific antibodies or antigens to diagnose infections, particularly diseases caused by microbes that are not easily detected by standard culture methods. We report here, for the first time, a poly(dimethylsiloxane) (PDMS)/glass slide hybrid microfluidic device that was used to manipulate the solution compartment and communication within the microchannel to establish sampler and indicator systems of CFT. Two types of on-chip CFT, solution-based and solid phase agar-based assays, were successfully demonstrated for biomarker carcinoembryonic antigen (CEA) and recombinant avian influenza A (rH7N9) virus protein detection. In addition, the feasibility of the on-chip CFT in assaying real biopsy was successfully demonstrated by specifically detecting rH7N9 and CEA in human serum. The results demonstrated that the miniaturized assay format significantly reduced the assay time and sample consumption. Exemption from protein immobilization, blocking, complicated washing steps and expensive enzyme/fluorescein conjugates highlights the merits of on-chip CFT over ELISA. Most attractively, the on-chip agar-based CFT results can be imaged and analysed by smartphone, strengthening its point-of-care application potential. We anticipate that the on-chip CFT reported herein will be a useful supplemental or back-up tool for on-chip immunoassays such as ELISA for disease diagnosis and food inspection. PMID:27016440

  13. Complement sensitivity of Entamoeba histolytica and various nonpathogenic amoeba species.

    PubMed

    Förster, B; Ebert, F; Horstmann, R D

    1994-12-01

    Culture forms of the potentially pathogenic Entamoeba histolytica were compared to those of the nonpathogenic species of E. dispar, E. hartmanni, E. coli, Endolimax nana, and E. moshkovskii regarding the sensitivity to lysis by human complement activated through the alternative pathway. E. dispar was found unique in its complement resistance; all other nonpathogenic isolates resembled E. histolytica in that they were complement sensitive. Thus, a state of complement sensitivity is not a particular property of potentially pathogenic amoebae. PMID:7716404

  14. Complementing asteroseismology with 4MOST spectroscopy

    NASA Astrophysics Data System (ADS)

    de Jong, R. S.; 4MOST Consortium; 4MOST Spectroscopy Consortium

    2016-09-01

    4MOST is a wide-field, high-multiplex spectroscopic survey facility under development for the VISTA telescope of the European Southern Observatory (ESO). Its main science drivers are in the areas of galactic archeology, high-energy physics, galaxy evolution and cosmology. 4MOST will in particular provide the spectroscopic complements to the large area surveys coming from space missions like Gaia, eROSITA, Euclid, and PLATO. 4MOST will have an unique operations concept in which 5-years public surveys from both the consortium and the ESO community will be combined and observed in parallel during each exposure, resulting in more than 25 million spectra of targets spread over a large fraction of the southern sky. As a dedicated spectroscopic survey facility with a large field-of-view, a high multiplex that can be reconfigured quickly, and with a broad wavelength coverage, 4MOST is particularly well suited to complement the upcoming asteroseismology space missions like TESS and PLATO. Here we show that, by dedicating the observing time during twilight and poor observing conditions to bright stars, 4MOST will obtain resolution {R>18 000} spectra of nearly all stars brighter than ˜ 12th magnitude at Dec < 30o every ˜ 2 years. 4MOST is also expected to spectroscopically complement any fainter asteroseismology target to be observed with PLATO. These observations will provide a chemical characterization of nearly all stars to be observed with the TESS and PLATO missions and place any planets found in a full chemo-dynamical context of the star formation history of the Galaxy, yield very accurate ages and masses for all stars that can be characterized with asteroseismology, and allow removal of contaminants from target samples (e.g., spectroscopic binaries).

  15. VISUALIZATION OF MOLECULAR INTERACTIONS BY FLUORESCENCE COMPLEMENTATION

    PubMed Central

    Kerppola, Tom K.

    2008-01-01

    The visualization of protein complexes in living cells enables validation of protein interactions in their normal environment and determination of their subcellular localization. The bimolecular fluorescence complementation (BiFC) assay has been used to visualize interactions among multiple proteins in many cell types and organisms. This assay is based on the association between two fluorescent-protein fragments when they are brought together by an interaction between proteins fused to the fragments. Modified forms of this assay have been used to visualize the competition between alternative interaction partners and the covalent modification of proteins by ubiquitin family peptides. PMID:16625152

  16. Complement-independent binding of microorganisms to primate erythrocytes in vitro by cross-linked monoclonal antibodies via complement receptor 1.

    PubMed Central

    Powers, J H; Buster, B L; Reist, C J; Martin, E; Bridges, M; Sutherland, W M; Taylor, R P; Scheld, W M

    1995-01-01

    Under certain circumstances, soluble antigens, particulate antigens, and/or microorganisms have been shown to bind to primate erythrocytes via complement receptor 1 (CR1) in the presence of specific antibodies and complement. This immune adherence reaction, specific for CR1, can lead to neutralization of antigens in the circulation and their subsequent clearance from the blood. The present experiments utilized cross-linked monoclonal antibody complexes (heteropolymers) with specificity for both CR1 and either 35S-labeled herpes simplex virus capsid or Haemophilus influenzae as prototype viral and bacterial particulate antigens, respectively. In each case, the respective specific heteropolymers facilitated binding of the target antigens (> or = 70 to 90%) in vitro to erythrocytes in the absence of complement. Several experimental protocols were employed to demonstrate that heteropolymers mediate specific, rapid (> or = 30 s), and quantitative binding of prototypical particulate pathogens to human and monkey erythrocytes but not to sheep erythrocytes, which lack CR1. These results extend the potential use of the erythrocyte-heteropolymer system to the neutralization and clearance of particulate viral and bacterial pathogens from the blood. PMID:7890390

  17. A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

    PubMed

    Powers, Jason G; Sit, Tim L; Qu, Feng; Morris, T Jack; Kim, Kook-Hyung; Lommel, Steven A

    2008-07-01

    The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed. PMID:18533829

  18. A versatile assay for the identification of RNA silencing suppressors based on complementation of viral movement.

    PubMed

    Powers, Jason G; Sit, Tim L; Qu, Feng; Morris, T Jack; Kim, Kook-Hyung; Lommel, Steven A

    2008-07-01

    The cell-to-cell movement of Turnip crinkle virus (TCV) in Nicotiana benthamiana requires the presence of its coat protein (CP), a known suppressor of RNA silencing. RNA transcripts of a TCV construct containing a reporter gene (green fluorescent protein) (TCV-sGFP) in place of the CP open reading frame generated foci of three to five cells. TCV CP delivered in trans by Agrobacterium tumefaciens infiltration potentiated movement of TCV-sGFP and increased foci diameter, on average, by a factor of four. Deletion of the TCV movement proteins in TCV-sGFP (construct TCVDelta92-sGFP) abolished the movement complementation ability of TCV CP. Other known suppressors of RNA silencing from a wide spectrum of viruses also complemented the movement of TCV-sGFP when delivered in trans by Agrobacterium tumefaciens. These include suppressors from nonplant viruses with no known plant movement function, demonstrating that this assay is based solely on RNA silencing suppression. While the TCV-sGFP construct is primarily used as an infectious RNA transcript, it was also subcloned for direct expression from Agrobacterium tumefaciens for simple quantification of suppressor activity based on fluorescence levels in whole leaves. Thus, this system provides the flexibility to assay for suppressor activity in either the cytoplasm or nucleus, depending on the construct employed.

  19. The Production of Complement Clauses in Children with Language Impairment

    ERIC Educational Resources Information Center

    Steel, Gillian; Rose, Miranda; Eadie, Patricia

    2016-01-01

    Purpose: The purpose of this research was to provide a comprehensive description of complement-clause production in children with language impairment. Complement clauses were examined with respect to types of complement structure produced, verb use, and both semantic and syntactic accuracy. Method: A group of 17 children with language impairment…

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

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5240 Complement components immunological test system. (a) Identification. A complement components... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Complement components immunological test...

  1. False Belief, Complementation Language, and Contextual Bias in Preschoolers

    ERIC Educational Resources Information Center

    Ng, Lisa; Cheung, Him; Xiao, Wen

    2010-01-01

    In the present study, we address two questions concerning the relation between children's false belief and their understanding of complex object complements. The first question is whether the previously demonstrated association between tensed complements and false belief generalizes to infinitival complements (de Villiers & Pyers, 2002). The…

  2. Attenuation of Vaccinia Virus.

    PubMed

    Yakubitskiy, S N; Kolosova, I V; Maksyutov, R A; Shchelkunov, S N

    2015-01-01

    Since 1980, in the post-smallpox vaccination era the human population has become increasingly susceptible compared to a generation ago to not only the variola (smallpox) virus, but also other zoonotic orthopoxviruses. The need for safer vaccines against orthopoxviruses is even greater now. The Lister vaccine strain (LIVP) of vaccinia virus was used as a parental virus for generating a recombinant 1421ABJCN clone defective in five virulence genes encoding hemagglutinin (A56R), the IFN-γ-binding protein (B8R), thymidine kinase (J2R), the complement-binding protein (C3L), and the Bcl-2-like inhibitor of apoptosis (N1L). We found that disruption of these loci does not affect replication in mammalian cell cultures. The isogenic recombinant strain 1421ABJCN exhibits a reduced inflammatory response and attenuated neurovirulence relative to LIVP. Virus titers of 1421ABJCN were 3 lg lower versus the parent VACV LIVP when administered by the intracerebral route in new-born mice. In a subcutaneous mouse model, 1421ABJCN displayed levels of VACV-neutralizing antibodies comparable to those of LIVP and conferred protective immunity against lethal challenge by the ectromelia virus. The VACV mutant holds promise as a safe live vaccine strain for preventing smallpox and other orthopoxvirus infections. PMID:26798498

  3. Distance effects during polyprotein processing in the complementation between defective FMDV RNAs.

    PubMed

    Moreno, Elena; Perales, Celia

    2016-07-01

    Passage of foot-and-mouth disease virus (FMDV) in BHK-21 cells resulted in the segmentation of the viral genome into two defective RNAs lacking part of either the L- or the capsid-coding region. The two RNAs are infectious by complementation. Electroporation of L-defective RNA in BHK-21 cells resulted in the accumulation of the precursor P3 located away from the deleted sequence. Expression of L in trans led to the processing of P3, indicating that there is a connection between L protease activity and the secondary cleavages carried out by 3C protease within P3. These results suggest that the complementation mechanism between defective RNAs is not restricted to supplying the L and capsid proteins but that distance effects on polyprotein processing events are also implicated. PMID:27073008

  4. Structure of complement receptor 2 in complex with its C3d ligand.

    PubMed

    Szakonyi, G; Guthridge, J M; Li, D; Young, K; Holers, V M; Chen, X S

    2001-06-01

    Complement receptor 2 (CR2/CD21) is an important receptor that amplifies B lymphocyte activation by bridging the innate and adaptive immune systems. CR2 ligands include complement C3d and Epstein-Barr virus glycoprotein 350/220. We describe the x-ray structure of this CR2 domain in complex with C3d at 2.0 angstroms. The structure reveals extensive main chain interactions between C3d and only one short consensus repeat (SCR) of CR2 and substantial SCR side-side packing. These results provide a detailed understanding of receptor-ligand interactions in this protein family and reveal potential target sites for molecular drug design. PMID:11387479

  5. Prevalence of herpes simplex virus disease in a professional population.

    PubMed

    Brooks, S L; Rowe, N H; Drach, J C; Shipman, C; Young, S K

    1981-01-01

    By virtue of occupation, dentists are frequently exposed to the herpes simplex virus. The risk of infection by the virus was evaluated by assessing disease experience as shown by history compared with the results of complement fixing or antibody titration tests, or both.

  6. H-ficolin binds Aspergillus fumigatus leading to activation of the lectin complement pathway and modulation of lung epithelial immune responses.

    PubMed

    Bidula, Stefan; Sexton, Darren W; Yates, Matthew; Abdolrasouli, Alireza; Shah, Anand; Wallis, Russell; Reed, Anna; Armstrong-James, Darius; Schelenz, Silke

    2015-10-01

    Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H-Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H-ficolin in fungal defence. Additionally, we investigated the presence of H-ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H-Ficolin exhibited demonstrable binding to A. fumigatus conidia via l-fucose, d-mannose and N-acetylglucosamine residues in a calcium- and pH-dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H-ficolin opsonization manifested an increase in interleukin-8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen-activated protein kinase MAPK kinase 1/2, p38 MAPK and c-Jun N-terminal kinase. Finally, H-ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H-ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H-ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus-host interactions and modulated immune responses.

  7. Complement analysis 2016: Clinical indications, laboratory diagnostics and quality control.

    PubMed

    Prohászka, Zoltán; Nilsson, Bo; Frazer-Abel, Ashley; Kirschfink, Michael

    2016-11-01

    In recent years, complement analysis of body fluids and biopsies, going far beyond C3 and C4, has significantly enhanced our understanding of the disease process. Such expanded complement analysis allows for a more precise differential diagnosis and for critical monitoring of complement-targeted therapy. These changes are a result of the growing understanding of the involvement of complement in a diverse set of disorders. To appreciate the importance of proper complement analysis, it is important to understand the role it plays in disease. Historically, it was the absence of complement as manifested in severe infection that was noted. Since then complement has been connected to a variety of inflammatory disorders, such as autoimmune diseases and hereditary angioedema. While the role of complement in the rejection of renal grafts has been known longer, the significant impact of complement. In certain nephropathies has now led to the reclassification of some rare kidney diseases and an increased role for complement analysis in diagnosis. Even more unexpected is that complement has also been implicated in neural, ophtalmological and dermatological disorders. With this level of involvement in some varied and impactful health issues proper complement testing is clearly important; however, analysis of the complement system varies widely among laboratories. Except for a few proteins, such as C3 and C4, there are neither well-characterized standard preparations nor calibrated assays available. This is especially true for the inter-laboratory variation of tests which assess classical, alternative, or lectin pathway function. In addition, there is a need for the standardization of the measurement of complement activation products that are so critical in determining whether clinically relevant complement activation has occurred in vivo. Finally, autoantibodies to complement proteins (e.g. anti-C1q), C3 and C4 convertases (C3 and C4 nephritic factor) or to regulatory proteins

  8. Complement analysis 2016: Clinical indications, laboratory diagnostics and quality control.

    PubMed

    Prohászka, Zoltán; Nilsson, Bo; Frazer-Abel, Ashley; Kirschfink, Michael

    2016-11-01

    In recent years, complement analysis of body fluids and biopsies, going far beyond C3 and C4, has significantly enhanced our understanding of the disease process. Such expanded complement analysis allows for a more precise differential diagnosis and for critical monitoring of complement-targeted therapy. These changes are a result of the growing understanding of the involvement of complement in a diverse set of disorders. To appreciate the importance of proper complement analysis, it is important to understand the role it plays in disease. Historically, it was the absence of complement as manifested in severe infection that was noted. Since then complement has been connected to a variety of inflammatory disorders, such as autoimmune diseases and hereditary angioedema. While the role of complement in the rejection of renal grafts has been known longer, the significant impact of complement. In certain nephropathies has now led to the reclassification of some rare kidney diseases and an increased role for complement analysis in diagnosis. Even more unexpected is that complement has also been implicated in neural, ophtalmological and dermatological disorders. With this level of involvement in some varied and impactful health issues proper complement testing is clearly important; however, analysis of the complement system varies widely among laboratories. Except for a few proteins, such as C3 and C4, there are neither well-characterized standard preparations nor calibrated assays available. This is especially true for the inter-laboratory variation of tests which assess classical, alternative, or lectin pathway function. In addition, there is a need for the standardization of the measurement of complement activation products that are so critical in determining whether clinically relevant complement activation has occurred in vivo. Finally, autoantibodies to complement proteins (e.g. anti-C1q), C3 and C4 convertases (C3 and C4 nephritic factor) or to regulatory proteins

  9. Therapeutic inhibition of the early phase of complement activation.

    PubMed

    Roos, Anja; Ramwadhdoebé, Tamara H; Nauta, Alma J; Hack, C Erik; Daha, Mohamed R

    2002-09-01

    The complement system is a key component of innate immunity against invading pathogens. However, undesired activation of complement is involved in inflammation and associated tissue damage in a number of pathological conditions, such as ischemia/reperfusion injury, autoimmune diseases, and rejection of allo- and xenografts. During recent years, various therapeutically active complement inhibitors have been developed. In vivo studies using these inhibitors underscored the value of complement inhibition in the prevention of tissue damage. The currently available complement inhibitors mainly target the effector phase of the complement system that is common to all three activation pathways. Such a complete block of complement activation breaks the innate anti-microbial barrier, thereby increasing the risk for infection. Therefore, the development of potent complement inhibitors that interfere in the recognition phase of a specific complement activation pathway will generate important novel possibilities for treatment. The present review is focused on molecules that are able to inhibit the function of C1q and MBL, the recognition units of the classical pathway and the lectin pathway of complement, respectively. The potential value of these molecules for the development of therapeutically active complement inhibitors is discussed.

  10. The Complement System and Antibody-Mediated Transplant Rejection.

    PubMed

    Stites, Erik; Le Quintrec, Moglie; Thurman, Joshua M

    2015-12-15

    Complement activation is an important cause of tissue injury in patients with Ab-mediated rejection (AMR) of transplanted organs. Complement activation triggers a strong inflammatory response, and it also generates tissue-bound and soluble fragments that are clinically useful markers of inflammation. The detection of complement proteins deposited within transplanted tissues has become an indispensible biomarker of AMR, and several assays have recently been developed to measure complement activation by Abs reactive to specific donor HLA expressed within the transplant. Complement inhibitors have entered clinical use and have shown efficacy for the treatment of AMR. New methods of detecting complement activation within transplanted organs will improve our ability to diagnose and monitor AMR, and they will also help guide the use of complement inhibitory drugs.

  11. The Complement System and Antibody-Mediated Transplant Rejection.

    PubMed

    Stites, Erik; Le Quintrec, Moglie; Thurman, Joshua M

    2015-12-15

    Complement activation is an important cause of tissue injury in patients with Ab-mediated rejection (AMR) of transplanted organs. Complement activation triggers a strong inflammatory response, and it also generates tissue-bound and soluble fragments that are clinically useful markers of inflammation. The detection of complement proteins deposited within transplanted tissues has become an indispensible biomarker of AMR, and several assays have recently been developed to measure complement activation by Abs reactive to specific donor HLA expressed within the transplant. Complement inhibitors have entered clinical use and have shown efficacy for the treatment of AMR. New methods of detecting complement activation within transplanted organs will improve our ability to diagnose and monitor AMR, and they will also help guide the use of complement inhibitory drugs. PMID:26637661

  12. Targeted complement inhibition and microvasculature in transplants: a therapeutic perspective.

    PubMed

    Khan, M A; Hsu, J L; Assiri, A M; Broering, D C

    2016-02-01

    Active complement mediators play a key role in graft-versus-host diseases, but little attention has been given to the angiogenic balance and complement modulation during allograft acceptance. The complement cascade releases the powerful proinflammatory mediators C3a and C5a anaphylatoxins, C3b, C5b opsonins and terminal membrane attack complex into tissues, which are deleterious if unchecked. Blocking complement mediators has been considered to be a promising approach in the modern drug discovery plan, and a significant number of therapeutic alternatives have been developed to dampen complement activation and protect host cells. Numerous immune cells, especially macrophages, develop both anaphylatoxin and opsonin receptors on their cell surface and their binding affects the macrophage phenotype and their angiogenic properties. This review discusses the mechanism that complement contributes to angiogenic injury, and the development of future therapeutic targets by antagonizing activated complement mediators to preserve microvasculature in rejecting the transplanted organ.

  13. Immune evasion by herpes simplex virus type 1, strategies for virus survival.

    PubMed Central

    Friedman, Harvey M.

    2003-01-01

    Many viruses capable of persistent or recurrent infections have evolved strategies to evade host immunity. Viral evasion molecules target components of innate and acquired immunity, including complement proteins, natural killer cells, MHC Class I or Class II molecules and antibody. Our work focuses on HSV-1 glycoproteins gC and gE that impair antibody and complement responses. gC inhibits complement activation by binding C3b and blocking activities mediated by this pivotal complement protein, while gE binds the IgG Fc domain, blocking Fc-mediated activities, including complement activation and antibody-dependent cellular cytotoxicity. HSV-1 mutant viruses that lack the ability to bind C3b, IgG Fc, or both are much less virulent than wild-type virus in a murine model. These HSV-1 immunoevasins help explain the virus' ability to produce recurrent infections despite intact immunity. Strategies to prevent immune evasion may be required to develop successful HSV vaccines. PMID:12813914

  14. Acidosis activates complement system in vitro.

    PubMed Central

    Emeis, M; Sonntag, J; Willam, C; Strauss, E; Walka, M M; Obladen, M

    1998-01-01

    We investigated the in vitro effect of different forms of acidosis (pH 7.0) on the formation of anaphylatoxins C3a and C5a. Metabolic acidosis due to addition of hydrochloric acid (10 micromol/ml blood) or lactic acid (5.5 micromol/ml) to heparin blood (N=12) caused significant activation of C3a and C5a compared to control (both p=0.002). Respiratory acidosis activated C3a (p=0.007) and C5a (p=0.003) compared to normocapnic controls. Making blood samples with lactic acidosis hypocapnic resulted in a median pH of 7.37. In this respiratory compensated metabolic acidosis, C3a and C5a were not increased. These experiments show that acidosis itself and not lactate trigger for activation of complement components C3 and C5. PMID:9927235

  15. Variola virus immune evasion proteins.

    PubMed

    Dunlop, Lance R; Oehlberg, Katherine A; Reid, Jeremy J; Avci, Dilek; Rosengard, Ariella M

    2003-09-01

    Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.

  16. The Expression Profile of Complement Components in Podocytes.

    PubMed

    Li, Xuejuan; Ding, Fangrui; Zhang, Xiaoyan; Li, Baihong; Ding, Jie

    2016-01-01

    Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors. PMID:27043537

  17. The Expression Profile of Complement Components in Podocytes

    PubMed Central

    Li, Xuejuan; Ding, Fangrui; Zhang, Xiaoyan; Li, Baihong; Ding, Jie

    2016-01-01

    Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors. PMID:27043537

  18. A vital role for complement in heart disease.

    PubMed

    Lappegård, Knut T; Garred, Peter; Jonasson, Lena; Espevik, Terje; Aukrust, Pål; Yndestad, Arne; Mollnes, Tom E; Hovland, Anders

    2014-10-01

    Heart diseases are common and significant contributors to worldwide mortality and morbidity. During recent years complement mediated inflammation has been shown to be an important player in a variety of heart diseases. Despite some negative results from clinical trials using complement inhibitors, emerging evidence points to an association between the complement system and heart diseases. Thus, complement seems to be important in coronary heart disease as well as in heart failure, where several studies underscore the prognostic importance of complement activation. Furthermore, patients with atrial fibrillation often share risk factors both with coronary heart disease and heart failure, and there is some evidence implicating complement activation in atrial fibrillation. Moreover, Chagas heart disease, a protozoal infection, is an important cause of heart failure in Latin America, and the complement system is crucial for the protozoa-host interaction. Thus, complement activation appears to be involved in the pathophysiology of a diverse range of cardiac conditions. Determination of the exact role of complement in the various heart diseases will hopefully help to identify patients that might benefit from therapeutic complement intervention.

  19. The complement system in systemic lupus erythematosus: an update.

    PubMed

    Leffler, Jonatan; Bengtsson, Anders A; Blom, Anna M

    2014-09-01

    The complement system plays a major role in the autoimmune disease, systemic lupus erythematosus (SLE). However, the role of complement in SLE is complex since it may both prevent and exacerbate the disease. In this review, we explore the latest findings in complement-focused research in SLE. C1q deficiency is the strongest genetic risk factor for SLE, although such deficiency is very rare. Various recently discovered genetic associations include mutations in the complement receptors 2 and 3 as well as complement inhibitors, the latter related to earlier onset of nephritis. Further, autoantibodies are a distinct feature of SLE that are produced as the result of an adaptive immune response and how complement can affect that response is also being reviewed. SLE generates numerous disease manifestations involving contributions from complement such as glomerulonephritis and the increased risk of thrombosis. Furthermore, since most of the complement system is present in plasma, complement is very accessible and may be suitable as biomarker for diagnosis or monitoring of disease activity. This review highlights the many roles of complement for SLE pathogenesis and how research has progressed during recent years.

  20. Complement-mediated antiinflammatory effect of bisbenzylisoquinoline alkaloid fangchinoline.

    PubMed

    Hristova, M; Istatkova, R

    1999-11-01

    Complement-mediated mode of action of bisbenzylisoquinoline alkaloid fangchinoline was investigated in vivo and in vitro. The application of fangchinoline intraperitoneally (i.p.) to complement normal mice, strain ICR, inhibited the complement activity in serum and peritoneal exudate. The substance activated serum complement of C5-deficient DBA/2 mice. Fangchinoline was able to provoke local inflammatory reaction in both strains after subcutaneous (s.c.) injection. The alkaloid suppressed paw swelling induced by live Candida albicans in ICR and DBA/2 mice. Its effect depended on the dose and time of injection prior to inflammatory reaction. The in vitro experiments proved the interference of fangchinoline action with post-C5 reactions. The substance augmented C5-convertase formation and functional activity. These results are in correspondence with our previous investigations, proving the complement-mediated action of fangchinoline. The antiinflammatory effect could be a consequence of the caused complement exhaustion. PMID:11962544

  1. Complement-Mediated Regulation of Metabolism and Basic Cellular Processes.

    PubMed

    Hess, Christoph; Kemper, Claudia

    2016-08-16

    Complement is well appreciated as a critical arm of innate immunity. It is required for the removal of invading pathogens and works by directly destroying them through the activation of innate and adaptive immune cells. However, complement activation and function is not confined to the extracellular space but also occurs within cells. Recent work indicates that complement activation regulates key metabolic pathways and thus can impact fundamental cellular processes, such as survival, proliferation, and autophagy. Newly identified functions of complement include a key role in shaping metabolic reprogramming, which underlies T cell effector differentiation, and a role as a nexus for interactions with other effector systems, in particular the inflammasome and Notch transcription-factor networks. This review focuses on the contributions of complement to basic processes of the cell, in particular the integration of complement with cellular metabolism and the potential implications in infection and other disease settings. PMID:27533012

  2. Complement, a target for therapy in inflammatory and degenerative diseases.

    PubMed

    Morgan, B Paul; Harris, Claire L

    2015-12-01

    The complement system is a key innate immune defence against infection and an important driver of inflammation; however, these very properties can also cause harm. Inappropriate or uncontrolled activation of complement can cause local and/or systemic inflammation, tissue damage and disease. Complement provides numerous options for drug development as it is a proteolytic cascade that involves nine specific proteases, unique multimolecular activation and lytic complexes, an arsenal of natural inhibitors, and numerous receptors that bind to activation fragments. Drug design is facilitated by the increasingly detailed structural understanding of the molecules involved in the complement system. Only two anti-complement drugs are currently on the market, but many more are being developed for diseases that include infectious, inflammatory, degenerative, traumatic and neoplastic disorders. In this Review, we describe the history, current landscape and future directions for anti-complement therapies.

  3. Inventing Viruses.

    PubMed

    Summers, William C

    2014-11-01

    In the nineteenth century, "virus" commonly meant an agent (usually unknown) that caused disease in inoculation experiments. By the 1890s, however, some disease-causing agents were found to pass through filters that retained the common bacteria. Such an agent was called "filterable virus," the best known being the virus that caused tobacco mosaic disease. By the 1920s there were many examples of filterable viruses, but no clear understanding of their nature. However, by the 1930s, the term "filterable virus" was being abandoned in favor of simply "virus," meaning an agent other than bacteria. Visualization of viruses by the electron microscope in the late 1930s finally settled their particulate nature. This article describes the ever-changing concept of "virus" and how virologists talked about viruses. These changes reflected their invention and reinvention of the concept of a virus as it was revised in light of new knowledge, new scientific values and interests, and new hegemonic technologies.

  4. Inventing Viruses.

    PubMed

    Summers, William C

    2014-11-01

    In the nineteenth century, "virus" commonly meant an agent (usually unknown) that caused disease in inoculation experiments. By the 1890s, however, some disease-causing agents were found to pass through filters that retained the common bacteria. Such an agent was called "filterable virus," the best known being the virus that caused tobacco mosaic disease. By the 1920s there were many examples of filterable viruses, but no clear understanding of their nature. However, by the 1930s, the term "filterable virus" was being abandoned in favor of simply "virus," meaning an agent other than bacteria. Visualization of viruses by the electron microscope in the late 1930s finally settled their particulate nature. This article describes the ever-changing concept of "virus" and how virologists talked about viruses. These changes reflected their invention and reinvention of the concept of a virus as it was revised in light of new knowledge, new scientific values and interests, and new hegemonic technologies. PMID:26958713

  5. Expansion of Viral Host Range through Complementation and Recombination in Transgenic Plants.

    PubMed Central

    Schoelz, JE; Wintermantel, WM

    1993-01-01

    We have shown previously that gene VI of cauliflower mosaic virus (CaMV) strain D4 governs systemic infection of Nicotiana bigelovii and that transgenic N. bigelovii expressing the D4 gene VI product can complement at least one CaMV isolate for long-distance transport. We have now found that DNA of two other isolates of CaMV recombine with the gene VI coding sequence present in the transgenic plants. The formation of recombinant viruses occurs as a consequence of CaMV replication, involving two template switches during reverse transcription of the CaMV RNA to DNA. The first template switch occurs at the 5[prime] end of the 35S RNA to the gene VI mRNA produced by the transgenic plants. A second switch occurs at the 5[prime] end of the gene VI mRNA back to the 35S RNA. We also demonstrate that CaMV can acquire sequences from transgenic plants that alter the symptomatology and host range of the virus, an observation that may have important risk assessment implications for strategies using pathogen-derived resistance to protect plants against virus diseases. PMID:12271051

  6. CRP-mediated activation of complement in vivo: assessment by measuring circulating complement-C-reactive protein complexes.

    PubMed

    Wolbink, G J; Brouwer, M C; Buysmann, S; ten Berge, I J; Hack, C E

    1996-07-01

    The in vivo function of C-reactive protein (CRP) is unknown. Among the in vitro functions assigned to CRP is the ability to activate complement via the classical pathway. To date, there is no evidence supporting that CRP exerts this function in vivo. We here show a novel approach to assess CRP-mediated complement activation in vivo, which is based on the property that activated complement factors C3 and C4 fix to CRP during complement activation induced by this acute phase protein. We developed specific ELISAs for complexes between CRP and C4b, C4d, C3b, or C3d. We established that in vitro complement-CRP complexes were formed only during CRP-dependent activation, and not during activation by other activators, even in the presence of high CRP levels. Circulating levels of complement-CRP complexes were undetectable in normal donors, but significantly increased in nine patients following implantation of a renal allograft. Importantly, levels of complement-CRP complexes did not change in these patients upon a bolus infusion of mAb OKT3, which induces activation of the classical complement pathway, demonstrating in vivo that complement-CRP complexes are not formed during CRP-independent activation of complement, even when CRP is elevated. We conclude that measurement of complement-CRP complexes provides a suitable tool to study CRP-mediated activation of complement in vivo. Furthermore, increased levels of these complexes occur in clinical samples, indicating that CRP may induce activation of complement in vivo.

  7. A Molecular Insight into Complement Evasion by the Staphylococcal Complement Inhibitor Protein Family1

    PubMed Central

    Ricklin, Daniel; Tzekou, Apostolia; Garcia, Brandon L.; Hammel, Michal; McWhorter, William J.; Sfyroera, Georgia; Wu, You-Qiang; Holers, V. Michael; Herbert, Andrew P.; Barlow, Paul N.; Geisbrecht, Brian V.; Lambris, John D.

    2010-01-01

    Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its β-chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies. PMID:19625656

  8. Characterization of the Uukuniemi virus group (Phlebovirus: Bunyaviridae): evidence for seven distinct species.

    PubMed

    Palacios, Gustavo; Savji, Nazir; Travassos da Rosa, Amelia; Guzman, Hilda; Yu, Xuejie; Desai, Aaloki; Rosen, Gail Emilia; Hutchison, Stephen; Lipkin, W Ian; Tesh, Robert

    2013-03-01

    Evolutionary insights into the phleboviruses are limited because of an imprecise classification scheme based on partial nucleotide sequences and scattered antigenic relationships. In this report, the serologic and phylogenetic relationships of the Uukuniemi group viruses and their relationships with other recently characterized tick-borne phleboviruses are described using full-length genome sequences. We propose that the viruses currently included in the Uukuniemi virus group be assigned to five different species as follows: Uukuniemi virus, EgAn 1825-61 virus, Fin V707 virus, Chizé virus, and Zaliv Terpenia virus would be classified into the Uukuniemi species; Murre virus, RML-105-105355 virus, and Sunday Canyon virus would be classified into a Murre virus species; and Grand Arbaud virus, Precarious Point virus, and Manawa virus would each be given individual species status. Although limited sequence similarity was detected between current members of the Uukuniemi group and Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus, a clear serological reaction was observed between some of them, indicating that SFTSV and Heartland virus should be considered part of the Uukuniemi virus group. Moreover, based on the genomic diversity of the phleboviruses and given the low correlation observed between complement fixation titers and genetic distance, we propose a system for classification of the Bunyaviridae based on genetic as well as serological data. Finally, the recent descriptions of SFTSV and Heartland virus also indicate that the public health importance of the Uukuniemi group viruses must be reevaluated. PMID:23283959

  9. Characterization of the Uukuniemi Virus Group (Phlebovirus: Bunyaviridae): Evidence for Seven Distinct Species

    PubMed Central

    Savji, Nazir; Travassos da Rosa, Amelia; Guzman, Hilda; Yu, Xuejie; Desai, Aaloki; Rosen, Gail Emilia; Hutchison, Stephen; Lipkin, W. Ian; Tesh, Robert

    2013-01-01

    Evolutionary insights into the phleboviruses are limited because of an imprecise classification scheme based on partial nucleotide sequences and scattered antigenic relationships. In this report, the serologic and phylogenetic relationships of the Uukuniemi group viruses and their relationships with other recently characterized tick-borne phleboviruses are described using full-length genome sequences. We propose that the viruses currently included in the Uukuniemi virus group be assigned to five different species as follows: Uukuniemi virus, EgAn 1825-61 virus, Fin V707 virus, Chizé virus, and Zaliv Terpenia virus would be classified into the Uukuniemi species; Murre virus, RML-105-105355 virus, and Sunday Canyon virus would be classified into a Murre virus species; and Grand Arbaud virus, Precarious Point virus, and Manawa virus would each be given individual species status. Although limited sequence similarity was detected between current members of the Uukuniemi group and Severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus, a clear serological reaction was observed between some of them, indicating that SFTSV and Heartland virus should be considered part of the Uukuniemi virus group. Moreover, based on the genomic diversity of the phleboviruses and given the low correlation observed between complement fixation titers and genetic distance, we propose a system for classification of the Bunyaviridae based on genetic as well as serological data. Finally, the recent descriptions of SFTSV and Heartland virus also indicate that the public health importance of the Uukuniemi group viruses must be reevaluated. PMID:23283959

  10. Complement factor H–related hybrid protein deregulates complement in dense deposit disease

    PubMed Central

    Chen, Qian; Wiesener, Michael; Eberhardt, Hannes U.; Hartmann, Andrea; Uzonyi, Barbara; Kirschfink, Michael; Amann, Kerstin; Buettner, Maike; Goodship, Tim; Hugo, Christian; Skerka, Christine; Zipfel, Peter F.

    2013-01-01

    The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H–related (CFHR) gene cluster. This deletion resulted in expression of a hybrid CFHR2-CFHR5 plasma protein. The recombinant hybrid protein stabilized the C3 convertase and reduced factor H–mediated convertase decay. One patient was refractory to plasma replacement and exchange therapy, as evidenced by the hybrid protein quickly returning to pretreatment plasma levels. Subsequently, complement inhibitors were tested on serum from the patient for their ability to block activity of CFHR2-CFHR5. Soluble CR1 restored defective C3 convertase regulation; however, neither eculizumab nor tagged compstatin had any effect. Our findings provide insight into the importance of CFHR proteins for C3 convertase regulation and identify a genetic variation in the CFHR gene cluster that promotes C3G-DDD. Monitoring copy number and sequence variations in the CFHR gene cluster in C3G-DDD and kidney patients with C3G-DDD variations will help guide treatment strategies. PMID:24334459

  11. Expression of complement 3 and complement 5 in newt limb and lens regeneration.

    PubMed

    Kimura, Yuko; Madhavan, Mayur; Call, Mindy K; Santiago, William; Tsonis, Panagiotis A; Lambris, John D; Del Rio-Tsonis, Katia

    2003-03-01

    Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species. PMID:12594255

  12. Initiation and Regulation of Complement during Hemolytic Transfusion Reactions

    PubMed Central

    Stowell, Sean R.; Winkler, Anne M.; Maier, Cheryl L.; Arthur, C. Maridith; Smith, Nicole H.; Girard-Pierce, Kathryn R.; Cummings, Richard D.; Zimring, James C.; Hendrickson, Jeanne E.

    2012-01-01

    Hemolytic transfusion reactions represent one of the most common causes of transfusion-related mortality. Although many factors influence hemolytic transfusion reactions, complement activation represents one of the most common features associated with fatality. In this paper we will focus on the role of complement in initiating and regulating hemolytic transfusion reactions and will discuss potential strategies aimed at mitigating or favorably modulating complement during incompatible red blood cell transfusions. PMID:23118779

  13. Enteric adenovirus type 40: complementation of the E4 defect in Ad2 dl808.

    PubMed

    Mautner, V; Mackay, N

    1991-07-01

    The enteric adenovirus type 40 cannot be passaged in HeLa cells, but will grow productively in cells that express the E1B region of adenovirus types 2 or 5. Even in such permissive cells, the lytic cycle is prolonged, there is an abnormal pattern of E1B early gene expression and a failure to switch off host cell functions, suggesting that other gene functions might be impaired in Ad40. For Ad2, E4 ORF 6 and ORF 3 proteins are known to have an essential role in progressing from the early to the late phase of lytic infection and the shutoff of host functions requires an interaction between the E4 ORF 6 34K protein and the E1B 55K protein. To test whether E4 functions of Ad40 are impaired, complementation tests have been made between Ad40 and the E4 deletion mutant Ad2 dl808, which lacks all but ORF 1 of the E4 region. In HeLa and Vero cells, Ad40 complements dl808 to levels equivalent to an Ad2 wild-type infection, as demonstrated by measuring virion packaged DNA, virus titration, and viral protein synthesis. Surprisingly, Ad2 dl808 fails to reciprocally complement Ad40. The results show that Ad40 produces functional E4 ORF 6 and/or ORF 3 activity, and that their expression precedes DNA replication.

  14. Transfected lymphocyte extracts of patients with urological tumours: complement temperature-sensitive adenovirus mutants in vitro.

    PubMed

    Ongrádi, J; Csata, S; Farkas, J; Nász, I; Bendinelli, M

    1994-01-01

    Patients with renal or bladder cancers exhibit a unique association with adenovirus (Ad) infections. About 60% of them contain antibodies to Ad early antigens. Both in their tumour cells and peripheral blood lymphocytes (PBL) they have detectable early Ad antigens known to be involved in malignant cell transformation. Transfection of tumour cell extracts resulted in complementing temperature-sensitive (ts) Ad mutants at nonpermissive temperatures (39 degrees C) indicating that some cells of the tumour mass possess active functions for Ad. Only 4 to 18% of control subjects were positive in these tests. Here we studied whether lymphocytes might be involved in tumourigenesis by Ad. PBL extracts of patients were transfected into HEp-2 culture cells, which were subsequently superinfected with Ad-5 ts18 and ts19 mutants at 39 degrees C. Titration of virus yields indicated complementation in 76% of patients with renal and bladder cancers in contrast to 20% of control individuals. Complementing ability of lymphocytes which had been prestimulated with phytohaemagglutinin (PHA) approached that of tumour extracts. It means that both specimens contain advanced functions in contrast to resting lymphocytes. Lymphocytes are nonpermissive for latently carried Ad infections. Expression, possible transfer of early Ad gene products via frequent contacts with tissue cells can result in removal of tumour suppressor gene products from complexes regulating cell cycle negatively. Further interaction with hormone-sensitive protooncogenes explains tissue, age and gender specificity of urological malignancies. These phenomena suggest an important cofactorial role for Ad in kidney and bladder tumours.

  15. Complement alternative pathway genetic variation and Dengue infection in the Thai population.

    PubMed

    Kraivong, R; Vasanawathana, S; Limpitikul, W; Malasit, P; Tangthawornchaikul, N; Botto, M; Screaton, G R; Mongkolsapaya, J; Pickering, M C

    2013-11-01

    Dengue disease is a mosquito-borne infection caused by Dengue virus. Infection may be asymptomatic or variably manifest as mild Dengue fever (DF) to the most severe form, Dengue haemorrhagic fever (DHF). Mechanisms that influence disease severity are not understood. Complement, an integral component of the immune system, is activated during Dengue infection and the degree of activation increases with disease severity. Activation of the complement alternative pathway is influenced by polymorphisms within activation (factor B rs12614/rs641153, C3 rs2230199) and regulatory [complement factor H (CFH) rs800292] proteins, collectively termed a complotype. Here, we tested the hypothesis that the complotype influences disease severity during secondary Dengue infection. In addition to the complotype, we also assessed two other disease-associated CFH polymorphisms (rs1061170, rs3753394) and a structural polymorphism within the CFH protein family. We did not detect any significant association between the examined polymorphisms and Dengue infection severity in the Thai population. However, the minor allele frequencies of the factor B and C3 polymorphisms were less than 10%, so our study was not sufficiently powered to detect an association at these loci. We were also unable to detect a direct interaction between CFH and Dengue NS1 using both recombinant NS1 and DV2-infected culture supernatants. We conclude that the complotype does not influence secondary Dengue infection severity in the Thai population.

  16. Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode.

    PubMed

    Forneris, Federico; Wu, Jin; Xue, Xiaoguang; Ricklin, Daniel; Lin, Zhuoer; Sfyroera, Georgia; Tzekou, Apostolia; Volokhina, Elena; Granneman, Joke Cm; Hauhart, Richard; Bertram, Paula; Liszewski, M Kathryn; Atkinson, John P; Lambris, John D; Gros, Piet

    2016-05-17

    Regulators of complement activation (RCA) inhibit complement-induced immune responses on healthy host tissues. We present crystal structures of human RCA (MCP, DAF, and CR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologous CCP domains (i-iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations in CCP domains explain the diverse C3b-binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease-related mutations and immune evasion. PMID:27013439

  17. Complement system in dermatological diseases - fire under the skin.

    PubMed

    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.

  18. Complement--tapping into new sites and effector systems.

    PubMed

    Kolev, Martin; Le Friec, Gaelle; Kemper, Claudia

    2014-12-01

    Complement is traditionally known to be a system of serum proteins that provide protection against pathogens through direct cell lysis and the mobilization of innate and adaptive immunity. However, recent work indicates that the complement system has additional physiological roles beyond those in host defence. In this Opinion article, we describe the new modes and locations of complement activation that enable it to interact with other cell effector systems, such as growth factor receptors, inflammasomes and metabolic pathways. We propose that the location of complement activation dictates its function.

  19. 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

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

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

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

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  2. Complement binding to Leishmania donovani promastigotes (LD)

    SciTech Connect

    Puentes, S.M.; Bates, P.A.; Dwyer, D.M.; Joiner, K.A.

    1986-03-01

    To study the binding and processing of C3 on LD, parasites in various phases of growth were incubated in human serum deficient in complement component 8 containing /sup 125/I-C3. Uptake of /sup 125/I-C3 is rapid, peaking at 1.7-2.1 x 10/sup 6/ C3 molecules bound per parasite at 15 minutes for all growth phases, and decreases thereafter with continued incubation. One half of total C3 bound is spontaneously released by 90 minutes of incubation with all LD phases and occurs at a similar rate for LD washed free of serum and incubated at 37/sup 0/ C in buffer. As assessed by SDS-PAGE autoradiography, C3 on the surface of LD is present as C3b (36 to 50%) and iC3b (50 to 65%), linked covalently via a bond resistant to hydroxylamine treatment, presumably an amide linkage. Immunoblot analysis of purified membranes from serum-incubated LD, using rabbit antibody to C3 and LD surface constituents, strongly suggests that a major C3 acceptor is the LD acid phosphatase (AP). These results, in conjunction with recent studies, suggest a previously unrecognized role of AP as a C3 acceptor and, thus, as a molecule potentially involved in parasite binding and uptake.

  3. Altmetrics - a complement to conventional metrics.

    PubMed

    Melero, Remedios

    2015-01-01

    Emerging metrics based on article-level does not exclude traditional metrics based on citations to the journal, but complements them. Both can be employed in conjunction to offer a richer picture of an article use from immediate to long terms. Article-level metrics (ALM) is the result of the aggregation of different data sources and the collection of content from multiple social network services. Sources used for the aggregation can be broken down into five categories: usage, captures, mentions, social media and citations. Data sources depend on the tool, but they include classic metrics indicators based on citations, academic social networks (Mendeley, CiteULike, Delicious) and social media (Facebook, Twitter, blogs, or Youtube, among others). Altmetrics is not synonymous with alternative metrics. Altmetrics are normally early available and allow to assess the social impact of scholarly outputs, almost at the real time. This paper overviews briefly the meaning of altmetrics and describes some of the existing tools used to apply this new metrics: Public Library of Science--Article-Level Metrics, Altmetric, Impactstory and Plum.

  4. Altmetrics – a complement to conventional metrics

    PubMed Central

    Melero, Remedios

    2015-01-01

    Emerging metrics based on article-level does not exclude traditional metrics based on citations to the journal, but complements them. Both can be employed in conjunction to offer a richer picture of an article use from immediate to long terms. Article-level metrics (ALM) is the result of the aggregation of different data sources and the collection of content from multiple social network services. Sources used for the aggregation can be broken down into five categories: usage, captures, mentions, social media and citations. Data sources depend on the tool, but they include classic metrics indicators based on citations, academic social networks (Mendeley, CiteULike, Delicious) and social media (Facebook, Twitter, blogs, or Youtube, among others). Altmetrics is not synonymous with alternative metrics. Altmetrics are normally early available and allow to assess the social impact of scholarly outputs, almost at the real time. This paper overviews briefly the meaning of altmetrics and describes some of the existing tools used to apply this new metrics: Public Library of Science - Article-Level Metrics, Altmetric, Impactstory and Plum. PMID:26110028

  5. Altmetrics - a complement to conventional metrics.

    PubMed

    Melero, Remedios

    2015-01-01

    Emerging metrics based on article-level does not exclude traditional metrics based on citations to the journal, but complements them. Both can be employed in conjunction to offer a richer picture of an article use from immediate to long terms. Article-level metrics (ALM) is the result of the aggregation of different data sources and the collection of content from multiple social network services. Sources used for the aggregation can be broken down into five categories: usage, captures, mentions, social media and citations. Data sources depend on the tool, but they include classic metrics indicators based on citations, academic social networks (Mendeley, CiteULike, Delicious) and social media (Facebook, Twitter, blogs, or Youtube, among others). Altmetrics is not synonymous with alternative metrics. Altmetrics are normally early available and allow to assess the social impact of scholarly outputs, almost at the real time. This paper overviews briefly the meaning of altmetrics and describes some of the existing tools used to apply this new metrics: Public Library of Science--Article-Level Metrics, Altmetric, Impactstory and Plum. PMID:26110028

  6. A one`s complement cache memory

    SciTech Connect

    Yang, Q.; Adina, S.

    1994-12-31

    Most of today`s microprocessors have an on-chip cache to reduce average memory access latency. These on-chip caches generally have low associativity and small sizes. Cache line conflicts are the main source of cache misses which are essential to overall system performance. This paper introduces an innovative, conflict-free cache design, called one`s complement cache. By means of parallel computation of cache addresses and memory addresses of data, the new design does not increase critical hit time of cache accesses. Cache misses caused by line interferences are minimized by means of evenly distributing data items referenced by program loops across all sets in a cache. Evenly distribution of data in the cache is achieved by making the number of sets in the cache a prime or an odd number thereby the chance of related data being mapped to a same set is small. Trace-driven simulations are used to evaluate the performance of the new design. Performance results on a set of programs from SPEC92 benchmarks show that the new design improves cache performance over the conventional set-associative cache by about 100% with negligibly additional hardware cost.

  7. Complement dependent cytotoxicity (CDC) in chronic lymphocytic leukemia (CLL): Ofatumumab enhances alemtuzumab CDC and reveals cells resistant to activated complement

    PubMed Central

    Baig, Nisar A.; Taylor, Ronald P.; Lindorfer, Margaret A.; Church, Amy K.; LaPlant, Betsy R.; Pavey, Emily S.; Nowakowski, Grzegorz S.; Zent, Clive S.

    2016-01-01

    Complement dependent cytotoxicity (CDC) is an important mechanism of action for monoclonal antibodies (mAb) used in the treatment of chronic lymphocytic leukemia (CLL). We hypothesized that alemtuzumab (ALM) mediated CDC would be increased by addition of ofatumumab (OFA). CLL cells from 21 previously untreated patients with progressive disease were tested in vitro for mAb binding, complement activation, and CDC. The subpopulation of CDC resistant CLL cells was examined for levels of C3b and C5b-9 binding, and expression of complement regulatory proteins. OFA significantly increased complement activation and CDC in ALM-treated CLL cells suggesting that combining ALM and OFA could improve clinical outcome in patients with CLL. Approximately 10% of CLL cells were resistant to CDC because of lower levels of complement activation or decreased cytotoxicity of activated complement. Improvement of clinical responses will require determining the mechanisms of CDC resistance and developing methods to overcome this problem. PMID:22475085

  8. Generation of infectious virus particles from inducible transgenic genomes.

    PubMed

    Wernet, Mathias F; Klovstad, Martha; Clandinin, Thomas R

    2014-02-01

    Arboviruses like dengue virus, yellow fever virus, and West Nile virus are enveloped particles spread by mosquitoes, infecting millions of humans per year, with neither effective vaccines, nor specific antiviral therapies [1,2]. Previous studies of infection and virus replication utilize either purified virus particles or deficient genomes that do not complete the viral life cycle [1,2]. Here we describe transgenic Drosophila strains expressing trans-complementing genomes (referred to as 'replicons') from the arbovirus Sindbis [2]. We use this binary system to produce, for the first time in any metazoan, infectious virus particles through self-assembly from transgenes. Such cell-type specific particle 'launching' could serve as an attractive alternative for the development of virus-based tools and the study of virus biology in specific tissues.

  9. The Structure-Function Relationships of Complement Receptor Type 2 (CR2; CD21).

    PubMed

    Hannan, Jonathan Paul

    2016-01-01

    Human complement receptor type 2 (CR2; CD21) is a surface-associated glycoprotein which binds to a variety of endogenous ligands, including the complement component C3 fragments iC3b, C3dg and C3d, the low-affinity IgE receptor CD23, and the type I cytokine, interferon-alpha. CR2 links the innate complement-mediated immune response to pathogens and foreign antigens with the adaptive immune response by binding to C3d that is covalently attached to targets, and which results in a cell signalling phenomenon that lowers the threshold for B cell activation. Variations or deletions of the CR2 gene in humans, or the Cr2 gene in mice associate with a variety of autoimmune and inflammatory conditions. A number of infectious agents including Epstein-Barr virus (EBV), Human Immunodeficiency Virus (HIV) and prions also bind to CR2 either directly or indirectly by means of C3d-targeted immune complexes. In this review we discuss the interactions that CR2 undertakes with its best characterized ligands C3d, CD23 and the EBV gp350/220 envelope protein. To date only a single physiologically relevant complex of CR2 with one of its ligands, C3d, has been elucidated. By contrast, the interactions with CD23 and EBV gp350/220, while being important from physiologic and disease-associated standpoints, respectively, are only incompletely understood. A detailed knowledge of the structure-function relationships that CR2 undergoes with its ligands is necessary to understand the implications of using recombinant CR2 in therapeutic or imaging agents, or alternatively targeting CR2 to down-regulate the antibody mediated immune response in cases of autoimmunity.

  10. The Structure-Function Relationships of Complement Receptor Type 2 (CR2; CD21).

    PubMed

    Hannan, Jonathan Paul

    2016-01-01

    Human complement receptor type 2 (CR2; CD21) is a surface-associated glycoprotein which binds to a variety of endogenous ligands, including the complement component C3 fragments iC3b, C3dg and C3d, the low-affinity IgE receptor CD23, and the type I cytokine, interferon-alpha. CR2 links the innate complement-mediated immune response to pathogens and foreign antigens with the adaptive immune response by binding to C3d that is covalently attached to targets, and which results in a cell signalling phenomenon that lowers the threshold for B cell activation. Variations or deletions of the CR2 gene in humans, or the Cr2 gene in mice associate with a variety of autoimmune and inflammatory conditions. A number of infectious agents including Epstein-Barr virus (EBV), Human Immunodeficiency Virus (HIV) and prions also bind to CR2 either directly or indirectly by means of C3d-targeted immune complexes. In this review we discuss the interactions that CR2 undertakes with its best characterized ligands C3d, CD23 and the EBV gp350/220 envelope protein. To date only a single physiologically relevant complex of CR2 with one of its ligands, C3d, has been elucidated. By contrast, the interactions with CD23 and EBV gp350/220, while being important from physiologic and disease-associated standpoints, respectively, are only incompletely understood. A detailed knowledge of the structure-function relationships that CR2 undergoes with its ligands is necessary to understand the implications of using recombinant CR2 in therapeutic or imaging agents, or alternatively targeting CR2 to down-regulate the antibody mediated immune response in cases of autoimmunity. PMID:26916158

  11. C1q binding to dengue virus decreases levels of infection and inflammatory molecules transcription in THP-1 cells.

    PubMed

    Douradinha, Bruno; McBurney, Sean P; Soares de Melo, Klecia M; Smith, Amanda P; Krishna, Neel K; Barratt-Boyes, Simon M; Evans, Jared D; Nascimento, Eduardo J M; Marques, Ernesto T A

    2014-01-22

    Dengue virus infection elicits a spectrum of clinical presentations ranging from asymptomatic to severe disease. The mechanisms leading to severe dengue are not known, however it has been reported that the complement system is hyper-activated in severe dengue. Screening of complement proteins demonstrated that C1q, a pattern recognition molecule, can bind directly to dengue virus envelope protein and to whole dengue virus serotype 2. Incubation of dengue virus serotype 2 with C1q prior to infection of THP-1 cells led to decreased virus infectivity and modulation of mRNA expression of immunoregulatory molecules suggesting reduced inflammatory responses.

  12. Minor Role of Plasminogen in Complement Activation on Cell Surfaces

    PubMed Central

    Hyvärinen, Satu; Jokiranta, T. Sakari

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation. PMID:26637181

  13. Complement-Coagulation Cross-Talk: A Potential Mediator of the Physiological Activation of Complement by Low pH

    PubMed Central

    Kenawy, Hany Ibrahim; Boral, Ismet; Bevington, Alan

    2015-01-01

    The complement system is a major constituent of the innate immune system. It not only bridges innate and adaptive arms of the immune system but also links the immune system with the coagulation system. Current understanding of the role of complement has extended far beyond fighting of infections, and now encompasses maintenance of homeostasis, tissue regeneration, and pathophysiology of multiple diseases. It has been known for many years that complement activation is strongly pH sensitive, but only relatively recently has the physiological significance of this been appreciated. Most complement assays are carried out at the physiological pH 7.4. However, pH in some extracellular compartments, for example, renal tubular fluid in parts of the tubule, and extracellular fluid at inflammation loci, is sufficiently acidic to activate complement. The exact molecular mechanism of this activation is still unclear, but possible cross-talk between the contact system (intrinsic pathway) and complement may exist at low pH with subsequent complement activation. The current article reviews the published data on the effect of pH on the contact system and complement activity, the nature of the pH sensor molecules, and the clinical implications of these effects. Of particular interest is chronic kidney disease (CKD) accompanied by metabolic acidosis, in which therapeutic alkalinization of urine has been shown significantly to reduce tubular complement activation products, an effect, which may have important implications for slowing progression of CKD. PMID:25999953

  14. Complement and membrane-bound complement regulatory proteins as biomarkers and therapeutic targets for autoimmune inflammatory disorders, RA and SLE.

    PubMed

    Das, Nibhriti

    2015-11-01

    Complement system is a major effecter system of the innate immunity that bridges with adaptive immunity. The system consists of about 40 humoral and cell surface proteins that include zymogens, receptors and regulators. The zymogens get activated in a cascade fashion by antigen-antibody complex, antigen alone or by polymannans, respectively, by the classical, alternative and mannose binding lectin (MBL) pathways. The ongoing research on complement regulators and complement receptors suggest key role of these proteins in the initiation, regulation and effecter mechanisms of the innate and adaptive immunity. Although, the complement system provides the first line of defence against the invading pathogens, its aberrant uncontrolled activation causes extensive self tissue injury. A large number of humoral and cell surface complement regulatory protein keep the system well-regulated in healthy individuals. Complement profiling had brought important information on the pathophysiology of several infectious and chronic inflammatory disorders. In view of the diversity of the clinical disorders involving abnormal complement activity or regulation, which include both acute and chronic diseases that affect a wide range of organs, diverse yet specifically tailored therapeutic approaches may be needed to shift complement back into balance. This brief review discusses on the complement system, its functions and its importance as biomarkers and therapeutic targets for autoimmune diseases with focus on SLE and RA.

  15. Down-regulation of complement receptors on the surface of host monocyte even as in vitro complement pathway blocking interferes in dengue infection.

    PubMed

    Marinho, Cintia Ferreira; Azeredo, Elzinandes Leal; Torrentes-Carvalho, Amanda; Marins-Dos-Santos, Alessandro; Kubelka, Claire Fernandes; de Souza, Luiz José; Cunha, Rivaldo Venâncio; de-Oliveira-Pinto, Luzia Maria

    2014-01-01

    In dengue virus (DENV) infection, complement system (CS) activation appears to have protective and pathogenic effects. In severe dengue fever (DF), the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b), CR4 (CD11c) and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b) or CR3 (CD11b/CD18) reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b) or CR3 (CD11b/CD18) blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b) pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

  16. Down-Regulation of Complement Receptors on the Surface of Host Monocyte Even as In Vitro Complement Pathway Blocking Interferes in Dengue Infection

    PubMed Central

    Marinho, Cintia Ferreira; Azeredo, Elzinandes Leal; Torrentes-Carvalho, Amanda; Marins-Dos-Santos, Alessandro; Kubelka, Claire Fernandes; de Souza, Luiz José; Cunha, Rivaldo Venâncio; de-Oliveira-Pinto, Luzia Maria

    2014-01-01

    In dengue virus (DENV) infection, complement system (CS) activation appears to have protective and pathogenic effects. In severe dengue fever (DF), the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b), CR4 (CD11c) and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b) or CR3 (CD11b/CD18) reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b) or CR3 (CD11b/CD18) blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b) pathways could have implications for the treatment of viral infection by antiviral-related mechanisms. PMID:25061945

  17. The complement system of the duck.

    PubMed

    Koppenheffer, T L; Chan, S W; Higgins, D A

    1999-02-01

    Antibody (Ab)-dependent and-independent activation of the duck complement (C') system were studied. Ab-independent C' activity exhibited characteristics similar to those of the mammalian alternative C' pathway (ACP), including the selective lysis of rabbit erythrocytes (RRBC), a requirement for Mg2+, but not Ca2+, depletion of activity by zymosan, and lack of sensitivity to the mammalian C1 inhibitor carrageenan. Measurement of C' activity using antisera against sheep erythrocytes (SRBC) revealed that duck Abs activate C' by a pathway resembling the mammalian classical pathway (CCP) requiring both Ca2+ and Mg2+. Ab-dependent and-independent activities were further distinguishable by their kinetics of lysis and sensitivities to heat. Duck Abs were also found to activate C' in normal and carrageenan-treated serum by a mechanism that requires only Mg2+, and thus resembles the ACP. However, this Ab-dependent ACP-like activity exhibits patterns of ionic strength dependence and ontogeny which are clearly different from those of the conventional ACP and CCP. These findings indicate that duck C' can be activated by three mechanisms: Ab-mediated activation of the CCP, and Ab-mediated and Ab-independent activation of the ACP. Duck Ab responses to SRBC and RRBC were followed by direct agglutination, antiglobulin agglutination, and activation of the CCP and ACP. While the C'-activating abilities of duck anti-SRBC Abs persisted through a 3-month programme of inoculation, the anti-RRBC response lost its ability to activate C' after 2 weeks. PMID:16147546

  18. How antibodies use complement to regulate antibody responses.

    PubMed

    Sörman, Anna; Zhang, Lu; Ding, Zhoujie; Heyman, Birgitta

    2014-10-01

    Antibodies, forming immune complexes with their specific antigen, can cause complete suppression or several 100-fold enhancement of the antibody response. Immune complexes containing IgG and IgM may activate complement and in such situations also complement components will be part of the immune complex. Here, we review experimental data on how antibodies via the complement system upregulate specific antibody responses. Current data suggest that murine IgG1, IgG2a, and IgG2b upregulate antibody responses primarily via Fc-receptors and not via complement. In contrast, IgM and IgG3 act via complement and require the presence of complement receptors 1 and 2 (CR1/2) expressed on both B cells and follicular dendritic cells. Complement plays a crucial role for antibody responses not only to antigen complexed to antibodies, but also to antigen administered alone. Lack of C1q, but not of Factor B or MBL, severely impairs antibody responses suggesting involvement of the classical pathway. In spite of this, normal antibody responses are found in mice lacking several activators of the classical pathway (complement activating natural IgM, serum amyloid P component (SAP), specific intracellular adhesion molecule-grabbing non-integrin R1 (SIGN-R1) or C-reactive protein. Possible explanations to these observations will be discussed.

  19. Immunoconglutinin and complement changes in children with acute nephritis

    PubMed Central

    Ngu, J. L.; Soothill, J. F.

    1969-01-01

    Immunoconglutinin and electrophoretically altered forms of complement are detectable only after the fall in complement levels in acute nephritis, and may occur even when the fall is not noted. The delay between the postulated initiating streptococcal infection and the development of immunoconglutinin is longer than would be expected. The immunopathological significance of these findings is discussed. PMID:4189125

  20. Complement inhibitors to treat IgM-mediated autoimmune hemolysis

    PubMed Central

    Wouters, Diana; Zeerleder, Sacha

    2015-01-01

    Complement activation in autoimmune hemolytic anemia may exacerbate extravascular hemolysis and may occasionally result in intravascular hemolysis. IgM autoantibodies as characteristically found in cold autoantibody autoimmune hemolytic anemia, in cold agglutinin disease but also in a considerable percentage of patients with warm autoantibodies are very likely to activate complement in vivo. Therapy of IgM-mediated autoimmune hemolytic anemia mainly aims to decrease autoantibody production. However, most of these treatments require time to become effective and will not stop immediate ongoing complement-mediated hemolysis nor prevent hemolysis of transfused red blood cells. Therefore pharmacological inhibition of the complement system might be a suitable approach to halt or at least attenuate ongoing hemolysis and improve the recovery of red blood cell transfusion in autoimmune hemolytic anemia. In recent years, several complement inhibitors have become available in the clinic, some of them with proven efficacy in autoimmune hemolytic anemia. In the present review, we give a short introduction on the pathogenesis of autoimmune hemolytic anemia, followed by an overview on the complement system with a special focus on its regulation. Finally, we will discuss complement inhibitors with regard to their potential efficacy to halt or attenuate hemolysis in complement-mediated autoimmune hemolytic anemia. PMID:26521297

  1. Assessing reprogramming by chimera formation and tetraploid complementation.

    PubMed

    Li, Xin; Xia, Bao-long; Li, Wei; Zhou, Qi

    2015-01-01

    Pluripotent stem cells can be evaluated by pluripotent markers expression, embryoid body aggregation, teratoma formation, chimera contribution and even more, tetraploid complementation. Whether iPS cells in general are functionally equivalent to normal ESCs is difficult to establish. Here, we present the detailed procedure for chimera formation and tetraploid complementation, the most stringent criterion, to assessing pluripotency.

  2. Maximality and Idealized Cognitive Models: The Complementation of Spanish "Tener."

    ERIC Educational Resources Information Center

    Hilferty, Joseph; Valenzuela, Javier

    2001-01-01

    Discusses the bare-noun phrase (NP) complementation pattern of the Spanish verb "tener" (have). Shows that the maximality of the complement NP is dependent upon three factors: (1) idiosyncratic valence requirements; (2) encyclopedic knowledge related to possession; and (3) contextualized semantic construal. (Author/VWL)

  3. Production of Infinitival Complements by Children with Specific Language Impairment

    ERIC Educational Resources Information Center

    Arndt, Karen Barako; Schuele, C. Melanie

    2012-01-01

    The purpose of this study was to explore the production of infinitival complements by children with specific language impairment (SLI) as compared with mean length of utterance (MLU)-matched children in an effort to clarify inconsistencies in the literature. Spontaneous language samples were analysed for infinitival complements (reduced…

  4. Complement inhibitors to treat IgM-mediated autoimmune hemolysis.

    PubMed

    Wouters, Diana; Zeerleder, Sacha

    2015-11-01

    Complement activation in autoimmune hemolytic anemia may exacerbate extravascular hemolysis and may occasionally result in intravascular hemolysis. IgM autoantibodies as characteristically found in cold autoantibody autoimmune hemolytic anemia, in cold agglutinin disease but also in a considerable percentage of patients with warm autoantibodies are very likely to activate complement in vivo. Therapy of IgM-mediated autoimmune hemolytic anemia mainly aims to decrease autoantibody production. However, most of these treatments require time to become effective and will not stop immediate ongoing complement-mediated hemolysis nor prevent hemolysis of transfused red blood cells. Therefore pharmacological inhibition of the complement system might be a suitable approach to halt or at least attenuate ongoing hemolysis and improve the recovery of red blood cell transfusion in autoimmune hemolytic anemia. In recent years, several complement inhibitors have become available in the clinic, some of them with proven efficacy in autoimmune hemolytic anemia. In the present review, we give a short introduction on the pathogenesis of autoimmune hemolytic anemia, followed by an overview on the complement system with a special focus on its regulation. Finally, we will discuss complement inhibitors with regard to their potential efficacy to halt or attenuate hemolysis in complement-mediated autoimmune hemolytic anemia.

  5. The emerging role of complement inhibitors in transplantation.

    PubMed

    Frémeaux-Bacchi, Véronique; Legendre, Christophe M

    2015-11-01

    The role of complement in the biology of kidney transplantation is becoming more and more significant, especially but not only because we now have access to drugs inhibiting complement. After describing the main characteristics of complement biology, both activation of the complement cascade and the many regulatory factors, we will review the precise role of complement in kidney transplant biology. Complement activation has been involved in ischemia-reperfusion injury, in the recurrence of several diseases such as atypical hemolytic uremic syndrome, C3 glomerulopathies, and antiphospholipid syndrome, as well as the process of antibody-mediated rejection, either acute or chronic. There are many potentially interesting drugs interfering with complement inhibition that have been or may be studied in kidney transplantation. Currently, the bulk of data concerns eculizumab, a monoclonal antibody blocking the complement cascade at the C5. Its efficacy has been demonstrated in the treatment and prevention of recurrence of atypical hemolytic uremic syndrome with an overall good safety profile. Although it has been reported to be efficacious to prevent antibody-mediated rejection, properly designed trials are currently being performed to state this efficacy. In addition, randomized trials are, in the process, regarding the prevention of ischemia-reperfusion injury after kidney transplantation.

  6. A Viral Noncoding RNA Complements a Weakened Viral RNA Silencing Suppressor and Promotes Efficient Systemic Host Infection

    PubMed Central

    Flobinus, Alyssa; Hleibieh, Kamal; Klein, Elodie; Ratti, Claudio; Bouzoubaa, Salah; Gilmer, David

    2016-01-01

    Systemic movement of beet necrotic yellow vein virus (BNYVV) in Beta macrocarpa depends on viral RNA3, whereas in Nicotiana benthamiana this RNA is dispensable. RNA3 contains a coremin motif of 20 nucleotides essential for the stabilization of noncoding RNA3 (ncRNA3) and for long-distance movement in Beta species. Coremin mutants that are unable to accumulate ncRNA3 also do not achieve systemic movement in Beta species. A mutant virus carrying a mutation in the p14 viral suppressor of RNA silencing (VSR), unable to move long distances, can be complemented with the ncRNA3 in the lesion phenotype, viral RNA accumulation, and systemic spread. Analyses of the BNYVV VSR mechanism of action led to the identification of the RNA-dependent RNA polymerase 6 (RDR6) pathway as a target of the virus VSR and the assignment of a VSR function to the ncRNA3. PMID:27782046

  7. [Renal risks of dietary complements: a forgotten cause].

    PubMed

    Dori, Olympia; Humbert, Antoine; Burnier, Michel; Teta, Daniel

    2014-02-26

    The use of dietary complements like vitamins, minerals, trace elements, proteins, aminoacids and plant-derived agents is prevalent in the general population, in order to promote health and treat diseases. Dietary complements are considered as safe natural products and are easily available without prescription. However, these can lead to severe renal toxicity, especially in cases of unknown pre-existing chronic kidney disease (CKD). In particular, Chinese herbs including aristolochic acid, high doses of vitamine C, creatine and protein complements may lead to acute and chronic renal failure, sometimes irreversible. Dietary complement toxicity should be suspected in any case of unexplained renal impairement. In the case of pre-existing CKD, the use of potentially nephrotoxic dietary complements should be screened for.

  8. Novel Evasion Mechanisms of the Classical Complement Pathway.

    PubMed

    Garcia, Brandon L; Zwarthoff, Seline A; Rooijakkers, Suzan H M; Geisbrecht, Brian V

    2016-09-15

    Complement is a network of soluble and cell surface-associated proteins that gives rise to a self-amplifying, yet tightly regulated system with fundamental roles in immune surveillance and clearance. Complement becomes activated on the surface of nonself cells by one of three initiating mechanisms known as the classical, lectin, and alternative pathways. Evasion of complement function is a hallmark of invasive pathogens and hematophagous organisms. Although many complement-inhibition strategies hinge on hijacking activities of endogenous complement regulatory proteins, an increasing number of uniquely evolved evasion molecules have been discovered over the past decade. In this review, we focus on several recent investigations that revealed mechanistically distinct inhibitors of the classical pathway. Because the classical pathway is an important and specific mediator of various autoimmune and inflammatory disorders, in-depth knowledge of novel evasion mechanisms could direct future development of therapeutic anti-inflammatory molecules. PMID:27591336

  9. Recent Developments in Low Molecular Weight Complement Inhibitors

    PubMed Central

    Qu, Hongchang; Ricklin, Daniel; Lambris, John D.

    2009-01-01

    As a key part of the innate immune system, complement plays an important role not only in defending invading pathogens but also in many other biological processes. Inappropriate or excessive activation of complement has been linked to many autoimmune, inflammatory, and neurodegenerative diseases, as well as ischemia-reperfusion injury and cancer. A wide array of low molecular weight complement inhibitors has been developed to target various components of the complement cascade. Their efficacy has been demonstrated in numerous in vitro and in vivo experiments. Though none of these inhibitors has reached the market so far, some of them have entered clinical trials and displayed promising results. This review provides a brief overview of the currently developed low molecular weight complement inhibitors, including short peptides and synthetic small molecules, with an emphasis on those targeting components C1 and C3, and the anaphylatoxin receptors. PMID:19800693

  10. Novel Evasion Mechanisms of the Classical Complement Pathway.

    PubMed

    Garcia, Brandon L; Zwarthoff, Seline A; Rooijakkers, Suzan H M; Geisbrecht, Brian V

    2016-09-15

    Complement is a network of soluble and cell surface-associated proteins that gives rise to a self-amplifying, yet tightly regulated system with fundamental roles in immune surveillance and clearance. Complement becomes activated on the surface of nonself cells by one of three initiating mechanisms known as the classical, lectin, and alternative pathways. Evasion of complement function is a hallmark of invasive pathogens and hematophagous organisms. Although many complement-inhibition strategies hinge on hijacking activities of endogenous complement regulatory proteins, an increasing number of uniquely evolved evasion molecules have been discovered over the past decade. In this review, we focus on several recent investigations that revealed mechanistically distinct inhibitors of the classical pathway. Because the classical pathway is an important and specific mediator of various autoimmune and inflammatory disorders, in-depth knowledge of novel evasion mechanisms could direct future development of therapeutic anti-inflammatory molecules.

  11. [Renal risks of dietary complements: a forgotten cause].

    PubMed

    Dori, Olympia; Humbert, Antoine; Burnier, Michel; Teta, Daniel

    2014-02-26

    The use of dietary complements like vitamins, minerals, trace elements, proteins, aminoacids and plant-derived agents is prevalent in the general population, in order to promote health and treat diseases. Dietary complements are considered as safe natural products and are easily available without prescription. However, these can lead to severe renal toxicity, especially in cases of unknown pre-existing chronic kidney disease (CKD). In particular, Chinese herbs including aristolochic acid, high doses of vitamine C, creatine and protein complements may lead to acute and chronic renal failure, sometimes irreversible. Dietary complement toxicity should be suspected in any case of unexplained renal impairement. In the case of pre-existing CKD, the use of potentially nephrotoxic dietary complements should be screened for. PMID:24665660

  12. The complement system in ischemia-reperfusion injuries.

    PubMed

    Gorsuch, William B; Chrysanthou, Elvina; Schwaeble, Wilhelm J; Stahl, Gregory L

    2012-11-01

    Tissue injury and inflammation following ischemia and reperfusion of various organs have been recognized for many years. Many reviews have been written over the last several decades outlining the role of complement in ischemia/reperfusion injury. This short review provides a current state of the art knowledge on the complement pathways activated, complement components involved and a review of the clinical biologics/inhibitors used in the clinical setting of ischemia/reperfusion. This is not a complete review of the complement system in ischemia and reperfusion injury but will give the reader an updated view point of the field, potential clinical use of complement inhibitors, and the future studies needed to advance the field.

  13. Complement regulators in human disease: lessons from modern genetics.

    PubMed

    K Liszewski, M; Atkinson, J P

    2015-03-01

    First identified in human serum in the late 19th century as a 'complement' to antibodies in mediating bacterial lysis, the complement system emerged more than a billion years ago probably as the first humoral immune system. The contemporary complement system consists of nearly 60 proteins in three activation pathways (classical, alternative and lectin) and a terminal cytolytic pathway common to all. Modern molecular biology and genetics have not only led to further elucidation of the structure of complement system components, but have also revealed function-altering rare variants and common polymorphisms, particularly in regulators of the alternative pathway, that predispose to human disease by creating 'hyperinflammatory complement phenotypes'. To treat these 'complementopathies', a monoclonal antibody against the initiator of the membrane attack complex, C5, has received approval for use. Additional therapeutic reagents are on the horizon.

  14. Physicochemical signatures of nanoparticle-dependent complement activation

    NASA Astrophysics Data System (ADS)

    Thomas, Dennis G.; Chikkagoudar, Satish; Heredia-Langner, Alejandro; Tardiff, Mark F.; Xu, Zhixiang; Hourcade, Dennis E.; Pham, Christine T. N.; Lanza, Gregory M.; Weinberger, Kilian Q.; Baker, Nathan A.

    2014-01-01

    Nanoparticles are potentially powerful therapeutic tools that have the capacity to target drug payloads and imaging agents. However, some nanoparticles can activate complement, a branch of the innate immune system, and cause adverse side-effects. Recently, we employed an in vitro hemolysis assay to measure the serum complement activity of perfluorocarbon nanoparticles that differed by size, surface charge, and surface chemistry, quantifying the nanoparticle-dependent complement activity using a metric called Residual Hemolytic Activity (RHA). In the present work, we have used a decision tree learning algorithm to derive the rules for estimating nanoparticle-dependent complement response based on the data generated from the hemolytic assay studies. Our results indicate that physicochemical properties of nanoparticles, namely, size, polydispersity index, zeta potential, and mole percentage of the active surface ligand of a nanoparticle, can serve as good descriptors for prediction of nanoparticle-dependent complement activation in the decision tree modeling framework.

  15. Protease-dependent mechanisms of complement evasion by bacterial pathogens

    PubMed Central

    Potempa, Michal; Potempa, Jan

    2012-01-01

    The human immune system has evolved a variety of mechanisms for the primary task of neutralizing and eliminating microbial intruders. As the first line of defense, the complement system is responsible for rapid recognition and opsonisation of bacteria, presentation to phagocytes and bacterial cell killing by direct lysis. All successful human pathogens have mechanisms of circumventing the antibacterial activity of the complement system and escaping this stage of the immune response. One of the ways in which pathogens achieve this is the deployment of proteases. Based on the increasing number of recent publications in this area, it appears that proteolytic inactivation of the antibacterial activities of the complement system is a common strategy of avoiding targeting by this arm of host innate immune defense. In this review, we focus on those bacteria that deploy proteases capable of degrading complement system components into non-functional fragments, thus impairing complement-dependent antibacterial activity and facilitating pathogen survival inside the host. PMID:22944688

  16. Quantitative differences between complement factor-B phenotypes.

    PubMed

    Mortensen, J P; Lamm, L U

    1981-04-01

    In a study of 365 unrelated blood donors the structural polymorphism was determined by high-voltage electrophoresis followed by immunofixation with monospecific anti-Bf serum as described by Alper, Boenish & Watson (1972), and serum levels were measured by rocket-immunoelectrophoresis as described by Sjöholm (1975). We found that the mean level of Factor B in relation to structural phenotypes varies significantly in the order BfF greater than BfFS greater than BfS, though great variation was observed within types. A similar difference was also found with a functional assay of Factor B in agarose plates, although this method is less accurate. Concerning the biological functions, such as opsonization and bacteriolysis, it might be that individuals with the BfF allele are more able to withstand infectious agents than BfS subjects. The Bf polymorphism may therefore be transient, the BfF allele being in a process of replacing BfS by natural selection.

  17. Hide and Seek: How Lyme Disease Spirochetes Overcome Complement Attack

    PubMed Central

    Kraiczy, Peter

    2016-01-01

    Overcoming the first line of the innate immune system is a general hallmark of pathogenic microbes to avoid recognition and to enter the human host. In particular, spirochetes belonging to the Borrelia burgdorferi sensu lato complex have developed various means to counter the immune response and to successfully survive in diverse host environments for a prolonged period of time. In regard to complement resistance, Borrelia utilize a plethora of immune evasion strategies involves capturing of host-derived complement regulators, terminating complement activation as well as shedding of cell-destroying complement complexes to manipulate and to expeditiously inhibit human complement. Owing to their mode of action, the interacting surface-exposed proteins identified among B. burgdorferi sensu stricto (s.s.), Borrelia afzelii, Borrelia spielmanii, and Borrelia bavariensis can be classified into at least two major categories, namely, molecules that directly interfere with distinct complement components including BBK32, CspA, BGA66, BGA71, and a CD59-like protein or molecules, which indirectly counteract complement activation by binding various complement regulators such as Factor H, Factor H-like protein 1 (FHL-1), Factor H-related proteins FHR-1, FHR-2, or C4Bp. The latter group of genetically and structurally unrelated proteins has been collectively referred to as “complement regulator-acquiring surface proteins” and consists of CspA, CspZ, ErpA, ErpC, ErpP, and the as yet unidentified protein p43. This review focuses on the current knowledge of immune evasion mechanisms exhibited by Lyme disease spirochetes and highlights the role of complement-interfering, infection-associated molecules playing an important part in these processes. Deciphering the immune evasion strategies may provide novel avenues for improved diagnostic approaches and therapeutic interventions. PMID:27725820

  18. Zika Virus

    MedlinePlus

    Zika is a virus that is spread mostly by mosquitoes. A pregnant mother can pass it to ... through blood transfusions. There have been outbreaks of Zika virus in the United States, Africa, Southeast Asia, ...

  19. Zika Virus

    MedlinePlus

    ... Search The CDC Cancel Submit Search The CDC Zika Virus Note: Javascript is disabled or is not supported ... Areas with Zika Countries and territories with active Zika virus transmission... Mosquito Control Prevent mosquito bites, integrated mosquito ...

  20. Chikungunya Virus

    MedlinePlus

    ... traveling to countries with chikungunya virus, use insect repellent, wear long sleeves and pants, and stay in ... Chikungunya Prevention is key! Prevent Infection. Use mosquito repellent. Chikungunya Virus Distribution Chikungunya in the U.S. What's ...

  1. Complement Component C3 Binds to Activated Normal Platelets without Preceding Proteolytic Activation and Promotes Binding to Complement Receptor 1

    PubMed Central

    Hamad, Osama A.; Nilsson, Per H.; Wouters, Diana; Lambris, John D.; Ekdahl, Kristina N.; Nilsson, Bo

    2010-01-01

    It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor-activating peptide-activated platelets in lepirudin-anticoagulated platelet-rich plasma (PRP) and whole blood. However, inhibiting complement activation at the C1q or C3 level did not block the binding of C3 to activated platelets. Diluting PRP and chelating divalent cations also had no effect, further indicating that the deposition of complement components was independent of complement activation. Furthermore, washed, activated platelets bound added C1q and C3 to the same extent as platelets in PRP. The use of mAbs against different forms of C3 demonstrated that the bound C3 consisted of C3(H2O). Furthermore, exogenously added soluble complement receptor 1 was shown to bind to this form of platelet-bound C3. These observations indicate that there is no complement activation on the surface of platelets under physiological conditions. This situation is in direct contrast to a number of pathological conditions in which regulators of complement activation are lacking and thrombocytopenia and thrombotic disease are the ultimate result. However, the generation of C3(H2O) represents nonproteolytic activation of C3 and after factor I cleavage may act as a ligand for receptor binding. PMID:20139276

  2. Serum albumin 'camouflage' of plant virus based nanoparticles prevents their antibody recognition and enhances pharmacokinetics.

    PubMed

    Pitek, Andrzej S; Jameson, Slater A; Veliz, Frank A; Shukla, Sourabh; Steinmetz, Nicole F

    2016-05-01

    Plant virus-based nanoparticles (VNPs) are a novel class of nanocarriers with unique potential for biomedical applications. VNPs have many advantageous properties such as ease of manufacture and high degree of quality control. Their biocompatibility and biodegradability make them an attractive alternative to synthetic nanoparticles (NPs). Nevertheless, as with synthetic NPs, to be successful in drug delivery or imaging, the carriers need to overcome several biological barriers including innate immune recognition. Plasma opsonization can tag (V)NPs for clearance by the mononuclear phagocyte system (MPS), resulting in shortened circulation half lives and non-specific sequestration in non-targeted organs. PEG coatings have been traditionally used to 'shield' nanocarriers from immune surveillance. However, due to broad use of PEG in cosmetics and other industries, the prevalence of anti-PEG antibodies has been reported, which may limit the utility of PEGylation in nanomedicine. Alternative strategies are needed to tailor the in vivo properties of (plant virus-based) nanocarriers. We demonstrate the use of serum albumin (SA) as a viable alternative. SA conjugation to tobacco mosaic virus (TMV)-based nanocarriers results in a 'camouflage' effect more effective than PEG coatings. SA-'camouflaged' TMV particles exhibit decreased antibody recognition, as well as enhanced pharmacokinetics in a Balb/C mouse model. Therefore, SA-coatings may provide an alternative and improved coating technique to yield (plant virus-based) NPs with improved in vivo properties enhancing drug delivery and molecular imaging. PMID:26950168

  3. Serum albumin 'camouflage' of plant virus based nanoparticles prevents their antibody recognition and enhances pharmacokinetics.

    PubMed

    Pitek, Andrzej S; Jameson, Slater A; Veliz, Frank A; Shukla, Sourabh; Steinmetz, Nicole F

    2016-05-01

    Plant virus-based nanoparticles (VNPs) are a novel class of nanocarriers with unique potential for biomedical applications. VNPs have many advantageous properties such as ease of manufacture and high degree of quality control. Their biocompatibility and biodegradability make them an attractive alternative to synthetic nanoparticles (NPs). Nevertheless, as with synthetic NPs, to be successful in drug delivery or imaging, the carriers need to overcome several biological barriers including innate immune recognition. Plasma opsonization can tag (V)NPs for clearance by the mononuclear phagocyte system (MPS), resulting in shortened circulation half lives and non-specific sequestration in non-targeted organs. PEG coatings have been traditionally used to 'shield' nanocarriers from immune surveillance. However, due to broad use of PEG in cosmetics and other industries, the prevalence of anti-PEG antibodies has been reported, which may limit the utility of PEGylation in nanomedicine. Alternative strategies are needed to tailor the in vivo properties of (plant virus-based) nanocarriers. We demonstrate the use of serum albumin (SA) as a viable alternative. SA conjugation to tobacco mosaic virus (TMV)-based nanocarriers results in a 'camouflage' effect more effective than PEG coatings. SA-'camouflaged' TMV particles exhibit decreased antibody recognition, as well as enhanced pharmacokinetics in a Balb/C mouse model. Therefore, SA-coatings may provide an alternative and improved coating technique to yield (plant virus-based) NPs with improved in vivo properties enhancing drug delivery and molecular imaging.

  4. Antibody-dependent enhancement of dengue virus infection is inhibited by SA-17, a doxorubicin derivative.

    PubMed

    Ayala-Nuñez, Nilda V; Jarupathirun, Patsaporn; Kaptein, Suzanne J F; Neyts, Johan; Smit, Jolanda M

    2013-10-01

    Antibody-dependent enhancement (ADE) is thought to play a critical role in the exacerbation of dengue virus (DENV)-induced disease during a heterologous re-infection. Despite ADE's clinical impact, only a few antiviral compounds have been assessed for their anti-ADE activity. We reported earlier that SA-17, a doxorubicin derivative, efficiently inhibits the in vitro infection of DENV and yellow fever virus. Here we explored SA-17's mechanism of inhibition and investigated if the compound is active against ADE of DENV infection. Since enhanced infectivity stimulated by antibodies has been observed with standard and immature DENV, both types of virions were included in the study. We observed that SA-17 (i) inhibits DENV infection by preventing binding/entry to the cell and (ii) interferes with antibody-mediated infection of both standard and immature DENV2. SA-17 markedly reduced the infectivity of DENV2 in ADE conditions, with IC50s ranging from 0.26 to 2.89μM. The compound exerted its activity when added before, during, and after antibody-opsonization of standard and immature virus. Thus, molecules with the characteristics of SA-17 may be attractive antiviral agents since they can be used both to block DENV2 entry during primary and secondary infection and to inhibit ADE of standard and immature virus. PMID:23994499

  5. Complement in disease: a defence system turning offensive.

    PubMed

    Ricklin, Daniel; Reis, Edimara S; Lambris, John D

    2016-07-01

    Although the complement system is primarily perceived as a host defence system, a more versatile, yet potentially more harmful side of this innate immune pathway as an inflammatory mediator also exists. The activities that define the ability of the complement system to control microbial threats and eliminate cellular debris - such as sensing molecular danger patterns, generating immediate effectors, and extensively coordinating with other defence pathways - can quickly turn complement from a defence system to an aggressor that drives immune and inflammatory diseases. These host-offensive actions become more pronounced with age and are exacerbated by a variety of genetic factors and autoimmune responses. Complement can also be activated inappropriately, for example in response to biomaterials or transplants. A wealth of research over the past two decades has led to an increasingly finely tuned understanding of complement activation, identified tipping points between physiological and pathological behaviour, and revealed avenues for therapeutic intervention. This Review summarizes our current view of the key activating, regulatory, and effector mechanisms of the complement system, highlighting important crosstalk connections, and, with an emphasis on kidney disease and transplantation, discusses the involvement of complement in clinical conditions and promising therapeutic approaches.

  6. 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. PMID:26306442

  7. Complement activation promotes muscle inflammation during modified muscle use

    NASA Technical Reports Server (NTRS)

    Frenette, J.; Cai, B.; Tidball, J. G.

    2000-01-01

    Modified muscle use can result in muscle inflammation that is triggered by unidentified events. In the present investigation, we tested whether the activation of the complement system is a component of muscle inflammation that results from changes in muscle loading. Modified rat hindlimb muscle loading was achieved by removing weight-bearing from the hindlimbs for 10 days followed by reloading through normal ambulation. Experimental animals were injected with the recombinant, soluble complement receptor sCR1 to inhibit complement activation. Assays for complement C4 or factor B in sera showed that sCR1 produced large reductions in the capacity for activation of the complement system through both the classical and alternative pathways. Analysis of complement C4 concentration in serum in untreated animals showed that the classical pathway was activated during the first 2 hours of reloading. Analysis of factor B concentration in untreated animals showed activation of the alternative pathway at 6 hours of reloading. Administration of sCR1 significantly attenuated the invasion of neutrophils (-49%) and ED1(+) macrophages (-52%) that occurred in nontreated animals after 6 hours of reloading. The presence of sCR1 also reduced significantly the degree of edema by 22% as compared to untreated animals. Together, these data show that increased muscle loading activated the complement system which then briefly contributes to the early recruitment of inflammatory cells during modified muscle loading.

  8. Bullous pemphigoid autoantibodies directly induce blister formation without complement activation.

    PubMed

    Ujiie, Hideyuki; Sasaoka, Tetsumasa; Izumi, Kentaro; Nishie, Wataru; Shinkuma, Satoru; Natsuga, Ken; Nakamura, Hideki; Shibaki, Akihiko; Shimizu, Hiroshi

    2014-11-01

    Complement activation and subsequent recruitment of inflammatory cells at the dermal/epidermal junction are thought to be essential for blister formation in bullous pemphigoid (BP), an autoimmune blistering disease induced by autoantibodies against type XVII collagen (COL17); however, this theory does not fully explain the pathological features of BP. Recently, the involvement of complement-independent pathways has been proposed. To directly address the question of the necessity of the complement activation in blister formation, we generated C3-deficient COL17-humanized mice. First, we show that passive transfer of autoantibodies from BP patients induced blister formation in neonatal C3-deficient COL17-humanized mice without complement activation. By using newly generated human and murine mAbs against the pathogenic noncollagenous 16A domain of COL17 with high (human IgG1, murine IgG2), low (murine IgG1), or no (human IgG4) complement activation abilities, we demonstrate that the deposition of Abs, and not complements, is relevant to the induction of blister formation in neonatal and adult mice. Notably, passive transfer of BP autoantibodies reduced the amount of COL17 in lesional mice skin, as observed in cultured normal human keratinocytes treated with the same Abs. Moreover, the COL17 depletion was associated with a ubiquitin/proteasome pathway. In conclusion, the COL17 depletion induced by BP autoantibodies, and not complement activation, is essential for the blister formation under our experimental system.

  9. 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.

  10. Complement activation and protein adsorption by carbon nanotubes.

    PubMed

    Salvador-Morales, Carolina; Flahaut, Emmanuel; Sim, Edith; Sloan, Jeremy; Green, Malcolm L H; Sim, Robert B

    2006-02-01

    As a first step to validate the use of carbon nanotubes as novel vaccine or drug delivery devices, their interaction with a part of the human immune system, complement, has been explored. Haemolytic assays were conducted to investigate the activation of the human serum complement system via the classical and alternative pathways. Western blot and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques were used to elucidate the mechanism of activation of complement via the classical pathway, and to analyse the interaction of complement and other plasma proteins with carbon nanotubes. We report for the first time that carbon nanotubes activate human complement via both classical and alternative pathways. We conclude that complement activation by nanotubes is consistent with reported adjuvant effects, and might also in various circumstances promote damaging effects of excessive complement activation, such as inflammation and granuloma formation. C1q binds directly to carbon nanotubes. Protein binding to carbon nanotubes is highly selective, since out of the many different proteins in plasma, very few bind to the carbon nanotubes. Fibrinogen and apolipoproteins (AI, AIV and CIII) were the proteins that bound to carbon nanotubes in greatest quantity.

  11. Surface-bound capsular polysaccharide of type Ia group B Streptococcus mediates C1 binding and activation of the classic complement pathway

    SciTech Connect

    Levy, N.J.; Kasper, D.L.

    1986-06-01

    The role of surface-bound type Ia group B Streptococcus (GBS) capsular polysaccharide in anti-body-independent binding of C1 and activation of the classic component pathway was investigated. In a radiolabeled bacterial-polymorphonuclear leukocyte (PMN) association assay, a measure of bacterial opsonization, preincubation of /sup 3/H-type Ia GBS with purified F(ab')/sub 2/ to the organism blocked the association of the bacteria with PMN', and the inhibitory effect was dose dependent. The specificity of F(ab')/sub 2/ blocking was shown after adsorption of F(ab')/sub 2/ with type Ia polysaccharide-sensitized erythrocytes. Polysaccharide-adsorbed F(ab')/sub 2/ had a 70% decrease in ability to block the association of bacteria with PMN. Neuraminidase digestion removed 80% of the terminal sialic acid residues from the native polysaccharide. These neuraminidase-digested organisms had a 72% decrease in binding and transfer of purified C1 compared with non-enzyme-treated organisms. Type Ia capsular polysaccharide bound to sheep erythrocytes promoted classic complement pathway-mediated hemolysis of the cells. The role of C1 inhibitor (INH) in modulation of C1 activation by the organisms was investigated. The possibility existed that the C1 INH could be bound by the bacteria, allowing C1 activation to occur in the fluid phase. The inhibitor was purified from human serum, and its activity was measured before and after incubation with type Ia GBS. The organisms had no effect on C1 INH activity. Thus surface-bound capsular polysacchardie of type Ia GBS mediates C1 binding and classic pathway activation, and this does not involve the C1 INH.

  12. Complement protein C1q bound to apoptotic cells suppresses human macrophage and dendritic cell-mediated Th17 and Th1 T cell subset proliferation

    PubMed Central

    Clarke, Elizabeth V.; Weist, Brian M.; Walsh, Craig M.; Tenner, Andrea J.

    2015-01-01

    A complete genetic deficiency of the complement protein C1q results in SLE with nearly 100% penetrance in humans, but the molecular mechanisms responsible for this association have not yet been fully determined. C1q opsonizes ACs for enhanced ingestion by phagocytes, such as Mϕ and iDCs, avoiding the extracellular release of inflammatory DAMPs upon loss of the membrane integrity of the dying cell. We previously showed that human monocyte-derived Mϕ and DCs ingesting autologous, C1q-bound LALs (C1q-polarized Mϕ and C1q-polarized DCs), enhance the production of anti-inflammatory cytokines, and reduce proinflammatory cytokines relative to Mϕ or DC ingesting LAL alone. Here, we show that C1q-polarized Mϕ have elevated PD-L1 and PD-L2 and suppressed surface CD40, and C1q-polarized DCs have higher surface PD-L2 and less CD86 relative to Mϕ or DC ingesting LAL alone, respectively. In an MLR, C1q-polarized Mϕ reduced allogeneic and autologous Th17 and Th1 subset proliferation and demonstrated a trend toward increased Treg proliferation relative to Mϕ ingesting LAL alone. Moreover, relative to DC ingesting AC in the absence of C1q, C1q-polarized DCs decreased autologous Th17 and Th1 proliferation. These data demonstrate that a functional consequence of C1q-polarized Mϕ and DC is the regulation of Teff activation, thereby “sculpting” the adaptive immune system to avoid autoimmunity, while clearing dying cells. It is noteworthy that these studies identify novel target pathways for therapeutic intervention in SLE and other autoimmune diseases. PMID:25381385

  13. Surface antigen expression and complement susceptibility of differentiated neuroblastoma clones.

    PubMed

    Chen, S; Caragine, T; Cheung, N K; Tomlinson, S

    2000-03-01

    Human neuroblastoma cell lines typically consist of heterogenous subpopulations of cells that are morphologically and biochemically distinct. The cell types are characterized as neuroblastic (N-type), substrate-adherent Schwann-like (S-type), or intermediate (I). These cell types can undergo spontaneous or induced transdifferentiation in vitro. We investigated the complement sensitivity of different neuroblastoma cell lines and of matched sets of cloned N- and S-type neuroblastoma cell lines. Human neuroblastoma cell lines that consisted predominantly of a neuroblastic phenotype were shown to be significantly more susceptible to human complement-mediated lysis than cell lines of other cancer types. Complement sensitivity of neuroblastoma cell lines was correlated with low levels of CD59, decay-accelerating factor, and membrane cofactor protein expression. We found that cloned S-type neuroblastoma cells were much more resistant to complement-mediated lysis than cloned N-type cells. The increased complement resistance of S-type cells was shown to be due to increased expression of membrane-bound complement inhibitors. CD59 was the single most important protein in providing S-type cells with protection from complement lysis. S-type cells were also found to express lower levels of GD2, a target antigen for a complement activating monoclonal antibody currently in clinical trials for neuroblastoma immunotherapy. The ability of S-type cells to evade complement, and the ability of S-type cells to differentiate into the more tumorigenic N-type cells, may represent a mechanism of tumor survival and regrowth, a phenomenon often observed with this cancer.

  14. Role of the lectin complement pathway in kidney transplantation.

    PubMed

    Farrar, Conrad A; Zhou, Wuding; Sacks, Steven H

    2016-10-01

    In the last 15 years two major advances in the role of complement in the kidney transplant have come about. The first is that ischaemia reperfusion injury and its profound effect on transplant outcome is dependent on the terminal product of complement activation, C5b-9. The second key observation relates to the function of the small biologically active fragments C3a and C5a released by complement activation in increasing antigen presentation and priming the T cell response that results in transplant rejection. In both cases local synthesis of C3 principally by the renal tubule cells plays an essential role that overshadows the role of the circulating pool of C3 generated largely by hepatocyte synthesis. More recent efforts have investigated the molecules expressed by renal tissue that can trigger complement activation. These have revealed a prominent effect of collectin-11 (CL-11), a soluble C-type lectin that is expressed in renal tissue and aligns with its major ligand L-fucose at sites of complement activation following ischaemic stress. Biochemical studies have shown that interaction between CL-11 and L-fucose results in complement activation by the lectin complement pathway, precisely targeting the innate immune response to the ischaemic tubule surface. Therapeutic approaches to reduce inflammatory and immune stimulation in ischaemic kidney have so far targeted C3 or its activation products and several are in clinical trials. The finding that lectin-fucose interaction is an important trigger of lectin pathway complement activation within the donor organ opens up further therapeutic targets where intervention could protect the donor kidney against complement. PMID:27286717

  15. Novel Interallelic Complementation at the his1 Locus of Yeast

    PubMed Central

    Lax, Carol; Fogel, Seymour

    1978-01-01

    In yeast, 17 histidine-requiring mutants derived from and interallelically complementary to his1–7 were analyzed. The genetic basis of the complementation response was elucidated by mitotic and meiotic gene conversion. Each allele probably carries an unaltered 7-site mutation and a unique second-site alteration. The second-site alterations appear to be clustered within the proximal and distal segments of the his1 structural gene. Models of intraallelic complementation are reviewed in light of the unique complementational response between a single-site mutant and a double mutant including the identical altered base sequence. PMID:365680

  16. Virus Maturation

    PubMed Central

    Veesler, David; Johnson, John E.

    2013-01-01

    We examined virus maturation of selected non-enveloped and enveloped ssRNA viruses; retroviruses; bacteriophages and herpes virus. Processes associated with maturation in the RNA viruses range from subtle (noda and picornaviruses) to dramatic (tetraviruses and togaviruses). The elaborate assembly and maturation pathway of HIV is discussed in contrast to the less sophisticated but highly efficient processes associated with togaviruses. Bacteriophage assembly and maturation are discussed in general terms with specific examples chosen for emphasis. Finally the herpes viruses are compared with bacteriophages. The data support divergent evolution of noda, picorna and tetraviruses from a common ancestor and divergent evolution of alpha and flaviviruses from a common ancestor. Likewise, bacteriophages and herpes viruses almost certainly share a common ancestor in their evolution. Comparing all the viruses, we conclude that maturation is a convergent process that is required to solve conflicting requirements in biological dynamics and function. PMID:22404678

  17. Bivens arm virus: a new rhabdovirus isolated from Culicoides insignis in Florida and related to Tibrogargan virus of Australia.

    PubMed

    Gibbs, E P; Calisher, C H; Tesh, R B; Lazuick, J S; Bowen, R; Greiner, E C

    1989-02-01

    During field studies in 1981 on the transmission of bluetongue viruses in ruminants in Florida, a virus was isolated from Culicoides insignis collected near water buffalo (Bubalus bubalis) recently imported from Trinidad. Electron microscopy showed that this isolate, for which the name Bivens Arm virus is proposed, has rhabdovirus morphology. Serologic comparisons were made with recognized rhabdoviruses from terrestrial vertebrates and hematophagous arthropods. Indirect fluorescent antibody, complement fixation and neutralization tests indicated antigenic reactivity between Bivens Arm virus and two rhabdoviruses found only in Australia, Tibrogargan and Coastal Plains viruses. The Australian isolates cause subclinical infections in cattle and water buffalo and are believed to be transmitted by Culicoides. Initially, it was thought that Bivens Arm virus may have been introduced to Florida with the water buffalo from Trinidad, but a serologic survey of cattle serum, collected before the importation of the buffalo revealed antibody to the virus in cattle on farms located in diverse areas of Florida.

  18. Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

    PubMed

    Sharp, Julia A; Hair, Pamela S; Pallera, Haree K; Kumar, Parvathi S; Mauriello, Clifford T; Nyalwidhe, Julius O; Phelps, Cody A; Park, Dalnam; Thielens, Nicole M; Pascal, Stephen M; Chen, Waldon; Duffy, Diane M; Lattanzio, Frank A; Cunnion, Kenji M; Krishna, Neel K

    2015-01-01

    The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases. PMID:26196285

  19. Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses.

    PubMed

    Flipse, Jacky; Diosa-Toro, Mayra A; Hoornweg, Tabitha E; van de Pol, Denise P I; Urcuqui-Inchima, Silvio; Smit, Jolanda M

    2016-01-01

    The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts' antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in primary human macrophages. No differences were observed in the number of bound and internalized DENV particles following infection in the absence and presence of enhancing concentrations of antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong effects on infection, virus production and immune response. Importantly, and in contrast to other studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive to interferon. Additionally, this study gives insight in how human macrophages interact and respond to viral infections and the tight regulation thereof under various conditions of infection. PMID:27380892

  20. Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses

    PubMed Central

    Flipse, Jacky; Diosa-Toro, Mayra A.; Hoornweg, Tabitha E.; van de Pol, Denise P. I.; Urcuqui-Inchima, Silvio; Smit, Jolanda M.

    2016-01-01

    The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts’ antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in primary human macrophages. No differences were observed in the number of bound and internalized DENV particles following infection in the absence and presence of enhancing concentrations of antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong effects on infection, virus production and immune response. Importantly, and in contrast to other studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive to interferon. Additionally, this study gives insight in how human macrophages interact and respond to viral infections and the tight regulation thereof under various conditions of infection. PMID:27380892

  1. Mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing.

    PubMed Central

    Merino, S; Camprubí, S; Albertí, S; Benedí, V J; Tomás, J M

    1992-01-01

    The different mechanisms of Klebsiella pneumoniae resistance to complement-mediated killing were investigated by using different strains and isogenic mutants previously characterized for their surface components. We found that strains from serotypes whose K antigen masks the lipopolysaccharide (LPS) molecules (such as serotypes K1, K10, and K16) fail to activate complement, while strains with smooth LPS exposed at the cell surface (with or without K antigen) activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b binds far from the cell membrane and that the lytic final complex C5b-9 (membrane attack complex) is not formed. Isogenic rough mutants (K+ or K-) are serum sensitive because they bind C3b close to the cell membrane and the lytic complex (C5b-9) is formed. Images PMID:1587619

  2. Complement - a key system for immune surveillance and homeostasis

    PubMed Central

    Ricklin, Daniel; Hajishengallis, George; Yang, Kun; Lambris, John D.

    2010-01-01

    Nearly a century after the significance of the human complement system was recognized we have come to realize that its versatile functions extend far beyond the elimination of microbes. Indeed, complement acts as a rapid and efficient immune surveillance system that has distinct effects on healthy and altered host cells and foreign intruders. By eliminating cellular debris and infectious microbes, orchestrating immune responses, and sending `danger' signals, complement contributes substantially to homeostasis, but it may also take action against healthy cells if not properly controlled. This review describes our updated view of the function, structure, and dynamics of the complement network, highlights its interconnection with immunity at large and with other endogenous pathways, and illustrates its dual role in homeostasis and disease. PMID:20720586

  3. The role of the complement system in acute kidney injury.

    PubMed

    McCullough, James W; Renner, Brandon; Thurman, Joshua M

    2013-11-01

    Acute kidney injury is a common and severe clinical problem. Patients who develop acute kidney injury are at increased risk of death despite supportive measures such as hemodialysis. Research in recent years has shown that tissue inflammation is central to the pathogenesis of renal injury, even after nonimmune insults such as ischemia/reperfusion and toxins. Examination of clinical samples and preclinical models has shown that activation of the complement system is a critical cause of acute kidney injury. Furthermore, complement activation within the injured kidney is a proximal trigger of many downstream inflammatory events within the renal parenchyma that exacerbate injury to the kidney. Complement activation also may account for the systemic inflammatory events that contribute to remote organ injury and patient mortality. Complement inhibitory drugs have now entered clinical use and may provide an important new therapeutic approach for patients suffering from, or at high risk of developing, acute kidney injury.

  4. Complementing T-cell Function: An Inhibitory Role of the Complement System in T-cell-Mediated Antitumor Immunity.

    PubMed

    Peng, Weiyi; McKenzie, Jodi A; Hwu, Patrick

    2016-09-01

    New data from Wang and colleagues show that complement C3 suppresses the function of CD8(+) tumor-infiltrating T cells by inhibiting IL10 production, and targeting the complement receptors C3aR and C5aR enhances the antitumor activity of immune checkpoint blockade. Their results not only define a new role of complement receptors as T-cell coinhibitory receptors, but also are useful in the development of novel strategies to improve the effectiveness of cancer immunotherapy. Cancer Discov; 6(9); 953-5. ©2016 AACR.See related article by Wang et al., p. 1022. PMID:27587467

  5. Polysomnographic correlates of inflammatory complement components in young healthy males.

    PubMed

    Hussain, M Ejaz; Golam Sarwar, Abu Hasnath M; Alam, Mohd Shoeb; Noohu, Majumi M; Zannat, Wassilatul; Pandi-Perumal, Seithikurippu R; Bahammam, Ahmed S; Manzar, Md Dilshad

    2016-01-01

    A growing body of evidence has delineated the predominant role of humoral mediators of inflammation in linking sleep with immunity. Nonetheless, characterization of the relationship between complement components with inflammatory functions and objective sleep measures has not been performed. In this study we investigated the relationships between objective measures of sleep and complement components with inflammatory functions. Thirty-six healthy male university students (age, 23.94±4.23 years; BMI, 23.44±2.67 kg/m(2)) completed the study. An RMS Quest 32 polysomnograph (PSG) was used for sleep recording. Non-fasting blood was collected before subjects went to bed on the second night in the sleep laboratory to estimate complement component 3 (C-3), complement component 4 (C-4), complement factor-H (Factor-H), C1-inhibitor (C1INH), complement factor I (CFI) and other inflammatory mediators, such as IL-6 and sICAM-1. Multiple linear regression analysis was used to assess the association between PSG sleep measures and inflammatory mediators. Higher values of C-3 and lower values of sICAM-1, C1INH, and CFI (adjusted model, R2=0.211, p<0.041) predicted longer sleep duration. Lower C-3 (adjusted model, R2=0.078, p<0.055) predicted higher N1 (%). Higher levels of C1INH and CFI and lower values of C-4 (model adjusted R2=0.269, p<0.008) predicted higher N3 (%). Higher C-3, higher C-4, lower IL-6, lower C1INH and lower CFI (model adjusted R2=0.296, p<0.007) predicted higher REM (%). Poor sleep measures were associated with increased levels of pro-inflammatory complement components and decreased anti-inflammatory complement components. PMID:27656278

  6. Protective responses to sublytic complement in the retinal pigment epithelium.

    PubMed

    Tan, Li Xuan; Toops, Kimberly A; Lakkaraju, Aparna

    2016-08-01

    The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4(-/-) Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4(-/-) mice, indicating that they could be effective therapeutic approaches for macular degenerations. PMID:27432952

  7. Von Willebrand factor regulates complement on endothelial cells.

    PubMed

    Noone, Damien G; Riedl, Magdalena; Pluthero, Fred G; Bowman, Mackenzie L; Liszewski, M Kathryn; Lu, Lily; Quan, Yi; Balgobin, Steve; Schneppenheim, Reinhard; Schneppenheim, Sonja; Budde, Ulrich; James, Paula; Atkinson, John P; Palaniyar, Nades; Kahr, Walter H A; Licht, Christoph

    2016-07-01

    Atypical hemolytic uremic syndrome and thrombotic thrombocytopenic purpura have traditionally been considered separate entities. Defects in the regulation of the complement alternative pathway occur in atypical hemolytic uremic syndrome, and defects in the cleavage of von Willebrand factor (VWF)-multimers arise in thrombotic thrombocytopenic purpura. However, recent studies suggest that both entities are related as defects in the disease-causing pathways overlap or show functional interactions. Here we investigate the possible functional link of VWF-multimers and the complement system on endothelial cells. Blood outgrowth endothelial cells (BOECs) were obtained from 3 healthy individuals and 2 patients with Type 3 von Willebrand disease lacking VWF. Cells were exposed to a standardized complement challenge via the combination of classical and alternative pathway activation and 50% normal human serum resulting in complement fixation to the endothelial surface. Under these conditions we found the expected release of VWF-multimers causing platelet adhesion onto BOECs from healthy individuals. Importantly, in BOECs derived from patients with von Willebrand disease complement C3c deposition and cytotoxicity were more pronounced than on BOECs derived from normal individuals. This is of particular importance as primary glomerular endothelial cells display a heterogeneous expression pattern of VWF with overall reduced VWF abundance. Thus, our results support a mechanistic link between VWF-multimers and the complement system. However, our findings also identify VWF as a new complement regulator on vascular endothelial cells and suggest that VWF has a protective effect on endothelial cells and complement-mediated injury. PMID:27236750

  8. Antigenic variation among parainfluenza type 1 (Sendai) viruses: analysis of 6/94 virus.

    PubMed

    Lief, F S; Loh, W; Meulen, V T; Koprowski, H

    1975-01-01

    6/94 virus, isolated originally from a multiple sclerosis (MS) patient, was compared antigenically with related parainfluenza type 1 strains. These included two Sendai strains of mouse and two Sendai strains of reported human origin as well as the HA2 strain. By standard hemagglutination inhibition (HI) or hemadsorption neutralization (HAD-N) tests or by the complement-fixation (CF) cross-block titration test for detecting surface antigens, 6/94 virus and the Sendai virus strains were indistinguishable from each other but distinct from the HA2 strain. By the kinetic HI test, however, 6/94 virus could be readily differentiated from the Sendai viruses isolated from mice and more closely resembled those recovered from man.

  9. The Role of Complement System in Septic Shock

    PubMed Central

    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. PMID:23049598

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

    PubMed Central

    Salvadori, Maurizio; Rosso, Giuseppina; Bertoni, Elisabetta

    2015-01-01

    Complement cascade is involved in several renal diseases and in renal transplantation. The different components of the complement cascade might represent an optimal target for innovative therapies. In the first section of the paper the authors review the physiopathology of complement involvement in renal diseases and transplantation. In some cases this led to a reclassification of renal diseases moving from a histopathological to a physiopathological classification. The principal issues afforded are: renal diseases with complement over activation, renal diseases with complement dysregulation, progression of renal diseases and renal transplantation. In the second section the authors discuss the several complement components that could represent a therapeutic target. Even if only the anti C5 monoclonal antibody is on the market, many targets as C1, C3, C5a and C5aR are the object of national or international trials. In addition, many molecules proved to be effective in vitro or in preclinical trials and are waiting to move to human trials in the future. PMID:25949931

  11. Activation of human complement by immunoglobulin G antigranulocyte antibody.

    PubMed Central

    Rustagi, P K; Currie, M S; Logue, G L

    1982-01-01

    The ability of antigranulocyte antibody to fix the third component of complement (C3) to the granulocyte surface was investigated by an assay that quantitates the binding of monoclonal anti-C3 antibody to paraformaldehyde-fixed cells preincubated with Felty's syndrome serum in the presence of human complement. The sera from 7 of 13 patients with Felty's syndrome bound two to three times as much C3 to granulocytes as sera from patients with uncomplicated rheumatoid arthritis. The complement-activating ability of Felty's syndrome serum seemed to reside in the monomeric IgG-containing serum fraction. For those sera capable of activating complement, the amount of C3 fixed to granulocytes was proportional to the amount of granulocyte-binding IgG present in the serum. Thus, complement fixation appeared to be a consequence of the binding of antigranulocyte antibody to the cell surface. These studies suggest a role for complement-mediated injury in the pathophysiology of immune granulocytopenia, as has been demonstrated for immune hemolytic anemia and immune thrombocytopenia. PMID:7174786

  12. Staphylococcal proteases aid in evasion of the human complement system.

    PubMed

    Jusko, Monika; Potempa, Jan; Kantyka, Tomasz; Bielecka, Ewa; Miller, Halie K; Kalinska, Magdalena; Dubin, Grzegorz; Garred, Peter; Shaw, Lindsey N; Blom, Anna M

    2014-01-01

    Staphylococcus aureus is an opportunistic pathogen that presents severe health care concerns due to the prevalence of multiple antibiotic-resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component by aureolysin (Aur). We demonstrate here that four major extracellular proteases of S. aureus are potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8 and the metalloproteinase Aur resulted in a drastic decrease in the hemolytic activity of serum, whereas two staphylococcal serine proteases D and E, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureus mutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureus appear to be important for pathogen-mediated evasion of the human complement system.

  13. Exploitation of complement regulatory proteins by Borrelia and Francisella.

    PubMed

    Madar, Marian; Bencurova, Elena; Mlynarcik, Patrik; Almeida, André M; Soares, Renata; Bhide, Katarina; Pulzova, Lucia; Kovac, Andrej; Coelho, Ana V; Bhide, Mangesh

    2015-06-01

    Pathogens have developed sophisticated mechanisms of complement evasion such as binding to the host complement regulatory proteins (CRPs) on their surface or expression of CRP mimicking molecules. The ability of pathogens to evade the complement system has been correlated with pathogenesis and host selectivity. Hitherto, little work has been undertaken to determine whether Borrelia and Francisella exploit various CRPs to block complement attack. Seventeen Borrelia (twelve species) and six Francisella (three subspecies) strains were used to assess their ability to bind human, sheep and cattle CRPs or mimic membrane associated complement regulators. A series of experiments including affinity ligand binding experiments, pull-down assays and mass spectrometry based protein identification, revealed an array of CRP binding proteins of Borrelia and Francisella. Unlike Francisella, Borrelia strains were able to bind multiple human CRPs. Three strains of Borrelia (SKT-4, SKT-2 and HO14) showed the presence of a human CD46-homologous motif, indicating their ability to possess putative human CD46 mimicking molecules. Similarly, five strains of Borrelia and two strains of Francisella may have surface proteins with human CD59-homologous motifs. Among ovine and bovine CRPs, the only CRP bound by Francisella (LVS, Tul4 strain) was vitronectin, while ovine C4BP, ovine factor H and bovine factor H were bound to Borrelia strains SKT-2, DN127 and Co53. This study presents an array of proteins of Borrelia and Francisella that bind CRPs or may mimic membrane-CRPs, thus enabling multiphasic complement evasion strategies of these pathogens.

  14. Complement Activation-Related Pseudoallergy Caused by Nanomedicines and its Testing In Vitro and In Vivo

    NASA Astrophysics Data System (ADS)

    Szebeni, Janos; Urbanics, Rudolf

    Nanotechnology has has been giving birth to a variety of therapeutic and diagnostic products, referred to as nanomedicines (NM), whose successes are based on improved efficacy and/or diminished toxicity. However, these benefits are not without a price. The introduction into the clinics of many NM revealed the presence of an acute immune response to the particles, manifested in hypersensitivity reactions (HSR). The phenomenon is often due to the structural similarity of reactogenic NM to viruses, which may trigger the nonspecific arm of humoral immunity, the complement (C) system to an immediate eliminatory response. The clinical manifestations of this reaction, called C activation-related pseudoallergy (CARPA), include cardiopulmonary distress, which is a safety risk for NM, particularly in the case of cardiac patients with atopic constitution. Thus, understanding CARPA and ways of its prediction and prevention represents an important challenge in NM R&D.

  15. Expanding the Repertoire of Modified Vaccinia Ankara-Based Vaccine Vectors via Genetic Complementation Strategies

    PubMed Central

    Garber, David A.; O'Mara, Leigh A.; Zhao, Jun; Gangadhara, Sailaja; An, InChul; Feinberg, Mark B.

    2009-01-01

    Background Modified Vaccinia virus Ankara (MVA) is a safe, highly attenuated orthopoxvirus that is being developed as a recombinant vaccine vector for immunization against a number of infectious diseases and cancers. However, the expression by MVA vectors of large numbers of poxvirus antigens, which display immunodominance over vectored antigens-of-interest for the priming of T cell responses, and the induction of vector-neutralizing antibodies, which curtail the efficacy of subsequent booster immunizations, remain as significant impediments to the overall utility of such vaccines. Thus, genetic approaches that enable the derivation of MVA vectors that are antigenically less complex may allow for rational improvement of MVA-based vaccines. Principal Findings We have developed a genetic complementation system that enables the deletion of essential viral genes from the MVA genome, thereby allowing us to generate MVA vaccine vectors that are antigenically less complex. Using this system, we deleted the essential uracil-DNA-glycosylase (udg) gene from MVA and propagated this otherwise replication-defective variant on a complementing cell line that constitutively expresses the poxvirus udg gene and that was derived from a newly identified continuous cell line that is permissive for growth of wild type MVA. The resulting virus, MVAΔudg, does not replicate its DNA genome or express late viral gene products during infection of non-complementing cells in culture. As proof-of-concept for immunological ‘focusing’, we demonstrate that immunization of mice with MVAΔudg elicits CD8+ T cell responses that are directed against a restricted repertoire of vector antigens, as compared to immunization with parental MVA. Immunization of rhesus macaques with MVAΔudg-gag, a udg− recombinant virus that expresses an HIV subtype-B consensus gag transgene, elicited significantly higher frequencies of Gag-specific CD8 and CD4 T cells following both primary (2–4-fold) and booster (2

  16. Structural Basis for Antagonism by Suramin of Heparin Binding to Vaccinia Complement Protein

    SciTech Connect

    Ganesh, Vannakambadi K.; Muthuvel, Suresh Kumar; Smith, Scott A.; Kotwal, Girish J.; Murthy, Krishna H.M.

    2010-07-19

    Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complex with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.

  17. Optimization of the virus concentration method using polyethyleneimine-conjugated magnetic beads and its application to the detection of human hepatitis A, B and C viruses.

    PubMed

    Uchida, Eriko; Kogi, Mieko; Oshizawa, Tadashi; Furuta, Birei; Satoh, Koei; Iwata, Akiko; Murata, Mitsuhiro; Hikata, Mikio; Yamaguchi, Teruhide

    2007-07-01

    To enhance the sensitivity of virus detection by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR), a novel virus concentration method using polyethyleneimine (PEI)-conjugated magnetic beads was developed in our previous study. However, several viruses could not be concentrated by this method. In this paper, the conditions of virus concentration were optimized to concentrate a wide range of viruses more efficiently. The PEI beads adsorbed viruses more efficiently than other cationic polymers, and the optimum virus concentration was obtained under weak acidic conditions. Mass spectrometric analysis revealed that several serum proteins, such as complement type 3, complement type 4 and immunoglobulin M (IgM), were co-adsorbed by the PEI beads, suggesting that the beads may adsorb viruses not only by direct adsorption, but also via immune complex formation. This hypothesis was confirmed by the result that poliovirus, which PEI beads could not adsorb directly, could be concentrated by the beads via immune complex formation. On the other hand, hepatitis A (HAV) and hepatitis C (HCV) viruses were adsorbed directly by PEI beads almost completely. Like poliovirus, hepatitis B virus (HBV) was concentrated efficiently by the addition of anti-HBV IgM. In conclusion, virus concentration using PEI beads is a useful method to concentrate a wide range of viruses and can be used to enhance the sensitivity of detection of HAV, HBV and HCV.

  18. Complementation of NADPH oxidase in p67-phox-deficient CGD patients p67-phox/p40-phox interaction.

    PubMed

    Vergnaud, S; Paclet, M H; El Benna, J; Pocidalo, M A; Morel, F

    2000-02-01

    Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.

  19. Genetic complementation in heterokaryons of human fibroblasts defective in cobalamin metabolism.

    PubMed Central

    Gravel, R A; Mahoney, M J; Ruddle, F H; Rosenberg, L E

    1975-01-01

    Inherited methylmalonicacidemia due to deficiency of methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase; EC 5.4.99.2) activity results from at least three classes of biochemically distinct defects affecting cobalamin (Cbl: vitamin B12) metabolism (cbl A, cbl B, and cbl C mutants) and a fourth class producing a defective mutase apoenzyme. We have obtained genetic evidence in support of this biochemical heterogeneity, using heterokaryons prepared by Sendai-virus-mediated cell fusion. Nine fibroblast lines from patients with defective Cbl metabolism (4 cbl A, 3 cbl B, and 2 cbl C), two from patients with defective mutase apoenzyme, and two from controls were fused in pairwise combinations and tested for functional mutase holoenzyme using a radioautographic procedure which detects [14C]propionate incorporation into trichloroacetic-acid-precipitable material in fibroblast monolayers in situ. Each of the mutants incorporates negligible radioactivity compared to control cells. Activity is also negligible when different mutants are mixed without virus or when homokaryons are produced by self-fusion. Heterokaryons produced by fusing members of each of the four mutant classes with representatives of any other class recover the ability to incorporate [14C]propionate to levels comparable to those of control cells. However, heterokaryons produced between members of the same class fail to complement in all cases. We conclude that the mutants with defective Cbl metabolism (cbl A, cbl B, cbl C) comprise three complementation groups, that a fourth group corresponds to mutase apoenzyme deficiency, and that all four classes of mutations are recessively inherited. Images PMID:1059104

  20. Complement Activation in Patients with Focal Segmental Glomerulosclerosis

    PubMed Central

    Thurman, Joshua M.; Wong, Maria; Renner, Brandon; Frazer-Abel, Ashley; Giclas, Patricia C.; Joy, Melanie S.; Jalal, Diana; Radeva, Milena K.; Gassman, Jennifer; Gipson, Debbie S.; Kaskel, Frederick; Friedman, Aaron; Trachtman, Howard

    2015-01-01

    Background Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process. Study Design Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed. Setting and Participants We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis. Outcomes Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR). Measurements Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine. Results Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study. Limitations Limited number of patients with samples from all time-points. Conclusions The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of

  1. Chronic rabies virus infection of cell cultures.

    PubMed

    Wiktor, T J; Clark, H F

    1972-12-01

    Exposure of both mammalian and reptilian cells in tissue culture to different strains of fixed rabies virus resulted in a carrier type of infection. No cytopathic effect was observed in either type of culture; infected cultures could be maintained by cell transfer for unlimited numbers of passages. A consistent pattern of cyclically rising and falling levels of viral infection was observed by fluorescent-antibody staining techniques and by titration of released infectious virus. Resistance to super-infection by vesicular stomatis virus and the production of an interferon-like substance by infected cells indicated that the maintenance of a carrier type of infection may be interferon-mediated. The degree of susceptibility of rabies-infected cells to immunolysis by antirabies antibody in the presence of complement was found to be correlated with the amount of virus maturation occurring by budding through the cell membrane and not with the presence of immunofluorescent antigen in the cytoplasm of infected cells.

  2. Complementation for an essential ancillary non-structural protein function across parvovirus genera.

    PubMed

    Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter

    2014-11-01

    Parvoviruses encode a small number of ancillary proteins that differ substantially between genera. Within the genus Protoparvovirus, minute virus of mice (MVM) encodes three isoforms of its ancillary protein NS2, while human bocavirus 1 (HBoV1), in the genus Bocaparvovirus, encodes an NP1 protein that is unrelated in primary sequence to MVM NS2. To search for functional overlap between NS2 and NP1, we generated murine A9 cell populations that inducibly express HBoV1 NP1. These were used to test whether NP1 expression could complement specific defects resulting from depletion of MVM NS2 isoforms. NP1 induction had little impact on cell viability or cell cycle progression in uninfected cells, and was unable to complement late defects in MVM virion production associated with low NS2 levels. However, NP1 did relocate to MVM replication centers, and supports both the normal expansion of these foci and overcomes the early paralysis of DNA replication in NS2-null infections.

  3. Physiological Transgene Regulation and Functional Complementation of a Neurological Disease Gene Deficiency in Neurons

    PubMed Central

    Peruzzi, Pier Paolo; Lawler, Sean E; Senior, Steve L; Dmitrieva, Nina; Edser, Pauline AH; Gianni, Davide; Chiocca, E Antonio; Wade-Martins, Richard

    2009-01-01

    The microtubule-associated protein tau (MAPT) and α-synuclein (SNCA) genes play central roles in neurodegenerative disorders. Mutations in each gene cause familial disease, whereas common genetic variation at both loci contributes to susceptibility to sporadic neurodegenerative disease. Here, we demonstrate exquisite gene regulation of the human MAPT and SNCA transgene loci and functional complementation in neuronal cell cultures and organotypic brain slices using the herpes simplex virus type 1 (HSV-1) amplicon-based infectious bacterial artificial chromosome (iBAC) vector to express complete loci >100 kb. Cell cultures transduced by iBAC vectors carrying a 143 kb MAPT or 135 kb SNCA locus expressed the human loci similar to the endogenous gene. We focused on analysis of the iBAC-MAPT vector carrying the complete MAPT locus. On transduction into neuronal cultures, multiple MAPT transcripts were expressed from iBAC-MAPT under strict developmental and cell type–specific control. In primary neurons from Mapt−/− mice, the iBAC-MAPT vector expressed the human tau protein, as detected by enzyme-linked immunosorbent assay and immunocytochemistry, and restored sensitivity of Mapt−/− neurons to Aβ peptide treatment in dissociated neuronal cultures and in organotypic slice cultures. The faithful retention of gene expression and phenotype complementation by the system provides a novel method to analyze neurological disease genes. PMID:19352323

  4. Zika virus.

    PubMed

    2016-02-10

    Essential facts Zika virus disease is caused by a virus that is transmitted by the Aedes mosquito. While it generally causes a mild illness, there is increasing concern that it is harmful in pregnancy and can cause congenital abnormalities in infants born to women infected with the virus. There is no antiviral treatment or vaccine currently available. The best form of prevention is protection against mosquito bites.

  5. 21 CFR 866.5260 - Complement C3b inactivator immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  6. 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...

  7. 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...

  8. 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...

  9. Ebola virus.

    PubMed

    Streether, L A

    1999-01-01

    Ebola virus was first identified as a filovirus in 1976, following epidemics of severe haemorrhagic fever in sub-Saharan Africa. Further outbreaks have occurred since, but, despite extensive and continued investigations, the natural reservoir for the virus remains unknown. The mortality rate is high and there is no cure for Ebola virus infection. Molecular technology is proving useful in extending our knowledge of the virus. Identification of the host reservoir, control and prevention of further outbreaks, rapid diagnosis of infection, and vaccine development remain areas of continued interest in the fight against this biosafety level-four pathogen.

  10. Virus Crystallography

    NASA Astrophysics Data System (ADS)

    Fry, Elizabeth; Logan, Derek; Stuart, David

    Crystallography provides a means of visualizing intact virus particles as well as their isolated constituent proteins and enzymes (1-3) at near-atomic resolution, and is thus an extraordinarily powerful tool in the pursuit of a fuller understanding of the functioning of these simple biological systems. We have already expanded our knowledge of virus evolution, assembly, antigenic variation, and host-cell interactions; further studies will no doubt reveal much more. Although the rewards are enormous, an intact virus structure determination is not a trivial undertaking and entails a significant scaling up in terms of time and resources through all stages of data collection and processing compared to a traditional protein crystallographic structure determination. It is the methodology required for such studies that will be the focus of this chapter. The computational requirements were satisfied in the late 1970s, and when combined with the introduction of phase improvement techniques utilizing the virus symmetry (4,5), the application of crystallography to these massive macromolecular assemblies became feasible. This led to the determination of the first virus structure (the small RNA plant virus, tomato bushy stunt virus), by Harrison and coworkers in 1978 (6). The structures of two other plant viruses followed rapidly (7,8). In the 1980s, a major focus of attention was a family of animal RNA viruses; the Picornaviridae.

  11. Molecular intercommunication between the complement and coagulation systems.

    PubMed

    Amara, Umme; Flierl, Michael A; Rittirsch, Daniel; Klos, Andreas; Chen, Hui; Acker, Barbara; Brückner, Uwe B; Nilsson, Bo; Gebhard, Florian; Lambris, John D; Huber-Lang, Markus

    2010-11-01

    The complement system as well as the coagulation system has fundamental clinical implications in the context of life-threatening tissue injury and inflammation. Associations between both cascades have been proposed, but the precise molecular mechanisms remain unknown. The current study reports multiple links for various factors of the coagulation and fibrinolysis cascades with the central complement components C3 and C5 in vitro and ex vivo. Thrombin, human coagulation factors (F) XIa, Xa, and IXa, and plasmin were all found to effectively cleave C3 and C5. Mass spectrometric analyses identified the cleavage products as C3a and C5a, displaying identical molecular weights as the native anaphylatoxins C3a and C5a. Cleavage products also exhibited robust chemoattraction of human mast cells and neutrophils, respectively. Enzymatic activity for C3 cleavage by the investigated clotting and fibrinolysis factors is defined in the following order: FXa > plasmin > thrombin > FIXa > FXIa > control. Furthermore, FXa-induced cleavage of C3 was significantly suppressed in the presence of the selective FXa inhibitors fondaparinux and enoxaparin in a concentration-dependent manner. Addition of FXa to human serum or plasma activated complement ex vivo, represented by the generation of C3a, C5a, and the terminal complement complex, and decreased complement hemolytic serum activity that defines exact serum concentration that results in complement-mediated lysis of 50% of sensitized sheep erythrocytes. Furthermore, in plasma from patients with multiple injuries (n = 12), a very early appearance and correlation of coagulation (thrombin-antithrombin complexes) and the complement activation product C5a was found. The present data suggest that coagulation/fibrinolysis proteases may act as natural C3 and C5 convertases, generating biologically active anaphylatoxins, linking both cascades via multiple direct interactions in terms of a complex serine protease system.

  12. Live Virus Smallpox Vaccine

    MedlinePlus

    ... Index SMALLPOX FACT SHEET The Live Virus Smallpox Vaccine The vaccinia virus is the "live virus" used ... cannot cause smallpox. What is a "live virus" vaccine? A "live virus" vaccine is a vaccine that ...

  13. Complement-dependent cytotoxicity in rats bearing human adenovirus type 12-induced primary retinoblastoma-like tumor in the eye.

    PubMed

    Nishida, T; Mukai, N; Solish, S P

    1981-01-01

    Using an animal model of retinoblastoma in inbred rats and cultured human adenovirus type 12-induced retinoblastoma-like tumor cells (RAO 188), complement-dependent cytotoxicity was determined by measuring release of 3H-uridine labelled RNA. Sera from rats in which tumors did not grow after adenovirus type 12 inoculation had higher cytotoxicity against RAO 188 cells than sera from rats bearing primary adenovirus type 12-induced retinoblastoma-like tumor. These results showed that the rat which could raise antibodies against adenovirus type 12-induced retinoblastoma-like tumor cells did not allow the tumor growth in the eye after virus inoculation.

  14. Studies on activation and levels of haemolytic complement of buffalo (Bubalus bubalis). 1. Classical complement pathway.

    PubMed

    Jain, A; Goel, M C

    1989-12-01

    Optimum conditions for haemolytic complement (HC) assay in buffalo serum were standardized. In all, 11 indicator systems of red blood cells (RBC) and haemolysins were investigated. Maximum HC CH50 titre was obtained with rabbit RBC sensitized with goat haemolysin. The effect of pH, Ca2+ and Mg2+ concentration, ionic strength, time and temperature were studied. Of all the variables, ionic strength influenced the HC activity most significantly. The standard system for titrating the HC consisted of rabbit RBC sensitized with goat haemolysin, sucrose-veronal buffer with pH 7.5, ionic strength 0.023 M and Ca2+ and Mg2+ concentrations 6 x 10(-4) and 2 x 10(-3) M, respectively. Incubation at 37 degrees C for 2 h gave highest haemolytic activity. With this protocol 5-7-fold higher HC activity was recorded than with prestandardized conditions. Levels of HC were determined in the sera of 98 buffaloes aged from 1 month to 12 years. The lowest mean CH50 units of 401 +/- 0.35 per ml were recorded in buffalo calves below 3 months of age. The mean HC levels increased with age, reaching peak values of 2349 +/- 62.25 CH50 units/ml in 2-3-year-old buffalo. Animals in the age group 5-12 years had significantly decreased (P less than 0.05) mean HC levels of 1545 +/- 68.94.

  15. Structural homology of complement protein C6 with other channel-forming proteins of complement.

    PubMed Central

    Chakravarti, D N; Chakravarti, B; Parra, C A; Muller-Eberhard, H J

    1989-01-01

    The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9. In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance. Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11. The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303. The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor. The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure. PMID:2468158

  16. LPS induces pulp progenitor cell recruitment via complement activation.

    PubMed

    Chmilewsky, F; Jeanneau, C; Laurent, P; About, I

    2015-01-01

    Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS.

  17. The effect of complement depletion on lung clearance of bacteria.

    PubMed

    Gross, G N; Rehm, S R; Pierce, A K

    1978-08-01

    We have investigated the effect of hypocomplementemia on early pulmonary clearance of four species of bacteria. The experiments were performed in an inbred animal model to minimize immunologic variability. Complement was depleted by cobra venom factor, and activity in serum was monitored with a phagocytic assay. Bacterial specific antibodies were examined by an indirect radioimmunoassay, and animals with high levels of activity were excluded from anaysis. 4 h after aerosolization with Streptococcus pneumoniae, complement-depleted animals had cleared only 75% of the initial number of organisms, whereas saline-treated controls cleared 91% (P less than 0.01). Aerosolization with Pseudomonas aeruginosa was followed at 4 h by a twofold greater growth of organisms in the complement-depleted animals (446% of original deposition) as compared to the saline-treated controls (211% of original deposition) (P less than 0.02). Clearance of Klebsiella pneumoniae and Staphylococcus aureus were similar in complement-depleted animals and saline-treated controls. These experiments suggest that hypocomplementemia predisposes to bacterial pneumonia and may explain the high incidence of pulmonary infections in patients having impaired complement activity. Our results further indicate that varying defense mechanisms may be involved with clearing the lung of differing bacterial species. PMID:27534

  18. 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

  19. Physicochemical signatures of nanoparticle-dependent complement activation

    SciTech Connect

    Thomas, Dennis G.; Chikkagoudar, Satish; Heredia-Langner, Alejandro; Tardiff, Mark F.; Xu, Zhixiang; Hourcade, Dennis; Pham, Christine; Lanza, Gregory M.; Weinberger, Kilian Q.; Baker, Nathan A.

    2014-03-21

    Nanoparticles are potentially powerful therapeutic tools that have the capacity to target drug payloads and imaging agents. However, some nanoparticles can activate complement, a branch of the innate immune system, and cause adverse side-effects. Recently, we developed an in vitro hemolytic assay protocol for measuring the nanoparticle-dependent complement activity of serum samples and applied this protocol to several nanoparticle formulations that differed in size, surface charge, and surface chemistry; quantifying the nanoparticle-dependent complement activity using a metric called Residual Hemolytic Activity (RHA). In the present work, we have used a decision tree learning algorithm to derive the rules for estimating nanoparticle-dependent complement response based on the data generated from the hemolytic assay studies. Our results indicate that physicochemical properties of nanoparticles, namely, size, polydispersity index, zeta potential, and mole percentage of the active surface ligand of a nanoparticle, can serve as good descriptors for prediction of nanoparticle-dependent complement activation in the decision tree modeling framework. The robustness and predictability of the model can be improved by training the model with additional data points that are uniformly distributed in the RHA/physicochemical descriptor space and by incorporating instability effects on nanoparticle physicochemical properties into the model.

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

    PubMed Central

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

    2015-01-01

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

  1. Decay-accelerating factor (CD55), a glycosylphosphatidylinositol-anchored complement regulatory protein, is a receptor for several echoviruses.

    PubMed Central

    Bergelson, J M; Chan, M; Solomon, K R; St John, N F; Lin, H; Finberg, R W

    1994-01-01

    Echoviruses are human pathogens belonging to the picornavirus family. Decay-accelerating factor (DAF) is a glycosylphosphatidylinositol (GPI)-anchored surface protein that protects cells from lysis by autologous complement. Anti-DAF monoclonal antibodies prevented echovirus 7 attachment to susceptible cells and protected cells from infection. HeLa cells specifically lost the capacity to bind echovirus 7 when treated with phosphatidylinositol-specific phospholipase C, an enzyme that releases GPI-anchored proteins from the cell surface, indicating that the virus receptor, like DAF, is a GPI-anchored protein. Although Chinese hamster ovary cells do not bind echovirus 7, transfectants expressing human DAF bound virus efficiently, and binding was prevented by pretreatment with an anti-DAF monoclonal antibody. Anti-DAF antibodies prevented infection by at least six echovirus serotypes. These results indicate that DAF is the receptor mediating attachment and infection by several echoviruses. Images PMID:7517044

  2. Novel complementation cell lines derived from human lung carcinoma A549 cells support the growth of E1-deleted adenovirus vectors.

    PubMed

    Imler, J L; Chartier, C; Dreyer, D; Dieterle, A; Sainte-Marie, M; Faure, T; Pavirani, A; Mehtali, M

    1996-01-01

    Replication-defective E1-deleted adenoviruses are attractive vectors for gene therapy or live vaccines. However, manufacturing methods required for their pharmaceutical development are not optimized. For example, the generation of E1-deleted adenovirus vectors relies on the complementation functions present in 293 cells. However, 293 cells are prone to the generation of replication competent particles as a result of recombination events between the viral DNA and the integrated adenovirus sequences present in the cell line. We report here that human lung A549 cells transformed with constitutive or inducible E1-expression vectors support the replication of E1-deficient adenoviruses. E1A transcription was elevated in most of the cell lines, and E1A proteins were expressed at levels similar to those of 293 cells. However, the levels of expression of E1A did not correlate with the efficiencies of complementation of E1-deleted viruses in A549 clones, since some clones complemented replication in the absence of induction of E1A expression. In addition, complementation of E1-deficient adenoviruses did not require expression of the E1B 55-kDa protein. Although these cell lines contain the coding and cis-acting regulatory sequences of the structural protein IX gene, they are not able to complement viruses in which this gene has been deleted. In contrast to 293 cells, such new complementation cell lines do not contain the left end of the adenoviral genome and thus represent a significant improvement over the currently used 293 cells, in which a single recombination event is sufficient to yield replication competent adenovirus. PMID:8929914

  3. Oncolytic and immunotherapeutic vaccinia induces antibody-mediated complement-dependent cancer cell lysis in humans.

    PubMed

    Kim, Mi Kyung; Breitbach, Caroline J; Moon, Anne; Heo, Jeong; Lee, Yu Kyoung; Cho, Mong; Lee, Jun Woo; Kim, Seong-Geun; Kang, Dae Hwan; Bell, John C; Park, Byeong Ho; Kirn, David H; Hwang, Tae-Ho

    2013-05-15

    Oncolytic viruses cause direct cytolysis and cancer-specific immunity in preclinical models. The goal of this study was to demonstrate induction of functional anticancer immunity that can lyse target cancer cells in humans. Pexa-Vec (pexastimogene devacirepvec; JX-594) is a targeted oncolytic and immunotherapeutic vaccinia virus engineered to express human granulocyte-macrophage colony-stimulating factor (GM-CSF). Pexa-Vec demonstrated replication, GM-CSF expression, and tumor responses in previous phase 1 trials. We now evaluated whether Pexa-Vec induced functional anticancer immunity both in the rabbit VX2 tumor model and in patients with diverse solid tumor types in phase 1. Antibody-mediated complement-dependent cancer cell cytotoxicity (CDC) was induced by intravenous Pexa-Vec in rabbits; transfer of serum from Pexa-Vec-treated animals to tumor-bearing animals resulted in tumor necrosis and improved survival. In patients with diverse tumor types treated on a phase 1 trial, CDC developed within 4 to 8 weeks in most patients; normal cells were resistant to the cytotoxic effects. T lymphocyte activation in patients was evidenced by antibody class switching. We determined that patients with the longest survival duration had the highest CDC activity, and identified candidate target tumor cell antigens. Thus, we demonstrated that Pexa-Vec induced polyclonal antibody-mediated CDC against multiple tumor antigens both in rabbits and in patients with diverse solid tumor types.

  4. Role of complement in porphyrin-induced photosensitivity