Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

PubMed Central

Näf, Dieter; Kupfer, Gary M.; Suliman, Ahmed; Lambert, Kathleen; D’Andrea, Alan D.

1998-01-01

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability. PMID:9742112

Factor H: A Complement Regulator in Health and Disease, and a Mediator of Cellular Interactions

PubMed Central

Kopp, Anne; Hebecker, Mario; Svobodová, Eliška; Józsi, Mihály

2012-01-01

Complement is an essential part of innate immunity as it participates in host defense against infections, disposal of cellular debris and apoptotic cells, inflammatory processes and modulation of adaptive immune responses. Several soluble and membrane-bound regulators protect the host from the potentially deleterious effects of uncontrolled and misdirected complement activation. Factor H is a major soluble regulator of the alternative complement pathway, but it can also bind to host cells and tissues, protecting them from complement attack. Interactions of factor H with various endogenous ligands, such as pentraxins, extracellular matrix proteins and DNA are important in limiting local complement-mediated inflammation. Impaired regulatory as well as ligand and cell recognition functions of factor H, caused by mutations or autoantibodies, are associated with the kidney diseases: atypical hemolytic uremic syndrome and dense deposit disease and the eye disorder: age-related macular degeneration. In addition, factor H binds to receptors on host cells and is involved in adhesion, phagocytosis and modulation of cell activation. In this review we discuss current concepts on the physiological and pathophysiological roles of factor H in light of new data and recent developments in our understanding of the versatile roles of factor H as an inhibitor of complement activation and inflammation, as well as a mediator of cellular interactions. A detailed knowledge of the functions of factor H in health and disease is expected to unravel novel therapeutic intervention possibilities and to facilitate the development or improvement of therapies. PMID:24970127

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21)

PubMed Central

Khandelwal, Sanjay; Lee, Grace M.; Hester, C. Garren; Poncz, Mortimer; McKenzie, Steven E.; Sachais, Bruce S.; Rauova, Lubica; Kelsoe, Garnett; Cines, Douglas B.; Frank, Michael

2016-01-01

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes. PMID:27412887

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

PubMed

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

2017-02-01

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

Older Adult Learners: A Comparison of Active and Non-Active Learners

ERIC Educational Resources Information Center

Sloane-Seale, Atlanta; Kops, Bill

2007-01-01

This paper reports on a 2004 follow-up study conducted in partnership with the University of Manitoba Continuing Education Division and local senior's organizations. The partnership was formed in 2002-03 to promote applied research on lifelong learning and older adults, develop new and complement existing educational activities, and explore new…

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-01-01

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

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

PubMed

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

2015-04-21

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

The in vivo mechanism of action of CD20 monoclonal antibodies depends on local tumor burden

PubMed Central

Boross, Peter; Jansen, J.H. Marco; de Haij, Simone; Beurskens, Frank J.; van der Poel, Cees E.; Bevaart, Lisette; Nederend, Maaike; Golay, Josée; van de Winkel, Jan G.J.; Parren, Paul W.H.I.; Leusen, Jeanette H.W.

2011-01-01

Background CD20 monoclonal antibodies are widely used in clinical practice. Antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity and direct cell death have been suggested to be important effector functions for CD20 antibodies. However, their specific contributions to the in vivo mechanism of action of CD20 immunotherapy have not been well defined. Design and Methods Here we studied the in vivo mechanism of action of type I (rituximab and ofatumumab) and type II (HuMab-11B8) CD20 antibodies in a peritoneal, syngeneic, mouse model with EL4-CD20 cells using low and high tumor burden. Results Interestingly, we observed striking differences in the in vivo mechanism of action of CD20 antibodies dependent on tumor load. In conditions of low tumor burden, complement was sufficient for tumor killing both for type I and type II CD20 antibodies. In contrast, in conditions of high tumor burden, activating FcγR (specifically FcγRIII), active complement and complement receptor 3 were all essential for tumor killing. Our data suggest that complement-enhanced antibody-dependent cellular cytotoxicity may critically affect tumor killing by CD20 antibodies in vivo. The type II CD20 antibody 11B8, which is a poor inducer of complement activation, was ineffective against high tumor burden. Conclusions Tumor burden affects the in vivo mechanism of action of CD20 antibodies. Low tumor load can be eliminated by complement alone, whereas elimination of high tumor load requires multiple effector mechanisms. PMID:21880632

Molecular mechanisms of inflammation and tissue injury after major trauma-is complement the "bad guy"?

PubMed Central

2011-01-01

Trauma represents the leading cause of death among young people in industrialized countries. Recent clinical and experimental studies have brought increasing evidence for activation of the innate immune system in contributing to the pathogenesis of trauma-induced sequelae and adverse outcome. As the "first line of defense", the complement system represents a potent effector arm of innate immunity, and has been implicated in mediating the early posttraumatic inflammatory response. Despite its generic beneficial functions, including pathogen elimination and immediate response to danger signals, complement activation may exert detrimental effects after trauma, in terms of mounting an "innocent bystander" attack on host tissue. Posttraumatic ischemia/reperfusion injuries represent the classic entity of complement-mediated tissue damage, adding to the "antigenic load" by exacerbation of local and systemic inflammation and release of toxic mediators. These pathophysiological sequelae have been shown to sustain the systemic inflammatory response syndrome after major trauma, and can ultimately contribute to remote organ injury and death. Numerous experimental models have been designed in recent years with the aim of mimicking the inflammatory reaction after trauma and to allow the testing of new pharmacological approaches, including the emergent concept of site-targeted complement inhibition. The present review provides an overview on the current understanding of the cellular and molecular mechanisms of complement activation after major trauma, with an emphasis of emerging therapeutic concepts which may provide the rationale for a "bench-to-bedside" approach in the design of future pharmacological strategies. PMID:22129197

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

PubMed Central

Goodrum, K J

1987-01-01

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

[The complement system as a main actor in the pathogenesis of obstetric antiphospholipid syndrome].

PubMed

Alijotas-Reig, Jaume

2010-01-23

Pregnancy losses are the main obstetrical complications of the obstetric antiphospholipid syndrome (obstetric-APS). Classically, they have been strongly attributed to thrombosis and further placental infarcts. But in some cases is not possible to show evidence of decidual thrombosis or placental vasculopathy, and sometimes inflammatory signs are present. Besides, the prevalence of systemic thrombosis is low in obstetric APS patients. Some cases have low plasma C4/C3 levels. Animal models show a local inflammatory mechanism. The beta2-glycoprotein-I/anti-beta2-glycoprotein-I complexes activate both, classical and alternative complement pathways. Complement proteins may injure trophoblast cells, recruiting and activating monocytes and neutrophils. Free radicals and proteolytic enzymes could also attack trophoblastic cells. In addition, an amplifier loop between the tissue factor, inflammatory cells and complement proteins could exist. Overall, these diverse mechanisms may explain both, inflammatory and thrombophilic placental alterations. In the end, the role played in this binomial by certain pro-inflammatory cytokines, mainly TNF-alpha, remains to clarify. Copyright 2009 Elsevier España, S.L. All rights reserved.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-05-08

Dysregulation of iron homeostasis may be a pathogenic factor in age-related macular degeneration (AMD). Meanwhile, the formation of complement-containing deposits under the retinal pigment epithelial (RPE) cell layer is a pathognomonic feature of AMD. In this study, we investigated the molecular mechanisms by which complement component 3 (C3), a central protein in the complement cascade, is up-regulated by iron in RPE cells. Modulation of TGF-β signaling, involving ERK1/2, SMAD3, and CCAAT/enhancer-binding protein-δ, is responsible for iron-induced C3 expression. The differential effects of spatially distinct SMAD3 phosphorylation sites at the linker region and at the C terminus determined the up-regulation of C3. Pharmacologic inhibition of either ERK1/2 or SMAD3 phosphorylation decreased iron-induced C3 expression levels. Knockdown of SMAD3 blocked the iron-induced up-regulation and nuclear accumulation of CCAAT/enhancer-binding protein-δ, a transcription factor that has been shown previously to bind the basic leucine zipper 1 domain in the C3 promoter. We show herein that mutation of this domain reduced iron-induced C3 promoter activity. In vivo studies support our in vitro finding of iron-induced C3 up-regulation. Mice with a mosaic pattern of RPE-specific iron overload demonstrated co-localization of iron-induced ferritin and C3d deposits. Humans with aceruloplasminemia causing RPE iron overload had increased RPE C3d deposition. The molecular events in the iron-C3 pathway represent therapeutic targets for AMD or other diseases exacerbated by iron-induced local complement dysregulation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

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

2015-01-01

Dysregulation of iron homeostasis may be a pathogenic factor in age-related macular degeneration (AMD). Meanwhile, the formation of complement-containing deposits under the retinal pigment epithelial (RPE) cell layer is a pathognomonic feature of AMD. In this study, we investigated the molecular mechanisms by which complement component 3 (C3), a central protein in the complement cascade, is up-regulated by iron in RPE cells. Modulation of TGF-β signaling, involving ERK1/2, SMAD3, and CCAAT/enhancer-binding protein-δ, is responsible for iron-induced C3 expression. The differential effects of spatially distinct SMAD3 phosphorylation sites at the linker region and at the C terminus determined the up-regulation of C3. Pharmacologic inhibition of either ERK1/2 or SMAD3 phosphorylation decreased iron-induced C3 expression levels. Knockdown of SMAD3 blocked the iron-induced up-regulation and nuclear accumulation of CCAAT/enhancer-binding protein-δ, a transcription factor that has been shown previously to bind the basic leucine zipper 1 domain in the C3 promoter. We show herein that mutation of this domain reduced iron-induced C3 promoter activity. In vivo studies support our in vitro finding of iron-induced C3 up-regulation. Mice with a mosaic pattern of RPE-specific iron overload demonstrated co-localization of iron-induced ferritin and C3d deposits. Humans with aceruloplasminemia causing RPE iron overload had increased RPE C3d deposition. The molecular events in the iron-C3 pathway represent therapeutic targets for AMD or other diseases exacerbated by iron-induced local complement dysregulation. PMID:25802332

A Novel fry1 Allele Reveals the Existence of a Mutant Phenotype Unrelated to 5′->3′ Exoribonuclease (XRN) Activities in Arabidopsis thaliana Roots

PubMed Central

Hirsch, Judith; Estavillo, Gonzalo M.; Javot, Hélène; Chiarenza, Serge; Mallory, Allison C.; Maizel, Alexis; Declerck, Marie; Pogson, Barry J.; Vaucheret, Hervé; Crespi, Martin; Desnos, Thierry; Thibaud, Marie-Christine; Nussaume, Laurent; Marin, Elena

2011-01-01

Background Mutations in the FRY1/SAL1 Arabidopsis locus are highly pleiotropic, affecting drought tolerance, leaf shape and root growth. FRY1 encodes a nucleotide phosphatase that in vitro has inositol polyphosphate 1-phosphatase and 3′,(2′),5′-bisphosphate nucleotide phosphatase activities. It is not clear which activity mediates each of the diverse biological functions of FRY1 in planta. Principal Findings A fry1 mutant was identified in a genetic screen for Arabidopsis mutants deregulated in the expression of Pi High affinity Transporter 1;4 (PHT1;4). Histological analysis revealed that, in roots, FRY1 expression was restricted to the stele and meristems. The fry1 mutant displayed an altered root architecture phenotype and an increased drought tolerance. All of the phenotypes analyzed were complemented with the AHL gene encoding a protein that converts 3′-polyadenosine 5′-phosphate (PAP) into AMP and Pi. PAP is known to inhibit exoribonucleases (XRN) in vitro. Accordingly, an xrn triple mutant with mutations in all three XRNs shared the fry1 drought tolerance and root architecture phenotypes. Interestingly these two traits were also complemented by grafting, revealing that drought tolerance was primarily conferred by the rosette and that the root architecture can be complemented by long-distance regulation derived from leaves. By contrast, PHT1 expression was not altered in xrn mutants or in grafting experiments. Thus, PHT1 up-regulation probably resulted from a local depletion of Pi in the fry1 stele. This hypothesis is supported by the identification of other genes modulated by Pi deficiency in the stele, which are found induced in a fry1 background. Conclusions/Significance Our results indicate that the 3′,(2′),5′-bisphosphate nucleotide phosphatase activity of FRY1 is involved in long-distance as well as local regulatory activities in roots. The local up-regulation of PHT1 genes transcription in roots likely results from local depletion of Pi and is independent of the XRNs. PMID:21304819

Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants.

PubMed

Henquet, Maurice; Heinhuis, Bas; Borst, Jan Willem; Eigenhuijsen, Jochem; Schreuder, Mariëlle; Bosch, Dirk; van der Krol, Alexander

2010-08-01

In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment.

Complement Inhibitors from Scabies Mites Promote Streptococcal Growth – A Novel Mechanism in Infected Epidermis?

PubMed Central

Mika, Angela; Reynolds, Simone L.; Pickering, Darren; McMillan, David; Sriprakash, Kadaba S.; Kemp, David J.; Fischer, Katja

2012-01-01

Background Scabies is highly prevalent in socially disadvantaged communities such as indigenous populations and in developing countries. Generalized itching causes discomfort to the patient; however, serious complications can occur as a result of secondary bacterial pyoderma, commonly caused by Streptococcus pyogenes (GAS) or Staphylococcus aureus. In the tropics, skin damage due to scabies mite infestations has been postulated to be an important link in the pathogenesis of disease associated with acute rheumatic fever and heart disease, poststreptococcal glomerulonephritis and systemic sepsis. Treatment of scabies decreases the prevalence of infections by bacteria. This study aims to identify the molecular mechanisms underlying the link between scabies and GAS infections. Methodology/Principal Findings GAS bacteria were pre-incubated with blood containing active complement, phagocytes and antibodies against the bacteria, and subsequently tested for viability by plate counts. Initial experiments were done with serum from an individual previously exposed to GAS with naturally acquired anti-GAS antibodies. The protocol was optimized for large-scale testing of low-opsonic whole blood from non-exposed human donors by supplementing with a standard dose of heat inactivated human sera previously exposed to GAS. This allowed an extension of the dataset to two additional donors and four proteins tested at a range of concentrations. Shown first is the effect of scabies mite complement inhibitors on human complement using ELISA-based complement activation assays. Six purified recombinant mite proteins tested at a concentration of 50 µg/ml blocked all three complement activation pathways. Further we demonstrate in human whole blood assays that each of four scabies mite complement inhibitors tested increased GAS survival rates by 2–15 fold. Conclusions/Significance We propose that local complement inhibition plays an important role in the development of pyoderma in scabies infested skin. This molecular link between scabies and bacterial infections may provide new avenues to develop alternative treatment options against this neglected disease. PMID:22815998

Proteflazid® and local immunity in diseases caused by human papillomavirus, herpesvirus and mixed urogenital infections.

PubMed

Kaminsky, Vjacheslav; Chernyshov, Viktor; Grynevych, Oleksandr; Benyuk, Vasil; Kornatskaya, Alla; Shalko, Miroslava; Usevich, Igor; Revenko, Oleg; Shepetko, Maxim; Solomakha, Ludmila

2017-03-21

Reporting of clinical trials results for Proteflazid® in the drug formulation suppositories and vaginal swabs soaked in the solution of the drug to the local immunity of the female reproductive tract. The aim of study was to examine the state of local immunity in the reproductive tract of women with sexually transmitted diseases caused by human papillomavirus, herpes viruses (Type 1, 2) and mixed infection (herpes viruses + chlamydia). The trials involved 216 women with viral sexually transmitted diseases: Cervical Dysplasia associated with papillomavirus infection (HPV) (Group 1); Herpes genitalis type 1 (HSV- 1) and type 2 (HSV-1) (Group 2); mixed infection - HSV-1, HSV-2 and chlamydia (Group 3). Treatment results have confirmed that Proteflazid® contributes to sustainable performance improvement of basic factors of local immunity - sIgA, lysozyme and complement component C3 in the cervical mucus for all three groups of women. Proteflazid® enhances level of local immunity markers (sIgA, lysozyme, C3 complement component) and improves their ratios. Also it intensifies anticontagious activity of mucosal protection and female reproductive system as whole, during treatment diseases caused by human papillomavirus, herpesvirus and mixed urogenital infections (herpesvirus and chlamydia).

Burn wound sepsis may be promoted by a failure of local antibacterial host defenses.

PubMed Central

Deitch, E A; Bridges, R M; Dobke, M; McDonald, J C

1987-01-01

Little attention has been focused on the local burn wound environment, even though burn wound sepsis is a common cause of death in the burn victim. To characterize the effect of the local burn wound environment on neutrophil function and metabolism, the opsonic activity of blister fluid specimens against Pseudomonas aeruginosa was measured as was the effect of blister fluid on control neutrophil oxygen consumption using preopsonized zymosan and f-met-leu-phe (FMLP) as stimuli. Blister fluid did not support the killing of P. aeruginosa by normal neutrophils as well as normal serum. Additionally, blister fluid inhibited zymosan-stimulated, but not FMLP-stimulated, neutrophil oxygen consumption. The inhibitory effect of blister fluid on zymosan-stimulated oxygen consumption correlated with the extent of complement activation, measured as C3d or C3AI (p less than 0.01). That blister fluid did not inhibit the FMLP-mediated respiratory burst supports the concept that the blister fluid inhibitory effect on the zymosan-mediated respiratory burst was mediated through the complement receptor. These findings that blister fluid can affect the bactericidal and metabolic activity of normal neutrophils support the concept that cellular function can be altered by the microenvironment in which the cells are bathed. This potential impairment of host defenses within the burn wound could predispose the burn victim to burn wound sepsis. PMID:3115207

Subcutaneous injection of water-soluble multi-walled carbon nanotubes in tumor-bearing mice boosts the host immune activity

NASA Astrophysics Data System (ADS)

Meng, Jie; Yang, Man; Jia, Fumin; Kong, Hua; Zhang, Weiqi; Wang, Chaoying; Xing, Jianmin; Xie, Sishen; Xu, Haiyan

2010-04-01

The immunological responses induced by oxidized water-soluble multi-walled carbon nanotubes on a hepatocarcinoma tumor-bearing mice model via a local administration of subcutaneous injection were investigated. Experimental results show that the subcutaneously injected carbon nanotubes induced significant activation of the complement system, promoted inflammatory cytokines' production and stimulated macrophages' phagocytosis and activation. All of these responses increased the general activity of the host immune system and inhibited the progression of tumor growth.

Real-time fMRI: a tool for local brain regulation.

PubMed

Caria, Andrea; Sitaram, Ranganatha; Birbaumer, Niels

2012-10-01

Real-time fMRI permits simultaneous measurement and observation of brain activity during an ongoing task. One of the most challenging applications of real-time fMRI in neuroscientific and clinical research is the possibility of acquiring volitional control of localized brain activity using real-time fMRI-based neurofeedback protocols. Real-time fMRI allows the experimenter to noninvasively manipulate brain activity as an independent variable to observe the effects on behavior. Real-time fMRI neurofeedback studies demonstrated that learned control of the local brain activity leads to specific changes in behavior. Here, the authors describe the implementation and application of real-time fMRI with particular emphasis on the self-regulation of local brain activity and the investigation of brain-function relationships. Real-time fMRI represents a promising new approach to cognitive neuroscience that could complement traditional neuroimaging techniques by providing more causal insights into the functional role of circumscribed brain regions in behavior.

Guinea pig complement potently measures vibriocidal activity of human antibodies in response to cholera vaccines.

PubMed

Kim, Kyoung Whun; Jeong, Soyoung; Ahn, Ki Bum; Yang, Jae Seung; Yun, Cheol-Heui; Han, Seung Hyun

2017-12-01

The vibriocidal assay using guinea pig complement is widely used for the evaluation of immune responses to cholera vaccines in human clinical trials. However, it is unclear why guinea pig complement has been used over human complement in the measurement of vibriocidal activity of human sera and there have not been comparison studies for the use of guinea pig complement over those from other species. Therefore, we comparatively investigated the effects of complements derived from human, guinea pig, rabbit, and sheep on vibriocidal activity. Complements from guinea pig, rabbit, and human showed concentration-dependent vibriocidal activity in the presence of quality control serum antibodies. Of these complements, guinea pig complement was the most sensitive and effective over a wide concentration range. When the vibriocidal activity of complements was measured in the absence of serum antibodies, human, sheep, and guinea pig complements showed vibriocidal activity up to 40-fold, 20-fold, and 1-fold dilution, respectively. For human pre- and post-vaccination sera, the most potent vibriocidal activity was observed when guinea pig complement was used. In addition, the highest fold-increases between pre- and post- vaccinated sera were obtained with guinea pig complement. Furthermore, human complement contained a higher amount of V. cholerae- and its lipopolysaccharide-specific antibodies than guinea pig complement. Collectively, these results suggest that guinea pig complements are suitable for vibriocidal assays due to their high sensitivity and effectiveness to human sera.

Using animal models to determine the significance of complement activation in Alzheimer's disease

PubMed Central

Loeffler, David A

2004-01-01

Complement inflammation is a major inflammatory mechanism whose function is to promote the removal of microorganisms and the processing of immune complexes. Numerous studies have provided evidence for an increase in this process in areas of pathology in the Alzheimer's disease (AD) brain. Because complement activation proteins have been demonstrated in vitro to exert both neuroprotective and neurotoxic effects, the significance of this process in the development and progression of AD is unclear. Studies in animal models of AD, in which brain complement activation can be experimentally altered, should be of value for clarifying this issue. However, surprisingly little is known about complement activation in the transgenic animal models that are popular for studying this disorder. An optimal animal model for studying the significance of complement activation on Alzheimer's – related neuropathology should have complete complement activation associated with senile plaques, neurofibrillary tangles (if present), and dystrophic neurites. Other desirable features include both classical and alternative pathway activation, increased neuronal synthesis of native complement proteins, and evidence for an increase in complement activation prior to the development of extensive pathology. In order to determine the suitability of different animal models for studying the role of complement activation in AD, the extent of complement activation and its association with neuropathology in these models must be understood. PMID:15479474

[Local immune and oxidative status in exacerbated chronic apical periodontitis].

PubMed

Konoplya, A I; Goldobin, D D; Loktionov, A L

The aim of the study was to define local immune and oxidative changes in patients with exacerbated chronic apical periodontitis. These changes were assessed in saliva of 67 patients with the mean age of 31±2.5 before and after treatment. The study revealed disturbances in cytokines and complement system balance and activation of lipids peroxidation. Combination of Gepon or Vobenzim with Essentiale forte H and Kaskatol proved to be the most effective for correction of this imbalance.

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

PubMed Central

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

2011-01-01

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

Complement activation in the tubulointerstitium: AKI, CKD, and in between.

PubMed

Brar, Jyoti E; Quigg, Richard J

2014-10-01

Complement activation is actively regulated to prevent injudicious activation, such as on peritubular endothelia and basolateral aspects of tubules. Miao et al. studied mice in which the key complement regulator, Crry, was deleted from tubular cells. This lacked functional consequence in unmanipulated animals. Yet, following ischemia-reperfusion, there was greater injury due to alternative pathway activation of C5. When the balance between complement activation and regulation is tipped towards the former, pathologic complement activation can ensue.

Can innate and autoimmune reactivity forecast early and advance stages of age-related macular degeneration?

PubMed

Adamus, Grazyna

2017-03-01

Age-related macular degeneration (AMD) is a major cause of central vision loss in persons over 55years of age in developed countries. AMD is a complex disease in which genetic, environmental and inflammatory factors influence its onset and progression. Elevation in serum anti-retinal autoantibodies, plasma and local activation of complement proteins of the alternative pathway, and increase in secretion of proinflammatory cytokines have been seen over the course of disease. Genetic studies of AMD patients confirmed that genetic variants affecting the alternative complement pathway have a major influence on AMD risk. Because the heterogeneity of this disease, there is no sufficient strategy to identify the disease onset and progression sole based eye examination, thus identification of reliable serological biomarkers for diagnosis, prognosis and response to treatment by sampling patient's blood is necessary. This review provides an outline of the current knowledge on possible serological (autoantibodies, complement factors, cytokines, chemokines) and related genetic biomarkers relevant to the pathology of AMD, and discusses their application for prediction of disease activity and prognosis in AMD. Copyright © 2017 Elsevier B.V. All rights reserved.

Persistent complement activation on tumor cells in breast cancer.

PubMed Central

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

1992-01-01

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

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

PubMed

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

2013-08-01

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

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

PubMed

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

2011-01-01

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

Complement Activation in Inflammatory Skin Diseases

PubMed Central

Giang, Jenny; Seelen, Marc A. J.; van Doorn, Martijn B. A.; Rissmann, Robert; Prens, Errol P.; Damman, Jeffrey

2018-01-01

The complement system is a fundamental part of the innate immune system, playing a crucial role in host defense against various pathogens, such as bacteria, viruses, and fungi. Activation of complement results in production of several molecules mediating chemotaxis, opsonization, and mast cell degranulation, which can contribute to the elimination of pathogenic organisms and inflammation. Furthermore, the complement system also has regulating properties in inflammatory and immune responses. Complement activity in diseases is rather complex and may involve both aberrant expression of complement and genetic deficiencies of complement components or regulators. The skin represents an active immune organ with complex interactions between cellular components and various mediators. Complement involvement has been associated with several skin diseases, such as psoriasis, lupus erythematosus, cutaneous vasculitis, urticaria, and bullous dermatoses. Several triggers including auto-antibodies and micro-organisms can activate complement, while on the other hand complement deficiencies can contribute to impaired immune complex clearance, leading to disease. This review provides an overview of the role of complement in inflammatory skin diseases and discusses complement factors as potential new targets for therapeutic intervention. PMID:29713318

Intracellular Localization, Interactions and Functions of Capsicum Chlorosis Virus Proteins

PubMed Central

Widana Gamage, Shirani M. K.; Dietzgen, Ralf G.

2017-01-01

Tospoviruses are among the most devastating viruses of horticultural and field crops. Capsicum chlorosis virus (CaCV) has emerged as an important pathogen of capsicum and tomato in Australia and South-east Asia. Present knowledge about CaCV protein functions in host cells is lacking. We determined intracellular localization and interactions of CaCV proteins by live plant cell imaging to gain insight into the associations of viral proteins during infection. Proteins were transiently expressed as fusions to autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana and capsicum. All viral proteins localized at least partially in the cell periphery suggestive of cytoplasmic replication and assembly of CaCV. Nucleocapsid (N) and non-structural movement (NSm) proteins localized exclusively in the cell periphery, while non-structural suppressor of silencing (NSs) protein and Gc and Gn glycoproteins accumulated in both the cell periphery and the nucleus. Nuclear localization of CaCV Gn and NSs is unique among tospoviruses. We validated nuclear localization of NSs by immunofluorescence in protoplasts. Bimolecular fluorescence complementation showed self-interactions of CaCV N, NSs and NSm, and heterotypic interactions of N with NSs and Gn. All interactions occurred in the cytoplasm, except NSs self-interaction was exclusively nuclear. Interactions of a tospoviral NSs protein with itself and with N had not been reported previously. Functionally, CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of silencing signal. Cell-to-cell movement activity of NSm was demonstrated by trans-complementation of a movement-defective tobamovirus replicon. CaCV NSm localized at plasmodesmata and its transient expression led to the formation of tubular structures that protruded from protoplasts. The D155 residue in the 30K-like movement protein-specific LxD/N50-70G motif of NSm was critical for plasmodesmata localization and movement activity. Compared to other tospoviruses, CaCV proteins have both conserved and unique properties in terms of in planta localization, interactions and protein functions which will effect viral multiplication and movement in host plants. PMID:28443083

Intracellular Localization, Interactions and Functions of Capsicum Chlorosis Virus Proteins.

PubMed

Widana Gamage, Shirani M K; Dietzgen, Ralf G

2017-01-01

Tospoviruses are among the most devastating viruses of horticultural and field crops. Capsicum chlorosis virus (CaCV) has emerged as an important pathogen of capsicum and tomato in Australia and South-east Asia. Present knowledge about CaCV protein functions in host cells is lacking. We determined intracellular localization and interactions of CaCV proteins by live plant cell imaging to gain insight into the associations of viral proteins during infection. Proteins were transiently expressed as fusions to autofluorescent proteins in leaf epidermal cells of Nicotiana benthamiana and capsicum. All viral proteins localized at least partially in the cell periphery suggestive of cytoplasmic replication and assembly of CaCV. Nucleocapsid (N) and non-structural movement (NSm) proteins localized exclusively in the cell periphery, while non-structural suppressor of silencing (NSs) protein and Gc and Gn glycoproteins accumulated in both the cell periphery and the nucleus. Nuclear localization of CaCV Gn and NSs is unique among tospoviruses. We validated nuclear localization of NSs by immunofluorescence in protoplasts. Bimolecular fluorescence complementation showed self-interactions of CaCV N, NSs and NSm, and heterotypic interactions of N with NSs and Gn. All interactions occurred in the cytoplasm, except NSs self-interaction was exclusively nuclear. Interactions of a tospoviral NSs protein with itself and with N had not been reported previously. Functionally, CaCV NSs showed strong local and systemic RNA silencing suppressor activity and appears to delay short-distance spread of silencing signal. Cell-to-cell movement activity of NSm was demonstrated by trans -complementation of a movement-defective tobamovirus replicon. CaCV NSm localized at plasmodesmata and its transient expression led to the formation of tubular structures that protruded from protoplasts. The D 155 residue in the 30K-like movement protein-specific LxD/N 50-70 G motif of NSm was critical for plasmodesmata localization and movement activity. Compared to other tospoviruses, CaCV proteins have both conserved and unique properties in terms of in planta localization, interactions and protein functions which will effect viral multiplication and movement in host plants.

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

PubMed Central

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

2016-01-01

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

Crossing the Line: Transitioning from Auditor to Researcher

ERIC Educational Resources Information Center

Raiskums, Bernadine W.

2006-01-01

What does a very active and successful auditor/accountant do when she is about to retire with adequate means but inadequate interest in traveling or golfing? She might decide to expand her adjunct work as instructor at the local university. To complement her professional knowledge of accounting and auditing, she might decide to take a few courses…

An Induced Pluripotent Stem Cell Patient Specific Model of Complement Factor H (Y402H) Polymorphism Displays Characteristic Features of Age-Related Macular Degeneration and Indicates a Beneficial Role for UV Light Exposure.

PubMed

Hallam, Dean; Collin, Joseph; Bojic, Sanja; Chichagova, Valeria; Buskin, Adriana; Xu, Yaobo; Lafage, Lucia; Otten, Elsje G; Anyfantis, George; Mellough, Carla; Przyborski, Stefan; Alharthi, Sameer; Korolchuk, Viktor; Lotery, Andrew; Saretzki, Gabriele; McKibbin, Martin; Armstrong, Lyle; Steel, David; Kavanagh, David; Lako, Majlinda

2017-11-01

Age-related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease; however, these do not exist for the dry form. Complement factor H polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, RPE damage, and visual decline. We have derived and characterized induced pluripotent stem cell (iPSC) lines from two subjects without AMD and low-risk genotype and two patients with advanced AMD and high-risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H, factor I, and factor H-like protein 1. The iPSC RPE cells derived from high-risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy, and deposition of "drüsen"-like deposits. The low- and high-risk RPE cells respond differently to intermittent exposure to UV light, which leads to an improvement in cellular and functional phenotype only in the high-risk AMD-RPE cells. Taken together, our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. Stem Cells 2017;35:2305-2320. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

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

PubMed

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

2017-11-05

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

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2013-01-01

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

Complement Depletion Protects Lupus-prone Mice from Ischemia-reperfusion-initiated Organ Injury

DTIC Science & Technology

2012-10-25

injury, we sought to evaluate whether complement inhibition mitigates organ damage. We found that complement deple- tion with cobra venom factor... venom factor and C5a receptor antagonist were able to protect mice from local tissue damage, treatment with C5a receptor antagonist was not able to...Complement depletion or blockage of the complement pathway using molecules such as cobra venom factor (CVF) (24, 33) and C5a receptor antagonists (C5aRA

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

PubMed

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

2007-01-01

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

COMPLEMENT FIXATION IN DISEASED TISSUES

PubMed Central

Burkholder, Peter M.

1961-01-01

An immunohistologic complement fixation test has been used in an effort to detect immune complexes in sections of kidney from rats injected with rabbit anti-rat kidney serum and in sections of biopsied kidneys from four humans with membranous glomerulonephritis. Sections of the rat and human kidneys were treated with fluorescein-conjugated anti-rabbit globulin or antihuman globulin respectively. Adjacent sections in each case were incubated first with fresh guinea pig serum and then in a second step were treated with fluorescein-conjugated antibodies against fixed guinea pig complement to detect sites of fixation of the complement. It was demonstrated that the sites of rabbit globulin in glomerular capillary walls of the rat kidneys and the sites of localized human globulin in thickened glomerular capillary walls and swollen glomerular endothelial cells of the human kidneys were the same sites in which guinea pig complement was fixed in vitro. It was concluded from these studies that rabbit nephrotoxic antibodies localize in rat glomeruli in complement-fixing antigen-antibody complexes. Furthermore, it was concluded that the deposits of human globulin in the glomeruli of the human kidneys behaved like antibody globulin in complement-fixing antigen-antibody complexes. The significance of demonstrating complement-fixing immune complexes in certain diseased tissues is discussed in regard to determination of the causative role of allergic reactions in disease. PMID:19867205

Protection of host cells by complement regulators.

PubMed

Schmidt, Christoph Q; Lambris, John D; Ricklin, Daniel

2016-11-01

The complement cascade is an ancient immune-surveillance system that not only provides protection from pathogen invasion but has also evolved to participate in physiological processes to maintain tissue homeostasis. The alternative pathway (AP) of complement activation is the evolutionarily oldest part of this innate immune cascade. It is unique in that it is continuously activated at a low level and arbitrarily probes foreign, modified-self, and also unaltered self-structures. This indiscriminate activation necessitates the presence of preformed regulators on autologous surfaces to spare self-cells from the undirected nature of AP activation. Although the other two canonical complement activation routes, the classical and lectin pathways, initiate the cascade more specifically through pattern recognition, their activity still needs to be tightly controlled to avoid excessive reactivity. It is the perpetual duty of complement regulators to protect the self from damage inflicted by inadequate complement activation. Here, we review the role of complement regulators as preformed mediators of defense, explain their common and specialized functions, and discuss selected cases in which alterations in complement regulators lead to disease. Finally, rational engineering approaches using natural complement inhibitors as potential therapeutics are highlighted. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Dengue virus induces increased activity of the complement alternative pathway in infected cells.

PubMed

Cabezas, Sheila; Bracho, Gustavo; L Aloia, Amanda; Adamson, Penelope J; Bonder, Claudine S; Smith, Justine R; Gordon, David L; Carr, Jillian M

2018-05-09

Severe dengue virus (DENV) infection is associated with overactivity of the complement alternative pathway (AP) in patient studies. Here, the molecular changes in components of the AP during DENV infection in vitro are investigated. mRNA for factor H (FH) a major negative regulator of the AP, is significantly increased in DENV-infected endothelial cells (EC) and macrophages but in contrast production of extracellular FH protein is not. This discord is not seen for the AP activator, factor B (FB), with DENV induction of both FB mRNA and protein, nor with Toll-like receptor 3 or 4 stimulation of EC and macrophages, which induces both FH and FB mRNA and protein. Surface bound and intracellular FH protein is however induced by DENV, but only in DENV antigen-positive cells, while in two other DENV-susceptible immortalised cell lines (ARPE-19 and HREC) FH protein is induced both intracellularly and extracellularly by DENV infection. Regardless of the cell type, there is an imbalance in AP components and an increase in markers of complement AP activity associated with DENV-infected cells - with lower FH relative to FB protein, increased ability to promote AP-mediated lytic activity and increased deposition of complement component C3b on the surface of DENV-infected cells. For EC in particular, these changes are predicted to result in higher complement activity in the local cellular microenvironment, with the potential to induce functional changes that may result in increased vascular permeability, a hallmark of dengue disease. IMPORTANCE Dengue virus (DENV) is a significant human viral pathogen with global medical and economic impact. DENV may cause serious and life-threatening disease with increased vascular permeability and plasma leakage. The pathogenic mechanisms underlying these features remain unclear; however overactivity of the complement alternative pathway has been suggested to play a role. In this study we investigate the molecular events that may be responsible for this observed alternative pathway overactivity and provide novel findings of changes in the complement system in response to DENV infection in primary cell types that are a major target for DENV infection (macrophages) and pathogenesis (endothelial cells) in vivo Our results suggest a new dimension of cellular events that may influence endothelial cell barrier function during DENV infection that could expand strategies for developing therapeutics to prevent or control DENV-mediated vascular disease. Copyright © 2018 American Society for Microbiology.

Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

PubMed

Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

2013-10-01

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

Gold nanoparticle-assisted all optical localized stimulation and monitoring of Ca2+ signaling in neurons

PubMed Central

Lavoie-Cardinal, Flavie; Salesse, Charleen; Bergeron, Éric; Meunier, Michel; De Koninck, Paul

2016-01-01

Light-assisted manipulation of cells to control membrane activity or intracellular signaling has become a major avenue in life sciences. However, the ability to perform subcellular light stimulation to investigate localized signaling has been limited. Here, we introduce an all optical method for the stimulation and the monitoring of localized Ca2+ signaling in neurons that takes advantage of plasmonic excitation of gold nanoparticles (AuNPs). We show with confocal microscopy that 800 nm laser pulse application onto a neuron decorated with a few AuNPs triggers a transient increase in free Ca2+, measured optically with GCaMP6s. We show that action potentials, measured electrophysiologically, can be induced with this approach. We demonstrate activation of local Ca2+ transients and Ca2+ signaling via CaMKII in dendritic domains, by illuminating a single or few functionalized AuNPs specifically targeting genetically-modified neurons. This NP-Assisted Localized Optical Stimulation (NALOS) provides a new complement to light-dependent methods for controlling neuronal activity and cell signaling. PMID:26857748

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

PubMed

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

2015-06-01

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

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

PubMed Central

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

2017-01-01

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

The structural requirements for immunoglobulin aggregates to localize in germinal centres.

PubMed Central

Embling, P H; Evans, H; Guttierez, C; Holborow, E J; Johns, P; Johnson, P M; Papamichail, M; Stanworth, D R

1978-01-01

The capacity of non-heat-aggregated monoclonal human immunoglobulins of different classes, to localize in murine splenic germinal centres within 24 h of intravenous injection has been investigated. It has been shown that at least trimerization of polyclonal IgG must occur before any germinal centre trapping is manifest. Studies of complement fixation by these IgG preparations in vivo, together with studies of the germinal centre trapping of various monoclonal immunoglobulins, have indicated that the sole structural requirement for germinal centre localization of immunoglobulin aggregates is the ability to fix complement. Results suggest that immunoglobulin aggregates are transported to germinal centres via membrane C3 receptors of mobile cells, and then are released with loss of complement to become fixed to dendritic macrophages by a separate mechanism. PMID:363602

Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants.

PubMed Central

Felser, J M; Straus, S E; Ostrove, J M

1987-01-01

Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene. PMID:3023701

Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

PubMed

Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

2015-08-01

While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.

Non-specific adsorption of complement proteins affects complement activation pathways of gold nanomaterials.

PubMed

Quach, Quang Huy; Kah, James Chen Yong

2017-04-01

The complement system is a key humoral component of innate immunity, serving as the first line of defense against intruders, including foreign synthetic nanomaterials. Although gold nanomaterials (AuNMs) are widely used in nanomedicine, their immunological response is not well understood. Using AuNMs of three shapes commonly used in biomedical applications: spherical gold nanoparticles, gold nanostars and gold nanorods, we demonstrated that AuNMs activated whole complement system, leading to the formation of SC5b-9 complex. All three complement pathways were simultaneously activated by all the AuNMs. Recognition molecules of the complement system interacted with all AuNMs in vitro, except for l-ficolin, but the correlation between these interactions and corresponding complement pathway activation was only observed in the classical and alternative pathways. We also observed the mediating role of complement activation in cellular uptake of all AuNMs by human U937 promonocytic cells, which expresses complement receptors. Taken together, our results highlighted the potential immunological challenges for clinical applications of AuNMs that were often overlooked.

The contributions of managed care plans to public health practice: evidence from the nation's largest local health departments.

PubMed Central

Mays, G. P.; Halverson, P. K.; Stevens, R.

2001-01-01

OBJECTIVE: The authors examine the extent and nature of managed care plans participating in local public health activities. METHODS: In 1998, the authors surveyed the directors of all US local health departments serving jurisdictions of at least 100,000 residents to collect information about public health activities performed in their jurisdictions and about organizations participating in the activities. Multivariate logistic and linear regression models were used to examine organizational and market characteristics associated with managed care plan participation in public health activities. RESULTS: Managed care plans were reported to participate in public health activities in 164 (46%) of the jurisdictions surveyed, and to contribute to 13% of the public health activities performed in the average jurisdiction. Plans appeared most likely to participate in public health activities involving the delivery or management of personal health services and the exchange of health-related information. Managed care participation was more likely to occur in jurisdictions with higher HMO penetration, fewer competing plans, and larger proportions of plans enrolling Medicaid recipients. Participation was positively associated with the overall scope and perceived effectiveness of local public health activities. CONCLUSIONS: Although plans participate in a narrow range of activities, these contributions may complement the work of public health agencies. PMID:11889275

Making In-Class Skills Training More Effective: The Scope for Interactive Videos to Complement the Delivery of Practical Pedestrian Training

ERIC Educational Resources Information Center

Hammond, James; Cherrett, Tom; Waterson, Ben

2015-01-01

Skills and awareness of young pedestrians can be improved with on-street practical pedestrian training, often delivered in schools in the UK by local authorities with the intention of improving road safety. This training is often supplemented by in-class paper-based worksheet activities that are seen to be less effective than practical training in…

Complement inhibition in pre-clinical models of periodontitis and prospects for clinical application.

PubMed

Hajishengallis, George; Hajishengallis, Evlambia; Kajikawa, Tetsuhiro; Wang, Baomei; Yancopoulou, Despina; Ricklin, Daniel; Lambris, John D

2016-06-01

Periodontitis is a dysbiotic inflammatory disease leading to the destruction of the tooth-supporting tissues. Current therapies are not always effective and this prevalent oral disease continues to be a significant health and economic burden. Early clinical studies have associated periodontitis with elevated complement activity. Consistently, subsequent genetic and pharmacological studies in rodents have implicated the central complement component C3 and downstream signaling pathways in periodontal host-microbe interactions that promote dysbiosis and inflammatory bone loss. This review discusses these mechanistic advances and moreover focuses on the compstatin family of C3 inhibitors as a novel approach to treat periodontitis. In this regard, local application of the current lead analog Cp40 was recently shown to block both inducible and naturally occurring periodontitis in non-human primates. These promising results from non-human primate studies and the parallel development of Cp40 for clinical use highlight the feasibility for developing an adjunctive, C3-targeted therapy for human periodontitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nonflowering Plants Possess a Unique Folate-Dependent Phenylalanine Hydroxylase That Is Localized in Chloroplasts[W

PubMed Central

Pribat, Anne; Noiriel, Alexandre; Morse, Alison M.; Davis, John M.; Fouquet, Romain; Loizeau, Karen; Ravanel, Stéphane; Frank, Wolfgang; Haas, Richard; Reski, Ralf; Bedair, Mohamed; Sumner, Lloyd W.; Hanson, Andrew D.

2010-01-01

Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism. PMID:20959559

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

PubMed Central

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

2009-01-01

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

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

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

PubMed

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

2015-11-01

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

The Complement System in Dialysis: A Forgotten Story?

PubMed Central

Poppelaars, Felix; Faria, Bernardo; Gaya da Costa, Mariana; Franssen, Casper F. M.; van Son, Willem J.; Berger, Stefan P.; Daha, Mohamed R.; Seelen, Marc A.

2018-01-01

Significant advances have lead to a greater understanding of the role of the complement system within nephrology. The success of the first clinically approved complement inhibitor has created renewed appreciation of complement-targeting therapeutics. Several clinical trials are currently underway to evaluate the therapeutic potential of complement inhibition in renal diseases and kidney transplantation. Although, complement has been known to be activated during dialysis for over four decades, this area of research has been neglected in recent years. Despite significant progress in biocompatibility of hemodialysis (HD) membranes and peritoneal dialysis (PD) fluids, complement activation remains an undesired effect and relevant issue. Short-term effects of complement activation include promoting inflammation and coagulation. In addition, long-term complications of dialysis, such as infection, fibrosis and cardiovascular events, are linked to the complement system. These results suggest that interventions targeting the complement system in dialysis could improve biocompatibility, dialysis efficacy, and long-term outcome. Combined with the clinical availability to safely target complement in patients, the question is not if we should inhibit complement in dialysis, but when and how. The purpose of this review is to summarize previous findings and provide a comprehensive overview of the role of the complement system in both HD and PD. PMID:29422906

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

PubMed

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

2016-08-01

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

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

DTIC Science & Technology

1977-01-12

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

Complement

MedlinePlus

... activity (CH50, CH100) looks at the overall activity of the complement system. In most cases, other tests that are more ... Church SE, Fremeaux-Bacchi V, Roumenina LT. Complement system part I - molecular mechanisms of activation and regulation. Front Immunol . 2015;6:262. ...

Foetal Ureaplasma parvum bacteraemia as a function of gestation-dependent complement insufficiency: Evidence from a sheep model of pregnancy.

PubMed

Kemp, Matthew W; Ahmed, Shatha; Beeton, Michael L; Payne, Matthew S; Saito, Masatoshi; Miura, Yuichiro; Usuda, Haruo; Kallapur, Suhas G; Kramer, Boris W; Stock, Sarah J; Jobe, Alan H; Newnham, John P; Spiller, Owen B

2017-01-01

Complement is a central defence against sepsis, and increasing complement insufficiency in neonates of greater prematurity may predispose to increased sepsis. Ureaplasma spp. are the most frequently cultured bacteria from preterm blood samples. A sheep model of intrauterine Ureaplasma parvum infection was used to examine in vivo Ureaplasma bacteraemia at early and late gestational ages. Complement function and Ureaplasma killing assays were used to determine the correlation between complement potency and bactericidal activity of sera ex vivo. Ureaplasma was cultured from 50% of 95-day gestation lamb cord blood samples compared to 10% of 125-day gestation lambs. Bactericidal activity increased with increased gestational age, and a direct correlation between functional complement activity and bactericidal activity (R 2 =.86; P<.001) was found for 95-day gestational lambs. Ureaplasma bacteraemia in vivo was confined to early preterm lambs with low complement function, but Ureaplasma infection itself did not diminish complement levels. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Guilty as charged: all available evidence implicates complement's role in fetal demise.

PubMed

Girardi, Guillermina

2008-03-01

Appropriate complement inhibition is an absolute requirement for normal pregancy. Uncontrolled complement activation in the maternal-fetal interface leads to fetal death. Here we show that complement activation is a crucial and early mediator of pregnancy loss in two different mouse models of pregnancy loss. Using a mouse model of fetal loss and growth restriction (IUGR) induced by antiphospholipid antibodies (aPL), we examined the role of complement activation in fetal loss and IUGR. We found that C5a-C5aR interaction and neutrophils are key mediators of fetal injury. Treatment with heparin, the standard therapy for pregnant patients with aPL, prevents complement activation and protects mice from pregnancy complications induced by aPL, and anticoagulants that do not inhibit complement do not protect pregnancies. In an antibody-independent mouse model of spontaneous miscarriage and IUGR (CBA/JxDBA/2) we also identified C5a as an essential mediator. Complement activation caused dysregulation of the angiogenic factors required for normal placental development. In CBA/JxDBA/2 mice, we observed inflammatory infiltrates in placentas, functional deficiency of free vascular endothelial growth factor (VEGF), elevated levels of soluble VEGF receptor-1 (sVEGFR-1, also known as sFlt-1; a potent anti-angiogenic molecule), and defective placental development. Inhibition of complement activation blocked the increase in sVEGFR-1 and rescued pregnancies. Our studies in antibody-dependent and antibody-independent models of pregnancy complications identified complement activation as the key mediator of damage and will allow development of new interventions to prevent pregnancy loss and IUGR.

Expression of recombinant CD59 with an N-terminal peptide epitope facilitates analysis of residues contributing to its complement-inhibitory function.

PubMed

Zhou, Q; Zhao, J; Hüsler, T; Sims, P J

1996-10-01

CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immunodominant epitopes of CD59 are known to be sensitive to disruption of native tertiary structure, complicating immunological measurement of expressed mutant constructs for structure function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of this complement inhibitor, independent of CD59 antigen, an 11-residue peptide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted before the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cell lines expressing 0 to > 10(5) CD59/cell. The TAG-CD59 fusion protein was confirmed to be GPI-anchored, N-glycosylated and showed identical complement-inhibitory function to wild-type CD59, lacking the TAG peptide sequence. Using this construct, the contribution of each of four surface-localized aromatic residues (4Y, 47F, 61Y, and 62Y) to CD59's complement-inhibitory function was examined. These assays revealed normal surface expression with complete loss of complement-inhibitory function in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 62Y --> S mutants retained approximately 40% of function of wild-type CD59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surface aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function.

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

PubMed

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

2018-05-01

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

Anti-complement activities of human breast-milk.

PubMed

Ogundele, M O

1999-08-01

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

In Situ complement activation and T-cell immunity in leprosy spectrum: An immunohistological study on leprosy lesional skin.

PubMed

Bahia El Idrissi, Nawal; Iyer, Anand M; Ramaglia, Valeria; Rosa, Patricia S; Soares, Cleverson T; Baas, Frank; Das, Pranab K

2017-01-01

Mycobacterium leprae (M. leprae) infection causes nerve damage and the condition worsens often during and long after treatment. Clearance of bacterial antigens including lipoarabinomannan (LAM) during and after treatment in leprosy patients is slow. We previously demonstrated that M. leprae LAM damages peripheral nerves by in situ generation of the membrane attack complex (MAC). Investigating the role of complement activation in skin lesions of leprosy patients might provide insight into the dynamics of in situ immune reactivity and the destructive pathology of M. leprae. In this study, we analyzed in skin lesions of leprosy patients, whether M. leprae antigen LAM deposition correlates with the deposition of complement activation products MAC and C3d on nerves and cells in the surrounding tissue. Skin biopsies of paucibacillary (n = 7), multibacillary leprosy patients (n = 7), and patients with erythema nodosum leprosum (ENL) (n = 6) or reversal reaction (RR) (n = 4) and controls (n = 5) were analyzed. The percentage of C3d, MAC and LAM deposition was significantly higher in the skin biopsies of multibacillary compared to paucibacillary patients (p = <0.05, p = <0.001 and p = <0.001 respectively), with a significant association between LAM and C3d or MAC in the skin biopsies of leprosy patients (r = 0.9578, p< 0.0001 and r = 0.8585, p<0.0001 respectively). In skin lesions of multibacillary patients, MAC deposition was found on axons and co-localizing with LAM. In skin lesions of paucibacillary patients, we found C3d positive T-cells in and surrounding granulomas, but hardly any MAC deposition. In addition, MAC immunoreactivity was increased in both ENL and RR skin lesions compared to non-reactional leprosy patients (p = <0.01 and p = <0.01 respectively). The present findings demonstrate that complement is deposited in skin lesions of leprosy patients, suggesting that inflammation driven by complement activation might contribute to nerve damage in the lesions of these patients. This should be regarded as an important factor in M. leprae nerve damage pathology.

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

PubMed Central

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

1984-01-01

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

Formation, clearance, deposition, pathogenicity, and identification of biopharmaceutical-related immune complexes: review and case studies.

PubMed

Rojko, Jennifer L; Evans, Mark G; Price, Shari A; Han, Bora; Waine, Gary; DeWitte, Mark; Haynes, Jill; Freimark, Bruce; Martin, Pauline; Raymond, James T; Evering, Winston; Rebelatto, Marlon C; Schenck, Emanuel; Horvath, Christopher

2014-06-01

Vascular inflammation, infusion reactions, glomerulopathies, and other potentially adverse effects may be observed in laboratory animals, including monkeys, on toxicity studies of therapeutic monoclonal antibodies and recombinant human protein drugs. Histopathologic and immunohistochemical (IHC) evaluation suggests these effects may be mediated by deposition of immune complexes (ICs) containing the drug, endogenous immunoglobulin, and/or complement components in the affected tissues. ICs may be observed in glomerulus, blood vessels, synovium, lung, liver, skin, eye, choroid plexus, or other tissues or bound to neutrophils, monocytes/macrophages, or platelets. IC deposition may activate complement, kinin, and/or coagulation/fibrinolytic pathways and result in a systemic proinflammatory response. IC clearance is biphasic in humans and monkeys (first from plasma to liver and/or spleen, second from liver or spleen). IC deposition/clearance is affected by IC composition, immunomodulation, and/or complement activation. Case studies are presented from toxicity study monkeys or rats and indicate IHC-IC deposition patterns similar to those predicted by experimental studies of IC-mediated reactions to heterologous protein administration to monkeys and other species. The IHC-staining patterns are consistent with findings associated with generalized and localized IC-associated pathology in humans. However, manifestations of immunogenicity in preclinical species are generally not considered predictive to humans. © 2014 by The Author(s).

Complement is activated in progressive multiple sclerosis cortical grey matter lesions.

PubMed

Watkins, Lewis M; Neal, James W; Loveless, Sam; Michailidou, Iliana; Ramaglia, Valeria; Rees, Mark I; Reynolds, Richard; Robertson, Neil P; Morgan, B Paul; Howell, Owain W

2016-06-22

The symptoms of multiple sclerosis (MS) are caused by damage to myelin and nerve cells in the brain and spinal cord. Inflammation is tightly linked with neurodegeneration, and it is the accumulation of neurodegeneration that underlies increasing neurological disability in progressive MS. Determining pathological mechanisms at play in MS grey matter is therefore a key to our understanding of disease progression. We analysed complement expression and activation by immunocytochemistry and in situ hybridisation in frozen or formalin-fixed paraffin-embedded post-mortem tissue blocks from 22 progressive MS cases and made comparisons to inflammatory central nervous system disease and non-neurological disease controls. Expression of the transcript for C1qA was noted in neurons and the activation fragment and opsonin C3b-labelled neurons and glia in the MS cortical and deep grey matter. The density of immunostained cells positive for the classical complement pathway protein C1q and the alternative complement pathway activation fragment Bb was significantly increased in cortical grey matter lesions in comparison to control grey matter. The number of cells immunostained for the membrane attack complex was elevated in cortical lesions, indicating complement activation to completion. The numbers of classical (C1-inhibitor) and alternative (factor H) pathway regulator-positive cells were unchanged between MS and controls, whilst complement anaphylatoxin receptor-bearing microglia in the MS cortex were found closely apposed to cortical neurons. Complement immunopositive neurons displayed an altered nuclear morphology, indicative of cell stress/damage, supporting our finding of significant neurodegeneration in cortical grey matter lesions. Complement is activated in the MS cortical grey matter lesions in areas of elevated numbers of complement receptor-positive microglia and suggests that complement over-activation may contribute to the worsening pathology that underlies the irreversible progression of MS.

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.

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

PubMed

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

2010-09-01

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

Complement in Non-Antibody-Mediated Kidney Diseases

PubMed Central

Angeletti, Andrea; Reyes-Bahamonde, Joselyn; Cravedi, Paolo; Campbell, Kirk N.

2017-01-01

The complement system is part of the innate immune response that plays important roles in protecting the host from foreign pathogens. The complement components and relative fragment deposition have long been recognized to be strongly involved also in the pathogenesis of autoantibody-related kidney glomerulopathies, leading to direct glomerular injury and recruitment of infiltrating inflammation pathways. More recently, unregulated complement activation has been shown to be associated with progression of non-antibody-mediated kidney diseases, including focal segmental glomerulosclerosis, C3 glomerular disease, thrombotic microangiopathies, or general fibrosis generation in progressive chronic kidney diseases. Some of the specific mechanisms associated with complement activation in these diseases were recently clarified, showing a dominant role of alternative activation pathway. Over the last decade, a growing number of anticomplement agents have been developed, and some of them are being approved for clinical use or already in use. Therefore, anticomplement therapies represent a realistic choice of therapeutic approaches for complement-related diseases. Herein, we review the complement system activation, regulatory mechanisms, their involvement in non-antibody-mediated glomerular diseases, and the recent advances in complement-targeting agents as potential therapeutic strategies. PMID:28748184

Activated complement components and complement activator molecules on the surface of cell‐derived microparticles in patients with rheumatoid arthritis and healthy individuals

PubMed Central

Biró, Éva; Nieuwland, Rienk; Tak, Paul P; Pronk, Loes M; Schaap, Marianne C L; Sturk, Augueste; Hack, C Erik

2007-01-01

Objectives In vitro, microparticles can activate complement via the classical pathway. If demonstrable ex vivo, this mechanism may contribute to the pathogenesis of rheumatoid arthritis (RA). We therefore investigated the presence of activated complement components and complement activator molecules on the surface of cell‐derived microparticles of RA patients and healthy individuals. Methods Microparticles from synovial fluid (n = 8) and plasma (n = 9) of 10 RA patients and plasma of sex‐ and age‐matched healthy individuals (n = 10) were analysed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C‐reactive protein (CRP), serum amyloid P component (SAP), immunoglobulin (Ig) M, IgG). Results Microparticles with bound C1q, C4, and/or C3 were abundant in RA synovial fluid, while in RA and control plasma much lower levels were present. Microparticles with bound C1q correlated with those with bound C3 in synovial fluid (r = 0.961, p = 0.0001), and with those with bound C4 in plasma (RA: r = 0.908, p = 0.0007; control: r = 0.632, p = 0.0498), indicating classical pathway activation. In synovial fluid, microparticles with IgM and IgG correlated with those with C1q (r = 0.728, p = 0.0408; r = 0.952, p = 0.0003, respectively), and in plasma, microparticles with CRP correlated with those with C1q (RA: r = 0.903, p = 0.0021; control: r = 0.683, p = 0.0296), implicating IgG and IgM in the classical pathway activation in RA synovial fluid, and CRP in the low level classical pathway activation in plasma. Conclusions This study demonstrates the presence of bound complement components and activator molecules on microparticles ex vivo, and supports their role in low grade complement activation in plasma and increased complement activation in RA synovial fluid. PMID:17261534

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

PubMed

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

2015-08-01

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

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-03-17

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

Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa

PubMed Central

Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

2010-01-01

Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum. PMID:20421194

Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

PubMed

Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

2010-06-01

Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

Keeping It All Going-Complement Meets Metabolism.

PubMed

Kolev, Martin; Kemper, Claudia

2017-01-01

The complement system is an evolutionary old and crucial component of innate immunity, which is key to the detection and removal of invading pathogens. It was initially discovered as a liver-derived sentinel system circulating in serum, the lymph, and interstitial fluids that mediate the opsonization and lytic killing of bacteria, fungi, and viruses and the initiation of the general inflammatory responses. Although work performed specifically in the last five decades identified complement also as a critical instructor of adaptive immunity-indicating that complement's function is likely broader than initially anticipated-the dominant opinion among researchers and clinicians was that the key complement functions were in principle defined. However, there is now a growing realization that complement activity goes well beyond "classic" immune functions and that this system is also required for normal (neuronal) development and activity and general cell and tissue integrity and homeostasis. Furthermore, the recent discovery that complement activation is not confined to the extracellular space but occurs within cells led to the surprising understanding that complement is involved in the regulation of basic processes of the cell, particularly those of metabolic nature-mostly via novel crosstalks between complement and intracellular sensor, and effector, pathways that had been overlooked because of their spatial separation. These paradigm shifts in the field led to a renaissance in complement research and provide new platforms to now better understand the molecular pathways underlying the wide-reaching effects of complement functions in immunity and beyond. In this review, we will cover the current knowledge about complement's emerging relationship with the cellular metabolism machinery with a focus on the functional differences between serum-circulating versus intracellularly active complement during normal cell survival and induction of effector functions. We will also discuss how taking a closer look into the evolution of key complement components not only made the functional connection between complement and metabolism rather "predictable" but how it may also give clues for the discovery of additional roles for complement in basic cellular processes.

Physicochemical characterization and study of in vitro interactions of pH-sensitive liposomes with the complement system.

PubMed

Carmo, Vildete A S; De Oliveira, Mônica C; Reis, Eduardo C O; Guimarães, Tânia M P D; Vilela, José M C; Andrade, Margareth S; Michalick, Marilene S M; Cardoso, Valbert N

2008-01-01

Complement activation is an important step in the acceleration of liposome clearance. The anaphylatoxins released following complement activation may motivate a wide variety of physiologic changes. We performed physicochemical characterization and in vitro studies of the interaction of complement system with both noncirculating and long-circulating pH-sensitive and nonpH-sensitive liposomes. The liposomes were characterized by diameter, zeta potential, and atomic force microscopy (AFM). The study of liposome interactions with complement system was conducted using hemolytic assay in rat serum. All liposomes presented a similar mean diameter (between 99.8 and 124.3 nm). The zeta potential was negative in all liposome preparations, except in liposomes modified with aminopoly (ethyleneglycol) 2000-distearoylphosphatidylethanolamine (aPEG(2000)-DSPE), which presented positive zeta potential. Atomic force microscopy images showed that non-long-circulating pH-sensitive liposomes are prone to vesicles aggregation. Non-pH-sensitive liposomes complement system activates, while pH-sensitive liposomes showed to be poor complement activators in rat serum.

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

PubMed

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

2014-01-15

Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

Complementation of a red-light-indifferent cyanobacterial mutant.

PubMed Central

Chiang, G G; Schaefer, M R; Grossman, A R

1992-01-01

Many cyanobacteria alter their phycobilisome composition in response to changes in light wavelength in a process termed complementary chromatic adaptation. Mutant strains FdR1 and FdR2 of the filamentous cyanobacterium Fremyella diplosiphon are characterized by aberrant chromatic adaptation. Instead of adjusting to different wavelengths of light, FdR1 and FdR2 behave as if they are always in green light; they do not respond to red light. We have previously reported complementation of FdR1 by conjugal transfer of a wild-type genomic library. The complementing DNA has now been localized by genetic analysis to a region on the rescued genomic subclone that contains a gene designated rcaC. This region of DNA is also able to complement FdR2. Southern blot analysis of genomic DNA from FdR1 and FdR2 indicates that these strains harbor DNA insertions within the rcaC sequence that may have resulted from the activity of transposable genetic elements. The predicted amino acid sequence of RcaC shares strong identity to response regulators of bacterial two-component regulatory systems. This relationship is discussed in the context of the signal-transduction pathway mediating regulation of genes encoding phycobilisome polypeptides during chromatic adaptation. Images PMID:1409650

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

PubMed Central

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

2016-01-01

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

The Microbiome and Complement Activation: A Mechanistic Model for Preterm Birth

PubMed Central

Dunn, Alexis B.; Dunlop, Anne L.; Hogue, Carol J.; Miller, Andrew; Corwin, Elizabeth J.

2018-01-01

Preterm Birth (PTB, < 37 completed weeks' gestation) is one of the leading obstetrical problems in the United States affecting approximately 1 of every 9 births. Even more concerning are the persistent racial disparities in PTB with particularly high rates in African Americans. There are several recognized pathophysiologic pathways to PTB, including infection and/or exaggerated systemic or local inflammation. Intrauterine infection is a causal factor linked to PTB, thought to result most commonly from inflammatory processes triggered by microbial invasion of bacteria ascending from the vaginal microbiome. Trials to treat various infections have shown limited efficacy in reducing PTB risk, suggesting that other complex mechanisms, including those associated with inflammation, may be involved in the relationship between microbes, infection, and PTB. A key mediator of the inflammatory response, and recently shown to be associated with PTB, is the complement system, an innate defense mechanism involved in both normal physiologic processes that occur during pregnancy implantation, as well as processes that promote the elimination of pathogenic microbes. The purpose of this paper is to present a mechanistic model of inflammation-associated PTB, which hypothesizes a relationship between the microbiome and dysregulation of the complement system. Exploring the relationships between the microbial environment and complement biomarkers may elucidate a potentially modifiable biological pathway to preterm birth. PMID:28073296

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

PubMed

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

2014-08-01

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

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

PubMed Central

2017-01-01

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

Cold activation of complement for monitoring the response to interferon in patients with chronic hepatitis C.

PubMed

Akahane, Y; Miyazaki, Y; Naitoh, S; Takeda, K; Tsuda, F; Okamoto, H; Itoh, K; Miyakawa, Y; Mayumi, M

1996-02-01

Because of its specific association with hepatitis C virus (HCV) infection, the cold activation of complement is an easy and inexpensive indicator of HCV viremia. It was evaluated for eligibility as a marker of response to interferon in patients with hepatitis C. The cold activation of complement was determined by the loss or decrease of hemolytic activity with the microtitration method in sera that had been stored at 4 degrees C overnight. We observed the loss of hemolytic activity by the cold activation of complement in 236 (72%) and a decrease in 56 (17%) of 327 sera from patients with HCV-associated chronic liver disease, which was much more (p < 0.001) that in 1 (1%) and 13 (14%), respectively, of 49 sera from patients with chronic liver disease associated with hepatitis B virus infection. Interferon-alpha (total dose 516 x 10(6) units) or interferon-alpha 2b (774 x 10(6) units) was given to 67 patients with chronic hepatitis C, of whom 56 had the cold activation of complement. The response to interferon was evaluated by the clearance of serum HCV RNA at 6 months after the completion of therapy. The cold activation of complement disappeared in 18 patients, of whom 15 (86%) responded. It persisted or fluctuated in the remaining 38 patients, only six (16%) of whom responded to interferon (p < 0.001). The cold activation of complement once disappeared at the completion of interferon and then reappeared in patients who relapsed after completing interferon therapy. These results indicate that the cold activation of complement may be associated with the presence of HCV in blood and a lower rate of durable response after completion of interferon therapy.

SALO, a novel classical pathway complement inhibitor from saliva of the sand fly Lutzomyia longipalpis

PubMed Central

Ferreira, Viviana P.; Fazito Vale, Vladimir; Pangburn, Michael K.; Abdeladhim, Maha; Ferreira Mendes-Sousa, Antonio; Coutinho-Abreu, Iliano V.; Rasouli, Manoochehr; Brandt, Elizabeth A.; Meneses, Claudio; Lima, Kolyvan Ferreira; Nascimento Araújo, Ricardo; Horácio Pereira, Marcos; Kotsyfakis, Michalis; Oliveira, Fabiano; Kamhawi, Shaden; Ribeiro, Jose M. C.; Gontijo, Nelder F.; Collin, Nicolas; Valenzuela, Jesus G.

2016-01-01

Blood-feeding insects inject potent salivary components including complement inhibitors into their host’s skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11 kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. PMID:26758086

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Complement activation and interleukin response in major abdominal surgery.

PubMed

Kvarnström, A L; Sarbinowski, R T; Bengtson, J-P; Jacobsson, L M; Bengtsson, A L

2012-05-01

The objective of this study was to evaluate whether major abdominal surgery leads to complement activation and interleukin response and whether the kind of anaesthesia influence complement activation and the release of inflammatory interleukins. The study design was prospective and randomised. Fifty patients undergoing open major colorectal surgery due to cancer disease or inflammatory bowel disease were studied. Twenty-five patients were given total intravenous anaesthesia (TIVA) with propofol and remifentanil, and 25 patients were given inhalational anaesthesia with sevoflurane and fentanyl. To determine complement activation (C3a and SC5b-9) and the release of pro- and anti-inflammatory interleukins (tumour necrosis factor-a (TNF-a)), interleukin-1b (IL-1b), IL-6, IL-8, IL-4 and IL-10), blood samples were drawn preoperatively, 60 minutes after start of surgery, 30 minutes after end of surgery and 24 hours postoperatively. Complement was activated and pro-inflammatory interleukins (IL-6 and IL-8) and anti-inflammatory interleukins (IL-10) were released during major colorectal surgery. There was no significant difference between TIVA and inhalational anaesthesia regarding complement activation and cytokine release. Major colorectal surgery leads to activation of the complement cascade and the release of both pro-inflammatory and anti-inflammatory cytokines. There are no significant differences between total intravenous anaesthesia (TIVA) with propofol and remifentanil and inhalational anaesthesia with sevoflurane and fentanyl regarding complement activation and the release of pro- and anti-inflammatory interleukins. © 2012 The Authors. Scandinavian Journal of Immunology © 2012 Blackwell Publishing Ltd. Scandinavian Journal of Immunology.

The DNA Helicase Activity of BLM Is Necessary for the Correction of the Genomic Instability of Bloom Syndrome Cells

PubMed Central

Neff, Norma F.; Ellis, Nathan A.; Ye, Tian Zhang; Noonan, James; Huang, Kelly; Sanz, Maureen; Proytcheva, Maria

1999-01-01

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern. PMID:10069810

A Genetic and Molecular Analysis of the 46c Chromosomal Region Surrounding the Fmrfamide Neuropeptide Gene in Drosophila Melanogaster

PubMed Central

O'Brien, M. A.; Roberts, M. S.; Taghert, P. H.

1994-01-01

We have analyzed the FMRFamide neuropeptide gene region of Drosophila melanogaster. This gene maps to the 46C region of chromosome 2R; this interval previously was not well characterized. For this genetic and molecular analysis, we have used X-ray mutagenesis, EMS mutagenesis, and the recently reported local P element transposition method. We identified four overlapping deletions, two of which have proximal breakpoints that define a 50-60-kb region surrounding the FMRFamide gene in 46C. To this small region, we mapped three lethal complementation groups; 10 additional lethal complementation groups were mapped to more distal regions of 46CD. One of these groups corresponds to even-skipped, the other 12 are previously unidentified. Using various lines of evidence we excluded the possibility that FMRFamide corresponds to any of the three lethal complementation groups mapping to its immediate 50-60-kb vicinity. The positions of two of the three lethal complementation groups were identified with P elements using a local transposition scheme. The third lethal complementation group was excluded as being FMRFamide mutants by sequence analysis and by immunocytochemistry with proFMRFamide precursor-specific antibodies. This analysis has (1) provided a genetic map of the 46CD chromosomal region and a detailed molecular map of a portion of the 46C region and (2) provided additional evidence of the utility of local transposition for targeting nearby genes. PMID:8056304

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

PubMed Central

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

2015-01-01

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

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

PubMed

Hovingh, Elise S; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion

PubMed Central

Hovingh, Elise S.; van den Broek, Bryan; Jongerius, Ilse

2016-01-01

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed. PMID:28066340

Solving the Schroedinger Equation of Atoms and Molecules without Analytical Integration Based on the Free Iterative-Complement-Interaction Wave Function

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

Nakatsuji, H.; Nakashima, H.; Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510

2007-12-14

A local Schroedinger equation (LSE) method is proposed for solving the Schroedinger equation (SE) of general atoms and molecules without doing analytic integrations over the complement functions of the free ICI (iterative-complement-interaction) wave functions. Since the free ICI wave function is potentially exact, we can assume a flatness of its local energy. The variational principle is not applicable because the analytic integrations over the free ICI complement functions are very difficult for general atoms and molecules. The LSE method is applied to several 2 to 5 electron atoms and molecules, giving an accuracy of 10{sup -5} Hartree in total energy.more » The potential energy curves of H{sub 2} and LiH molecules are calculated precisely with the free ICI LSE method. The results show the high potentiality of the free ICI LSE method for developing accurate predictive quantum chemistry with the solutions of the SE.« less

Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

PubMed Central

Einfinger, Katrin; Badrnya, Sigrun; Furtmüller, Margareta; Handschuh, Daniela; Lindner, Herbert; Geiger, Margarethe

2015-01-01

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles. PMID:26580551

The role of complement system in septic shock.

PubMed

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

2012-01-01

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

Protective immune responses against West Nile virus are primed by distinct complement activation pathways.

PubMed

Mehlhop, Erin; Diamond, Michael S

2006-05-15

West Nile virus (WNV) causes a severe infection of the central nervous system in several vertebrate animals including humans. Prior studies have shown that complement plays a critical role in controlling WNV infection in complement (C) 3(-/-) and complement receptor 1/2(-/-) mice. Here, we dissect the contributions of the individual complement activation pathways to the protection from WNV disease. Genetic deficiencies in C1q, C4, factor B, or factor D all resulted in increased mortality in mice, suggesting that all activation pathways function together to limit WNV spread. In the absence of alternative pathway complement activation, WNV disseminated into the central nervous system at earlier times and was associated with reduced CD8+ T cell responses yet near normal anti-WNV antibody profiles. Animals lacking the classical and lectin pathways had deficits in both B and T cell responses to WNV. Finally, and somewhat surprisingly, C1q was required for productive infection in the spleen but not for development of adaptive immune responses after WNV infection. Our results suggest that individual pathways of complement activation control WNV infection by priming adaptive immune responses through distinct mechanisms.

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

PubMed Central

Carter, Angela M.

2012-01-01

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

Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM.

PubMed

Goetz, Lindsey; Laskowski, Jennifer; Renner, Brandon; Pickering, Matthew C; Kulik, Liudmila; Klawitter, Jelena; Stites, Erik; Christians, Uwe; van der Vlag, Johan; Ravichandran, Kameswaran; Holers, V Michael; Thurman, Joshua M

2018-05-01

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM-mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild-type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed Central

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

2012-01-01

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

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

PubMed Central

Renner, Brandon; Klawitter, Jelena; Goldberg, Ryan; McCullough, James W.; Ferreira, Viviana P.; Cooper, James E.; Christians, Uwe

2013-01-01

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases. PMID:24092930

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

PubMed Central

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

1996-01-01

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

Complement in autoimmune diseases.

PubMed

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

2017-02-01

The complement system is an ancient and evolutionary conserved element of the innate immune mechanism. It comprises of more than 20 serum proteins most of which are synthesized in the liver. These proteins are synthesized as inactive precursor proteins which are activated by appropriate stimuli. The activated forms of these proteins act as proteases and cleave other components successively in amplification pathways leading to exponential generation of final effectors. Three major pathways of complement pathways have been described, namely the classical, alternative and lectin pathways which are activated by different stimuli. However, all the 3 pathways converge on Complement C3. Cleavage of C3 and C5 successively leads to the production of the membrane attack complex which is final common effector. Excessive and uncontrolled activation of the complement has been implicated in the host of autoimmune diseases. But the complement has also been bemusedly described as the proverbial "double edged sword". On one hand, complement is the final effector of tissue injury in autoimmune diseases and on the other, deficiencies of some components of the complement can result in autoimmune diseases. Currently available tools such as enzyme based immunoassays for functional assessment of complement pathways, flow cytometry, next generation sequencing and proteomics-based approaches provide an exciting opportunity to study this ancient yet mysterious element of innate immunity. Copyright © 2017 Elsevier B.V. All rights reserved.

Classical Complement Pathway Activation in the Kidneys of Women With Preeclampsia.

PubMed

Penning, Marlies; Chua, Jamie S; van Kooten, Cees; Zandbergen, Malu; Buurma, Aletta; Schutte, Joke; Bruijn, Jan Anthonie; Khankin, Eliyahu V; Bloemenkamp, Kitty; Karumanchi, S Ananth; Baelde, Hans

2015-07-01

A growing body of evidence suggests that complement dysregulation plays a role in the pathogenesis of preeclampsia. The kidney is one of the major organs affected in preeclampsia. Because the kidney is highly susceptible to complement activation, we hypothesized that preeclampsia is associated with renal complement activation. We performed a nationwide search for renal autopsy material in the Netherlands using a computerized database (PALGA). Renal tissue was obtained from 11 women with preeclampsia, 25 pregnant controls, and 14 nonpregnant controls with hypertension. The samples were immunostained for C4d, C1q, mannose-binding lectin, properdin, C3d, C5b-9, IgA, IgG, and IgM. Preeclampsia was significantly associated with renal C4d-a stable marker of complement activation-and the classical pathway marker C1q. In addition, the prevalence of IgM was significantly higher in the kidneys of the preeclamptic women. No other complement markers studied differed between the groups. Our findings in human samples were validated using a soluble fms-like tyrosine kinase 1 mouse model of preeclampsia. The kidneys in the soluble fms-like tyrosine kinase 1-injected mice had significantly more C4 deposits than the control mice. The association between preeclampsia and renal C4d, C1q, and IgM levels suggests that the classical complement pathway is involved in the renal injury in preeclampsia. Moreover, our finding that soluble fms-like tyrosine kinase 1-injected mice develop excess C4 deposits indicates that angiogenic dysregulation may play a role in complement activation within the kidney. We suggest that inhibiting complement activation may be beneficial for preventing the renal manifestations of preeclampsia. © 2015 American Heart Association, Inc.

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

PubMed

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

2016-12-01

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

Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

PubMed

Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

2015-02-01

sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

Plasma Exosomes Contribute to Microvascular Damage in Diabetic Retinopathy (DR) by Activating Classical Complement Pathway.

PubMed

Huang, Chao; Fisher, Kiera P; Hammer, Sandra S; Navitskaya, Svetlana; Blanchard, Gary J; Busik, Julia V

2018-06-04

Diabetic Retinopathy (DR) is a micro-vascular complication of diabetes and is the leading cause of vision loss in working-age adults. Recent studies have implicated the complement system as an emerging player in development of vascular damage and progression of DR. However, the role and activation of the complement system in DR is not well understood. Exosomes, small vesicles that are secreted into the extracellular environment, have a cargo of complement proteins in plasma suggesting that they can participate in causing vascular damage associated with DR. We demonstrate that IgG-laden exosomes in plasma activate the classical complement pathway, and that the quantity of these exosomes is increased in diabetes. Moreover, we show that lack of IgG in exosomes results in a reduction of retinal vascular damage in diabetic mice. Together, the results of this study demonstrate that complement activation by IgG-laden plasma exosomes could contribute to the development of DR. © 2018 by the American Diabetes Association.

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

PubMed Central

Kunchithapautham, Kannan; Atkinson, Carl; Rohrer, Bärbel

2014-01-01

Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD. PMID:24711457

Functional analysis of the putative peroxidase domain of FANCA, the Fanconi anemia complementation group A protein.

PubMed

Ren, J; Youssoufian, H

2001-01-01

Fanconi anemia (FA) is an autosomal recessive disorder manifested by chromosomal breakage, birth defects, and susceptibility to bone marrow failure and cancer. At least seven complementation groups have been identified, and the genes defective in four groups have been cloned. The most common subtype is complementation group A. Although the normal functions of the gene products defective in FA cells are not completely understood, a clue to the function of the FA group A gene product (FANCA) was provided by the detection of limited homology in the amino terminal region to a class of heme peroxidases. We evaluated this hypothesis by mutagenesis and functional complementation studies. We substituted alanine residues for the most conserved FANCA residues in the putative peroxidase domain and tested their effects on known biochemical and cellular functions of FANCA. While the substitution mutants were comparable to wild-type FANCA with regard to their stability, subcellular localization, and interaction with FANCG, only the Trp(183)-to-Ala substitution (W183A) abolished the ability of FANCA to complement the sensitivity of FA group A cells to mitomycin C. By contrast, TUNEL assays for apoptosis after exposure to H2O2 showed no differences between parental FA group A cells, cells complemented with wild-type FANCA, and cells complemented with the W183A of FANCA. Moreover, semiquantitative RT-PCR analysis for the expression of the peroxide-sensitive heme oxygenase gene showed appropriate induction after H2O2 exposure. Thus, W183A appears to be essential for the in vivo activity of FANCA in a manner independent of its interaction with FANCG. Moreover, neither wild-type FANCA nor the W183A mutation appears to alter the peroxide-induced apoptosisor peroxide-sensing ability of FA group A cells. Copyright 2001 Academic Press.

Tax-Exempt Hospitals' Investments in Community Health and Local Public Health Spending: Patterns and Relationships.

PubMed

Singh, Simone R; Young, Gary J

2017-12-01

To investigate whether tax-exempt hospitals' investments in community health are associated with patterns of governmental public health spending focusing specifically on the relationship between hospitals' community benefit expenditures and the spending patterns of local health departments (LHDs). We combined data on tax-exempt hospitals' community benefit spending with data on spending by the corresponding LHD that served the county in which a hospital was located. Data were available for 2 years, 2009 and 2013. Generalized linear regressions were estimated with indicators of hospital community benefit spending as the dependent variable and LHD spending as the key independent variable. Hospital community benefit spending was unrelated to how much local public health agencies spent, per capita, on public health in their communities. Patterns of local public health spending do not appear to impact the investments of tax-exempt hospitals in community health activities. Opportunities may, however, exist for a more active engagement between the public and private sector to ensure that the expenditures of all stakeholders involved in community health improvement efforts complement one another. © Health Research and Educational Trust.

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

PubMed

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

2010-11-23

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

Complement in Lupus Nephritis: New Perspectives.

PubMed

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

2015-09-01

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. 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. 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 trials support the use of complement-targeted therapy in the treatment of SLE.

Regulation of complement C3 and C4 synthesis in human peritoneal mesothelial cells by peritoneal dialysis fluid

PubMed Central

TANG, S; LEUNG, J C K; CHAN, L Y Y; TSANG, A W L; CHEN, C X R; ZHOU, W; LAI, K N; SACKS, S H

2004-01-01

Although complement is activated in the peritoneal cavity during chronic peritoneal dialysis (PD), little is known about its role in peritoneal defence and injury related to long-term PD. We examined the impact of glucose and commercial peritoneal dialysis solutions on complement expression in HPMCs obtained by primary culture from omental tissues of consented patients undergoing elective abdominal surgery. Constitutive expression of C3 and C4 mRNA in HPMCs was up-regulated upon exposure to 75 mm glucose in a time-dependent manner. C3 and C4 protein was secreted in both apical and basolateral directions. Glucose doses beyond 100 mm markedly down-regulated C3 and C4 expression, and stimulated LDH release dose-dependently. Such cytotoxic effects were attenuated using equivalent doses of mannitol instead of glucose. Treatment with conventional lactate-buffered dialysis solution gave rise to down-regulation of C3 and C4 expression, and heightened LDH release in HPMCs. These effects correlated with the glucose strength of the solution, persisted despite replacement with a bicarbonate-buffered solution, aggravated by glycated albumin, and were partially abrogated by supplementation with 10% fetal bovine serum in the culture system. Our findings suggest that the artificial conditions imposed by PD lead to alterations in local complement synthesis that have implications for the role of the peritoneal mesothelium in both inflammation and defence. PMID:15030518

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

PubMed Central

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

2013-01-01

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

Complement factor B expression profile in a spontaneous uveitis model.

PubMed

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

2010-12-01

Equine recurrent uveitis serves as a spontaneous model for human autoimmune uveitis. Unpredictable relapses and ongoing inflammation in the eyes of diseased horses as well as in humans lead to destruction of the retina and finally result in blindness. However, the molecular mechanisms leading to inflammation and retinal degeneration are not well understood. An initial screening for differentially regulated proteins in sera of uveitic cases compared to healthy controls revealed an increase of the alternative pathway complement component factor B in ERU cases. To determine the activation status of the complement system, sera were subsequently examined for complement split products. We could demonstrate a significant higher concentration of the activation products B/Ba, B/Bb, Bb neoantigen, iC3b and C3d in uveitic condition compared to healthy controls, whereas for C5b-9 no differences were detected. Additionally, we investigated complement activation directly in the retina by immunohistochemistry, since it is the main target organ of this autoimmune disease. Interestingly, infiltrating cells co-expressed activated factor Bb neoantigen, complement split product C3d as well as CD68, a macrophage marker. In this study, we could demonstrate activation of the complement system both systemically as well as in the eye, the target organ of spontaneous recurrent uveitis. Based on these novel findings, we postulate a novel role for macrophages in connection with complement synthesis at the site of inflammation. Copyright © 2010 Elsevier GmbH. All rights reserved.

Neighbourhood Built Environment Influences on Physical Activity among Adults: A Systematized Review of Qualitative Evidence

PubMed Central

Salvo, Grazia; Doyle-Baker, Patricia K.; McCormack, Gavin R.

2018-01-01

Qualitative studies can provide important information about how and why the built environment impacts physical activity decision-making—information that is important for informing local urban policies. We undertook a systematized literature review to synthesize findings from qualitative studies exploring how the built environment influences physical activity in adults. Our review included 36 peer-reviewed qualitative studies published from 1998 onwards. Our findings complemented existing quantitative evidence and provided additional insight into how functional, aesthetic, destination, and safety built characteristics influence physical activity decision-making. Sociodemographic characteristics (age, sex, ethnicity, and socioeconomic status) also impacted the BE’s influence on physical activity. Our review findings reinforce the need for synergy between transportation planning, urban design, landscape architecture, road engineering, parks and recreation, bylaw enforcement, and public health to be involved in creating neighbourhood environments that support physical activity. Our findings support a need for local neighbourhood citizens and associations with representation from individuals and groups with different sociodemographic backgrounds to have input into neighbourhood environment planning process. PMID:29724048

Probable systemic lupus erythematosus with cell-bound complement activation products (CB-CAPS).

PubMed

Lamichhane, D; Weinstein, A

2016-08-01

Complement activation is a key feature of systemic lupus erythematosus (SLE). Detection of cell-bound complement activation products (CB-CAPS) occurs more frequently than serum hypocomplementemia in definite lupus. We describe a patient with normocomplementemic probable SLE who did not fulfill ACR classification criteria for lupus, but the diagnosis was supported by the presence of CB-CAPS. © The Author(s) 2016.

Complement activating properties of complexes containing rheumatoid factor in synovial fluids and sera from patients with rheumatoid arthritis.

PubMed Central

Elson, C J; Carter, S D; Cottrell, B J; Scott, D G; Bacon, P A; Wallington, T B

1985-01-01

The relationship between complexes containing rheumatoid factor and complexes activating complement was examined in synovial fluids and sera from patients with rheumatoid arthritis (RA). In each case this was performed by quantifying the amount of rheumatoid factor bound by solid phase Fab'2 anti-C3 and/or solid phase conglutinin. Both anti-C3 coated and conglutinin coated microtitre plates bound high levels of complexes containing rheumatoid factor from sera of RA patients with vasculitis. Unexpectedly, these complexes were detected in synovial fluids from only a minority of RA patients with synovitis. However, RA synovial fluids did contain other complexes as shown by the presence of complement consuming activity, C1q binding material and immunoglobulin attaching to conglutinin. It is considered that in RA synovial fluids the complexes containing RF and those activating complement are not necessarily the same whilst in vasculitic sera the complexes containing rheumatoid factor also activate complement. PMID:3978872

Demonstration of monolignol β-glucosidase activity of rice Os4BGlu14, Os4BGlu16 and Os4BGlu18 in Arabidopsis thaliana bglu45 mutant.

PubMed

Baiya, Supaporn; Mahong, Bancha; Lee, Sang-Kyu; Jeon, Jong-Seong; Ketudat Cairns, James R

2018-06-01

The glycoside hydrolase family 1 members Os4BGlu14, Os4BGlu16, and Os4BGlu18 were proposed to be rice monolignol β-glucosidases. In vitro studies demonstrated that the Os4BGlu16 and Os4BGlu18 hydrolyze the monolignol glucosides coniferin and syringin with high efficiency compared to other substrates. The replacement of the conserved catalytic acid/base glutamate residue by a nonionizable glutamine residue in Os4BGlu14 suggested that it may be inactive as a β-glucosidase. Here, we investigated the activities of Os4BGlu14, Os4BGlu16, and Os4BGlu18 in planta by recombinant expression of their genes in the Arabidopsis bglu45-2 (monolignol β-glucosidase) mutant and analysis of monolignol glucosides by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MSMS). The bglu45-2 line exhibits elevated monolignol glucoside levels, but lower amounts of coniferin, syringin, and p-coumaryl alcohol glucoside were seen in Arabidopsis bglu45-2 rescued lines complemented by the Os4BGlu14, Os4BGlu16, and Os4BGlu18 genes. These data suggest that the bglu45-2 mutant has a broader effect on monolignols than previously reported and that the Os4BGlu14, Os4BGlu16 and Os4BGlu18 proteins act as monolignol β-glucosidases to complement the defect. An OsBGlu16-GFP fusion protein localized to the cell wall. This apoplastic localization and the effect of these enzymes on monolignol glucoside levels suggest monolignol glucosides from the vacuole may meet the monolignol β-glucosidases, despite their different localization. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Assessment of Complement Activation by Nanoparticles: Development of a SPR Based Method and Comparison with Current High Throughput Methods.

PubMed

Coty, Jean-Baptiste; Noiray, Magali; Vauthier, Christine

2018-04-26

A Surface Plasmon Resonance chip (SPR) was developed to study the activation of complement system triggered by nanomaterials in contact with human serum, which is an important concern today to warrant safety of nanomedicines. The developed chip was tested for its specificity in complex medium and its longevity of use. It was then employed to assess the release of complement fragments upon incubation of nanoparticles in serum. A comparison was made with other current methods assessing complement activation (μC-IE, ELISA). The SPR chip was found to give a consistent response for C3a release upon activation by nanoparticles. Results were similar to those obtained by μC-IE. However, ELISA detection of iC3b fragments showed an explained high non-specific background. The impact of sample preparation preceding the analysis was assessed with the newly develop SPR method. The removal of nanoparticles before analysis showed an important modification in the obtained response, possibly leading to false negative results. The SPR chip developed in this work allows for an automated assessment of complement activation triggered by nanoparticles with possibility of multiplexed analysis. The design of the chip proved to give consistent results of complement activation by nanoparticles.

Complement Evasion by Pathogenic Leptospira.

PubMed

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

2016-01-01

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

Complement Evasion by Pathogenic Leptospira

PubMed Central

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

2016-01-01

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

C1q-Mediated Complement Activation and C3 Opsonization Trigger Recognition of Stealth Poly(2-methyl-2-oxazoline)-Coated Silica Nanoparticles by Human Phagocytes.

PubMed

Tavano, Regina; Gabrielli, Luca; Lubian, Elisa; Fedeli, Chiara; Visentin, Silvia; Polverino De Laureto, Patrizia; Arrigoni, Giorgio; Geffner-Smith, Alessandra; Chen, Fangfang; Simberg, Dmitri; Morgese, Giulia; Benetti, Edmondo M; Wu, Linping; Moghimi, Seyed Moein; Mancin, Fabrizio; Papini, Emanuele

2018-05-23

Poly(2-methyl-2-oxazoline) (PMOXA) is an alternative promising polymer to poly(ethylene glycol) (PEG) for design and engineering of macrophage-evading nanoparticles (NPs). Although PMOXA-engineered NPs have shown comparable pharmacokinetics and in vivo performance to PEGylated stealth NPs in the murine model, its interaction with elements of the human innate immune system has not been studied. From a translational angle, we studied the interaction of fully characterized PMOXA-coated vinyltriethoxysilane-derived organically modified silica NPs (PMOXA-coated NPs) of approximately 100 nm in diameter with human complement system, blood leukocytes, and macrophages and compared their performance with PEGylated and uncoated NP counterparts. Through detailed immunological and proteomic profiling, we show that PMOXA-coated NPs extensively trigger complement activation in human sera exclusively through the classical pathway. Complement activation is initiated by the sensing molecule C1q, where C1q binds with high affinity ( K d = 11 ± 1 nM) to NP surfaces independent of immunoglobulin binding. C1q-mediated complement activation accelerates PMOXA opsonization with the third complement protein (C3) through the amplification loop of the alternative pathway. This promoted NP recognition by human blood leukocytes and monocyte-derived macrophages. The macrophage capture of PMOXA-coated NPs correlates with sera donor variability in complement activation and opsonization but not with other major corona proteins, including clusterin and a wide range of apolipoproteins. In contrast to these observations, PMOXA-coated NPs poorly activated the murine complement system and were marginally recognized by mouse macrophages. These studies provide important insights into compatibility of engineered NPs with elements of the human innate immune system for translational steps.

Coagulation cascade and complement system in systemic lupus erythematosus

PubMed Central

Liang, Yan; Xie, Shang-Bo; Wu, Chang-Hao; Hu, Yuan; Zhang, Qin; Li, Si; Fan, Yin-Guang; Leng, Rui-Xue; Pan, Hai-Feng; Xiong, Hua-Bao; Ye, Dong-Qing

2018-01-01

This study was conducted to (1) characterize coagulation cascade and complement system in systemic lupus erythematosus (SLE); (2) evaluate the associations between coagulation cascade, complement system, inflammatory response and SLE disease severity; (3) test the diagnostic value of a combination of D-dimer and C4 for lupus activity. Transcriptomics, proteomics and metabolomics were performed in 24 SLE patients and 24 healthy controls. The levels of ten coagulations, seven complements and three cytokines were measured in 112 SLE patients. Clinical data were collected from 2025 SLE patients. The analysis of multi-omics data revealed the common links for the components of coagulation cascade and complement system. The results of ELISA showed coagulation cascade and complement system had an interaction effect on SLE disease severity, this effect was pronounced among patients with excess inflammation. The analysis of clinical data revealed a combination of D-dimer and C4 provided good diagnostic performance for lupus activity. This study suggested that coagulation cascade and complement system become ‘partners in crime’, contributing to SLE disease severity and identified the diagnostic value of D-dimer combined with C4for lupus activity. PMID:29599912

Structure of C3b reveals conformational changes that underlie complement activity.

PubMed

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

2006-11-09

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

Unc-51 controls active zone density and protein composition by downregulating ERK signaling.

PubMed

Wairkar, Yogesh P; Toda, Hirofumi; Mochizuki, Hiroaki; Furukubo-Tokunaga, Katsuo; Tomoda, Toshifumi; Diantonio, Aaron

2009-01-14

Efficient synaptic transmission requires the apposition of neurotransmitter release sites opposite clusters of postsynaptic neurotransmitter receptors. Transmitter is released at active zones, which are composed of a large complex of proteins necessary for synaptic development and function. Many active zone proteins have been identified, but little is known of the mechanisms that ensure that each active zone receives the proper complement of proteins. Here we use a genetic analysis in Drosophila to demonstrate that the serine threonine kinase Unc-51 acts in the presynaptic motoneuron to regulate the localization of the active zone protein Bruchpilot opposite to glutamate receptors at each synapse. In the absence of Unc-51, many glutamate receptor clusters are unapposed to Bruchpilot, and ultrastructural analysis demonstrates that fewer active zones contain dense body T-bars. In addition to the presence of these aberrant synapses, there is also a decrease in the density of all synapses. This decrease in synaptic density and abnormal active zone composition is associated with impaired evoked transmitter release. Mechanistically, Unc-51 inhibits the activity of the MAP kinase ERK to promote synaptic development. In the unc-51 mutant, increased ERK activity leads to the decrease in synaptic density and the absence of Bruchpilot from many synapses. Hence, activated ERK negatively regulates synapse formation, resulting in either the absence of active zones or the formation of active zones without their proper complement of proteins. The Unc-51-dependent inhibition of ERK activity provides a potential mechanism for synapse-specific control of active zone protein composition and release probability.

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

PubMed

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

2009-11-01

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

Increased Global Interaction Across Functional Brain Modules During Cognitive Emotion Regulation.

PubMed

Brandl, Felix; Mulej Bratec, Satja; Xie, Xiyao; Wohlschläger, Afra M; Riedl, Valentin; Meng, Chun; Sorg, Christian

2017-07-13

Cognitive emotion regulation (CER) enables humans to flexibly modulate their emotions. While local theories of CER neurobiology suggest interactions between specialized local brain circuits underlying CER, e.g., in subparts of amygdala and medial prefrontal cortices (mPFC), global theories hypothesize global interaction increases among larger functional brain modules comprising local circuits. We tested the global CER hypothesis using graph-based whole-brain network analysis of functional MRI data during aversive emotional processing with and without CER. During CER, global between-module interaction across stable functional network modules increased. Global interaction increase was particularly driven by subregions of amygdala and cuneus-nodes of highest nodal participation-that overlapped with CER-specific local activations, and by mPFC and posterior cingulate as relevant connector hubs. Results provide evidence for the global nature of human CER, complementing functional specialization of embedded local brain circuits during successful CER. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

PubMed Central

Brocklebank, Vicky

2017-01-01

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

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

PubMed

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

2016-06-15

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

Complement evasion by Bordetella pertussis: implications for improving current vaccines.

PubMed

Jongerius, Ilse; Schuijt, Tim J; Mooi, Frits R; Pinelli, Elena

2015-04-01

Bordetella pertussis causes whooping cough or pertussis, a highly contagious disease of the respiratory tract. Despite high vaccination coverage, reported cases of pertussis are rising worldwide and it has become clear that the current vaccines must be improved. In addition to the well-known protective role of antibodies and T cells during B. pertussis infection, innate immune responses such as the complement system play an essential role in B. pertussis killing. In order to evade this complement activation and colonize the human host, B. pertussis expresses several molecules that inhibit complement activation. Interestingly, one of the known complement evasion proteins, autotransporter Vag8, is highly expressed in the recently emerged B. pertussis isolates. Here, we describe the current knowledge on how B. pertussis evades complement-mediated killing. In addition, we compare this to complement evasion strategies used by other bacterial species. Finally, we discuss the consequences of complement evasion by B. pertussis on adaptive immunity and how identification of the bacterial molecules and the mechanisms involved in complement evasion might help improve pertussis vaccines.

Dengue-Immune Humans Have Higher Levels of Complement-Independent Enhancing Antibody than Complement-Dependent Neutralizing Antibody.

PubMed

Yamanaka, Atsushi; Konishi, Eiji

2017-09-25

Dengue is the most important arboviral disease worldwide. We previously reported that most inhabitants of dengue-endemic countries who are naturally immune to the disease have infection-enhancing antibodies whose in vitro activity does not decrease in the presence of complement (complement-independent enhancing antibodies, or CiEAb). Here, we compared levels of CiEAb and complement-dependent neutralizing antibodies (CdNAb) in dengue-immune humans. A typical antibody dose-response pattern obtained in our assay system to measure the balance between neutralizing and enhancing antibodies showed both neutralizing and enhancing activities depending on serum dilution factor. The addition of complement to the assay system increased the activity of neutralizing antibodies at lower dilutions, indicating the presence of CdNAb. In contrast, similar dose-response curves were obtained with and without complement at higher dilutions, indicating higher levels of CiEAb than CdNAb. For experimental support for the higher CiEAb levels, a cocktail of mouse monoclonal antibodies against dengue virus type 1 was prepared. The antibody dose-response curves obtained in this assay, with or without complement, were similar to those obtained with human serum samples when a high proportion of D1-V-3H12 (an antibody exhibiting only enhancing activity and thus a model for CiEAb) was used in the cocktail. This study revealed higher-level induction of CiEAb than CdNAb in humans naturally infected with dengue viruses.

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

PubMed

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

2014-01-31

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

Complement factor H family proteins in their non-canonical role as modulators of cellular functions.

PubMed

Józsi, Mihály; Schneider, Andrea E; Kárpáti, Éva; Sándor, Noémi

2018-01-04

Complement factor H is a major regulator of the alternative pathway of the complement system. The factor H-related proteins are less characterized, but recent data indicate that they rather promote complement activation. These proteins have some common ligands with factor H and have both overlapping and distinct functions depending on domain composition and the degree of conservation of amino acid sequence. Factor H and some of the factor H-related proteins also appear in a non-canonical function that is beyond their role in the modulation of complement activation. This review covers our current understanding on this emerging role of factor H family proteins in modulating the activation and function of various cells by binding to receptors or receptor ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.

Targeting the complement system for the management of retinal inflammatory and degenerative diseases.

PubMed

Xu, Heping; Chen, Mei

2016-09-15

The retina, an immune privileged tissue, has specialized immune defense mechanisms against noxious insults that may exist in diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), uveoretinitis and glaucoma. The defense system consists of retinal innate immune cells (including microglia, perivascular macrophages, and a small population of dendritic cells) and the complement system. Under normal aging conditions, retinal innate immune cells and the complement system undergo a low-grade activation (parainflammation) which is important for retinal homeostasis. In disease states such as AMD and DR, the parainflammatory response is dysregulated and develops into detrimental chronic inflammation. Complement activation in the retina is an important part of chronic inflammation and may contribute to retinal pathology in these disease states. Here, we review the evidence that supports the role of uncontrolled or dysregulated complement activation in various retinal degenerative and angiogenic conditions. We also discuss current strategies that are used to develop complement-based therapies for retinal diseases such as AMD. The potential benefits of complement inhibition in DR, uveoretinitis and glaucoma are also discussed, as well as the need for further research to better understand the mechanisms of complement-mediated retinal damage in these disease states. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Bothrops asper snake venom and its metalloproteinase BaP-1 activate the complement system. Role in leucocyte recruitment.

PubMed Central

Farsky, S H; Gonçalves, L R; Gutiérrez, J M; Correa, A P; Rucavado, A; Gasque, P; Tambourgi, D V

2000-01-01

The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins--phospholipases and metalloproteinase--activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boyden's chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP-1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s) derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1) did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s) which can cause direct activation of C5a. PMID:11200361

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

PubMed

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

2014-12-01

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

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

PubMed Central

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

1982-01-01

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

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

PubMed Central

Jäckel, Sven; Saffarzadeh, Mona; Langer, Florian

2017-01-01

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

Cobra venom factor immunoconjugates: effects of carbohydrate-directed versus amino group-directed conjugation.

PubMed

Zara, J; Pomato, N; McCabe, R P; Bredehorst, R; Vogel, C W

1995-01-01

Human IgM monoclonal antibody 16-88, derived from patients immunized with autologous colon carcinoma cells, was derivatized with two different cross-linkers, S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), which is carbohydrate-directed, and N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), which is amino group-directed. Two antibody functions, antigen binding and complement activation, were assayed upon derivatization with TPCH and SPDP. TPCH allowed for extensive modification (up to 17 TPCH molecules per antibody) without impairment of antigen binding activity, while this function was significantly compromised upon derivatization with SPDP. Antibody molecules derivatized with 16 SPDP residues showed almost complete loss of their antigen binding function. The complement activating ability of antibody 16-88 was significantly decreased after derivatization with TPCH or SPDP. In the case of SPDP derivatization, this decrease of the complement activating ability is predominantly a consequence of the impaired binding function. Upon conjugation of cobra venom factor (CVF), a nontoxic 137-kDa glycoprotein which is capable of activating the alternative pathway of complement, the antigen binding activity of SPDP-derivatized antibody was further compromised, whereas that of TPCH-derivatized antibody remained unaffected even after attachment of three or four CVF molecules per antibody. In both conjugates CVF retained good functional activity. CVF was slightly more active when attached to SPDP-derivatized antibody, suggesting a better accessibility of amino group-coupled CVF for its interaction with other complement proteins. These results indicate that carbohydrate-directed conjugation compromises the antibody function of complement activation, but allows for the generation of immunoconjugates with unimpaired antigen binding capability.(ABSTRACT TRUNCATED AT 250 WORDS)

A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood.

PubMed

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

2017-02-08

Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood.

A versatile assay to determine bacterial and host factors contributing to opsonophagocytotic killing in hirudin-anticoagulated whole blood

PubMed Central

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

2017-01-01

Most bacteria entering the bloodstream will be eliminated through complement activation on the bacterial surface and opsonophagocytosis. However, when these protective innate immune systems do not work optimally, or when bacteria are equipped with immune evasion mechanisms that prevent killing, this can lead to serious infections such as bacteremia and meningitis, which is associated with high morbidity and mortality. In order to study the complement evasion mechanisms of bacteria and the capacity of human blood to opsonize and kill bacteria, we developed a versatile whole blood killing assay wherein both phagocyte function and complement activity can easily be monitored and modulated. In this assay we use a selective thrombin inhibitor hirudin to fully preserve complement activity of whole blood. This assay allows controlled analysis of the requirements for active complement by replacing or heat-inactivating plasma, phagocyte function and bacterial immune evasion mechanisms that contribute to survival in human blood. PMID:28176849

Anti-complementary constituents of Houttuynia cordata and their targets in complement activation cascade.

PubMed

Jiang, Yun; Lu, Yan; Zhang, Yun-Yi; Chen, Dao-Feng

2014-01-01

Activity-guided fractionation for complement inhibitors led to the isolation of 23 known compounds from Houttuynia cordata Thunb. Seven flavonoids, two alkaloids, one coumarin and two phenols showed anti-complementary activity. Preliminary inhibitory mechanism of four flavonoids, including quercitrin, afzelin, isoquercitrin and quercetin in the complement activation cascade were examined for the first time. The results indicated that the target components of flavonols are different from those of flavonosides, and the glycoside moieties may be necessary to block C3 and C4 components.

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

PubMed

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

2015-01-01

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

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

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

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with onemore » GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.« less

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay.

PubMed

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil; Kim, Dong-Myung; Yoo, Tae Hyeon; Kim, Yong-Sung

2015-11-27

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Function of Serum Complement in Drinking Water Arsenic Toxicity

PubMed Central

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

2012-01-01

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

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

Pleiotropic Effects of Cell Wall Amidase LytA on Streptococcus pneumoniae Sensitivity to the Host Immune Response

PubMed Central

Ramos-Sevillano, Elisa; Urzainqui, Ana; Campuzano, Susana; Moscoso, Miriam; González-Camacho, Fernando; Domenech, Mirian; Rodríguez de Córdoba, Santiago; Sánchez-Madrid, Francisco; Brown, Jeremy S.; García, Ernesto

2014-01-01

The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia. PMID:25404032

Complement deposition induced by binding of anti-contactin-1 auto-antibodies is modified by immunoglobulins.

PubMed

Appeltshauser, Luise; Weishaupt, Andreas; Sommer, Claudia; Doppler, Kathrin

2017-01-01

Inflammatory neuropathies associated with auto-antibodies against paranodal proteins like contactin-1 are reported to respond poorly to treatment with intravenous immunoglobulins (IVIG). A reason might be that IVIG interacts with the complement pathway and these auto-antibodies often belong to the IgG4 subclass that does not activate complement. However, some patients do show a response to IVIG, especially at the beginning of the disease. This corresponds with the finding of coexisting IgG subclasses IgG1, IgG2 and IgG3. We therefore aimed to investigate complement deposition and activation by samples of three patients with anti-contactin-1 IgG auto-antibodies of different subclasses as a potential predictor for response to IVIG. Complement deposition and activation was measured by cell binding and ELISA based assays, and the effect of IVIG on complement deposition was assessed by addition of different concentrations of IVIG. Binding of anti-contactin-1 auto-antibodies of all three patients induced complement deposition and activation with the strongest effect shown by the serum of a patient with predominance of IgG3 auto-antibodies. IVIG led to a reduction of complement deposition in a dose-dependent manner, but did not reduce binding of auto-antibodies to contactin-1. We conclude that complement deposition may contribute to the pathophysiology of anti-contactin-1 associated neuropathy, particularly in patients with predominance of the IgG3 subclass. The proportion of different auto-antibody subclasses may be a predictor for the response to IVIG in patients with auto-antibodies against paranodal proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway.

PubMed

Rosbjerg, Anne; Genster, Ninette; Pilely, Katrine; Garred, Peter

2017-01-01

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition molecules that bind specific patterns on microbial surfaces, a group of associated proteases that initiates the complement cascade, and a group of proteins that interact in proteolytic complexes or the terminal pore-forming complex. In addition, various regulatory proteins are important for controlling the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system. Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination.

Increased C3 production in human monocytes after stimulation with Candida albicans is suppressed by granulocyte-macrophage colony-stimulating factor.

PubMed Central

Høgåsen, A K; Abrahamsen, T G

1993-01-01

Activation of the complement system is an important part of host resistance against fungal infections. When human monocytes, cultured for 2 days or more, were treated in vitro with Candida albicans for 24 h, an enhancement of their biosynthesis of the complement components C3 and factor B was found. However, when C. albicans was administered to freshly isolated monocytes, a consistent stimulation of factor B biosynthesis occurred, while the C3 production was increased in about 50% of the donors. C. albicans also induced the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from the cultured cells, apparently in larger amounts in the donors in whom no stimulation of C3 production was found. An antibody to GM-CSF administered with the yeast at the initiation of the monocyte culture caused an increase in the C3 production. Furthermore, when monocytes were treated with recombinant human GM-CSF either at the same time as or 4 days prior to the addition of C. albicans, the increase in C3 production was suppressed or neutralized, while factor B biosynthesis was unaffected. Taken together, these results indicate that monocytes respond to C. albicans with an increased production of complement factors. This may be an important mechanism both for opsonization of the fungus and for initiation of an inflammatory reaction. At an inflammatory site, this complement response may be suppressed by locally produced GM-CSF. PMID:8478067

Lutein and atherosclerosis: Belfast versus Toulouse revisited.

PubMed

Howard, A N; Thurnham, D I

2017-01-01

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

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.

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

PubMed Central

Granoff, Dan M.

2009-01-01

Killing of Neisseria meningitidis can result from complement-mediated bactericidal activity (SBA) or opsonophagocytosis (OPA), or a combination of the two mechanisms. While SBA titers ≥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 ≥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. PMID:19477054

ROLES OF ADIPOCYTES AND FIBROBLASTS IN ACTIVATION OF THE ALTERNATIVE PATHWAY OF COMPLEMENT IN INFLAMMATORY ARTHRITIS IN MICE

PubMed Central

Arend, William P.; Mehta, Gaurav; Antonioli, Alexandra H.; Takahashi, Minoru; Takahashi, Kazue; Stahl, Gregory L.; Holers, V. Michael; Banda, Nirmal K.

2013-01-01

The complement system is involved in mediation of joint damage in rheumatoid arthritis, with evidence suggesting activation of both the classical and alternative pathways (AP). The AP is both necessary and sufficient to mediate collagen antibody-induced arthritis (CAIA), an experimental animal model of immune complex (IC)-induced joint disease. The AP in mice is dependent on MASP-1/3 cleavage of pro-factor D (pro-FD) into mature FD. The objectives of the present study were to determine the cells synthesizing MASP-1/3 and pro-FD in synovial tissue. CAIA was studied in wild-type C57BL/6 mice, and the localization of mRNA and protein for FD and MASP-1/3 in synovial adipose tissue (SAT) and fibroblast-like synoviocytes (FLS) was determined using various techniques, including laser capture micro-dissection (LCM). SAT was the sole source of mRNA for pro-FD. Cultured differentiated 3T3 adipocytes, a surrogate for SAT, produced pro-FD but no mature FD. FLS were the main source of MASP-1/3 mRNA and protein. Using cartilage micro-particles (CMP) coated with anti-collagen mAb and serum from MASP-1/3−/− mice as a source of factor B, pro-FD in 3T3 supernatants was cleaved into mature FD by MASP-1/3 in FLS supernatants. The mature FD was eluted from the CMP, and was not present in the supernatants from the incubation with CMP, indicating that cleavage of pro-FD into mature FD by MASP-1 occurred on the CMP. These results demonstrate that pathogenic activation of the AP may occur in the joint through IC adherent to cartilage and the local production of necessary AP proteins by adipocytes and FLS. PMID:23650618

Neutrophil extracellular traps can activate alternative complement pathways.

PubMed

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

2015-09-01

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

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

PubMed

Wada, Takehiko; Nangaku, Masaomi

2013-09-01

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

Early Complementopathy after Multiple Injuries in Humans

PubMed Central

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

2012-01-01

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

Musa paradisica RCI complements AtRCI and confers Na+ tolerance and K+ sensitivity in Arabidopsis.

PubMed

Liu, Bing; Feng, Dongru; Zhang, Bipei; Mu, Peiqiang; Zhang, Yang; He, Yanming; Qi, Kangbiao; Wang, Jinfa; Wang, Hongbin

2012-03-01

The mechanisms involved in Na⁺/K⁺ uptake and extrusion are important in plant salt tolerance. In this study, we investigated the physiological role of a plasma membrane (PM)-localized protein, MpRCI, from plantain in transgenic Arabidopsis under NaCl and KCl stress and determined its effect on PM fluidity and H⁺-ATPase activity. The MpRCI gene exhibited high homology to the AtRCI2 gene family in Arabidopsis and was therefore able to complement for loss of the yeast AtRCI2-related PMP3 gene. Results of phenotypic espial and atomic emission spectrophotometer (AES) assays indicated that MpRCI overexpression in the AtRCI2A knockout mutant with reduced shoot Na⁺ and increased K⁺ exhibited increased Na⁺-tolerance and K⁺-sensitivity under NaCl or KCl treatments, respectively. Furthermore, comparisons of PM fluidity and H⁺-ATPase activity in shoots, with expression or absence of MpRCI/AtRCI2A expression under NaCl or KCl stress, showed MpRCI maintained PM fluidity and H⁺-ATPase activity under stress conditions. Results suggest that MpRCI plays an essential role in Na⁺/K⁺ flux in plant cells. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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

2011-09-01

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

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

ERIC Educational Resources Information Center

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

2008-01-01

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

Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy.

PubMed

Xu, Jijun; Zhang, Lingjun; Xie, Mian; Li, Yan; Huang, Ping; Saunders, Thomas L; Fox, David A; Rosenquist, Richard; Lin, Feng

2018-06-15

Chemotherapy-induced peripheral neuropathy (CIPN) is a painful and debilitating side effect of cancer chemotherapy with an unclear pathogenesis. Consequently, the available therapies for this neuropathic pain syndrome are inadequate, leading to a significantly reduced quality of life in many patients. Complement, a key component of the innate immune system, has been associated with neuroinflammation, a potentially important trigger of some types of neuropathic pain. However, the role of complement in CIPN remains unclear. To address this issue, we developed a C3 knockout (KO) rat model and induced CIPN in these KO rats and wild-type littermates via the i.p. administration of paclitaxel, a chemotherapeutic agent associated with CIPN. We then compared the severity of mechanical allodynia, complement activation, and intradermal nerve fiber loss between the groups. We found that 1) i.p. paclitaxel administration activated complement in wild-type rats, 2) paclitaxel-induced mechanical allodynia was significantly reduced in C3 KO rats, and 3) the paclitaxel-induced loss of intradermal nerve fibers was markedly attenuated in C3 KO rats. In in vitro studies, we found that paclitaxel-treated rat neuronal cells activated complement, leading to cellular injury. Our findings demonstrate a previously unknown but pivotal role of complement in CIPN and suggest that complement may be a new target for the development of novel therapeutics to manage this painful disease. Copyright © 2018 by The American Association of Immunologists, Inc.

Activated microglia proliferate at neurites of mutant huntingtin-expressing neurons

PubMed Central

Kraft, Andrew D.; Kaltenbach, Linda S.; Lo, Donald C.; Harry, G. Jean

2011-01-01

In Huntington's disease (HD), mutated huntingtin (mhtt) causes striatal neurodegeneration which is paralleled by elevated microglia cell numbers. In vitro cortico-striatal slice and primary neuronal culture models, in which neuronal expression of mhtt fragments drives HD-like neurotoxicity, were employed to examine wild type microglia during both the initiation and progression of neuronal pathology. As neuronal pathology progressed, microglia initially localized in the vicinity of neurons expressing mhtt fragments increased in number, demonstrated morphological evidence of activation, and expressed the proliferation marker, Ki67. These microglia were positioned along irregular neurites, but did not localize with mhtt inclusions nor exacerbate mhtt fragment-induced neurotoxicity. Prior to neuronal pathology, microglia upregulated Iba1, signaling a functional shift. With neurodegeneration, interleukin-6 and complement component 1q were increased. The results suggest a stimulatory, proliferative signal for microglia present at the onset of mhtt fragment-induced neurodegeneration. Thus, microglia effect a localized inflammatory response to neuronal mhtt expression that may serve to direct microglial removal of dysfunctional neurites or aberrant synapses, as is required for reparative actions in vivo. PMID:21482444

The Gly-54-->Asp allelic form of human mannose-binding protein (MBP) fails to bind MBP-associated serine protease.

PubMed Central

Matsushita, M; Ezekowitz, R A; Fujita, T

1995-01-01

The human mannose-binding protein (MBP) is a pattern recognition molecule that appears to play a role in initial host defence. MBP activates the complement cascade and it may act as an opsonin both in the absence and in the presence of complement. A number of distinct MBP allelic forms exist in different population groups. An allele that occurs in 5-7% of Caucasians was identified by an inability to activate the complement system. A homozygous mutation at base pair 230 of the MBP gene results in a Gly-to-Asp substitution at the fifth collagen repeat. It appears that the resultant protein, MBPD, is able to form high-order multimers that bind bacteria but do not support complement activation. Recently a novel serine protease, the MBP-associated serine protease (MASP), has been described. MBP-MASP complexes circulate in serum and result in the direct activation of a novel complement pathway (lectin pathway) in the absence of the first complement components. In this study we demonstrate that MASP and its proenzyme proMASP are unable to bind to recombinant (r)MBPD. This lack of a MASP-rMBPD association corresponds to a failure of the Gly-54-->Asp form of MBP to activate complement. Our results provide a biochemical basis for the functional deficit in the Gly-54-->Asp allelic form of MBP and suggest that the proMASP/MASP binding site maps to the fifth collagen repeat of MBP. Images Figure 1 PMID:7487919

Innate immune humoral factors, C1q and factor H, with differential pattern recognition properties, alter macrophage response to carbon nanotubes.

PubMed

Pondman, Kirsten M; Pednekar, Lina; Paudyal, Basudev; Tsolaki, Anthony G; Kouser, Lubna; Khan, Haseeb A; Shamji, Mohamed H; Ten Haken, Bennie; Stenbeck, Gudrun; Sim, Robert B; Kishore, Uday

2015-11-01

Interaction between the complement system and carbon nanotubes (CNTs) can modify their intended biomedical applications. Pristine and derivatised CNTs can activate complement primarily via the classical pathway which enhances uptake of CNTs and suppresses pro-inflammatory response by immune cells. Here, we report that the interaction of C1q, the classical pathway recognition molecule, with CNTs involves charge pattern and classical pathway activation that is partly inhibited by factor H, a complement regulator. C1q and its globular modules, but not factor H, enhanced uptake of CNTs by macrophages and modulated the pro-inflammatory immune response. Thus, soluble complement factors can interact differentially with CNTs and alter the immune response even without complement activation. Coating CNTs with recombinant C1q globular heads offers a novel way of controlling classical pathway activation in nanotherapeutics. Surprisingly, the globular heads also enhance clearance by phagocytes and down-regulate inflammation, suggesting unexpected complexity in receptor interaction. Carbon nanotubes (CNTs) maybe useful in the clinical setting as targeting drug carriers. However, it is also well known that they can interact and activate the complement system, which may have a negative impact on the applicability of CNTs. In this study, the authors functionalized multi-walled CNT (MWNT), and investigated the interaction with the complement pathway. These studies are important so as to gain further understanding of the underlying mechanism in preparation for future use of CNTs in the clinical setting. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

In vitro C3 mRNA expression in Pemphigus vulgaris: complement activation is increased by IL-1alpha and TNF-alpha.

PubMed

Feliciani, C; Toto, P; Amerio, P

1999-01-01

Pemphigus vulgaris (PV) is a potentially life-threatening disease, characterized immunohistologically by IgG deposits and complement activation on the surface of keratinocytes. Complement activation has been implicated in the pathogenesis with C3 deposits in about 90% of patients. In order to further elucidate the role of complement in PV and to define which cytokines play a role in C3 mRNA expression, we performed an in vitro study in human keratinocytes. Normal human epidermal keratinocytes (NHuK) were incubated with PV serum and C3 mRNA was measured. We previously had shown that IL-1alpha and TNF-alpha are expressed in PV in vivo and in vitro. Since cytokines are able to modulate complement activation, mRNA expression was evaluated in a similar experiment after pretreatment using antibodies against IL-1alpha and TNF-alpha. Incubation of NHuK with PV sera caused their detachment from the plates after 20-30 minutes with a complete acantholysis within 12 hours. An early C3 mRNA expression was seen after 30 minutes with a peak level after 1 hour. Blocking studies, using antibodies against human IL-1alpha and TNF-alpha in NHuK together with PV-IgG, showed reduction of in vitro induced acantholysis and inhibition of C3 mRNA expression. This study supports the hypothesis that complement C3 is important in PV acantholysis and that complement activation is increased by IL-1alpha and TNF-alpha.

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

PubMed

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

2017-01-01

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

The Effect of Ionic Strength on the Haemolytic Activity of Complement

PubMed Central

Wardlaw, A. C.; Walker, H. G.

1963-01-01

The haemolytic activity of guinea-pig complement has been measured in isotonic solutions of various ionic strengths in the range 0.034–0.28 and shown to be maximum at an ionic strength close to 0.08. Haemolytic activity was virtually abolished at ionic strength 0.034, while at 0.28, the complement titre was only about 20 per cent of the value found at the physiological ionic strength 0.155. NaCl, KCl, LiBr and K2SO4 were the electrolytes used to provide ionic strength, and sucrose, mannitol and inositol the non-electrolytes used to maintain isotonicity. Nine permutations of the four electrolytes with the three non-electrolytes were tested and gave similar results. Human and rabbit complements also showed optimum haemolytic activity at ionic strength 0.08–0.10. PMID:13998876

Micrurus snake venoms activate human complement system and generate anaphylatoxins

PubMed Central

2012-01-01

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

Immunological response to nitroglycerin-loaded shear-responsive liposomes in vitro and in vivo.

PubMed

Buscema, Marzia; Matviykiv, Sofiya; Mészáros, Tamás; Gerganova, Gabriela; Weinberger, Andreas; Mettal, Ute; Mueller, Dennis; Neuhaus, Frederik; Stalder, Etienne; Ishikawa, Takashi; Urbanics, Rudolf; Saxer, Till; Pfohl, Thomas; Szebeni, János; Zumbuehl, Andreas; Müller, Bert

2017-10-28

Liposomes formulated from the 1,3-diamidophospholipid Pad-PC-Pad are shear-responsive and thus promising nano-containers to specifically release a vasodilator at stenotic arteries. The recommended preclinical safety tests for therapeutic liposomes of nanometer size include the in vitro assessment of complement activation and the evaluation of the associated risk of complement activation-related pseudo-allergy (CARPA) in vivo. For this reason, we measured complement activation by Pad-PC-Pad formulations in human and porcine sera, along with the nanopharmaceutical-mediated cardiopulmonary responses in pigs. The evaluated formulations comprised of Pad-PC-Pad liposomes, with and without polyethylene glycol on the surface of the liposomes, and nitroglycerin as a model vasodilator. The nitroglycerin incorporation efficiency ranged from 25% to 50%. In human sera, liposome formulations with 20mg/mL phospholipid gave rise to complement activation, mainly via the alternative pathway, as reflected by the rises in SC5b-9 and Bb protein complex concentrations. Formulations having a factor of ten lower phospholipid content did not result in measurable complement activation. The weak complement activation induced by Pad-PC-Pad liposomal formulations was confirmed by the results obtained by performing an in vivo study in a porcine model, where hemodynamic parameters were monitored continuously. Our study suggests that, compared to FDA-approved liposomal drugs, Pad-PC-Pad exhibits less or similar risks of CARPA. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Micrurus snake venoms activate human complement system and generate anaphylatoxins.

PubMed

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

2012-01-16

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

CsMAP34, a teleost MAP with dual role: A promoter of MASP-assisted complement activation and a regulator of immune cell activity.

PubMed

Li, Mo-Fei; Li, Jun; Sun, Li

2016-12-23

In teleost fish, the immune functions of mannan-binding lectin (MBL) associated protein (MAP) and MBL associated serine protease (MASP) are scarcely investigated. In the present study, we examined the biological properties both MAP (CsMAP34) and MASP (CsMASP1) molecules from tongue sole (Cynoglossus semilaevis). We found that CsMAP34 and CsMASP1 expressions occurred in nine different tissues and were upregulated by bacterial challenge. CsMAP34 protein was detected in blood, especially during bacterial infection. Recombinant CsMAP34 (rCsMAP34) bound C. semilaevis MBL (rCsBML) when the latter was activated by bacteria, while recombinant CsMASP1 (rCsMASP1) bound activated rCsBML only in the presence of rCsMAP34. rCsMAP34 stimulated the hemolytic and bactericidal activities of serum complement, whereas anti-CsMAP34 antibody blocked complement activities. Knockdown of CsMASP1 in C. semilaevis resulted in significant inhibition of complement activities. Furthermore, rCsMAP34 interacted directly with peripheral blood leukocytes (PBL) and enhanced the respiratory burst, acid phosphatase activity, chemotactic activity, and gene expression of PBL. These results indicate for the first time that a teleost MAP acts one hand as a regulator that promotes the lectin pathway of complement activation via its ability to recruit MBL to MASP, and other hand as a modulator of immune cell activity.

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

PubMed

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

2015-06-01

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

Novel Evasion Mechanisms of the Classical Complement Pathway

PubMed Central

Garcia, Brandon L.; Zwarthoff, Seline A.; Rooijakkers, Suzan H. M.; Geisbrecht, Brian V.

2016-01-01

Complement is a network of soluble and cell surface-associated proteins which 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 ‘non-self’ cells by one of three initiating mechanisms known as the classical, lectin, or alternative pathways. Evasion of complement function is a hallmark of invasive pathogens and hematophagous organisms. While 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 which have 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

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. Copyright © 2016 by The American Association of Immunologists, Inc.

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway

PubMed Central

Rosbjerg, Anne; Genster, Ninette; Pilely, Katrine; Garred, Peter

2017-01-01

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition molecules that bind specific patterns on microbial surfaces, a group of associated proteases that initiates the complement cascade, and a group of proteins that interact in proteolytic complexes or the terminal pore-forming complex. In addition, various regulatory proteins are important for controlling the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system. Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination. PMID:28553281

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

PubMed

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

1998-10-01

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

Kinetics of leukocyte sequestration in the lungs of acutely septic primates: A study using sup 111 In-labeled autologous leukocytes

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

Hangen, D.H.; Segall, G.M.; Harney, E.W.

1990-03-01

To further clarify the role of leukocytes in the pathogenesis of ARDS, we studied the localization and kinetics of leukocyte migration using 111In-labeled autologous white cell scans ({sup 111}In wbc scans) in four primates made acutely septic with infusions of Escherichia coli. Whole body images were obtained with a gamma camera and were acquired on computer every 15 min beginning immediately after the E. coli infusion. Simultaneous measurements of C5a and peripheral blood leukocyte count were also obtained. Within 5 min of initiating sepsis, three major events occurred: complement activation as measured by the production of C5a, a profound fallmore » in peripheral leukocyte count, and a significant increase in the sequestration of leukocytes in the lungs. The pulmonary sequestration reached a peak at 15 min with a mean of 152% of baseline activity. This sequestration consisted of a population that was predominantly neutrophils. Damage to the pulmonary capillary endothelium was demonstrated by an increase in extravascular lung water. The results support a role for neutrophils and complement as mediators in the pathogenesis of ARDS.« less

Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes.

PubMed

Garcia, Nahuel A; Moncayo-Arlandi, Javier; Sepulveda, Pilar; Diez-Juan, Antonio

2016-03-01

Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship, which is essential for maintaining the metabolic requirements of the heart. Little is known about the mechanisms that regulate nutrient flow from ECs to associated CMs, especially in situations of acute stress when local active processes are required to regulate endothelial transport. We examined whether CM-derived exosomes can modulate glucose transport and metabolism in ECs. In conditions of glucose deprivation, CMs increase the synthesis and secretion of exosomes. These exosomes are loaded with functional glucose transporters and glycolytic enzymes, which are internalized by ECs, leading to increased glucose uptake, glycolytic activity, and pyruvate production in recipient cells. These findings establish CM-derived exosomes as key components of the cardio-endothelial communication system which, through intercellular protein complementation, would allow a rapid response from ECs to increase glucose transport and a putative uptake of metabolic fuels from blood to CMs. This CM-EC protein complementation process might have implications for metabolic regulation in health and disease. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

Specificity of EIA immunoassay for complement factor Bb testing.

PubMed

Pavlov, Igor Y; De Forest, Nikol; Delgado, Julio C

2011-01-01

During the alternative complement pathway activation, factor B is cleaved in two fragments, Ba and Bb. Concentration of those fragments is about 2 logs lower than of factor B present in the blood, which makes fragment detection challenging because of potential cross-reactivity. Lack of information on Bb assay cross-reactivity stimulated the authors to investigate this issue. We ran 109 healthy donor EDTA plasmas and 80 sera samples with both factor B immunodiffusion (The Binding Site) and Quidel Bb EIA assays. During the study it was shown that physiological concentrations of gently purified factor B demonstrated approximately 0.15% cross-reactivity in the Quidel Bb EIA assay. We also observed that Bb concentration in serum is higher than in plasma due to complement activation during clot formation which let us use sera as samples representing complement activated state. Our study demonstrated that despite the potential 0.15% cross-reactivity between endogenous factor B and cleaved Bb molecule, measuring plasma concentrations of factor Bb is adequate to evaluate the activation of the alternative complement pathway.

A targeted complement-dependent strategy to improve the outcome of mAb therapy, and characterization in a murine model of metastatic cancer

PubMed Central

Elvington, Michelle; Huang, Yuxiang; Morgan, B. Paul; Qiao, Fei; van Rooijen, Nico; Atkinson, Carl

2012-01-01

Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer. PMID:22442351

The Surface-Exposed Protein SntA Contributes to Complement Evasion in Zoonotic Streptococcus suis.

PubMed

Deng, Simin; Xu, Tong; Fang, Qiong; Yu, Lei; Zhu, Jiaqi; Chen, Long; Liu, Jiahui; Zhou, Rui

2018-01-01

Streptococcus suis is an emerging zoonotic pathogen causing streptococcal toxic shock like syndrome (STSLS), meningitis, septicemia, and even sudden death in human and pigs. Serious septicemia indicates this bacterium can evade the host complement surveillance. In our previous study, a functionally unknown protein SntA of S. suis has been identified as a heme-binding protein, and contributes to virulence in pigs. SntA can interact with the host antioxidant protein AOP2 and consequently inhibit its antioxidant activity. In the present study, SntA is identified as a cell wall anchored protein that functions as an important player in S. suis complement evasion. The C3 deposition and membrane attack complex (MAC) formation on the surface of sntA -deleted mutant strain Δ sntA are demonstrated to be significantly higher than the parental strain SC-19 and the complementary strain CΔ sntA . The abilities of anti-phagocytosis, survival in blood, and in vivo colonization of Δ sntA are obviously reduced. SntA can interact with C1q and inhibit hemolytic activity via the classical pathway. Complement activation assays reveal that SntA can also directly activate classical and lectin pathways, resulting in complement consumption. These two complement evasion strategies may be crucial for the pathogenesis of this zoonotic pathogen. Concerning that SntA is a bifunctional 2',3'-cyclic nucleotide 2'-phosphodiesterase/3'-nucleotidase in many species of Gram-positive bacteria, these complement evasion strategies may have common biological significance.

21 CFR 866.5320 - Properdin factor B immuno-logical test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... involvement of the alternative to the classical pathway of activation of complement (a group of plasma... the skin). Other diseases in which the alternate pathway of complement activation has been implicated...

21 CFR 866.5320 - Properdin factor B immuno-logical test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... involvement of the alternative to the classical pathway of activation of complement (a group of plasma... the skin). Other diseases in which the alternate pathway of complement activation has been implicated...

21 CFR 866.5320 - Properdin factor B immuno-logical test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... involvement of the alternative to the classical pathway of activation of complement (a group of plasma... the skin). Other diseases in which the alternate pathway of complement activation has been implicated...

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

PubMed

Giuntini, Serena; Beernink, Peter T; Reason, Donald C; Granoff, Dan M

2012-01-01

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

Innate Immune Mechanisms in Transplant Allograft Vasculopathy

PubMed Central

Jane-wit, D; Fang, C; Goldstein, DR

2016-01-01

Purpose of Review Allograft vasculopathy (AV) is the leading cause of late allograft loss following solid organ transplantation. Ischemia reperfusion injury (IRI) and donor specific antibody (DSA)-induced complement activation confer heightened risk for AV via numerous innate immune mechanisms including MyD88, HMGB1, and complement induced non-canonical NF-kB signaling. Recent Findings The role of MyD88, a signal adaptor downstream of the toll-like receptors (TLR), has been defined in an experimental heart transplant model, which demonstrated that recipient MyD88 enhanced AV. Importantly, triggering receptor on myeloid receptor 1(Trem1), a MyD88 amplifying signal, was present in rejecting human cardiac transplant biopsies and enhanced the development of AV in mice. HMGB1, a nuclear protein that activates TLRs, also enhanced the development of AV. Complement activation elicits assembly of membrane attack complexes (MAC) on endothelial cells which activate non-canonical NF-kB signaling, a novel complement effector pathway that induces pro-inflammatory genes and potentiates endothelial cell mediated alloimmune T cell activation, processes which enhance AV. Summary Innate immune mediators including HMGB1, MyD88, and non-canonical NFκB signaling via complement activation contribute to AV. These pathways represent potential therapeutic targets to reduce AV after solid organ transplantation. PMID:27077602

Evolution and diversity of the complement system of poikilothermic vertebrates.

PubMed

Sunyer, J O; Lambris, J D

1998-12-01

In mammals the complement system plays an important role in innate and acquired host defense mechanisms against infection and in various immunoregulatory processes. The complement system is an ancient defense mechanism that is already present in the invertebrate deuterostomes. In these species as well as in agnathans (the most primitive vertebrate species), both the alternative and lectin pathway of complement activation are already present, and the complement system appears to be involved mainly in opsonization of foreign material. With the emergence of immunoglobulins in cartilaginous fish, the classical and lytic pathways first appear. The rest of the poikilothermic species, from teleosts to reptilians, appear to contain a well-developed complement system resembling that of homeothermic vertebrates. However, important differences remain. Unlike homeotherms, several species of poikilotherms have recently been shown to possess multiple forms of complement components (C3 and factor B) that are structurally and functionally more diverse than those of higher vertebrates. It is noteworthy that the multiple forms of C3 that have been characterized in several teleost fish are able to bind with varying efficiencies to various complement-activating surfaces. We hypothesize that this diversity has allowed these animals to expand their innate capacity for immune recognition.

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

PubMed

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

2016-12-01

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

Use of Phage Display to Identify Novel Mineralocorticoid Receptor-Interacting Proteins

PubMed Central

Yang, Jun; Fuller, Peter J.; Morgan, James; Shibata, Hirotaka; McDonnell, Donald P.; Clyne, Colin D.

2014-01-01

The mineralocorticoid receptor (MR) plays a central role in salt and water homeostasis via the kidney; however, inappropriate activation of the MR in the heart can lead to heart failure. A selective MR modulator that antagonizes MR signaling in the heart but not the kidney would provide the cardiovascular protection of current MR antagonists but allow for normal electrolyte balance. The development of such a pharmaceutical requires an understanding of coregulators and their tissue-selective interactions with the MR, which is currently limited by the small repertoire of MR coregulators described in the literature. To identify potential novel MR coregulators, we used T7 phage display to screen tissue-selective cDNA libraries for MR-interacting proteins. Thirty MR binding peptides were identified, from which three were chosen for further characterization based on their nuclear localization and their interaction with other MR-interacting proteins or, in the case of x-ray repair cross-complementing protein 6, its known status as an androgen receptor coregulator. Eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 modulated MR-mediated transcription in a ligand-, cell- and/or promoter-specific manner and colocalized with the MR upon agonist treatment when imaged using immunofluorescence microscopy. These results highlight the utility of phage display for rapid and sensitive screening of MR binding proteins and suggest that eukaryotic elongation factor 1A1, structure-specific recognition protein 1, and x-ray repair cross-complementing protein 6 may be potential MR coactivators whose activity is dependent on the ligand, cellular context, and target gene promoter. PMID:25000480

Dendritic protein synthesis in the normal and diseased brain

PubMed Central

Swanger, Sharon A.; Bassell, Gary J.

2015-01-01

Synaptic activity is a spatially-limited process that requires a precise, yet dynamic, complement of proteins within the synaptic micro-domain. The maintenance and regulation of these synaptic proteins is regulated, in part, by local mRNA translation in dendrites. Protein synthesis within the postsynaptic compartment allows neurons tight spatial and temporal control of synaptic protein expression, which is critical for proper functioning of synapses and neural circuits. In this review, we discuss the identity of proteins synthesized within dendrites, the receptor-mediated mechanisms regulating their synthesis, and the possible roles for these locally synthesized proteins. We also explore how our current understanding of dendritic protein synthesis in the hippocampus can be applied to new brain regions and to understanding the pathological mechanisms underlying varied neurological diseases. PMID:23262237

A De Novo Deletion in the Regulators of Complement Activation Cluster Producing a Hybrid Complement Factor H/Complement Factor H-Related 3 Gene in Atypical Hemolytic Uremic Syndrome.

PubMed

Challis, Rachel C; Araujo, Geisilaine S R; Wong, Edwin K S; Anderson, Holly E; Awan, Atif; Dorman, Anthony M; Waldron, Mary; Wilson, Valerie; Brocklebank, Vicky; Strain, Lisa; Morgan, B Paul; Harris, Claire L; Marchbank, Kevin J; Goodship, Timothy H J; Kavanagh, David

2016-06-01

The regulators of complement activation cluster at chromosome 1q32 contains the complement factor H (CFH) and five complement factor H-related (CFHR) genes. This area of the genome arose from several large genomic duplications, and these low-copy repeats can cause genome instability in this region. Genomic disorders affecting these genes have been described in atypical hemolytic uremic syndrome, arising commonly through nonallelic homologous recombination. We describe a novel CFH/CFHR3 hybrid gene secondary to a de novo 6.3-kb deletion that arose through microhomology-mediated end joining rather than nonallelic homologous recombination. We confirmed a transcript from this hybrid gene and showed a secreted protein product that lacks the recognition domain of factor H and exhibits impaired cell surface complement regulation. The fact that the formation of this hybrid gene arose as a de novo event suggests that this cluster is a dynamic area of the genome in which additional genomic disorders may arise. Copyright © 2016 by the American Society of Nephrology.

Complement system and immunological mediators: Their involvements in the induced inflammatory process by Androctonus australis hector venom and its toxic components.

PubMed

Bekkari, Nadjia; Martin-Eauclaire, Marie-France; Laraba-Djebari, Fatima

2015-01-01

Androctonus australis hector scorpion venom is well known by its high toxicity, it induces massive release of neurotransmitters that lead to pathophysiological disorders in cardiovascular, neuro-hormonal and immune systems. Previous studies have shown the relationship between the severity of scorpion envenoming and immune system activation. This study was assessed to investigate the involvement of complement system and inflammatory mediators after sublethal injection of Aah venom, its toxic fraction (FtoxG50) and its main toxins (AahI and AahII) into NMRI mice. The Activation complement system by the venom is also compared to that induced of lipopolysaccharides (LPS). Obtained results showed that seric complement system (CS) is activated by the venom and by its toxic components; this activation is more pronounced into liver tissue when toxic components (FtoxG50, AahI or AahII) are used. Increase of cytokine levels (IL1β, TNFα and ICAM) into hepatic tissue induced by AahI or AahII neurotoxins is correlated with tissue alterations. Aprotinin, a non specific inhibitor of complement system seems to be able to reduce CS consumption and to restore partially the induced tissue damage by venom. The mechanisms by which toxic fraction or LPS induced the activation of complement system seem to be different. Sensitivity of hepatic tissue is more pronounced after FtoxG50 injection; however lung tissue is more sensible to LPS than FoxG50. Copyright © 2015 Elsevier GmbH. All rights reserved.

The lectin pathway in renal disease: old concept and new insights.

PubMed

Gaya da Costa, Mariana; Poppelaars, Felix; Berger, Stefan P; Daha, Mohamed R; Seelen, Marc A

2018-04-26

The complement system is composed of a network of at least 40 proteins, which significantly contributes to health and disease. The lectin pathway (LP) is one of three pathways that can activate the complement system. Next to protection of the host against pathogens, the LP has been shown to play a crucial role in multiple renal diseases as well as during renal replacement therapy. Therefore, several complement-targeted drugs are currently being explored in clinical trials. Among these complement inhibitors, specific LP inhibitors are also being tested in renal abnormalities such as in immunoglobulin A nephropathy and lupus nephritis. Using various in vitro models, Yaseen et al. (Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement component 3 (C3) in absence of C4 and/or C2. FASEB J 2017; 31: 2210-2219) showed that Mannan-associated serine protease2 can directly activate C3 thereby bypassing C2 and C4 in the activation of the LP. These new findings broaden our understanding of the mechanisms of complement activation and could potentially impact our strategies to inhibit the LP in renal diseases. In support of these findings, we present data of human renal biopsies, demonstrating the occurrence of the LP bypass mechanism in vivo. In conclusion, this review provides a detailed overview of the LP and clarifies the recently described bypass mechanism and its relevance. Finally, we speculate on the role of the C4 bypass mechanism in other renal diseases.

Cardiac Sirt1 mediates the cardioprotective effect of caloric restriction by suppressing local complement system activation after ischemia-reperfusion.

PubMed

Yamamoto, Tsunehisa; Tamaki, Kayoko; Shirakawa, Kohsuke; Ito, Kentaro; Yan, Xiaoxiang; Katsumata, Yoshinori; Anzai, Atsushi; Matsuhashi, Tomohiro; Endo, Jin; Inaba, Takaaki; Tsubota, Kazuo; Sano, Motoaki; Fukuda, Keiichi; Shinmura, Ken

2016-04-15

Caloric restriction (CR) confers cardioprotection against ischemia-reperfusion (I/R) injury. We previously found the essential roles of endothelial nitric oxide synthase in the development of CR-induced cardioprotection and Sirt1 activation during CR (Shinmura K, Tamaki K, Ito K, Yan X, Yamamoto T, Katsumata Y, Matsuhashi T, Sano M, Fukuda K, Suematsu M, Ishii I. Indispensable role of endothelial nitric oxide synthase in caloric restriction-induced cardioprotection against ischemia-reperfusion injury.Am J Physiol Heart Circ Physiol 308: H894-H903, 2015). However, the exact mechanism by which Sirt1 in cardiomyocytes mediates the cardioprotective effect of CR remains undetermined. We subjected cardiomyocyte-specific Sirt1 knockout (CM-Sirt1(-/-)) mice and the corresponding control mice to either 3-mo ad libitum feeding or CR (-40%). Isolated perfused hearts were subjected to 25-min global ischemia, followed by 60-min reperfusion. The recovery of left ventricle function after I/R was improved, and total lactate dehydrogenase release into the perfusate during reperfusion was attenuated in the control mice treated with CR, but a similar cardioprotective effect of CR was not observed in the CM-Sirt1(-/-)mice. The expression levels of cardiac complement component 3 (C3) at baseline and the accumulation of C3 and its fragments in the ischemia-reperfused myocardium were attenuated by CR in the control mice, but not in the CM-Sirt1(-/-)mice. Resveratrol treatment also attenuated the expression levels of C3 protein in cultured neonatal rat ventricular cardiomyocytes. Moreover, the degree of myocardial I/R injury in conventional C3 knockout (C3(-/-)) mice treated with CR was similar to that in the ad libitum-fed C3(-/-)mice, although the expression levels of Sirt1 were enhanced by CR. These results demonstrate that cardiac Sirt1 plays an essential role in CR-induced cardioprotection against I/R injury by suppressing cardiac C3 expression. This is the first report suggesting that cardiac Sirt1 regulates the local complement system during CR. Copyright © 2016 the American Physiological Society.

Smallpox Inhibitor of Complement Enzymes (SPICE): Dissecting Functional Sites and Abrogating Activity1

PubMed Central

Liszewski, M. Kathryn; Leung, Marilyn K.; Hauhart, Richard; Fang, Celia J.; Bertram, Paula; Atkinson, John P.

2010-01-01

Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host’s immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity ~200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent. PMID:19667083

Interaction of extremophilic archaeal viruses with human and mouse complement system and viral biodistribution in mice

PubMed Central

Wu, Linping; Uldahl, Kristine Buch; Chen, Fangfang; Benasutti, Halli; Logvinski, Deborah; Vu, Vivian; Banda, Nirmal K.; Peng, Xu; Simberg, Dmitri; Moghimi, Seyed Moein

2017-01-01

Archaeal viruses offer exceptional biophysical properties for modification and exploration of their potential in bionanotechnology, bioengineering and nanotherapeutic developments. However, the interaction of archaeal viruses with elements of the innate immune system has not been explored, which is a necessary prerequisite if their potential for biomedical applications to be realized. Here we show complement activation through lectin (via direct binding of MBL/MASPs) and alternative pathways by two extremophilic archaeal viruses (Sulfolobus monocaudavirus 1 and Sulfolobus spindle-shaped virus 2) in human serum. We further show some differences in initiation of complement activation pathways between these viruses. Since, Sulfolobus monocaudavirus 1 was capable of directly triggering the alternative pathway, we also demonstrate that the complement regulator factor H has no affinity for the viral surface, but factor H deposition is purely C3-dependent. This suggests that unlike some virulent pathogens Sulfolobus monocaudavirus 1 does not acquire factor H for protection. Complement activation with Sulfolobus monocaudavirus 1 also proceeds in murine sera through MBL-A/C as well as factor D-dependent manner, but C3 deficiency has no overall effect on viral clearance by organs of the reticuloendothelial system on intravenous injection. However, splenic deposition was significantly higher in C3 knockout animals compared with the corresponding wild type mice. We discuss the potential application of these viruses in biomedicine in relation to their complement activating properties. PMID:28846925

Classroom Active Learning Complemented by an Online Discussion Forum to Teach Sustainability

ERIC Educational Resources Information Center

Dengler, Mary

2008-01-01

This paper identifies some of the pedagogical benefits of an active learning course delivery complemented by an online discussion forum to teach sustainability by evaluating the case of a geography master's course. The potential benefits and some challenges of an active learning course delivery to teach sustainability in geography and related…

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

PubMed

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

2013-08-01

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

Exopolysaccharides Isolated from Hydrothermal Vent Bacteria Can Modulate the Complement System

PubMed Central

Courtois, Anthony; Berthou, Christian; Guézennec, Jean

2014-01-01

The complement system is involved in the defence against bacterial infection, or in the elimination of tumour cells. However, disturbances in this system contributes to the pathogenesis of various inflammatory diseases. The efficiency of therapeutic anti-tumour antibodies is enhanced when the complement system is stimulated. In contrast, cancer cells are able to inhibit the complement system and thus proliferate. Some marine molecules are currently being developed as new drugs for use in humans. Among them, known exopolyssacharides (EPSs) generally originate from fungi, but few studies have been performed on bacterial EPSs and even fewer on EPSs extracted from deep-sea hydrothermal vent microbes. For use in humans, these high molecular weight EPSs must be depolymerised. Furthermore, the over-sulphation of EPSs can modify their biological activity. The aim of this study was to investigate the immunodulation of the complement system by either native or over-sulphated low molecular weight EPSs isolated from vent bacteria in order to find pro or anti-activators of complement. PMID:24736648

Method of monaural localization of the acoustic source direction from the standpoint of the active perception theory

NASA Astrophysics Data System (ADS)

Gai, V. E.; Polyakov, I. V.; Krasheninnikov, M. S.; Koshurina, A. A.; Dorofeev, R. A.

2017-01-01

Currently, the scientific and educational center of the “Transport” of NNSTU performs work on the creation of the universal rescue vehicle. This vehicle is a robot, and intended to reduce the number of human victims in accidents on offshore oil platforms. An actual problem is the development of a method for determining the location of a person overboard in low visibility conditions, when a traditional vision is not efficient. One of the most important sensory robot systems is the acoustic sensor system, because it is omnidirectional and does not require finding of an acoustic source in visibility scope. Features of the acoustic sensor robot system can complement the capabilities of the video sensor in the solution of the problem of localization of a person or some event in the environment. This paper describes the method of determination of the direction of the acoustic source using just one microphone. The proposed method is based on the active perception theory.

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

PubMed

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

2018-01-01

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

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

PubMed Central

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

2018-01-01

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

Polysaccharides from Sargassum thunbergii: Monthly variations and anti-complement and anti-tumour activities.

PubMed

Jin, Weihua; Liu, Ge; Zhong, Weihong; Sun, Chaomin; Zhang, Quanbin

2017-12-01

Monthly variations of polysaccharides from Sargassum thunbergii and their anti-complement and anti-tumour activities were investigated. It was observed that an increase in fucose and total sugar contents occurred during the growth period (from early April to mid-June), accompanied by a decrease in molar ratios of other monosaccharides to fucose. The highest yields were obtained from early July to early September, which was in accordance with the significant increase in molar ratio of glucose to fucose and decrease in molar ratio of other monosaccharides to fucose. And the above results suggested that S. Thunbergii synthesized large amount of laminaran, the storage substance of brown algae, during the senescence period. However, sulfate contents were relatively stable in the life cycle of S. thunbergii. These results suggested that S. thunbergii synthesized complex sulfated heteropolysacchairdes during inactive period, while during other periods, it synthesized more sulfated galactofucan. All polysaccharides showed anti-complement activity, suggesting that the harvesting time did not influence the anti-complement activities. In the anti-tumour assay in vitro, the polysaccharides taken during the senescence period had much lower anti-tumour activity, suggesting that fucoidan, but not laminaran, determined the anti-tumour activities. Therefore, polysaccharides from S. thunbergii might have great potential in anti-complement and anti-tumour application. Copyright © 2017 Elsevier B.V. All rights reserved.

Sublytic complement protects prostate cancer cells from tumour necrosis factor-α-induced cell death.

PubMed

Liu, L; Li, W; Li, Z; Kirschfink, M

2012-08-01

Inflammation is a critical component of tumour progression. Although complement and tumour necrosis factor (TNF)-α potentially exert significant anti-tumour effects, both mediators may also promote tumour progression. It has been demonstrated that sublytic complement confers resistance on tumour cells not only against lytic complement, but also other danger molecules such as perforin. In low concentrations, TNF promotes survival of malignant cells rather than exerting cytotoxic activity. In this study, we tested if sublytic complement is able to interfere with TNF-mediated tumour cell killing. Our results demonstrate that either subcytotoxic concentrations of TNF or sublytic complement rescue prostate carcinoma cells (DU145) from TNF-α-mediated cell death. Upon pretreatment with low-dose TNF-α, but not upon pre-exposure to sublytic complement, TNF resistance was associated with the down-regulation of TNF receptor 1 (TNF-R1) expression. Complement-induced protection against TNF-mediated apoptosis accompanied the induction of anti-apoptotic proteins [B cell leukaemia/lymphoma (Bcl)-2 and Bcl-xL] at an early stage followed by inhibition of the TNF-induced decrease in the amount of Bcl-2 and Bcl-xL. Cell protection also accompanied the inhibition of caspase-8 activation, poly (ADP-ribose) polymerase (PARP)-1 cleavage and the activation of nuclear factor (NF)-κB. Our data extend our current view on the induction of tumour cell resistance against cytotoxic mediators supporting the role of the tumour microenvironment in mediating protection against the anti-cancer immune response. © 2012 The Authors. Clinical and Experimental Immunology © 2012 British Society for Immunology.

Disulfide proteomics of rice cultured cells in response to OsRacl and probenazole-related immune signaling pathway in rice.

PubMed

Morino, Kazuko; Kimizu, Mayumi; Fujiwara, Masayuki

2016-01-01

Reactive oxygen species (ROS) production is an early event in the immune response of plants. ROS production affects the redox-based modification of cysteine residues in redox proteins, which contribute to protein functions such as enzymatic activity, protein-protein interactions, oligomerization, and intracellular localization. Thus, the sensitivity of cysteine residues to changes in the cellular redox status is critical to the immune response of plants. We used disulfide proteomics to identify immune response-related redox proteins. Total protein was extracted from rice cultured cells expressing constitutively active or dominant-negative OsRacl, which is a key regulator of the immune response in rice, and from rice cultured cells that were treated with probenazole, which is an activator of the plant immune response, in the presence of the thiol group-specific fluorescent probe monobromobimane (mBBr), which was a tag for reduced proteins in a differential display two-dimensional gel electrophoresis. The mBBr fluorescence was detected by using a charge-coupled device system, and total protein spots were detected using Coomassie brilliant blue staining. Both of the protein spots were analyzed by gel image software and identified using MS spectrometry. The possible disulfide bonds were identified using the disulfide bond prediction software. Subcellular localization and bimolecular fluorescence complementation analysis were performed in one of the identified proteins: Oryza sativa cold shock protein 2 (OsCSP2). We identified seven proteins carrying potential redox-sensitive cysteine residues. Two proteins of them were oxidized in cultured cells expressing DN-OsRac1, which indicates that these two proteins would be inactivated through the inhibition of OsRac1 signaling pathway. One of the two oxidized proteins, OsCSP2, contains 197 amino acid residues and six cysteine residues. Site-directed mutagenesis of these cysteine residues revealed that a Cys 140 mutation causes mislocalization of a green fluorescent protein fusion protein in the root cells of rice. Bimolecular fluorescence complementation analysis revealed that OsCSP2 is localized in the nucleus as a homo dimer in rice root cells. The findings of the study indicate that redox-sensitive cysteine modification would contribute to the immune response in rice.

Coupling complement regulators to immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo

PubMed Central

HARRIS, C L; WILLIAMS, A S; LINTON, S M; MORGAN, B P

2002-01-01

Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59-Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function. PMID:12165074

Coating of human decay accelerating factor (hDAF) onto medical devices to improve biocompatibility.

PubMed

Watkins, N J; Braidley, P; Bray, C J; Savill, C M; White, D J

1997-12-01

In passing blood through an artificial circulatory system, the blood is exposed to surfaces that result in activation of the complement system. The consequences of the activation of complement can be extremely serious for the patient ranging from mild discomfort to respiratory distress and even anaphylaxis. An entirely novel approach was to express recombinant GPI anchored human decay accelerating factor (hDAF) using the baculovirus system and then coat the recombinant protein onto the surfaces of these materials to reduce complement activation. Expression of hDAF in Sf9 cells was shown by ELISA, FACS analysis, and Western blot. Functional activity was tested by CH50 assay. For the coating experiments a small scale model of a cardiovascular bypass circuit constructed from COBE tubing was used. hDAF was either coated onto the circuit using adsorption or covalently linked via the photoreactive crosslinker, p-azidobenzoyl hydrazide. After coating, heparinised human blood was pumped around the circuit and samples were collected into EDTA collection tubes at different time points. Complement activation was measured using a Quidel C3a-des-arg EIA. The photolinked circuits gave a reduction in C3a production of 20-50%, compared to 10-20% seen with an absorbed hDAF circuit. Furthermore, the inhibition of complement was seen over the whole time scale of the photolinked circuit, 60-90 min, whilst in the adsorbed circuit inhibition was not seen to a significant degree after 60 min. The time scale of a standard cardiac bypass is 45-90 min, therefore, the photolinked circuit results are encouraging, as significant inhibition of complement activation is seen within this time frame.

Anticomplementary activity of horse IgG and F(ab')2 antivenoms.

PubMed

Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V

2014-03-01

Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs.

GABA(A) receptors in visual and auditory cortex and neural activity changes during basic visual stimulation.

PubMed

Qin, Pengmin; Duncan, Niall W; Wiebking, Christine; Gravel, Paul; Lyttelton, Oliver; Hayes, Dave J; Verhaeghe, Jeroen; Kostikov, Alexey; Schirrmacher, Ralf; Reader, Andrew J; Northoff, Georg

2012-01-01

Recent imaging studies have demonstrated that levels of resting γ-aminobutyric acid (GABA) in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABA(A) receptors, in the changes in brain activity between the eyes closed (EC) and eyes open (EO) state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: an EO and EC block design, allowing the modeling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [(18)F]Flumazenil PET to measure GABA(A) receptor binding potentials. It was demonstrated that the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABA(A) receptor binding potential in the visual cortex also predicted the change in functional connectivity between the visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABA(A) receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

Oscillatory brain activity differentially reflects false belief understanding and complementation syntax processing.

PubMed

Guan, Yao; Farrar, M Jeffrey; Keil, Andreas

2018-02-01

False belief understanding (FBU) enables people to consider conflicting beliefs about the same situation. While language has been demonstrated to be a correlate of FBU, there is still controversy about the extent to which a specific aspect of language, complementation syntax, is a necessary condition for FBU. The present study tested an important notion from the debate proposing that complementation syntax task is redundant to FBU measures. Specifically, we examined electrophysiological correlates of false belief, false complementation, and their respective true conditions in adults using electroencephalography (EEG), focusing on indices of oscillatory brain activity and large-scale connectivity. The results showed strong modulation of parieto-occipital alpha (8-12 Hz) and beta (13-20 Hz) power by the experimental manipulations, with heightened sustained alpha power reflective of effortful internal processing observed in the false compared to the true conditions and reliable beta power reductions sensitive to mentalizing and/or syntactic demands in the belief versus the complementation conditions. In addition, higher coupling between parieto-occipital regions and widespread frontal sites in the beta band was found for the false-belief condition selectively. The result of divergence in beta oscillatory activity and in connectivity between false belief and false complementation does not support the redundancy hypothesis.

Localizing Ground Penetrating RADAR: A Step Towards Robust Autonomous Ground Vehicle Localization

DTIC Science & Technology

2016-07-14

localization designed to complement existing approaches with a low sensitivity to failure modes of LIDAR, camera, and GPS/INS sensors due to its low...the detailed design and results from highway testing, which uses a simple heuristic for fusing LGPR estimates with a GPS/INS system. Cross-track... designed to enable a priori map-based local- ization. LGPR offers complementary capabilities to tradi- tional optics-based approaches to map-based

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. Copyright © 2012 Elsevier Inc. All rights reserved.

Mannose Binding Lectin Is Required for Alphavirus-Induced Arthritis/Myositis

PubMed Central

Whitmore, Alan C.; Blevins, Lance K.; Hueston, Linda; Fraser, Robert J.; Herrero, Lara J.; Ramirez, Ruben; Smith, Paul N.; Mahalingam, Suresh; Heise, Mark T.

2012-01-01

Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3−/− mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis. PMID:22457620

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

PubMed

Zhu, Li; Zhai, Ya-Ling; Wang, Feng-Mei; Hou, Ping; Lv, Ji-Cheng; Xu, Da-Min; Shi, Su-Fang; Liu, Li-Jun; Yu, Feng; Zhao, Ming-Hui; Novak, Jan; Gharavi, Ali G; Zhang, Hong

2015-05-01

Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1-5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3-1 deletion (CFHR3-1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype-phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3-1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN. Copyright © 2015 by the American Society of Nephrology.

[Dynamics of complement hemolytic activity in experimental Ebola infection].

PubMed

Zabavichene, N M; Chepurnov, A A

2004-01-01

The dynamic hemolytic activity of complements (HAC) was investigated in blood of guinea pigs in lethal and non-lethal Ebola infection. The increasing HAC dynamic activity in the animal blood was found to correlate with the infection lethal course. HAC as observed in animals with lethal infection was sweepingly increasing after they, were infected with Ebola virus, and yet after 15 hours from the infection time the complement activity parameters topped 2-fold the basic values in 100% of guinea pigs. They began to be dropping by the end of day 1, their decrease reached, when the incubation time was over (days 3-4 after infection) the basic value, after which they continued to go down to the zero value in 2-3 days before the lethal outcome. The described phenomenon, like the phenomenon of accelerated death, was even more pronounced, when the animals were infected after a single immunization by activated Ebola virus. In case, guinea pigs were infected by a non-lethal Ebola virus strain, the compliment synthesis was observed to be activated only at the end of the incubation period; the process was accompanied with a gradual raise and with a plateau-type or wave-type increase of the complement during the treatment time--it was equally accompanied with normalizing activity parameters during recovery. The detected specificity could be important in prognosticating a disease outcome. A reliable correlation was demonstrated between the complement hemolytic activity and the level of circulating immune complexes in blood of experimental animals, which can be traced both in lethal and non-lethal infection.

Effective implementation of the weak Galerkin finite element methods for the biharmonic equation

DOE PAGES

Mu, Lin; Wang, Junping; Ye, Xiu

2017-07-06

The weak Galerkin (WG) methods have been introduced in [11, 12, 17] for solving the biharmonic equation. The purpose of this paper is to develop an algorithm to implement the WG methods effectively. This can be achieved by eliminating local unknowns to obtain a global system with significant reduction of size. In fact this reduced global system is equivalent to the Schur complements of the WG methods. The unknowns of the Schur complement of the WG method are those defined on the element boundaries. The equivalence of theWG method and its Schur complement is established. The numerical results demonstrate themore » effectiveness of this new implementation technique.« less

Effective implementation of the weak Galerkin finite element methods for the biharmonic equation

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

Mu, Lin; Wang, Junping; Ye, Xiu

The weak Galerkin (WG) methods have been introduced in [11, 12, 17] for solving the biharmonic equation. The purpose of this paper is to develop an algorithm to implement the WG methods effectively. This can be achieved by eliminating local unknowns to obtain a global system with significant reduction of size. In fact this reduced global system is equivalent to the Schur complements of the WG methods. The unknowns of the Schur complement of the WG method are those defined on the element boundaries. The equivalence of theWG method and its Schur complement is established. The numerical results demonstrate themore » effectiveness of this new implementation technique.« less

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

PubMed

Ratelade, Julien; Verkman, A S

2014-11-01

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

Elderberry and Elderflower Extracts, Phenolic Compounds, and Metabolites and Their Effect on Complement, RAW 264.7 Macrophages and Dendritic Cells

PubMed Central

Ho, Giang Thanh Thi; Wangensteen, Helle; Barsett, Hilde

2017-01-01

Modulation of complement activity and inhibition of nitric oxide (NO) production by macrophages and dendritic cells may have therapeutic value in inflammatory diseases. Elderberry and elderflower extracts, constituents, and metabolites were investigated for their effects on the complement system, and on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages and murine dendritic D2SC/I cells. The EtOH crude extracts from elderberry and elderflower and the isolated anthocyanins and procyanidins possessed strong complement fixating activity and strong inhibitory activity on NO production in RAW cells and dendritic cells. Phenolic compounds in the range of 0.1–100 µM showed a dose-dependent inhibition of NO production, with quercetin, rutin, and kaempferol as the most potent ones. Among the metabolites, caffeic acid and 3,4-dihydroxyphenylacetic acid showed the strongest inhibitory effects on NO production in both cell lines, without having cytotoxic effect. Only 4-methylcatechol was cytotoxic at the highest tested concentration (100 µM). Elderberry and elderflower constituents may possess inflammatory modulating activity, which increases their nutritional value. PMID:28282861

Elderberry and Elderflower Extracts, Phenolic Compounds, and Metabolites and Their Effect on Complement, RAW 264.7 Macrophages and Dendritic Cells.

PubMed

Ho, Giang Thanh Thi; Wangensteen, Helle; Barsett, Hilde

2017-03-08

Modulation of complement activity and inhibition of nitric oxide (NO) production by macrophages and dendritic cells may have therapeutic value in inflammatory diseases. Elderberry and elderflower extracts, constituents, and metabolites were investigated for their effects on the complement system, and on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages and murine dendritic D2SC/I cells. The EtOH crude extracts from elderberry and elderflower and the isolated anthocyanins and procyanidins possessed strong complement fixating activity and strong inhibitory activity on NO production in RAW cells and dendritic cells. Phenolic compounds in the range of 0.1-100 µM showed a dose-dependent inhibition of NO production, with quercetin, rutin, and kaempferol as the most potent ones. Among the metabolites, caffeic acid and 3,4-dihydroxyphenylacetic acid showed the strongest inhibitory effects on NO production in both cell lines, without having cytotoxic effect. Only 4-methylcatechol was cytotoxic at the highest tested concentration (100 µM). Elderberry and elderflower constituents may possess inflammatory modulating activity, which increases their nutritional value.

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

PubMed Central

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

1991-01-01

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

Preparation of Low Molecular Weight Chondroitin Sulfates, Screening of a High Anti-Complement Capacity of Low Molecular Weight Chondroitin Sulfate and Its Biological Activity Studies in Attenuating Osteoarthritis.

PubMed

Li, Lian; Li, Yan; Feng, Danyang; Xu, Linghua; Yin, Fengxin; Zang, Hengchang; Liu, Chunhui; Wang, Fengshan

2016-10-11

Chondroitin sulfate (CS) plays important roles in the complement system. However, the CS structure is complicated due to different sources and the number and positions of sulfate groups. The objective of this study was to prepare different low molecular weight chondroitin sulfates (LMWCSs) and to investigate the biological activity in anti-complement capacity. A series of LMWCSs was prepared from different sources and characterized by ultraviolet-visible (UV) spectroscopy, high-performance liquid chromatography (HPLC), size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) and nuclear magnetic resonance (NMR) spectroscopy. Hemolytic, anti-complement 3 deposition capacity and cell viability assays were carried out to investigate the biological activities in vitro. The results showed that LMWCS prepared from shark cartilage with the oxidative degradation method (LMWCS-S-O) had the best anti-complement capacity. LMWCS-S-O could inhibit the alternative pathway of the complement system and protect chondrocytes from cell death. The attenuating effect of LMWCS-S-O on Osteoarthritis (OA) was investigated by destabilization of the medial meniscus (DMM) model in vivo. Functional wind-up, histological and C5b-9 analyses were used to evaluate the treatment effect on the OA model. In vivo results showed that LMWCS-S-O could attenuate OA. LMWCS-S-O with a high content of ΔDi-2,6diS and ΔDi-6S could be used for attenuating OA through regulating the complement system.

Calcineurin inhibitor-induced complement system activation via ERK1/2 signalling is inhibited by SOCS-3 in human renal tubule cells.

PubMed

Loeschenberger, Beatrix; Niess, Lea; Würzner, Reinhard; Schwelberger, Hubert; Eder, Iris E; Puhr, Martin; Guenther, Julia; Troppmair, Jakob; Rudnicki, Michael; Neuwirt, Hannes

2018-02-01

One factor that significantly contributes to renal allograft loss is chronic calcineurin inhibitor (CNI) nephrotoxicity (CIN). Among other factors, the complement (C-) system has been proposed to be involved CIN development. Hence, we investigated the impact of CNIs on intracellular signalling and the effects on the C-system in human renal tubule cells. In a qPCR array, CNI treatment upregulated C-factors and downregulated SOCS-3 and the complement inhibitors CD46 and CD55. Additionally, ERK1/-2 was required for these regulations. Following knock-down and overexpression of SOCS-3, we found that SOCS-3 inhibits ERK1/-2 signalling. Finally, we assessed terminal complement complex formation, cell viability and apoptosis. Terminal complement complex formation was induced by CNIs. Cell viability was significantly decreased, whereas apoptosis was increased. Both effects were reversed under complement component-depleted conditions. In vivo, increased ERK1/-2 phosphorylation and SOCS-3 downregulation were observed at the time of transplantation in renal allograft patients who developed a progressive decline of renal function in the follow-up compared to stable patients. The progressive cohort also had lower total C3 levels, suggesting higher complement activity at baseline. In conclusion, our data suggest that SOCS-3 inhibits CNI-induced ERK1/-2 signalling, thereby blunting the negative control of C-system activation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2015-12-30

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

Methanol extract of grain dust shows complement fixing activity and other characteristics similar to tannic acid.

PubMed Central

Skea, D; Broder, I

1986-01-01

We have found several similarities between tannic acid and grain dust extract prepared with methanol. Both formed a precipitate with IgG, and these interactions were inhibited by albumin. In addition, both preparations fixed complement; this activity was heat stable and was removed by prior adsorption of the preparations with hide powder. Adsorption with polyvinyl polypyrrolidone reduced the complement-fixing activity of tannic acid but not that of the methanol grain dust extract. The similarities between tannic acid and the methanol grain dust extract are consistent with the presence of a tannin or tanninlike material in grain dust. Images FIGURE 1. PMID:3709479

Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System.

PubMed

Weinberger, Katherine; Collazo, Norberto; Aguillón, Juan Carlos; Molina, María Carmen; Rosas, Carlos; Peña, Jaime; Pizarro, Javier; Maldonado, Ismael; Cattan, Pedro E; Apt, Werner; Ferreira, Arturo

2017-02-08

Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans , and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects. © The American Society of Tropical Medicine and Hygiene.

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

PubMed

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

2016-05-01

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

An ecoimmunological approach to study evolutionary and ancient links between coagulation, complement and Innate immunity

PubMed Central

Kasetty, Gopinath; Alyafei, Saud; Smeds, Emanuel; Salo-Ahen, Outi M. H.; Hansson, Stefan R.; Egesten, Arne; Herwald, Heiko

2018-01-01

ABSTRACT Coagulation, complement, and innate immunity are tightly interwoven and form an alliance that can be traced back to early eukaryotic evolution. Here we employed an ecoimmunological approach using Tissue Factor Pathway Inhibitor (TFPI)-1-derived peptides from the different classes of vertebrates (i.e. fish, reptile, bird, and mammals) and tested whether they can boost killing of various human bacterial pathogens in plasma. We found signs of species-specific conservation and diversification during evolution in these peptides that significantly impact their antibacterial activity. Though all peptides tested executed bactericidal activity in mammalian plasma (with the exception of rodents), no killing was observed in plasma from birds, reptiles, and fish, pointing to a crucial role for the classical pathway of the complement system. We also observed an interference of these peptides with the human intrinsic pathway of coagulation though, unlike complement activation, this mechanism appears not to be evolutionary conserved. PMID:29473457

A connectionist model for diagnostic problem solving

NASA Technical Reports Server (NTRS)

Peng, Yun; Reggia, James A.

1989-01-01

A competition-based connectionist model for solving diagnostic problems is described. The problems considered are computationally difficult in that (1) multiple disorders may occur simultaneously and (2) a global optimum in the space exponential to the total number of possible disorders is sought as a solution. The diagnostic problem is treated as a nonlinear optimization problem, and global optimization criteria are decomposed into local criteria governing node activation updating in the connectionist model. Nodes representing disorders compete with each other to account for each individual manifestation, yet complement each other to account for all manifestations through parallel node interactions. When equilibrium is reached, the network settles into a locally optimal state. Three randomly generated examples of diagnostic problems, each of which has 1024 cases, were tested, and the decomposition plus competition plus resettling approach yielded very high accuracy.

Genotypic and phenotypic diversity of Lactobacillus rhamnosus clinical isolates, their comparison with strain GG and their recognition by complement system

PubMed Central

Douillard, François P.; Ritari, Jarmo; Paulin, Lars; Järvinen, Hanna M.; Rasinkangas, Pia; Haapasalo, Karita; Meri, Seppo; Jarva, Hanna; de Vos, Willem M.

2017-01-01

Lactobacillus rhamnosus strains are ubiquitous in fermented foods, and in the human body where they are commensals naturally present in the normal microbiota composition of gut, vagina and skin. However, in some cases, Lactobacillus spp. have been implicated in bacteremia. The aim of the study was to examine the genomic and immunological properties of 16 clinical blood isolates of L. rhamnosus and to compare them to the well-studied L. rhamnosus probiotic strain GG. Blood cultures from bacteremic patients were collected at the Helsinki University Hospital laboratory in 2005–2011 and L. rhamnosus strains were isolated and characterized by genomic sequencing. The capacity of the L. rhamnosus strains to activate serum complement was studied using immunological assays for complement factor C3a and the terminal pathway complement complex (TCC). Binding of complement regulators factor H and C4bp was also determined using radioligand assays. Furthermore, the isolated strains were evaluated for their ability to aggregate platelets and to form biofilms in vitro. Genomic comparison between the clinical L. rhamnosus strains showed them to be clearly different from L. rhamnosus GG and to cluster in two distinct lineages. All L. rhamnosus strains activated complement in serum and none of them bound complement regulators. Four out of 16 clinical blood isolates induced platelet aggregation and/or formed more biofilms than L. rhamnosus GG, which did not display platelet aggregation activity nor showed strong biofilm formation. These findings suggest that clinical L. rhamnosus isolates show considerable heterogeneity but are clearly different from L. rhamnosus GG at the genomic level. All L. rhamnosus strains are still normally recognized by the human complement system. PMID:28493885

THE GLOMERULAR MESANGIUM

PubMed Central

Mauer, S. Michael; Sutherland, David E. R.; Howard, Richard J.; Fish, Alfred J.; Najarian, John S.; Michael, Alfred F.

1973-01-01

A mechanism of immune glomerular injury is described based on the fixation of antibody (Ab) to an antigen (Ag) that has localized in the glomerular mesangium. Rabbits were given, intravenously (i.v.), aggregated human IgG (AHIgG) or albumin (AHSA) and 10 h later, when the Ag by immunofluorescent microscopy was present in the mesangium, a kidney was removed and transplanted into a normal rabbit. The recipient then received, i.v., rabbit anti-HIgG or anti-HSA. Within minutes of Ab infusion, glomeruli of the donor kidney had polymorphonuclear (PMN) infiltration that over the next few hours became marked and was associated with glomerular cell swelling. At 24 h a decrease in PMN's and early mesangial proliferation was seen. By 3 days there was marked mesangial hypercellularity and increased mesangial matrix. Within minutes after Ab administration rabbit IgG, C3, and fibrin were seen in the glomerular mesangium. There was a fall in complement titer by 1 min after Ab infusion that was due to complement consumption by the donor kidney. Complement then returned to normal levels by 48 h. Significant glomerular injury did not occur (a) in the recipient's own kidney, (b) from Ag administration and transplantation without recipient Ab administration, or (c) from transplantation and Ab administration without prior Ag administration. These studies demonstrated that Ag localized in the glomerular mesangium can react with circulating Ab and complement resulting in severe glomerular injury. PMID:4570015

Factor H-related proteins.

PubMed

Józsi, Mihály; Meri, Seppo

2014-01-01

Factor H-related proteins (CFHRs) are plasma glycoproteins related in structure and antigenicity to each other and to the complement inhibitory protein factor H. Such proteins are found in most mammals but their number and domain composition vary. This chapter summarizes our current knowledge on the human factor H-related proteins. In contrast to factor H, they have no strong complement inhibitory activity, although for some of them regulatory or complement modulatory activity has been reported. A common feature of CFHRs is that they bind to the C3b component of complement. Novel links between CFHRs and various diseases (C3 glomerulopathies, atypical hemolytic uremic syndrome and age-related macular degeneration) have been revealed in recent years, but we are still far from understanding their biological function.

Hemocompatibility evaluation in vitro of methoxy polyethyleneglycol-polycaprolactone copolymer solutions.

PubMed

Hu, Qian; Zhang, Yi; Wang, Changyong; Xu, Jiake; Wu, Jianping; Liu, Zonghua; Xue, Wei

2016-03-01

Amphiphilic block copolymer methoxy polyethyleneglycol-polycaprolactone (mPEG-PCL) has attracted interest in the biomedical field, due to its water solubility and biodegradability. Nevertheless, the blood safety of mPEG-PCL copolymers has not been investigated in detail. Because mPEG-PCL copolymers introduced in vivo would inevitably interact with blood tissue, an investigation of possible interactions of mPEG-PCL with key blood components is crucial. We studied the effects of two mPEG-PCL copolymer solutions on blood coagulation, the morphology and lysis of human red blood cells (RBCs), the structure of plasma fibrinogen, complement activation, and platelet aggregation. We found that higher concentrations of the mPEG-PCL copolymers impaired blood clotting, and the copolymers had little impact on the morphology or lysis of RBCs. From the spectroscopy results, the copolymers affected the local microstructure of fibrinogen. The copolymers significantly activated the complement system in a concentration-dependent way. At higher concentrations, the copolymers impaired platelet aggregation, which may have been mediated by an inhibition of the arachidonic acid pathway. These findings provide important information that may be useful for the molecular design and biomedical applications of mPEG-PCL copolymers. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 802-812, 2016. © 2016 Wiley Periodicals, Inc.

The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line.

PubMed Central

Beck, P J; Orlean, P; Albright, C; Robbins, P W; Gething, M J; Sambrook, J F

1990-01-01

The Saccharomyces cerevisiae DPM1 gene product, dolichol-phosphate-mannose (Dol-P-Man) synthase, is involved in the coupled processes of synthesis and membrane translocation of Dol-P-Man. Dol-P-Man is the lipid-linked sugar donor of the last four mannose residues that are added to the core oligosaccharide transferred to protein during N-linked glycosylation in the endoplasmic reticulum. We present evidence that the S. cerevisiae gene DPM1, when stably transfected into a mutant Chinese hamster ovary cell line, B4-2-1, is able to correct the glycosylation defect of the cells. Evidence for complementation includes (i) fluorescence-activated cell sorter analysis of differential lectin binding to cell surface glycoproteins, (ii) restoration of Dol-P-Man synthase enzymatic activity in crude cell lysates, (iii) isolation and high-performance liquid chromatography fractionation of the lipid-linked oligosaccharides synthesized in the transfected and control cell lines, and (iv) the restoration of endoglycosidase H sensitivity to the oligosaccharides transferred to a specific glycoprotein synthesized in the DPM1 CHO transfectants. Indirect immunofluorescence with a primary antibody directed against the DPM1 protein shows a reticular staining pattern of protein localization in transfected hamster and monkey cell lines. Images PMID:2201896

Exploring the Innate Immune System: Using Complement-Medicated Cell Lysis in the Classroom

ERIC Educational Resources Information Center

Fuller, Kevin G.

2008-01-01

The protein complement pathway comprises an important part of the innate immunity. The use of serum to demonstrate complement-mediated destruction across a series of bacterial dilutions allows an instructor to introduce a number of important biological concepts such as bacterial growth, activation cascades, and adaptive versus innate immunity.

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

PubMed

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

2016-03-23

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

Complement factor H in host defense and immune evasion.

PubMed

Parente, Raffaella; Clark, Simon J; Inforzato, Antonio; Day, Anthony J

2017-05-01

Complement is the major humoral component of the innate immune system. It recognizes pathogen- and damage-associated molecular patterns, and initiates the immune response in coordination with innate and adaptive immunity. When activated, the complement system unleashes powerful cytotoxic and inflammatory mechanisms, and thus its tight control is crucial to prevent damage to host tissues and allow restoration of immune homeostasis. Factor H is the major soluble inhibitor of complement, where its binding to self markers (i.e., particular glycan structures) prevents complement activation and amplification on host surfaces. Not surprisingly, mutations and polymorphisms that affect recognition of self by factor H are associated with diseases of complement dysregulation, such as age-related macular degeneration and atypical haemolytic uremic syndrome. In addition, pathogens (i.e., non-self) and cancer cells (i.e., altered-self) can hijack factor H to evade the immune response. Here we review recent (and not so recent) literature on the structure and function of factor H, including the emerging roles of this protein in the pathophysiology of infectious diseases and cancer.

Complement-fixing Activity of Fulvic Acid from Shilajit and Other Natural Sources

PubMed Central

Schepetkin, Igor A.; Xie, Gang; Jutila, Mark A.; Quinn, Mark T.

2008-01-01

Shilajit has been used traditionally in folk medicine for treatment of a variety of disorders, including syndromes involving excessive complement activation. Extracts of Shilajit contain significant amounts of fulvic acid (FA), and it has been suggested that FA is responsible for many therapeutic properties of Shilajit. However, little is known regarding physical and chemical properties of Shilajit extracts, and nothing is known about their effects on the complement system. To address this issue, we fractionated extracts of commercial Shilajit using anion exchange and size-exclusion chromatography. One neutral (S-I) and two acidic (S-II and S-III) fractions were isolated, characterized, and compared with standardized FA samples. The most abundant fraction (S-II) was further fractionated into three sub-fractions (S-II-1 to S-II-3). The van Krevelen diagram showed that the Shilajit fractions are products of polysaccharide degradation, and all fractions, except S-II-3, contained type II arabinogalactan. All Shilajit fractions exhibited dose-dependent complement-fixing activity in vitro with high potency. Furthermore, we found a strong correlation between complement-fixing activity and carboxylic group content in the Shilajit fractions and other FA sources. These data provide a molecular basis to explain at least part of the beneficial therapeutic properties of Shilajit and other humic extracts. PMID:19107845

Evaluation of Pasteurella multocida serotype B:2 resistance to immune serum and complement system

PubMed Central

Ataei Kachooei, Saeed; Ranjbar, Mohammad Mehdi; Ataei Kachooei, Saba

2017-01-01

Members of gram-negative bacteria family Pasteurellaceae, include a large number of important economically human and veterinary pathogens. Organisms belonging to the family can colonize in mucosal surfaces of the respiratory, alimentary, genital tracts and cause diseases in various mammals, birds, and reptiles. Hemorrhagic septicemia is an acute disease of cattle and buffaloes in tropical countries caused by Pasteurella multocida serotype B:2. In the present study, the possible bactericidal activity of immune calf sera in the presence and absence of complement system was investigated. The results showed that P. multocida B:2 is highly resistant to positive serum, containing high levels of IgG and IgM obtained from calves after vaccination, and complement activity in normal fresh calf serum. This organism also grew rapidly in the normal fresh calf serum and the mixture of positive serum as well as normal fresh calf serum. As a control test an E. coli strain was subjected to the same experiment and found completely sensitive to the bactericidal activity of complement in calf and guinea pig fresh sera. Results were indicative of the presence of inhibitory mechanism(s) in P. multocida B:2 against bactericidal activity of immune calf serum and complement system. PMID:29085604

Dimerization of the bacterial effector protein AvrBs3 in the plant cell cytoplasm prior to nuclear import.

PubMed

Gürlebeck, Doreen; Szurek, Boris; Bonas, Ulla

2005-04-01

The effector protein AvrBs3 from the bacterial phytopathogen Xanthomonas campestris pv. vesicatoria is translocated into the plant cell where it specifically induces hypertrophy symptoms or the hypersensitive reaction. Activity of AvrBs3 depends on nuclear localization signals (NLSs) and an acidic activation domain, suggesting a role in regulation of plant transcription. Here, we show that AvrBs3 dimerizes in the plant cell prior to its nuclear import. AvrBs3 deletion derivatives were tested in the yeast two-hybrid system revealing that the repeat region, which confers specific recognition in resistant plants and is crucial for virulence function, is also essential for the self-interaction. GST pull-down assays showed that the AvrBs3-AvrBs3 interaction occurs independent of plant proteins. Coexpression of two different inactive mutant AvrBs3 derivatives in Bs3-resistant pepper plants resulted in 'trans-complementation', i.e., the induction of a hypersensitive reaction. This clearly indicates that AvrBs3-dimerization occurs in planta. Interestingly, 'trans-complementation' was not observed in susceptible plants suggesting that wild-type homodimers are needed for the AvrBs3 virulence function in plants. Furthermore, a green fluorescent protein (GFP) fusion of AvrBs3 deleted in the NLSs (AvrBs3DeltaNLS-GFP), normally localized in the cytoplasm, was imported into the nucleus upon coexpression with wild-type AvrBs3 in Nicotiana benthamiana. Thus, AvrBs3 dimerization takes place in the cytoplasm of the plant cell prior to nuclear import. Given the fact that dimerization is a common feature of transcriptional regulators, our data are consistent with the idea that AvrBs3 manipulates expression of plant genes involved in the establishment of compatible and incompatible interactions.

PASSIVE HEMOLYSIS BY SERUM AND COBRA VENOM FACTOR: A NEW MECHANISM INDUCING MEMBRANE DAMAGE BY COMPLEMENT*

PubMed Central

Pickering, R. J.; Wolfson, M. R.; Good, R. A.; Gewurz, H.

1969-01-01

The studies presented here indicate that activation of the complement (C′) system by a foreign protein will cause membrane injury and passive lysis of unsensitized erythrocytes present at the time of the reaction. These observations suggest that in addition to the classical antibody-C′-induced cytolysis, there are alternative pathways or mechanisms for activation and participation of the terminal C′ components in the production of cell membrane injury. We have shown that a substance derived from cobra venom and eluted from a single protein band on polyacrylamide can promote lysis of unsensitized autologous or heterologous erythrocytes in the presence of fresh guinea pig serum and that this lysis-inducing activity and C′-inhibiting activity appear to reside in the same fractions. The lytic activity is prevented by several agents known to impair classical C′3 activity, but is unaffected by certain procedures which interfere with the function of C′ components C′1 and C′2, a suggestion that this reaction involves chiefly C′3-C′9. Further, the cobra venom (CV) factor depletes C′ activity in cobra serum, and the CV factor (with its 5S serum cofactor) converts purified C′3 to its inactive form,1 indicating that the reaction of this complex with the complement system occurs without participation of antibody. Therefore, since the lysis-inducing and C′-inhibiting activity of the CV factor appear to result from similar interactions with the complement system, these observations suggest that cell membrane damage and cell lysis can be accomplished through activation of the complement system by a mechanism involving little or no participation of classical antibody or C′ components C′1, 4, or 2. Images PMID:4978744

Positioning soundscape research and management.

PubMed

Andringa, Tjeerd C; Weber, Miriam; Payne, Sarah R; Krijnders, J D Dirkjan; Dixon, Maxwell N; v d Linden, Robert; de Kock, Eveline G L; Lanser, J Jolie L

2013-10-01

This paper is an outcome of a workshop that addressed the question how soundscape research can improve its impact on the local level. It addresses a number of topics by complementing existing approaches and practices with possible future approaches and practices. The paper starts with an analysis of the role of sound annoyance and suboptimal soundscapes on the lives of individuals and concludes that a good soundscape, or more generally a good sensescape, is at the same time pleasant as well as conducive for the adoption of healthy habits. To maintain or improve sensescape quality, urban planning needs improved design tools that allow for a more holistic optimization and an active role of the local stakeholders. Associated with this is a gradual development from government to governance in which optimization of the soundscape at a local (administrative or geographic) level is directly influenced by the users of spaces. The paper concludes that soundscape research can have a greater impact by helping urban planners design for health and pleasant experiences as well as developing tools for improved citizen involvement in local optimization.

The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease

PubMed Central

Sugihara, T; Kobori, A; Imaeda, H; Tsujikawa, T; Amagase, K; Takeuchi, K; Fujiyama, Y; Andoh, A

2010-01-01

Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa α-chain linked to a 70-kDa β-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of IκBα. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17. PMID:20089077

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

PubMed

Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

2018-05-01

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

Microfibrillar associated protein 4 mfap4 genes in catfish play a novel role in innate immune responses

USDA-ARS?s Scientific Manuscript database

The lectin pathway of the complement system is characterized by two groups of soluble pattern recognition molecules, mannose-binding lectins (MBLs) and ficolins. These molecules recognize and bind carbohydrates in pathogens and activate complement leading to opsonization, leukocyte activation, and d...

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

PubMed

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

2016-05-01

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

Combined roles of human IgG subclass, alternative complement pathway activation, and epitope density in the bactericidal activity of antibodies to meningococcal factor h binding protein.

PubMed

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

2012-01-01

Meningococcal vaccines containing factor H binding protein (fHbp) are in clinical development. fHbp binds human fH, which enables the meningococcus to resist complement-mediated bacteriolysis. Previously, we found that chimeric human IgG1 mouse anti-fHbp monoclonal antibodies (MAbs) had human complement-mediated bactericidal activity only if the MAb inhibited fH binding. Since IgG subclasses differ in their ability to activate complement, we investigated the role of human IgG subclasses on antibody functional activity. We constructed chimeric MAbs in which three different murine fHbp-specific binding domains were each paired with human IgG1, IgG2, or IgG3. Against a wild-type group B isolate, all three IgG3 MAbs, irrespective of their ability to inhibit fH binding, had bactericidal activity that was >5-fold higher than the respective IgG1 MAbs, while the IgG2 MAbs had the least activity. Against a mutant with increased fHbp expression, the anti-fHbp MAbs elicited greater C4b deposition (classical pathway) and greater bactericidal activity than against the wild-type strain, and the IgG1 MAbs had similar or greater activity than the respective IgG3 MAbs. The bactericidal activity against both wild-type and mutant strains also was dependent, in part, on activation of the alternative complement pathway. Thus, at lower epitope density in the wild-type strain, the IgG3 anti-fHbp MAbs had the greatest bactericidal activity. At a higher epitope density in the mutant, the IgG1 MAbs had similar or greater bactericidal activity than the IgG3 MAbs, and the activity was less dependent on the inhibition of fH binding than at a lower epitope density.

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

PubMed

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

2006-07-01

The role of the complement system in the pathogenesis of systemic diseases is very ambivalent. In systemic lupus erythematosus (SLE), many abnormalities in the activation of the complement system have been reported. The most important antibodies formed against the complement system in SLE are the ones associated with the C1q component. The aim of this study was to assess separately the anti-C1q antibodies and C1q component in the serum from 65 patients with SLE, then in individuals with (n=33) and without (n=32) lupus nephritis and with active (n=36) and nonactive (n=29) form of the disease (European Consensus Lupus Activity Measurement, ECLAM>3, ECLAM

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

PubMed Central

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

1998-01-01

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

Visceral perfusion abnormalities following complement activation. Clues to the mediators of organ ischemia in trauma and sepsis. First place winner: Conrad Jobst Award.

PubMed

Schirmer, W J; Schirmer, J M; Naff, G B; Fry, D E

1988-12-01

Complement, activated during infection and injury, has been implicated as a mediator of microvascular injury and obstruction. This study examines how two potent activators of complement, zymosan, and cobra venom factor (CVF), affect systemic and visceral perfusion. Rats were injected with either saline (1 ml/kg), zymosan (5 mg/kg) or CVF (5 units/kg) at t = 0 and 30 minutes. Thermodilution cardiac output, mean arterial pressure, heart rate, systemic vascular resistance, and hematocrit were determined at t = 2 hours. Effective hepatic and renal blood flows, by clearance of galactose and p-aminohippurate respectively, were determined over the next hour. The per cent change in total hemolytic complement from t = 0 to t = 3 hours was determined by immune hemolysis of sheep erythrocytes. There was no difference in systemic hemodynamic parameters between the three groups. Hepatic blood flow was depressed in both the zymosan (3.83 +/- 0.23 ml/min/100 g) and CVF (3.72 +/- 0.20 ml/min/100 g) groups compared with controls (4.62 +/- 0.19 ml/min/100 g, P less than 0.05). Renal blood flow in the zymosan-treated group (6.40 +/- 0.24 ml/min/100 g) increased over control (4.80 +/- 0.40 ml/min/100 g, P less than 0.05) but was unchanged in the CVF group (5.06 +/- 0.23 ml/min/100 g). The amount of complement activated correlated with the change in hepatic (r = -0.419, P less than 0.05) but not renal (r = -0.008, P = 0.917) flow. Complement activation may occupy a proximal position in the pathogenesis of hepatic ischemia associated with trauma and sepsis.

Trypanosoma cruzi H+-ATPase 1 (TcHA1) and 2 (TcHA2) genes complement yeast mutants defective in H+ pumps and encode plasma membrane P-type H+-ATPases with different enzymatic properties.

PubMed

Luo, Shuhong; Scott, David A; Docampo, Roberto

2002-11-15

Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H(+)-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi: TcHA1 is expressed maximally in epimastigotes, whereas TcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H(+)-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H(+)-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1- and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in the T. cruzi life cycle.

PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes.

PubMed

Yu, Nancy Y; Wagner, James R; Laird, Matthew R; Melli, Gabor; Rey, Sébastien; Lo, Raymond; Dao, Phuong; Sahinalp, S Cenk; Ester, Martin; Foster, Leonard J; Brinkman, Fiona S L

2010-07-01

PSORTb has remained the most precise bacterial protein subcellular localization (SCL) predictor since it was first made available in 2003. However, the recall needs to be improved and no accurate SCL predictors yet make predictions for archaea, nor differentiate important localization subcategories, such as proteins targeted to a host cell or bacterial hyperstructures/organelles. Such improvements should preferably be encompassed in a freely available web-based predictor that can also be used as a standalone program. We developed PSORTb version 3.0 with improved recall, higher proteome-scale prediction coverage, and new refined localization subcategories. It is the first SCL predictor specifically geared for all prokaryotes, including archaea and bacteria with atypical membrane/cell wall topologies. It features an improved standalone program, with a new batch results delivery system complementing its web interface. We evaluated the most accurate SCL predictors using 5-fold cross validation plus we performed an independent proteomics analysis, showing that PSORTb 3.0 is the most accurate but can benefit from being complemented by Proteome Analyst predictions. http://www.psort.org/psortb (download open source software or use the web interface). psort-mail@sfu.ca Supplementary data are available at Bioinformatics online.

The dynamics of error processing in the human brain as reflected by high-gamma activity in noninvasive and intracranial EEG.

PubMed

Völker, Martin; Fiederer, Lukas D J; Berberich, Sofie; Hammer, Jiří; Behncke, Joos; Kršek, Pavel; Tomášek, Martin; Marusič, Petr; Reinacher, Peter C; Coenen, Volker A; Helias, Moritz; Schulze-Bonhage, Andreas; Burgard, Wolfram; Ball, Tonio

2018-06-01

Error detection in motor behavior is a fundamental cognitive function heavily relying on local cortical information processing. Neural activity in the high-gamma frequency band (HGB) closely reflects such local cortical processing, but little is known about its role in error processing, particularly in the healthy human brain. Here we characterize the error-related response of the human brain based on data obtained with noninvasive EEG optimized for HGB mapping in 31 healthy subjects (15 females, 16 males), and additional intracranial EEG data from 9 epilepsy patients (4 females, 5 males). Our findings reveal a multiscale picture of the global and local dynamics of error-related HGB activity in the human brain. On the global level as reflected in the noninvasive EEG, the error-related response started with an early component dominated by anterior brain regions, followed by a shift to parietal regions, and a subsequent phase characterized by sustained parietal HGB activity. This phase lasted for more than 1 s after the error onset. On the local level reflected in the intracranial EEG, a cascade of both transient and sustained error-related responses involved an even more extended network, spanning beyond frontal and parietal regions to the insula and the hippocampus. HGB mapping appeared especially well suited to investigate late, sustained components of the error response, possibly linked to downstream functional stages such as error-related learning and behavioral adaptation. Our findings establish the basic spatio-temporal properties of HGB activity as a neural correlate of error processing, complementing traditional error-related potential studies. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The Integrated Library System: The University of Alabama in Birmingham.

ERIC Educational Resources Information Center

Nelms, Doyal E.; Stephens, Jerry W.

1980-01-01

This discussion of the approach taken in developing and integrating the available local and national systems into one unified management system examines and illustrates how the home-grown acquisitions module, the shared cataloging of OCLC and local circulation/inventory control module under development all interface and complement each other.…

Deposition of mannose-binding lectin and ficolins and activation of the lectin pathway of complement on the surface of polyurethane tubing used for cardiopulmonary bypass.

PubMed

Eppa, Łukasz; Pągowska-Klimek, Izabela; Świerzko, Anna S; Moll, Maciej; Krajewski, Wojciech R; Cedzyński, Maciej

2018-04-01

The artificial surface used for cardiopulmonary bypass (CPB) is a crucial factor activating the complement system and thus contributing to the generation of a systemic inflammatory response. The activation of classical and alternative pathways on this artificial surface is well known. In contrast, lectin pathway (LP) activation has not been fully investigated, although noted during CPB in several studies. Moreover, we have recently proved the contribution of the LP to the generation of the systemic inflammatory response syndrome after pediatric cardiac surgery. The aim of this study was to assess LP-mediated complement activation on the surface of polyurethane CPB circuit tubing (noncoated Chalice ® ), used for CPB procedures in children with congenital heart disease. We found deposition of mannose-binding lectin, ficolin-1, -2, and -3 on the surface of unused tubing and on tubing used for CPB from a small minority of patients. Furthermore, we observed deposition of complement C4 activation products on tubing used for CPB and previously unused tubing after incubation with normal serum. The latter finding indicates LP activation in vitro on the polyurethane surface. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1202-1208, 2018. © 2017 Wiley Periodicals, Inc.

Assessing Fishing and Marine Biodiversity Changes Using Fishers' Perceptions: The Spanish Mediterranean and Gulf of Cadiz Case Study

PubMed Central

Coll, Marta; Carreras, Marta; Ciércoles, Cristina; Cornax, Maria-José; Gorelli, Giulia; Morote, Elvira; Sáez, Raquel

2014-01-01

Background The expansion of fishing activities has intensively transformed marine ecosystems worldwide. However, available time series do not frequently cover historical periods. Methodology Fishers' perceptions were used to complement data and characterise changes in fishing activity and exploited ecosystems in the Spanish Mediterranean Sea and Gulf of Cadiz. Fishers' interviews were conducted in 27 fishing harbours of the area, and included 64 fishers from ages between 20 to >70 years old to capture the experiences and memories of various generations. Results are discussed in comparison with available independent information using stock assessments and international convention lists. Principal Findings According to fishers, fishing activity substantially evolved in the area with time, expanding towards deeper grounds and towards areas more distant from the coast. The maximum amount of catch ever caught and the weight of the largest species ever captured inversely declined with time. Fishers (70%) cited specific fishing grounds where depletion occurred. They documented ecological changes of marine biodiversity during the last half of the century: 94% reported the decline of commercially important fish and invertebrates and 61% listed species that could have been extirpated, with frequent mentions to cartilaginous fish. Declines and extirpations were in line with available quantitative evaluations from stock assessments and international conventions, and were likely linked to fishing impacts. Conversely, half of interviewed fishers claimed that several species had proliferated, such as cephalopods, jellyfish, and small-sized fish. These changes were likely related to trophic cascades due to fishing and due to climate change effects. The species composition of depletions, local extinctions and proliferations showed differences by region suggesting that regional dynamics are important when analysing biodiversity changes. Conclusions/Significance Using fishers' perceptions, fishing and ecological changes in the study area were documented. The recovery of local ecological knowledge provides valuable information complementing quantitative monitoring and evaluation surveys. PMID:24465644

Assessing fishing and marine biodiversity changes using fishers' perceptions: the Spanish Mediterranean and Gulf of Cadiz case study.

PubMed

Coll, Marta; Carreras, Marta; Ciércoles, Cristina; Cornax, Maria-José; Gorelli, Giulia; Morote, Elvira; Sáez, Raquel

2014-01-01

The expansion of fishing activities has intensively transformed marine ecosystems worldwide. However, available time series do not frequently cover historical periods. Fishers' perceptions were used to complement data and characterise changes in fishing activity and exploited ecosystems in the Spanish Mediterranean Sea and Gulf of Cadiz. Fishers' interviews were conducted in 27 fishing harbours of the area, and included 64 fishers from ages between 20 to >70 years old to capture the experiences and memories of various generations. Results are discussed in comparison with available independent information using stock assessments and international convention lists. According to fishers, fishing activity substantially evolved in the area with time, expanding towards deeper grounds and towards areas more distant from the coast. The maximum amount of catch ever caught and the weight of the largest species ever captured inversely declined with time. Fishers (70%) cited specific fishing grounds where depletion occurred. They documented ecological changes of marine biodiversity during the last half of the century: 94% reported the decline of commercially important fish and invertebrates and 61% listed species that could have been extirpated, with frequent mentions to cartilaginous fish. Declines and extirpations were in line with available quantitative evaluations from stock assessments and international conventions, and were likely linked to fishing impacts. Conversely, half of interviewed fishers claimed that several species had proliferated, such as cephalopods, jellyfish, and small-sized fish. These changes were likely related to trophic cascades due to fishing and due to climate change effects. The species composition of depletions, local extinctions and proliferations showed differences by region suggesting that regional dynamics are important when analysing biodiversity changes. Using fishers' perceptions, fishing and ecological changes in the study area were documented. The recovery of local ecological knowledge provides valuable information complementing quantitative monitoring and evaluation surveys.

A teleost CD46 is involved in the regulation of complement activation and pathogen infection.

PubMed

Li, Mo-Fei; Sui, Zhi-Hai; Sun, Li

2017-11-03

In mammals, CD46 is involved in the inactivation of complement by factor I (FI). In teleost, study on the function of CD46 is very limited. In this study, we examined the immunological property of a CD46 molecule (CsCD46) from tongue sole, a teleost species with important economic value. We found that recombinant CsCD46 (rCsCD46) interacted with FI and inhibited complement activation in an FI-dependent manner. rCsCD46 also interacted with bacterial pathogens via a different mechanism to that responsible for the FI interaction, involving different rCsCD46 sites. Cellular study showed that CsCD46 was expressed on peripheral blood leukocytes (PBL) and protected the cells against the killing effect of complement. When the CsCD46 on PBL was blocked by antibody before incubation of the cells with bacterial pathogens, cellular infection was significantly reduced. Consistently, when tongue sole were infected with bacterial pathogens in the presence of rCsCD46, tissue dissemination and survival of the pathogens were significantly inhibited. These results provide the first evidence to indicate that CD46 in teleosts negatively regulates complement activation via FI and protects host cells from complement-induced damage, and that CD46 is required for optimal bacterial infection probably by serving as a receptor for the bacteria.

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

PubMed

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

2017-05-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of more than two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology, such as acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases, which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small-molecule inhibitors, small-molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study, we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies, we identified 45 small molecules that putatively bind C3b near ligand-guided functional hot spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand that guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small-molecule complement inhibitors and, to our knowledge, provides the first demonstration of cheminformatics-based, complement-directed drug discovery. Copyright © 2017 by The American Association of Immunologists, Inc.

Identification of C3b-binding Small Molecule Complement Inhibitors Using Cheminformatics

PubMed Central

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

2017-01-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of over two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology which include acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small molecule inhibitors, small molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies we identified 45 small molecules which putatively bind C3b near ligand-guided functional hot-spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand which guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small molecule complement inhibitors, and to our knowledge, provides the first demonstration of cheminformatics-based complement-directed drug discovery. PMID:28298523

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

PubMed Central

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

2017-01-01

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

Phylogenetic aspects of the complement system.

PubMed

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

2001-01-01

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

Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

PubMed

Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

2006-05-01

The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

Preparation of Low Molecular Weight Chondroitin Sulfates, Screening of a High Anti-Complement Capacity of Low Molecular Weight Chondroitin Sulfate and Its Biological Activity Studies in Attenuating Osteoarthritis

PubMed Central

Li, Lian; Li, Yan; Feng, Danyang; Xu, Linghua; Yin, Fengxin; Zang, Hengchang; Liu, Chunhui; Wang, Fengshan

2016-01-01

Chondroitin sulfate (CS) plays important roles in the complement system. However, the CS structure is complicated due to different sources and the number and positions of sulfate groups. The objective of this study was to prepare different low molecular weight chondroitin sulfates (LMWCSs) and to investigate the biological activity in anti-complement capacity. A series of LMWCSs was prepared from different sources and characterized by ultraviolet-visible (UV) spectroscopy, high-performance liquid chromatography (HPLC), size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) and nuclear magnetic resonance (NMR) spectroscopy. Hemolytic, anti-complement 3 deposition capacity and cell viability assays were carried out to investigate the biological activities in vitro. The results showed that LMWCS prepared from shark cartilage with the oxidative degradation method (LMWCS-S-O) had the best anti-complement capacity. LMWCS-S-O could inhibit the alternative pathway of the complement system and protect chondrocytes from cell death. The attenuating effect of LMWCS-S-O on Osteoarthritis (OA) was investigated by destabilization of the medial meniscus (DMM) model in vivo. Functional wind-up, histological and C5b-9 analyses were used to evaluate the treatment effect on the OA model. In vivo results showed that LMWCS-S-O could attenuate OA. LMWCS-S-O with a high content of ΔDi-2,6diS and ΔDi-6S could be used for attenuating OA through regulating the complement system. PMID:27727159

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

PubMed

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

2014-12-01

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

Activation of the classical pathway of complement by binding of bovine lactoferrin to unencapsulated Streptococcus agalactiae.

PubMed Central

Rainard, P

1993-01-01

The ability of lactoferrin (Lf) bound to Streptococcus agalactiae to interfere with the deposition of complement components on the bacterial surface was investigated by enzyme-linked immunosorbent assay (ELISA). By using a strain of S. agalactiae which activates the alternative pathway of complement in the absence of antibodies, it was found that pretreatment of bacteria with Lf shortened the lag phase preceding the deposition of C3 on bacteria. The kinetics of C3 deposition was comparable to that obtained by adding antibodies against S. agalactiae to agammaglobulinaemic precolostral calf serum (PCS) heated at 56 degrees for 3 min to inactivate the alternative pathway. Accelerated C3 deposition did not occur in the absence of Ca2+ ions. Deposition of C4 on bacteria occurred only when either antibodies or Lf were added to PCS. These results demonstrate that the interaction of lactoferrin with bacteria activated the classical pathway of complement in the absence of antibodies. The binding of purified C1q to bacteria was promoted in a dose-dependent manner by Lf, suggesting that recruitment of classical pathway of complement resulted from the interaction of C1q with Lf adsorbed to the bacterial surface. Phagocytosis of bacteria opsonized with heated PCS (at 56 degrees for 3 min) and Lf was comparable to that occurring in the presence of heated PCS and antibodies. In conclusion, Lf was able to substitute for antibodies in order to activate the classical pathway of complement and to opsonize unencapsulated S. agalactiae efficiently. PMID:8406591

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

Is complement good, bad, or both? New functions of the complement factors associated with inflammation mechanisms in the central nervous system.

PubMed

Tahtouh, Muriel; Croq, Françoise; Lefebvre, Christophe; Pestel, Joël

2009-09-01

The complement system is well known as an enzyme cascade that helps to defend against infections. Indeed, this ancestral system bridges innate and adaptive immunity. Its implication in diseases of the central nervous system (CNS), has led to an increased number of studies. Complement activation in the CNS has been generally considered to contribute to tissue damage. However, recent studies suggest that complement may be neuroprotective, and can participate in maintenance and repair of the adult brain. Here, we will review this dual role of complement proteins and some of their functional interactions with part of the chemokine and cytokine network associated with the protection of CNS integrity.

Muscarinic Acetylcholine Receptor Localization and Activation Effects on Ganglion Response Properties

PubMed Central

Renna, Jordan M.; Amthor, Franklin R.; Keyser, Kent T.

2010-01-01

Purpose. The activation and blockade of muscarinic acetylcholine receptors (mAChRs) affects retinal ganglion cell light responses and firing rates. This study was undertaken to identify the full complement of mAChRs expressed in the rabbit retina and to assess mAChR distribution and the functional effects of mAChR activation and blockade on retinal response properties. Methods. RT-PCR, Western blot analysis, and immunohistochemistry were used to identify the complement and distribution of mAChRs in the rabbit retina. Extracellular electrophysiology was used to determine the effects of the activation or blockade of mAChRs on ganglion cell response properties. Results. RT-PCR of whole neural retina resulted in the amplification of mRNA transcripts for the m1 to m5 mAChR subtypes. Western blot and immunohistochemical analyses confirmed that all five mAChR subtypes were expressed by subpopulations of bipolar, amacrine, and ganglion cells in the rabbit retina, including subsets of cells in cholinergic and glycinergic circuits. Nonspecific muscarinic activation and blockade resulted in the class-specific modulation of maintained ganglion cell firing rates and light responses. Conclusions. The expression of mAChR subtypes on subsets of bipolar, amacrine, and ganglion cells provides a substrate for both enhancement and suppression of retinal responses via activation by cholinergic agents. Thus, the muscarinic cholinergic system in the retina may contribute to the modulation of complex stimuli. Understanding the distribution and function of mAChRs in the retina has the potential to provide important insights into the visual changes that are caused by decreased ACh in the retinas of Alzheimer's patients and the potential visual effects of anticholinergic treatments for ocular diseases. PMID:20042645

In vitro immunomodulatory activity of plants used by the Tacana ethnic group in Bolivia.

PubMed

Deharo, E; Baelmans, R; Gimenez, A; Quenevo, C; Bourdy, G

2004-09-01

One hundred and seventy-eight ethanolic plant extracts from the pharmacopoeia of the Tacana, an ethnic group from Bolivia, were screened for immunomodulatory activity using complement cascade inhibition and ADP-induced platelet aggregation inhibition assays. Six impaired both complement pathways (classical and alternative): stem bark from Astronium urundeuvea (Anacardiaceae), Cochlospermum vitifolium (Cochlospermaceae), Terminalia amazonica (Combretaceae), Triplaris americana (Polygonaceae), Uncaria tomentosa (Rubiaceae) and Euterpe precatoria (Arecaceae) roots. Inhibition of complement cascade was independent of essential ion complexation, and was not due to direct hemolytic activity on target red blood cells. For A. urundeuvea, C. vitifolium, and T. amazonica, anti-inflammatory activity relied on cyclo-oxygenase inhibition. Four of these species (A. urundeuva, T. americana, U. tomentosa and E. precatoria) are used traditionally to treat inflammatory processes.

Protective responses to sublytic complement in the retinal pigment epithelium

PubMed Central

Tan, Li Xuan; Toops, Kimberly A.; Lakkaraju, Aparna

2016-01-01

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

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

PubMed

Barata, Lidia; Miwa, Takashi; Sato, Sayaka; Kim, David; Mohammed, Imran; Song, Wen-Chao

2013-03-15

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

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

PubMed Central

1988-01-01

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

Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes

PubMed Central

Degn, Søren E.; Kjaer, Troels R.; Kidmose, Rune T.; Jensen, Lisbeth; Hansen, Annette G.; Tekin, Mustafa; Jensenius, Jens C.; Andersen, Gregers R.; Thiel, Steffen

2014-01-01

Defining mechanisms governing translation of molecular binding events into immune activation is central to understanding immune function. In the lectin pathway of complement, the pattern recognition molecules (PRMs) mannan-binding lectin (MBL) and ficolins complexed with the MBL-associated serine proteases (MASP)-1 and MASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 was recently found to be the exclusive activator of MASP-2 under physiological conditions, yet the predominant oligomeric forms of MBL carry only a single MASP homodimer. This prompted us to investigate whether activation of MASP-2 by MASP-1 occurs through PRM-driven juxtaposition on ligand surfaces. We demonstrate that intercomplex activation occurs between discrete PRM/MASP complexes. PRM ligand binding does not directly escort the transition of MASP from zymogen to active enzyme in the PRM/MASP complex; rather, clustering of PRM/MASP complexes directly causes activation. Our results support a clustering-based mechanism of activation, fundamentally different from the conformational model suggested for the classical pathway of complement. PMID:25197071

L3:PHI.CMD.P13.02 Support for CILC L1 Milestone Using STAR-CCM+

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

Slattery, Stuart R.; Gurecky, William L.

2016-10-07

This report documents work performed to support Consortium for the Advanced Simulation of LWRs (CASL) modeling of Chalk River Unidentified Deposit (CRUD) Induced Power Shift (CIPS) and CRUD Induced Local Corrosion (CILC) using the Cicada package. The work documented here is intended to complement current and future CIPS and CILC modeling activities in CASL. We provide tools for crud and corrosion-related simulation and analysis by developing a better understanding of the interplay between the coupled physics that describe the phenomena at different time and length scales. We intend to use these models to better inform future simulation capability and development.

Behavior‐dependent activity patterns of GABAergic long‐range projecting neurons in the rat hippocampus

PubMed Central

Micklem, Ben; Borhegyi, Zsolt; Swiejkowski, Daniel A.; Valenti, Ornella; Viney, Tim J.; Kotzadimitriou, Dimitrios; Klausberger, Thomas

2017-01-01

ABSTRACT Long‐range glutamatergic and GABAergic projections participate in temporal coordination of neuronal activity in distributed cortical areas. In the hippocampus, GABAergic neurons project to the medial septum and retrohippocampal areas. Many GABAergic projection cells express somatostatin (SOM+) and, together with locally terminating SOM+ bistratified and O‐LM cells, contribute to dendritic inhibition of pyramidal cells. We tested the hypothesis that diversity in SOM+ cells reflects temporal specialization during behavior using extracellular single cell recording and juxtacellular neurobiotin‐labeling in freely moving rats. We have demonstrated that rare GABAergic projection neurons discharge rhythmically and are remarkably diverse. During sharp wave‐ripples, most projection cells, including a novel SOM+ GABAergic back‐projecting cell, increased their activity similar to bistratified cells, but unlike O‐LM cells. During movement, most projection cells discharged along the descending slope of theta cycles, but some fired at the trough jointly with bistratified and O‐LM cells. The specialization of hippocampal SOM+ projection neurons complements the action of local interneurons in differentially phasing inputs from the CA3 area to CA1 pyramidal cell dendrites during sleep and wakefulness. Our observations suggest that GABAergic projection cells mediate the behavior‐ and network state‐dependent binding of neuronal assemblies amongst functionally‐related brain regions by transmitting local rhythmic entrainment of neurons in CA1 to neuronal populations in other areas. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc. PMID:27997999

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

PubMed

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

2017-12-01

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

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

PubMed

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

2016-04-01

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

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

PubMed Central

Moore, Gregory L; Chen, Hsing; Karki, Sher

2010-01-01

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complementenhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens. PMID:20150767

Complement-fixing activity of fulvic acid from Shilajit and other natural sources.

PubMed

Schepetkin, Igor A; Xie, Gang; Jutila, Mark A; Quinn, Mark T

2009-03-01

Shilajit has been used traditionally in folk medicine for the treatment of a variety of disorders, including syndromes involving excessive complement activation. Extracts of Shilajit contain significant amounts of fulvic acid (FA), and it has been suggested that FA is responsible for many therapeutic properties of Shilajit. However, little is known regarding the physical and chemical properties of Shilajit extracts, and nothing is known about their effects on the complement system. To address this issue, extracts of commercial Shilajit were fractionated using anion exchange and size-exclusion chromatography. One neutral (S-I) and two acidic (S-II and S-III) fractions were isolated, characterized and compared with standardized FA samples. The most abundant fraction (S-II) was further fractionated into three sub-fractions (S-II-1 to S-II-3). The van Krevelen diagram showed that the Shilajit fractions are the products of polysaccharide degradation, and all fractions, except S-II-3, contained type II arabinogalactan. All Shilajit fractions exhibited dose-dependent complement-fixing activity in vitro with high potency. Furthermore, a strong correlation was found between the complement-fixing activity and carboxylic group content in the Shilajit fractions and other FA sources. These data provide a molecular basis to explain at least part of the beneficial therapeutic properties of Shilajit and other humic extracts. (c) 2008 John Wiley & Sons, Ltd.

Cluster Analysis Identifies Distinct Pathogenetic Patterns in C3 Glomerulopathies/Immune Complex-Mediated Membranoproliferative GN.

PubMed

Iatropoulos, Paraskevas; Daina, Erica; Curreri, Manuela; Piras, Rossella; Valoti, Elisabetta; Mele, Caterina; Bresin, Elena; Gamba, Sara; Alberti, Marta; Breno, Matteo; Perna, Annalisa; Bettoni, Serena; Sabadini, Ettore; Murer, Luisa; Vivarelli, Marina; Noris, Marina; Remuzzi, Giuseppe

2018-01-01

Membranoproliferative GN (MPGN) was recently reclassified as alternative pathway complement-mediated C3 glomerulopathy (C3G) and immune complex-mediated membranoproliferative GN (IC-MPGN). However, genetic and acquired alternative pathway abnormalities are also observed in IC-MPGN. Here, we explored the presence of distinct disease entities characterized by specific pathophysiologic mechanisms. We performed unsupervised hierarchical clustering, a data-driven statistical approach, on histologic, genetic, and clinical data and data regarding serum/plasma complement parameters from 173 patients with C3G/IC-MPGN. This approach divided patients into four clusters, indicating the existence of four different pathogenetic patterns. Specifically, this analysis separated patients with fluid-phase complement activation (clusters 1-3) who had low serum C3 levels and a high prevalence of genetic and acquired alternative pathway abnormalities from patients with solid-phase complement activation (cluster 4) who had normal or mildly altered serum C3, late disease onset, and poor renal survival. In patients with fluid-phase complement activation, those in clusters 1 and 2 had massive activation of the alternative pathway, including activation of the terminal pathway, and the highest prevalence of subendothelial deposits, but those in cluster 2 had additional activation of the classic pathway and the highest prevalence of nephrotic syndrome at disease onset. Patients in cluster 3 had prevalent activation of C3 convertase and highly electron-dense intramembranous deposits. In addition, we provide a simple algorithm to assign patients with C3G/IC-MPGN to specific clusters. These distinct clusters may facilitate clarification of disease etiology, improve risk assessment for ESRD, and pave the way for personalized treatment. Copyright © 2018 by the American Society of Nephrology.

Complement in the Initiation and Evolution of Rheumatoid Arthritis

PubMed Central

Holers, V. Michael; Banda, Nirmal K.

2018-01-01

The complement system is a major component of the immune system and plays a central role in many protective immune processes, including circulating immune complex processing and clearance, recognition of foreign antigens, modulation of humoral and cellular immunity, removal of apoptotic and dead cells, and engagement of injury resolving and tissue regeneration processes. In stark contrast to these beneficial roles, however, inadequately controlled complement activation underlies the pathogenesis of human inflammatory and autoimmune diseases, including rheumatoid arthritis (RA) where the cartilage, bone, and synovium are targeted. Recent studies of this disease have demonstrated that the autoimmune response evolves over time in an asymptomatic preclinical phase that is associated with mucosal inflammation. Notably, experimental models of this disease have demonstrated that each of the three major complement activation pathways plays an important role in recognition of injured joint tissue, although the lectin and amplification pathways exhibit particularly impactful roles in the initiation and amplification of damage. Herein, we review the complement system and focus on its multi-factorial role in human patients with RA and experimental murine models. This understanding will be important to the successful integration of the emerging complement therapeutics pipeline into clinical care for patients with RA. PMID:29892280

Spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides and their ability to activate human complement system in vitro.

PubMed

Granja, Luiz Fernando Zmetek; Pinto, Lysianne; Almeida, Cátia Amancio; Alviano, Daniela Sales; Da Silva, Maria Helena; Ejzemberg, Regina; Alviano, Celuta Sales

2010-03-01

Complement activation by spores of Mucor ramosissimus, Mucor plumbeus and Mucor circinelloides was studied using absorbed human serum in the presence or absence of chelators (EGTA or EDTA). We found that the spore caused full complement activation when incubated with EGTA-Mg2+ or without chelators, indicating that the alternative pathway is mainly responsible for this response. In order to compare activation profiles from each species, ELISAs for C3 and C4 fragments, mannan binding lectin (MBL), C-reactive protein (CRP) and IgG studies were carried out. All proteins were present on the species tested. Immunofluorescence tests demonstrated the presence of C3 fragments on the surface of all samples, which were confluent throughout fungal surfaces. The same profile of C3, C4, MBL, CRP and IgG deposition, observed in all species, suggests a similar activation behavior for these species.

Chemical characterization and complement fixation of pectins from Cola cordifolia leaves.

PubMed

Austarheim, Ingvild; Christensen, Bjørn E; Aas, Hoai Thi Nguyen; Thöle, Christian; Diallo, Drissa; Paulsen, Berit S

2014-02-15

Defatted leaves from the medicinal tree Cola cordifolia were extracted with 50% EtOH, 50 °C and 100 °C water. The polysaccharide rich extracts were fractionated and the structure of the polysaccharides elucidated. Linkage analysis of the polysaccharides indicates a rhamnogalacturonan type I backbone where both Rha and parts of GalA are substituted in position 3, indicating a highly branched polymer with short side chains. The purified fractions were tested for complement fixation, macrophage stimulating activity and anti-adhesion activity towards Helicobacter pylori. Here we report on complex and polydisperse types of pectins (Mw: 3-1300 kDa) as well as the presence of low Mw (<3 kDa) acidic oligosaccharides. The fractions showed a moderate complement fixing activity and no macrophage activating effects after LPS removal. Anti-adhesion activity towards H. pylori was not found. Copyright © 2013 Elsevier Ltd. All rights reserved.

Virulence Associated Gene 8 of Bordetella pertussis Enhances Contact System Activity by Inhibiting the Regulatory Function of Complement Regulator C1 Inhibitor

PubMed Central

Hovingh, Elise S.; de Maat, Steven; Cloherty, Alexandra P. M.; Johnson, Steven; Pinelli, Elena; Maas, Coen; Jongerius, Ilse

2018-01-01

Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Whooping cough is currently re-emerging worldwide and, therefore, still poses a continuous global health threat. B. pertussis expresses several virulence factors that play a role in evading the human immune response. One of these virulence factors is virulence associated gene 8 (Vag8). Vag8 is a complement evasion molecule that mediates its effects by binding to the complement regulator C1 inhibitor (C1-INH). This regulatory protein is a fluid phase serine protease that controls proenzyme activation and enzyme activity of not only the complement system but also the contact system. Activation of the contact system results in the generation of bradykinin, a pro-inflammatory peptide. Here, the activation of the contact system by B. pertussis was explored. We demonstrate that recombinant as well as endogenous Vag8 enhanced contact system activity by binding C1-INH and attenuating its inhibitory function. Moreover, we show that B. pertussis itself is able to activate the contact system. This activation was dependent on Vag8 production as a Vag8 knockout B. pertussis strain was unable to activate the contact system. These findings show a previously overlooked interaction between the contact system and the respiratory pathogen B. pertussis. Activation of the contact system by B. pertussis may contribute to its pathogenicity and virulence. PMID:29915576

Virulence Associated Gene 8 of Bordetella pertussis Enhances Contact System Activity by Inhibiting the Regulatory Function of Complement Regulator C1 Inhibitor.

PubMed

Hovingh, Elise S; de Maat, Steven; Cloherty, Alexandra P M; Johnson, Steven; Pinelli, Elena; Maas, Coen; Jongerius, Ilse

2018-01-01

Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Whooping cough is currently re-emerging worldwide and, therefore, still poses a continuous global health threat. B. pertussis expresses several virulence factors that play a role in evading the human immune response. One of these virulence factors is virulence associated gene 8 (Vag8). Vag8 is a complement evasion molecule that mediates its effects by binding to the complement regulator C1 inhibitor (C1-INH). This regulatory protein is a fluid phase serine protease that controls proenzyme activation and enzyme activity of not only the complement system but also the contact system. Activation of the contact system results in the generation of bradykinin, a pro-inflammatory peptide. Here, the activation of the contact system by B. pertussis was explored. We demonstrate that recombinant as well as endogenous Vag8 enhanced contact system activity by binding C1-INH and attenuating its inhibitory function. Moreover, we show that B. pertussis itself is able to activate the contact system. This activation was dependent on Vag8 production as a Vag8 knockout B. pertussis strain was unable to activate the contact system. These findings show a previously overlooked interaction between the contact system and the respiratory pathogen B. pertussis . Activation of the contact system by B. pertussis may contribute to its pathogenicity and virulence.

Schooling and Local Environmental Knowledge: Do They Complement or Substitute Each Other?

ERIC Educational Resources Information Center

Reyes-Garcia, Victoria; Kightley, Eric; Ruiz-Mallen, Isabel; Fuentes-Pelaez, Nuria; Demps, Katie; Huanca, Tomas; Martinez-Rodriguez, Maria Ruth

2010-01-01

Schooling and the knowledge acquired at school have been considered both a cause of loss of indigenous knowledge (because it opens pathways to the non-indigenous world and worldviews) and a potential remedy to its demise (if educational curricula is aligned with indigenous realities by giving instruction in local languages and incorporating local…

Construction and characterization of an anti-CD20 mAb nanocomb with exceptionally excellent lymphoma-suppressing activity.

PubMed

Li, Hua-Fei; Wu, Cong; Chen, Ting; Zhang, Ge; Zhao, He; Ke, Chang-Hong; Xu, Zheng

2015-01-01

The CD20-directed monoclonal antibody rituximab (RTX) established a new era in the treatment of non-Hodgkin lymphoma (NHL); however, suboptimal response and/or resistance to RTX still limit its clinical merits. Although four effector mechanisms are validated to participate in CD20-based immunotherapy, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, caspase-dependent apoptosis, and lysosome-mediated programmed cell death (PCD), they could hardly be synchronously activated by any anti-CD20 mAb or mAb derivative until now. Herein, a novel mAb nanocomb (polyethylenimine polymer-RTX-tositumomab [PPRT nanocomb]) was firstly constructed through mass arming two different anti-CD20 mAbs (RTX and tositumomab) to one polymer by nanotechnology. Comparing with free mAbs, PPRT nanocomb possesses a comparable binding ability and reduced "off-rate" to surface CD20 of NHL cells. When treated by PPRT nanocomb, the caspase-dependent apoptosis was remarkably enhanced except for concurrently eliciting complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and lysosome-mediated PCD. Besides, "cross-cell link"-assisted homotypic adhesion by PPRT nanocomb further enhanced the susceptibility to PCD of lymphoma cells. Pharmacokinetic assays revealed that PPRT nanocomb experienced a relatively reduced clearance from peripheral blood compared with free antibodies. With the cooperation of all the abovementioned superiorities, PPRT nanocomb exhibits exceptionally excellent in vivo antitumor activities in both disseminated and localized human NHL xenotransplant models.

Construction and characterization of an anti-CD20 mAb nanocomb with exceptionally excellent lymphoma-suppressing activity

PubMed Central

Li, Hua-Fei; Wu, Cong; Chen, Ting; Zhang, Ge; Zhao, He; Ke, Chang-Hong; Xu, Zheng

2015-01-01

The CD20-directed monoclonal antibody rituximab (RTX) established a new era in the treatment of non-Hodgkin lymphoma (NHL); however, suboptimal response and/or resistance to RTX still limit its clinical merits. Although four effector mechanisms are validated to participate in CD20-based immunotherapy, including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, caspase-dependent apoptosis, and lysosome-mediated programmed cell death (PCD), they could hardly be synchronously activated by any anti-CD20 mAb or mAb derivative until now. Herein, a novel mAb nanocomb (polyethylenimine polymer–RTX–tositumomab [PPRT nanocomb]) was firstly constructed through mass arming two different anti-CD20 mAbs (RTX and tositumomab) to one polymer by nanotechnology. Comparing with free mAbs, PPRT nanocomb possesses a comparable binding ability and reduced “off-rate” to surface CD20 of NHL cells. When treated by PPRT nanocomb, the caspase-dependent apoptosis was remarkably enhanced except for concurrently eliciting complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and lysosome-mediated PCD. Besides, “cross-cell link”-assisted homotypic adhesion by PPRT nanocomb further enhanced the susceptibility to PCD of lymphoma cells. Pharmacokinetic assays revealed that PPRT nanocomb experienced a relatively reduced clearance from peripheral blood compared with free antibodies. With the cooperation of all the abovementioned superiorities, PPRT nanocomb exhibits exceptionally excellent in vivo antitumor activities in both disseminated and localized human NHL xenotransplant models. PMID:26257518

In-depth interviews with state public health practitioners on the United States National Physical Activity Plan

PubMed Central

2013-01-01

Background The United States National Physical Activity Plan (NPAP; 2010), the country’s first national plan for physical activity, provides strategies to increase population-level physical activity to complement the 2008 physical activity guidelines. This study examined state public health practitioner awareness, dissemination, use, challenges, and recommendations for the NPAP. Methods In 2011–2012, we interviewed 27 state practitioners from 25 states. Interviews were recorded and transcribed verbatim. Transcripts were coded using a standard protocol, verified and reconciled by an independent coder, and input into qualitative software to facilitate development of common themes. Results NPAP awareness was high among state practitioners; dissemination to local constituents varied. Development of state-level strategies and goals was the most frequently reported use of the NPAP. Some respondents noted the usefulness of the NPAP for coalitions and local practitioners. Challenges to the plan included implementation cost, complexity, and consistency with other policies. The most frequent recommendation made was to directly link examples of implementation activities to the plan. Conclusions These results provide early evidence of NPAP dissemination and use, along with challenges encountered and suggestions for future iterations. Public health is one of eight sectors in the NPAP. Further efforts are needed to understand uptake and use by other sectors, as well as to monitor long-term relevance, progress, and collaboration across sectors. PMID:23731829

Decreased complement mediated binding of antibody//sup 3/-dsDNA immune complexes to the red blood cells of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies

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

Taylor, R.P.; Horgan, C.; Buschbacher, R.

1983-06-01

The complement mediated binding of prepared antibody//sup 3/H-dsDNA immune complexes to the red blood cells obtained from a number of patient populations has been investigated. Patients with solid tumors have binding activity similar to that seen in a normal group of individuals. However, a significant fraction of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies have lowered binding activity compared with normal subjects. Quantitative studies indicate the lowered activity probably arises due to a decrease in complement receptors on the respective red blood cells. The potential importance and implications of these findings are briefly discussed.

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

PubMed Central

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

2016-01-01

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

Delineating the Importance of Serum Opsonins and the Bacterial Capsule in Affecting the Uptake and Killing of Burkholderia pseudomallei by Murine Neutrophils and Macrophages

PubMed Central

Mulye, Minal; Bechill, Michael P.; Grose, William; Ferreira, Viviana P.; Lafontaine, Eric R.; Wooten, R. Mark

2014-01-01

Infection of susceptible hosts by the encapsulated Gram-negative bacterium Burkholderia pseudomallei (Bp) causes melioidosis, with septic patients attaining mortality rates ≥40%. Due to its high infectivity through inhalation and limited effective therapies, Bp is considered a potential bioweapon. Thus, there is great interest in identifying immune effectors that effectively kill Bp. Our goal is to compare the relative abilities of murine macrophages and neutrophils to clear Bp, as well as determine the importance of serum opsonins and bacterial capsule. Our findings indicate that murine macrophages and neutrophils are inherently unable to clear either unopsonized Bp or the relatively-avirulent acapsular bacterium B. thailandensis (Bt). Opsonization of Bp and Bt with complement or pathogen-specific antibodies increases macrophage-uptake, but does not promote clearance, although antibody-binding enhances complement deposition. In contrast, complement opsonization of Bp and Bt causes enhanced uptake and killing by neutrophils, which is linked with rapid ROS induction against bacteria exhibiting a threshold level of complement deposition. Addition of bacteria-specific antibodies enhances complement deposition, but antibody-binding alone cannot elicit neutrophil clearance. Bp capsule provides some resistance to complement deposition, but is not anti-phagocytic or protective against reactive oxygen species (ROS)-killing. Macrophages were observed to efficiently clear Bp only after pre-activation with IFNγ, which is independent of serum- and/or antibody-opsonization. These studies indicate that antibody-enhanced complement activation is sufficient for neutrophil-clearance of Bp, whereas macrophages are ineffective at clearing serum-opsonized Bp unless pre-activated with IFNγ. This suggests that effective immune therapies would need to elicit both antibodies and Th1-adaptive responses for successful prevention/eradication of melioidosis. PMID:25144195

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

Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP▿

PubMed Central

Barbosa, Angela S.; Monaris, Denize; Silva, Ludmila B.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Cianciarullo, Aurora M.; Isaac, Lourdes; Abreu, Patricia A. E.

2010-01-01

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis. PMID:20404075

Susceptibility of pathogenic and nonpathogenic Naegleria ssp

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

Whiteman, L.Y.

1988-01-01

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

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

PubMed

Hoh Kam, Jaimie; Lenassi, Eva; Malik, Talat H; Pickering, Matthew C; Jeffery, Glen

2013-08-01

Complement component C3 is the central complement component and a key inflammatory protein activated in age-related macular degeneration (AMD). AMD is associated with genetic variation in complement proteins that results in enhanced activation of C3 through the complement alternative pathway. These include complement factor H (CFH), a negative regulator of C3 activation. Both C3 inhibition and/or CFH augmentation are potential therapeutic strategies in AMD. Herein, we examined retinal integrity in aged (12 months) mice deficient in both factors H and C3 (CFH(-/-).C3(-/-)), CFH alone (CFH(-/-)), or C3 alone (C3(-/-)), and wild-type mice (C57BL/6). Retinal function was assessed by electroretinography, and retinal morphological features were analyzed at light and electron microscope levels. Retinas were also stained for amyloid β (Aβ) deposition, inflammation, and macrophage accumulation. Contrary to expectation, electroretinograms of CFH(-/-).C3(-/-) mice displayed more severely reduced responses than those of other mice. All mutant strains showed significant photoreceptor loss and thickening of Bruch's membrane compared with wild-type C57BL/6, but these changes were greater in CFH(-/-).C3(-/-) mice. CFH(-/-).C3(-/-) mice had significantly more Aβ on Bruch's membrane, fewer macrophages, and high levels of retinal inflammation than the other groups. Our data show that both uncontrolled C3 activation (CFH(-/-)) and complete absence of C3 (CFH(-/-).C3(-/-) and C3(-/-)) negatively affect aged retinas. These findings suggest that strategies that inhibit C3 in AMD may be deleterious. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Complement inhibition by hydroxychloroquine prevents placental and fetal brain abnormalities in antiphospholipid syndrome.

PubMed

Bertolaccini, Maria Laura; Contento, Gregorio; Lennen, Ross; Sanna, Giovanni; Blower, Philip J; Ma, Michelle T; Sunassee, Kavitha; Girardi, Guillermina

2016-12-01

Placental ischemic disease and adverse pregnancy outcomes are frequently observed in patients with antiphospholipid syndrome (APS). Despite the administration of conventional antithrombotic treatment a significant number of women continue to experience adverse pregnancy outcomes, with uncertain prevention and management. Efforts to develop effective pharmacological strategies for refractory obstetric APS cases will be of significant clinical benefit for both mothers and fetuses. Although the antimalarial drug, hydroxychloroquine (HCQ) is increasingly used to treat pregnant women with APS, little is known about its efficacy and mechanism of action of HCQ. Because complement activation plays a crucial and causative role in placental ischemia and abnormal fetal brain development in APS we hypothesised that HCQ prevents these pregnancy complications through inhibition of complement activation. Using a mouse model of obstetric APS that closely resembles the clinical condition, we found that HCQ prevented fetal death and the placental metabolic changes -measured by proton magnetic resonance spectroscopy in APS-mice. Using 111 In labelled antiphospholipid antibodies (aPL) we identified the placenta and the fetal brain as the main organ targets in APS-mice. Using this same method, we found that HCQ does not inhibit aPL binding to tissues as was previously suggested from in vitro studies. While HCQ did not affect aPL binding to fetal brain it prevented fetal brain abnormal cortical development. HCQ prevented complement activation in vivo and in vitro. Complement C5a levels in serum samples from APS patients and APS-mice were lower after treatment with HCQ while the antibodies titres remained unchanged. HCQ prevented not only placental insufficiency but also abnormal fetal brain development in APS. By inhibiting complement activation, HCQ might also be an effective antithrombotic therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

PubMed

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

2017-03-01

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

Identification of hot spots in the variola virus complement inhibitor (SPICE) for human complement regulation.

PubMed

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

2008-04-01

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is approximately 90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

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

PubMed Central

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

2008-01-01

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is ∼90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement. PMID:18216095

Development of an Oral Barley Beta-Glucan Adjuvant That Augments the Tumoricidal Activity of Antibodies or Vaccines Used for the Immunotherapy of Breast Cancer

DTIC Science & Technology

2003-07-01

These Data provided strong evidence for the efficacy of an oral beta - glucan adjuvant for use in combination with anti-tumor antibodies such as...oral beta - glucan . The data showing that antibodies must activate complement and deposit iC3b on tumors means that antibodies that do not to activate complement would not benefit from oral beta - glucan .

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

PubMed Central

Becker, Elmer L.

1972-01-01

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

Plasma complement and vascular complement deposition in patients with coronary artery disease with and without inflammatory rheumatic diseases

PubMed Central

2017-01-01

Purpose Inflammatory rheumatic diseases (IRD) are associated with accelerated coronary artery disease (CAD), which may result from both systemic and vascular wall inflammation. There are indications that complement may be involved in the pathogenesis of CAD in Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA). This study aimed to evaluate the associations between circulating complement and complement activation products with mononuclear cell infiltrates (MCI, surrogate marker of vascular inflammation) in the aortic media and adventitia in IRDCAD and non-IRDCAD patients undergoing coronary artery bypass grafting (CABG). Furthermore, we compared complement activation product deposition patterns in rare aorta adventitial and medial biopsies from SLE, RA and non-IRD patients. Methods We examined plasma C3 (p-C3) and terminal complement complexes (p-TCC) in 28 IRDCAD (SLE = 3; RA = 25), 52 non-IRDCAD patients, and 32 IRDNo CAD (RA = 32) from the Feiring Heart Biopsy Study. Aortic biopsies taken from the CAD only patients during CABG were previously evaluated for adventitial MCIs. The rare aortic biopsies from 3 SLE, 3 RA and 3 non-IRDCAD were assessed for the presence of C3 and C3d using immunohistochemistry. Results IRDCAD patients had higher p-TCC than non-IRDCAD or IRDNo CAD patients (p<0.0001), but a similar p-C3 level (p = 0.42). Circulating C3 was associated with IRD duration (ρ, p-value: 0.46, 0.03). In multiple logistic regression analysis, IRD remained significantly related to the presence and size of MCI (p<0.05). C3 was present in all tissue samples. C3d was detected in the media of all patients and only in the adventitia of IRD patients (diffuse in all SLE and focal in one RA). Conclusion The independent association of IRD status with MCI and the observed C3d deposition supports the unique relationship between rheumatic disease, and, in particular, SLE with the complement system. Exaggerated systemic and vascular complement activation may accelerate CVD, serve as a CVD biomarker, and represent a target for new therapies. PMID:28362874

Dialysis in rats with acute renal failure: evaluation of three different dialyzer membranes.

PubMed

Kränzlin, B; Gretz, N; Kirschfink, M; Mujais, S K

1996-11-01

Exposure to complement-activating cellulosic dialysis membranes has been claimed to adversely affect the course of acute renal failure (ARF). To test this hypothesis, male Sprague-Dawley rats were allocated to 2 groups: in Group 1, ARF was induced by bilateral renal artery clamping whereas in Group 2, animals underwent a sham procedure. In each group, rats were further allocated to undergo hemodialysis with either a Cuprophan, a Hemophan, or a polyacrylonitrile minidialyzer on Days 4 and 8 after surgery, or no dialysis. Renal function was measured by inulin clearance on the days after dialysis. Additionally, total complement activity (CH50) was estimated on Days 1, 2, 4, and 8, and complement factor C3 was detected immunohistochemically. The degree of renal failure and the rate of recovery of renal function were similar in all the ARF groups irrespective of whether they had undergone dialysis or not, or of the type of the dialysis membrane. Furthermore, there were no significant differences in the course of CH50 or in the amount and distribution of complement factor C3 in the kidney tissue between the rats of Groups 1 and 2. Our findings refute the hypothesis that in ischemic ARF exposure to complement-activating cellulosic dialysis membranes impairs the recovery of renal function in rats.

Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

PubMed Central

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M.

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases. PMID:22514678

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

PubMed

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Complement activation and liver impairment in trichloroethylene-sensitized BALB/c mice.

PubMed

Zhang, Jiaxiang; Zha, Wansheng; Wang, Feng; Jiang, Tao; Xu, Shuhai; Yu, Junfeng; Zhou, Chengfan; Shen, Tong; Wu, Changhao; Zhu, Qixing

2013-01-01

Our recent studies have shown that trichloroethylene (TCE) was able to induce multisystem injuries in the form of occupational medicamentosa-like dermatitis, including skin, kidney, and liver damages. However, the role of complement activation in the immune-mediated liver injury is not known. This study examined the role of complement activation in the liver injury in a mouse model of TCE-induced sensitization. Treatment of female BALB/c mice with TCE under specific dosing protocols resulted in skin inflammation and sensitization. Skin edema and erythema occurred in TCE-sensitized groups. Trichloroethylene sensitization produced liver histopathological lesions, increased serum alanine aminotransferase, aspartate transaminase activities, and the relative liver weight. The concentrations of serum complement components C3a-desArg, C5a-desArg, and C5b-9 were significantly increased in 24-hour, 48-hour, and 72-hour sensitization-positive groups treated with TCE and peaked in the 72-hour sensitization-positive group. Depositions of C3a, C5a, and C5b-9 into the liver tissue were also revealed by immunohistochemistry. Immunofluorescence further verified high C5b-9 expression in 24-hour, 48-hour, and 72-hour sensitization-positive groups in response to TCE treatment. Reverse transcription-polymerase chain reaction detected C3 messenger RNA expression in the liver, and this was significantly increased in 24-hour and 48-hour sensitization-positive groups with a transient reduction at 72 hours. These results provide the first experimental evidence that complement activation may play a key role in the generation and progression of immune-mediated hepatic injury by exposure to TCE.

Molecular characterization of the rhesus rhadinovirus (RRV) ORF4 gene and the RRV complement control protein it encodes.

PubMed

Mark, Linda; Spiller, O Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A; Wong, Scott W; Damania, Blossom; Blom, Anna M; Blackbourn, David J

2007-04-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes▿

PubMed Central

Mark, Linda; Spiller, O. Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A.; Wong, Scott W.; Damania, Blossom; Blom, Anna M.; Blackbourn, David J.

2007-01-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model. PMID:17287274

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

PubMed

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

2016-12-01

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

Cytotoxic activity against human neuroblastoma and melanoma cells mediated by IgM antibodies derived from peripheral blood of healthy donors.

PubMed

Devarapu, Satish Kumar; Mamidi, Srinivas; Plöger, Frank; Dill, Othmar; Blixt, Ola; Kirschfink, Michael; Schwartz-Albiez, Reinhard

2016-06-15

A small percentage of healthy donors identified in the Western population carry antibodies in their peripheral blood which convey cytotoxic activity against certain human melanoma and neuroblastoma cell lines. We measured the cytotoxic activity of sera and plasmas from healthy donors on the human neuroblastoma cell line Kelly and various melanoma cell lines. Antibodies of IgM isotype, presumably belonging to the class of naturally occurring antibodies, exerted cytotoxic activity in a complement-dependent fashion. Apart from complement-dependent tumor cell lysis, we observed C3 opsonization in all tumor cell lines upon treatment with cytotoxic plasmas. Cell lines tested primarily expressed membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59 to various extents. Blocking of mCRPs by monoclonal antibodies enhanced cell lysis and opsonization, though some melanoma cells remained resistant to complement attack. Epitopes recognized by cytotoxic antibodies were represented by gangliosides such as GD2 and GD3, as evidenced by cellular sialidase pretreatment and enhanced expression of distinct gangliosides. It remains to be clarified why only a small fraction of healthy persons carry these antitumor cytotoxic antibodies. © 2016 UICC.

Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

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

Sjoewall, Christopher; Wetteroe, Jonas; Bengtsson, Torbjoern

2007-01-05

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fc{gamma} receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficientlymore » down-regulated at CRP levels >150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.« less

Molecular and functional characterization of CD59 from Nile tilapia (Oreochromis niloticus) involved in the immune response to Streptococcus agalactiae.

PubMed

Gan, Zhen; Wang, Bei; Zhou, Wei; Lu, Yishan; Zhu, Weiwei; Tang, Jufen; Jian, JiChang; Wu, Zaohe

2015-05-01

CD59, the major inhibitor of membrane attack complex, plays a crucial role in regulation of complement activation. In this paper, a CD59 gene of Nile tilapia, Oreochromis niloticus (designated as On-CD59) was cloned and its expression pattern under the stimulation of Streptococcus agalactiae was investigated. Sequence analysis showed main structural features required for complement-inhibitory activity were detected in the deduced amino acid sequence of On-CD59. In healthy Nile tilapia, the On-CD59 transcripts could be detected in all the examined tissues, with the most abundant expression in the brain. When immunized with inactivated S. agalactiae, there was a clear time-dependent expression pattern of On-CD59 in the skin, brain, head kidney, thymus and spleen, with quite different kinetic expressions. The assays for the complement-inhibitory activity suggested that recombinant On-CD59 protein had a species-selective inhibition of complement. Moreover, our works showed that recombinant On-CD59 protein may possess both binding activities to PGN and LTA and inhibiting activity of S. agalactiae. These findings indicated that On-CD59 may play important roles in the immune response to S. agalactiae in Nile tilapia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Complementation studies in Niemann-Pick disease type C indicate the existence of a second group.

PubMed Central

Steinberg, S J; Ward, C P; Fensom, A H

1994-01-01

Niemann-Pick disease type C is a clinically heterogeneous storage disorder with an unknown primary metabolic defect. We have undertaken somatic cell hybridisation experiments using skin fibroblast strains from 12 patients representing a wide clinical spectrum. Preliminary experiments using filipin staining of free cholesterol as a marker for complementation indicated the existence of one major group (group alpha) and one minor group (group beta) represented by one mutant strain. Subsequent experiments in which sphingomyelinase activity was measured as a marker for complementation using five mutant strains showing activity consistently < 40% control levels confirmed the existence of the second group. Images PMID:8071958

[Genetic study of bacteriophage phi81. I. Isolation, study of complementation and preliminary mapping of amber-mutants of bacteriophage phi81].

PubMed

Sineokiĭ, S P; Pogosov, V Z; Iankovskiĭ, N K; Krylov, V N

1976-01-01

123 Amber mutants of lambdoid bacteriophage phi81 are isolated and distributed into 19 complementation groups. Deletion mapping made possible to locate 5 gene groups on the genetic map of bacteriophage phi81 and to determine a region of possible location of mm' sticky ends on the prophage genetic map. A gene of phage phi81 is localized, which controls the adsorption specificity, and which functional similarity to a respective gene of phage phi80 is demonstrated.

Nonrenal and renal activity of systemic lupus erythematosus: a comparison of two anti-C1q and five anti-dsDNA assays and complement C3 and C4.

PubMed

Julkunen, Heikki; Ekblom-Kullberg, Susanne; Miettinen, Aaro

2012-08-01

Associations of different assays for antibodies to C1q (anti-C1q) and to dsDNA (anti-dsDNA) and of complements C3 and C4 with disease activity in patients with systemic lupus erythematosus (SLE) were studied. The clinical manifestations of 223 SLE patients were recorded, and the disease activity was assessed by the SLEDAI score. Anti-C1q were determined by two enzyme-linked immunosorbent assays (ELISA) and anti-dsDNA by a radioimmunoassay (RIA), a Crithidia immunofluorescence (IF) assay and three ELISA assays using human telomere DNA, plasmid DNA circles, or calf thymus DNA as antigens, respectively. Complement C3 and C4 were determined by nephelometry. Control sera were obtained from 98 blood donors. In patients with SLE, the prevalence of anti-C1q was 17-18% and that of anti-dsDNA was 36-69%. Anti-C1q, anti-dsDNA, and complement C3 and C4 correlated well with the overall activity of SLE (r = 0.323-0.351, 0.353-0.566, and -0.372-0.444, respectively; P < 0.001). Sensitivity, specificity, positive predictive value, and negative predictive value for active lupus nephritis among SLE patients were 40-44, 92, 29, and 91-92% for anti-C1q and 48-68, 29-66, 11-16, and 86-91% for anti-dsDNA, respectively. Patients with active nephritis had higher levels of anti-C1q and lower levels of C3 and C4 than patients with inactive nephritis (P = 0.003-0.018). The corresponding associations of anti-dsDNA were somewhat weaker (P = 0.023-0.198). Hematological parameters reflecting disease activity correlated clearly better with anti-dsDNA and complement C3 and C4 than with anti-C1q. Anti-C1q is inferior to anti-dsDNA as a diagnostic test in SLE and in the evaluation of overall clinical activity of the disease. Anti-C1q together with complement C3 and C4 may offer useful additional information to monitor lupus nephritis activity. There are no practical differences between different assays for anti-C1q and anti-dsDNA.

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

PubMed

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

2017-03-10

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

Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes

PubMed Central

Petersson, Frida; Kilsgård, Ola; Shannon, Oonagh

2018-01-01

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction. PMID:29385206

Platelet activation and aggregation by the opportunistic pathogen Cutibacterium (Propionibacterium) acnes.

PubMed

Petersson, Frida; Kilsgård, Ola; Shannon, Oonagh; Lood, Rolf

2018-01-01

Cutibacterium (Propionibacterium) acnes, considered a part of the skin microbiota, is one of the most commonly isolated anaerobic bacteria from medical implants in contact with plasma. However, the precise interaction of C. acnes with blood cells and plasma proteins has not been fully elucidated. Herein, we have investigated the molecular interaction of C. acnes with platelets and plasma proteins. We report that the ability of C. acnes to aggregate platelets is dependent on phylotype, with a significantly lower ability amongst type IB isolates, and the interaction of specific donor-dependent plasma proteins (or concentrations thereof) with C. acnes. Pretreatment of C. acnes with plasma reduces the lag time before aggregation demonstrating that pre-deposition of plasma proteins on C. acnes is an important step in platelet aggregation. Using mass spectrometry we identified several plasma proteins deposited on C. acnes, including IgG, fibrinogen and complement factors. Inhibition of IgG, fibrinogen or complement decreased C. acnes-mediated platelet aggregation, demonstrating the importance of these plasma proteins for aggregation. The interaction of C. acnes and platelets was visualized using fluorescence microscopy, verifying the presence of IgG and fibrinogen as components of the aggregates, and co-localization of C. acnes and platelets in the aggregates. Here, we have demonstrated the ability of C. acnes to activate and aggregate platelets in a bacterium and donor-specific fashion, as well as added mechanistic insights into this interaction.

Enduracidin Analogues with Altered Halogenation Patterns Produced by Genetically Engineered Strains of Streptomyces fungicidicus

PubMed Central

Yin, Xihou; Chen, Ying; Zhang, Ling; Wang, Yang; Zabriskie, T. Mark

2010-01-01

Enduracidins (1, 2) and ramoplanin (3) are structurally and functionally closely related lipodepsipeptide antibiotics. They are active against multidrug resistant Gram-positive pathogens, including MRSA. Each peptide contains one chlorinated non-proteinogenic amino acid residue, Cl2-Hpg or Cl-Hpg. To investigate the timing of halogenation, the importance of chlorination on bioactivity and bioavailability of enduracidin, and to probe the substrate specificity and portability of the ramoplanin halogenase, we constructed the mutant strain SfΔ30 in which the enduracidin halogenase gene orf30 had been deleted and complemented it with the ramoplanin counterpart orf20. We also expressed orf20 in the enduracidin wild type producer. Metabolite analysis revealed SfΔ30 produced the novel analogues dideschloroenduracidins A (4) and B (5), while the recombinant strains SfΔ30R20 and SfR20 produced monodeschloroenduracidins A (6) and B (7), and a trichlorinated enduracidin (8), respectively. In addition, orf30 self-complementation yielded the strain SfΔ30E30 which is capable of producing six peptides including 6 and 7. MS/MS analysis positioned the single chlorine atom in 6 at Hpg13 and localized the third chlorine atom in 8 to Hpg11. Biological evaluation of these enduracidin analogues indicated that all retained activity against Staphylococcus aureus. Our findings lay the foundation for further utilization of enduracidin and ramoplanin halogenases in combinatorial biosynthesis. PMID:20353165

The Group B Streptococcus–Secreted Protein CIP Interacts with C4, Preventing C3b Deposition via the Lectin and Classical Complement Pathways

PubMed Central

Pietrocola, Giampiero; Rindi, Simonetta; Rosini, Roberto; Buccato, Scilla

2016-01-01

The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for the development of anti-infective agents against this important pathogen. PMID:26608922

Eliminating Factor H-Binding Activity of Borrelia burgdorferi CspZ Combined with Virus-Like Particle Conjugation Enhances Its Efficacy as a Lyme Disease Vaccine.

PubMed

Marcinkiewicz, Ashley L; Lieknina, Ilva; Kotelovica, Svetlana; Yang, Xiuli; Kraiczy, Peter; Pal, Utpal; Lin, Yi-Pin; Tars, Kaspars

2018-01-01

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the most common tick-borne disease in the US and Europe. No potent human vaccine is currently available. The innate immune complement system is vital to host defense against pathogens, as complement activation on the surface of spirochetes results in bacterial killing. Complement system is inhibited by the complement regulator factor H (FH). To escape killing, B. burgdorferi produces an outer surface protein CspZ that binds FH to inhibit complement activation on the cell surface. Immunization with CspZ alone does not protect mice from infection, which we speculate is because FH-binding cloaks potentially protective epitopes. We modified CspZ by conjugating to virus-like particles (VLP-CspZ) and eliminating FH binding (modified VLP-CspZ) to increase immunogenicity. We observed greater bactericidal antibody titers in mice vaccinated with modified VLP-CspZ: A serum dilution of 1:395 (modified VLP-CspZ) vs 1:143 (VLP-CspZ) yielded 50% borreliacidal activity. Immunizing mice with modified VLP-CspZ cleared spirochete infection, as did passive transfer of elicited antibodies. This work developed a novel Lyme disease vaccine candidate by conjugating CspZ to VLP and eliminating FH-binding ability. Such a strategy of conjugating an antigen to a VLP and eliminating binding to the target ligand can serve as a general model for developing vaccines against other bacterial infectious agents.

Energy Deposition in the Body from External Sources to Chemically Trigger Cellular Responses in Desired Localized Regions

NASA Astrophysics Data System (ADS)

Ibsen, Stuart Duncan

One of the major challenges of modern chemotherapy is to deliver a therapeutic dose of active drug to the tumor tissue without causing systemic exposure. The realization of this goal could considerably reduce the negative side effects experienced by patients. The work conducted in this thesis looks at two different approaches to trigger drug activation with the use of external energy sources. This avoids the challenges of relying solely on biochemical and environmental differences as triggers. The two triggers used were low intensity focused ultrasound and 365 nm light delivered with a custom designed needle UV LED fiber optic system. Both can be localized within the body to spatially highlight just the tumor tissue creating a stark differentiation between it and the healthy tissue. The 365nm light based delivery scheme developed here was the first demonstration of a photoactivatable doxorubicin (DOX) prodrug called DOX-PCB. DOX-PCB was shown to be 200 times less toxic than DOX and could be activated to a fully therapeutic form upon exposure to 365nm light. The pharmacokinetics showed a circulation half life comparable to that of DOX and stability against in vivo metabolic degradation. The 365 nm light was shown to adequately irradiate a centimeter of tumor tissue and cause localized activation. In vivo tumors exposed to the light had significantly higher doses of DOX than unexposed control tumors in the same individual. The second delivery scheme made use of focused ultrasound to activate echogenic drug delivery vehicles. These vehicles were the first demonstration of encapsulating microbubbles within liposomes. Specially designed optical equipment documented that the microbubble was ultrasound responsive. The microbubble was shown to violently cavitate and rupture the outer liposome membrane releasing the payload contents. The three dimensional localization of activation was demonstrated in tissue phantoms. The strengths of these two delivery schemes could complement each other when used together. The delivery vehicle could achieve high doses of DOX-PCB within the tumor while the low toxicity prevents harm to the liver and spleen. The 365 nm light could then activate just the DOX-PCB found within the tumor itself causing localized cell death.

INFLAMMATORY MARKERS ASSOCIATED WITH TRAUMA AND INFECTION IN RED-TAILED HAWKS (BUTEO JAMAICENSIS) IN THE USA.

PubMed

Lee, Kelly A; Goetting, Valerie S; Tell, Lisa A

2015-10-01

Changes in inflammatory marker concentrations or activity can be used to monitor health and disease condition of domestic animals but have not been applied with the same frequency to wildlife. We measured concentrations or activity of six inflammatory markers (ceruloplasmin, haptoglobin, mannan-binding lectin-dependent complement [MBL/complement], unsaturated iron-binding capacity (UIBC) and total iron-binding capacity (TIBC), and plasma iron) in apparently healthy and sick or injured Red-tailed Hawks (Buteo jamaicensis). Haptoglobin and ceruloplasmin activities were consistently elevated in sick or injured hawks (2.1 and 2.5 times higher, respectively), and plasma iron concentrations decreased (0.46 times lower), relative to those of healthy birds. There were no differences between healthy and unhealthy hawks in TIBC and UIBC concentrations or MBL/complement activity. Therefore, haptoglobin, ceruloplasmin, and plasma iron would be useful inclusions in a panel of inflammatory markers for monitoring health in raptors.

The re-socialisation of migrants in a local community in Shanghai, China

NASA Astrophysics Data System (ADS)

Chang, Bo

2015-04-01

Following China's economic reforms in the early 1990s, the wave of internal North-to-South, West-to-East and rural-to-urban migration has still not subsided. The purpose of this study was to investigate how a local community in Shanghai supported migrants from other provinces in China in the process of their re-socialisation. By examining the component parts of re-socialisation (integration, assimilation and culturalisation), this paper analyses how the learning programmes and services provided in Shanghai's Zhabei District played a role in migrants' adaptation to their new community environment. The author conducted interviews with migrants of both rural and urban origin at two migrant clubs, and complemented her respondents' statements with formal and informal background research. Her findings indicate that participation in educational activity is only one aspect of migrants' re-socialisation. She demonstrates how educational activities merge into a larger community context and are mingled simultaneously with other activities which relate to employment, healthcare, setting up a business, etc. She argues that educational activity loses its backbone if the initial entry-level support given to migrants is not followed up with advanced development activities, such as providing migrants with lifelong learning opportunities tailored to their aptitudes and needs, motivating them to engage in learning which can serve as a pathway towards their career goals, and helping them improve their life circumstances.

Spread of activation and deactivation in the brain: does age matter?

PubMed Central

Gordon, Brian A.; Tse, Chun-Yu; Gratton, Gabriele; Fabiani, Monica

2014-01-01

Cross-sectional aging functional MRI results are sometimes difficult to interpret, as standard measures of activation and deactivation may confound variations in signal amplitude and spread, which however, may be differentially affected by age-related changes in various anatomical and physiological factors. To disentangle these two types of measures, here we propose a novel method to obtain independent estimates of the peak amplitude and spread of the BOLD signal in areas activated (task-positive) and deactivated (task-negative) by a Sternberg task, in 14 younger and 28 older adults. The peak measures indicated that, compared to younger adults, older adults had increased activation of the task-positive network, but similar levels of deactivation in the task-negative network. Measures of signal spread revealed that older adults had an increased spread of activation in task-positive areas, but a starkly reduced spread of deactivation in task-negative areas. These effects were consistent across regions within each network. Further, there was greater variability in the anatomical localization of peak points in older adults, leading to reduced cross-subject overlap. These results reveal factors that may confound the interpretation of studies of aging. Additionally, spread measures may be linked to local connectivity phenomena and could be particularly useful to analyze age-related deactivation patterns, complementing the results obtained with standard peak and region of interest analyses. PMID:25360115

Functional brain imaging predicts public health campaign success.

PubMed

Falk, Emily B; O'Donnell, Matthew Brook; Tompson, Steven; Gonzalez, Richard; Dal Cin, Sonya; Strecher, Victor; Cummings, Kenneth Michael; An, Lawrence

2016-02-01

Mass media can powerfully affect health decision-making. Pre-testing through focus groups or surveys is a standard, though inconsistent, predictor of effectiveness. Converging evidence demonstrates that activity within brain systems associated with self-related processing can predict individual behavior in response to health messages. Preliminary evidence also suggests that neural activity in small groups can forecast population-level campaign outcomes. Less is known about the psychological processes that link neural activity and population-level outcomes, or how these predictions are affected by message content. We exposed 50 smokers to antismoking messages and used their aggregated neural activity within a 'self-localizer' defined region of medial prefrontal cortex to predict the success of the same campaign messages at the population level (n = 400,000 emails). Results demonstrate that: (i) independently localized neural activity during health message exposure complements existing self-report data in predicting population-level campaign responses (model combined R(2) up to 0.65) and (ii) this relationship depends on message content-self-related neural processing predicts outcomes in response to strong negative arguments against smoking and not in response to compositionally similar neutral images. These data advance understanding of the psychological link between brain and large-scale behavior and may aid the construction of more effective media health campaigns. © The Author (2015). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-01-01

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

Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.

PubMed

Paulmurugan, R; Gambhir, S S

2003-04-01

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.

Monitoring Protein–Protein Interactions Using Split Synthetic Renilla Luciferase Protein-Fragment-Assisted Complementation

PubMed Central

Paulmurugan, R.; Gambhir, S. S.

2014-01-01

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein–protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor α through NFκB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein–protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network. PMID:12705589

A pore-forming protein implements VLR-activated complement cytotoxicity in lamprey.

PubMed

Wu, Fenfang; Feng, Bo; Ren, Yong; Wu, Di; Chen, Yue; Huang, Shengfeng; Chen, Shangwu; Xu, Anlong

2017-01-01

Lamprey is a basal vertebrate with a unique adaptive immune system, which uses variable lymphocyte receptors (VLRs) for antigen recognition. Our previous study has shown that lamprey possessed a distinctive complement pathway activated by VLR. In this study, we identified a natterin family member-lamprey pore-forming protein (LPFP) with a jacalin-like lectin domain and an aerolysin-like pore-forming domain. LPFP had a high affinity with mannan and could form oligomer in the presence of mannan. LPFP could deposit on the surface of target cells, form pore-like complex resembling a wheel with hub and spokes, and mediate powerful cytotoxicity on target cells. These pore-forming proteins along with VLRs and complement molecules were essential for the specific cytotoxicity against exogenous pathogens and tumor cells. This unique cytotoxicity implemented by LPFP might emerge before or in parallel with the IgG-based classical complement lytic pathway completed by polyC9.

Apolipoprotein J (clusterin) and Alzheimer's disease.

PubMed

Calero, M; Rostagno, A; Matsubara, E; Zlokovic, B; Frangione, B; Ghiso, J

2000-08-15

Apolipoprotein J (clusterin) is a ubiquitous multifunctional glycoprotein capable of interacting with a broad spectrum of molecules. In pathological conditions, it is an amyloid associated protein, co-localizing with fibrillar deposits in systemic and localized amyloid disorders. In Alzheimer's disease, the most frequent form of amyloidosis in humans and the major cause of dementia in the elderly, apoJ is present in amyloid plaques and cerebrovascular deposits but is rarely seen in NFT-containing neurons. ApoJ expression is up-regulated in a wide variety of insults and may represent a defense response against local damage to neurons. Four different mechanisms of action could be postulated to explain the role of apoJ as a neuroprotectant during cellular stress: (1) function as an anti-apoptotic signal, (2) protection against oxidative stress, (3) inhibition of the membrane attack complex of complement proteins locally activated as a result of inflammation, and (4) binding to hydrophobic regions of partially unfolded, stressed proteins, and therefore avoiding aggregation in a chaperone-like manner. This review focuses on the association of apoJ in biological fluids with Alzheimer's soluble Abeta. This interaction prevents Abeta aggregation and fibrillization and modulates its blood-brain barrier transport at the cerebrovascular endothelium. Copyright 2000 Wiley-Liss, Inc.

Knowledge-based design of a biosensor to quantify localized ERK activation in living cells

PubMed Central

Kummer, Lutz; Hsu, Chia-Wen; Dagliyan, Onur; MacNevin, Christopher; Kaufholz, Melanie; Zimmermann, Bastian; Dokholyan, Nikolay V.; Hahn, Klaus M.; Plückthun, Andreas

2014-01-01

Summary Investigation of protein activation in living cells is fundamental to understand how proteins are influenced by the full complement of upstream regulators they experience. We describe here the generation of a biosensor based on the Designed Ankyrin Repeat Protein (DARPin) binding scaffold suited for intracellular applications. Combining selection and evolution from libraries, knowledge-based design and efficient and rapid testing of conjugate candidates, we created an ERK activity biosensor by derivatizing a DARPin specific for phosphorylated ERK (pERK) with a solvatochromic merocyanine dye (mero87), whose fluorescence increases upon pERK binding. The biosensor specifically responded to pERK2, recognized by its conformation, but not to non-phosphorylated ERK2 or other closely related mitogen-activated kinases tested. Activated endogenous ERK was visualized in mouse embryo fibroblasts incubated in 2% serum, revealing greater activation in the nucleus, perinuclear regions, and especially the nucleoli. Activity was greatly reduced by the MEK1/2 inhibitor U0126. The DARPin-based biosensor will serve as useful tool for studying biological functions of ERK in vitro and in vivo. PMID:23790495

Targeting complement-mediated immunoregulation for cancer immunotherapy.

PubMed

Kolev, Martin; Markiewski, Maciej M

2018-06-01

Complement was initially discovered as an assembly of plasma proteins "complementing" the cytolytic activity of antibodies. However, our current knowledge places this complex system of several plasma proteins, receptors, and regulators in the center of innate immunity as a bridge between the initial innate responses and adaptive immune reactions. Consequently, complement appears to be pivotal for elimination of pathogens, not only as an early response defense, but by directing the subsequent adaptive immune response. The discovery of functional intracellular complement and its roles in cellular metabolism opened novel avenues for research and potential therapeutic implications. The recent studies demonstrating immunoregulatory functions of complement in the tumor microenvironment and the premetastatic niche shifted the paradigm on our understanding of functions of the complement system in regulating immunity. Several complement proteins, through their interaction with cells in the tumor microenvironment and in metastasis-targeted organs, contribute to modulating tumor growth, antitumor immunity, angiogenesis, and therefore, the overall progression of malignancy and, perhaps, responsiveness of cancer to different therapies. Here, we focus on recent progress in our understanding of immunostimulatory vs. immunoregulatory functions of complement and potential applications of these findings to the design of novel therapies for cancer patients. Copyright © 2018 Elsevier Ltd. All rights reserved.

Inactivation of complement by Loxosceles reclusa spider venom.

PubMed

Gebel, H M; Finke, J H; Elgert, K D; Cambell, B J; Barrett, J T

1979-07-01

Zymosan depletion of serum complement in guinea pigs rendered them highly resistant to lesion by Loxosceles reclusa spider venom. Guinea pigs deficient in C4 of the complement system are as sensitive to the venom as normal guinea pigs. The injection of 35 micrograms of whole recluse venom intradermally into guinea pigs lowered their complement level by 35.7%. Brown recluse spider venom in concentrations as slight as 0.02 micrograms protein/ml can totally inactivate one CH50 of guinea pig complement in vitro. Bee, scorpion, and other spider venoms had no influence on the hemolytic titer of complement. Fractionation of recluse spider venom by Sephadex G-200 filtration separated the complement-inactivating property of the venom into three major regions which could be distinguished on the basis of heat stability as well as size. None was neutralized by antivenom. Polyacrylamide gel electrophoresis of venom resolved the complement inactivators into five fractions. Complement inactivated by whole venom or the Sephadex fractions could be restored to hemolytic activity by supplements of fresh serum but not by heat-inactivated serum, pure C3, pure C5, or C3 and C5 in combination.

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

PubMed Central

Binks, Michael; Sriprakash, K. S.

2004-01-01

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

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

PubMed

Binks, Michael; Sriprakash, K S

2004-07-01

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

A bone substitute with high affinity for vitamin D-binding protein―relationship with niche of osteoclasts

PubMed Central

Ikeda, Tohru; Kasai, Michiyuki; Tatsukawa, Eri; Kamitakahara, Masanobu; Shibata, Yasuaki; Yokoi, Taishi; Nemoto, Takayuki K; Ioku, Koji

2014-01-01

The biological activity of osteoblasts and osteoclasts is regulated not only by hormones but also by local growth factors, which are expressed in neighbouring cells or included in bone matrix. Previously, we developed hydroxyapatite (HA) composed of rod-shaped particles using applied hydrothermal methods (HHA), and it revealed mild biodegradability and potent osteoclast homing activity. Here, we compared serum proteins adsorbed to HHA with those adsorbed to conventional HA composed of globular-shaped particles (CHA). The two ceramics adsorbed serum albumin and γ-globulin to similar extents, but affinity for γ-globulin was much greater than that to serum albumin. The chemotactic activity for macrophages of serum proteins adsorbed to HHA was significantly higher than that of serum proteins adsorbed to CHA. Quantitative proteomic analysis of adsorbed serum proteins revealed preferential binding of vitamin D-binding protein (DBP) and complements C3 and C4B with HHA. When implanted with the femur of 8-week-old rats, HHA contained significantly larger amount of DBP than CHA. The biological activity of DBP was analysed and it was found that the chemotactic activity for macrophages was weak. However, DBP-macrophage activating factor, which is generated by the digestion of sugar chains of DBP, stimulated osteoclastogenesis. These results confirm that the microstructure of hydroxyapatite largely affects the affinity for serum proteins, and suggest that DBP preferentially adsorbed to HA composed of rod-shaped particles influences its potent osteoclast homing activity and local bone metabolism. PMID:24286277

Activation of the lectin pathway of complement in experimental human keratitis with Pseudomonas aeruginosa.

PubMed

Osthoff, Michael; Brown, Karl D; Kong, David C M; Daniell, Mark; Eisen, Damon P

2014-01-01

Pseudomonas aeruginosa (P. aeruginosa) microbial keratitis (MK) is a sight-threatening disease. Previous animal studies have identified an important contribution of the complement system to the clearance of P. aeruginosa infection of the cornea. Mannose-binding lectin (MBL), a pattern recognition receptor of the lectin pathway of complement, has been implicated in the host defense against P. aeruginosa. However, studies addressing the role of the lectin pathway in P. aeruginosa MK are lacking. Hence, we sought to determine the activity of the lectin pathway in human MK caused by P. aeruginosa. Primary human corneal epithelial cells (HCECs) from cadaveric donors were exposed to two different P. aeruginosa strains. Gene expression of interleukin (IL)-6, IL-8, MBL, and other complement proteins was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MBL synthesis by enzyme-linked immunosorbent assay and intracellular flow cytometry. MBL gene expression was not detected in unchallenged HCECs. Exposure of HCECs to P. aeruginosa resulted in rapid induction of the transcriptional expression of MBL, IL-6, and IL-8. In addition, expression of several complement proteins of the classical and lectin pathways, but not the alternative pathway, were upregulated after 5 h of challenge, including MBL-associated serine protease 1. However, MBL protein secretion was not detectable 18 h after challenge with P. aeruginosa. MK due to P. aeruginosa triggers activation of MBL and the lectin pathway of complement. However, the physiologic relevance of this finding is unclear, as corresponding MBL oligomer production was not observed.

Activation of the lectin pathway of complement in experimental human keratitis with Pseudomonas aeruginosa

PubMed Central

Osthoff, Michael; Brown, Karl D.; Kong, David C.M.; Daniell, Mark

2014-01-01

Purpose Pseudomonas aeruginosa (P. aeruginosa) microbial keratitis (MK) is a sight-threatening disease. Previous animal studies have identified an important contribution of the complement system to the clearance of P. aeruginosa infection of the cornea. Mannose-binding lectin (MBL), a pattern recognition receptor of the lectin pathway of complement, has been implicated in the host defense against P. aeruginosa. However, studies addressing the role of the lectin pathway in P. aeruginosa MK are lacking. Hence, we sought to determine the activity of the lectin pathway in human MK caused by P. aeruginosa. Methods Primary human corneal epithelial cells (HCECs) from cadaveric donors were exposed to two different P. aeruginosa strains. Gene expression of interleukin (IL)-6, IL-8, MBL, and other complement proteins was determined by reverse transcription-polymerase chain reaction (RT–PCR) and MBL synthesis by enzyme-linked immunosorbent assay and intracellular flow cytometry. Results MBL gene expression was not detected in unchallenged HCECs. Exposure of HCECs to P. aeruginosa resulted in rapid induction of the transcriptional expression of MBL, IL-6, and IL-8. In addition, expression of several complement proteins of the classical and lectin pathways, but not the alternative pathway, were upregulated after 5 h of challenge, including MBL-associated serine protease 1. However, MBL protein secretion was not detectable 18 h after challenge with P. aeruginosa. Conclusions MK due to P. aeruginosa triggers activation of MBL and the lectin pathway of complement. However, the physiologic relevance of this finding is unclear, as corresponding MBL oligomer production was not observed. PMID:24426774

State of psychiatry in Italy 35 years after psychiatric reform. A critical appraisal of national and local data.

PubMed

Amaddeo, Francesco; Barbui, Corrado; Tansella, Michele

2012-08-01

Thirty-four years have elapsed since the passing of the Italian Law 180, the reform law that marked the transition from a hospital-based system of care to a model of community psychiatry that was designed to be an alternative to, rather than to complement, the old hospital-centred services. The main principle of Law 180 is that psychiatric patients have the right to be treated the same way as patients with other diseases and only voluntary treatments are allowed, with a few exceptions that are strictly regulated. The main features and consequences of the Italian reform are initially reviewed; national and local level experiences and epidemiological data are then analysed in order to highlight and disentangle the 'active ingredients' of the Italian experience. A public health attitude with the capacity to network good practice in service organization by giving voice to successful experiences and promoting health service research, apart from some local services, is still generally lacking. Furthermore, it is still difficult to provide an evidence-based reply to the question: can à l'Italienne community-care be exported elsewhere?

IMMUNOREACTIONS INVOLVING PLATELETS

PubMed Central

Shulman, N. Raphael

1958-01-01

Quantitative aspects of platelet agglutination and inhibition of clot retraction by the antibody of quinidine purpura were described. The reactions appeared to depend on formation of types of antibody-quinidine-platelet complexes which could fix complement but complement was not necessary for these reactions. Complement fixation was at least 10 times more sensitive than platelet agglutination or inhibition of clot retraction for measurement and detection of antibody activity. Although it has been considered that antibodies of drug purpura act as platelet lysins in the presence of complement and that direct lysis of platelets accounts for development of thrombocytopenia in drug purpura, the present study suggests that attachment of antibody produces a change in platelets which is manifested in vitro only by increased susceptibility to non-specific factors which can alter the stability of platelets in the absence of antibody. The attachment of antibody to platelets in vivo may only indirectly affect platelet survival. In contrast to human platelets, dog, rabbit, and guinea pig platelets, and normal or trypsin-treated human red cells did not agglutinate, fix complement, or adsorb antibody; and intact human endothelial cells did not fix complement or adsorb antibody. Rhesus monkey platelets were not agglutinated by the antibody but did adsorb antibody and fix complement although their activity in these reactions differed quantitatively from that of human platelets. Cinchonine could be substituted for quinidine in agglutination and inhibition of clot retraction reactions but quinine and cinchonidine could not. Attempts to cause passive anaphylaxis in guinea pigs with the antibody of quinidine purpura were not successful. PMID:13525580

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

PubMed Central

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

2014-01-01

Early-onset pre-eclampsia is characterized by decreased placental perfusion, new-onset hypertension, angiogenic imbalance, and endothelial dysfunction associated with excessive activation of the innate immune complement system. Although our previous studies demonstrated that inhibition of complement activation attenuates placental ischemia–induced hypertension using the rat reduced uterine perfusion pressure (RUPP) model, the important product(s) of complement activation has yet to be identified. We hypothesized that antagonism of receptors for complement activation products C3a and C5a would improve vascular function and attenuate RUPP hypertension. On gestational day (GD) 14, rats underwent sham surgery or vascular clip placement on ovarian arteries and abdominal aorta (RUPP). Rats were treated once daily with the C5a receptor antagonist (C5aRA), PMX51 (acetyl-F-[Orn-P-(D-Cha)-WR]), the C3a receptor antagonist (C3aRA), SB290157 (N2-[(2,2-diphenylethoxy)acetyl]-l-arginine), or vehicle from GD 14–18. Both the C3aRA and C5aRA attenuated placental ischemia–induced hypertension without affecting the decreased fetal weight or decreased concentration of free circulating vascular endothelial growth factor (VEGF) also present in this model. The C5aRA, but not the C3aRA, attenuated placental ischemia–induced increase in heart rate and impaired endothelial-dependent relaxation. The C3aRA abrogated the acute pressor response to C3a peptide injection, but it also unexpectedly attenuated the placental ischemia–induced increase in C3a, suggesting nonreceptor-mediated effects. Overall, these results indicate that both C3a and C5a are important products of complement activation that mediate the hypertension regardless of the reduction in free plasma VEGF. The mechanism by which C3a contributes to placental ischemia–induced hypertension appears to be distinct from that of C5a, and management of pregnancy-induced hypertension is likely to require a broad anti-inflammatory approach. PMID:25150279

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

PubMed Central

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

2011-01-01

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

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

PubMed

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

2016-04-01

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

The Structure-Activity Relationship between Marine Algae Polysaccharides and Anti-Complement Activity

PubMed Central

Jin, Weihua; Zhang, Wenjing; Liang, Hongze; Zhang, Quanbin

2015-01-01

In this study, 33 different polysaccharides were prepared to investigate the structure-activity relationships between the polysaccharides, mainly from marine algae, and anti-complement activity in the classical pathway. Factors considered included extraction methods, fractionations, molecular weight, molar ratio of galactose to fucose, sulfate, uronic acid (UA) content, linkage, branching, and the type of monosaccharide. It was shown that the larger the molecular weights, the better the activities. The molar ratio of galactose (Gal) to fucose (Fuc) was a positive factor at a concentration lower than 10 µg/mL, while it had no effect at a concentration more than 10 µg/mL. In addition, sulfate was necessary; however, the sulfate content, the sulfate pattern, linkage and branching had no effect at a concentration of more than 10 µg/mL. Moreover, the type of monosaccharide had no effect. Laminaran and UA fractions had no activity; however, they could reduce the activity by decreasing the effective concentration of the active composition when they were mixed with the active compositions. The effect of the extraction methods could not be determined. Finally, it was observed that sulfated galactofucan showed good anti-complement activity after separation. PMID:26712768

Evolutionary Analysis of Heterochromatin Protein Compatibility by Interspecies Complementation in Saccharomyces

PubMed Central

Zill, Oliver A.; Scannell, Devin R.; Kuei, Jeffrey; Sadhu, Meru; Rine, Jasper

2012-01-01

The genetic bases for species-specific traits are widely sought, but reliable experimental methods with which to identify functionally divergent genes are lacking. In the Saccharomyces genus, interspecies complementation tests can be used to evaluate functional conservation and divergence of biological pathways or networks. Silent information regulator (SIR) proteins in S. bayanus provide an ideal test case for this approach because they show remarkable divergence in sequence and paralog number from those found in the closely related S. cerevisiae. We identified genes required for silencing in S. bayanus using a genetic screen for silencing-defective mutants. Complementation tests in interspecies hybrids identified an evolutionarily conserved Sir-protein-based silencing machinery, as defined by two interspecies complementation groups (SIR2 and SIR3). However, recessive mutations in S. bayanus SIR4 isolated from this screen could not be complemented by S. cerevisiae SIR4, revealing species-specific functional divergence in the Sir4 protein despite conservation of the overall function of the Sir2/3/4 complex. A cladistic complementation series localized the occurrence of functional changes in SIR4 to the S. cerevisiae and S. paradoxus branches of the Saccharomyces phylogeny. Most of this functional divergence mapped to sequence changes in the Sir4 PAD. Finally, a hemizygosity modifier screen in the interspecies hybrids identified additional genes involved in S. bayanus silencing. Thus, interspecies complementation tests can be used to identify (1) mutations in genetically underexplored organisms, (2) loci that have functionally diverged between species, and (3) evolutionary events of functional consequence within a genus. PMID:22923378

Relative roles of general and complementation language in theory-of-mind development: evidence from Cantonese and English.

PubMed

Cheung, Him; Hsuan-Chih, Chen; Creed, Nikki; Ng, Lisa; Ping Wang, Sui; Mo, Lei

2004-01-01

Complex complements are clausal objects containing tensed verbs (e.g., that she cried) or infinitives (e.g., to cry), following main verbs of communication or mental activities (e.g., say, want). This research examined whether English- and Cantonese-speaking 4-year-olds' complement understanding uniquely predicts their representation of other minds (i.e., theory of mind). Results showed that neither meaning of main verbs (communication vs. desire) nor complement structure (tensed vs. infinitival) affected the correlation between complement understanding and theory of mind. More important, the correlation became insignificant after controlling for general language comprehension. These findings led to the conclusion that the syntax of complement per se does not contribute uniquely to theory-of-mind development; general language comprehension is a more important factor to consider. Copyright 2004 Society for Research in Child Development, Inc.

Complementation Studies of Bacteriophage λ O Amber Mutants by Allelic Forms of O Expressed from Plasmid, and O-P Interaction Phenotypes.

PubMed

Hayes, Sidney; Rajamanickam, Karthic; Hayes, Connie

2018-04-05

λ genes O and P are required for replication initiation from the bacteriophage λ origin site, ori λ, located within gene O . Questions have persisted for years about whether O-defects can indeed be complemented in trans . We show the effect of original null mutations in O and the influence of four origin mutations (three are in-frame deletions and one is a point mutation) on complementation. This is the first demonstration that O proteins with internal deletions can complement for O activity, and that expression of the N-terminal portion of gene P can completely prevent O complementation. We show that O-P co-expression can limit the lethal effect of P on cell growth. We explore the influence of the contiguous small RNA OOP on O complementation and P-lethality.

Evaluation of Non-Lethal Effects of N2 Bubbles on Marine Mammal Health and the Potential Role of Immune Activity in Facilitating the Development of Dive Related Injury

DTIC Science & Technology

2015-09-30

control for confounding effects of changing physiological status. Cortisol will be measured using a commercial enzyme immunoassay ( EIA ) and plasma...complement activity in plasma and serum has been tested using zymosan (Sigma Aldrich) which is a known activator of complement. Validation of the EIA ...expected to be underway by November 2015. Data collection for objective 3 will occur during project year 2. RESULTS EIA kits for seal

Local distinguishability of Dicke states in quantum secret sharing

NASA Astrophysics Data System (ADS)

Wang, Jing-Tao; Xu, Gang; Chen, Xiu-Bo; Sun, Xing-Ming; Jia, Heng-Yue

2017-03-01

We comprehensively investigate the local distinguishability of orthogonal Dicke states under local operations and classical communication (LOCC) from both qualitative and quantitative aspects. Based on our work, defects in the LOCC-quantum secret sharing (QSS) scheme can be complemented, and the information leakage can be quantified. For (k1 ,k2 , k , n)-threshold LOCC-QSS scheme, more intuitive formulas for unambiguous probability and guessing probability were established, which can be used for determining the parameter k1 and k2 directly.

STUDIES ON THE ANTIGENIC PROPERTIES OF COMPLEMENT

PubMed Central

Klein, Paul G.; Burkholder, Peter M.

1960-01-01

Sheep erythrocytes sensitized with amboceptor and persensitized thereafter with guinea pig complement are agglutinated by rabbit anti-guinea pig globulin and by immune sera obtained by injection of rabbits with fixed complement. In this agglutination neither C'1 nor C'2 takes part. Fixed C'4 acts as an agglutinogen. An additional agglutinogen, distinct from C'4, was found on persensitized cells. This additional agglutinogen appears to be distinct from hemolytically active C'3. PMID:14409703

[Complement deficiencies and meningococcal disease in The Netherlands].

PubMed

Swart, A G; Fijen, C A; te Bulte, M T; Daha, M R; Dankert, J; Kuijper, E J

1993-06-05

To determine the prevalence of complement system deficiencies in patients who have survived a Neisseria meningitidis infection. Retrospective. Reference laboratory for bacterial meningitis of the University of Amsterdam and the National Institute of Public Health and Environmental Protection. Out of the files of the laboratory 187 patients who had experienced a meningococcal infection in the Netherlands between 1959-1990 were selected in two groups according to the infecting bacterial strain: 97 patients with a serogroup X, Y, Z, W135, 29E, or non-groupable strains and 90 patients with an infection due to serogroup A or C. The patients were asked for their cooperation by their family doctor and one of us visited the patients at home to take blood samples. The complement activity was studied with a haemolysis in gel test and with an assay of haemolytic activity in free solution. Complement deficiency was present in 18% of the 187 patients who had experienced a meningococcal infection. The highest prevalence was found in patients older than 10 years who had developed infections due to serogroups X, Y, W135, or non-groupable strains (45%). Of the patients with a serogroup A or C infection, 3% had an complement deficiency. Of the complement deficiencies, 42% concerned a component of the alternative pathway, 12% a deficiency of C3, and 46% a component of the terminal route. The most commonly found deficiencies were properdin deficiency (39%) and C8 deficiency (18%). 30% of the complement deficient patients reported other family members having experienced meningitis. Recurrent meningitis was only observed in patients with terminal route deficiencies. We recommend that patients with a meningococcal infection due to serogroups X, Y, W135 or non-groupable strains should be screened for complement deficiency.

Malaria in rural Burkina Faso: local illness concepts, patterns of traditional treatment and influence on health-seeking behaviour.

PubMed

Beiersmann, Claudia; Sanou, Aboubakary; Wladarsch, Evelyn; De Allegri, Manuela; Kouyaté, Bocar; Müller, Olaf

2007-08-08

The literature on health care seeking behaviour in sub-Saharan Africa for children suffering from malaria is quite extensive. This literature, however, is predominantly quantitative and, inevitably, fails to explore how the local concepts of illness may affect people's choices. Understanding local concepts of illness and their influence on health care-seeking behaviour can complement existing knowledge and lead to the development of more effective malaria control interventions. In a rural area of Burkina Faso, four local concepts of illness resembling the biomedical picture of malaria were described according to symptoms, aetiology, and treatment. Data were collected through eight focus group discussions, 17 semi-structured interviews with key informants, and through the analysis of 100 verbal autopsy questionnaires of children under-five diagnosed with malaria. Sumaya, dusukun yelema, kono, and djoliban were identified as the four main local illness concepts resembling respectively uncomplicated malaria, respiratory distress syndrome, cerebral malaria, and severe anaemia. The local disease categorization was found to affect both treatment and provider choice. While sumaya is usually treated by a mix of traditional and modern methods, dusukun yelema and kono are preferably treated by traditional healers, and djoliban is preferably treated in modern health facilities. Besides the conceptualization of illness, poverty was found to be another important influencing factor of health care-seeking behaviour. The findings complement previous evidence on health care-seeking behaviour, by showing how local concepts of illness strongly influence treatment and choice of provider. Local concepts of illness need to be considered when developing specific malaria control programmes.

The Verticillium-specific protein VdSCP7 localizes to the plant nucleus and modulates immunity to fungal infections.

PubMed

Zhang, Lisha; Ni, Hao; Du, Xuan; Wang, Sheng; Ma, Xiao-Wei; Nürnberger, Thorsten; Guo, Hui-Shan; Hua, Chenlei

2017-07-01

Fungal pathogens secrete effector proteins to suppress plant basal defense for successful colonization. Resistant plants, however, can recognize effectors by cognate R proteins to induce effector-triggered immunity (ETI). By analyzing secretomes of the vascular fungal pathogen Verticillium dahliae, we identified a novel secreted protein VdSCP7 that targets the plant nucleus. The green fluorescent protein (GFP)-tagged VdSCP7 gene with either a mutated nuclear localization signal motif or with additional nuclear export signal was transiently expressed in Nicotiana benthamiana, and investigated for induction of plant immunity. The role of VdSCP7 in V. dahliae pathogenicity was characterized by gene knockout and complementation, and GFP labeling. Expression of the VdSCP7 gene in N. benthamiana activated both salicylic acid and jasmonate signaling, and altered the plant's susceptibility to the pathogens Botrytis cinerea and Phytophthora capsici. The immune response activated by VdSCP7 was highly dependent on its initial extracellular secretion and subsequent nuclear localization in plants. Knockout of the VdSCP7 gene significantly enhanced V. dahliae aggressiveness on cotton. GFP-labeled VdSCP7 is secreted by V. dahliae and accumulates in the plant nucleus. We conclude that VdSCP7 is a novel effector protein that targets the host nucleus to modulate plant immunity, and suggest that plants can recognize VdSCP7 to activate ETI during fungal infection. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Behavior-dependent activity patterns of GABAergic long-range projecting neurons in the rat hippocampus.

PubMed

Katona, Linda; Micklem, Ben; Borhegyi, Zsolt; Swiejkowski, Daniel A; Valenti, Ornella; Viney, Tim J; Kotzadimitriou, Dimitrios; Klausberger, Thomas; Somogyi, Peter

2017-04-01

Long-range glutamatergic and GABAergic projections participate in temporal coordination of neuronal activity in distributed cortical areas. In the hippocampus, GABAergic neurons project to the medial septum and retrohippocampal areas. Many GABAergic projection cells express somatostatin (SOM+) and, together with locally terminating SOM+ bistratified and O-LM cells, contribute to dendritic inhibition of pyramidal cells. We tested the hypothesis that diversity in SOM+ cells reflects temporal specialization during behavior using extracellular single cell recording and juxtacellular neurobiotin-labeling in freely moving rats. We have demonstrated that rare GABAergic projection neurons discharge rhythmically and are remarkably diverse. During sharp wave-ripples, most projection cells, including a novel SOM+ GABAergic back-projecting cell, increased their activity similar to bistratified cells, but unlike O-LM cells. During movement, most projection cells discharged along the descending slope of theta cycles, but some fired at the trough jointly with bistratified and O-LM cells. The specialization of hippocampal SOM+ projection neurons complements the action of local interneurons in differentially phasing inputs from the CA3 area to CA1 pyramidal cell dendrites during sleep and wakefulness. Our observations suggest that GABAergic projection cells mediate the behavior- and network state-dependent binding of neuronal assemblies amongst functionally-related brain regions by transmitting local rhythmic entrainment of neurons in CA1 to neuronal populations in other areas. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc.

A rapid microtiter plate serum bactericidal assay method for determining serum complement-mediated killing of Mannheimia haemolytica.

PubMed

Ayalew, Sahlu; Confer, Anthony W; Shrestha, Binu; Payton, Mark E

2012-05-01

In this study, we describe a rapid microtiter serum bactericidal assay (RMSBA) that can be used to measure the functionality of immune sera. It quantifies bactericidal activity of immune sera in the presence of complement against a homologous bacterium, M. haemolytica in this case. There is high correlation between data from RMSBA and standard complement-mediated bacterial killing assay (r=0.756; p<0.0001). The RMSBA activity of sera can be generated in less than 5 h instead of overnight incubation. RMSBA costs substantially less in terms of time, labor, and resources and is highly reproducible. Copyright © 2012 Elsevier B.V. All rights reserved.

The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

PubMed

Landeo-Ríos, Yazmín; Navas-Castillo, Jesús; Moriones, Enrique; Cañizares, M. Carmen

2017-11-24

To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus , family Closteroviridae ) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana . Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

Complement Factor B is the Downstream Effector of Toll-Like Receptors and Plays an Important Role in a Mouse Model of Severe Sepsis¶

PubMed Central

Zou, Lin; Feng, Yan; Li, Yan; Zhang, Ming; Chen, Chan; Cai, Jiayan; Gong, Yu; Wang, Larry; Thurman, Joshua M.; Wu, Xiaobo; Atkinson, John P.; Chao, Wei

2013-01-01

Severe sepsis involves massive activation of the innate immune system and leads to high mortality. Previous studies have demonstrated that various types of Toll-like receptors (TLRs) mediate a systemic inflammatory response and contribute to organ injury and mortality in animal models of severe sepsis. However, the downstream mechanisms responsible for TLR-mediated septic injury are poorly understood. Here, we show that activation of TLR2, TLR3 and TLR4 markedly enhanced complement factor B (cfB) synthesis and release by macrophages and cardiac cells. Polymicrobial sepsis, created by cecal ligation and puncture (CLP) in a mouse model, augmented cfB levels in the serum, peritoneal cavity and major organs including the kidney and heart. CLP also led to the alternative pathway (AP) activation, C3 fragment deposition in the kidney and heart, and cfB-dependent C3dg elevation. Bacteria isolated from septic mice activated the serum AP via a factor D-dependent manner. MyD88 deletion attenuated cfB/C3 up-regulation as well as cleavage induced by polymicrobial infection. Importantly, during sepsis, absence of cfB conferred a protective effect with improved survival and cardiac function, and markedly attenuated acute kidney injury. cfB deletion also led to increased neutrophil migratory function during the early phase of sepsis, decreased local and systemic bacterial load, attenuated cytokine production and reduced neutrophil reactive oxygen species production. Together, our data indicate that cfB acts as a downstream effector of TLR signaling and plays a critical role in the pathogenesis of severe bacterial sepsis. PMID:24154627

How the Pacific Islands Climate Education Partnership (PCEP) Has Collaboratively Increased Regional Collective Impacts on Climate Literacy Via Networks of Diverse Stakeholders Engaging in Multiple Reinforcing Activities

NASA Astrophysics Data System (ADS)

Sussman, A.

2016-12-01

The Pacific Islands Climate Education Partnership (PCEP) serves the U.S. Affiliated Pacific Island (USAPI) Region. The international entities served by PCEP are the state of Hawai`i (USA); three Freely Associated States (the Federated States of Micronesia, the Republic of the Marshall Islands, and the Republic of Palau), and three Territories (Guam, Commonwealth of Northern Mariana Islands, and American Samoa). Funded by NSF, the PCEP aims to educate the region's students and citizens in ways that exemplify modern science and indigenous environmental knowledge, address the urgency of climate change impacts, and focus on adaptation strategies that can increase resiliency with respect to climate change impacts. PCEP partners include universities, education nonprofits, state or country offices/ministries of education, local ecological nonprofits, and a variety of community organizations. Partners contribute and share expertise in climate science, local ecological knowledge, K-12 education in the Pacific island region, science and environmental education, community college education, learning science, indigenous navigation, and oceanography. Over the past six years, PCEP has engaged with regional school systems and communities in a wide variety of ways that complement and reinforce each other. Highlighted activities include improving country and state climate science education standards; focusing on place-based local ecological knowledge and skills in working with schools and communities; developing and disseminating formal education resources such as books and web resources that focus on local contexts and skills rather than contextually inappropriate mainland the textbooks; developing and implementing professional development for teachers; and supporting local ways of knowing by gathering and sharing local stories of climate change; and promoting an emphasis on climate adaptation strategies that increase resilience of natural environments and community systems.

Successful Reproduction Requires the Function of Arabidopsis YELLOW STRIPE-LIKE1 and YELLOW STRIPE-LIKE3 Metal-Nicotianamine Transporters in Both Vegetative and Reproductive Structures

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

Chu, H.; Chiecko, J; Punshon, T

2010-01-01

Several members of the Yellow Stripe-Like (YSL) family of proteins are transporters of metals that are bound to the metal chelator nicotianamine or the related set of mugineic acid family chelators known as phytosiderophores. Here, we examine the physiological functions of three closely related Arabidopsis (Arabidopsis thaliana) YSL family members, AtYSL1, AtYSL2, and AtYSL3, to elucidate their role(s) in the allocation of metals into various organs of Arabidopsis. We show that AtYSL3 and AtYSL1 are localized to the plasma membrane and function as iron transporters in yeast functional complementation assays. By using inflorescence grafting, we show that AtYSL1 and AtYSL3more » have dual roles in reproduction: their activity in the leaves is required for normal fertility and normal seed development, while activity in the inflorescences themselves is required for proper loading of metals into the seeds. We further demonstrate that the AtYSL1 and AtYSL2 proteins, when expressed from the AtYSL3 promoter, can only partially rescue the phenotypes of a ysl1ysl3 double mutant, suggesting that although these three YSL transporters are closely related and have similar patterns of expression, they have distinct activities in planta. In particular, neither AtYSL1 nor AtYSL2 is able to functionally complement the reproductive defects exhibited by ysl1ysl3 double mutant plants.« less

Mitochondrial and cytoplasmic isoleucyl-, glutamyl- and arginyl-tRNA synthetases of yeast are encoded by separate genes.

PubMed

Tzagoloff, A; Shtanko, A

1995-06-01

Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.

Transient Receptor Potential Channel 6 (TRPC6) Protects Podocytes during Complement-mediated Glomerular Disease*

PubMed Central

Kistler, Andreas D.; Singh, Geetika; Altintas, Mehmet M.; Yu, Hao; Fernandez, Isabel C.; Gu, Changkyu; Wilson, Cory; Srivastava, Sandeep Kumar; Dietrich, Alexander; Walz, Katherina; Kerjaschki, Dontscho; Ruiz, Phillip; Dryer, Stuart; Sever, Sanja; Dinda, Amit K.; Faul, Christian; Reiser, Jochen

2013-01-01

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis. PMID:24194522

Molecular Imaging of Phosphorylation Events for Drug Development

PubMed Central

Chan, C. T.; Paulmurugan, R.; Reeves, R. E.; Solow-Cordero, D.; Gambhir, S. S.

2014-01-01

Purpose Protein phosphorylation mediated by protein kinases controls numerous cellular processes. A genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system was developed for noninvasive monitoring phosphorylation events and efficacies of kinase inhibitors in cell culture and in small living subjects by optical bioluminescence imaging. Procedures An Akt sensor (AST) was constructed to monitor Akt phosphorylation and the effect of different PI-3K and Akt inhibitors. Specificity of AST was determined using a non-phosphorylable mutant sensor containing an alanine substitution (ASA). Results The PI-3K inhibitor LY294002 and Akt kinase inhibitor perifosine led to temporal- and dose-dependent increases in complemented FL activities in 293T human kidney cancer cells stably expressing AST (293T/AST) but not in 293T/ASA cells. Inhibition of endogenous Akt phosphorylation and kinase activities by perifosine also correlated with increase in complemented FL activities in 293T/AST cells but not in 293T/ASA cells. Treatment of nude mice bearing 293T/AST xenografts with perifosine led to a 2-fold increase in complemented FL activities compared to that of 293T/ASA xenografts. Our system was used to screen a small chemical library for novel modulators of Akt kinase activity. Conclusion This generalizable approach for noninvasive monitoring of phosphorylation events will accelerate the discovery and validation of novel kinase inhibitors and modulators of phosphorylation events. PMID:19048345

Platelet-activating factor mediates monocyte chemoattractant protein-1 expression in glomerular immune injury.

PubMed

Jocks, T; Freudenberg, J; Zahner, G; Stahl, R A

1998-01-01

These studies were designed to determine the possible role of platelet-activating factor (PAF) in the production of monocyte chemoattractant protein-1 (MCP-1) in glomerular immune injury. The glomerular lesion was induced in isolated perfused rat kidneys by a rabbit anti-rat-thymocyte serum (ATS) and rat serum (RS) as a complement source. Perfusion of kidneys with ATS and RS results in the selective binding of the antiserum to the glomerular mesangium with consecutive intraglomerular activation of complement. Antibody binding and complement activation induced a significant increase in glomerular MCP-1 mRNA levels when assessed by Northern blotting or RT-PCR. Decomplemented RS or non antibody rabbit IgG had only moderate effects on glomerular MCP-1 mRNA levels. The PAF receptor antagonist WEB 2170 almost completely blocked the ATS and RS induced MCP-1 mRNA levels. Perfusion of control kidneys with PAF increased MCP-1 mRNA expression, an effect which was blocked by WEB 2170. Glomerular MCP-1 protein formation, assessed by Western blotting, was stimulated following ATS and RS and PAF, respectively, was blocked by WEB 2170. These data show that PAF, derived from glomerular resident cells following antibody and complement induced injury, stimulates MCP-1 expression. In addition to the direct effects on leukocyte adhesion and activation PAF may mediate inflammatory cell influx in glomerular injuries due to the release of MCP-1.

Role of Ficolin-A and Lectin Complement Pathway in the Innate Defense against Pathogenic Aspergillus Species

PubMed Central

Bidula, Stefan; Kenawy, Hany; Ali, Youssif M.; Sexton, Darren; Schwaeble, Wilhelm J.

2013-01-01

Aspergillus species are saprophytic molds causing life-threatening invasive fungal infections in the immunocompromised host. Innate immune recognition, in particular, the mechanisms of opsonization and complement activation, has been reported to be an integral part of the defense against fungi. We have shown that the complement component ficolin-A significantly binds to Aspergillus conidia and hyphae in a concentration-dependent manner and was inhibited by N-acetylglucosamine and N-acetylgalactosamine. Calcium-independent binding to Aspergillus fumigatus and A. terreus was observed, but binding to A. flavus and A. niger was calcium dependent. Ficolin-A binding to conidia was increased under low-pH conditions, and opsonization led to enhanced binding of conidia to A549 airway epithelial cells. In investigations of the lectin pathway of complement activation, ficolin-A-opsonized conidia did not lead to lectin pathway-specific C4 deposition. In contrast, the collectin mannose binding lectin C (MBL-C) but not MBL-A led to efficient lectin pathway activation on A. fumigatus in the absence of ficolin-A. In addition, ficolin-A opsonization led to a modulation of the proinflammatory cytokine interleukin-8. We conclude that ficolin-A may play an important role in the innate defense against Aspergillus by opsonizing conidia, immobilizing this fungus through enhanced adherence to epithelial cells and modulation of inflammation. However, it appears that other immune pattern recognition molecules, i.e., those of the collectin MBL-C, are involved in the Aspergillus-lectin complement pathway activation rather than ficolin-A. PMID:23478320

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

PubMed

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

2018-03-01

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

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

PubMed Central

Li, Yujia; Parks, Griffith D.

2018-01-01

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

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

Progress and trends in complement therapeutics.

PubMed

Ricklin, Daniel; Lambris, John D

2013-01-01

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.

Progress and Trends in Complement Therapeutics.

PubMed

Ricklin, Daniel; Lambris, John D

2013-01-01

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.

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

Effect of anti-GM2 antibodies on rat sciatic nerve: electrophysiological and morphological study.

PubMed

Ortiz, Nicolau; Sabaté, M Mar; Garcia, Neus; Santafe, Manel M; Lanuza, M Angel; Tomàs, Marta; Tomàs, Josep

2009-03-31

We found that a monoclonal human IgM anti-GM2 was fixed in rat sciatic axons and Schwann cells and was able to activate human complement. The passive transfer of IgM and complement in sciatic nerves can induce an acute alteration in nerve conduction. When the transfer of IgM plus complement was repeated for 10 days, the compound action motor potential amplitude was very low and the morphological study showed axons and myelin damage. Without human complement, IgM can only slightly disorganize the myelin by separating some layers, probably by interfering with the functional role of gangliosides in the myelin package.

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

PubMed

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

1995-02-24

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

Oral Microbiome Link to Neurodegeneration in Glaucoma

PubMed Central

Astafurov, Konstantin; Elhawy, Eman; Ren, Lizhen; Dong, Cecilia Q.; Igboin, Christina; Hyman, Leslie; Griffen, Ann; Mittag, Thomas; Danias, John

2014-01-01

Background Glaucoma is a progressive optic nerve degenerative disease that often leads to blindness. Local inflammatory responses are implicated in the pathology of glaucoma. Although inflammatory episodes outside the CNS, such as those due to acute systemic infections, have been linked to central neurodegeneration, they do not appear to be relevant to glaucoma. Based on clinical observations, we hypothesized that chronic subclinical peripheral inflammation contributes to neurodegeneration in glaucoma. Methods Mouthwash specimens from patients with glaucoma and control subjects were analyzed for the amount of bacteria. To determine a possible pathogenic mechanism, low-dose subcutaneous lipopolysaccharide (LPS) was administered in two separate animal models of glaucoma. Glaucomatous neurodegeneration was assessed in the retina and optic nerve two months later. Changes in gene expression of toll-like receptor 4 (TLR4) signaling pathway and complement as well as changes in microglial numbers and morphology were analyzed in the retina and optic nerve. The effect of pharmacologic blockade of TLR4 with naloxone was determined. Findings Patients with glaucoma had higher bacterial oral counts compared to control subjects (p<0.017). Low-dose LPS administration in glaucoma animal models resulted in enhancement of axonal degeneration and neuronal loss. Microglial activation in the optic nerve and retina as well as upregulation of TLR4 signaling and complement system were observed. Pharmacologic blockade of TLR4 partially ameliorated the enhanced damage. Conclusions The above findings suggest that the oral microbiome contributes to glaucoma pathophysiology. A plausible mechanism by which increased bacterial loads can lead to neurodegeneration is provided by experiments in animal models of the disease and involves activation of microglia in the retina and optic nerve, mediated through TLR4 signaling and complement upregulation. The finding that commensal bacteria may play a role in the development and/or progression of glaucomatous pathology may also be relevant to other chronic neurodegenerative disorders. PMID:25180891

Comparative immune responses of corals to stressors associated with offshore reef-based tourist platforms

PubMed Central

Lamb, Joleah B; van Oppen, Madeleine J H; Willis, Bette L; Bourne, David G

2015-01-01

Abstract Unravelling the contributions of local anthropogenic and seasonal environmental factors in suppressing the coral immune system is important for prioritizing management actions at reefs exposed to high levels of human activities. Here, we monitor health of the model coral Acropora millepora adjacent to a high-use and an unused reef-based tourist platform, plus a nearby control site without a platform, over 7 months spanning a typical austral summer. Comparisons of temporal patterns in a range of biochemical and genetic immune parameters (Toll-like receptor signalling pathway, lectin–complement system, prophenoloxidase-activating system and green fluorescent protein-like proteins) among healthy, injured and diseased corals revealed that corals exhibit a diverse array of immune responses to environmental and anthropogenic stressors. In healthy corals at the control site, expression of genes involved in the Toll-like receptor signalling pathway (MAPK p38, MEKK1, cFos and ATF4/5) and complement system (C3 and Bf) was modulated by seasonal environmental factors in summer months. Corals at reef platform sites experienced additional stressors over the summer, as evidenced by increased expression of various immune genes, including MAPK p38 and MEKK1. Despite increased expression of immune genes, signs of white syndromes were detected in 31% of study corals near tourist platforms in the warmest summer month. Evidence that colonies developing disease showed reduced expression of genes involved in the complement pathway prior to disease onset suggests that their immune systems may have been compromised. Responses to disease and physical damage primarily involved the melanization cascade and GFP-like proteins, and appeared to be sufficient for recovery when summer heat stress subsided. Overall, seasonal and anthropogenic factors may have interacted synergistically to overwhelm the immune systems of corals near reef platforms, leading to increased disease prevalence in summer at these sites. PMID:27293717

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum

PubMed Central

Castiblanco-Valencia, Mónica M.; Fraga, Tatiana R.; Breda, Leandro C.D.; Vasconcellos, Sílvio A.; Figueira, Cláudio P.; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I.; Barbosa, Angela S.; Isaac, Lourdes

2017-01-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. PMID:26976804

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

The XX sex chromosome complement in mice is associated with increased spontaneous lupus compared with XY.

PubMed

Sasidhar, Manda V; Itoh, Noriko; Gold, Stefan M; Lawson, Gregory W; Voskuhl, Rhonda R

2012-08-01

Many autoimmune diseases are characterised by a female predominance. This may be caused by sex hormones, sex chromosomes or both. This report uses a transgenic mouse model to investigate how sex chromosome complement, not confounded by differences in gonadal type, might contribute to lupus pathogenesis. Transgenic NZM2328 mice were created by deletion of the Sry gene from the Y chromosome, thereby separating genetic from gonadal sex. Survival, renal histopathology and markers of immune activation were compared in mice carrying the XX versus the XY(-) sex chromosome complement, with each genotype being ovary bearing. Mice with XX sex chromosome complement compared with XY(-) exhibited poorer survival rates and increased kidney pathology. Splenic T lymphocytes from XX mice demonstrated upregulated X-linked CD40 ligand expression and higher levels of activation markers ex vivo. Increased MMP, TGF and IL-13 production was found, while IL-2 was lower in XX mice. An accumulation of splenic follicular B cells and peritoneal marginal zone B cells was observed, coupled with upregulated costimulatory marker expression on B cells in XX mice. These data show that the XX sex chromosome complement, compared with XY(-), is associated with accelerated spontaneous lupus.

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

PubMed Central

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

2016-01-01

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

Pathogenetic mechanisms in dengue haemorrhagic fever: Report of an international collaborative study*

PubMed Central

1973-01-01

In a study of 55 persons with dengue haemorrhagic fever—36 of whom showed the dengue shock syndrome—clinical, haematological, virological, and serological changes were correlated with serial measurements of complement components and immunopathological studies. Viruses dengue-1 or dengue-2 were isolated from the sera of 9 patients. Serological responses indicative of secondary dengue virus infections were observed in 53 patients; 2 (infants) had primary infections. During the acute phase of the disease, dengue antibody titres rose logarithmically. Marked depression of complement components, especially C3, was observed. Activation of both the classical and alternative complement pathways was demonstrated, with depression of both C4 and C3 proactivator levels in most instances, although in some cases it appeared that one mechanism was involved to a greater extent than the other. The level of depression of C3 was correlated with the severity of the disease. Relatively stable transferrin levels indicated that depletion of complement proteins was not primarily due to extravasation. Fibrinogen levels were depressed and fibrinogen split products were found in the plasma. The accumulated data provide further evidence of the central role that activated complement components play in the pathogenesis of dengue haemorrhagic fever. PMID:4575523

The Group B Streptococcus-Secreted Protein CIP Interacts with C4, Preventing C3b Deposition via the Lectin and Classical Complement Pathways.

PubMed

Pietrocola, Giampiero; Rindi, Simonetta; Rosini, Roberto; Buccato, Scilla; Speziale, Pietro; Margarit, Immaculada

2016-01-01

The group B Streptococcus (GBS) is a leading cause of neonatal invasive disease. GBS bacteria are surrounded by a thick capsular polysaccharide that is a potent inhibitor of complement deposition via the alternative pathway. Several of its surface molecules can however activate the classical and lectin complement pathways, rendering this species still vulnerable to phagocytic killing. In this study we have identified a novel secreted protein named complement interfering protein (CIP) that downregulates complement activation via the classical and lectin pathways, but not the alternative pathway. The CIP protein showed high affinity toward C4b and inhibited its interaction with C2, presumably preventing the formation of the C4bC2a convertase. Addition of recombinant CIP to GBS cip-negative bacteria resulted in decreased deposition of C3b on their surface and in diminished phagocytic killing in a whole-blood assay. Our data reveal a novel strategy exploited by GBS to counteract innate immunity and could be valuable for the development of anti-infective agents against this important pathogen. Copyright © 2015 by The American Association of Immunologists, Inc.

Evidence for a repair enzyme complex involving ERCC1 and complementing activities of ERCC4, ERCC11 and xeroderma pigmentosum group F.

PubMed Central

van Vuuren, A J; Appeldoorn, E; Odijk, H; Yasui, A; Jaspers, N G; Bootsma, D; Hoeijmakers, J H

1993-01-01

Nucleotide excision repair (NER), one of the major cellular DNA repair systems, removes a wide range of lesions in a multi-enzyme reaction. In man, a NER defect due to a mutation in one of at least 11 distinct genes, can give rise to the inherited repair disorders xeroderma pigmentosum (XP), Cockayne's syndrome or PIBIDS, a photosensitive form of the brittle hair disease trichothiodystrophy. Laboratory-induced NER-deficient mutants of cultured rodent cells have been classified into 11 complementation groups (CGs). Some of these have been shown to correspond with human disorders. In cell-free extracts prepared from rodent CGs 1-5 and 11, but not in a mutant from CG6, we find an impaired repair of damage induced in plasmids by UV light and N-acetoxy-acetylaminofluorene. Complementation analysis in vitro of rodent CGs is accomplished by pairwise mixing of mutant extracts. The results show that mutants from groups 2, 3, 5 and XP-A can complement all other CGs tested. However, selective non-complementation in vitro was observed in mutual mixtures of groups 1, 4, 11 and XP-F, suggesting that the complementing activities involved somehow affect each other. Depletion of wild-type human extracts from ERCC1 protein using specific anti-ERCC1 antibodies concomitantly removed the correcting activities for groups 4, 11 and XP-F, but not those for the other CGs. Furthermore, we find that 33 kDa ERCC1 protein sediments as a high mol. wt species of approximately 120 kDa in a native glycerol gradient.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8253091

Relationship among antineutrophil cytoplasmic antibody, blood urea nitrogen and complement in patients with eosinophilic granulomatosis polyangiitis (Churg-Strauss syndrome).

PubMed

Kawakami, Tamihiro; Kimura, Satoko; Takeuchi, Sora; Soma, Yoshinao

2013-07-01

Eosinophilic granulomatosis with polyangiitis (EGPA), also known as Churg-Strauss syndrome, is an antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis characterized by a history of asthma, hypereosinophilia. The prevalence of ANCA in EGPA is less common than in other ANCA-associated vasculitis. Increasing evidence of complement activation in the pathogenesis of ANCA-associated vasculitis has been provided by studies in animal models. We examined EGPA patients with cutaneous manifestations as an initial sign and investigated the correlations among clinical, serological and histopathological findings. We focused on differences among ANCA, blood urea nitrogen and complement levels such as complement 3 (C3), C4 and total complement hemolytic activity (CH50). We retrospectively investigated the records of 22 patients (11 male and 11 female) with EGPA admitted to our hospital from 1997-2012. Ten of the 22 patients (46%) were positive for serum myeloperoxidase (MPO)-ANCA. In contrast, all the patients were negative for serum proteinase 3 ANCA. There was a significantly positive correlation between serum CH50 and C4 levels in patients with EGPA. Serum blood urea nitrogen (BUN) levels differed significantly between MPO-ANCA-positive and -negative patients. Serum CH50 levels were higher in MPO-ANCA-positive patients compared to negative patients. Serum BUN levels were higher in elevated CH50 patients compared to normal and low CH50-negative patients. We propose that positive findings for MPO-ANCA with CH50 high activity may be a risk factor for developing renal insufficiency. Assuming there are correlations between the presence of ANCA and complements, earlier diagnosis based on initial efficacious treatment for EGPA. © 2013 Japanese Dermatological Association.

Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies

PubMed Central

Meulenbroek, Elisabeth M.; de Haas, Masja; Brouwer, Conny; Folman, Claudia; Zeerleder, Sacha S.; Wouters, Diana

2015-01-01

In autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal hemolytic anemia. Depending on whether IgG or IgM antibodies are involved, response to therapy is different. Proper identification of the isotype of the anti-erythrocyte autoantibodies is, therefore, crucial. However, detection of IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of complement C3 on a patient’s erythrocytes could be used for indirect detection of anti-erythrocyte IgM. To this end, we fractionated patients’ sera by size exclusion chromatography and tested which fractions yielded complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an IgM anti-erythrocyte component that could activate complement, even if no such autoantibody had been detected with any other test. This also included all tested patients with only IgG and C3 on their erythrocytes, who would previously have been classified as having an IgG-only mediated autoimmune hemolytic anemia. Depleting patients’ sera of either IgG or IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in autoimmune hemolytic anemia is mostly IgM-mediated and the presence of covalent C3 on patients’ erythrocytes can be taken as a footprint of the presence of anti-erythrocyte IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy. PMID:26354757

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

PubMed

Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin ( Ts -CRT), a Ca 2+ -binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts -CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts -CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts -CRT (r Ts -CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte-macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts -CRT on the surface of newborn larvae (NBL) of T. spiralis with anti- Ts -CRT antibody increased the C1q-mediated adherence of monocyte-macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis -expressed Ts -CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy

PubMed Central

Zhao, Limei; Shao, Shuai; Chen, Yi; Sun, Ximeng; Sun, Ran; Huang, Jingjing; Zhan, Bin; Zhu, Xinping

2017-01-01

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin (Ts-CRT), a Ca2+-binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts-CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts-CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts-CRT (rTs-CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte–macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts-CRT on the surface of newborn larvae (NBL) of T. spiralis with anti-Ts-CRT antibody increased the C1q-mediated adherence of monocyte–macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis-expressed Ts-CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages. PMID:28620388

Marked central nervous system pathology in CD59 knockout rats following passive transfer of Neuromyelitis optica immunoglobulin G.

PubMed

Yao, Xiaoming; Verkman, Alan S

2017-02-17

Neuromyelitis optica spectrum disorders (herein called NMO) is an inflammatory demyelinating disease of the central nervous system in which pathogenesis involves complement-dependent cytotoxicity (CDC) produced by immunoglobulin G autoantibodies targeting aquaporin-4 (AQP4-IgG) on astrocytes. We reported evidence previously, using CD59 -/- mice, that the membrane-associated complement inhibitor CD59 modulates CDC in NMO (Zhang and Verkman, J. Autoimmun. 53:67-77, 2014). Motivated by the observation that rats, unlike mice, have human-like complement activity, here we generated CD59 -/- rats to investigate the role of CD59 in NMO and to create NMO pathology by passive transfer of AQP4-IgG under conditions in which minimal pathology is produced in normal rats. CD59 -/- rats generated by CRISPR/Cas9 technology showed no overt phenotype at baseline except for mild hemolysis. CDC assays in astrocyte cultures and cerebellar slices from CD59 -/- rats showed much greater sensitivity to AQP4-IgG and complement than those from CD59 +/+ rats. Intracerebral administration of AQP4-IgG in CD59 -/- rats produced marked NMO pathology, with astrocytopathy, inflammation, deposition of activated complement, and demyelination, whereas identically treated CD59 +/+ rats showed minimal pathology. A single, intracisternal injection of AQP4-IgG in CD59 -/- rats produced hindlimb paralysis by 3 days, with inflammation and deposition of activated complement in spinal cord, optic nerves and brain periventricular and surface matter, with most marked astrocyte injury in cervical spinal cord. These results implicate an important role of CD59 in modulating NMO pathology in rats and demonstrate amplification of AQP4-IgG-induced NMO disease with CD59 knockout.

Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase Complex

PubMed Central

Chen, Yan; Carrington-Lawrence, Stacy D.; Bai, Ping; Weller, Sandra K.

2005-01-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface. PMID:15994803

Mutations in the putative zinc-binding motif of UL52 demonstrate a complex interdependence between the UL5 and UL52 subunits of the human herpes simplex virus type 1 helicase/primase complex.

PubMed

Chen, Yan; Carrington-Lawrence, Stacy D; Bai, Ping; Weller, Sandra K

2005-07-01

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase-primase (UL5/8/52) complex. UL5 contains seven motifs found in helicase superfamily 1, and UL52 contains conserved motifs found in primases. The contributions of each subunit to the biochemical activities of the complex, however, remain unclear. We have previously demonstrated that a mutation in the putative zinc finger at UL52 C terminus abrogates not only primase but also ATPase, helicase, and DNA-binding activities of a UL5/UL52 subcomplex, indicating a complex interdependence between the two subunits. To test this hypothesis and to further investigate the role of the zinc finger in the enzymatic activities of the helicase-primase, a series of mutations were constructed in this motif. They differed in their ability to complement a UL52 null virus: totally defective, partial complementation, and potentiating. In this study, four of these mutants were studied biochemically after expression and purification from insect cells infected with recombinant baculoviruses. All mutants show greatly reduced primase activity. Complementation-defective mutants exhibited severe defects in ATPase, helicase, and DNA-binding activities. Partially complementing mutants displayed intermediate levels of these activities, except that one showed a wild-type level of helicase activity. These data suggest that the UL52 zinc finger motif plays an important role in the activities of the helicase-primase complex. The observation that mutations in UL52 affected helicase, ATPase, and DNA-binding activities indicates that UL52 binding to DNA via the zinc finger may be necessary for loading UL5. Alternatively, UL5 and UL52 may share a DNA-binding interface.

k-Nearest neighbour local linear prediction of scalp EEG activity during intermittent photic stimulation.

PubMed

Erla, Silvia; Faes, Luca; Tranquillini, Enzo; Orrico, Daniele; Nollo, Giandomenico

2011-05-01

The characterization of the EEG response to photic stimulation (PS) is an important issue with significant clinical relevance. This study aims to quantify and map the complexity of the EEG during PS, where complexity is measured as the degree of unpredictability resulting from local linear prediction. EEG activity was recorded with eyes closed (EC) and eyes open (EO) during resting and PS at 5, 10, and 15 Hz in a group of 30 healthy subjects and in a case-report of a patient suffering from cerebral ischemia. The mean squared prediction error (MSPE) resulting from k-nearest neighbour local linear prediction was calculated in each condition as an index of EEG unpredictability. The linear or nonlinear nature of the system underlying EEG activity was evaluated quantifying MSPE as a function of the neighbourhood size during local linear prediction, and by surrogate data analysis as well. Unpredictability maps were obtained for each subject interpolating MSPE values over a schematic head representation. Results on healthy subjects evidenced: (i) the prevalence of linear mechanisms in the generation of EEG dynamics, (ii) the lower predictability of EO EEG, (iii) the desynchronization of oscillatory mechanisms during PS leading to increased EEG complexity, (iv) the entrainment of alpha rhythm during EC obtained by 10 Hz PS, and (v) differences of EEG predictability among different scalp regions. Ischemic patient showed different MSPE values in healthy and damaged regions. The EEG predictability decreased moving from the early acute stage to a stage of partial recovery. These results suggest that nonlinear prediction can be a useful tool to characterize EEG dynamics during PS protocols, and may consequently constitute a complement of quantitative EEG analysis in clinical applications. Copyright © 2010 IPEM. Published by Elsevier Ltd. All rights reserved.

Mode of complement activation by acidic heteroglycans from the leaves of Artemisia princeps PAMP.

PubMed

Yamada, H; Nagai, T; Cyong, J C; Otsuka, Y

1991-08-01

The mode of action of the anti-complementary acidic heteroglycans, AAF-IIb-2 and IIb-3 which consisted of rhamnogalacturonan core and arabinogalactan moieties, purified from the leaves of Artemisia princeps PAMP (Japanese name = Gaiyo) were investigated. The anti-complementary activities of AAF-IIb-2 and IIb-3 were reduced partially in the absence of Ca2+ ions. A marked consumption of C4 was observed to have occurred when serum was incubated with both polysaccharides in the presence of Ca2+ ions. AAF-IIb-2 showed more potent C4 consumption than IIb-3. After the incubation of the serum with AAF-IIb-2 in the absence of Ca2+ ions, a cleavage of C3 in the serum was detected by immunoelectrophoresis. AAF-IIb-2 showed more significant consumption of the complement than IIb-3 when rabbit erythrocytes were used in the assay system in the absence of Ca2+ ions. These results indicate that AAF-IIb-2 activates the complement via both the alternative and classical pathways, whereas IIb-3 mainly activates the complement via the classical pathway. The absorption of serum with Protein A-Sepharose results in a decrease of the activity of AAF-IIb-2 and IIb-3. However, the decrease of the activity was restored by the replacement of the immunoglobulin G (IgG) fraction after its recovery from the Protein A-Sepharose. These results suggest that IgG dependent mechanisms are both involved in the anti-complementary activity of AAF-IIb-2 and IIb-3.

Coagulation and complement system in critically ill patients.

PubMed

Helling, H; Stephan, B; Pindur, G

2015-01-01

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

Along-Strike Differences of the Main Himalayan Thrust and Deformation within the Indian Crust: Insights from Seismicity and Seismic Velocities in Bhutan and its Foreland

NASA Astrophysics Data System (ADS)

Diehl, T.; Singer, J.; Hetényi, G.; Kissling, E. H.; Clinton, J. F.

2015-12-01

The seismicity of Bhutan is characterized by the apparent lack of great earthquakes and a significantly lower activity compared to most other parts of the Himalayan arc. To better understand the underlying mechanisms of this anomalously low activity and to relate it with possible along-strike differences in the structure of the orogenic belt, a temporary network with up to 38 broadband seismometers was installed in Bhutan between January 2013 and November 2014. In this work we present a catalog of local and regional earthquakes detected and located with the GANSSER network complemented by regional stations in India, Bangladesh, and China. State-of-the-art data analysis and earthquake location procedures were applied to derive a high-precision earthquake catalog of Bhutan and surrounding regions. Focal mechanisms from regional moment tensor inversions and first-motion polarities complement the earthquake catalog. In the vicinity of the Shumar-Kuru Chu Spur in East Bhutan, seismicity forms a moderately dipping structure at about 12 km depth, which we associate with the Main Himalayan Thrust (MHT). North of 27.6°N the dip of the structure steepens, which can be interpreted as a ramp along the MHT. In West Bhutan seismicity occurs at depths of 20 to 40 km and receiver function images indicate that seismicity occurs in the underthrusting Indian crust rather than on the MHT. The highest seismic activity is clustered along the Goalpara Lineament, a dextral NE-SW striking shear zone in southwest Bhutan, which appears to connect to the western edge of the Shillong Plateau in the foreland. Focal depths indicate that this shear zone is located at depths of 20-30 km and therefore in the underthrusting Indian crust. Preliminary results of a 3D local earthquake tomography show substantial differences in the uppermost crust between east and west Bhutan. Consistent with our receiver function images, the results also indicate a thinning of the crustal root towards eastern Bhutan.

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

Scoring Opportunity or Hospital Pass? The Changing Role of Local Authorities in 14-19 Education and Training in England

ERIC Educational Resources Information Center

Payne, Jonathan

2010-01-01

In 2008, the UK Labour government unveiled plans to abolish the Learning and Skills Council and transfer funding for the education and training of 16-19-year-olds in England to local authorities (LAs). The transfer of 16-19 funding complements the responsibilities that LAs have already acquired in relation to the raising of the education and…

Allelic Variants of Complement Genes Associated with Dense Deposit Disease

PubMed Central

Abrera-Abeleda, Maria Asuncion; Nishimura, Carla; Frees, Kathy; Jones, Michael; Maga, Tara; Katz, Louis M.; Zhang, Yuzhou

2011-01-01

The alternative pathway of the complement cascade plays a role in the pathogenesis of dense deposit disease (DDD). Deficiency of complement factor H and mutations in CFH associate with the development of DDD, but it is unknown whether allelic variants in other complement genes also associate with this disease. We studied patients with DDD and identified previously unreported sequence alterations in several genes in addition to allelic variants and haplotypes common to patients with DDD. We found that the likelihood of developing DDD increases with the presence of two or more risk alleles in CFH and C3. To determine the functional consequence of this finding, we measured the activity of the alternative pathway in serum samples from phenotypically normal controls genotyped for variants in CFH and C3. Alternative pathway activity was higher in the presence of variants associated with DDD. Taken together, these data confirm that DDD is a complex genetic disease and may provide targets for the development of disease-specific therapies. PMID:21784901

Deficiency in Mannose-Binding Lectin-Associated Serine Protease-2 Does Not Increase Susceptibility to Trypanosoma cruzi Infection

PubMed Central

Ribeiro, Carolina H.; Lynch, Nicholas J.; Stover, Cordula M.; Ali, Youssif M.; Valck, Carolina; Noya-Leal, Francisca; Schwaeble, Wilhelm J.; Ferreira, Arturo

2015-01-01

Trypanosoma cruzi is the causative agent of Chagas' disease, a chronic illness affecting 10 million people around the world. The complement system plays an important role in fighting microbial infections. The recognition molecules of the lectin pathway of complement activation, mannose-binding lectin (MBL), ficolins, and CL-11, bind to specific carbohydrates on pathogens, triggering complement activation through MBL-associated serine protease-2 (MASP-2). Previous in vitro work showed that human MBL and ficolins contribute to T. cruzi lysis. However, MBL-deficient mice are only moderately compromised in their defense against the parasite, as they may still activate the lectin pathway through ficolins and CL-11. Here, we assessed MASP-2-deficient mice, the only presently available mouse line with total lectin pathway deficiency, for a phenotype in T. cruzi infection. Total absence of lectin pathway functional activity did not confer higher susceptibility to T. cruzi infection, suggesting that it plays a minor role in the immune response against this parasite. PMID:25548381

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity

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

Song, Yuan; Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, Canada N5V4T3; Wu, Keqiang

2010-05-28

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants.more » We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.« less

In-vivo detection of binary PKA network interactions upon activation of endogenous GPCRs

PubMed Central

Röck, Ruth; Bachmann, Verena; Bhang, Hyo-eun C; Malleshaiah, Mohan; Raffeiner, Philipp; Mayrhofer, Johanna E; Tschaikner, Philipp M; Bister, Klaus; Aanstad, Pia; Pomper, Martin G; Michnick, Stephen W; Stefan, Eduard

2015-01-01

Membrane receptor-sensed input signals affect and modulate intracellular protein-protein interactions (PPIs). Consequent changes occur to the compositions of protein complexes, protein localization and intermolecular binding affinities. Alterations of compartmentalized PPIs emanating from certain deregulated kinases are implicated in the manifestation of diseases such as cancer. Here we describe the application of a genetically encoded Protein-fragment Complementation Assay (PCA) based on the Renilla Luciferase (Rluc) enzyme to compare binary PPIs of the spatially and temporally controlled protein kinase A (PKA) network in diverse eukaryotic model systems. The simplicity and sensitivity of this cell-based reporter allows for real-time recordings of mutually exclusive PPIs of PKA upon activation of selected endogenous G protein-coupled receptors (GPCRs) in cancer cells, xenografts of mice, budding yeast, and zebrafish embryos. This extends the application spectrum of Rluc PCA for the quantification of PPI-based receptor-effector relationships in physiological and pathological model systems. PMID:26099953

[Haemophilia A and B in dogs].

PubMed

Mischke, R

2012-01-01

Based on own clinical experiences and the literature, this review article elaborates on aspects of aetiology, pathogenesis, clinical signs, diagnosis and therapy of haemophilia A and B in dogs. When compared to humans, dogs reveal more severe haemorrhagic symptoms at a defined residual factor activity (e.g. subcutaneous, intramuscular, and intraarticular haemorrhages after inappropriate trauma resulting in lameness and paralysis, excessive haemorrhage during second dentition, venal puncture, and surgery). Fortunately, genetic tests are now commercially available in Germany for selected breeds (haemophilia B in Rhodesian Ridgebacks; haemophilia A in Havaneses), which complement the conventional individual factor activity measurements and facilitate the detection of female carrier dogs. Treatment of bleeding crises is still mainly based on substitution therapy with fresh or fresh frozen plasma in addition to local haemostatic measures. In contrast, expectations regarding the timely clinical availability of gene therapy (particularly in humans) have not yet been fulfilled.

Travtek Evaluation Rental And Local User Study

DOT National Transportation Integrated Search

1997-12-01

Part 2 of the FHWA report on Road Safety Audits was prepared to provide the following: Additional detail on road safety organizations and the development of safety policies in Australia and New Zealand. This complements the information in Section 2 o...

Systemic inflammatory response following acute myocardial infarction

PubMed Central

Fang, Lu; Moore, Xiao-Lei; Dart, Anthony M; Wang, Le-Min

2015-01-01

Acute cardiomyocyte necrosis in the infarcted heart generates damage-associated molecular patterns, activating complement and toll-like receptor/interleukin-1 signaling, and triggering an intense inflammatory response. Inflammasomes also recognize danger signals and mediate sterile inflammatory response following acute myocardial infarction (AMI). Inflammatory response serves to repair the heart, but excessive inflammation leads to adverse left ventricular remodeling and heart failure. In addition to local inflammation, profound systemic inflammation response has been documented in patients with AMI, which includes elevation of circulating inflammatory cytokines, chemokines and cell adhesion molecules, and activation of peripheral leukocytes and platelets. The excessive inflammatory response could be caused by a deregulated immune system. AMI is also associated with bone marrow activation and spleen monocytopoiesis, which sustains a continuous supply of monocytes at the site of inflammation. Accumulating evidence has shown that systemic inflammation aggravates atherosclerosis and markers for systemic inflammation are predictors of adverse clinical outcomes (such as death, recurrent myocardial infarction, and heart failure) in patients with AMI. PMID:26089856

IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions.

PubMed

Lee, Chang-Han; Romain, Gabrielle; Yan, Wupeng; Watanabe, Makiko; Charab, Wissam; Todorova, Biliana; Lee, Jiwon; Triplett, Kendra; Donkor, Moses; Lungu, Oana I; Lux, Anja; Marshall, Nicholas; Lindorfer, Margaret A; Goff, Odile Richard-Le; Balbino, Bianca; Kang, Tae Hyun; Tanno, Hidetaka; Delidakis, George; Alford, Corrine; Taylor, Ronald P; Nimmerjahn, Falk; Varadarajan, Navin; Bruhns, Pierre; Zhang, Yan Jessie; Georgiou, George

2017-08-01

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Iofetamine hydrochloride I 123: a new radiopharmaceutical for cerebral perfusion imaging

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

Druckenbrod, R.W.; Williams, C.C.; Gelfand, M.J.

1989-01-01

Iofetamine hydrochloride I-123 permits cerebral blood perfusion imaging with single photon emission computed tomography (SPECT). SPECT is more widely available than positron emission tomography, and complements anatomic visualization with X-ray computed tomography (CT) or magnetic resonance imaging. Iofetamine is an amphetamine analog that is rapidly taken up by the lungs, then redistributed principally to the liver and brain. The precise mechanism of localization has not been determined, but is believed to result from nonspecific receptor binding. Brain uptake peaks at 30 minutes postinjection and remains relatively constant through 60 minutes. The drug is metabolized and excreted in the urine, withmore » negligible activity remaining at 48 hours. When compared with CT in stroke patients, visualization may be performed sooner after symptom onset and a larger zone of involvement may be evident with iofetamine. Localization of seizure foci and diagnosis of Alzheimer's disease may also be possible. As CT has revolutionized noninvasive imaging of brain anatomy, SPECT with iofetamine permits routine cerebral blood flow imaging. 36 references.« less

Evolution of the environmental justice movement: activism, formalization and differentiation

NASA Astrophysics Data System (ADS)

Colsa Perez, Alejandro; Grafton, Bernadette; Mohai, Paul; Hardin, Rebecca; Hintzen, Katy; Orvis, Sara

2015-10-01

To complement a recent flush of research on transnational environmental justice movements, we sought a deeper organizational history of what we understand as the contemporary environmental justice movement in the United States. We thus conducted in-depth interviews with 31 prominent environmental justice activists, scholars, and community leaders across the US. Today’s environmental justice groups have transitioned from specific local efforts to broader national and global mandates, and more sophisticated political, technological, and activist strategies. One of the most significant transformations has been the number of groups adopting formal legal status, and emerging as registered environmental justice organizations (REJOs) within complex partnerships. This article focuses on the emergence of REJOs, and describes the respondents’ views about the implications of this for more local grassroots groups. It reveals a central irony animating work across groups in today’s movement: legal formalization of many environmental justice organizations has made the movement increasingly internally differentiated, dynamic, and networked, even as the passage of actual national laws on environmental justice has proven elusive.

Local structure and defects in ion irradiated KTaO3

NASA Astrophysics Data System (ADS)

Zhang, F. X.; Xi, J.; Zhang, Y.; Tong, Yang; Xue, H.; Huang, R.; Trautmann, C.; Weber, W. J.

2018-04-01

The modification of the local structure in cubic perovskite KTaO3 irradiated with 3 MeV and 1.1 GeV Au ions is studied by Raman and x-ray absorption spectroscopy, complemented by density functional theory (DFT) calculations. In the case of irradiation with 3 MeV Au ions where displacement cascade processes are dominant, the Ta L3-edge x-ray absorption measurements suggest that a peak corresponding to the Ta-O bonds in the TaO6 octahedra splits, which is attributed to the formation of TaK antisite defects that are coupled with oxygen vacancies, V O. This finding is consistent with the DFT calculations. Under irradiation with 1.1 GeV ions, the intense ionization and electronic energy deposition lead to a blue shift and an intensity reduction of active Raman bands. In the case of sequential irradiations, extended x-ray absorption fine structure measurements reveal a decrease in concentration of coupled TaK-V O defects under subsequent irradiation with 1.1 GeV Au ions.

Effects of MASP-1 of the Complement System on Activation of Coagulation Factors and Plasma Clot Formation

PubMed Central

Hess, Katharina; Ajjan, Ramzi; Phoenix, Fladia; Dobó, József; Gál, Péter; Schroeder, Verena

2012-01-01

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation. PMID:22536427

New Milestones Ahead in Complement-Targeted Therapy

PubMed Central

Ricklin, Daniel; Lambris, John D.

2017-01-01

The complement system is a powerful effector arm of innate immunity that typically confers protection from microbial intruders and accumulating debris. In many clinical situations, however, the defensive functions of complement can turn against host cells and induce or exacerbate immune, inflammatory, and degenerative conditions. Although the value of inhibiting complement in a therapeutic context has long been recognized, bringing complement-targeted drugs into clinical use has proved challenging. This important milestone was finally reached a decade ago, yet the clinical availability of complement inhibitors has remained limited. Still, the positive long-term experience with complement drugs and their proven effectiveness in various diseases has reinvigorated interest and confidence in this approach. Indeed, a broad variety of clinical candidates that act at almost any level of the complement activation cascade are currently in clinical development, with several of them being evaluated in phase 2 and phase 3 trials. With antibody-related drugs dominating the panel of clinical candidates, the emergence of novel small-molecule, peptide, protein, and oligonucleotide-based inhibitors offers new options for drug targeting and administration. Whereas all the currently approved and many of the proposed indications for complement-targeted inhibitors belong to the rare disease spectrum, these drugs are increasingly being evaluated for more prevalent conditions. Fortunately, the growing experience from preclinical and clinical use of therapeutic complement inhibitors has enabled a more evidence-based assessment of suitable targets and rewarding indications as well as related technical and safety considerations. This review highlights recent concepts and developments in complement-targeted drug discovery, provides an overview of current and emerging treatment options, and discusses the new milestones ahead on the way to the next generation of clinically available complement therapeutics. PMID:27321574

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

PubMed

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

2018-05-09

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

Complement 3 activates the renal renin-angiotensin system by induction of epithelial-to-mesenchymal transition of the nephrotubulus in mice.

PubMed

Zhou, Xueli; Fukuda, Noboru; Matsuda, Hiroyuki; Endo, Morito; Wang, Xiaofei; Saito, Kosuke; Ueno, Takahiro; Matsumoto, Taro; Matsumoto, Koichi; Soma, Masayoshi; Kobayashi, Naohiko; Nishiyama, Akira

2013-10-01

We have demonstrated that mesenchymal cells from spontaneously hypertensive rats genetically express complement 3 (C3). Mature tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT) that is linked to the pathogenesis of renal fibrosis and injury. In this study, we investigated the contribution of C3 in EMT and in the renal renin-angiotensin (RA) systems associated with hypertension. C3a induced EMT in mouse TCMK-1 epithelial cells, which displayed increased expression of renin and Krüppel-like factor 5 (KLF5) and nuclear localization of liver X receptor α (LXRα). C3 and renin were strongly stained in the degenerated nephrotubulus and colocalized with LXRα and prorenin receptor in unilateral ureteral obstruction (UUO) kidneys from wild-type mice. In C3-deficient mice, hydronephrus and EMT were suppressed, with no expression of renin and C3. After UUO, systolic blood pressure was increased in wild-type but not C3-deficient mice. In wild-type mice, intrarenal angiotensin II (ANG II) levels were markedly higher in UUO kidneys than normal kidneys and decreased with aliskiren. There were no increases in intrarenal ANG II levels after UUO in C3-deficient mice. Thus C3 induces EMT and dedifferentiation of epithelial cells, which produce renin through induction of LXRα. These data indicate for the first time that C3 may be a primary factor to activate the renal RA systems to induce hypertension.

Impact of Short Interval SMS Digital Data on Wind Vector Determination for a Severe Local Storms Area

NASA Technical Reports Server (NTRS)

Peslen, C. A.

1979-01-01

The impact of 5 minute interval SMS-2 visible digital image data in analyzing severe local storms is examined using wind vectors derived from cloud tracking on time lapsed sequence of geosynchronous satellite images. The cloud tracking areas are located in the Central Plains, where on 6 May 1975, hail-producing thunderstorms occurred ahead of a well defined dry line. The results demonstrate that satellite-derived wind vectors and their associated divergence fields complement conventional meteorological analyses in describing the conditions preceding severe local storm development.

On the Local Convergence of Pattern Search

NASA Technical Reports Server (NTRS)

Dolan, Elizabeth D.; Lewis, Robert Michael; Torczon, Virginia; Bushnell, Dennis M. (Technical Monitor)

2000-01-01

We examine the local convergence properties of pattern search methods, complementing the previously established global convergence properties for this class of algorithms. We show that the step-length control parameter which appears in the definition of pattern search algorithms provides a reliable asymptotic measure of first-order stationarity. This gives an analytical justification for a traditional stopping criterion for pattern search methods. Using this measure of first-order stationarity, we analyze the behavior of pattern search in the neighborhood of an isolated local minimizer. We show that a recognizable subsequence converges r-linearly to the minimizer.

Male Sterile2 Encodes a Plastid-Localized Fatty Acyl Carrier Protein Reductase Required for Pollen Exine Development in Arabidopsis

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

Chen, W.; Shanklin, J.; Yu, X.-H.

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. Inmore » addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30 C), MS2 exhibits a K{sub m} for 16:0-Acyl Carrier Protein of 23.3 {+-} 4.0 {mu}m, a V{sub max} of 38.3 {+-} 4.5 nmol mg{sup -1} min{sup -1}, and a catalytic efficiency/K{sub m} of 1,873 m{sup -1} s{sup -1}. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.« less

Belimumab in the treatment of systemic lupus erythematosus: high disease activity predictors of response.

PubMed

van Vollenhoven, Ronald F; Petri, Michelle A; Cervera, Ricard; Roth, David A; Ji, Beulah N; Kleoudis, Christi S; Zhong, Z John; Freimuth, William

2012-08-01

To identify factors that predict response to belimumab treatment in the phase 3 BLISS trials of autoantibody-positive systemic lupus erythematosus (SLE) and further analyse clinical efficacy in various patient subsets. The BLISS trials compared belimumab 1 and 10 mg/kg versus placebo, all plus standard SLE therapy, over 52 or 76 weeks. Pooled subgroup analyses of week 52 SLE responder index rates (the primary endpoint in both trials) were performed based on demographic characteristics and baseline disease activity indicators. Pooled multivariate analysis was performed to determine predictors of response and treatment effect. Pooled univariate and multivariate analyses (N=1684) identified baseline factors associated with an increased benefit of belimumab versus placebo. These factors included the Safety Of Estrogens In Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) ≥10, low complement, anti-dsDNA positivity and corticosteroid use. Efficacy outcomes were assessed in the low complement/anti-dsDNA-positive and SELENA-SLEDAI ≥10 subgroups. Week 52 SLE Responder Index rates in the low complement/anti-dsDNA-positive subgroup were 31.7%, 41.5% (p=0.002) and 51.5% (p<0.001) with placebo and belimumab 1 mg/kg and 10 mg/kg, respectively; corresponding rates in the SELENA-SLEDAI ≥10 subgroup were 44.3%, 58.0% (p<0.001) and 63.2% (p<0.001). Further analysis of secondary endpoints in the low complement/anti-dsDNA-positive subgroup showed that compared with placebo, belimumab produced greater benefits regarding severe flares, corticosteroid use and health-related quality of life. These findings suggest that belimumab has greater therapeutic benefit than standard therapy alone in patients with higher disease activity, anti-dsDNA positivity, low complement or corticosteroid treatment at baseline. CLINICALTRIALS.GOV: identifiers NCT00424476 and NCT00410384.

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

PubMed Central

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

2012-01-01

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

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

PubMed Central

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

2009-01-01

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

Polyanion-Induced Self Association of Complement Factor H1

PubMed Central

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

2008-01-01

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

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

PubMed

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

2009-07-01

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

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

PubMed Central

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

2009-01-01

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

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

PubMed

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

2009-01-02

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

Myasthenia gravis: the role of complement at the neuromuscular junction.

PubMed

Howard, James F

2018-01-01

Generalized myasthenia gravis (gMG) is a rare autoimmune disorder characterized by skeletal muscle weakness caused by disrupted neurotransmission at the neuromuscular junction (NMJ). Approximately 74-88% of patients with gMG have acetylcholine receptor (AChR) autoantibodies. Complement plays an important role in innate and antibody-mediated immunity, and activation and amplification of complement results in the formation of membrane attack complexes (MACs), lipophilic proteins that damage cell membranes. The role of complement in gMG has been demonstrated in animal models and patients. Studies in animals lacking specific complement proteins have confirmed that MAC formation is required to induce experimental autoimmune MG (EAMG) and NMJ damage. Complement inhibition in EAMG models can prevent disease induction and reverse its progression. Patients with anti-AChR + MG have autoantibodies and MACs present at NMJs. Damaged NMJs are associated with more severe disease, fewer AChRs, and MACs in synaptic debris. Current MG therapies do not target complement directly. Eculizumab is a humanized monoclonal antibody that inhibits cleavage of complement protein C5, preventing MAC formation. Eculizumab treatment improved symptoms compared with placebo in a phase II study in patients with refractory gMG. Direct complement inhibition could preserve NMJ physiology and muscle function in patients with anti-AChR + gMG. © 2017 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

Cell lines that support replication of a novel herpes simplex virus 1 U{sub L}31 deletion mutant can properly target U{sub L}34 protein to the nuclear rim in the absence of U{sub L}31

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

Liang Li; Tanaka, Michiko; Kawaguchi, Yasushi

2004-11-10

Previous results indicated that the herpes simplex virus 1 (HSV-1) U{sub L}31 gene is necessary and sufficient for localization of the U{sub L}34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U{sub L}31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Veromore » cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U{sub L}31 gene. The replication of the U{sub L}31 deletion virus was restored on U{sub L}31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U{sub L}34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U{sub L}34 protein localized at the nuclear membrane in rabbit skin cells, and U{sub L}31 complementing CV1 cells infected with the U{sub L}31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U{sub L}34 protein to the nuclear membrane. We speculate that this function partially complements that of U{sub L}31 and may explain why U{sub L}31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.« less

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

Maternal and fetal alternative complement pathway activation in early severe preeclampsia.

PubMed

Hoffman, M Camille; Rumer, Kristen K; Kramer, Anita; Lynch, Anne M; Winn, Virginia D

2014-01-01

We sought to determine whether alternative complement activation fragment Bb (Bb) levels are elevated in the maternal, fetal, and placental blood in cases of severe preeclampsia (PE) compared with normotensive controls. This was a cross-sectional study of women admitted at ≥24 weeks gestation with or without severe PE. Maternal plasma was collected at the time of enrollment. Umbilical venous cord and intervillous space blood were collected at delivery. Plasma Bb levels were assessed using ELISA. Bb levels were compared between cases and controls. Median Bb levels were higher in the maternal plasma of severe PE subjects (n = 24) than in controls (n = 20), 1.45 ± 1.03 versus 0.65 ± 0.23 μg/mL, P < 0.001. In umbilical venous plasma, Bb levels were higher in severe PE subjects (n = 15) compared with controls (n = 15), 2.48 ± 1.40 versus 1.01 ± 0.57 μg/mL, P = 0.01. Activation fragment Bb is increased in the maternal and umbilical venous blood of cases of severe PE when compared with normotensive controls. These data provide support for alternative complement pathway involvement in the pathogenesis of severe PE and demonstrate that alternative complement activation occurs not only in the maternal but also in the fetal compartment. © 2013 John Wiley & Sons Ltd.

SIGN-R1 and complement factors are involved in the systemic clearance of radiation-induced apoptotic cells in whole-body irradiated mice

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

Park, Jin-Yeon; Loh, SoHee; Cho, Eun-hee

Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body γ-irradiation of mice increased caspase-3{sup +} apoptotic lymphocyte numbers in secondary lymphoid organs. Following γ-irradiation, SIGN-R1 and complements (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively activated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver ofmore » SIGN-R1 KO mice, followed by a significant increase of CD11b{sup +} cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after γ-irradiation. - Highlights: • Splenic SIGN-R1{sup +} macrophages are activated after γ-irradiation. • C3 and C4 levels increased and C3 was activated in the spleen after γ-irradiation. • SIGN-R1 mediated the systemic clearance of radiation-induced apoptotic cells in spleen and liver.« less

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum.

PubMed

Castiblanco-Valencia, Mónica M; Fraga, Tatiana R; Breda, Leandro C D; Vasconcellos, Sílvio A; Figueira, Cláudio P; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I; Barbosa, Angela S; Isaac, Lourdes

2016-05-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. Copyright © 2016 European Federation of Immunological Societies. All rights reserved.

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

PubMed Central

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

2015-01-01

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

Transcriptome Analysis of the Innate Immunity-Related Complement System in Spleen Tissue of Ctenopharyngodon idella Infected with Aeromonas hydrophila

PubMed Central

Dang, Yunfei; Xu, Xiaoyan; Shen, Yubang; Hu, Moyan; Zhang, Meng; Li, Lisen; Lv, Liqun; Li, Jiale

2016-01-01

The grass carp (Ctenopharyngodon idella) is an important commercial farmed herbivorous fish species in China, but is susceptible to Aeromonas hydrophila infections. In the present study, we performed de novo RNA-Seq sequencing of spleen tissue from specimens of a disease-resistant family, which were given intra-peritoneal injections containing PBS with or without a dose of A. hydrophila. The fish were sampled from the control group at 0 h, and from the experimental group at 4, 8, 12, 24, 48 and 72 h. 122.18 million clean reads were obtained from the normalized cDNA libraries; these were assembled into 425,260 contigs and then 191,795 transcripts. Of those, 52,668 transcripts were annotated with the NCBI Nr database, and 41,347 of the annotated transcripts were assigned into 90 functional groups. 20,569 unigenes were classified into six main categories, including 38 secondary KEGG pathways. 2,992 unigenes were used in the analysis of differentially expressed genes (DEGs). 89 of the putative DEGs were related to the immune system and 41 of them were involved in the complement and coagulation cascades pathway. This study provides insights into the complement and complement-related pathways involved in innate immunity, through expression profile analysis of the genomic resources in C. idella. We conclude that complement and complement-related genes play important roles during defense against A. hydrophila infection. The immune response is activated at 4 h after the bacterial injections, indicating that the complement pathways are activated at the early stage of bacterial infection. The study has improved our understanding of the immune response mechanisms in C. idella to bacterial pathogens. PMID:27383749

World Epidemiology Review, Number 79

DTIC Science & Technology

1977-02-09

currently in the same situation, paying tribute to the mentioned helminthiasis as a result of the basic influence of three factors: arrival of... helminthiasis in other states and, as a complement, to those actually infected locally and representing native threats, who are liable to increase the

Linkage specificity and role of properdin in activation of the alternative complement pathway by fungal glycans.

PubMed

Agarwal, Sarika; Specht, Charles A; Haibin, Huang; Ostroff, Gary R; Ram, Sanjay; Rice, Peter A; Levitz, Stuart M

2011-01-01

Fungal cell walls are predominantly composed of glucans, mannans, and chitin. Recognition of these glycans by the innate immune system is a critical component of host defenses against the mycoses. Complement, an important arm of innate immunity, plays a significant role in fungal pathogenesis, especially the alternative pathway (AP). Here we determine that the glycan monosaccharide composition and glycosidic linkages affect AP activation and C3 deposition. Furthermore, properdin, a positive regulator of the AP, contributes to these functions. AP activation by glycan particles that varied in composition and linkage was measured by C3a generation in serum treated with 10 mM EGTA and 10 mM Mg(2+) (Mg-EGTA-treated serum) (AP specific; properdin functional) or Mg-EGTA-treated serum that lacked functional properdin. Particles that contained either β1→3 or β1→6 glucans or both generated large and similar amounts of C3a when the AP was intact. Blocking properdin function resulted in 5- to 10-fold-less C3a production by particulate β1→3 glucans. However, particulate β1→6 glucans generated C3a via the AP only in the presence of intact properdin. Interestingly, zymosan and glucan-mannan particles (GMP), which contain both β-glucans and mannans, also required properdin to generate C3a. The β1→4 glycans chitin and chitosan minimally activated C3 even when properdin was functional. Finally, properdin binding to glucan particles (GP) and zymosan in serum required active C3. Properdin colocalized with bound C3, suggesting that in the presence of serum, properdin bound indirectly to glycans through C3 convertases. These findings provide a better understanding of how properdin facilitates AP activation by fungi through interaction with the cell wall components. Invasive fungal infections have increased in incidence with the widespread use of immunosuppressive therapy and invasive procedures. Activation of the complement system contributes to innate immunity against fungi by generating chemoattractants that recruit white blood cells and by coating the pathogen with complement fragments that "mark" them for phagocytosis. The fungal cell wall activates complement in an antibody-independent manner through the alternative pathway (AP). Properdin is a positive regulator of the AP. This study elucidates how the specificity of cell wall glycan linkages affects AP activation and the role properdin plays in this process. Particulate β1→3 glucans activated the AP even in the absence of properdin, while β1→6 glucans required properdin for AP activation. In contrast, the β1→4 glycans chitin and chitosan failed to activate the AP. These findings enhance our mechanistic understanding of how fungi activate complement and have implications for the use of glycans in biomedical applications.

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

PubMed

Swe, Pearl M; Fischer, Katja

2014-06-01

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

Species Specificity of Vaccinia Virus Complement Control Protein for the Bovine Classical Pathway Is Governed Primarily by Direct Interaction of Its Acidic Residues with Factor I

PubMed Central

Kumar, Jitendra; Yadav, Viveka Nand; Phulera, Swastik; Kamble, Ashish; Gautam, Avneesh Kumar; Panwar, Hemendra Singh

2017-01-01

ABSTRACT Poxviruses display species tropism—variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is ∼43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(H2O) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals. IMPORTANCE Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the complement classical pathway (CP). Because the virus encodes a soluble complement regulator, VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP and that this is primarily determined by its amino acids E108, E120, and E144, which interact with bovine serine protease factor I to inactivate bovine C4b—one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby the fitness of the virus in cattle. PMID:28724763

Hemorrhage-induced intestinal damage is complement independent in Helicobacter-hepaticus infected mice

PubMed Central

Hylton, Diana J.; Phillips, Lauren M.; Hoffman, Sara M.; Fleming, Sherry D.

2010-01-01

With over half of the world population infected, Helicobacter infection is an important public health issue associated with gastrointestinal cancers and inflammatory bowel disease. Animal studies indicate that complement and oxidative stress play a role in Helicobacter infections. Hemorrhage induces tissue damage which is attenuated by blockade of either complement activation or oxidative stress products. Therefore, we hypothesized that chronic Helicobacter hepaticus infection would modulate hemorrhage-induced intestinal damage and inflammation. To test this hypothesis, we examined hemorrhage-induced jejunal damage and inflammation in uninfected and H. hepaticus infected mice. H. hepaticus infection increased hemorrhage-induced mid-jejunal mucosal damage despite attenuating complement activation. In addition, infection alone increased chemokine secretion, changing the hemorrhage-induced neutrophil infiltration to a macrophage-mediated inflammatory response. The hemorrhage-induced macrophage infiltration correlated with increased secretion of tumor necrosis factor-α (TNF-α3) and nitric oxide (NO) in the infected mice. Together these data indicate that Helicobacter infection modulates the mechanism of hemorrhage-induced intestinal damage and inflammation from a complement-mediated response to a macrophage response with elevated TNF-α and NO. These data indicate that chronic, low level infections change the response to trauma and should be considered when designing and administering therapeutics. PMID:20220569

Exclusivity structures and graph representatives of local complementation orbits

NASA Astrophysics Data System (ADS)

Cabello, Adán; Parker, Matthew G.; Scarpa, Giannicola; Severini, Simone

2013-07-01

We describe a construction that maps any connected graph G on three or more vertices into a larger graph, H(G), whose independence number is strictly smaller than its Lovász number which is equal to its fractional packing number. The vertices of H(G) represent all possible events consistent with the stabilizer group of the graph state associated with G, and exclusive events are adjacent. Mathematically, the graph H(G) corresponds to the orbit of G under local complementation. Physically, the construction translates into graph-theoretic terms the connection between a graph state and a Bell inequality maximally violated by quantum mechanics. In the context of zero-error information theory, the construction suggests a protocol achieving the maximum rate of entanglement-assisted capacity, a quantum mechanical analogue of the Shannon capacity, for each H(G). The violation of the Bell inequality is expressed by the one-shot version of this capacity being strictly larger than the independence number. Finally, given the correspondence between graphs and exclusivity structures, we are able to compute the independence number for certain infinite families of graphs with the use of quantum non-locality, therefore highlighting an application of quantum theory in the proof of a purely combinatorial statement.

Complement dysregulation and disease: from genes and proteins to diagnostics and drugs.

PubMed

de Cordoba, Santiago Rodriguez; Tortajada, Agustin; Harris, Claire L; Morgan, B Paul

2012-11-01

During the last decade, numerous studies have associated genetic variations in complement components and regulators with a number of chronic and infectious diseases. The functional characterization of these complement protein variants, in addition to recent structural advances in understanding of the assembly, activation and regulation of the AP C3 convertase, have provided important insights into the pathogenic mechanisms involved in some of these complement related disorders. This knowledge has identified potential targets for complement inhibitory therapies which are demonstrating efficacy and generating considerable expectation in changing the natural history of these diseases. Comprehensive understanding of the genetic and non-genetic risk factors contributing to these disorders will also result in targeting of the right patient groups in a stratified medicine approach through better diagnostics and individually tailored treatments, thereby improving management of patients. Crown Copyright © 2012. Published by Elsevier GmbH. All rights reserved.

Autoantibodies against complement components in systemic lupus erythematosus - role in the pathogenesis and clinical manifestations.

PubMed

Hristova, M H; Stoyanova, V S

2017-12-01

Many complement structures and a number of additional factors, i.e. autoantibodies, receptors, hormones and cytokines, are implicated in the complex pathogenesis of systemic lupus erythematosus. Genetic defects in the complement as well as functional deficiency due to antibodies against its components lead to different pathological conditions, usually clinically presented. Among them hypocomplementemic urticarial vasculitis, different types of glomerulonephritis as dense deposit disease, IgA nephropathy, atypical haemolytic uremic syndrome and lupus nephritis are very common. These antibodies cause conformational changes leading to pathological activation or inhibition of complement with organ damage and/or limited capacity of the immune system to clear immune complexes and apoptotic debris. Finally, we summarize the role of complement antibodies in the pathogenesis of systemic lupus erythematosus and discuss the mechanism of some related clinical conditions such as infections, thyroiditis, thrombosis, acquired von Willebrand disease, etc.

p62/sequestosome-1 up-regulation promotes ABT-263-induced caspase-8 aggregation/activation on the autophagosome.

PubMed

Huang, Shengbing; Okamoto, Koichi; Yu, Chunrong; Sinicrope, Frank A

2013-11-22

Autophagy and apoptosis regulate cancer cell viability in response to cytotoxic stress; however, their functional relationship remains unclear. p62/sequestosome 1 is a multifunctional protein and a signaling hub that shuttles ubiquitinated proteins to the lysosome during autophagy. Autophagy inhibition up-regulates p62, and prior data suggest that p62 may mediate apoptosis. Here, we demonstrate that p62 can regulate a caspase-8-dependent apoptosis in response to the BH3 mimetic agent, ABT-263. Up-regulation of p62 was shown to enhance ABT-263-induced caspase-8 activation that was Bax-dependent and resulted from mitochondrial amplification. Dependence upon caspase-8 was confirmed using caspase-8-deficient cells and by caspase-8 siRNA. Ectopic wild-type p62, but not p62 mutants with loss of ability to promote apoptosis, was shown to co-localize with caspase-8 and to promote its self-aggregation in ABT-263-treated cells, shown using a bimolecular fluorescence complementation assay. Endogenous p62 co-localized with caspase-8 in the presence of ABT-263 plus an autophagy inhibitor. Caspase-8 was shown to interact and co-localize with the autophagosome marker, LC3II. Knockdown of p62 attenuated binding between caspase-8 and LC3II, whereas p62 overexpression enhanced the co-localization of caspase-8 aggregates with LC3. LC3 knockdown did not affect interaction between caspase-8 and p62, suggesting that p62 may facilitate caspase-8 translocation to the autophagosomal membrane. A direct activator of caspase-8, i.e., TRAIL, alone or combined with ABT-263, induced caspase-8 aggregation and co-localization with p62 that was associated with a synergistic drug interaction. Together, these results demonstrate that up-regulation of p62 can mediate apoptosis via caspase-8 in the setting of autophagy inhibition.

p62/Sequestosome-1 Up-regulation Promotes ABT-263-induced Caspase-8 Aggregation/Activation on the Autophagosome*

PubMed Central

Huang, Shengbing; Okamoto, Koichi; Yu, Chunrong; Sinicrope, Frank A.

2013-01-01

Autophagy and apoptosis regulate cancer cell viability in response to cytotoxic stress; however, their functional relationship remains unclear. p62/sequestosome 1 is a multifunctional protein and a signaling hub that shuttles ubiquitinated proteins to the lysosome during autophagy. Autophagy inhibition up-regulates p62, and prior data suggest that p62 may mediate apoptosis. Here, we demonstrate that p62 can regulate a caspase-8-dependent apoptosis in response to the BH3 mimetic agent, ABT-263. Up-regulation of p62 was shown to enhance ABT-263-induced caspase-8 activation that was Bax-dependent and resulted from mitochondrial amplification. Dependence upon caspase-8 was confirmed using caspase-8-deficient cells and by caspase-8 siRNA. Ectopic wild-type p62, but not p62 mutants with loss of ability to promote apoptosis, was shown to co-localize with caspase-8 and to promote its self-aggregation in ABT-263-treated cells, shown using a bimolecular fluorescence complementation assay. Endogenous p62 co-localized with caspase-8 in the presence of ABT-263 plus an autophagy inhibitor. Caspase-8 was shown to interact and co-localize with the autophagosome marker, LC3II. Knockdown of p62 attenuated binding between caspase-8 and LC3II, whereas p62 overexpression enhanced the co-localization of caspase-8 aggregates with LC3. LC3 knockdown did not affect interaction between caspase-8 and p62, suggesting that p62 may facilitate caspase-8 translocation to the autophagosomal membrane. A direct activator of caspase-8, i.e., TRAIL, alone or combined with ABT-263, induced caspase-8 aggregation and co-localization with p62 that was associated with a synergistic drug interaction. Together, these results demonstrate that up-regulation of p62 can mediate apoptosis via caspase-8 in the setting of autophagy inhibition. PMID:24121507

Clinical diagnostic and sociocultural dimensions of deliberate self-harm in Mumbai, India.

PubMed

Parkar, Shubhangi R; Dawani, Varsha; Weiss, Mitchell G

2006-04-01

Patients' accounts complement psychiatric assessment of deliberate self-harm (DSH). In this study we examined psychiatric disorders, and sociocultural and cross-cultural features of DSH. SCID diagnostic interviews and a locally adapted EMIC interview were used to study 196 patients after DSH at a general hospital in Mumbai, India. Major depression was the most common diagnosis (38.8%), followed by substance use disorders (16.8%), but 44.4% of patients did not meet criteria for an enduring Axis-I disorder (no diagnosis, V-code, or adjustment disorder). Psychache arising from patient-identified sociocultural contexts and stressors complements, but does not necessarily fulfill, criteria for explanatory psychiatric disorders.

Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme.

PubMed Central

Fu, C; Javedan, S; Moshiri, F; Maier, R J

1994-01-01

Nickel is an essential component of all H2-uptake hydrogenases. A fragment of DNA that complements a H2-uptake-deficient but nickel-cured mutant strain (JHK7) of Bradyrhizobium japonicum was isolated and sequenced. This 4.5-kb DNA fragment contains four open reading frames designated as ORF1, hupN, hupO, and hupP, which encode polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, respectively. The last three open reading frames (hupNOP) are most likely organized as an operon with a putative sigma 54-type promoter. Based on its hydropathy profile, HupN is predicted to be a transmembrane protein. It has 56% identity to the previously described HoxN (high-affinity nickel transport protein) of Alcaligenes eutrophus. A subclone (pJF23) containing the hupNOP genes excluding ORF1 completely complemented (in trans) strain JHK7 for hydrogenase activity in low nickel conditions. pJF26 containing only a functional hupN complemented the hydrogenase activity of mutant strain JHK7 to 30-55% of the wild-type level. Mutant strain JHK70, with a chromosomal deletion in hupP but with an intact hupNO, showed greater activities than pJF26-complemented JHK7 but still had lower activities than the wild type at all nickel levels tested. pJF25, containing the entire hupO and hupP, but without hupN (a portion of hupN was deleted), did not complement hydrogenase activity of mutant strain JHK7. The results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydrogenase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved in nickel transport by whole cells. The deleterious effects on hydrogenase expression are most pronounced by lack of the HupN product. PMID:8197192

Complement C1q formation of immune complexes with milk caseins and wheat glutens in schizophrenia

PubMed Central

Severance, Emily G.; Gressitt, Kristin; Halling, Meredith; Stallings, Cassie R.; Origoni, Andrea E.; Vaughan, Crystal; Khushalani, Sunil; Alaedini, Armin; Dupont, Didier; Dickerson, Faith B.; Yolken, Robert H.

2012-01-01

Immune system factors including complement pathway activation are increasingly linked to the etiology and pathophysiology of schizophrenia. Complement protein, C1q, binds to and helps to clear immune complexes composed of immunoglobulins coupled to antigens. The antigenic stimuli for C1q activation in schizophrenia are not known. Food sensitivities characterized by elevated IgG antibodies to bovine milk caseins and wheat glutens have been reported in individuals with schizophrenia. Here, we examined the extent to which these food products might comprise the antigen component of complement C1q immune complexes in individuals with recent onset schizophrenia (n=38), non-recent onset schizophrenia (n=61) and non-psychiatric controls (n=63). C1q seropositivity was significantly associated with both schizophrenia groups (recent onset, odds ratio (OR)=8.02, p≤0.008; non-recent onset, OR=3.15, p≤0.03) compared to controls (logistic regression models corrected for age, sex, race and smoking status). Casein- and/or gluten-IgG binding to C1q was significantly elevated in the non-recent onset group compared to controls (OR=4.36, p≤0.01). Significant amounts of C1q-casein/gluten-related immune complexes and C1q correlations with a marker for gastrointestinal inflammation in non-recent onset schizophrenia suggests a heightened rate of food antigens in the systemic circulation, perhaps via a disease-associated altered intestinal permeability. In individuals who are in the early stages of disease onset, C1q activation may reflect the formation of immune complexes with non-casein- or non-gluten-related antigens, the presence of C1q autoantibodies, and/or a dissociated state of immune complex components. In conclusion, complement activation may be a useful biomarker to diagnose schizophrenia early during the course of the disease. Future prospective studies should evaluate the impacts of casein- and gluten-free diets on C1q activation in schizophrenia. PMID:22801085

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

PubMed Central

Conde, Jonas Nascimento; da Silva, Emiliana Mandarano; Allonso, Diego; Coelho, Diego Rodrigues; Andrade, Iamara da Silva; de Medeiros, Luciano Neves; Menezes, Joice Lima; Barbosa, Angela Silva

2016-01-01

ABSTRACT Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system. PMID:27512066

Hospital Community Benefit in the Context of the Larger Public Health System: A State-Level Analysis of Hospital and Governmental Public Health Spending Across the United States.

PubMed

Singh, Simone R; Bakken, Erik; Kindig, David A; Young, Gary J

2016-01-01

Achieving meaningful population health improvements has become a priority for communities across the United States, yet funding to sustain multisector initiatives is frequently not available. One potential source of funding for population health initiatives is the community benefit expenditures that are required of nonprofit hospitals to maintain their tax-exempt status. In this article, we explore the importance of nonprofit hospitals' community benefit dollars as a funding source for population health. Hospitals' community benefit expenditures were obtained from their 2009 IRS (Internal Revenue Service) Form 990 Schedule H and complemented with data on state and local public health spending from the Association of State and Territorial Health Officials and the National Association of County & City Health Officials. Key measures included indicators of hospitals' community health spending and governmental public health spending, all aggregated to the state level. Univariate and bivariate statistics were used to describe how much hospitals spent on programs and activities for the community at large and to understand the relationship between hospitals' spending and the expenditures of state and local health departments. Tax-exempt hospitals spent a median of $130 per capita on community benefit activities, of which almost $11 went toward community health improvement and community-building activities. In comparison, median state and local health department spending amounted to $82 and $48 per capita, respectively. Hospitals' spending thus contributed an additional 9% to the resources available for population health to state and local health departments. Spending, however, varied widely by state and was unrelated to governmental public health spending. Moreover, adding hospitals' spending to the financial resources available to governmental public health agencies did not reduce existing inequalities in population health funding across states. Hospitals' community health investments represent an important source for public health activities, yet inequalities in the availability of funding across communities remain.

Spatiotemporal variability of urban growth factors: A global and local perspective on the megacity of Mumbai

NASA Astrophysics Data System (ADS)

Shafizadeh-Moghadam, Hossein; Helbich, Marco

2015-03-01

The rapid growth of megacities requires special attention among urban planners worldwide, and particularly in Mumbai, India, where growth is very pronounced. To cope with the planning challenges this will bring, developing a retrospective understanding of urban land-use dynamics and the underlying driving-forces behind urban growth is a key prerequisite. This research uses regression-based land-use change models - and in particular non-spatial logistic regression models (LR) and auto-logistic regression models (ALR) - for the Mumbai region over the period 1973-2010, in order to determine the drivers behind spatiotemporal urban expansion. Both global models are complemented by a local, spatial model, the so-called geographically weighted logistic regression (GWLR) model, one that explicitly permits variations in driving-forces across space. The study comes to two main conclusions. First, both global models suggest similar driving-forces behind urban growth over time, revealing that LRs and ALRs result in estimated coefficients with comparable magnitudes. Second, all the local coefficients show distinctive temporal and spatial variations. It is therefore concluded that GWLR aids our understanding of urban growth processes, and so can assist context-related planning and policymaking activities when seeking to secure a sustainable urban future.

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

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

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

2008-06-20

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

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo.

PubMed

Stefan, E; Aquin, S; Berger, N; Landry, C R; Nyfeler, B; Bouvier, M; Michnick, S W

2007-10-23

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Galpha(s) protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive beta-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.

[Cloning, expression and identification of functional fragment rC3B of human complement C3 in E. Coli].

PubMed

Gan, Hui; Zhou, Yong; Sun, Ping; Zhu, Xiao-Xia; Wang, Quan-Li; Zhan, Lin-Sheng

2007-08-01

This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.

Polar Localization of the NIP5;1 Boric Acid Channel Is Maintained by Endocytosis and Facilitates Boron Transport in Arabidopsis Roots

PubMed Central

Yoshinari, Akira; Shimada, Tomoo; Mitani-Ueno, Namiki

2017-01-01

Boron uptake in Arabidopsis thaliana is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that is located preferentially on the soil side of the plasma membrane in root cells. However, the mechanism underlying this polar localization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cells is mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region of NIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasma membrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1. Consistent with this, the polar localization was compromised in µ subunit mutants of the clathrin adaptor AP2. The Thr-to-Ala substitutions did not affect the boron transport activity of GFP-NIP5;1 in Xenopus laevis oocytes but did inhibit the ability to complement boron translocation to shoots and rescue growth defects in nip5;1-1 mutant plants under boron-limited conditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, is dependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots. PMID:28341806

Polar Localization of the NIP5;1 Boric Acid Channel Is Maintained by Endocytosis and Facilitates Boron Transport in Arabidopsis Roots.

PubMed

Wang, Sheliang; Yoshinari, Akira; Shimada, Tomoo; Hara-Nishimura, Ikuko; Mitani-Ueno, Namiki; Feng Ma, Jian; Naito, Satoshi; Takano, Junpei

2017-04-01

Boron uptake in Arabidopsis thaliana is mediated by nodulin 26-like intrinsic protein 5;1 (NIP5;1), a boric acid channel that is located preferentially on the soil side of the plasma membrane in root cells. However, the mechanism underlying this polar localization is poorly understood. Here, we show that the polar localization of NIP5;1 in epidermal and endodermal root cells is mediated by the phosphorylation of Thr residues in the conserved TPG (ThrProGly) repeat in the N-terminal region of NIP5;1. Although substitutions of Ala for three Thr residues in the TPG repeat did not affect lateral diffusion in the plasma membrane, these substitutions inhibited endocytosis and strongly compromised the polar localization of GFP-NIP5;1. Consistent with this, the polar localization was compromised in µ subunit mutants of the clathrin adaptor AP2. The Thr-to-Ala substitutions did not affect the boron transport activity of GFP-NIP5;1 in Xenopus laevis oocytes but did inhibit the ability to complement boron translocation to shoots and rescue growth defects in nip5;1-1 mutant plants under boron-limited conditions. These results demonstrate that the polar localization of NIP5;1 is maintained by clathrin-mediated endocytosis, is dependent on phosphorylation in the TPG repeat, and is necessary for the efficient transport of boron in roots. © 2017 American Society of Plant Biologists. All rights reserved.

Complement system studies in systemic lupus erythematosus (SLE)

PubMed

Teisberg, P

1975-01-01

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

Regulation of Complement and Contact System Activation via C1 Inhibitor Potentiation and Factor XIIa Activity Modulation by Sulfated Glycans – Structure-Activity Relationships

PubMed Central

Schoenfeld, Ann-Kathrin; Lahrsen, Eric; Alban, Susanne

2016-01-01

The serpin C1 inhibitor (C1-INH) is the only regulator of classical complement activation as well as the major regulator of the contact system. Its importance is demonstrated by hereditary angioedema (HAE), a severe disease with potentially life-threatening attacks due to deficiency or dysfunction of C1-INH. C1-INH replacement is the therapy of choice in HAE. In addition, C1-INH showed to have beneficial effects in other diseases characterized by inappropriate complement and contact system activation. Due to some limitations of its clinical application, there is a need for improving the efficacy of therapeutically applied C1-INH or to enhance the activity of endogenous C1-INH. Given the known potentiating effect of heparin on C1-INH, sulfated glycans (SG) may be such candidates. The aim of this study was to characterize suitable SG by evaluating structure-activity relationships. For this, more than 40 structurally distinct SG were examined for their effects on C1-INH, C1s and FXIIa. The SG turned out to potentiate the C1s inhibition by C1-INH without any direct influence on C1s. Their potentiating activity proved to depend on their degree of sulfation, molecular mass as well as glycan structure. In contrast, the SG had no effect on the FXIIa inhibition by C1-INH, but structure-dependently modulated the activity of FXIIa. Among the tested SG, β-1,3-glucan sulfates with a Mr ≤ 10 000 were identified as most promising lead candidates for the development of a glycan-based C1-INH amplifier. In conclusion, the obtained information on structural characteristics of SG favoring C1-INH potentiation represent an useful elementary basis for the development of compounds improving the potency of C1-INH in diseases and clinical situations characterized by inappropriate activation of complement and contact system. PMID:27783665

A Novel Cytosolic Isoform of Mitochondrial Trans-2-Enoyl-CoA Reductase Enhances Peroxisome Proliferator-Activated Receptor α Activity.

PubMed

Kim, Dong-Gyu; Yoo, Jae Cheal; Kim, Eunju; Lee, Young-Sun; Yarishkin, Oleg V; Lee, Da Yong; Lee, Kun Ho; Hong, Seong-Geun; Hwang, Eun Mi; Park, Jae-Yong

2014-06-01

Mitochondrial trans-2-enoyl-CoA reductase (MECR) is involved in mitochondrial synthesis of fatty acids and is highly expressed in mitochondria. MECR is also known as nuclear receptor binding factor-1, which was originally reported with yeast two-hybrid screening as a binding protein of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). However, MECR and PPARα are localized at different compartment, mitochondria, and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for functional interaction between MECR and PPARα. To identify the expression pattern of MECR and the cytosolic form of MECR (cMECR), we performed reverse transcription polymerase chain reaction (RT-PCR) with various tissue samples from Sprague-Dawley rats. To confirm the interaction between cMECR and PPARα, we performed several binding assays such as yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation. To observe subcellular localization of these proteins, immunocytochemistry was performed. A luciferase assay was used to measure PPARα activity. We provide evidence of an alternatively spliced variant of the rat MECR gene that yields cMECR. The cMECR lacks the N-terminal 76 amino acids of MECR and shows uniform distribution in the cytoplasm and nucleus of HeLa cells. cMECR directly bound PPARα in the nucleus and increased PPARα-dependent luciferase activity in HeLa cells. We found the cytosolic form of MECR (cMECR) was expressed in the cytosolic and/or nuclear region, directly binds with PPARα, and enhances PPARα activity.

Transformation of serum-susceptible Escherichia coli O111 with p16Slux plasmid to allow for real-time monitoring of complement-based inactivation of bacterial growth in bovine milk.

PubMed

Maye, S; Stanton, C; Fitzgerald, G F; Kelly, P M

2016-01-01

Complement activity has only recently been characterized in raw bovine milk. However, the activity of this component of the innate immune system was found to diminish as milk was subjected to heat or partitioning during cream separation. Detection of complement in milk relies on a bactericidal assay. This assay exploits the specific growth susceptibility of Escherichia coli O111 to the presence of complement. Practical application of the assay was demonstrated when a reduction in complement activity was recorded in the case of pasteurized and reduced-fat milks. This presented an opportunity to improve the functionality of the bactericidal assay by incorporating bioluminescence capability into the target organism. Following some adaptation, the strain was transformed by correctly integrating the p16Slux plasmid. Growth properties of the transformed strain of E. coli O111 were unaffected by the modification. The efficacy of the strain adaptation was correlated using the LINEST function analysis [r=0.966; standard error of prediction (SEy)=0.957] bioluminescence with that of bactericidal assay total plate counts within the range of 7.5 to 9.2 log cfu/mL using a combination of raw and processed milk samples. Importantly, the transformed E. coli O111 p16Slux strain could be identified in milk and broth samples using bioluminescence measurement, thus enabling the bactericidal assay-viability test to be monitored in real time throughout incubation. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Complement in Action: An Analysis of Patent Trends from 1976 Through 2011.

PubMed

Yang, Kun; Deangelis, Robert A; Reed, Janet E; Ricklin, Daniel; Lambris, John D

2013-01-01

Complement is an essential part of the innate immune response. It interacts with diverse endogenous pathways and contributes to the maintenance of homeostasis, the modulation of adaptive immune responses, and the development of various pathologies. The potential usefulness, in both research and clinical settings, of compounds that detect or modulate complement activity has resulted in thousands of publications on complement-related innovations in fields such as drug discovery, disease diagnosis and treatment, and immunoassays, among others. This study highlights the distribution and publication trends of patents related to the complement system that were granted by the United States Patent and Trademark Office from 1976 to the present day. A comparison to complement-related documents published by the World Intellectual Property Organization is also included. Statistical analyses revealed increasing diversity in complement-related research interests over time. More than half of the patents were found to focus on the discovery of inhibitors; interest in various inhibitor classes exhibited a remarkable transformation from chemical compounds early on to proteins and antibodies in more recent years. Among clinical applications, complement proteins and their modulators have been extensively patented for the diagnosis and treatment of eye diseases (especially age-related macular degeneration), graft rejection, cancer, sepsis, and a variety of other inflammatory and immune diseases. All of the patents discussed in this chapter, as well as those from other databases, are available from our newly constructed complement patent database: www.innateimmunity.us/patent .

Complement in action: an analysis of patent trends from 1976 through 2011.

PubMed

Yang, Kun; DeAngelis, Robert A; Reed, Janet E; Ricklin, Daniel; Lambris, John D

2013-01-01

Complement is an essential part of the innate immune response. It interacts with diverse endogenous pathways and contributes to the maintenance of homeostasis, the modulation of adaptive immune responses, and the development of various pathologies. The potential usefulness, in both research and clinical settings, of compounds that detect or modulate complement activity has resulted in thousands of publications on complement-related innovations in fields such as drug discovery, disease diagnosis and treatment, and immunoassays, among others. This study highlights the distribution and publication trends of patents related to the complement system that were granted by the United States Patent and Trademark Office from 1976 to the present day. A comparison to complement-related documents published by the World Intellectual Property Organization is also included. Statistical analyses revealed increasing diversity in complement-related research interests over time. More than half of the patents were found to focus on the discovery of inhibitors; interest in various inhibitor classes exhibited a remarkable transformation from chemical compounds early on to proteins and antibodies in more recent years. Among clinical applications, complement proteins and their modulators have been extensively patented for the diagnosis and treatment of eye diseases (especially age-related macular degeneration), graft rejection, cancer, sepsis, and a variety of other inflammatory and immune diseases. All of the patents discussed in this chapter, as well as those from other databases, are available from our newly constructed complement patent database: www.innateimmunity.us/patent.

Alternative complement activity in the egg cytosol of amphioxus Branchiostoma belcheri: evidence for the defense role of maternal complement components.

PubMed

Liang, Yujun; Zhang, Shicui; Wang, Zhiping

2009-01-01

The eggs in most invertebrates are fertilized externally, and therefore their resulting embryos are exposed to an environment full of microbes, many of which are pathogens capable of killing other organisms. How the developing embryos of invertebrates defend themselves against pathogenic attacks is an intriguing question to biologists, and remains largely unknown. Here we clearly demonstrated that the egg cytosol prepared from the newly fertilized eggs of amphioxus Branchiostoma belcheri, an invertebrate chordate, was able to inhibit the growth of both the Gram-negative bacterium Vibrio anguillarum and the Gram-positive bacterium Staphylococcus aureus. All findings point to that it is the complement system operating via the alternative pathway that is attributable to the bacteriostatic activity. This appears to be the first report providing the evidence for the functional role of the maternal complement components in the eggs of invertebrate species, paving the way for the study of maternal immunity in other invertebrate organisms whose eggs are fertilized in vitro. It also supports the notion that the early developing embryos share some defense mechanisms common with the adult species.

C-Reactive Protein as a Therapeutic Target in Age-Related Macular Degeneration.

PubMed

Molins, Blanca; Romero-Vázquez, Sara; Fuentes-Prior, Pablo; Adan, Alfredo; Dick, Andrew D

2018-01-01

Age-related macular degeneration (AMD), a retinal degenerative disease, is the leading cause of central vision loss among the elderly population in developed countries and an increasing global burden. The major risk is aging, compounded by other environmental factors and association with genetic variants for risk of progression. Although the etiology of AMD is not yet clearly understood, several pathogenic pathways have been proposed, including dysfunction of the retinal pigment epithelium, inflammation, and oxidative stress. The identification of AMD susceptibility genes encoding complement factors and the presence of complement and other inflammatory mediators in drusen, the hallmark deposits of AMD, support the concept that local inflammation and immune-mediated processes play a key role in AMD pathogenesis that may be accelerated through systemic immune activation. In this regard, increased levels of circulating C-reactive protein (CRP) have been associated with higher risk of AMD. Besides being a risk marker for AMD, CRP may also play a role in the progression of the disease as it has been identified in drusen, and we have recently found that its monomeric form (mCRP) induces blood retinal barrier disruption in vitro . In this review, we will address recent evidence that links CRP and AMD pathogenesis, which may open new therapeutic opportunities to prevent the progression of AMD.

Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

PubMed

Rastaldi, M P; Candiano, G; Musante, L; Bruschi, M; Armelloni, S; Rimoldi, L; Tardanico, R; Sanna-Cherchi, S; Cherchi, S Sanna; Ferrario, F; Montinaro, V; Haupt, R; Parodi, S; Carnevali, M L; Allegri, L; Camussi, G; Gesualdo, L; Scolari, F; Ghiggeri, G M

2006-08-01

Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

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

PubMed Central

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

2012-01-01

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

MO-DE-210-03: Ultrasound imaging is an attractive method for image guided radiation treatment (IGRT), by itself or to complement other imaging modalities

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

Ding, K.

Ultrasound imaging is an attractive method for image guided radiation treatment (IGRT), by itself or to complement other imaging modalities. It is inexpensive, portable and provides good soft tissue contrast. For challenging soft tissue targets such as pancreatic cancer, ultrasound imaging can be used in combination with pre-treatment MRI and/or CT to transfer important anatomical features for target localization at time of treatment. The non-invasive and non-ionizing nature of ultrasound imaging is particularly powerful for intra-fraction localization and monitoring. Recognizing these advantages, efforts are being made to incorporate novel robotic approaches to position and manipulate the ultrasound probe during irradiation.more » These recent enabling developments hold potential to bring ultrasound imaging to a new level of IGRT applications. However, many challenges, not limited to image registration, robotic deployment, probe interference and image acquisition rate, need to be addressed to realize the full potential of IGRT with ultrasound imaging. Learning Objectives: Understand the benefits and limitations in using ultrasound to augment MRI and/or CT for motion monitoring during radiation therapy delivery. Understanding passive and active robotic approaches to implement ultrasound imaging for intra-fraction monitoring. Understand issues of probe interference with radiotherapy treatment. Understand the critical clinical workflow for effective and reproducible IGRT using ultrasound guidance. The work of X.L. is supported in part by Elekta; J.W. and K.D. is supported in part by a NIH grant R01 CA161613 and by Elekta; D.H. is support in part by a NIH grant R41 CA174089.« less

The European Drought Observatory (EDO): Current State and Future Directions

NASA Astrophysics Data System (ADS)

Vogt, Jürgen; Sepulcre, Guadalupe; Magni, Diego; Valentini, Luana; Singleton, Andrew; Micale, Fabio; Barbosa, Paulo

2013-04-01

Europe has repeatedly been affected by droughts, resulting in considerable ecological and economic damage and climate change studies indicate a trend towards increasing climate variability most likely resulting in more frequent drought occurrences also in Europe. Against this background, the European Commission's Joint Research Centre (JRC) is developing methods and tools for assessing, monitoring and forecasting droughts in Europe and develops a European Drought Observatory (EDO) to complement and integrate national activities with a European view. At the core of the European Drought Observatory (EDO) is a portal, including a map server, a metadata catalogue, a media-monitor and analysis tools. The map server presents Europe-wide up-to-date information on the occurrence and severity of droughts, which is complemented by more detailed information provided by regional, national and local observatories through OGC compliant web mapping and web coverage services. In addition, time series of historical maps as well as graphs of the temporal evolution of drought indices for individual grid cells and administrative regions in Europe can be retrieved and analysed. Current work is focusing on validating the available products, developing combined indicators, improving the functionalities, extending the linkage to additional national and regional drought information systems and testing options for medium-range probabilistic drought forecasting across Europe. Longer-term goals include the development of long-range drought forecasting products, the analysis of drought hazard and risk, the monitoring of drought impact and the integration of EDO in a global drought information system. The talk will provide an overview on the development and state of EDO, the different products, and the ways to include a wide range of stakeholders (i.e. European, national river basin, and local authorities) in the development of the system as well as an outlook on the future developments.

Analysis of signal transduction in cell-free extracts and rafts of Xenopus eggs.

PubMed

Tokmakov, Alexander A; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

2010-05-01

Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCgamma activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs.

Effects of Pectic Polysaccharides Isolated from Leek on the Production of Reactive Oxygen and Nitrogen Species by Phagocytes

PubMed Central

Nikolova, Mariana; Ambrozova, Gabriela; Kratchanova, Maria; Denev, Petko; Kussovski, Veselin; Ciz, Milan

2013-01-01

Abstract The current survey investigates the effect of four polysaccharides isolated from fresh leek or alcohol insoluble substances (AIS) of leek on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) from phagocytes. The ability of the polysaccharides to activate serum complement was also investigated. Despite the lack of antioxidant activity, the pectic polysaccharides significantly decreased the production of ROS by human neutrophils. Polysaccharides isolated from AIS markedly activated RAW 264.7 macrophages for RNS production in a concentration-dependent manner. The Western blot analysis revealed that this effect was due to the stimulation of the inducible nitric oxide synthase protein expression of macrophages. The polysaccharides extracted from AIS with water showed the ability to fix serum complement, especially through the alternative pathway. It was found that the polysaccharide that has the highest complement-fixing effect is characterized by the highest content of uronic acids and the highest molecular weight. PMID:23905651

Complement anaphylatoxin C3a is a potent inducer of embryonic chick retina regeneration

PubMed Central

Haynes, Tracy; Luz-Madrigal, Agustin; Reis, Edimara S.; Echeverri Ruiz, Nancy P.; Grajales-Esquivel, Erika; Tzekou, Apostolia; Tsonis, Panagiotis A.; Lambris, John D.; Del Rio-Tsonis, Katia

2013-01-01

Identifying the initiation signals for tissue regeneration in vertebrates is one of the major challenges in regenerative biology. Much of the research thus far has indicated that certain growth factors have key roles. Here we show that complement fragment C3a is sufficient to induce complete regeneration of the embryonic chick retina from stem/progenitor cells present in the eye, independent of fibroblast growth factor receptor signaling. Instead, C3a induces retina regeneration via STAT3 activation, which in turn activates the injury- and inflammation-responsive factors, IL-6, IL-8 and TNF-α. This activation sets forth regulation of Wnt2b, Six3 and Sox2, genes associated with retina stem and progenitor cells. Thus, our results establish a mechanism for retina regeneration based on injury and inflammation signals. Furthermore, our results indicate a unique function for complement anaphylatoxins that implicate these molecules in the induction and complete regeneration of the retina, opening new avenues of experimentation in the field. PMID:23942241

Elevated factor H-related protein 1 and factor H pathogenic variants decrease complement regulation in IgA nephropathy.

PubMed

Tortajada, Agustín; Gutiérrez, Eduardo; Goicoechea de Jorge, Elena; Anter, Jaouad; Segarra, Alfons; Espinosa, Mario; Blasco, Miquel; Roman, Elena; Marco, Helena; Quintana, Luis F; Gutiérrez, Josué; Pinto, Sheila; Lopez-Trascasa, Margarita; Praga, Manuel; Rodriguez de Córdoba, Santiago

2017-10-01

IgA nephropathy (IgAN), a frequent cause of chronic kidney disease worldwide, is characterized by mesangial deposition of galactose-deficient IgA1-containing immune complexes. Complement involvement in IgAN pathogenesis is suggested by the glomerular deposition of complement components and the strong protection from IgAN development conferred by the deletion of the CFHR3 and CFHR1 genes (Δ CFHR3-CFHR1 ). Here we searched for correlations between clinical progression and levels of factor H (FH) and FH-related protein 1 (FHR-1) using well-characterized patient cohorts consisting of 112 patients with IgAN, 46 with non-complement-related autosomal dominant polycystic kidney disease (ADPKD), and 76 control individuals. Patients with either IgAN or ADPKD presented normal FH but abnormally elevated FHR-1 levels and FHR-1/FH ratios compared to control individuals. Highest FHR-1 levels and FHR-1/FH ratios are found in patients with IgAN with disease progression and in patients with ADPKD who have reached chronic kidney disease, suggesting that renal function impairment elevates the FHR-1/FH ratio, which may increase FHR-1/FH competition for activated C3 fragments. Interestingly, Δ CFHR3-CFHR1 homozygotes are protected from IgAN, but not from ADPKD, and we found five IgAN patients with low FH carrying CFH or CFI pathogenic variants. These data support a decreased FH activity in IgAN due to increased FHR-1/FH competition or pathogenic CFH variants. They also suggest that alternative pathway complement activation in patients with IgAN, initially triggered by galactose-deficient IgA1-containing immune complexes, may exacerbate in a vicious circle as renal function deterioration increase FHR-1 levels. Thus, a role of FHR-1 in IgAN pathogenesis is to compete with complement regulation by FH. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Interaction of Leptospira Elongation Factor Tu with Plasminogen and Complement Factor H: A Metabolic Leptospiral Protein with Moonlighting Activities

PubMed Central

Abe, Cecília M.; Monaris, Denize; Morais, Zenaide M.; Souza, Gisele O.; Vasconcellos, Sílvio A.; Isaac, Lourdes; Abreu, Patrícia A. E.; Barbosa, Angela S.

2013-01-01

The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities. PMID:24312361

Alternative complement pathway activation fragment Bb in early pregnancy as a predictor of preeclampsia

PubMed Central

Lynch, Anne M.; Murphy, James R.; Byers, Tim; Gibbs, Ronald S.; Neville, Margaret C.; Giclas, Patricia C.; Salmon, Jane E.; Holers, V. Michael

2008-01-01

OBJECTIVE Preeclampsia is a multisystem disease classically defined on the basis of hypertension and proteinuria. As shown in animal studies, complement activation is associated with inflammation in the placenta and adverse pregnancy outcomes. The association between complement activation in humans and adverse pregnancy outcomes is unclear. The purpose of this study was to determine whether elevated levels of the activation fragment Bb in early pregnancy are predictive of preeclampsia. STUDY DESIGN This prospective study of 701 women was conducted in Denver, CO. A single plasma sample was obtained from each woman before 20 weeks’ gestation. The cohort was followed up throughout pregnancy for the development of preeclampsia. Analysis included multivariate logistic regression to adjust for established risk factors for preeclampsia. RESULTS Preeclampsia developed in 4.6% of the cohort. Women with elevated Bb (90th or greater percentile) were substantially more likely to develop preeclampsia than women who had levels less than the 90th percentile (unadjusted relative risk [RR], 3.3, 95% confidence interval [CI] 1.6 to 7, P = .0009). Other significant risk factors for preeclampsia included nulliparity (RR, 2.1, 95% CI, 1–4), a high body mass index (P = .006 for trend), and maternal medical (preexisting maternal hypertension, type 1 diabetes, systemic lupus erythematosus) disease (RR, 4.4, 95% CI, 2–10). Significant risk factors among multiparous women included a history of hypertension in a previous pregnancy (RR, 5, 95% CI, 1.6 to 16) and a change of paternity (RR, 5.1, 95% CI, 1.6 to 15). Adjustment for risk factors did not attenuate the association between an elevated Bb and preeclampsia (adjusted odds ratio [OR], 3.8, 95% CI, 1.6 to 9, P = .002) in the cohort. After removing women with plasma obtained before 10 weeks, the adjusted OR of Bb in the top decile for preeclampsia was 6.1 (95% CI 2.2, 17, P = .0005). CONCLUSION The complement activation product Bb in early pregnancy is a biomarker for elevated risk of preeclampsia. This observation suggests that events linked to activation of complement in early pregnancy are associated with the pathogenesis of preeclampsia. PMID:18221926

Disease-linked mutations in factor H reveal pivotal role of cofactor activity in self-surface-selective regulation of complement activation.

PubMed

Kerr, Heather; Wong, Edwin; Makou, Elisavet; Yang, Yi; Marchbank, Kevin; Kavanagh, David; Richards, Anna; Herbert, Andrew P; Barlow, Paul N

2017-08-11

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes ( E S ) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on E S but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of E S PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on E S Conversely, PspCN boosted the CA, on E S , of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Complement activation as a bioequivalence issue relevant to the development of generic liposomes and other nanoparticulate drugs

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

Szebeni, Janos, E-mail: jszebeni2@gmail.com; Storm, Gert

Liposomes are known to activate the complement (C) system, which can lead in vivo to a hypersensitivity syndrome called C activation-related pseudoallergy (CARPA). CARPA has been getting increasing attention as a safety risk of i.v. therapy with liposomes, whose testing is now recommended in bioequivalence evaluations of generic liposomal drug candidates. This review highlights the adverse consequences of C activation, the unique symptoms of CARPA triggered by essentially all i.v. administered liposomal drugs, and the various features of vesicles influencing this adverse immune effect. For the case of Doxil, we also address the mechanism of C activation and the opsonization vs.more » long circulation (stealth) paradox. In reviewing the methods of assessing C activation and CARPA, we delineate the most sensitive porcine model and an algorithm for stepwise evaluation of the CARPA risk of i.v. liposomes, which are proposed for standardization for preclinical toxicology evaluation of liposomal and other nanoparticulate drug candidates. - Highlights: • Outlining of difficulties in generic development of liposomal drugs. • New regulatory requirements to evaluate CARPA in preclinical studies. • Review of complement activation by liposomes and its adverse consequences (CARPA). • Assays of C activation in vitro and CARPA in vivo, with the porcine test in focus. • Decision tree how to handle the risk of CARPA assessed by a battery of tests.« less