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.

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.

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

Hepatic macrophage complement receptor clearance function following injury.

PubMed

Cuddy, B G; Loegering, D J; Blumenstock, F A; Shah, D M

1986-03-01

Previous work has demonstrated that in vivo hepatic macrophage complement receptor clearance function is depressed following thermal injury. The present study was carried out to determine if complement receptor function depression is associated with other states of depressed host defense. Hepatic complement receptor clearance function was determined from the hepatic uptake of rat erythrocytes coated with antierythrocyte IgM (EIgM) in rats. Receptor function was determined following cannulation of a carotid artery, laparotomy plus enterotomy, hemorrhagic shock, trauma, thermal injury, acute bacteremia, acute endotoxemia, and injection of erythrocyte stroma, gelatinized lipid emulsion, or colloidal carbon. Hepatic uptake of EIgM was depressed following each of these experimental interventions except arterial cannulation. This effect was shown not to be due to a decrease in hepatic blood flow or depletion of complement and was therefore due to a depression in hepatic macrophage complement receptor clearance function. Thus, impairment of hepatic macrophage complement receptor function is associated with several states of depressed host defense.

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

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.

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.

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.

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.

Complement anaphylatoxins as immune regulators in cancer.

PubMed

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

2014-08-01

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

The complement system and toll-like receptors as integrated players in the pathophysiology of atherosclerosis.

PubMed

Hovland, Anders; Jonasson, Lena; Garred, Peter; Yndestad, Arne; Aukrust, Pål; Lappegård, Knut T; Espevik, Terje; Mollnes, Tom E

2015-08-01

Despite recent medical advances, atherosclerosis is a global burden accounting for numerous deaths and hospital admissions. Immune-mediated inflammation is a major component of the atherosclerotic process, but earlier research focus on adaptive immunity has gradually switched towards the role of innate immunity. The complement system and toll-like receptors (TLRs), and the crosstalk between them, may be of particular interest both with respect to pathogenesis and as therapeutic targets in atherosclerosis. Animal studies indicate that inhibition of C3a and C5a reduces atherosclerosis. In humans modified LDL-cholesterol activate complement and TLRs leading to downstream inflammation, and histopathological studies indicate that the innate immune system is present in atherosclerotic lesions. Moreover, clinical studies have demonstrated that both complement and TLRs are upregulated in atherosclerotic diseases, although interventional trials have this far been disappointing. However, based on recent research showing an intimate interplay between complement and TLRs we propose a model in which combined inhibition of both complement and TLRs may represent a potent anti-inflammatory therapeutic approach to reduce atherosclerosis. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

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.

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.

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

M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator.

PubMed

Bahia El Idrissi, Nawal; Das, Pranab K; Fluiter, Kees; Rosa, Patricia S; Vreijling, Jeroen; Troost, Dirk; Morgan, B Paul; Baas, Frank; Ramaglia, Valeria

2015-05-01

Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients.

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.

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

PubMed

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

2018-05-09

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

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2013-01-01

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

The 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.

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.

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

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

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

PubMed Central

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

1980-01-01

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

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

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.

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins.

PubMed

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; Mohana-Borges, Ronaldo

2016-11-01

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. 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. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Prepregnancy obesity and complement system activation in early pregnancy and the subsequent development of preeclampsia.

PubMed

Lynch, Anne M; Eckel, Robert H; Murphy, James R; Gibbs, Ronald S; West, Nancy A; Giclas, Patricia C; Salmon, Jane E; Holers, V Michael

2012-05-01

We hypothesized that women who are obese before they become pregnant and also have elevations of complement Bb and C3a in the top quartile in early pregnancy would have the highest risk of preeclampsia compared with a referent group of women who were not obese and had levels of complement less than the top quartile. This was a prospective study of 1013 women recruited at less than 20 weeks' gestation. An EDTA-plasma sample was obtained, and complement fragments were measured using enzyme-linked immunosorbent assays. The data were analyzed using univariable and multivariable logistic regression analysis. Women who were obese with levels of Bb or C3a in the top quartile were 10.0 (95% confidence interval, 3.3-30) and 8.8 (95% confidence interval, 3-24) times, respectively, more likely to develop preeclampsia compared with the referent group. We demonstrate a combined impact of obesity and elevated complement on the development of preeclampsia. Copyright © 2012. Published by Mosby, Inc.

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

PubMed

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

2014-06-01

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

Expression of complement membrane regulators membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59) in human malignant gliomas.

PubMed Central

Mäenpää, A.; Junnikkala, S.; Hakulinen, J.; Timonen, T.; Meri, S.

1996-01-01

Gliomas are malignant brain tumors, which, despite recent progress in surgical and radiological treatment, still have a poor prognosis. Since gliomas apparently resist immunological clearance mechanisms, we became interested in examining bow gliomas resist killing by the human complement system. The resistance of human cells to complement-mediated damage is, in large part, mediated by specific inhibitors of complement:membrane cofactor protein (CD46), decay-accelerating factor (CD55), and protectin (CD59). In the present study we examined the expression of complement regulators in 14 human glioma tumors and in 7 glioma cell lines (U251, U87, HS683, U373, U138, U118, and H2). Protectin was found to be strongly expressed by all glioma tumors and cell lines. Northern blotting analysis demonstrated the typical pattern of four to five protectin mRNAs in the glioma cells. Except for blood vessels, the expression of decay-accelerating factor was weak or absent in the tumors in situ, whereas in the cell lines its expression varied, ranging from negative to intermediate. Membrane cofactor protein was moderately expressed by all the cell lines but only weakly in the tumors. Cell-killing experiments demonstrated that the glioma cell lines were exceptionally resistant to C-mediated lysis. Five of the seven cell lines (U373, HS683, U118, U138, and H2) resisted complement lysis under conditions where most other cell lines were sensitive to killing. Neutralization experiments using specific monoclonal antibodies indicated that protectin was functionally the most important complement regulator in the glioma cells. The killing of the U87 and U251 cells could be significantly increased by a blocking anti-protectin monoclonal antibody, whereas for the other cell lines only moderate or no response was observed. The H2 cell line resisted killing by all antibodies and by complement. These results show that protectin is the most important complement regulator on human glioma cells. The exceptional complement resistance of some glioma cell lines suggests that they may utilize other, hitherto less well characterized, mechanisms to resist complement killing. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 PMID:8644856

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.

[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.

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.

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

PubMed Central

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

1980-01-01

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

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.

Phylogeny and immune evasion: a putative correlation for cerebral Pseudallescheria/Scedosporium infections.

PubMed

Rainer, Johannes; Rambach, Günter; Kaltseis, Josef; Hagleitner, Magdalena; Heiss, Silvia; Speth, Cornelia

2011-10-01

Representatives of the genus Pseudallescheria (anamorph: Scedosporium) are saprobes and the aetiologic agent of invasive mycosis in humans. After dissemination, the central nervous system (CNS) is one of the most affected organs. Prerequisites for the survival of Pseudallescheria/Scedosporium in the host are the ability to acquire nutrients and to evade the immune attack. The cleavage of complement compounds via the secretion of fungal proteases might meet both challenges since proteolytic degradation of proteins can provide nutrients and destroy the complement factors, a fast and effective immune weapon in the CNS. Therefore, we studied the capacity of different Pseudallescheria/Scedosporium species to degrade key elements of the complement cascade in the cerebrospinal fluid and investigated a correlation with the phylogenetic background. The majority of the Pseudallescheria apiosperma isolates tested were demonstrated to efficiently eliminate proteins like complement factors C3 and C1q, thus affecting two main components of a functional complement cascade, presumably by proteolytic degradation, and using them as nutrient source. In contrast, the tested strains of Pseudallescheria boydii have no or only weak capacity to eliminate these complement proteins. We hypothesise that the ability of Pseudallescheria/Scedosporium strains to acquire nutrients and to undermine the complement attack is at least partly phylogenetically determined. © 2011 Blackwell Verlag GmbH.

The Manuscript That We Finished: Structural Separation Reduces the Cost of Complement Coercion

ERIC Educational Resources Information Center

Lowder, Matthew W.; Gordon, Peter C.

2015-01-01

Two eye-tracking experiments examined the effects of sentence structure on the processing of complement coercion, in which an event-selecting verb combines with a complement that represents an entity (e.g., "began the memo"). Previous work has demonstrated that these expressions impose a processing cost, which has been attributed to the…

Expanding the Evidence Base: Comparing Randomized Controlled Trials and Observational Studies of Statins.

PubMed

Atar, Dan; Ong, Seleen; Lansberg, Peter J

2015-01-01

It is widely accepted that randomized controlled trials (RCTs) are the gold standard for demonstrating the efficacy of a given therapy (results under ideal conditions). Observational studies, on the other hand, can complement this by demonstrating effectiveness (results under real-world conditions). To examine the role that observational studies can play in complementing data from RCTs, we reviewed published studies for statins, a class of drugs that have been widely used to reduce the risk of cardiovascular (CV) events by lowering low-density lipoprotein cholesterol levels. RCTs have consistently demonstrated the benefits of statin treatment in terms of CV risk reduction and have demonstrated that more intensive statin therapy has incremental benefits over less intensive treatment. Observational studies of statin use in 'real-world' populations have served to augment the evidence base generated from statin RCTs in preselected populations of patients who are often at high CV risk and have led to similar safety and efficacy findings. They have also raised questions about factors affecting medication adherence, under-treatment, switching between statins, and failure to reach low-density lipoprotein cholesterol target levels, questions for which the answers could lead to improved patient care.

CFHR1-Modified Neural Stem Cells Ameliorated Brain Injury in a Mouse Model of Neuromyelitis Optica Spectrum Disorders.

PubMed

Shi, Kaibin; Wang, Zhen; Liu, Yuanchu; Gong, Ye; Fu, Ying; Li, Shaowu; Wood, Kristofer; Hao, Junwei; Zhang, Guang-Xian; Shi, Fu-Dong; Yan, Yaping

2016-11-01

A major hurdle for effective stem cell therapy is ongoing inflammation in the target organ. Reconditioning the lesion microenvironment may be an effective way to promote stem cell therapy. In this study, we showed that engineered neural stem cells (NSCs) with complement factor H-related protein 1, a complement inhibitor protein, can attenuate inflammatory infiltration and immune-mediated damage of astrocytes, an important pathogenic progress in patients with neuromyelitis optica spectrum disorders. Furthermore, we demonstrated that transplantation of the complement factor H-related protein 1-modified NSCs effectively blocked the complement activation cascade and inhibited formation of the membrane attack complex, thus contributing to the protection of endogenous and transplanted NSC-differentiated astrocytes. Therefore, manipulation of the lesion microenvironment contributes to a more effective cell replacement therapeutic strategy for autoimmune diseases of the CNS. Copyright © 2016 by The American Association of Immunologists, Inc.

STUDIES ON SCRUB TYPHUS

PubMed Central

Smadel, Joseph E.; Rights, Fred L.; Jackson, Elizabeth B.

1946-01-01

A complement-fixing antigen specific for scrub typhus occurs in the body fluids and tissues of infected mice, white rats, and cotton rats. The specific serological substance is demonstrable only in those animals which develop a rapidly fatal disease after an incubation period of a few days. Such an experimental infection is induced in mice and rats by the intravenous injection of suspensions of yolk sac rich in R. orientalis. Ether extraction is an important step in the preparation of a complement-fixing antigen from tissues of mice dying with scrub typhus. The Imphal No. 8 and Calcutta strains of R. orientalis are indistinguishable on the basis of complement fixation and cross-immunity tests. The complement-fixing antigen in body fluids of infected mice and rats and in our preparations of tissues from such animals occurs as a soluble antigen. Under the proper conditions the soluble antigen can be stored or dehydrated without loss of serological activity. PMID:19871518

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

PubMed Central

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

1967-01-01

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

Binding of Soluble Yeast β-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

PubMed Central

Bose, Nandita; Chan, Anissa S. H.; Guerrero, Faimola; Maristany, Carolyn M.; Qiu, Xiaohong; Walsh, Richard M.; Ertelt, Kathleen E.; Jonas, Adria Bykowski; Gorden, Keith B.; Dudney, Christine M.; Wurst, Lindsay R.; Danielson, Michael E.; Elmasry, Natalie; Magee, Andrew S.; Patchen, Myra L.; Vasilakos, John P.

2013-01-01

The immunomodulatory properties of yeast β-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate β-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble β-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast β-glucan, this study evaluated and characterized the binding of soluble β-glucan to human neutrophils and monocytes. The results demonstrated that soluble β-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble β-glucan in these cells. Binding of soluble β-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble β-glucan was demonstrated by detection of iC3b, the complement opsonin on β-glucan-bound cells, as well as by the direct binding of iC3b to β-glucan in the absence of cells. Binding of β-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding. PMID:23964276

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

PubMed Central

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

2014-01-01

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

Complement-fixing properties of antinuclear antibodies distinguish drug-induced lupus from systemic lupus erythematosus.

PubMed

Rubin, R L; Teodorescu, M; Beutner, E H; Plunkett, R W

2004-01-01

The immunofluorescence antinuclear antibody (ANA) test has been widely used to monitor autoimmune disease, but its value for diagnostic purposes is compromised by low specificity and high prevalence in disease-free individuals. The capacity of autoantibodies to fix serum complement proteins when bound to antigen is an important effector function because this property is associated with acute and chronic inflammatory processes. The current study evaluates the complement-fixing properties of antinuclear antibodies (CANA) in three well-defined and clinically-related patient groups: systemic lupus erythematosus (SLE), drug-induced lupus (DIL) and drug-induced autoimmunity (DIA). Of 20 patients diagnosed with SLE, 90% displayed complement-fixing ANA while this feature was present in only two of 18 patients with DIL and no patients with DIA without associated disease even though the mean ANA titres were similar among these patient groups. CANA was significantly correlated with anti-Sm activity. Because SLE but not DIL or DIA can be a life-threatening disease associated with complement consumption in vivo, these results demonstrate that measurement of CANA is a diagnostically useful tool and may have immunopathologic implications.

Solution-Space Screening of a Hypersonic Endurance Demonstrator

NASA Technical Reports Server (NTRS)

Chudoba, Bernd; Coleman, Gary; Oza, Amit; Gonzalez, Lex; Czysz, Paul

2012-01-01

This report documents a parametric sizing study performed to develop a program strategy for research and development and procurement of a feasible next-generation hypersonic air-breathing endurance demonstrator. Overall project focus has been on complementing technical and managerial decision-making during the earliest conceptual design phase towards minimization of operational, technical, and managerial risks.

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.

l-Alanine Auxotrophy of Lactobacillus johnsonii as Demonstrated by Physiological, Genomic, and Gene Complementation Approaches

PubMed Central

van der Kaaij, Hengameh; Desiere, Frank; Mollet, Beat; Germond, Jacques-Edouard

2004-01-01

Using a chemically defined medium without l-alanine, Lactobacillus johnsonii was demonstrated to be strictly auxotrophic for that amino acid. A comparative genetic analysis showed that all known genes involved in l-alanine biosynthesis are absent from the genome of L. johnsonii. This auxotrophy was complemented by heterologous expression of the Bacillus subtilis l-alanine dehydrogenase. PMID:15006820

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

The Role of Complement in Cnidarian-Dinoflagellate Symbiosis and Immune Challenge in the Sea Anemone Aiptasia pallida

PubMed Central

Poole, Angela Z.; Kitchen, Sheila A.; Weis, Virginia M.

2016-01-01

The complement system is an innate immune pathway that in vertebrates, is responsible for initial recognition and ultimately phagocytosis and destruction of microbes. Several complement molecules including C3, Factor B, and mannose binding lectin associated serine proteases (MASP) have been characterized in invertebrates and while most studies have focused on their conserved role in defense against pathogens, little is known about their role in managing beneficial microbes. The purpose of this study was to (1) characterize complement pathway genes in the symbiotic sea anemone Aiptasia pallida, (2) investigate the evolution of complement genes in invertebrates, and (3) examine the potential dual role of complement genes Factor B and MASP in the onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge using qPCR based studies. The results demonstrate that A. pallida has multiple Factor B genes (Ap_Bf-1, Ap_Bf-2a, and Ap_Bf-2b) and one MASP gene (Ap_MASP). Phylogenetic analysis indicates that the evolutionary history of complement genes is complex, and there have been many gene duplications or gene loss events, even within members of the same phylum. Gene expression analyses revealed a potential role for complement in both onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge. Specifically, Ap_Bf-1 and Ap_MASP are significantly upregulated in the light at the onset of symbiosis and in response to challenge with the pathogen Serratia marcescens suggesting that they play a role in the initial recognition of both beneficial and harmful microbes. Ap_Bf-2b in contrast, was generally downregulated during the onset and maintenance of symbiosis and in response to challenge with S. marcescens. Therefore, the exact role of Ap_Bf-2b in response to microbes remains unclear, but the results suggest that the presence of microbes leads to repressed expression. Together, these results indicate functional divergence between Ap_Bf-1 and Ap_Bf-2b, and that Ap_Bf-1 and Ap_MASP may be functioning together in an ancestral hybrid of the lectin and alternative complement pathways. Overall, this study provides information on the role of the complement system in a basal metazoan and its role in host-microbe interactions. PMID:27148208

The Role of Complement in Cnidarian-Dinoflagellate Symbiosis and Immune Challenge in the Sea Anemone Aiptasia pallida.

PubMed

Poole, Angela Z; Kitchen, Sheila A; Weis, Virginia M

2016-01-01

The complement system is an innate immune pathway that in vertebrates, is responsible for initial recognition and ultimately phagocytosis and destruction of microbes. Several complement molecules including C3, Factor B, and mannose binding lectin associated serine proteases (MASP) have been characterized in invertebrates and while most studies have focused on their conserved role in defense against pathogens, little is known about their role in managing beneficial microbes. The purpose of this study was to (1) characterize complement pathway genes in the symbiotic sea anemone Aiptasia pallida, (2) investigate the evolution of complement genes in invertebrates, and (3) examine the potential dual role of complement genes Factor B and MASP in the onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge using qPCR based studies. The results demonstrate that A. pallida has multiple Factor B genes (Ap_Bf-1, Ap_Bf-2a, and Ap_Bf-2b) and one MASP gene (Ap_MASP). Phylogenetic analysis indicates that the evolutionary history of complement genes is complex, and there have been many gene duplications or gene loss events, even within members of the same phylum. Gene expression analyses revealed a potential role for complement in both onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge. Specifically, Ap_Bf-1 and Ap_MASP are significantly upregulated in the light at the onset of symbiosis and in response to challenge with the pathogen Serratia marcescens suggesting that they play a role in the initial recognition of both beneficial and harmful microbes. Ap_Bf-2b in contrast, was generally downregulated during the onset and maintenance of symbiosis and in response to challenge with S. marcescens. Therefore, the exact role of Ap_Bf-2b in response to microbes remains unclear, but the results suggest that the presence of microbes leads to repressed expression. Together, these results indicate functional divergence between Ap_Bf-1 and Ap_Bf-2b, and that Ap_Bf-1 and Ap_MASP may be functioning together in an ancestral hybrid of the lectin and alternative complement pathways. Overall, this study provides information on the role of the complement system in a basal metazoan and its role in host-microbe interactions.

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.

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.

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation.

PubMed

Lekowski, R; Collard, C D; Reenstra, W R; Stahl, G L

2001-02-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress.

Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation

PubMed Central

Lekowski, Robert; Collard, Charles D.; Reenstra, Wende R.; Stahl, Gregory L.

2001-01-01

Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O2, 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 ± 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (≤ 100 μmol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC50 = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC50 ≈ 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress. PMID:11266613

Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complement-mediated virus neutralization.

PubMed

Ji, Xin; Olinger, Gene G; Aris, Sheena; Chen, Ying; Gewurz, Henry; Spear, Gregory T

2005-09-01

Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.

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.

Identification of a Naegleria fowleri Membrane Protein Reactive with Anti-Human CD59 Antibody

PubMed Central

Fritzinger, Angela E.; Toney, Denise M.; MacLean, Rebecca C.; Marciano-Cabral, Francine

2006-01-01

Naegleria fowleri, the causative agent of primary amebic meningoencephalitis, is resistant to complement lysis. The presence of a complement regulatory protein on the surface of N. fowleri was investigated. Southern blot and Northern blot analyses demonstrated hybridization of a radiolabeled cDNA probe for CD59 to genomic DNA and RNA, respectively, from pathogenic N. fowleri. An 18-kDa immunoreactive protein was detected on the membrane of N. fowleri by Western immunoblot and immunofluorescence analyses with monoclonal antibodies for human CD59. Complement component C9 immunoprecipitated with the N. fowleri “CD59-like” protein from amebae incubated with normal human serum. In contrast, a gene or protein similar to CD59 was not detected in nonpathogenic, complement-sensitive N. gruberi amebae. Collectively, our studies suggest that a protein reactive with antibodies to human CD59 is present on the surface of N. fowleri amebae and may play a role in resistance to lysis by cytolytic proteins. PMID:16428768

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

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.

Donor Brain Death Exacerbates Complement-Dependent Ischemia Reperfusion Injury in Transplanted Hearts

PubMed Central

Atkinson, Carl; Floerchinger, Bernhard; Qiao, Fei; Casey, Sarah; Williamson, Tucker; Moseley, Ellen; Stoica, Serban; Goddard, Martin; Ge, Xupeng; Tullius, Stefan G.; Tomlinson, Stephen

2013-01-01

Background Brain death (BD) can immunologically prime the donor organ and is thought to lead to exacerbated ischemia reperfusion injury (IRI) post-transplantation. Using a newly developed mouse model of BD, we investigated the effect of donor BD on post transplant cardiac IRI. We further investigated the therapeutic effect of a targeted complement inhibitor in recipients of BD donor hearts, and addressed the clinical relevance of these studies by analysis of human heart biopsies from BD and domino (living) donors. Methods and Results Hearts from living or brain dead donor C57BL/6 mice were transplanted into C57BL/6 or BALB/c recipients. Recipient mice were treated with the complement inhibitor CR2-Crry or vehicle control (n=6). Isografts were analyzed 48 hours post-transplant for injury, inflammation and complement deposition, and allografts monitored for graft survival. Human cardiac biopsies were analyzed for complement deposition and inflammatory cell infiltration. In the murine model, donor BD exacerbated IRI and graft rejection as demonstrated by increased myocardial injury, serum cardiac troponin, cellular infiltration, inflammatory chemokine and cytokine levels, complement deposition, and decreased graft survival. CR2-Crry treatment of recipients significantly reduced all measured outcomes in grafts from both BD and living donors compared to controls. Analysis of human samples documented the relevance of our experimental findings and revealed exacerbated complement deposition and inflammation in grafts from BD donors compared to grafts from living donors. Conclusions BD exacerbates post-transplant cardiac IRI in mice and humans, and decreases survival of mouse allografts. Further, targeted complement inhibition in recipient mice ameliorates BD-exacerbated IRI. PMID:23443736

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

A Viral Receptor Complementation Strategy to Overcome CAV-2 Tropism for Efficient Retrograde Targeting of Neurons.

PubMed

Li, Shu-Jing; Vaughan, Alexander; Sturgill, James Fitzhugh; Kepecs, Adam

2018-06-06

Retrogradely transported neurotropic viruses enable genetic access to neurons based on their long-range projections and have become indispensable tools for linking neural connectivity with function. A major limitation of viral techniques is that they rely on cell-type-specific molecules for uptake and transport. Consequently, viruses fail to infect variable subsets of neurons depending on the complement of surface receptors expressed (viral tropism). We report a receptor complementation strategy to overcome this by potentiating neurons for the infection of the virus of interest-in this case, canine adenovirus type-2 (CAV-2). We designed AAV vectors for expressing the coxsackievirus and adenovirus receptor (CAR) throughout candidate projection neurons. CAR expression greatly increased retrograde-labeling rates, which we demonstrate for several long-range projections, including some resistant to other retrograde-labeling techniques. Our results demonstrate a receptor complementation strategy to abrogate endogenous viral tropism and thereby facilitate efficient retrograde targeting for functional analysis of neural circuits. Copyright © 2018 Elsevier Inc. All rights reserved.

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.

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

PubMed Central

2012-01-01

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

Defining the genetics of thrombotic microangiopathies.

PubMed

Vieira-Martins, Paula; El Sissy, Carine; Bordereau, Pauline; Gruber, Aurelia; Rosain, Jeremie; Fremeaux-Bacchi, Veronique

2016-04-01

The spectrum of the thrombotic microangiopathies (TMA) encompasses a heterogeneous group of disorders with hereditary and acquired forms. Endothelial cell injury in the microvasculature is common to all TMAs, whatever the pathophysiological process. In this review we describe genetic mutations characteristic of certain TMAs and review their contributions to disease. Recent identification of novel pathologic mutations has been enabled by exome studies. The monogenic forms of TMA are more frequently caused by recessive alterations in von Willebrand factor cleaving protease ADAMST13, leading to congenital thrombotic thrombocytopenic purpura, or cobalamine C and DGKE genes, leading to an atypical hemolytic-uremic syndrome (aHUS)-like TMA. aHUS, whether idiopathic or linked to a known complement amplifying condition, is a TMA that primarily affects kidney function. It often results from a combination of an underlying genetic susceptibility with environmental factors activating the alternative complement pathway. Pathogenic variants in at least five complement genes coding for complement factor H (CFH) complement factor I (CFI), MCP (CD46), C3 and complement factor B (CFB) have been demonstrated to increase the risk of developing aHUS, but several more genes have been implicated. A new challenge is to separate disease-associated genetic variants from the broader background of variants or polymorphisms present in all human genomes that are rare, potentially functional, but may or may not be pathogenic. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface

PubMed Central

Medina, Eva; van Rooijen, Willemien J.; Spaan, András N.; van Kessel, Kok P. M.; Höök, Magnus; Rooijakkers, Suzan H. M.

2013-01-01

Upon contact with human plasma, bacteria are rapidly recognized by the complement system that labels their surface for uptake and clearance by phagocytic cells. Staphylococcus aureus secretes the 16 kD Extracellular fibrinogen binding protein (Efb) that binds two different plasma proteins using separate domains: the Efb N-terminus binds to fibrinogen, while the C-terminus binds complement C3. In this study, we show that Efb blocks phagocytosis of S. aureus by human neutrophils. In vitro, we demonstrate that Efb blocks phagocytosis in plasma and in human whole blood. Using a mouse peritonitis model we show that Efb effectively blocks phagocytosis in vivo, either as a purified protein or when produced endogenously by S. aureus. Mutational analysis revealed that Efb requires both its fibrinogen and complement binding residues for phagocytic escape. Using confocal and transmission electron microscopy we show that Efb attracts fibrinogen to the surface of complement-labeled S. aureus generating a ‘capsule’-like shield. This thick layer of fibrinogen shields both surface-bound C3b and antibodies from recognition by phagocytic receptors. This information is critical for future vaccination attempts, since opsonizing antibodies may not function in the presence of Efb. Altogether we discover that Efb from S. aureus uniquely escapes phagocytosis by forming a bridge between a complement and coagulation protein. PMID:24348255

Exploring photoinactivation of microbial biofilms using laser scanning microscopy and confined two-photon excitation.

PubMed

Thomsen, Hanna; Graf, Fabrice E; Farewell, Anne; Ericson, Marica B

2018-05-21

One pertinent complication in bacterial infection is the growth of biofilms, i.e., communities of surface-adhered bacteria resilient to antibiotics. Photodynamic inactivation has been proposed as an alternative to antibiotic treatment; however, novel techniques complementing standard efficacy measures are required. Herein, we present an approach employing multiphoton microscopy complemented with Airyscan super-resolution microscopy, to visualize the distribution of curcumin in Staphylococcus epidermidis biofilms. The effects of complexation of curcumin with hydroxypropyl-γ-cyclodextrin (HPγCD) were studied. It was shown that HPγCD-curcumin demonstrated higher bioavailability in the biofilms compared to curcumin, without affecting the subcellular uptake. Spectral quantification following photodynamic inactivation demonstrates a method for monitoring elimination of biofilms in real time using noninvasive 3D imaging. Additionally, spatially confined two-photon inactivation was demonstrated for the first time in biofilms. These results support the feasibility of advanced optical microscopy as a sensitive tool for evaluating treatment efficacy in biofilms towards improved mechanistic studies of photodynamic inactivation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Study on recognition technology of complementary image

NASA Astrophysics Data System (ADS)

Liu, Chengxiang; Hu, Xuejuan; Jian, Yaobo; Zhang, Li

2006-11-01

Complementation image is often used as a guard technology in the trademark and paper currency. The key point of recognizing this kind of images is judging the complementary effect of complementation printing. The perspective images are usually not clear and legible, so it is difficult to recognize them. In this paper, a new method is proposed. Firstly, capture the image by reflex. Secondly, find the same norm to man-made pair printing. Lastly, judge the true and false of paper currency by the complementary effect of complementation printing. This is the purpose of inspecting the false. Theoretic analysis and simulation results reveal that the effect of man-made pair printing is good, the method has advantages such as simplicity, high calculating speed, and good robust to different RMB. The experiment results reveal that the conclusion is reasonable, and demonstrates that this approach is effective.

Verbal Complementizers in Arabic

ERIC Educational Resources Information Center

Ahmed, Hossam Eldin Ibrahim

2015-01-01

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

AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7.

PubMed

Meyer, Benjamin H; Shams-Eldin, Hosam; Albers, Sonja-Verena

2017-01-01

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D 100 ), IV (F 220 ) and V (F 264 ) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

Studies on the serological relationships between avian pox, sheep pox, goat pox and vaccinia viruses

PubMed Central

Uppal, P. K.; Nilakantan, P. R.

1970-01-01

By using neutralization, complement fixation and immunogel-diffusion tests, it has been demonstrated that cross-reactions occur between various avian pox viruses and between sheep pox and goat pox viruses. No such reactions were demonstrated between avian pox viruses and vaccinia virus or between avian pox and sheep pox and goat pox viruses. Furthermore, no serological relationship was demonstrable between vaccinia virus and sheep pox and goat pox viruses. PMID:4989854

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

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

PubMed

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

2002-06-25

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30-40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges.

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

PubMed Central

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

2002-01-01

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30–40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges. PMID:12034872

Studies of Dynamic Protein-Protein Interactions in Bacteria Using Renilla Luciferase Complementation Are Undermined by Nonspecific Enzyme Inhibition

PubMed Central

Hatzios, Stavroula K.; Ringgaard, Simon; Davis, Brigid M.; Waldor, Matthew K.

2012-01-01

The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology. PMID:22905225

Studies of dynamic protein-protein interactions in bacteria using Renilla luciferase complementation are undermined by nonspecific enzyme inhibition.

PubMed

Hatzios, Stavroula K; Ringgaard, Simon; Davis, Brigid M; Waldor, Matthew K

2012-01-01

The luciferase protein fragment complementation assay is a powerful tool for studying protein-protein interactions. Two inactive fragments of luciferase are genetically fused to interacting proteins, and when these two proteins interact, the luciferase fragments can reversibly associate and reconstitute enzyme activity. Though this technology has been used extensively in live eukaryotic cells, split luciferase complementation has not yet been applied to studies of dynamic protein-protein interactions in live bacteria. As proof of concept and to develop a new tool for studies of bacterial chemotaxis, fragments of Renilla luciferase (Rluc) were fused to the chemotaxis-associated response regulator CheY3 and its phosphatase CheZ in the enteric pathogen Vibrio cholerae. Luciferase activity was dependent on the presence of both CheY3 and CheZ fusion proteins, demonstrating the specificity of the assay. Furthermore, enzyme activity was markedly reduced in V. cholerae chemotaxis mutants, suggesting that this approach can measure defects in chemotactic signaling. However, attempts to measure changes in dynamic CheY3-CheZ interactions in response to various chemoeffectors were undermined by nonspecific inhibition of the full-length luciferase. These observations reveal an unexpected limitation of split Rluc complementation that may have implications for existing data and highlight the need for great caution when evaluating small molecule effects on dynamic protein-protein interactions using the split luciferase technology.

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.

Targeted organ generation using Mixl1-inducible mouse pluripotent stem cells in blastocyst complementation.

PubMed

Kobayashi, Toshihiro; Kato-Itoh, Megumi; Nakauchi, Hiromitsu

2015-01-15

Generation of functional organs from patients' own cells is one of the ultimate goals of regenerative medicine. As a novel approach to creation of organs from pluripotent stem cells (PSCs), we employed blastocyst complementation in organogenesis-disabled animals and successfully generated PSC-derived pancreas and kidneys. Blastocyst complementation, which exploits the capacity of PSCs to participate in forming chimeras, does not, however, exclude contribution of PSCs to the development of tissues-including neural cells or germ cells-other than those specifically targeted by disabling of organogenesis. This fact provokes ethical controversy if human PSCs are to be used. In this study, we demonstrated that forced expression of Mix-like protein 1 (encoded by Mixl1) can be used to guide contribution of mouse embryonic stem cells to endodermal organs after blastocyst injection. We then succeeded in applying this method to generate functional pancreas in pancreatogenesis-disabled Pdx1 knockout mice using a newly developed tetraploid-based organ-complementation method. These findings hold promise for targeted organ generation from patients' own PSCs in livestock animals.

Studies of Antigens for Complement Fixation and Gel Diffusion Tests in the Diagnosis of Infections Caused by Brucella ovis and Other Brucella

PubMed Central

Myers, Donald M.; Jones, Lois M.; Varela-Diaz, Victor M.

1972-01-01

Sonically treated and saline-extracted antigens of Brucella ovis, B. canis, B. abortus, and B. melitensis were compared in gel diffusion, complement fixation, and serum absorption tests. All the sonically extracted antigens showed cross-reactions with sera from animals infected or immunized with these species, whereas the saline-extracted antigens were specific for the surface of the rough or smooth colonial phase of the species or strain. The saline-extracted antigens of B. ovis and B. melitensis were both eluted as a single peak in the void volume by Sephadex G-200 column chromatography, in gel diffusion had staining characteristics of lipoproteins, but in immunoelectrophoresis showed distinct mobility patterns. Serological activity for both gel diffusion and complement fixation tests was demonstrated in the immunoglobulin G-containing fraction of sera taken from sheep 12 to 412 days after infection with B. ovis. The gel diffusion test with saline extract of B. ovis is as sensitive as the complement fixation test for the diagnosis of ram epididymitis and is more practical. Images PMID:4624210

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

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.

Anti-GK1 antibodies damage Taenia crassiceps cysticerci through complement activation.

PubMed

Núñez, Guadalupe; Villalobos, Nelly; Herrera, Cinthia P; Navarrete-Perea, José; Méndez, Adriana; Martinez-Maya, José J; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Aguilar, Laura; Del Arenal, Irene P

2018-06-06

Taeniasis-cysticercosis, a zoonosis caused by Taenia solium, is prevalent in underdeveloped countries, where marginalization promotes its continued transmission. Pig cysticercosis, an essential stage for transmission, is preventable by vaccination. An efficient multiepitope vaccine against pig cysticercosis, S3Pvac, was developed. Previous studies showed that antibodies against one of the S3Pvac components, GK-1, are capable of damaging T. solium cysticerci, inhibiting their ability to transform into the adult stage in golden hamster gut. This study is aimed to evaluate one of the mechanisms that could mediate anti-GK-1 antibody-dependent protection. To this end, pig anti-GK-1 antibodies were produced and purified by using protein A. Proteomic analysis showed that the induced antibodies recognized the respective native cysticercal protein KE7 (Bobes et al. Infect Immun 85:e00395-17, 2017) and two additional T. solium proteins (endophilin B1 and Gp50). A new procedure to evaluate cysticercus viability, based on quantifying the cytochrome c released after parasite damage, was developed. Taenia crassiceps cysticerci were cultured in the presence of differing amounts of anti-GK-1 antibody and complement in a saturating concentration, along with the respective controls. Cysticercus viability was assessed by recording parasite motility, trypan blue exclusion, and cytochrome c levels in cysticercal soluble extract. Anti-GK-1 antibody significantly increased cysticercus damage as measured by all three methods. Parasite evaluation by electron microscopy after treatment with anti-GK-1 antibody plus complement demonstrated cysticercus damage as shorter, capsule-severed microtrichia; a decrease in glycocalyx length with respect to untreated cysts; and disaggregated desmosomes. These results demonstrate that anti-GK-1 antibodies damage cysticerci through classic complement activation.

Sexual dimorphism in mammalian autosomal gene regulation is determined not only by Sry but by sex chromosome complement as well.

PubMed

Wijchers, Patrick J; Yandim, Cihangir; Panousopoulou, Eleni; Ahmad, Mushfika; Harker, Nicky; Saveliev, Alexander; Burgoyne, Paul S; Festenstein, Richard

2010-09-14

Differences between males and females are normally attributed to developmental and hormonal differences between the sexes. Here, we demonstrate differences between males and females in gene silencing using a heterochromatin-sensitive reporter gene. Using "sex-reversal" mouse models with varying sex chromosome complements, we found that this differential gene silencing was determined by X chromosome complement, rather than sex. Genome-wide transcription profiling showed that the expression of hundreds of autosomal genes was also sensitive to sex chromosome complement. These genome-wide analyses also uncovered a role for Sry in modulating autosomal gene expression in a sex chromosome complement-specific manner. The identification of this additional layer in the establishment of sexual dimorphisms has implications for understanding sexual dimorphisms in physiology and disease. Copyright © 2010 Elsevier Inc. All rights reserved.

Characterization of RbmD (glycosyltransferase in ribostamycin gene cluster) through neomycin production reconstituted from the engineered Streptomyces fradiae BS1.

PubMed

Nepal, Keshav Kumar; Oh, Tae-Jin; Subba, Bimala; Yoo, Jin Cheol; Sohng, Jae Kyung

2009-01-31

Amino acid homology analysis predicted that rbmD, a putative glycosyltransferase from Streptomyces ribosidificus ATCC 21294, has the highest homology with neoD in neomycin biosynthesis. S. fradiae BS1, in which the production of neomycin was abolished, was generated by disruption of the neoD gene in the neomycin producer S. fradiae. The restoration of neomycin by self complementation suggested that there was no polar effect in the mutant. In addition, S. fradiae BS6 was created with complementation by rbmD in S. fradiae BS1, and secondary metabolite analysis by ESI/MS, LC/MS and MS/MS showed the restoration of neomycin production in S. fradiae BS6. These gene inactivation and complementation studies suggested that, like neoD, rbmD functions as a 2-N-acetlyglucosaminyltransferase and demonstrated the potential for the generation of novel aminoglycoside antibiotics using glycosyltransferases in vivo.

Microinjection of cytoplasm as a test of complementation in Paramecium

PubMed Central

1982-01-01

Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard- to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species. PMID:7061597

THE INHIBITION OF PLASMIN, PLASMA KALLIKREIN, PLASMA PERMEABILITY FACTOR, AND THE C'1r SUBCOMPONENT OF THE FIRST COMPONENT OF COMPLEMENT BY SERUM C'1 ESTERASE INHIBITOR

PubMed Central

Ratnoff, Oscar D.; Pensky, Jack; Ogston, Derek; Naff, George B.

1969-01-01

The fraction of human serum designated as C'1 esterase inhibitor is known to inhibit the action of C'1 esterase, a plasma kallikrein, and PF/Dil, an enzyme in plasma enhancing cutaneous vascular permeability. In the present study, C'1 esterase inhibitor has been found to block the actions of plasmin and the C'1r subcomponent of the first component of complement, and to retard the generation of PF/Dil. No inhibition of blood clotting or of the generation of plasmin was demonstrable. PMID:4178758

Simulating History to Understand International Politics

ERIC Educational Resources Information Center

Weir, Kimberly; Baranowski, Michael

2011-01-01

To understand world politics, one must appreciate the context in which international systems develop and operate. Pedagogy studies demonstrate that the more active students are in their learning, the more they learn. As such, using computer simulations can complement and enhance classroom instruction. CIVILIZATION is a computer simulation game…

Discriminating the hemolytic risk of blood type A plasmas using the complement hemolysis using human erythrocytes (CHUHE) assay.

PubMed

Cunnion, Kenji M; Hair, Pamela S; Krishna, Neel K; Sass, Megan A; Enos, Clinton W; Whitley, Pamela H; Maes, Lanne Y; Goldberg, Corinne L

2017-03-01

The agglutination-based cross-matching method is sensitive for antibody binding to red blood cells but is only partially predictive of complement-mediated hemolysis, which is important in many acute hemolytic transfusion reactions. Here, we describe complement hemolysis using human erythrocytes (CHUHE) assays that directly evaluate complement-mediated hemolysis between individual serum-plasma and red blood cell combinations. The CHUHE assay is used to evaluate correlations between agglutination titers and complement-mediated hemolysis as well as the hemolytic potential of plasma from type A blood donors. Plasma or serum from each type A blood donor was incubated with AB or B red blood cells in the CHUHE assay and measured for free hemoglobin release. CHUHE assays for serum or plasma demonstrate a wide, dynamic range and high sensitivity for complement-mediated hemolysis for individual serum/plasma and red blood cell combinations. CHUHE results suggest that agglutination assays alone are only moderately predictive of complement-mediated hemolysis. CHUHE results also suggest that plasma from particular type A blood donors produce minimal complement-mediated hemolysis, whereas plasma from other type A blood donors produce moderate to high-level complement-mediated hemolysis, depending on the red blood cell donor. The current results indicate that the CHUHE assay can be used to assess complement-mediated hemolysis for plasma or serum from a type A blood donor, providing additional risk discrimination over agglutination titers alone. © 2016 AABB.

Contemporary Films' Mini Course on Film Study.

ERIC Educational Resources Information Center

Schillaci, Peter

This minicourse on film study can be a unit in English, in arts, or in the humanities. It can help to launch a film study course or complement an introduction to theater. Whatever form it takes, it helps to build a bridge to the student's media environment. Part one, the language of images, utilizes four films which demonstrate the basic elements…

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

Biomedical Research Experiences for Biology Majors at a Small College

ERIC Educational Resources Information Center

Stover, Shawn K.; Mabry, Michelle L.

2010-01-01

A program-level assessment of the biology curriculum at a small liberal arts college validates a previous study demonstrating success in achieving learning outcomes related to content knowledge and communication skills. Furthermore, research opportunities have been provided to complement pedagogical strategies and give students a more complete…

The Educational and Economic Value of Online Learning for Museums

ERIC Educational Resources Information Center

Crow, William B.; Din, Herminia

2010-01-01

Given the increasingly interactive, dynamic, and cost-effective online learning platforms that are available, as well as studies that demonstrate the efficacy of online and blended learning, museums should consider how these new formats may complement their existing educational programs. Besides offering new possibilities for education, online…

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

PubMed

Prechl, József; Papp, Krisztián; Hérincs, Zoltán; Péterfy, Hajna; Lóránd, Veronika; Szittner, Zoltán; Estonba, Andone; Rovero, Paolo; Paolini, Ilaria; Del Amo, Jokin; Uribarri, Maria; Alcaro, Maria Claudia; Ruiz-Larrañaga, Otsanda; Migliorini, Paola; Czirják, László

2016-01-01

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

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.

Neuron-derived IgG protects neurons from complement-dependent cytotoxicity.

PubMed

Zhang, Jie; Niu, Na; Li, Bingjie; McNutt, Michael A

2013-12-01

Passive immunity of the nervous system has traditionally been thought to be predominantly due to the blood-brain barrier. This concept must now be revisited based on the existence of neuron-derived IgG. The conventional concept is that IgG is produced solely by mature B lymphocytes, but it has now been found to be synthesized by murine and human neurons. However, the function of this endogenous IgG is poorly understood. In this study, we confirm IgG production by rat cortical neurons at the protein and mRNA levels, with 69.0 ± 5.8% of cortical neurons IgG-positive. Injury to primary-culture neurons was induced by complement leading to increases in IgG production. Blockage of neuron-derived IgG resulted in more neuronal death and early apoptosis in the presence of complement. In addition, FcγRI was found in microglia and astrocytes. Expression of FcγR I in microglia was increased by exposure to neuron-derived IgG. Release of NO from microglia triggered by complement was attenuated by neuron-derived IgG, and this attenuation could be reversed by IgG neutralization. These data demonstrate that neuron-derived IgG is protective of neurons against injury induced by complement and microglial activation. IgG appears to play an important role in maintaining the stability of the nervous system.

Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes.

PubMed

Yang, C; Jones, J L; Barnum, S R

1993-09-01

In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells, demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.

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

XY sex chromosome complement, compared with XX, in the CNS confers greater neurodegeneration during experimental autoimmune encephalomyelitis

PubMed Central

Du, Sienmi; Itoh, Noriko; Askarinam, Sahar; Hill, Haley; Arnold, Arthur P.; Voskuhl, Rhonda R.

2014-01-01

Women are more susceptible to multiple sclerosis (MS) and have more robust immune responses than men. However, men with MS tend to demonstrate a more progressive disease course than women, suggesting a disconnect between the severity of an immune attack and the CNS response to a given immune attack. We have previously shown in an MS model, experimental autoimmune encephalomyelitis, that autoantigen-sensitized XX lymph node cells, compared with XY, are more encephalitogenic. These studies demonstrated an effect of sex chromosomes in the induction of immune responses, but did not address a potential role of sex chromosomes in the CNS response to immune-mediated injury. Here, we examined this possibility using XX versus XY bone marrow chimeras reconstituted with a common immune system of one sex chromosomal type. We found that experimental autoimmune encephalomyelitis mice with an XY sex chromosome complement in the CNS, compared with XX, demonstrated greater clinical disease severity with more neuropathology in the spinal cord, cerebellum, and cerebral cortex. A candidate gene on the X chromosome, toll-like receptor 7, was then examined. Toll-like receptor 7 expression in cortical neurons was higher in mice with XY compared with mice with XX CNS, consistent with the known neurodegenerative role for toll-like receptor 7 in neurons. These results suggest that sex chromosome effects on neurodegeneration in the CNS run counter to effects on immune responses, and may bear relevance to the clinical enigma of greater MS susceptibility in women but faster disability progression in men. This is a demonstration of a direct effect of sex chromosome complement on neurodegeneration in a neurological disease. PMID:24550311

XY sex chromosome complement, compared with XX, in the CNS confers greater neurodegeneration during experimental autoimmune encephalomyelitis.

PubMed

Du, Sienmi; Itoh, Noriko; Askarinam, Sahar; Hill, Haley; Arnold, Arthur P; Voskuhl, Rhonda R

2014-02-18

Women are more susceptible to multiple sclerosis (MS) and have more robust immune responses than men. However, men with MS tend to demonstrate a more progressive disease course than women, suggesting a disconnect between the severity of an immune attack and the CNS response to a given immune attack. We have previously shown in an MS model, experimental autoimmune encephalomyelitis, that autoantigen-sensitized XX lymph node cells, compared with XY, are more encephalitogenic. These studies demonstrated an effect of sex chromosomes in the induction of immune responses, but did not address a potential role of sex chromosomes in the CNS response to immune-mediated injury. Here, we examined this possibility using XX versus XY bone marrow chimeras reconstituted with a common immune system of one sex chromosomal type. We found that experimental autoimmune encephalomyelitis mice with an XY sex chromosome complement in the CNS, compared with XX, demonstrated greater clinical disease severity with more neuropathology in the spinal cord, cerebellum, and cerebral cortex. A candidate gene on the X chromosome, toll-like receptor 7, was then examined. Toll-like receptor 7 expression in cortical neurons was higher in mice with XY compared with mice with XX CNS, consistent with the known neurodegenerative role for toll-like receptor 7 in neurons. These results suggest that sex chromosome effects on neurodegeneration in the CNS run counter to effects on immune responses, and may bear relevance to the clinical enigma of greater MS susceptibility in women but faster disability progression in men. This is a demonstration of a direct effect of sex chromosome complement on neurodegeneration in a neurological disease.

Favorite Demonstrations for College Science

ERIC Educational Resources Information Center

Shmaefsky, Brian

2004-01-01

Peer-reviewed, classroom-tested, and tailored specifically for introductory science courses, Favorite Demonstrations is a complement to every college instructor's lesson plans. The book is an all-in-one compilation of 36 popular classroom demonstrations published since 1993 in the "Favorite Demonstration" column of NSTA's Journal of College …

Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

USDA-ARS?s Scientific Manuscript database

Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

Immune response and histology of humoral rejection in kidney transplantation.

PubMed

González-Molina, Miguel; Ruiz-Esteban, Pedro; Caballero, Abelardo; Burgos, Dolores; Cabello, Mercedes; Leon, Miriam; Fuentes, Laura; Hernandez, Domingo

2016-01-01

The adaptive immune response forms the basis of allograft rejection. Its weapons are direct cellular cytotoxicity, identified from the beginning of organ transplantation, and/or antibodies, limited to hyperacute rejection by preformed antibodies and not as an allogenic response. This resulted in allogenic response being thought for decades to have just a cellular origin. But the experimental studies by Gorer demonstrating tissue damage in allografts due to antibodies secreted by B lymphocytes activated against polymorphic molecules were disregarded. The special coexistence of binding and unbinding between antibodies and antigens of the endothelial cell membranes has been the cause of the delay in demonstrating the humoral allogenic response. The endothelium, the target tissue of antibodies, has a high turnover, and antigen-antibody binding is non-covalent. If endothelial cells are attacked by the humoral response, immunoglobulins are rapidly removed from their surface by shedding and/or internalization, as well as degrading the components of the complement system by the action of MCP, DAF and CD59. Thus, the presence of complement proteins in the membrane of endothelial cells is transient. In fact, the acute form of antibody-mediated rejection was not demonstrated until C4d complement fragment deposition was identified, which is the only component that binds covalently to endothelial cells. This review examines the relationship between humoral immune response and the types of acute and chronic histological lesion shown on biopsy of the transplanted organ. Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

The alpha-TIF (VP16) homologue (ETIF) of equine herpesvirus 1 is essential for secondary envelopment and virus egress.

PubMed

von Einem, Jens; Schumacher, Daniel; O'Callaghan, Dennis J; Osterrieder, Nikolaus

2006-03-01

The equine herpesvirus 1 (EHV-1) alpha-trans-inducing factor homologue (ETIF; VP16-E) is a 60-kDa virion component encoded by gene 12 (ORF12) that transactivates the immediate-early gene promoter. Here we report on the function of EHV-1 ETIF in the context of viral infection. An ETIF-null mutant from EHV-1 strain RacL11 (vL11deltaETIF) was constructed and analyzed. After transfection of vL11deltaETIF DNA into RK13 cells, no infectious virus could be reconstituted, and only single infected cells or small foci containing up to eight infected cells were detected. In contrast, after transfection of vL11deltaETIF DNA into a complementing cell line, infectious virus could be recovered, indicating the requirement of ETIF for productive virus infection. The growth defect of vL11deltaETIF could largely be restored by propagation on the complementing cell line, and growth on the complementing cell line resulted in incorporation of ETIF in mature and secreted virions. Low- and high-multiplicity infections of RK13 cells with phenotypically complemented vL11deltaETIF virus resulted in titers of virus progeny similar to those used for infection, suggesting that input ETIF from infection was recycled. Ultrastructural studies of vL11deltaETIF-infected cells demonstrated a marked defect in secondary envelopment at cytoplasmic membranes, resulting in very few enveloped virions in transport vesicles or extracellular space. Taken together, our results demonstrate that ETIF has an essential function in the replication cycle of EHV-1, and its main role appears to be in secondary envelopment.

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

Development and Use of a Serum Bactericidal Assay Using Pooled Human Complement To Assess Responses to a Meningococcal Group A Conjugate Vaccine in African Toddlers

PubMed Central

Lynn, Freyja; Mocca, Brian; Borrow, Ray; Findlow, Helen; Hassan-King, Musa; Preziosi, Marie-Pierre; Idoko, Olubukola; Sow, Samba; Kulkarni, Prasad; LaForce, F. Marc

2014-01-01

A meningococcal group A polysaccharide (PS) conjugate vaccine (PsA-TT) has been developed for African countries affected by epidemic meningitis caused by Neisseria meningitidis. Complement-mediated serum bactericidal antibody (SBA) assays are used to assess protective immune responses to meningococcal vaccination. Human complement (hC′) was used in early studies demonstrating antibody-mediated protection against disease, but it is difficult to obtain and standardize. We developed and evaluated a method for sourcing hC′ and then used the SBA assay with hC′ (hSBA) to measure bactericidal responses to PsA-TT vaccination in 12- to 23-month-old African children. Sera with active complement from 100 unvaccinated blood donors were tested for intrinsic bactericidal activity, SBA titer using rabbit complement (rSBA), and anti-group A PS antibody concentration. Performance criteria and pooling strategies were examined and then verified by comparisons of three independently prepared hC′ lots in two laboratories. hSBA titers of clinical trial sera were then determined using this complement sourcing method. Two different functional antibody tests were necessary for screening hC′. hSBA titers determined using three independent lots of pooled hC′ were within expected assay variation among lots and between laboratories. In African toddlers, PsA-TT elicited higher hSBA titers than meningococcal polysaccharide or Hib vaccines. PsA-TT immunization or PS challenge of PsA-TT-primed subjects resulted in vigorous hSBA memory responses, and titers persisted in boosted groups for over a year. Quantifying SBA using pooled hC′ is feasible and showed that PsA-TT was highly immunogenic in African toddlers. PMID:24671551

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

Influence of heat inactivation of human serum on the opsonization of Streptococcus mutans.

PubMed

Moore, M A; Hakki, Z W; Gregory, R L; Gfell, L E; Kim-Park, W K; Kowolik, M J

1997-12-15

Phagocytosis of bacteria, such as Streptococcus mutans, is important to host defense. One mechanism by which phagocytosis can be enhanced is by antibody or complement-mediated opsonization of bacteria. Many studies utilize opsonization of bacteria to enhance a cellular response, but little information has been found examining methodology or validity of the opsonization process following the denaturization of the serum. Human serum was inactivated by heat in order to disrupt the classical and alternative pathways of the complement cascade. S. mutans isolated from human subjects were opsonized with heat-inactivated human serum before exposing them to viable neutrophils in vitro. Luminol-dependent chemiluminescence (CL) was used to measure neutrophil activation. Human serum used to opsonize the bacteria was denatured by incubation at 57 degrees C for intervals of 30 and 60 min to inactivate complement. The results from the opsonization data indicated that there was significantly increased CL with 60-min inactivation of the serum (34% increase in mean integration mV.min; p < or = 0.05) over the nonopsonized control. This indicated a successful opsonization of the bacteria. In addition, the data demonstrate that the inactivation of serum requires a minimum of 60 min at 57 degrees C to disrupt the complement cascade, while 30- and 15-min inactivations produced no significant increase in CL activity over the control. Standard sandwich ELISA assays, detecting complement binding to S. mutans, confirmed successful heat inactivation of serum showing a significant decrease (p < or = 0.001) in complement binding to S. mutans after 30 min, but could not explain the increased CL response after 60-min heat deactivation of the serum.

Chickpea transcription factor CaTLP1 interacts with protein kinases, modulates ROS accumulation and promotes ABA-mediated stomatal closure

PubMed Central

Wardhan, Vijay; Pandey, Aarti; Chakraborty, Subhra; Chakraborty, Niranjan

2016-01-01

Tubby and Tubby-like proteins (TLPs), in mammals, play critical roles in neural development, while its function in plants is largely unknown. We previously demonstrated that the chickpea TLP, CaTLP1, participates in osmotic stress response and might be associated with ABA-dependent network. However, how CaTLP1 is connected to ABA signaling remains unclear. The CaTLP1 was found to be engaged in ABA-mediated gene expression and stomatal closure. Complementation of the yeast yap1 mutant with CaTLP1 revealed its role in ROS scavenging. Furthermore, complementation of Arabidopsis attlp2 mutant displayed enhanced stress tolerance, indicating the functional conservation of TLPs across the species. The presence of ABA-responsive element along with other motifs in the proximal promoter regions of TLPs firmly established their involvement in stress signalling pathways. The CaTLP1 promoter driven GUS expression was restricted to the vegetative organs, especially stem and rosette leaves. Global protein expression profiling of wild-type, attlp2 and complemented Arabidopsis plants revealed 95 differentially expressed proteins, presumably involved in maintaining physiological and biological processes under dehydration. Immunoprecipitation assay revealed that protein kinases are most likely to interact with CaTLP1. This study provides the first demonstration that the TLPs act as module for ABA-mediated stomatal closure possibly via interaction with protein kinase. PMID:27934866

Scabies Mite Peritrophins Are Potential Targets of Human Host Innate Immunity

PubMed Central

Holt, Deborah C.; Kemp, Dave J.; Fischer, Katja

2011-01-01

Background Pruritic scabies lesions caused by Sarcoptes scabiei burrowing in the stratum corneum of human skin facilitate opportunistic bacterial infections. Emerging resistance to current therapeutics emphasizes the need to identify novel targets for protective intervention. We have characterized several protein families located in the mite gut as crucial factors for host-parasite interactions. Among these multiple proteins inhibit human complement, presumably to avoid complement-mediated damage of gut epithelial cells. Peritrophins are major components of the peritrophic matrix often found in the gut of arthropods. We hypothesized that a peritrophin, if abundant in the scabies mite gut, could be an activator of complement. Methodology/Principal Findings A novel full length scabies mite peritrophin (SsPTP1) was identified in a cDNA library from scabies mites. The amino acid sequence revealed four putative chitin binding domains (CBD). Recombinant expression of one CBD of the highly repetitive SsPTP1 sequence as TSP-hexaHis-fusion protein resulted in soluble protein, which demonstrated chitin binding activity in affinity chromatography assays. Antibodies against a recombinant SsPTP1 fragment were used to immunohistochemically localize native SsPTP1 in the mite gut and in fecal pellets within the upper epidermis, co-localizing with serum components such as host IgG and complement. Enzymatic deglycosylation confirmed strong N- and O-glycosylation of the native peritrophin. Serum incubation followed by immunoblotting with a monoclonal antibody against mannan binding lectin (MBL), the recognition molecule of the lectin pathway of human complement activation, indicated that MBL may specifically bind to glycosylated SsPTP1. Conclusions/Significance This study adds a new aspect to the accumulating evidence that complement plays a major role in scabies mite biology. It identifies a novel peritrophin localized in the mite gut as a potential target of the lectin pathway of the complement cascade. These initial findings indicate a novel role of scabies mite peritrophins in triggering a host innate immune response within the mite gut. PMID:21980545

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.

Practical Ways to Integrate Literature into Spanish for International Business Courses

ERIC Educational Resources Information Center

Torres, German

2004-01-01

In general, language for business courses do not include the study of literary texts, despite significant research that demonstrates the value of literature as a way to gain competence in a foreign culture. Carefully selected, adapted, and programmed as a culture complement to the main textbook, literary texts can be a valuable resource in the…

Distinction in Doctoral Education: Using Bourdieu's Tools to Assess the Socialization of Doctoral Students

ERIC Educational Resources Information Center

Gopaul, Bryan

2011-01-01

This conceptual article uses the tools of Pierre Bourdieu (1977, 1986, 1990) to examine the socialization of doctoral students by suggesting that the processes of doctoral study highlight inequities among students. Using Young's (1990) social justice approach as a framework to complement the ideas of Bourdieu, I demonstrate how aspects of academic…

Genetic analysis of indefinite division in human cells: Evidence for a cell senescence-related gene(s) on human chromosome 4

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

Yi Ning; Ledbetter, D.H.; Smith, J.R.

1991-07-01

Earlier studies had demonstrated that fusion of normal with immortal human cells yielded hybrids having limited division potential. This indicated that the phenotype of limited proliferation (cellular senescence) is dominant and that immortal cells result from recessive changes in normal growth-regulatory genes. In additional studies, the authors exploited the fact that the immortal phenotype is recessive and, by fusing various immortal human cell lines with each other, identified four complementation groups for indefinite division. Assignment of cell lines to specific groups allowed us to take a focused approach to identify the chromosomes and genes involved in growth regulation that havemore » been modified in immortal cells. They report here that introduction of a normal human chromosome 4 into three immortal cell lines (HeLa, J82, T98G) assigned to complementation group B resulted in loss of proliferation and reversal of the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. This result suggests that a gene(s) involved in cellular senescence and normal growth regulation resides on chromosome 4.« less

Chromosomal abnormalities in human sperm

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

Martin, R.H.

1985-01-01

The ability to analyze human sperm chromosome complements after penetration of zona pellucida-free hamster eggs provides the first opportunity to study the frequency and type of chromosomal abnormalities in human gametes. Two large-scale studies have provided information on normal men. We have studied 1,426 sperm complements from 45 normal men and found an abnormality rate of 8.9%. Brandriff et al. (5) found 8.1% abnormal complements in 909 sperm from 4 men. The distribution of numerical and structural abnormalities was markedly dissimilar in the 2 studies. The frequency of aneuploidy was 5% in our sample and only 1.6% in Brandriff's, perhapsmore » reflecting individual variability among donors. The frequency of 24,YY sperm was low: 0/1,426 and 1/909. This suggests that the estimates of nondisjunction based on fluorescent Y body data (1% to 5%) are not accurate. We have also studied men at increased risk of sperm chromosomal abnormalities. The frequency of chromosomally unbalanced sperm in 6 men heterozygous for structural abnormalities varied dramatically: 77% for t11;22, 32% for t6;14, 19% for t5;18, 13% for t14;21, and 0% for inv 3 and 7. We have also studied 13 cancer patients before and after radiotherapy and demonstrated a significant dose-dependent increase of sperm chromosome abnormalities (numerical and structural) 36 months after radiation treatment.« less

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum.

PubMed

Robbins, Jonathan A; Absalon, Sabrina; Streva, Vincent A; Dvorin, Jeffrey D

2017-06-13

All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G 1 -, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G 1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways. IMPORTANCE Human infection by the eukaryotic parasite Plasmodium falciparum causes malaria. Most well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs) to promote essential cell division processes. Remarkably, there are no identifiable cyclins that are predicted to control the cell cycle in the malaria parasite genome. Thus, our knowledge regarding the basic mechanisms of the malaria parasite cell cycle remains unsatisfactory. We demonstrate that P. falciparum Cyc1 (PfCyc1), a transcriptional cyclin homolog, complements a cell cycle cyclin-deficient yeast strain but not a transcriptional cyclin-deficient strain. We show that PfCyc1 forms a complex in the parasite with PfMRK and the P. falciparum MAT1 homolog. PfCyc1 is essential and nonredundant in blood-stage P. falciparum PfCyc1 knockdown causes a stage-specific arrest after nuclear division, demonstrating morphologically aberrant cytokinesis. This work demonstrates a conserved PfCyc1/PfMAT1/PfMRK complex in malaria and suggests that it functions as a schizont stage-specific regulator of the P. falciparum life cycle. Copyright © 2017 Robbins et al.

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

Complement Membrane Attack and Tumorigenesis: A SYSTEMS BIOLOGY APPROACH.

PubMed

Towner, Laurence D; Wheat, Richard A; Hughes, Timothy R; Morgan, B Paul

2016-07-15

Tumor development driven by inflammation is now an established phenomenon, but the role that complement plays remains uncertain. Recent evidence has suggested that various components of the complement (C) cascade may influence tumor development in disparate ways; however, little attention has been paid to that of the membrane attack complex (MAC). This is despite abundant evidence documenting the effects of this complex on cell behavior, including cell activation, protection from/induction of apoptosis, release of inflammatory cytokines, growth factors, and ECM components and regulators, and the triggering of the NLRP3 inflammasome. Here we present a novel approach to this issue by using global gene expression studies in conjunction with a systems biology analysis. Using network analysis of MAC-responsive expression changes, we demonstrate a cluster of co-regulated genes known to have impact in the extracellular space and on the supporting stroma and with well characterized tumor-promoting roles. Network analysis highlighted the central role for EGF receptor activation in mediating the observed responses to MAC exposure. Overall, the study sheds light on the mechanisms by which sublytic MAC causes tumor cell responses and exposes a gene expression signature that implicates MAC as a driver of tumor progression. These findings have implications for understanding of the roles of complement and the MAC in tumor development and progression, which in turn will inform future therapeutic strategies in cancer. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Expression of Human Complement Factor H Prevents Age-Related Macular Degeneration–Like Retina Damage and Kidney Abnormalities in Aged Cfh Knockout Mice

PubMed Central

Ding, Jin-Dong; Kelly, Una; Landowski, Michael; Toomey, Christopher B.; Groelle, Marybeth; Miller, Chelsey; Smith, Stephanie G.; Klingeborn, Mikael; Singhapricha, Terry; Jiang, Haixiang; Frank, Michael M.; Bowes Rickman, Catherine

2016-01-01

Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh−/−) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh−/− mice, and transgenics had a thicker outer nuclear layer and less sub–retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets. PMID:25447048

Variola virus immune evasion proteins.

PubMed

Dunlop, Lance R; Oehlberg, Katherine A; Reid, Jeremy J; Avci, Dilek; Rosengard, Ariella M

2003-09-01

Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.

Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening.

PubMed

Schallmey, Marcus; Ly, Anh; Wang, Chunxia; Meglei, Gabriela; Voget, Sonja; Streit, Wolfgang R; Driscoll, Brian T; Charles, Trevor C

2011-08-01

We previously reported the construction of metagenomic libraries in the IncP cosmid vector pRK7813, enabling heterologous expression of these broad-host-range libraries in multiple bacterial hosts. Expressing these libraries in Sinorhizobium meliloti, we have successfully complemented associated phenotypes of polyhydroxyalkanoate synthesis mutants. DNA sequence analysis of three clones indicates that the complementing genes are homologous to, but substantially different from, known polyhydroxyalkanaote synthase-encoding genes. Thus we have demonstrated the ability to isolate diverse genes for polyhydroxyalkanaote synthesis by functional complementation of defined mutants. Such genes might be of use in the engineering of more efficient systems for the industrial production of bioplastics. The use of functional complementation will also provide a vehicle to probe the genetics of polyhydroxyalkanaote metabolism and its relation to carbon availability in complex microbial assemblages. 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

ERIC Educational Resources Information Center

Smith, Michael B.

2009-01-01

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

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.

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

Innate immune performance and steroid hormone profiles of pregnant versus nonpregnant cottonmouth snakes (Agkistrodon piscivorus).

PubMed

Graham, Sean P; Earley, Ryan L; Guyer, Craig; Mendonça, Mary T

2011-12-01

Squamates (lizards and snakes) have independently evolved viviparity over 100 times, and exhibit a wide range of maternal investment in developing embryos from the extremes of lecithotrophic oviparity to matrotrophic viviparity. This group therefore provides excellent comparative opportunities for studying endocrine and immune involvement during pregnancy, and their possible interactions. We studied the cottonmouth (Agkistrodon piscivorus), since they exhibit limited placentation (e.g., ovoviviparity), allowing comparison with squamate species hypothesized to require considerable maternal immune modulation due to the presence of a more extensive placental connection. Furthermore, the cottonmouth's biennial reproductive cycle provides an opportunity for simultaneously comparing pregnant and non-pregnant females in the wild. We document significantly elevated concentrations of progesterone (P4) and significantly lower concentrations of estradiol (E2) in pregnant females relative to non-pregnant females. Pregnant females had lower plasma bacteria lysis capacity relative to non-pregnant females. This functional measure of innate immunity is a proxy for complement performance, and we also determined significant correlations between P4 and decreased complement performance in pregnant females. These findings are consistent with studies that have determined P4's role in complement modulation during pregnancy in mammals, and thus this study joins a growing number of studies that have demonstrated convergent and/or conserved physiological mechanisms regulating viviparous reproduction in vertebrates. Copyright © 2011. Published by Elsevier Inc.

Monospecific high-affinity and complement activating anti-GM1 antibodies are determinants in experimental axonal neuropathy.

PubMed

Notturno, Francesca; Del Boccio, Piero; Luciani, Mirella; Caporale, Christina Michaela; Pieragostino, Damiana; Prencipe, Vincenza; Sacchetta, Paolo; Uncini, Antonino

2010-06-15

It has been difficult to replicate consistently the experimental model of axonal Guillain-Barré syndrome (GBS). We immunized rabbits with two lipo-oligosaccharides (LOS1 and LOS2) derived from the same C. jejuni strain and purified in a slightly different way. LOS1 did not contain proteins whereas several proteins were present in LOS2. In spite of a robust anti-GM1 antibody response in all animals the neuropathy developed only in rabbits immunized with LOS1. To explain this discrepancy we investigated fine specificity, affinity and ability to activate the complement of anti-GM1 antibodies. Only rabbits immunized with LOS1 showed monospecific high-affinity antibodies which activated more effectively the complement. Although it is not well understood how monospecific high-affinity antibodies are induced these are crucial for the induction of experimental axonal neuropathy. Only a strict adherence to the protocols demonstrated to be successful may guarantee the reproducibility and increase the confidence in the animal model as a reliable tool for the study of the human axonal GBS. Copyright 2010 Elsevier B.V. All rights reserved.

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

PubMed

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

2007-01-01

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

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement.

PubMed

Caswell, Clayton C; Han, Runlin; Hovis, Kelley M; Ciborowski, Pawel; Keene, Douglas R; Marconi, Richard T; Lukomski, Slawomir

2008-02-01

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.

The Impact of Social-Cognitive Stress on Speech Variability, Determinism, and Stability in Adults Who Do and Do Not Stutter

ERIC Educational Resources Information Center

Jackson, Eric S.; Tiede, Mark; Beal, Deryk; Whalen, D. H.

2016-01-01

Purpose: This study examined the impact of social-cognitive stress on sentence-level speech variability, determinism, and stability in adults who stutter (AWS) and adults who do not stutter (AWNS). We demonstrated that complementing the spatiotemporal index (STI) with recurrence quantification analysis (RQA) provides a novel approach to both…

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.

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

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

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.

Critical role of LuxS in the virulence of Campylobacter jejuni in a guinea pig model of abortion.

PubMed

Plummer, Paul; Sahin, Orhan; Burrough, Eric; Sippy, Rachel; Mou, Kathy; Rabenold, Jessica; Yaeger, Mike; Zhang, Qijing

2012-02-01

Previous studies on Campylobacter jejuni have demonstrated the role of LuxS in motility, cytolethal distending toxin production, agglutination, and intestinal colonization; however, its direct involvement in virulence has not been reported. In this study, we demonstrate a direct role of luxS in the virulence of C. jejuni in two different animal hosts. The IA3902 strain, a highly virulent sheep abortion strain recently described by our laboratory, along with its isogenic luxS mutant and luxS complement strains, was inoculated by the oral route into both a pregnant guinea pig virulence model and a chicken colonization model. In both cases, the IA3902 luxS mutant demonstrated a complete loss of ability to colonize the intestinal tract. In the pregnant model, the mutant also failed to induce abortion, while the wild-type strain was highly abortifacient. Genetic complementation of the luxS gene fully restored the virulent phenotype in both models. Interestingly, when the organism was inoculated into guinea pigs by the intraperitoneal route, no difference in virulence (abortion induction) was observed between the luxS mutant and the wild-type strain, suggesting that the defect in virulence following oral inoculation is likely associated with a defect in colonization and/or translocation of the organism out of the intestine. These studies provide the first direct evidence that LuxS plays an important role in the virulence of C. jejuni using an in vivo model of natural disease.

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

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/

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

PubMed Central

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

2012-01-01

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

Study of the plant COPII vesicle coat subunits by functional complementation of yeast Saccharomyces cerevisiae mutants.

PubMed

De Craene, Johan-Owen; Courte, Fanny; Rinaldi, Bruno; Fitterer, Chantal; Herranz, Mari Carmen; Schmitt-Keichinger, Corinne; Ritzenthaler, Christophe; Friant, Sylvie

2014-01-01

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-07-01

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

Improved understanding of geologic CO{sub 2} storage processes requires risk-driven field experiments

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

Oldenburg, C.M.

2011-06-01

The need for risk-driven field experiments for CO{sub 2} geologic storage processes to complement ongoing pilot-scale demonstrations is discussed. These risk-driven field experiments would be aimed at understanding the circumstances under which things can go wrong with a CO{sub 2} capture and storage (CCS) project and cause it to fail, as distinguished from accomplishing this end using demonstration and industrial scale sites. Such risk-driven tests would complement risk-assessment efforts that have already been carried out by providing opportunities to validate risk models. In addition to experimenting with high-risk scenarios, these controlled field experiments could help validate monitoring approaches to improvemore » performance assessment and guide development of mitigation strategies.« less

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

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

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

1994-09-01

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

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

PubMed

McGonigal, Rhona; Cunningham, Madeleine E; Yao, Denggao; Barrie, Jennifer A; Sankaranarayanan, Sethu; Fewou, Simon N; Furukawa, Koichi; Yednock, Ted A; Willison, Hugh J

2016-03-02

Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

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.

Structure-function mapping of BbCRASP-1, the key complement factor H and FHL-1 binding protein of Borrelia burgdorferi.

PubMed

Cordes, Frank S; Kraiczy, Peter; Roversi, Pietro; Simon, Markus M; Brade, Volker; Jahraus, Oliver; Wallis, Russell; Goodstadt, Leo; Ponting, Chris P; Skerka, Christine; Zipfel, Peter F; Wallich, Reinhard; Lea, Susan M

2006-05-01

Borrelia burgdorferi, a spirochaete transmitted to human hosts during feeding of infected Ixodes ticks, is the causative agent of Lyme disease, the most frequent vector-borne disease in Eurasia and North America. Sporadically Lyme disease develops into a chronic, multisystemic disorder. Serum-resistant B. burgdorferi strains bind complement factor H (FH) and FH-like protein 1 (FHL-1) on the spirochaete surface. This binding is dependent on the expression of proteins termed complement-regulator acquiring surface proteins (CRASPs). The atomic structure of BbCRASP-1, the key FHL-1/FH-binding protein of B. burgdorferi, has recently been determined. Our analysis indicates that its protein topology apparently evolved to provide a high affinity interaction site for FH/FHL-1 and leads to an atomic-level hypothesis for the functioning of BbCRASP-1. This work demonstrates that pathogens interact with complement regulators in ways that are distinct from the mechanisms used by the host and are thus obvious targets for drug design.

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.

Functional analyses of rare genetic variants in complement component C9 identified in patients with age-related macular degeneration.

PubMed

Kremlitzka, Mariann; Geerlings, Maartje J; de Jong, Sarah; Bakker, Bjorn; Nilsson, Sara C; Fauser, Sascha; Hoyng, Carel B; de Jong, Eiko K; den Hollander, Anneke I; Blom, Anna M

2018-05-14

Age-related macular degeneration (AMD) is a progressive disease of the central retina and the leading cause of irreversible vision loss in the western world. The involvement of abnormal complement activation in AMD has been suggested by association of variants in genes encoding complement proteins with disease development. A low-frequency variant (p.P167S) in the complement component C9 (C9) gene was recently shown to be highly associated with AMD, however its functional outcome remains largely unexplored. In this study, we reveal five novel rare genetic variants (p.M45L, p.F62S, p.G126R, p.T170I and p.A529T) in C9 in AMD patients, and evaluate their functional effects in vitro together with the previously identified (p.R118W and p.P167S) C9 variants.Our results demonstrate that the concentration of C9 is significantly elevated in patients' sera carrying the p.M45L, p.F62S, p.P167S and p.A529T variants compared to non-carrier controls. However, no difference can be observed in soluble terminal complement complex levels between the carrier and non-carrier groups. Comparing the polymerization of the C9 variants we reveal that the p.P167S mutant spontaneously aggregates, while the other mutant proteins (except for C9 p.A529T) fail to polymerize in the presence of zinc. Altered polymerization of the p.F62S and p.P167S proteins associated with decreased lysis of sheep erythrocytes and ARPE-19 cells by carriers' sera. Our data suggest that the analysed C9 variants affect only the secretion and polymerization of C9, without influencing its classical lytic activity. Future studies need to be performed to understand the implications of the altered polymerization of C9 in AMD pathology.

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

Microwave Power Transmission System Studies. Volume 1: Executive Summary

NASA Technical Reports Server (NTRS)

Maynard, O. E.; Brown, W. C.; Edwards, A.; Meltz, G.; Haley, J. T.; Howell, J. M.; Nathan, A.

1975-01-01

A study of microwave power generation, transmission, reception and control was conducted as a part of a program to demonstrate the feasibility of power transmission from geosynchronous orbit. A summary is presented of results concerning design approaches, estimated costs (ROM), critical technology, associated ground and orbital test programs with emphasis on dc to rf conversion, transmitting antenna, phase control, mechanical systems, flight operations, ground power receiving-rectifying antenna with systems analysis, and evaluation. Recommendations for early further in-depth studies complementing the technology program are included.

Dispersion of Nanoparticles in Different Media Importantly Determines the Composition of Their Protein Corona.

PubMed

Strojan, Klemen; Leonardi, Adrijana; Bregar, Vladimir B; Križaj, Igor; Svete, Jurij; Pavlin, Mojca

2017-01-01

Protein corona of nanoparticles (NPs), which forms when these particles come in to contact with protein-containing fluids, is considered as an overlooked factor in nanomedicine. Through numerous studies it has been becoming increasingly evident that it importantly dictates the interaction of NPs with their surroundings. Several factors that determine the compositions of NPs protein corona have been identified in recent years, but one has remained largely ignored-the composition of media used for dispersion of NPs. Here, we determined the effect of dispersion media on the composition of protein corona of polyacrylic acid-coated cobalt ferrite NPs (PAA NPs) and silica NPs. Our results confirmed some of the basic premises such as NPs type-dependent specificity of the protein corona. But more importantly, we demonstrated the effect of the dispersion media on the protein corona composition. The differences between constituents of the media used for dispersion of NPs, such as divalent ions and macromolecules were responsible for the differences in protein corona composition formed in the presence of fetal bovine serum (FBS). Our results suggest that the protein corona composition is a complex function of the constituents present in the media used for dispersion of NPs. Regardless of the dispersion media and FBS concentration, majority of proteins from either PAA NPs or silica NPs coronas were involved in the process of transport and hemostasis. Interestingly, corona of silica NPs contained three complement system related proteins: complement factor H, complement C3 and complement C4 while PAA NPs bound only one immune system related protein, α-2-glycoprotein. Importantly, relative abundance of complement C3 protein in corona of silica NPs was increased when NPs were dispersed in NaCl, which further implies the relevance of dispersion media used to prepare NPs.

Dispersion of Nanoparticles in Different Media Importantly Determines the Composition of Their Protein Corona

PubMed Central

Strojan, Klemen; Leonardi, Adrijana; Bregar, Vladimir B.; Križaj, Igor; Svete, Jurij; Pavlin, Mojca

2017-01-01

Protein corona of nanoparticles (NPs), which forms when these particles come in to contact with protein-containing fluids, is considered as an overlooked factor in nanomedicine. Through numerous studies it has been becoming increasingly evident that it importantly dictates the interaction of NPs with their surroundings. Several factors that determine the compositions of NPs protein corona have been identified in recent years, but one has remained largely ignored—the composition of media used for dispersion of NPs. Here, we determined the effect of dispersion media on the composition of protein corona of polyacrylic acid-coated cobalt ferrite NPs (PAA NPs) and silica NPs. Our results confirmed some of the basic premises such as NPs type-dependent specificity of the protein corona. But more importantly, we demonstrated the effect of the dispersion media on the protein corona composition. The differences between constituents of the media used for dispersion of NPs, such as divalent ions and macromolecules were responsible for the differences in protein corona composition formed in the presence of fetal bovine serum (FBS). Our results suggest that the protein corona composition is a complex function of the constituents present in the media used for dispersion of NPs. Regardless of the dispersion media and FBS concentration, majority of proteins from either PAA NPs or silica NPs coronas were involved in the process of transport and hemostasis. Interestingly, corona of silica NPs contained three complement system related proteins: complement factor H, complement C3 and complement C4 while PAA NPs bound only one immune system related protein, α-2-glycoprotein. Importantly, relative abundance of complement C3 protein in corona of silica NPs was increased when NPs were dispersed in NaCl, which further implies the relevance of dispersion media used to prepare NPs. PMID:28052135

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.

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.

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.

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.

Association between anti-beta2 glycoprotein I antibodies and renal glomerular C4d deposition in lupus nephritis patients with glomerular microthrombosis: a prospective study of 155 cases.

PubMed

Shen, Y; Chen, X-W; Sun, C-Y; Dai, M; Yan, Y-C; Yang, C-D

2010-09-01

Glomerular microthrombosis (GMT) is a common vascular change in patients with lupus nephritis (LN). The mechanism underlying GMT is still unknown. In our previous study, we found that the level of IgG anti-beta2 glycoprotein I (beta2GPI) antibodies was higher in the LN-GMT group than in the LN-non-GMT group, which indicated that anti-beta2GPI antibodies may play a role in GMT formation. Many studies have demonstrated that the activation of the classical complement pathway may play a critical role in fetal loss and aPL-induced thrombosis formation. To investigate whether complement activation plays a role in GMT formation and to evaluate its relationship with aPL, we prospectively investigated deposition of C4d in 155 renal biopsy specimens of LN patients. The results revealed a strong relationship between the intensity of glomerular C4d staining and the presence of microthrombi (p < 0.001). The detection rate of IgG anti-beta2GPI antibodies was higher in the LN-GMT group than in the LN-non-GMT group (p < 0.05). Further, the intensity of glomerular C4d staining was significantly related with IgG anti-beta2GPI antibodies (p < 0.05). The results of our study suggest that anti-beta2GPI antibodies may play a role in GMT formation, and this process might involve complement activation.

The relevance of coagulation factor X protection of adenoviruses in human sera

PubMed Central

Duffy, M R; Doszpoly, A; Turner, G; Nicklin, S A; Baker, A H

2016-01-01

Intravenous delivery of adenoviruses is the optimal route for many gene therapy applications. Once in the blood, coagulation factor X (FX) binds to the adenovirus capsid and protects the virion from natural antibody and classical complement-mediated neutralisation in mice. However, to date, no studies have examined the relevance of this FX/viral immune protective mechanism in human samples. In this study, we assessed the effects of blocking FX on adenovirus type 5 (Ad5) activity in the presence of human serum. FX prevented human IgM binding directly to the virus. In individual human sera samples (n=25), approximately half of those screened inhibited adenovirus transduction only when the Ad5–FX interaction was blocked, demonstrating that FX protected the virus from neutralising components in a large proportion of human sera. In contrast, the remainder of sera tested had no inhibitory effects on Ad5 transduction and FX armament was not required for effective gene transfer. In human sera in which FX had a protective role, Ad5 induced lower levels of complement activation in the presence of FX. We therefore demonstrate for the first time the importance of Ad–FX protection in human samples and highlight subject variability and species-specific differences as key considerations for adenoviral gene therapy. PMID:27014840

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.

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

Complement system biomarkers in epilepsy.

PubMed

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

2018-05-24

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

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.

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.

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

PubMed

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

2014-01-01

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

The Structure of Jarai Clauses and Noun Phrases

ERIC Educational Resources Information Center

Jensen, Joshua Martin

2013-01-01

This dissertation provides a syntactic account for the Jarai noun phrase and for the three regions of the Jarai clause: the operator domain, the inflectional domain, and the theta domain. Within the noun phrase, I argue that demonstrative-final word order involves phrasal movement of the demonstrative's complement into Spec,D, where it…

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

PubMed Central

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

2017-01-01

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

Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation.

PubMed

Grandvalet, Cosette; Assad-García, Juan Simón; Chu-Ky, Son; Tollot, Marie; Guzzo, Jean; Gresti, Joseph; Tourdot-Maréchal, Raphaëlle

2008-09-01

Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were grown in the presence of ethanol or at low pH, suggesting transcriptional regulation of the cfa gene under different stress conditions. In contrast to Escherichia coli, only one functional promoter was identified upstream of the cfa gene of O. oeni. The function of the cfa gene was confirmed by complementation of a cfa-deficient E. coli strain. Nevertheless, the complementation remained partial because the conversion percentage of unsaturated fatty acids into CFA of the complemented strain was much lower than that of the wild-type strain. Moreover, a prevalence of cycC19 : 0 was observed in the membrane of the complemented strain. This could be due to a specific affinity of the CFA synthase from O. oeni. In spite of this partial complementation, the complemented strain of E. coli totally recovered its viability after ethanol shock (10 %, v/v) whereas its viability was only partly recovered after an acid shock at pH 3.0.

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.

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

PpsA-mediated alternative pathway to complement RNase E essentiality in Escherichia coli.

PubMed

Tamura, Masaru; Honda, Naoko; Fujimoto, Hirofumi; Cohen, Stanley N; Kato, Atsushi

2016-07-01

Escherichia coli cells require RNase E, encoded by the essential gene rne, to propagate. The growth properties on different carbon sources of E. coli cells undergoing suppression of RNase E production suggested that reduction in RNase E is associated with decreased expression of phosphoenolpyruvate synthetase (PpsA), which converts pyruvate to phosphoenolpyruvate during gluconeogenesis. Western blotting and genetic complementation confirmed the role of RNase E in PpsA expression. Adventitious ppsA overexpression from a multicopy plasmid was sufficient to restore colony formation of ∆rne E. coli on minimal media containing glycerol or succinate as the sole carbon source. Complementation of ∆rne by ppsA overproduction was observed during growth on solid media but was only partial, and bacteria showed slowed cell division and grew as filamentous chains. We found that restoration of colony-forming ability by ppsA complementation occurred independent of the presence of endogenous RNase G or second-site suppressors of RNase E essentiality. Our investigations demonstrate the role of phosphoryl transfer catalyzable by PpsA as a determinant of RNase E essentiality in E. coli.

Targeting α-synuclein oligomers by protein-fragment complementation for drug discovery in synucleinopathies.

PubMed

Moussaud, Simon; Malany, Siobhan; Mehta, Alka; Vasile, Stefan; Smith, Layton H; McLean, Pamela J

2015-05-01

Reducing the burden of α-synuclein oligomeric species represents a promising approach for disease-modifying therapies against synucleinopathies such as Parkinson's disease and dementia with Lewy bodies. However, the lack of efficient drug discovery strategies that specifically target α-synuclein oligomers has been a limitation to drug discovery programs. Here we describe an innovative strategy that harnesses the power of bimolecular protein-fragment complementation to monitor synuclein-synuclein interactions. We have developed two robust models to monitor α-synuclein oligomerization by generating novel stable cell lines expressing α-synuclein fusion proteins for either fluorescent or bioluminescent protein-fragment complementation under the tetracycline-controlled transcriptional activation system. A pilot screen was performed resulting in the identification of two potential hits, a p38 MAPK inhibitor and a casein kinase 2 inhibitor, thereby demonstrating the suitability of our protein-fragment complementation assay for the measurement of α-synuclein oligomerization in living cells at high throughput. The application of the strategy described herein to monitor α-synuclein oligomer formation in living cells with high throughput will facilitate drug discovery efforts for disease-modifying therapies against synucleinopathies and other proteinopathies.

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity

PubMed Central

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane. PMID:19531344

Morin hydrate augments phagocytosis mechanism and inhibits LPS induced autophagic signaling in murine macrophage.

PubMed

Jakhar, Rekha; Paul, Souren; Chauhan, Anil Kumar; Kang, Sun Chul

2014-10-01

Morin, a natural flavonoid that is the primary bioactive constituent of the family Moraceae, has been found to be associated with many therapeutic properties. In this study, we evaluated the immunomodulatory activities of increasing concentration of morin hydrate in vitro. Three different concentrations of morin hydrate (5, 10, and 15μM) were used to evaluate their effect on splenocyte proliferation, phagocytic activity of macrophages, cytokine secretion and complement inhibition. We also evaluated the role of morin hydrate on lipopolysaccharide (LPS) induced autophagy. Our study demonstrated that morin hydrate elicited a significant increase in splenocyte proliferation, phagocytic capacity and suppressed the production of cytokines and nitric oxide in activated macrophages. Humoral immunity measured by anti-complement activity showed an increase in inhibition of the complement system after the addition of morin hydrate, where morin hydrate at 15μM concentration induced a significant inhibition. Depending on our results, we can also conclude that morin hydrate protects macrophages from LPS induced autophagic cell death. Our findings suggest that morin hydrate represents a structurally diverse class of flavonoid and this structural variability can profoundly affect its cell-type specificity and its biological activities. Supplementation of immune cells with morin hydrate has an upregulating and immunoprotective effect that shows potential as a countermeasure to the immune dysfunction and suggests an interesting use in inflammation related diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity.

PubMed

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.

MuSK induced experimental autoimmune myasthenia gravis does not require IgG1 antibody to MuSK.

PubMed

Küçükerden, Melike; Huda, Ruksana; Tüzün, Erdem; Yılmaz, Abdullah; Skriapa, Lamprini; Trakas, Nikos; Strait, Richard T; Finkelman, Fred D; Kabadayı, Sevil; Zisimopoulou, Paraskevi; Tzartos, Socrates; Christadoss, Premkumar

2016-06-15

Sera of myasthenia gravis (MG) patients with muscle-specific receptor kinase-antibody (MuSK-Ab) predominantly display the non-complement fixing IgG4 isotype. Similarly, mouse IgG1, which is the analog of human IgG4, is the predominant isotype in mice with experimental autoimmune myasthenia gravis (EAMG) induced by MuSK immunization. The present study was performed to determine whether IgG1 anti-MuSK antibody is required for immunized mice to develop EAMG. Results demonstrated a significant correlation between clinical severity of EAMG and levels of MuSK-binding IgG1+, IgG2+ and IgG3+ peripheral blood B cells in MuSK-immunized wild-type (WT) mice. Moreover, MuSK-immunized IgG1 knockout (KO) and WT mice showed similar EAMG severity, serum MuSK-Ab levels, muscle acetylcholine receptor concentrations, neuromuscular junction immunoglobulin and complement deposit ratios. IgG1 and IgG3 were the predominant anti-MuSK isotypes in WT and IgG1 KO mice, respectively. These observations demonstrate that non-IgG1 isotypes can mediate MuSK-EAMG pathogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism

PubMed Central

Williams, Emily A.; Mba Medie, Felix; Bosserman, Rachel E.; Johnson, Benjamin K.; Reyna, Cristal; Ferrell, Micah J.; Champion, Matthew M.; Abramovitch, Robert B.

2016-01-01

ABSTRACT Mycobacterial pathogens use the ESAT-6 system 1 (Esx-1) exporter to promote virulence. Previously, we used gene disruption and complementation to conclude that the MMAR_0039 gene in Mycobacterium marinum is required to promote Esx-1 export. Here we applied molecular genetics, proteomics, and whole-genome sequencing to demonstrate that the MMAR_0039 gene is not required for Esx-1 secretion or virulence. These findings suggest that we initially observed an indirect mechanism of genetic complementation. We identified a spontaneous nonsense mutation in a known Esx-1-associated gene which causes a loss of Esx-1 activity. We show that the Esx-1 function was restored by nonsense suppression. Moreover, we identified a polar mutation in the ppsC gene which reduced cellular impermeability but did not impact cytotoxicity in macrophages. Our studies reveal insight into Esx-1 export, nonsense suppression, and cell envelope lipid biogenesis. PMID:27789543

Trichloroethylene (TCE) in tree cores to complement a subsurface investigation on residential property near a former electroplating facility.

PubMed

Wilcox, Jeffrey D; Johnson, Kathy M

2016-10-01

Tree cores were collected and analyzed for trichloroethylene (TCE) on a private property between a former electroplating facility in Asheville, North Carolina (USA), and a contaminated wetland/spring complex. TCE was detected in 16 of 31 trees, the locations of which were largely consistent with a "plume core" delineated by a more detailed subsurface investigation nearly 2 years later. Concentrations in tree cores and nearby soil borings were not correlated, perhaps due to heterogeneities in both geologic and tree root structure, spatial and temporal variability in transpiration rates, or interferences caused by other contaminants at the site. Several tree cores without TCE provided evidence for significantly lower TCE concentrations in shallow groundwater along the margins of the contaminated spring complex in an area with limited accessibility. This study demonstrates that tree core analyses can complement a more extensive subsurface investigation, particularly in residential or ecologically sensitive areas.

Quantifying Intrinsic Specificity: A Potential Complement to Affinity in Drug Screening

NASA Astrophysics Data System (ADS)

Wang, Jin; Zheng, Xiliang; Yang, Yongliang; Drueckhammer, Dale; Yang, Wei; Verkhivker, Gennardy; Wang, Erkang

2007-11-01

We report here the investigation of a novel description of specificity in protein-ligand binding based on energy landscape theory. We define a new term, intrinsic specificity ratio (ISR), which describes the level of discrimination in binding free energies of the native basin for a protein-ligand complex from the weaker binding states of the same ligand. We discuss the relationship between the intrinsic specificity we defined here and the conventional definition of specificity. In a docking study of molecules with the enzyme COX-2, we demonstrate a statistical correspondence between ISR value and geometrical shapes of the small molecules binding to COX-2. We further observe that the known selective (nonselective) inhibitors of COX-2 have higher (lower) ISR values. We suggest that intrinsic specificity ratio may be a useful new criterion and a complement to affinity in drug screening and in searching for potential drug lead compounds.

Complement Involvement in Periodontitis: Molecular Mechanisms and Rational Therapeutic Approaches.

PubMed

Hajishengallis, George; Maekawa, Tomoki; Abe, Toshiharu; Hajishengallis, Evlambia; Lambris, John D

2015-01-01

The complement system is a network of interacting fluid-phase and cell surface-associated molecules that trigger, amplify, and regulate immune and inflammatory signaling pathways. Dysregulation of this finely balanced network can destabilize host-microbe homeostasis and cause inflammatory tissue damage. Evidence from clinical and animal model-based studies suggests that complement is implicated in the pathogenesis of periodontitis, a polymicrobial community-induced chronic inflammatory disease that destroys the tooth-supporting tissues. This review discusses molecular mechanisms of complement involvement in the dysbiotic transformation of the periodontal microbiome and the resulting destructive inflammation, culminating in loss of periodontal bone support. These mechanistic studies have additionally identified potential therapeutic targets. In this regard, interventional studies in preclinical models have provided proof-of-concept for using complement inhibitors for the treatment of human periodontitis.

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

PubMed

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

2014-05-01

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

Complement and the control of HIV infection: an evolving story.

PubMed

Frank, Michael M; Hester, Christopher; Jiang, Haixiang

2014-05-01

Thirty years ago, investigators isolated and later determined the structure of HIV-1 and its envelope proteins. Using techniques that were effective with other viruses, they prepared vaccines designed to generate antibody or T-cell responses, but they were ineffective in clinical trials. In this article, we consider the role of complement in host defense against enveloped viruses, the role it might play in the antibody response and why complement has not controlled HIV-1 infection. Complement consists of a large group of cell-bound and plasma proteins that are an integral part of the innate immune system. They provide a first line of defense against microbes and also play a role in the immune response. Here we review the studies of complement-mediated HIV destruction and the role of complement in the HIV antibody response. HIV-1 has evolved a complex defense to prevent complement-mediated killing reviewed here. As part of these studies, we have discovered that HIV-1 envelope, on administration into animals, is rapidly broken down into small peptides that may prove to be very inefficient at provident the type of antigenic stimulation that leads to an effective immune response. Improving complement binding and stabilizing envelope may improve the vaccine response.

The effect of storage temperature on the biological activity of extracellular vesicles for the complement system.

PubMed

Park, Sang June; Jeon, Hyungtaek; Yoo, Seung-Min; Lee, Myung-Shin

2018-05-10

Extracellular vesicles (EVs) are mediators of intercellular communication by transporting cargo containing proteins, lipids, mRNA, and miRNA. There is increasing evidence that EVs have various roles in regulating migration, invasion, stemness, survival, and immune functions. Previously, we have found that EVs from Kaposi's sarcoma-associated herpesvirus (KSHV)-infected human endothelial cells have the potential to activate the complement system. Although many studies have shown that the physical properties of EVs can be changed by their storage condition, there have been few studies for the stability of biological activity of EVs in various storage conditions. In this study, we investigated various conditions to identify the best conditions to store EVs with functional stability for 25 d. Furthermore, the correlation between the function and other characteristics of EVs, including the expression of EV markers, size distribution, and particle number, were also analyzed. Our results demonstrated that storage temperature is an important factor to maintain the activity of EVs and would be useful information for basic research and clinical application using EVs.

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

Language and false belief: evidence for general, not specific, effects in cantonese-speaking preschoolers.

PubMed

Tardif, Twila; So, Catherine Wing-Chee; Kaciroti, Niko

2007-03-01

Two studies were conducted with Cantonese-speaking preschoolers examining J. de Villiers's (1995) hypothesis that syntactic complements play a unique role in the acquisition of false belief (FB). In Study 1, the authors found a positive correlation between FB and syntactic complements in 72 four- to six-year-old Cantonese-speaking preschoolers. Study 2 followed 72 three- to five-year-old Cantonese-speaking children who initially failed an FB screening task and were then tested on general language abilities, short-term memory, inhibition, nonverbal IQ, and on FB and complement tasks. Once age and initial FB understanding were controlled for in both multiple regression and hierarchical linear modeling analyses, complements no longer uniquely predicted FB. Instead, individual differences in general language abilities and short-term memory contributed to the variation in both complements and FB.

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

PubMed

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

2016-04-12

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

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

Complement Protein C1q Modulates Neurite Outgrowth In Vitro and Spinal Cord Axon Regeneration In Vivo

PubMed Central

Peterson, Sheri L.; Nguyen, Hal X.; Mendez, Oscar A.

2015-01-01

Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI. PMID:25762679

Mitochondrial Telomeres as Molecular Markers for Identification of the Opportunistic Yeast Pathogen Candida parapsilosis

PubMed Central

Nosek, Jozef; Tomáška, L'ubomír; Ryčovská, Adriana; Fukuhara, Hiroshi

2002-01-01

Recent studies have demonstrated that a large number of organisms carry linear mitochondrial DNA molecules possessing specialized telomeric structures at their ends. Based on this specific structural feature of linear mitochondrial genomes, we have developed an approach for identification of the opportunistic yeast pathogen Candida parapsilosis. The strategy for identification of C. parapsilosis strains is based on PCR amplification of specific DNA sequences derived from the mitochondrial telomere region. This assay is complemented by immunodetection of a protein component of mitochondrial telomeres. The results demonstrate that mitochondrial telomeres represent specific molecular markers with potential applications in yeast diagnostics and taxonomy. PMID:11923346

Syntactic and Semantic Coordination in Finite Complement-Clause Constructions: A Diary-Based Case Study

ERIC Educational Resources Information Center

Köymen, Bahar; Lieven, Elena; Brandt, Silke

2016-01-01

This study investigates the coordination of matrix and subordinate clauses within finite complement-clause constructions. The data come from diary and audio recordings which include the utterances produced by an American English-speaking child, L, between the ages 1;08 and 3;05. We extracted all the finite complement-clause constructions that L…

Contrasting Complement Control, Temporal Adjunct Control and Controlled Verbal Gerund Subjects in ASD: The Role of Contextual Cues in Reference Assignment.

PubMed

Janke, Vikki; Perovic, Alexandra

2017-01-01

This study examines two complex syntactic dependencies (complement control and sentence-final temporal adjunct control) and one pragmatic dependency (controlled verbal gerund subjects) in children with ASD. Sixteen high-functioning (HFA) children (aged 6-16) with a diagnosis of autism and no language impairment, matched on age, gender and non-verbal MA to one TD control group, and on age, gender and verbal MA to another TD control group, undertook three picture-selection tasks. Task 1 measured their base-line interpretations of the empty categories ( ec ). Task 2 preceded these sentence sets with a weakly established topic cueing an alternative referent and Task 3 with a strongly established topic cueing an alternative referent. In complement control (Ron persuaded Hermione ec to kick the ball) and sentence-final temporal adjunct control (Harry tapped Luna while ec feeding the owl), the reference of the ec is argued to be related obligatorily to the object and subject respectively. In controlled verbal-gerund subjects (VGS) ( ec Rowing the boat clumsily made Luna seasick), the ec 's reference is resolved pragmatically. Referent choices across the three tasks were compared. TD children chose the object uniformly in complement control across all tasks but in adjunct control, preferences shifted toward the object in Task 3. In controlled VGSs, they exhibited a strong preference for an internal-referent interpretation in Task 1, which shifted in the direction of the cues in Tasks 2 and 3. HFA children gave a mixed performance. They patterned with their TD counterparts on complement control and controlled VGSs but performed marginally differently on adjunct control: no TD groups were influenced by the weakly established topic in Task 2 but all groups were influenced by the strongly established topic in Task 3. HFA children were less influenced than the TD children, resulting in their making fewer object choices overall but revealing parallel patterns of performance. In this first study of three sub-types of control in ASD, we demonstrate that HFA children consult the same pragmatic cues to the same degree as TD children, in spite of the diverse pragmatic deficits reported for this population.

Contrasting Complement Control, Temporal Adjunct Control and Controlled Verbal Gerund Subjects in ASD: The Role of Contextual Cues in Reference Assignment

PubMed Central

Janke, Vikki; Perovic, Alexandra

2017-01-01

This study examines two complex syntactic dependencies (complement control and sentence-final temporal adjunct control) and one pragmatic dependency (controlled verbal gerund subjects) in children with ASD. Sixteen high-functioning (HFA) children (aged 6–16) with a diagnosis of autism and no language impairment, matched on age, gender and non-verbal MA to one TD control group, and on age, gender and verbal MA to another TD control group, undertook three picture-selection tasks. Task 1 measured their base-line interpretations of the empty categories (ec). Task 2 preceded these sentence sets with a weakly established topic cueing an alternative referent and Task 3 with a strongly established topic cueing an alternative referent. In complement control (Ron persuaded Hermione ec to kick the ball) and sentence-final temporal adjunct control (Harry tapped Luna while ec feeding the owl), the reference of the ec is argued to be related obligatorily to the object and subject respectively. In controlled verbal-gerund subjects (VGS) (ec Rowing the boat clumsily made Luna seasick), the ec's reference is resolved pragmatically. Referent choices across the three tasks were compared. TD children chose the object uniformly in complement control across all tasks but in adjunct control, preferences shifted toward the object in Task 3. In controlled VGSs, they exhibited a strong preference for an internal-referent interpretation in Task 1, which shifted in the direction of the cues in Tasks 2 and 3. HFA children gave a mixed performance. They patterned with their TD counterparts on complement control and controlled VGSs but performed marginally differently on adjunct control: no TD groups were influenced by the weakly established topic in Task 2 but all groups were influenced by the strongly established topic in Task 3. HFA children were less influenced than the TD children, resulting in their making fewer object choices overall but revealing parallel patterns of performance. In this first study of three sub-types of control in ASD, we demonstrate that HFA children consult the same pragmatic cues to the same degree as TD children, in spite of the diverse pragmatic deficits reported for this population. PMID:28400743

STUDIES ON THE ANTIGENIC PROPERTIES OF COMPLEMENT

PubMed Central

Klein, Paul G.; Burkholder, Peter M.

1960-01-01

Evidence is presented to show that guinea pig complement fixed on sensitized sheep red cells acts as a specific agglutinogen. Agglutinating antibodies that react with cell-fixed complement can be produced by immunizing rabbits with a complex of stromata-amboceptor-complement or with guinea pig serum globulin. These agglutinins can be removed by precipitation with guinea pig serum. They are, therefore, distinct from immunoconglutinins. PMID:14409702

EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785.

PubMed

Dertli, Enes; Mayer, Melinda J; Colquhoun, Ian J; Narbad, Arjan

2016-07-01

Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked βGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

[Atypical hemolytic uremic syndrome].

PubMed

Blasco Pelicano, Miquel; Rodríguez de Córdoba, Santiago; Campistol Plana, Josep M

2015-11-20

The hemolytic uremic syndrome (HUS) is a clinical entity characterized by thrombocytopenia, non-immune hemolytic anemia and renal impairment. Kidney pathology shows thrombotic microangiopathy (TMA) with endothelial cell injury leading to thrombotic occlusion of arterioles and capillaries. Traditionally, HUS was classified in 2 forms: Typical HUS, most frequently occurring in children and caused by Shiga-toxin-producing bacteria, and atypical HUS (aHUS). aHUS is associated with mutations in complement genes in 50-60% of patients and has worse prognosis, with the majority of patients developing end stage renal disease. After kidney transplantation HUS may develop as a recurrence of aHUS or as de novo disease. Over the last years, many studies have demonstrated that complement dysregulation underlies the endothelial damage that triggers the development of TMA in most of these patients. Advances in our understanding of the pathogenic mechanisms of aHUS, together with the availability of novel therapeutic options, will enable better strategies for the early diagnosis and etiological treatment, which are changing the natural history of aHUS. This review summarizes the aHUS clinical entity and describes the role of complement dysregulation in the pathogenesis of aHUS. Finally, we review the differential diagnosis and the therapeutic options available to patients with aHUS. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

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

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

Fusobacterium nucleatum binding to complement regulatory protein CD46 modulates the expression and secretion of cytokines and matrix metalloproteinases by oral epithelial cells.

PubMed

Mahtout, Hayette; Chandad, Fatiha; Rojo, Jose M; Grenier, Daniel

2011-02-01

Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum-bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.

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

PubMed Central

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

2010-01-01

Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues. PMID:19825847

Discovery of potential protein biomarkers of lung adenocarcinoma in bronchoalveolar lavage fluid by SWATH MS data-independent acquisition and targeted data extraction.

PubMed

Ortea, I; Rodríguez-Ariza, A; Chicano-Gálvez, E; Arenas Vacas, M S; Jurado Gámez, B

2016-04-14

Lung cancer currently ranks as the neoplasia with the highest global mortality rate. Although some improvements have been introduced in recent years, new advances in diagnosis are required in order to increase survival rates. New mildly invasive endoscopy-based diagnostic techniques include the collection of bronchoalveolar lavage fluid (BALF), which is discarded after using a portion of the fluid for standard pathological procedures. BALF proteomic analysis can contribute to clinical practice with more sensitive biomarkers, and can complement cytohistological studies by aiding in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. The range of quantitative proteomics methodologies used for biomarker discovery is currently being broadened with the introduction of data-independent acquisition (DIA) analysis-related approaches that address the massive quantitation of the components of a proteome. Here we report for the first time a DIA-based quantitative proteomics study using BALF as the source for the discovery of potential lung cancer biomarkers. The results have been encouraging in terms of the number of identified and quantified proteins. A panel of candidate protein biomarkers for adenocarcinoma in BALF is reported; this points to the activation of the complement network as being strongly over-represented and suggests this pathway as a potential target for lung cancer research. In addition, the results reported for haptoglobin, complement C4-A, and glutathione S-transferase pi are consistent with previous studies, which indicates that these proteins deserve further consideration as potential lung cancer biomarkers in BALF. Our study demonstrates that the analysis of BALF proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), combining a simple sample pre-treatment and SWATH DIA MS, is a useful method for the discovery of potential lung cancer biomarkers. Bronchoalveolar lavage fluid (BALF) analysis can contribute to clinical practice with more sensitive biomarkers, thus complementing cytohistological studies in order to aid in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. Here we report a panel of candidate protein biomarkers for adenocarcinoma in BALF. Forty-four proteins showed a fold-change higher than 3.75 among adenocarcinoma patients compared with controls. This report is the first DIA-based quantitative proteomics study to use bronchoalveolar lavage fluid (BALF) as a matrix for discovering potential biomarkers. The results are encouraging in terms of the number of identified and quantified proteins, demonstrating that the analysis of BALF proteins by a SWATH approach is a useful method for the discovery of potential biomarkers of pulmonary diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta.

PubMed

Nicolay, Nils H; Carter, Rebecca; Hatch, Stephanie B; Schultz, Niklas; Prevo, Remko; McKenna, W Gillies; Helleday, Thomas; Sharma, Ricky A

2012-11-01

DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt's lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control.

Complement Inhibition in the Immunotherapy of Breast Cancer

DTIC Science & Technology

2014-03-01

found to promote tumor growth by modulating anti-tumor immunity in a syngeneic model of cervical cancer 15. However, its role in metastatic spread of...previous study in a model of HPV -induced cancer , we demonstrated that C5a acts as a potent chemoattractant of MDSCs to the primary tumors 15. Thus, we...Gupta, G. P., Massague J. Cancer metastasis: building a framework. Cell 2006, 127(4): 679-695. 3. Fidler, I. J. The pathogenesis of cancer metastasis

Non-structural proteins P17 and P33 are involved in the assembly of the internal membrane-containing virus PRD1

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

Karttunen, Jenni; Mäntynen, Sari; Ihalainen, Teemu O.

2015-08-15

Bacteriophage PRD1, which has been studied intensively at the structural and functional levels, still has some gene products with unknown functions and certain aspects of the PRD1 assembly process have remained unsolved. In this study, we demonstrate that the phage-encoded non-structural proteins P17 and P33, either individually or together, complement the defect in a temperature-sensitive GroES mutant of Escherichia coli for host growth and PRD1 propagation. Confocal microscopy of fluorescent fusion proteins revealed co-localisation between P33 and P17 as well as between P33 and the host chaperonin GroEL. A fluorescence recovery after photobleaching assay demonstrated that the diffusion of themore » P33 fluorescent fusion protein was substantially slower in E. coli than theoretically calculated, presumably resulting from intermolecular interactions. Our results indicate that P33 and P17 function in procapsid assembly, possibly in association with the host chaperonin complex GroEL/GroES. - Highlights: • Two non-structural proteins of PRD1 are involved in the virus assembly. • P17 and P33 complement the defect in GroES of Escherichia coli. • P33 co-localises with GroEL and P17 in the bacterium. • Slow motion of P33 in the bacterium suggests association with cellular components.« less

Quantification and Mood Distribution in Spanish Complements: On the Negative Features of "Poco/a/s" in Spanish

ERIC Educational Resources Information Center

Laskurain-Ibarluzea, Patxi

2015-01-01

This paper studies mood distribution in the complement of Spanish assertive matrices when the matrix subject is modified by the quantifier "poco/a/s". The focus of this study is solely complement clauses, and adjectival and adverbial clauses are not considered. Following Mejías-Bikandi's (1994, 1998) account that the distribution of mood…

A widespread plant-fungal-bacterial symbiosis promotes plant biodiversity, plant nutrition and seedling recruitment.

PubMed

van der Heijden, Marcel G A; de Bruin, Susanne; Luckerhoff, Ludo; van Logtestijn, Richard S P; Schlaeppi, Klaus

2016-02-01

Highly diverse microbial assemblages colonize plant roots. It is still poorly understood whether different members of this root microbiome act synergistically by supplying different services (for example, different limiting nutrients) to plants and plant communities. In order to test this, we manipulated the presence of two widespread plant root symbionts, arbuscular mycorrhizal fungi and nitrogen-fixing rhizobia bacteria in model grassland communities established in axenic microcosms. Here, we demonstrate that both symbionts complement each other resulting in increased plant diversity, enhanced seedling recruitment and improved nutrient acquisition compared with a single symbiont situation. Legume seedlings obtained up to 15-fold higher productivity if they formed an association with both symbionts, opposed to productivity they reached with only one symbiont. Our results reveal the importance of functional diversity of symbionts and demonstrate that different members of the root microbiome can complement each other in acquiring different limiting nutrients and in driving important ecosystem functions.

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

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

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

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.

The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Pro-Convertase

PubMed Central

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

2014-01-01

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. While the majority of staphylococcal complement inhibitors act on the alternative pathway (AP) to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical (CP) and lectin (LP) pathways. We screened a collection of recombinant, secreted staphylococcal proteins to determine if S. aureus produces other molecules that inhibit either the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 pro-convertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits the two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion. PMID:25381436

Complement deficiency predisposes for meningitis due to nongroupable meningococci and Neisseria-related bacteria.

PubMed

Fijen, C A; Kuijper, E J; Tjia, H G; Daha, M R; Dankert, J

1994-05-01

Nongroupable meningococci or bacteria related to the genus Neisseria rarely cause meningitis. Complement deficiency has been identified as a major predisposing factor for meningococcal disease. To assess whether patients with meningitis due to such strains have a complement deficiency, we studied 12 persons. Six patients had meningitis due to nongroupable strains of meningococci, and six patients had meningitis due to Moraxella species or Acinetobacter species. Inherited complement component C7 or C8 deficiency was found in two persons who had had meningitis due to nongroupable meningococci, and one C8-deficient person had had meningitis caused by Moraxella osloensis. Hypocomplementemia resulting from CSF drain-associated shunt nephritis was found in one person with meningitis due to Moraxella nonliquefaciens and in one person with meningitis due to Acinetobacter lwoffi. This rather high frequency of inherited or acquired complement deficiencies among patients with meningitis due to nongroupable meningococci, Moraxella species, and Acinetobacter species justifies the recommendation that such patients must be studied for complement deficiency.

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

Electrostatic Steering Accelerates C3d:CR2 Association.

PubMed

Mohan, Rohith R; Huber, Gary A; Morikis, Dimitrios

2016-08-25

Electrostatic effects are ubiquitous in protein interactions and are found to be pervasive in the complement system as well. The interaction between complement fragment C3d and complement receptor 2 (CR2) has evolved to become a link between innate and adaptive immunity. Electrostatic interactions have been suggested to be the driving factor for the association of the C3d:CR2 complex. In this study, we investigate the effects of ionic strength and mutagenesis on the association of C3d:CR2 through Brownian dynamics simulations. We demonstrate that the formation of the C3d:CR2 complex is ionic strength-dependent, suggesting the presence of long-range electrostatic steering that accelerates the complex formation. Electrostatic steering occurs through the interaction of an acidic surface patch in C3d and the positively charged CR2 and is supported by the effects of mutations within the acidic patch of C3d that slow or diminish association. Our data are in agreement with previous experimental mutagenesis and binding studies and computational studies. Although the C3d acidic patch may be locally destabilizing because of unfavorable Coulombic interactions of like charges, it contributes to the acceleration of association. Therefore, acceleration of function through electrostatic steering takes precedence to stability. The site of interaction between C3d and CR2 has been the target for delivery of CR2-bound nanoparticle, antibody, and small molecule biomarkers, as well as potential therapeutics. A detailed knowledge of the physicochemical basis of C3d:CR2 association may be necessary to accelerate biomarker and drug discovery efforts.

Students' conceptual difficulties in hydrodynamics

NASA Astrophysics Data System (ADS)

Suarez, Alvaro; Kahan, Sandra; Zavala, Genaro; Marti, Arturo C.

2017-12-01

We describe a study on the conceptual difficulties faced by college students in understanding hydrodynamics of ideal fluids. This study was based on responses obtained in hundreds of written exams complemented with several oral interviews, which were held with first-year engineering and science university students. Their responses allowed us to identify a series of misconceptions unreported in the literature so far. The study findings demonstrate that the most critical difficulties arise from the students' inability to establish a link between the kinematics and dynamics of moving fluids, and from a lack of understanding regarding how different regions of a system interact.

Downregulation of membrane complement inhibitors CD55 and CD59 by siRNA sensitises uterine serous carcinoma overexpressing Her2/neu to complement and antibody-dependent cell cytotoxicity in vitro: implications for trastuzumab-based immunotherapy.

PubMed

Bellone, S; Roque, D; Cocco, E; Gasparrini, S; Bortolomai, I; Buza, N; Abu-Khalaf, M; Silasi, D-A; Ratner, E; Azodi, M; Schwartz, P E; Rutherford, T J; Pecorelli, S; Santin, A D

2012-04-24

We evaluated the expression of CD46, CD55 and CD59 membrane-bound complement-regulatory proteins (mCRPs) in primary uterine serous carcinoma (USC) and the ability of small interfering RNA (siRNA) against these mCRPs to sensitise USC to complement-dependent cytotoxicity (CDC) and antibody (trastuzumab)-dependent cellular cytotoxicity (ADCC) in vitro. Membrane-bound complement-regulatory proteins expression was evaluated using real-time PCR (RT-PCR) and flow cytometry, whereas Her2/neu expression and c-erbB2 gene amplification were assessed using immunohistochemistry, flow cytometry and fluorescent in-situ hybridisation. The biological effect of siRNA-mediated knockdown of mCRPs on HER2/neu-overexpressing USC cell lines was evaluated in CDC and ADCC 4-h chromium-release assays. High expression of mCRPs was found in USC cell lines when compared with normal endometrial cells (P<0.05). RT-PCR and FACS analyses demonstrated that anti-mCRP siRNAs were effective in reducing CD46, CD55 and CD59 expression on USC (P<0.05). Baseline complement-dependent cytotoxicity (CDC) against USC cell lines was low (mean ± s.e.m.=6.8 ± 0.9%) but significantly increased upon CD55 and CD59 knockdown (11.6 ± 0.8% and 10.7 ± 0.9%, respectively, P<0.05). Importantly, in the absence of complement, both CD55 and CD59, but not CD46, knockdowns significantly augmented ADCC against USC overexpressing Her2/neu. Uterine serous carcinoma express high levels of the mCRPs CD46, CD55 and CD59. Small interfering RNA inhibition of CD55 and CD59, but not CD46, sensitises USC to both CDC and ADCC in vitro, and if specifically targeted to tumour cells, may significantly increase trastuzumab-mediated therapeutic effect in vivo.

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.

THE PATHOPHYSIOLOGY OF GEOGRAPHIC ATROPHY SECONDARY TO AGE-RELATED MACULAR DEGENERATION AND THE COMPLEMENT PATHWAY AS A THERAPEUTIC TARGET

PubMed Central

Schmidt-Erfurth, Ursula; van Lookeren Campagne, Menno; Henry, Erin C.; Brittain, Christopher

2017-01-01

Purpose: Geographic atrophy (GA) is an advanced, vision-threatening form of age-related macular degeneration (AMD) affecting approximately five million individuals worldwide. To date, there are no approved therapeutics for GA treatment; however, several are in clinical trials. This review focuses on the pathophysiology of GA, particularly the role of complement cascade dysregulation and emerging therapies targeting the complement cascade. Methods: Primary literature search on PubMed for GA, complement cascade in age-related macular degeneration. ClinicalTrials.gov was searched for natural history studies in GA and clinical trials of drugs targeting the complement cascade for GA. Results: Cumulative damage to the retina by aging, environmental stress, and other factors triggers inflammation via multiple pathways, including the complement cascade. When regulatory components in these pathways are compromised, as with several GA-linked genetic risk factors in the complement cascade, chronic inflammation can ultimately lead to the retinal cell death characteristic of GA. Complement inhibition has been identified as a key candidate for therapeutic intervention, and drugs targeting the complement pathway are currently in clinical trials. Conclusion: The complement cascade is a strategic target for GA therapy. Further research, including on natural history and genetics, is crucial to expand the understanding of GA pathophysiology and identify effective therapeutic targets. PMID:27902638

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.

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

PubMed

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

2015-11-01

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

De Novo Chromosome Copy Number Variation in Fanconi Anemia-Associated Hematopoietic Defects

DTIC Science & Technology

2013-04-01

We have confirmed re-expression of FANCA , FANCD2, and FANCG in the FA-A + FANCA , FA-D2 + FANCD2, and FA-G + FANCG cells, respectively. We have also...confirmed restoration of FANCD2 and FANCI monoubiquitination and functional complementation of the FA-A + FANCA and FA-D2 + FANCD2 cells. In... gene products BRCA1 and BRCA2 function cooperatively in the FA-BRCA pathway to repair damaged DNA. Recent studies have demonstrated that the FA-BRCA

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

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.

Production of Infinitival Complements by Children with Specific Language Impairment

ERIC Educational Resources Information Center

Arndt, Karen Barako; Schuele, C. Melanie

2012-01-01

The purpose of this study was to explore the production of infinitival complements by children with specific language impairment (SLI) as compared with mean length of utterance (MLU)-matched children in an effort to clarify inconsistencies in the literature. Spontaneous language samples were analysed for infinitival complements (reduced…

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.

Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism

PubMed Central

2015-01-01

Background Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. Results ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids, cofactors and vitamins. Conclusions The ECemble method is able to hierarchically assign high quality enzyme annotations to genomic and metagenomic data. This study demonstrated the real application of ECemble to understand the indispensable role played by microbe-encoded enzymes in the healthy functioning of human metabolic systems. PMID:26099921

Application of a hierarchical enzyme classification method reveals the role of gut microbiome in human metabolism.

PubMed

Mohammed, Akram; Guda, Chittibabu

2015-01-01

Enzymes are known as the molecular machines that drive the metabolism of an organism; hence identification of the full enzyme complement of an organism is essential to build the metabolic blueprint of that species as well as to understand the interplay of multiple species in an ecosystem. Experimental characterization of the enzymatic reactions of all enzymes in a genome is a tedious and expensive task. The problem is more pronounced in the metagenomic samples where even the species are not adequately cultured or characterized. Enzymes encoded by the gut microbiota play an essential role in the host metabolism; thus, warranting the need to accurately identify and annotate the full enzyme complements of species in the genomic and metagenomic projects. To fulfill this need, we develop and apply a method called ECemble, an ensemble approach to identify enzymes and enzyme classes and study the human gut metabolic pathways. ECemble method uses an ensemble of machine-learning methods to accurately model and predict enzymes from protein sequences and also identifies the enzyme classes and subclasses at the finest resolution. A tenfold cross-validation result shows accuracy between 97 and 99% at different levels in the hierarchy of enzyme classification, which is superior to comparable methods. We applied ECemble to predict the entire complements of enzymes from ten sequenced proteomes including the human proteome. We also applied this method to predict enzymes encoded by the human gut microbiome from gut metagenomic samples, and to study the role played by the microbe-derived enzymes in the human metabolism. After mapping the known and predicted enzymes to canonical human pathways, we identified 48 pathways that have at least one bacteria-encoded enzyme, which demonstrates the complementary role of gut microbiome in human gut metabolism. These pathways are primarily involved in metabolizing dietary nutrients such as carbohydrates, amino acids, lipids, cofactors and vitamins. The ECemble method is able to hierarchically assign high quality enzyme annotations to genomic and metagenomic data. This study demonstrated the real application of ECemble to understand the indispensable role played by microbe-encoded enzymes in the healthy functioning of human metabolic systems.

Regulation of age-related macular degeneration-like pathology by complement factor H

PubMed Central

Toomey, Christopher B.; Kelly, Una; Saban, Daniel R.; Bowes Rickman, Catherine

2015-01-01

Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh+/− and Cfh−/− mice fed a high-fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (sub-RPE) deposit formation, specifically basal laminar deposits, following high-fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch’s membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh+/− and Cfh−/− mice, RPE damage accompanied by loss of vision occurred only in old Cfh+/− mice. We demonstrate that such pathology is a function of excess complement activation in Cfh+/− mice versus complement deficiency in Cfh−/− animals. Due to the CFH-dependent increase in sub-RPE deposit height, we interrogated the potential of CFH as a previously unidentified regulator of Bruch’s membrane lipoprotein binding and show, using human Bruch’s membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Thus, advanced age, high-fat diet, and decreased CFH induce sub-RPE deposit formation leading to complement activation, which contributes to RPE damage and visual function impairment. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD. PMID:25991857

There Is a Method to the Madness: Strategies to Study Host Complement Evasion by Lyme Disease and Relapsing Fever Spirochetes.

PubMed

Marcinkiewicz, Ashley L; Kraiczy, Peter; Lin, Yi-Pin

2017-01-01

Lyme disease and relapsing fever are caused by various Borrelia species. Lyme disease borreliae , the most common vector-borne pathogens in both the U.S. and Europe, are transmitted by Ixodes ticks and disseminate from the site of tick bites to tissues leading to erythema migrans skin rash, arthritis, carditis, and neuroborreliosis. Relapsing fever borreliae , carried by ticks and lice, trigger reoccurring fever episodes. Following transmission, spirochetes survive in the blood to induce bacteremia at the early stages of infection, which is thought to promote evasion of the host complement system. The complement system acts as an important innate immune defense mechanism in humans and vertebrates. Upon activation, the cleaved complement components form complexes on the pathogen surface to eventually promote bacteriolysis. The complement system is negatively modulated by a number of functionally diverse regulators to avoid tissue damage. To evade and inhibit the complement system, spirochetes are capable of binding complement components and regulators. Complement inhibition results in bacterial survival in serum (serum resistance) and is thought to promote bloodstream survival, which facilitates spirochete dissemination and disease manifestations. In this review, we discuss current methodologies to elucidate the mechanisms of Borrelia spp. that promote serum resistance and bloodstream survival, as well as novel methods to study factors responsible for bloodstream survival of Lyme disease borreliae that can be applied to relapsing fever borreliae . Understanding the mechanisms these pathogens utilize to evade the complement system will ultimately aid in the development of novel therapeutic strategies and disease prevention to improve human health.

A radar-enabled collaborative sensor network integrating COTS technology for surveillance and tracking.

PubMed

Kozma, Robert; Wang, Lan; Iftekharuddin, Khan; McCracken, Ernest; Khan, Muhammad; Islam, Khandakar; Bhurtel, Sushil R; Demirer, R Murat

2012-01-01

The feasibility of using Commercial Off-The-Shelf (COTS) sensor nodes is studied in a distributed network, aiming at dynamic surveillance and tracking of ground targets. Data acquisition by low-cost (<$50 US) miniature low-power radar through a wireless mote is described. We demonstrate the detection, ranging and velocity estimation, classification and tracking capabilities of the mini-radar, and compare results to simulations and manual measurements. Furthermore, we supplement the radar output with other sensor modalities, such as acoustic and vibration sensors. This method provides innovative solutions for detecting, identifying, and tracking vehicles and dismounts over a wide area in noisy conditions. This study presents a step towards distributed intelligent decision support and demonstrates effectiveness of small cheap sensors, which can complement advanced technologies in certain real-life scenarios.

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

Homologous recombination mediates S-phase-dependent radioresistance in cells deficient in DNA polymerase eta

PubMed Central

Sharma, Ricky A.

2012-01-01

DNA polymerase eta (pol η) is the only DNA polymerase causally linked to carcinogenesis in humans. Inherited deficiency of pol η in the variant form of xeroderma pigmentosum (XPV) predisposes to UV-light-induced skin cancer. Pol η-deficient cells demonstrate increased sensitivity to cisplatin and oxaliplatin chemotherapy. We have found that XP30R0 fibroblasts derived from a patient with XPV are more resistant to cell kill by ionising radiation (IR) than the same cells complemented with wild-type pol η. This phenomenon has been confirmed in Burkitt’s lymphoma cells, which either expressed wild-type pol η or harboured a pol η deletion. Pol η deficiency was associated with accumulation of cells in S-phase, which persisted after IR. Cells deficient in pol η demonstrated increased homologous recombination (HR)-directed repair of double strand breaks created by IR. Depletion of the HR protein, X-ray repair cross-complementing protein 3 (XRCC3), abrogated the radioresistance observed in pol η-deficient cells as compared with pol η-complemented cells. These findings suggest that HR mediates S-phase-dependent radioresistance associated with pol η deficiency. We propose that pol η protein levels in tumours may potentially be used to identify patients who require treatment with chemo-radiotherapy rather than radiotherapy alone for adequate tumour control. PMID:22822095

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

PubMed Central

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

2002-01-01

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

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

PubMed

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

2002-06-01

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

Lack of association of CFD polymorphisms with advanced age-related macular degeneration.

PubMed

Zeng, Jiexi; Chen, Yuhong; Tong, Zongzhong; Zhou, Xinrong; Zhao, Chao; Wang, Kevin; Hughes, Guy; Kasuga, Daniel; Bedell, Matthew; Lee, Clara; Ferreyra, Henry; Kozak, Igor; Haw, Weldon; Guan, Jean; Shaw, Robert; Stevenson, William; Weishaar, Paul D; Nelson, Mark H; Tang, Luosheng; Zhang, Kang

2010-11-03

Age-related macular degeneration (AMD) is the most common cause of irreversible central vision loss worldwide. Research has linked AMD susceptibility with dysregulation of the complement cascade. Typically, complement factor H (CFH), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3) are associated with AMD. In this paper, we investigated the association between complement factor D (CFD), another factor of the complement system, and advanced AMD in a Caucasian population. Six single nucleotide polymorphisms (SNPs), rs1683564, rs35186399, rs1683563, rs3826945, rs34337649, and rs1651896, across the region covering CFD, were chosen for this study. One hundred and seventy-eight patients with advanced AMD and 161 age-matched normal controls were genotyped. Potential positive signals were further tested in another independent 445 advanced AMD patients and 190 controls. χ2 tests were performed to compare the allele frequencies between case and control groups. None of the six SNPs of CFD was found to be significantly associated with advanced AMD in our study. Our findings suggest that CFD may not play a major role in the genetic susceptibility to AMD because no association was found between the six SNPs analyzed in the CFD region and advanced AMD.

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 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

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.

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

Trichinella spiralis Paramyosin Binds to C8 and C9 and Protects the Tissue-Dwelling Nematode from Being Attacked by Host Complement

PubMed Central

Zhang, Zhifei; Yang, Jing; Wei, Junfei; Yang, Yaping; Chen, Xiaoqin; Zhao, Xi; Gu, Yuan; Cui, Shijuan; Zhu, Xinping

2011-01-01

Background Paramyosin is a thick myofibrillar protein found exclusively in invertebrates. Evidence suggested that paramyosin from helminths serves not only as a structural protein but also as an immunomodulatory agent. We previously reported that recombinant Trichinella spiralis paramyosin (Ts-Pmy) elicited a partial protective immunity in mice. In this study, the ability of Ts-Pmy to bind host complement components and protect against host complement attack was investigated. Methods and Findings In this study, the transcriptional and protein expression levels of Ts-Pmy were determined in T. spiralis newborn larva (NBL), muscle larva (ML) and adult worm developmental stages by RT-PCR and western blot analysis. Expression of Ts-Pmy at the outer membrane was observed in NBL and adult worms using immunogold electron microscopy and immunofluorescence staining. Functional analysis revealed that recombinant Ts-Pmy(rTs-Pmy) strongly bound to complement components C8 and C9 and inhibited the polymerization of C9 during the formation of the membrane attack complex (MAC). rTs-Pmy also inhibited the lysis of rabbit erythrocytes (ER) elicited by an alternative pathway-activated complement from guinea pig serum. Inhibition of native Ts-Pmy on the surface of NBL with a specific antiserum reduced larvae viability when under the attack of complement in vitro. In vivo passive transfer of anti-Ts-Pmy antiserum and complement-treated larvae into mice also significantly reduced the number of larvae that developed to ML. Conclusion These studies suggest that the outer membrane form of T. spiralis paramyosin plays an important role in the evasion of the host complement attack. PMID:21750743

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

PubMed

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

2017-04-01

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

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

Combined application of imaging techniques for the characterization and authentication of ancient weapons

NASA Astrophysics Data System (ADS)

Salvemini, Filomena; Grazzi, Francesco; Kardjilov, Nikolay; Wieder, Frank; Manke, Ingo; Edge, David; Williams, Alan; Zoppi, Marco

2017-05-01

Non-invasive experimental methods play an important role in the field of cultural heritage. Benefiting from the technical progress in recent years, neutron imaging has been demonstrated to complement effectively studies based on surface analysis, allowing for a non-invasive characterization of the whole three-dimensional volume. This study focuses on a kris and a kanjar, two weapons from ancient Asia, to show the potential of the combined use of X-ray and neutron imaging techniques for the characterisation of the manufacturing methods and the authentication of objects of cultural and historical interest.

Electrostatic Steering Accelerates C3d:CR2 Association

PubMed Central

2016-01-01

Electrostatic effects are ubiquitous in protein interactions and are found to be pervasive in the complement system as well. The interaction between complement fragment C3d and complement receptor 2 (CR2) has evolved to become a link between innate and adaptive immunity. Electrostatic interactions have been suggested to be the driving factor for the association of the C3d:CR2 complex. In this study, we investigate the effects of ionic strength and mutagenesis on the association of C3d:CR2 through Brownian dynamics simulations. We demonstrate that the formation of the C3d:CR2 complex is ionic strength-dependent, suggesting the presence of long-range electrostatic steering that accelerates the complex formation. Electrostatic steering occurs through the interaction of an acidic surface patch in C3d and the positively charged CR2 and is supported by the effects of mutations within the acidic patch of C3d that slow or diminish association. Our data are in agreement with previous experimental mutagenesis and binding studies and computational studies. Although the C3d acidic patch may be locally destabilizing because of unfavorable Coulombic interactions of like charges, it contributes to the acceleration of association. Therefore, acceleration of function through electrostatic steering takes precedence to stability. The site of interaction between C3d and CR2 has been the target for delivery of CR2-bound nanoparticle, antibody, and small molecule biomarkers, as well as potential therapeutics. A detailed knowledge of the physicochemical basis of C3d:CR2 association may be necessary to accelerate biomarker and drug discovery efforts. PMID:27092816

EEG oscillations and recognition memory: theta correlates of memory retrieval and decision making.

PubMed

Jacobs, Joshua; Hwang, Grace; Curran, Tim; Kahana, Michael J

2006-08-15

Studies of memory retrieval have identified electroencephalographic (EEG) correlates of a test item's old-new status, reaction time, and memory load. In the current study, we used a multivariate analysis to disentangle the effects of these correlated variables. During retrieval, power of left-parietal theta (4-8 Hz) oscillations increased in proportion to how well a test item was remembered, and theta in central regions correlated with decision making. We also studied how these oscillatory dynamics complemented event-related potentials. These findings are the first to demonstrate that distinct patterns of theta oscillations can simultaneously relate to different aspects of behavior.

A widespread plant-fungal-bacterial symbiosis promotes plant biodiversity, plant nutrition and seedling recruitment

PubMed Central

van der Heijden, Marcel GA; Bruin, Susanne de; Luckerhoff, Ludo; van Logtestijn, Richard SP; Schlaeppi, Klaus

2016-01-01

Highly diverse microbial assemblages colonize plant roots. It is still poorly understood whether different members of this root microbiome act synergistically by supplying different services (for example, different limiting nutrients) to plants and plant communities. In order to test this, we manipulated the presence of two widespread plant root symbionts, arbuscular mycorrhizal fungi and nitrogen-fixing rhizobia bacteria in model grassland communities established in axenic microcosms. Here, we demonstrate that both symbionts complement each other resulting in increased plant diversity, enhanced seedling recruitment and improved nutrient acquisition compared with a single symbiont situation. Legume seedlings obtained up to 15-fold higher productivity if they formed an association with both symbionts, opposed to productivity they reached with only one symbiont. Our results reveal the importance of functional diversity of symbionts and demonstrate that different members of the root microbiome can complement each other in acquiring different limiting nutrients and in driving important ecosystem functions. PMID:26172208

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.

[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.

The prognostic performance of the complement system in septic patients in emergency department: a cohort study.

PubMed

Zhao, Xin; Chen, Yun-Xia; Li, Chun-Sheng

2015-01-01

To investigate the prognostic performance of complement components in septic patients, complement 3, membrane attack complex (MAC) and mannose-binding lectin were measured and compared among adult patients with sepsis, severe sepsis and septic shock, as well as between in-hospital nonsurvivors and survivors. The prognostic value of complement components was compared with mortality in emergency department sepsis (MEDS) score. Median complement 3, MAC and mannose-binding lectin increased directly with the sepsis, severe sepsis and septic shock groups, and were significantly higher in nonsurvivors than in survivors. MEDS and MAC independently predicted in-hospital mortality. The prognostic performance of MAC was superior to MEDS as analyzed by receiver operating characteristic curve and area under the curve.

Language and theory of mind in autism spectrum disorder: the relationship between complement syntax and false belief task performance.

PubMed

Lind, Sophie E; Bowler, Dermot M

2009-06-01

This study aimed to test the hypothesis that children with autism spectrum disorder (ASD) use their knowledge of complement syntax as a means of "hacking out" solutions to false belief tasks, despite lacking a representational theory of mind (ToM). Participants completed a "memory for complements" task, a measure of receptive vocabulary, and traditional location change and unexpected contents false belief tasks. Consistent with predictions, the correlation between complement syntax score and location change task performance was significantly stronger within the ASD group than within the comparison group. However, contrary to predictions, complement syntax score was not significantly correlated with unexpected contents task performance within either group. Possible explanations for this pattern of results are considered.

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.

Complement Set Reference after Implicitly Small Quantities: An Event-Related Potentials Study

ERIC Educational Resources Information Center

Ingram, Joanne; Ferguson, Heather J.

2018-01-01

An anaphoric reference to the complement-set is a reference to the set that does not fulfil the predicate of the preceding sentence. Preferred reference to the complement-set has been found in eye movements when a character's implicit desire for a high amount has been denied using a negative emotion. We recorded event-related potentials to examine…

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

Theory of mind in SLI revisited: links with syntax, comparisons with ASD.

PubMed

Durrleman, Stephanie; Burnel, Morgane; Reboul, Anne

2017-11-01

According to the linguistic determinism approach, knowledge of sentential complements such as: John says that the earth is flat plays a crucial role in theory of mind (ToM) development by providing a means to represent explicitly people's mental attitudes and beliefs. This approach predicts that mastery of complements determines successful belief reasoning across explicit ToM tasks, even low-verbal ones, and across populations. (1) To investigate the link between a low-verbal ToM-task and complements in Specific Language Impairment (SLI), (2) To determine whether this population shows similar ToM performance to that of children with Autism Spectrum Disorder (ASD) or those with Typical Development (TD) once these groups are matched on competency for complements, (3) To explore whether complements conveying a falsehood without jeopardizing the veracity of the entire sentence, such as complements of verbs of communication, are more crucial for belief attribution than complements which do not have this property, namely complements of verbs of perception, (?John sees that the earth is flat). Children with SLI (n = 20), with ASD (n = 34) and TD (n = 30) completed sentence-picture-matching tasks assessing complementation with communication and perception verbs, as well as a picture-sequencing task assessing ToM. Children were furthermore evaluated for general grammatical and lexical abilities and non-verbal IQ. Results reveal that competency on complements relates to ToM performance with a low-verbal task in SLI, and that SLI, ASD and TD groups of equivalent performance on complements also perform similarly for ToM. Results further suggest that complements with an independent truth-value are the only ones to show a significant relation to ToM performance after teasing out the impact of non-verbal reasoning. This study suggests that clinical groups of different aetiologies as well as TD children perform comparably for ToM once they have similar complementation skills. Findings further highlight that specific types of complements, namely those with an independent truth value, relate in a special way to mentalizing. Future work should determine whether these specific structures could be effective in ToM remediation programmes. © 2017 Royal College of Speech and Language Therapists.

A new web-based framework development for fuzzy multi-criteria group decision-making.

PubMed

Hanine, Mohamed; Boutkhoum, Omar; Tikniouine, Abdessadek; Agouti, Tarik

2016-01-01

Fuzzy multi-criteria group decision making (FMCGDM) process is usually used when a group of decision-makers faces imprecise data or linguistic variables to solve the problems. However, this process contains many methods that require many time-consuming calculations depending on the number of criteria, alternatives and decision-makers in order to reach the optimal solution. In this study, a web-based FMCGDM framework that offers decision-makers a fast and reliable response service is proposed. The proposed framework includes commonly used tools for multi-criteria decision-making problems such as fuzzy Delphi, fuzzy AHP and fuzzy TOPSIS methods. The integration of these methods enables taking advantages of the strengths and complements each method's weakness. Finally, a case study of location selection for landfill waste in Morocco is performed to demonstrate how this framework can facilitate decision-making process. The results demonstrate that the proposed framework can successfully accomplish the goal of this study.

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.

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.

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

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.

Second-harmonic phonon spectroscopy of α -quartz

NASA Astrophysics Data System (ADS)

Winta, Christopher J.; Gewinner, Sandy; Schöllkopf, Wieland; Wolf, Martin; Paarmann, Alexander

2018-03-01

We demonstrate midinfrared second-harmonic generation as a highly sensitive phonon spectroscopy technique that we exemplify using α -quartz (SiO2) as a model system. A midinfrared free-electron laser provides direct access to optical phonon resonances ranging from 350 to 1400 cm-1 . While the extremely wide tunability and high peak fields of a free-electron laser promote nonlinear spectroscopic studies—complemented by simultaneous linear reflectivity measurements—azimuthal scans reveal crystallographic symmetry information of the sample. Additionally, temperature-dependent measurements show how damping rates increase, phonon modes shift spectrally and in certain cases disappear completely when approaching Tc=846 K where quartz undergoes a structural phase transition from trigonal α -quartz to hexagonal β -quartz, demonstrating the technique's potential for studies of phase transitions.

Hha Controls Escherichia coli O157:H7 Biofilm Formation by Differential Regulation of Global Transcriptional Regulators FlhDC and CsgD

PubMed Central

Bearson, Bradley L.

2013-01-01

Although molecular mechanisms promoting adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation are not fully understood. In this study, we demonstrate that biofilm formation in EHEC O157:H7 strain 86-24 is highly repressed compared to that in an isogenic hha mutant. The hha mutant produced large quantities of biofilm compared to the wild-type strain at 30°C and 37°C. Complementation of the hha mutant reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production. While swimming motility and expression of the flagellar gene fliC were significantly reduced, the expression of csgA (encoding curlin of curli fimbriae) and the ability to bind Congo red were significantly enhanced. The expression of both fliC and csgA and the phenotypes of motility and curli production affected by these two genes, respectively, were restored to wild-type levels in the complemented hha mutant. The csgA deletion abolished biofilm formation in the hha mutant and wild-type strain, and csgA complementation restored biofilm formation to these strains, indicating the importance of csgA and curli in biofilm formation. The regulatory effects of Hha on flagellar and curli gene expression appear to occur via the induction and repression of FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant, respectively. In gel shift assays Hha interacted with flhDC and csgD promoters. In conclusion, Hha regulates biofilm formation in EHEC O157:H7 by differential regulation of FlhDC and CsgD, the global regulators of motility and curli production, respectively. PMID:23377937

Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD.

PubMed

Sharma, Vijay K; Bearson, Bradley L

2013-04-01

Although molecular mechanisms promoting adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation are not fully understood. In this study, we demonstrate that biofilm formation in EHEC O157:H7 strain 86-24 is highly repressed compared to that in an isogenic hha mutant. The hha mutant produced large quantities of biofilm compared to the wild-type strain at 30°C and 37°C. Complementation of the hha mutant reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production. While swimming motility and expression of the flagellar gene fliC were significantly reduced, the expression of csgA (encoding curlin of curli fimbriae) and the ability to bind Congo red were significantly enhanced. The expression of both fliC and csgA and the phenotypes of motility and curli production affected by these two genes, respectively, were restored to wild-type levels in the complemented hha mutant. The csgA deletion abolished biofilm formation in the hha mutant and wild-type strain, and csgA complementation restored biofilm formation to these strains, indicating the importance of csgA and curli in biofilm formation. The regulatory effects of Hha on flagellar and curli gene expression appear to occur via the induction and repression of FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant, respectively. In gel shift assays Hha interacted with flhDC and csgD promoters. In conclusion, Hha regulates biofilm formation in EHEC O157:H7 by differential regulation of FlhDC and CsgD, the global regulators of motility and curli production, respectively.

A Radar-Enabled Collaborative Sensor Network Integrating COTS Technology for Surveillance and Tracking

PubMed Central

Kozma, Robert; Wang, Lan; Iftekharuddin, Khan; McCracken, Ernest; Khan, Muhammad; Islam, Khandakar; Bhurtel, Sushil R.; Demirer, R. Murat

2012-01-01

The feasibility of using Commercial Off-The-Shelf (COTS) sensor nodes is studied in a distributed network, aiming at dynamic surveillance and tracking of ground targets. Data acquisition by low-cost (<$50 US) miniature low-power radar through a wireless mote is described. We demonstrate the detection, ranging and velocity estimation, classification and tracking capabilities of the mini-radar, and compare results to simulations and manual measurements. Furthermore, we supplement the radar output with other sensor modalities, such as acoustic and vibration sensors. This method provides innovative solutions for detecting, identifying, and tracking vehicles and dismounts over a wide area in noisy conditions. This study presents a step towards distributed intelligent decision support and demonstrates effectiveness of small cheap sensors, which can complement advanced technologies in certain real-life scenarios. PMID:22438713

Latent cytomegalovirus infection in blood donors

PubMed Central

Diosi, Peter; Moldovan, Eva; Tomescu, Nicholas

1969-01-01

Twenty-one out of 32 apparently healthy blood donors aged 21 to 65 years yielded positive complement fixation tests with a cytomegalovirus antigen, at titres ranging from 1:8 to 1:64. Virus was present in leucocyte cultures of fresh peripheral blood of two seropositive subjects from a total of 35 donors examined. Plasma and 48-hour stored blood specimens failed to disclose virus in culture. Viruria could not be demonstrated, and there was no evidence of recent illness in the study group. These findings suggest that subclinical viraemia is not uncommon in blood donors. PMID:4311727

Intravenous immunoglobulins – understanding properties and mechanisms

PubMed Central

Durandy, A; Kaveri, S V; Kuijpers, T W; Basta, M; Miescher, S; Ravetch, J V; Rieben, R

2009-01-01

High-dose intravenous immunoglobulin (IVIg) preparations are used currently for the treatment of autoimmune or inflammatory diseases. Despite numerous studies demonstrating efficacy, the precise mode of action of IVIg remains unclear. Paradoxically, IgG can exert both pro- and anti-inflammatory activities, depending on its concentration. The proinflammatory activity of low-dose IVIg requires complement activation or binding of the Fc fragment of IgG to IgG-specific receptors (FcγR) on innate immune effector cells. In contrast, when administered in high concentrations, IVIg has anti-inflammatory properties. How this anti-inflammatory effect is mediated has not yet been elucidated fully, and several mutually non-exclusive mechanisms have been proposed. This paper represents the proceedings of a session entitled ‘IVIg – Understanding properties and mechanisms’ at the 6th International Immunoglobulin Symposium that was held in Interlaken on 26–28 March 2009. The presentations addressed how IgG may affect the cellular compartment, evidence for IVIg-mediated scavenging of complement fragments, the role of the dimeric fraction of IVIg, the anti-inflammatory properties of the minor fraction of sialylated IgG molecules, and the genetic organization and variation in FcγRs. These findings demonstrate the considerable progress that has been made in understanding the mechanisms of action of IVIgs, and may influence future perspectives in the field of Ig therapy. PMID:19883419

The effects of increasing dietary levels of soy protein concentrate (SPC) on the immune responses and disease resistance (furunculosis) of vaccinated and non-vaccinated Atlantic salmon (Salmo salar L.) parr.

PubMed

Metochis, Christoforos P; Spanos, I; Auchinachie, N; Crampton, V O; Bell, J G; Adams, A; Thompson, K D

2016-12-01

Juvenile salmon, with an initial weight of 9 g, were fed three experimental diets, formulated to replace 35 (SPC35), 58 (SPC58) and 80 (SPC80) of high quality fishmeal (FM) with soy protein concentrate (SPC) in quadruplicate tanks. Higher dietary SPC inclusion was combined with increased supplementation of methionine, lysine, threonine and phosphorus. The experiment was carried out for 177 days. On day 92 salmon in each tank were bulk weighed. Post weighing eighty salmon from each tank were redistributed in two sets of 12 tanks. Salmon from the first set of tanks were vaccinated, while the second group was injected with phosphate buffer saline (PBS). Salmon were sampled on day 92 (pre-vaccination), day 94 (2 days post vaccination [dpv]/PBS injection [dpPBSinj]) and day 154 (62 dpv/dpPBSinj) of the trial for the assessment of their immune responses, prior to the performance of salmon bulk weights for each tank. On day 154, fish from each tank were again bulk weighed and then seventeen salmon per tank were redistributed in two sets of twelve tanks and intra-peritoneally infected with Aeromonas salmonicida. At Day 154, SPC80 demonstrated lower performance (weight gain, specific growth rate and thermal growth coefficient and feed conversion ratio) compared to SPC35 salmon. Reduced classical and total complement activities for salmon fed diets with over 58% of protein from SPC, were demonstrated prior to vaccination. Reduced alternative complement activity was detected for both SPC58 and SPC80 salmon at 2 dpv and for the SPC80 group at 62 dpv. Total and classical complement activities demonstrated no differences among the dietary groups after vaccination. Numerical increases in classical complement activity were apparent upon increased dietary SPC levels. Increased phagocytic activity (% phagocytosis and phagocytic index) was exhibited for the SPC58 group compared to SPC35 salmon at 62 dpPBSinj. No differences in serum lysozyme activity, total IgM, specific antibodies, protein, glucose and HKM respiratory burst were detected among the dietary groups at any timepoint or state. Mortalities as a result of the experimental infection only occurred in PBS-injected fish. No differences in mortality levels were demonstrated among the dietary groups. SPC58 diet supported both good growth and health in juvenile Atlantic salmon while SPC80 diet did not compromise salmon' immunity or resistance to intraperitoneally inflicted furunculosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

In Vitro and in Vivo Molecular Imaging of Estrogen Receptor α and β Homo- and Heterodimerization: Exploration of New Modes of Receptor Regulation

PubMed Central

Tamrazi, Anobel; Massoud, Tarik F.; Katzenellenbogen, John A.; Gambhir, Sanjiv S.

2011-01-01

Estrogen receptor (ER) biology reflects the actions of estrogens through the two receptors, ERα and ERβ, although little is known regarding the preference for formation of ER homo- vs. heterodimers, and how this is affected by the level of ligand occupancy and preferential ligand affinity for one of the ER subtypes. In this report, we use a split optical reporter-protein complementation system to demonstrate the physical interaction between ERα and ERβ in response to different ER ligands in cells and, for the first time, by in vivo imaging in living animals. The genetically encoded reporter vectors constructed with the ligand-binding domains of ERα and ERβ, fused to split firefly or Renilla luciferase (Fluc or hRluc) fragments, were used for this study. This molecular proteomic technique was used to detect ERα/ERα or ERβ/ERβ homodimerization, or ERα/ERβ heterodimerization induced by ER subtype-selective and nonselective ligands, and selective ER modulators (SERM), as well as in dimers in which one mutant monomer was unable to bind estradiol. The SERM-bound ERα and ERβ form the strongest dimers, and subtype-preferential homodimerization was seen with ERα-selective ligands (methyl piperidino pyrazole/propyl pyrazole triol) and the ERβ-selective ligands (diarylpropionitrile/tetrahydrochrysene/genistein). We also demonstrated that a single ligand-bound monomer can form homo- or heterodimers with an apo-monomer. Xenografts of human embryonic kidney 293T cells imaged in living mice by bioluminescence showed real-time ligand induction of ERα/ERβ heterodimerization and reversal of dimerization upon ligand withdrawal. The results from this study demonstrate the value of the split luciferase-based complementation system for studying ER-subtype interactions in cells and for evaluating them in living animals by noninvasive imaging. They also probe what combinations of ERα and ERβ dimers might be the mediators of the effects of different types of ER ligands given at different doses. PMID:22052998

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.

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.

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

PubMed

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

2018-01-01

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

The effect of chronic consumption of red wine on cardiovascular disease risk factors in postmenopausal women.

PubMed

Naissides, Mary; Mamo, John C L; James, Anthony P; Pal, Sebely

2006-04-01

Moderate red wine has been shown to reduce cardiovascular disease (CVD) risk, however the effects on certain CVD risk factors are unclear. In this study we have investigated the effects of dealcoholised red wine (DRW) and full-complement red wine (RW) on several cardiovascular risk factors in mildly hypercholesterolaemic postmenopausal women. To elucidate whether the chronic consumption of red wine polyphenols improves risk factors associated with CVD in hypercholesterolaemic postmenopausal women. Forty-five hypercholesterolaemic postmenopausal women were randomly assigned to consume 400 mL/day of either water, DRW or RW for 6 weeks following a 4-week washout. Fasting measures of lipids, lipoproteins, insulin and glucose were taken at 0 and 6 weeks. DRW consumption had no effect of fasting concentrations of lipids, lipoproteins, insulin and glucose. However, chronic consumption of RW significantly reduced fasting LDL cholesterol concentrations by 8% and increased HDL cholesterol concentrations by 17% in hypercholesterolaemic postmenopausal women. Collectively, regular consumption of full-complement red wine reduces CVD risk by improving fasting lipid levels in hypercholesterolaemic postmenopausal women. This study uniquely demonstrated the LDL cholesterol-lowering effects of red wine in individuals at high CVD risk, which has not previously been shown.

T cell-independent and T cell-dependent immunoglobulin G responses to polyomavirus infection are impaired in complement receptor 2-deficient mice.

PubMed

Szomolanyi-Tsuda, Eva; Seedhom, Mina O; Carroll, Michael C; Garcea, Robert L

2006-08-15

Polyomavirus (PyV) infection induces protective T cell-independent (TI) IgM and IgG antibody responses in T cell-deficient mice, but these responses are not generated by immunization with viral proteins or virus like particles. We hypothesized that innate signals contribute to the generation of isotype-switched antiviral antibody responses. We studied the role of complement receptor (CR2) engagement in TI and T cell-dependent (TD) antibody responses to PyV using CR2-deficient mice. Antiviral IgG responses were reduced by 80-40% in CR2-/- mice compared to wild type. Adoptive transfer experiments demonstrated the need for CR2 not only in TD, but also in TI IgG responses to PyV. Transfer of CR2-/- B lymphocytes to SCID mice resulted in TI antiviral IgG responses that corresponded to 10% of that seen in wild-type B cell-reconstituted mice. Thus, our studies revealed a profound dependence of TI and TD antiviral antibody responses on CR2-mediated signals in PyV-infected mice, where the viral antigen is abundant and persistent.

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

Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

PubMed Central

Joseph, Joan; Fernández-Lloris, Raquel; Pezzat, Elías; Saubi, Narcís; Cardona, Pere-Joan; Mothe, Beatriz; Gatell, Josep Maria

2010-01-01

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors. PMID:20617151

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

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

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

1994-09-01

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

Language and Theory of Mind in Autism Spectrum Disorder: The Relationship between Complement Syntax and False Belief Task Performance

ERIC Educational Resources Information Center

Lind, Sophie E.; Bowler, Dermot M.

2009-01-01

This study aimed to test the hypothesis that children with autism spectrum disorder (ASD) use their knowledge of complement syntax as a means of "hacking out" solutions to false belief tasks, despite lacking a representational theory of mind (ToM). Participants completed a "memory for complements" task, a measure of receptive vocabulary, and…

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

PubMed

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

2010-10-01

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

Functional basis for complement evasion by staphylococcal superantigen-like 7

PubMed Central

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

2010-01-01

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

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

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.

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.

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

PubMed Central

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

2014-01-01

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

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum.

PubMed

El Bissati, Kamal; Downie, Megan J; Kim, Seong-Kyoun; Horowitz, Michael; Carter, Nicola; Ullman, Buddy; Ben Mamoun, Choukri

2008-10-01

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.

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

Glucose-6-Phosphate Dehydrogenase Deficiency Mimicking Atypical Hemolytic Uremic Syndrome.

PubMed

Walsh, Patrick R; Johnson, Sally; Brocklebank, Vicky; Salvatore, Jacobo; Christian, Martin; Kavanagh, David

2018-02-01

A 4-year-old boy presented with nonimmune hemolysis, thrombocytopenia, and acute kidney injury. Investigations for an underlying cause failed to identify a definitive cause and a putative diagnosis of complement-mediated atypical hemolytic uremic syndrome (aHUS) was made. The patient was started initially on plasma exchange and subsequently eculizumab therapy, after which his kidney function rapidly improved. While on eculizumab therapy, despite adequate complement blockade, he presented 2 more times with hemolytic anemia and thrombocytopenia, but without renal involvement. Genetic analysis did not uncover a mutation in any known aHUS gene (CFH, CFI, CFB, C3, CD46, THBD, INF2, and DGKE) and anti-factor H antibodies were undetectable. Whole-exome sequencing was undertaken to identify a cause for the eculizumab resistance. This revealed a pathogenic variant in G6PD (glucose-6-phosphate dehydrogenase), which was confirmed by functional analysis demonstrating decreased erythrocyte G6PD activity. Eculizumab therapy was withdrawn. Complement-mediated aHUS is a diagnosis of exclusion and this case highlights the diagnostic difficulty that remains without an immediately available biomarker for confirmation. This case of G6PD deficiency presented with a phenotype clinically indistinguishable from complement-mediated aHUS. We recommend that G6PD deficiency be included in the differential diagnosis of patients presenting with aHUS and suggest measuring erythrocyte G6PD concentrations in these patients. Copyright © 2017. Published by Elsevier Inc.

Demonstrating Proof by Contrapositive and Contradiction through Physical Analogs.

ERIC Educational Resources Information Center

Kaiser, Mark J.

1993-01-01

Presents examples where mathematical and physical reasoning complement each other in interpreting and analyzing some basic science concepts using proof by contradiction and contrapositive. Examples involve the rotation of the moon, the stability of electrons and protons, the electron's orbit about the nucleus, and the earth's liquid core. (MDH)

Synchronous E-Learning: Reflections and Design Considerations

ERIC Educational Resources Information Center

Tabak, Filiz; Rampal, Rohit

2014-01-01

This paper is a personal reflection on the design, development, and delivery of online synchronous conferencing as a pedagogical tool complementing traditional, face-to-face content delivery and learning. The purpose of the paper is to demonstrate how instructors can combine collaborative and virtual learning principles in course design. In…

[The representation of physical pain in art and the Greek escultural group of the Laocoonte].

PubMed

Roqué, M H; Ruival, C; Roqué, C M

2006-01-01

It makes reference to the symptoms and signs of external pain and internal man suffering, masterly represented on marble by greek sculptors of Ancient Greece. A demonstration of the importance of literature and sculpture as an humanistic complement for teaching History of Medicine.

Expanding Possibilities through Metaphor: Breaking Biases to Improve Crisis Management

ERIC Educational Resources Information Center

Cirka, Carol C.; Corrigall, Elizabeth A.

2010-01-01

In this article, we demonstrate that an exercise using metaphors to overcome cognitive biases helped students to proactively imagine and prepare for an expanded set of potential crises. The exercise complements traditional textbook approaches to crisis management and incorporates creativity skill building in a realistic context. Learning outcomes…

TROPIX: A solar electric propulsion flight experiment

NASA Technical Reports Server (NTRS)

Hickman, J. Mark; Hillard, G. Barry; Oleson, Steven R.

1993-01-01

The Transfer Orbit Plasma Interaction Experiment (TROPIX) is a proposed scientific experiment and flight demonstration of a solar electric propulsion vehicle. Its mission goals are to significantly increase our knowledge of Earth's magnetosphere and its associated plasma environment and to demonstrate an operational solar electric upper stage (SEUS) for small launch vehicles. The scientific investigations and flight demonstration technology experiments are uniquely interrelated because of the spacecraft's interaction with the surrounding environment. The data obtained will complement previous studies of the Earth's magnetosphere and space plasma environment by supplying the knowledge necessary to attain the strategic objectives of the NASA Office of Space Science. This first operational use of a primary ion propulsion vehicle, designed to withstand the harsh environments from low Earth orbit to geosynchronous Earth orbit, may lead to the development of a new class of electric propulsion upper stages or space-based transfer vehicles and may improve future spacecraft design and safety.

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.

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

PubMed

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

2015-12-01

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

Mutations participating in interallelic complementation in propionic acidemia

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

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

1994-07-01

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

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

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.

Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis.

PubMed

Skjoedt, Mikkel-Ole; Palarasah, Yaseelan; Rasmussen, Karina; Vitved, Lars; Salomonsen, Jan; Kliem, Anette; Hansen, Soren; Koch, Claus; Skjodt, Karsten

2010-01-01

The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intestinalis. The CioMBLs display similarities with vertebrate MBLs and comprise a collagen-like region, alpha-helical coiled-coils and a carbohydrate recognition domain (CRD) with conserved residues involved in calcium and carbohydrate binding. Structural analysis revealed an oligomerization through interchain disulphide bridges between N-terminal cysteine residues and cysteines located between the neck region and the CRD. RT-PCR showed a tissue specific expression of CioMBL in the gut and by immunohistochemistry analysis we also demonstrated that CioMBL co-localize with an MBL-associated serine protease in the epithelia cells lining the stomach and intestine. In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway.

Checkout/demonstration application program for the SEL 840MP Multi-Processing Control System: Version 1 (MPCS/1)

NASA Technical Reports Server (NTRS)

Anderson, W. F.; Conway, J. R.; Keller, L. C.

1972-01-01

The characteristics of the application program were developed to verify and demonstrate the SEL 840MP Multi-Processing Control System - Version I (MPCS/1). The application program emphasizes the display support and task control capabilities. The application program is further intended to be used as an aid to familization with MPCS/1. It complements the information provided in the MPCS/1 Users Guide, Volume I and II.

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.

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.

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

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.

Production and Evaluation of a Purified Adenovirus Group-Specific (Hexon) Antigen for Use in the Diagnostic Complement Fixation Test

PubMed Central

Dowdle, W. R.; Lambriex, M.; Hierholzer, J. C.

1971-01-01

A simple procedure for the production of large volumes of purified adenovirus group-specific complement-fixing (CF) (hexon) antigen by selective adsorption to and elution from CaHPO4 is described. Results of immunodiffusion tests, electrophoresis, electron microscopy, and tests for hemagglutination and infectivity indicate that the purified antigen consisted of a single virus component (hexon). The purified product contained little host materials. Unlike the crude virus harvest usually employed for serodiagnostic CF tests, the purified antigen demonstrated no anticomplementary activity and did not develop such activity during storage. The purified antigen was equal to or slightly more sensitive than crude virus harvests for serodiagnosis of adenovirus infections. Images PMID:4325021

Glutamine Deprivation Causes Enhanced Plating Efficiency of a Herpes Simplex Virus Type 1 ICP0-Null Mutant ▿

PubMed Central

Bringhurst, Ryan M.; Dominguez, Antonia A.; Schaffer, Priscilla A.

2008-01-01

Isoleucine deprivation of cellular monolayers prior to infection has been reported to result in partial complementation of a herpes simplex virus type 1 (HSV-1) ICP0 null (ICP0−) mutant. We now report that glutamine deprivation alone is able to enhance the plating efficiency of an ICP0− virus and that isoleucine deprivation has little or no effect. Because a low glutamine level is associated with stress and because stress is known to induce reactivation, low levels of glutamine may be relevant to the reactivation of HSV-1 from latency. Additionally, we demonstrate that arginine and methionine deprivation result in partial complementation of the ICP0− virus. PMID:18768961

Assessment of posterior vaginal wall prolapse: comparison of physical findings to cystodefecoperitoneography.

PubMed

Altman, Daniel; López, Annika; Kierkegaard, Jonas; Zetterström, Jan; Falconer, Christian; Pollack, Johan; Mellgren, Anders

2005-01-01

The aim of the present study was to compare clinical and radiological findings when assessing posterior vaginal wall prolapse. Defecography can be used to complement the clinical evaluation in patients with posterior vaginal wall prolapse. Further development of the defecography technique, using contrast medium in the urinary bladder and intraperitoneally, have resulted in cystodefecoperitoneography (CDP). Thirty-eight women underwent clinical examination using the pelvic organ prolapse quantification system (POP-Q) followed by CDP. All patients answered a standardized bowel function questionnaire. Statistical analysis measuring correlation between POP-Q and CDP using Pearson's correlation coefficient (r) and Spearman's rank order correlation coefficient (rs) demonstrated a poor to moderate correlation, r=0.49 and rs=0.55. Although there was a strong association between large rectoceles (>3 cm) at CDP and symptoms of rectal emptying difficulties (p<0.001), severity and prevalence of bowel dysfunction showed poor coherence with clinical prolapse staging and findings at radiological imaging. Vaginal topography and POP-Q staging predict neither radiological size nor visceral involvement in posterior vaginal wall prolapse. Radiological evaluation may therefore be a useful complement in selected patients.

Genetic relatedness of Brazilian Colletotrichum truncatum isolates assessed by vegetative compatibility groups and RAPD analysis.

PubMed

Sant'Anna, Juliane R; Miyamoto, Cláudia T; Rosada, Lúcia J; Franco, Claudinéia C S; Kaneshima, Edilson N; Castro-Prado, Marialba A A

2010-01-01

The genetic variation among nine soybean-originating isolates of Colletotrichum truncatum from different Brazilian states was studied. Nitrate non-utilizing (nit) mutants were obtained with potassium chlorate and used to characterize vegetative compatibility reactions, heterokaryosis and RAPD profile. Based on pairings of nit mutants from the different isolates, five vegetative complementation groups (VCG) were identified, and barriers to the formation of heterokaryons were observed among isolates derived from the same geographic area. No complementation was observed among any of the nit mutants recovered from the isolate A, which was designed heterokaryon-self-incompatible. Based on RAPD analysis, a polymorphism was detected among the wild isolate C and their nit1 and NitM mutants. RAPD amplification, with five different primers, also showed polymorphic profiles among Brazilian C. truncatum isolates. Dendrogram analysis resulted in a similarity degree ranging between 0.331 and 0.882 among isolates and identified three RAPD groups. Despite the lack of a correlation between the RAPD analysis and the vegetative compatibility grouping, results demonstrated the potential of VCG analysis to differentiate C. truncatum isolates genotypically similar when compared by RAPD.

Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling.

PubMed

Yuan, Yinyin; Failmezger, Henrik; Rueda, Oscar M; Ali, H Raza; Gräf, Stefan; Chin, Suet-Feung; Schwarz, Roland F; Curtis, Christina; Dunning, Mark J; Bardwell, Helen; Johnson, Nicola; Doyle, Sarah; Turashvili, Gulisa; Provenzano, Elena; Aparicio, Sam; Caldas, Carlos; Markowetz, Florian

2012-10-24

Solid tumors are heterogeneous tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Furthermore, tissue architecture is generally not reflected in molecular assays, rendering this rich information underused. To address these challenges, we developed a computational approach based on standard hematoxylin and eosin-stained tissue sections and demonstrated its power in a discovery and validation cohort of 323 and 241 breast tumors, respectively. To deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy number profiles between samples. We next devised a predictor for survival in estrogen receptor-negative breast cancer that integrated both image-based and gene expression analyses and significantly outperformed classifiers that use single data types, such as microarray expression signatures. Image processing also allowed us to describe and validate an independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative, image-based method could benefit any large-scale cancer study by refining and complementing molecular assays of tumor samples.

3β-Hydroxysterol Δ24-Reductase on the Surface of Hepatitis C Virus-Related Hepatocellular Carcinoma Cells Can Be a Target for Molecular Targeting Therapy

PubMed Central

Saito, Makoto; Takano, Takashi; Nishimura, Tomohiro; Kohara, Michinori; Tsukiyama-Kohara, Kyoko

2015-01-01

In our previous study, we demonstrated that 3β-hydroxysterol Δ24-reductase (DHCR24) was overexpressed in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), and that its expression was induced by HCV. Using a monoclonal antibody against DHCR24 (2-152a MAb), we found that DHCR24 was specifically expressed on the surface of HCC cell lines. Based on these findings, we aimed to establish a novel targeting strategy using 2-152a MAb to treat HCV-related HCC. In the present study, we examined the antitumor activity of 2-152a MAb. In the presence of complement, HCC-derived HuH-7 cells were killed by treatment with 2-152a MAb, which was mediated by complement-dependent cytotoxicity (CDC). In addition, the antigen recognition domain of 2-152a MAb was responsible for the unique anti-HCV activity. These findings demonstrate the feasibility of using 2-152a MAb for antibody therapy against HCV-related HCC. In addition, surface DHCR24 on HCC cells exhibited a functional property, agonist-induced internalization. We showed that 2-152a MAb-mediated binding of a cytotoxic agent (a saponin-conjugated secondary antibody) to surface DHCR24 led to significant cytotoxicity. This suggests that surface DHCR24 on HCC cells can function as a carrier for internalization. Therefore, surface DHCR24 could be a valuable target for HCV-related HCC therapy, and 2-152a MAb appears to be useful for this targeted therapy. PMID:25875901

Optical 1's and 2's complement devices using lithium-niobate-based waveguide

NASA Astrophysics Data System (ADS)

Pal, Amrindra; Kumar, Santosh; Sharma, Sandeep

2016-12-01

Optical 1's and 2's complement devices are proposed with the help of lithium-niobate-based Mach-Zehnder interferometers. It has a powerful capability of switching an optical signal from one port to the other port with the help of an electrical control signal. The paper includes the optical conversion scheme using sets of optical switches. 2's complement is common in computer systems and is used in binary subtraction and logical manipulation. The operation of the circuits is studied theoretically and analyzed through numerical simulations. The truth table of these complement methods is verified with the beam propagation method and MATLAB® simulation results.

Complement Activation and STAT4 Expression Are Associated with Early Inflammation in Diabetic Wounds

PubMed Central

Cunnion, Kenji M.; Krishna, Neel K.; Pallera, Haree K.; Pineros-Fernandez, Angela; Rivera, Magdielis Gregory; Hair, Pamela S.; Lassiter, Brittany P.; Huyck, Ryan; Clements, Mary A.; Hood, Antoinette F.; Rodeheaver, George T.; Nadler, Jerry L.; Dobrian, Anca D.

2017-01-01

Diabetic non-healing wounds are a major clinical problem. The mechanisms leading to poor wound healing in diabetes are multifactorial but unresolved inflammation may be a major contributing factor. The complement system (CS) is the most potent inflammatory cascade in humans and contributes to poor wound healing in animal models. Signal transducer and activator of transcription 4 (STAT4) is a transcription factor expressed in immune and adipose cells and contributes to upregulation of some inflammatory chemokines and cytokines. Persistent CS and STAT4 expression in diabetic wounds may thus contribute to chronic inflammation and delayed healing. The purpose of this study was to characterize CS and STAT4 in early diabetic wounds using db/db mice as a diabetic skin wound model. The CS was found to be activated early in the diabetic wounds as demonstrated by increased anaphylatoxin C5a in wound fluid and C3-fragment deposition by immunostaining. These changes were associated with a 76% increase in nucleated cells in the wounds of db/db mice vs. controls. The novel classical CS inhibitor, Peptide Inhibitor of Complement C1 (PIC1) reduced inflammation when added directly or saturated in an acellular skin scaffold, as reflected by reduced CS components and leukocyte infiltration. A significant increase in expression of STAT4 and the downstream macrophage chemokine CCL2 and its receptor CCR2 were also found in the early wounds of db/db mice compared to non-diabetic controls. These studies provide evidence for two new promising targets to reduce unresolved inflammation and to improve healing of diabetic skin wounds. PMID:28107529

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

PubMed

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

2003-01-01

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

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.

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

PubMed

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

2018-04-01

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

Complement factor H: spatial and temporal expression and localization in the eye.

PubMed

Mandal, Md Nawajes A; Ayyagari, Radha

2006-09-01

Complement factor H (CFH) is a component of the mammalian complement system, which regulates the alternative pathway of complement activation and protects the host cell from inappropriate complement activation. CFH is a key regulator of innate immunity, and CFH deficiency leads to membranoproliferative glomerulonephritis type II. A variation in human CFH, Y402H, has been shown to be associated with an increased risk for age-related macular degeneration. The authors describe studies on the spatial and temporal expression of the CFH gene and localization of this protein in ocular tissues to gain insight into its role in the eye. CFH expression in human and mouse tissues was studied by quantitative RT-PCR and Western blot analysis, and localization of CFH was studied by immunohistochemical analysis followed by fluorescence microscopy. In human and mouse, CFH expression was found to be similar to the highest level of expression in the liver. In ocular tissue, CFH was detected in the distalmost optic nerve (3 mm) cut from the scleral surface of the eyeball, sclera, RPE-choroid, retina, lens, and ciliary body. In mouse, Cfh expression was observed from early embryonic stages, and in the eye its expression increased with age. A significant level of CFH expression is maintained in different ocular tissues during development and aging. Sustained high levels of CFH expression in eye tissues suggest that this protein may play a role in protecting these tissues from indiscriminate complement activation and inflammatory insult.

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

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

PubMed

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

2018-02-01

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

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

Diagnosis of cystocele--the correlation between clinical and radiological evaluation.

PubMed

Altman, Daniel; Mellgren, Anders; Kierkegaard, Jonas; Zetterström, Jan; Falconer, Christian; López, Annika

2004-01-01

In patients with genital prolapse involving several compartments simultaneously, radiologic investigation can be used to complement the clinical assessment. Contrast medium in the urinary bladder enables visualization of the bladder base at cystodefecoperitoneography (CDP). The aim of the present study was to evaluate the correlation between clinical examination using the Pelvic Organ Prolapse Quantification system (POP-Q) and CDP. Thirty-three women underwent clinical assessment and CDP. Statistical analysis using Pearson's correlation coefficient ( r) demonstrated a wide variability between the current definition of cystocele at CDP and POP-Q ( r=0.67). An attempt to provide an alternative definition of cystocele at CDP had a similar outcome ( r=0.63). The present study demonstrates a moderate correlation between clinical and radiologic findings in patients with anterior vaginal wall prolapse. It does not support the use of bladder contrast at radiologic investigation in the routine preoperative assessment of patients with genital prolapse.

Graphene Quantum Capacitors for High Frequency Tunable Analog Applications.

PubMed

Moldovan, Clara F; Vitale, Wolfgang A; Sharma, Pankaj; Tamagnone, Michele; Mosig, Juan R; Ionescu, Adrian M

2016-08-10

Graphene quantum capacitors (GQC) are demonstrated to be enablers of radio-frequency (RF) functions through voltage-tuning of their capacitance. We show that GQC complements MEMS and MOSFETs in terms of performance for high frequency analog applications and tunability. We propose a CMOS compatible fabrication process and report the first experimental assessment of their performance at microwaves frequencies (up to 10 GHz), demonstrating experimental GQCs in the pF range with a tuning ratio of 1.34:1 within 1.25 V, and Q-factors up to 12 at 1 GHz. The figures of merit of graphene variable capacitors are studied in detail from 150 to 350 K. Furthermore, we describe a systematic, graphene specific approach to optimize their performance and predict the figures of merit achieved if such a methodology is applied.

SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline.

PubMed

Baud, A; Aymé, L; Gonnet, F; Salard, I; Gohon, Y; Jolivet, P; Brodolin, K; Da Silva, P; Giuliani, A; Sclavi, B; Chardot, T; Mercère, P; Roblin, P; Daniel, R

2017-05-01

Synchrotron X-ray footprinting complements the techniques commonly used to define the structure of molecules such as crystallography, small-angle X-ray scattering and nuclear magnetic resonance. It is remarkably useful in probing the structure and interactions of proteins with lipids, nucleic acids or with other proteins in solution, often better reflecting the in vivo state dynamics. To date, most X-ray footprinting studies have been carried out at the National Synchrotron Light Source, USA, and at the European Synchrotron Radiation Facility in Grenoble, France. This work presents X-ray footprinting of biomolecules performed for the first time at the X-ray Metrology beamline at the SOLEIL synchrotron radiation source. The installation at this beamline of a stopped-flow apparatus for sample delivery, an irradiation capillary and an automatic sample collector enabled the X-ray footprinting study of the structure of the soluble protein factor H (FH) from the human complement system as well as of the lipid-associated hydrophobic protein S3 oleosin from plant seed. Mass spectrometry analysis showed that the structural integrity of both proteins was not affected by the short exposition to the oxygen radicals produced during the irradiation. Irradiated molecules were subsequently analysed using high-resolution mass spectrometry to identify and locate oxidized amino acids. Moreover, the analyses of FH in its free state and in complex with complement C3b protein have allowed us to create a map of reactive solvent-exposed residues on the surface of FH and to observe the changes in oxidation of FH residues upon C3b binding. Studies of the solvent accessibility of the S3 oleosin show that X-ray footprinting offers also a unique approach to studying the structure of proteins embedded within membranes or lipid bodies. All the biomolecular applications reported herein demonstrate that the Metrology beamline at SOLEIL can be successfully used for synchrotron X-ray footprinting of biomolecules.

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

PubMed

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

2015-11-01

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

Complement Inhibition Alleviates Paraquat-Induced Acute Lung Injury

PubMed Central

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

2011-01-01

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

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

Children's understanding of the addition/subtraction complement principle.

PubMed

Torbeyns, Joke; Peters, Greet; De Smedt, Bert; Ghesquière, Pol; Verschaffel, Lieven

2016-09-01

In the last decades, children's understanding of mathematical principles has become an important research topic. Different from the commutativity and inversion principles, only few studies have focused on children's understanding of the addition/subtraction complement principle (if a - b = c, then c + b = a), mainly relying on verbal techniques. This contribution aimed at deepening our understanding of children's knowledge of the addition/subtraction complement principle, combining verbal and non-verbal techniques. Participants were 67 third and fourth graders (9- to 10-year-olds). Children solved two tasks in which verbal reports as well as accuracy and speed data were collected. These two tasks differed only in the order of the problems and the instructions. In the looking-back task, children were told that sometimes the preceding problem might help to answer the next problem. In the baseline task, no helpful preceding items were offered. The looking-back task included 10 trigger-target problem pairs on the complement relation. Children verbally reported looking back on about 40% of all target problems in the looking-back task; the target problems were also solved faster and more accurately than in the baseline task. These results suggest that children used their understanding of the complement principle. The verbal and non-verbal data were highly correlated. This study complements previous work on children's understanding of mathematical principles by highlighting interindividual differences in 9- to 10-year-olds' understanding of the complement principle and indicating the potential of combining verbal and non-verbal techniques to investigate (the acquisition of) this understanding. © 2016 The British Psychological Society.

Parasitic scabies mites and associated bacteria joining forces against host complement defence.

PubMed

Swe, P M; Reynolds, S L; Fischer, K

2014-11-01

Scabies is a ubiquitous and contagious skin disease caused by the parasitic mite Sarcoptes scabiei Epidemiological studies have identified scabies as a causative agent for secondary skin infections caused by Staphylococcus aureus and Streptococcus pyogenes. This is an important notion, as such bacterial infections can lead to serious downstream life-threatening complications. As the complement system is the first line of host defence that confronts invading pathogens, both the mite and bacteria produce a large array of molecules that inhibit the complement cascades. It is hypothesised that scabies mite complement inhibitors may play an important role in providing a favourable micro-environment for the establishment of secondary bacterial infections. This review aims to bring together the current literature on complement inhibition by scabies mites and bacteria associated with scabies and to discuss the proposed molecular link between scabies and bacterial co-infections. © 2014 John Wiley & Sons Ltd.

Critical Revolutionary Pedagogy Spiced by Pedagogical Love

ERIC Educational Resources Information Center

FitzSimmons, Robert; Uusiautti, Satu

2013-01-01

The latest incidents demonstrating human beings' inhumanity to their fellow human beings have given impetus to dissect the connection between critical revolutionary pedagogy and the idea of pedagogical love. In this essay we attempt to answer the following questions: How do these two pedagogies complement each other? What can they offer for…

Verb Position and the Order of Adverbials in German.

ERIC Educational Resources Information Center

Dean, O. C., Jr.

Recent work in word-order typology has demonstrated that the dominant order of verbs and objects (or complements) correlates well with the general ordering tendencies of languages. The work on German reported in this paper suggests, however, that certain traits, such as the order of adverbials, are influenced not only by general ordering…

Surprising Resists

ERIC Educational Resources Information Center

Morton, Stephie

2007-01-01

In this article, the author discusses an art adventure with her third, fourth, and fifth grade enrichment kids to the Fort Collins Museum of Contemporary Art in Colorado. The author demonstrates and teaches her students how to use the art tissue paper and oil pastel complementing the creative spirit of the Jaune Quick-to-See Smith work presented…

Constructive Alignment in Economics Teaching: A Reflection on Effective Implementation

ERIC Educational Resources Information Center

McCann, Michael

2017-01-01

The typical approach to student-centred learning in Economics has focused on innovation within the classroom, with little thought given to how this complements teaching and learning and, crucially, assessment. This paper reflects on the implementation of constructive alignment in a final year managerial economics course. It demonstrates how it is…

Tints, Shades and Frost

ERIC Educational Resources Information Center

Sterling, Joan

2009-01-01

This article describes a classroom art project inspired by the work of Robert Frost, one of the most acclaimed and beloved American poets of all time. Using tints and shades in a composition, this project demonstrates how quality literature may be incorporated into elementary art lessons in a very useful way, making art an important complement to…

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell

PubMed Central

Wang, Qianli; Zhang, Junping; Guo, Zhongwu

2007-01-01

To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine. PMID:17892942

Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell.

PubMed

Wang, Qianli; Zhang, Junping; Guo, Zhongwu

2007-12-15

To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine.

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

PubMed Central

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

2009-01-01

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

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.

Genetics Home Reference: Fanconi anemia

MedlinePlus

... D1 Genetic Testing Registry: Fanconi anemia, complementation group D2 Genetic Testing Registry: Fanconi anemia, complementation group E ... ANEMIA, COMPLEMENTATION GROUP D1 FANCONI ANEMIA, COMPLEMENTATION GROUP D2 FANCONI ANEMIA, COMPLEMENTATION GROUP E FANCONI ANEMIA, COMPLEMENTATION ...

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.

Ballistic near-field heat transport in dense many-body systems

NASA Astrophysics Data System (ADS)

Latella, Ivan; Biehs, Svend-Age; Messina, Riccardo; Rodriguez, Alejandro W.; Ben-Abdallah, Philippe

2018-01-01

Radiative heat transport mediated by near-field interactions is known to be superdiffusive in dilute, many-body systems. Here we use a generalized Landauer theory of radiative heat transfer in many-body planar systems to demonstrate a nonmonotonic transition from superdiffusive to ballistic transport in dense systems. We show that such a transition is associated to a change of the polarization of dominant modes. Our findings are complemented by a quantitative study of the relaxation dynamics of the system in the different regimes of heat transport. This result could have important consequences on thermal management at nanoscale of many-body systems.

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.

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.

DrsG from Streptococcus dysgalactiae subsp. equisimilis Inhibits the Antimicrobial Peptide LL-37

PubMed Central

Smyth, Danielle; Cameron, Ainslie; Davies, Mark R.; McNeilly, Celia; Hafner, Louise; Sriprakash, Kadaba S.

2014-01-01

SIC and DRS are related proteins present in only 4 of the >200 Streptococcus pyogenes emm types. These proteins inhibit complement-mediated lysis and/or the activity of certain antimicrobial peptides (AMPs). A gene encoding a homologue of these proteins, herein called DrsG, has been identified in the related bacterium Streptococcus dysgalactiae subsp. equisimilis. Here we show that geographically dispersed isolates representing 14 of 50 emm types examined possess variants of drsG. However, not all isolates within the drsG-positive emm types possess the gene. Sequence comparisons also revealed a high degree of conservation in different S. dysgalactiae subsp. equisimilis emm types. To examine the biological activity of DrsG, recombinant versions of two major DrsG variants, DrsGS and DrsGL, were expressed and purified. Western blot analysis using antisera raised to these proteins demonstrated both variants to be expressed and secreted into culture supernatants. Unlike SIC, but similar to DRS, DrsG does not inhibit complement-mediated lysis. However, like both SIC and DRS, DrsG is a ligand of the cathelicidin LL-37 and is inhibitory to its bactericidal activity in in vitro assays. Conservation of prolines in the C-terminal region also suggests that these residues are important in the biology of this family of proteins. This is the first report demonstrating the activity of an AMP-inhibitory protein in S. dysgalactiae subsp. equisimilis and suggests that inhibition of AMP activity is the primary function of this family of proteins. The acquisition of the complement-inhibitory activity of SIC may reflect its continuing evolution. PMID:24664506

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.

Sulfurtransferase and thioredoxin specifically interact as demonstrated by bimolecular fluorescence complementation analysis and biochemical tests.

PubMed

Henne, Melina; König, Nicolas; Triulzi, Tiziana; Baroni, Sara; Forlani, Fabio; Scheibe, Renate; Papenbrock, Jutta

2015-01-01

Sulfurtransferases (Strs) and thioredoxins (Trxs) are members of large protein families. Trxs are disulfide reductases and play an important role in redox-related cellular processes. They interact with a broad range of proteins. Strs catalyze the transfer of a sulfur atom from a suitable sulfur donor to nucleophilic sulfur acceptors in vitro, but the physiological roles of these enzymes are not well defined. Several studies in different organisms demonstrate protein-protein interactions of Strs with members of the Trx family. We are interested in investigating the specificity of the interaction between Str and Trx isoforms. In order to use the bimolecular fluorescence complementation (BiFC), several Str and Trx sequences from Arabidopsis thaliana were cloned into the pUC-SPYNE and pUC-SPYCE split-YFP vectors, respectively. Each couple of plasmids containing the sequences for the putative interaction partners were transformed into Arabidopsis protoplasts and screened using a confocal laser scanning microscope. Compartment- and partner-specific interactions could be observed in transformed protoplasts. Replacement of cysteine residues in the redox-active site of Trxs abolished the interaction signal. Therefore, the redox site is not only involved in the redox reaction but also responsible for the interaction with partner proteins. Biochemical assays support a specific interaction among Strs and certain Trxs. Based on the results obtained, the interaction of Strs and Trxs indicates a role of Strs in the maintenance of the cellular redox homeostasis.

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

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

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.

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

Protein domains connect cell cycle stimulation directly to initiation of DNA replication.

PubMed Central

Gjørup, O V; Rose, P E; Holman, P S; Bockus, B J; Schaffhausen, B S

1994-01-01

Polyoma large T antigen (LT) is the only viral gene product required for viral DNA replication. LT can be divided into two domains, one N-terminal (NT) spanning residues 1-260 and one C-terminal (CT) comprising approximately residues 264-785. NT is known to immortalize primary cells in a manner dependent on binding of pRB/p107. Here a CT construct comprising residues 264-785 was shown to have independent function in DNA replication. CT is entirely sufficient for driving viral DNA replication in vivo in growing mouse cells at a level approaching that of full-length LT. In contrast, CT is strikingly deficient for replication in serum-starved cells. However, this deficiency can be complemented by coexpression of NT. BrdUrd incorporation in transfected, starved cells showed that NT was sufficient for inducing S phase, suggesting a mechanism for complementation. By contrast, CT was unable to induce S phase when tested in the same assay. NT also promotes phosphorylation of sites in CT that are likely to be important for replication. Other DNA tumor virus gene products such as adenovirus E1A 12S and human papillomavirus 16 E7 could also complement CT for replication. Although NT, E1A 12S, and E7 all bind the retinoblastoma gene product (pRB) and p107, genetic analysis demonstrates an additional function, independent of that binding, is responsible for complementation. Images PMID:7991595

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.

A perspective of computational fluid dynamics

NASA Technical Reports Server (NTRS)

Kutler, P.

1986-01-01

Computational fluid dynamics (CFD) is maturing, and is at a stage in its technological life cycle in which it is now routinely applied to some rather complicated problems; it is starting to create an impact on the design cycle of aerospace flight vehicles and their components. CFD is also being used to better understand the fluid physics of flows heretofore not understood, such as three-dimensional separation. CFD is also being used to complement and is being complemented by experiments. In this paper, the primary and secondary pacing items that govern CFD in the past are reviewed and updated. The future prospects of CFD are explored which will offer people working in the discipline challenges that should extend the technological life cycle to further increase the capabilities of a proven demonstrated technology.

Effect of chlorphenesin on localized hemolysis in gel assay.

PubMed

Fukui, G M; Berger, F M; Chandlee, G C; Goldenbaum, E G

1968-10-01

Chlorphenesin, a simple glycerol ether, when added to Jerne plates greatly reduced the number of hemolytic plaques. This effect appeared to be related to dose, and was clearly demonstrable with antibody-forming spleen cells from mice that had been immunized either with sheep red blood cells or with penicillin G conjugated with Keyhole limpet hemocyanin. Chlorphenesin did not affect the antigen, destroy complement, or interfere with the interaction of complement and the antigen-antibody complexes. Incubation of spleen cell suspensions with chlorphenesin prior to plating was more effective in reducing the number of plaques than was addition of the substance to the plates. It may act by reducing the ability of antibodies to react with antigens or by affecting the release of antibodies from the spleen cells.

Effect of Chlorphenesin on Localized Hemolysis in Gel Assay

PubMed Central

Fukui, G. M.; Berger, F. M.; Chandlee, G. C.; Goldenbaum, E. G.

1968-01-01

Chlorphenesin, a simple glycerol ether, when added to Jerne plates greatly reduced the number of hemolytic plaques. This effect appeared to be related to dose, and was clearly demonstrable with antibody-forming spleen cells from mice that had been immunized either with sheep red blood cells or with penicillin G conjugated with Keyhole limpet hemocyanin. Chlorphenesin did not affect the antigen, destroy complement, or interfere with the interaction of complement and the antigen-antibody complexes. Incubation of spleen cell suspensions with chlorphenesin prior to plating was more effective in reducing the number of plaques than was addition of the substance to the plates. It may act by reducing the ability of antibodies to react with antigens or by affecting the release of antibodies from the spleen cells. PMID:5685993

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

PubMed

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

2014-09-01

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

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.

Identification and characterization of properdin in amphioxus: Implications for a functional alternative complement pathway in the basal chordate.

PubMed

Gao, Zhan; Ma, Zengyu; Qu, Baozhen; Jiao, Deyan; Zhang, Shicui

2017-06-01

A complement system operating via the alternative pathway similar to that of vertebrates has been demonstrated in the primitive chordate amphioxus. However, the factor P (fP), a positive regulator of the alternative pathway, remains elusive in amphioxus to date. In this study, we identified and characterized a properdin gene in the amphioxus B. japonicum, BjfP, which represents an archetype of vertebrate properdins. Real-time PCR analysis showed that the BjfP was ubiquitously expressed and its expression was significantly up-regulated following the challenge with bacteria or lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Recombinant BjfP (rBjfP) and its truncated proteins including rTSR1-3, rTSR4-6 and rTSR7-8, were all capable of interacting with both Gram-negative and positive bacteria as well as LPS and LTA. Moreover, rBjfP, rTSR1-3 and rTSR4-6 could also specifically bind to C3b. Importantly, both rTSR1-3 and rTSR4-6 could inhibit the binding of rBjfP to C3b, and thus suppress the activation of the alternative pathway of complement, suggesting the involvement of BjfP in the alternative pathway. This is the first report showing that a properdin protein in invertebrates plays similar roles to vertebrate properdins. Collectively, these data suggest that BjfP might represent the ancient molecule from which vertebrate properdins evolved. Copyright © 2017 Elsevier Ltd. All rights reserved.

Increased taxon sampling reveals thousands of hidden orthologs in flatworms

PubMed Central

2017-01-01

Gains and losses shape the gene complement of animal lineages and are a fundamental aspect of genomic evolution. Acquiring a comprehensive view of the evolution of gene repertoires is limited by the intrinsic limitations of common sequence similarity searches and available databases. Thus, a subset of the gene complement of an organism consists of hidden orthologs, i.e., those with no apparent homology to sequenced animal lineages—mistakenly considered new genes—but actually representing rapidly evolving orthologs or undetected paralogs. Here, we describe Leapfrog, a simple automated BLAST pipeline that leverages increased taxon sampling to overcome long evolutionary distances and identify putative hidden orthologs in large transcriptomic databases by transitive homology. As a case study, we used 35 transcriptomes of 29 flatworm lineages to recover 3427 putative hidden orthologs, some unidentified by OrthoFinder and HaMStR, two common orthogroup inference algorithms. Unexpectedly, we do not observe a correlation between the number of putative hidden orthologs in a lineage and its “average” evolutionary rate. Hidden orthologs do not show unusual sequence composition biases that might account for systematic errors in sequence similarity searches. Instead, gene duplication with divergence of one paralog and weak positive selection appear to underlie hidden orthology in Platyhelminthes. By using Leapfrog, we identify key centrosome-related genes and homeodomain classes previously reported as absent in free-living flatworms, e.g., planarians. Altogether, our findings demonstrate that hidden orthologs comprise a significant proportion of the gene repertoire in flatworms, qualifying the impact of gene losses and gains in gene complement evolution. PMID:28400424

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector.

PubMed

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis -specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector

PubMed Central

Sharma, Pratibha; Teymournejad, Omid; Rikihisa, Yasuko

2017-01-01

Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs) technology to interrupt type IV secretion system (T4SS) effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1), which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria. PMID:28638803

Urease Activity Represents an Alternative Pathway for Mycobacterium tuberculosis Nitrogen Metabolism

PubMed Central

Lin, Wenwei; Mathys, Vanessa; Ang, Emily Lei Yin; Koh, Vanessa Hui Qi; Martínez Gómez, Julia María; Ang, Michelle Lay Teng; Zainul Rahim, Siti Zarina; Tan, Mai Ping; Pethe, Kevin

2012-01-01

Urease represents a critical virulence factor for some bacterial species through its alkalizing effect, which helps neutralize the acidic microenvironment of the pathogen. In addition, urease serves as a nitrogen source provider for bacterial growth. Pathogenic mycobacteria express a functional urease, but its role during infection has yet to be characterized. In this study, we constructed a urease-deficient Mycobacterium tuberculosis strain and confirmed the alkalizing effect of the urease activity within the mycobacterium-containing vacuole in resting macrophages but not in the more acidic phagolysosomal compartment of activated macrophages. However, the urease-mediated alkalizing effect did not confer any growth advantage on M. tuberculosis in macrophages, as evidenced by comparable growth profiles for the mutant, wild-type (WT), and complemented strains. In contrast, the urease-deficient mutant exhibited impaired in vitro growth compared to the WT and complemented strains when urea was the sole source of nitrogen. Substantial amounts of ammonia were produced by the WT and complemented strains, but not with the urease-deficient mutant, which represents the actual nitrogen source for mycobacterial growth. However, the urease-deficient mutant displayed parental colonization profiles in the lungs, spleen, and liver in mice. Together, our data demonstrate a role for the urease activity in M. tuberculosis nitrogen metabolism that could be crucial for the pathogen's survival in nutrient-limited microenvironments where urea is the sole nitrogen source. Our work supports the notion that M. tuberculosis virulence correlates with its unique metabolic versatility and ability to utilize virtually any carbon and nitrogen sources available in its environment. PMID:22645285

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

PubMed

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

2016-03-08

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

Dynamic phenotypic restructuring of the CD4 and CD8 T-cell subsets with age in healthy humans: a compartmental model analysis.

PubMed

Jackola, D R; Hallgren, H M

1998-11-16

In healthy humans, phenotypic restructuring occurs with age within the CD3+ T-lymphocyte complement. This is characterized by a non-linear decrease of the percentage of 'naive' (CD45RA+) cells and a corresponding non-linear increase of the percentage of 'memory' (CD45R0+) cells among both the CD4+ and CD8+ T-cell subsets. We devised a simple compartmental model to study the age-dependent kinetics of phenotypic restructuring. We also derived differential equations whose parameters determined yearly gains minus losses of the percentage and absolute numbers of circulating naive cells, yearly gains minus losses of the percentage and absolute numbers of circulating memory cells, and the yearly rate of conversion of naive to memory cells. Solutions of these evaluative differential equations demonstrate the following: (1) the memory cell complement 'resides' within its compartment for a longer time than the naive cell complement within its compartment for both CD4 and CD8 cells; (2) the average, annual 'turnover rate' is the same for CD4 and CD8 naive cells. In contrast, the average, annual 'turnover rate' for memory CD8 cells is 1.5 times that of memory CD4 cells; (3) the average, annual conversion rate of CD4 naive cells to memory cells is twice that of the CD8 conversion rate; (4) a transition in dynamic restructuring occurs during the third decade of life that is due to these differences in turnover and conversion rates, between and from naive to memory cells.

Complement Protein C1q Binds to Hyaluronic Acid in the Malignant Pleural Mesothelioma Microenvironment and Promotes Tumor Growth

PubMed Central

Agostinis, Chiara; Vidergar, Romana; Belmonte, Beatrice; Mangogna, Alessandro; Amadio, Leonardo; Geri, Pietro; Borelli, Violetta; Zanconati, Fabrizio; Tedesco, Francesco; Confalonieri, Marco; Tripodo, Claudio; Kishore, Uday; Bulla, Roberta

2017-01-01

C1q is the first recognition subcomponent of the complement classical pathway, which acts toward the clearance of pathogens and apoptotic cells. C1q is also known to modulate a range of functions of immune and non-immune cells, and has been shown to be involved in placental development and sensorial synaptic pruning. We have recently shown that C1q can promote tumor by encouraging their adhesion, migration, and proliferation in addition to angiogenesis and metastasis. In this study, we have examined the role of human C1q in the microenvironment of malignant pleural mesothelioma (MPM), a rare form of cancer commonly associated with exposure to asbestos. We found that C1q was highly expressed in all MPM histotypes, particularly in epithelioid rather than in sarcomatoid histotype. C1q avidly bound high and low molecular weight hyaluronic acid (HA) via its globular domain. C1q bound to HA was able to induce adhesion and proliferation of mesothelioma cells (MES) via enhancement of ERK1/2, SAPK/JNK, and p38 phosphorylation; however, it did not activate the complement cascade. Consistent with the modular organization of the globular domain, we demonstrated that C1q may bind to HA through ghA module, whereas it may interact with human MES through the ghC. In conclusion, C1q highly expressed in MPM binds to HA and enhances the tumor growth promoting cell adhesion and proliferation. These data can help develop novel diagnostic markers and molecular targets for MPM. PMID:29209316

Complement Protein C1q Binds to Hyaluronic Acid in the Malignant Pleural Mesothelioma Microenvironment and Promotes Tumor Growth.

PubMed

Agostinis, Chiara; Vidergar, Romana; Belmonte, Beatrice; Mangogna, Alessandro; Amadio, Leonardo; Geri, Pietro; Borelli, Violetta; Zanconati, Fabrizio; Tedesco, Francesco; Confalonieri, Marco; Tripodo, Claudio; Kishore, Uday; Bulla, Roberta

2017-01-01

C1q is the first recognition subcomponent of the complement classical pathway, which acts toward the clearance of pathogens and apoptotic cells. C1q is also known to modulate a range of functions of immune and non-immune cells, and has been shown to be involved in placental development and sensorial synaptic pruning. We have recently shown that C1q can promote tumor by encouraging their adhesion, migration, and proliferation in addition to angiogenesis and metastasis. In this study, we have examined the role of human C1q in the microenvironment of malignant pleural mesothelioma (MPM), a rare form of cancer commonly associated with exposure to asbestos. We found that C1q was highly expressed in all MPM histotypes, particularly in epithelioid rather than in sarcomatoid histotype. C1q avidly bound high and low molecular weight hyaluronic acid (HA) via its globular domain. C1q bound to HA was able to induce adhesion and proliferation of mesothelioma cells (MES) via enhancement of ERK1/2, SAPK/JNK, and p38 phosphorylation; however, it did not activate the complement cascade. Consistent with the modular organization of the globular domain, we demonstrated that C1q may bind to HA through ghA module, whereas it may interact with human MES through the ghC. In conclusion, C1q highly expressed in MPM binds to HA and enhances the tumor growth promoting cell adhesion and proliferation. These data can help develop novel diagnostic markers and molecular targets for MPM.

Engineering new mycobacterial vaccine design for HIV–TB pediatric vaccine vectored by lysine auxotroph of BCG

PubMed Central

Saubi, Narcís; Gea-Mallorquí, Ester; Ferrer, Pau; Hurtado, Carmen; Sánchez-Úbeda, Sara; Eto, Yoshiki; Gatell, Josep M; Hanke, Tomáš; Joseph, Joan

2014-01-01

In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)–mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA2auxo. We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA2auxo vaccine strain. The BCG.HIVA2auxo vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice–compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on “double” auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth. PMID:26015961

A comparative analysis of the encapsulated end-organs of mammalian skeletal muscles and of their sensory nerve endings.

PubMed

Banks, R W; Hulliger, M; Saed, H H; Stacey, M J

2009-06-01

The encapsulated sensory endings of mammalian skeletal muscles are all mechanoreceptors. At the most basic functional level they serve as length sensors (muscle spindle primary and secondary endings), tension sensors (tendon organs), and pressure or vibration sensors (lamellated corpuscles). At a higher functional level, the differing roles of individual muscles in, for example, postural adjustment and locomotion might be expected to be reflected in characteristic complements of the various end-organs, their sensory endings and afferent nerve fibres. This has previously been demonstrated with regard to the number of muscle-spindle capsules; however, information on the other types of end-organ, as well as the complements of primary and secondary endings of the spindles themselves, is sporadic and inconclusive regarding their comparative provision in different muscles. Our general conclusion that muscle-specific variability in the provision of encapsulated sensory endings does exist demonstrates the necessity for the acquisition of more data of this type if we are to understand the underlying adaptive relationships between motor control and the structure and function of skeletal muscle. The present quantitative and comparative analysis of encapsulated muscle afferents is based on teased, silver-impregnated preparations. We begin with a statistical analysis of the number and distribution of muscle-spindle afferents in hind-limb muscles of the cat, particularly tenuissimus. We show that: (i) taking account of the necessity for at least one primary ending to be present, muscles differ significantly in the mean number of additional afferents per spindle capsule; (ii) the frequency of occurrence of spindles with different sensory complements is consistent with a stochastic, rather than deterministic, developmental process; and (iii) notwithstanding the previous finding, there is a differential distribution of spindles intramuscularly such that the more complex ones tend to be located closer to the main divisions of the nerve. Next, based on a sample of tendon organs from several hind-foot muscles of the cat, we demonstrate the existence in at least a large proportion of tendon organs of a structural substrate to account for multiple spike-initiation sites and pacemaker switching, namely the distribution of sensory terminals supplied by the different first-order branches of the Ib afferent to separate, parallel, tendinous compartments of individual tendon organs. We then show that the numbers of spindles, tendon organs and paciniform corpuscles vary independently in a sample of (mainly) hind-foot muscles of the cat. Grouping muscles by anatomical region in the cat indicated the existence of a gradual proximo-distal decline in the overall average size of the afferent complement of muscle spindles from axial through hind limb to intrinsic foot muscles, but with considerable muscle-specific variability. Finally, we present some comparative data on muscle-spindle afferent complements of rat, rabbit and guinea pig, one particularly notable feature being the high incidence of multiple primary endings in the rat.

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

PubMed Central

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

2011-01-01

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

Measurement of nurses' attitudes and knowledge regarding acute care older patients: Psychometrics of the OPACS-US combined with the KOP-Q.

PubMed

Dikken, Jeroen; Hoogerduijn, Jita G; Lagerwey, Mary D; Shortridge-Baggett, Lillie; Klaassen, Sharon; Schuurmans, Marieke J

In clinical practice, identifying positive and negative attitudes toward older patients is very important to improve quality of care provided to them. The Older People in Acute Care Survey - United States (OPACS-US) is an instrument measuring hospital nurses attitudes regarding older patients. However, psychometrics have never been assessed. Furthermore, knowledge being related to attitude and behavior should also be measured complementing the OPACS-US. The purpose of this study was to assess structural validity and reliability of the OPACS-US and assess whether the OPACS-US can be complemented with the Knowledge about Older Patients-Quiz (KOP-Q). A multicenter cross sectional design was conducted. Registered nurses (n = 130, mean age 39,9 years; working experience 14,6 years) working in four general hospitals were included in the study. Nurses completed the OPACS-US section A: practice experiences, B: general opinion and the KOP-Q online. Findings demonstrated that the OPACS-US is a valid and reliable survey instrument that measures practice experiences and general opinion. Furthermore, the OPACS-US can be combined with the KOP-Q adding a knowledge construct, and is ready for use within education and/or quality improvement programs in the USA. Copyright © 2017 Elsevier Inc. All rights reserved.

ROLE OF TYROSINE-SULFATED PROTEINS IN RETINAL STRUCTURE AND FUNCTION

PubMed Central

Kanan, Y.; Al-Ubaidi, M.R.

2014-01-01

The extracellular matrix (ECM) plays a significant role in cellular and retinal health. The study of retinal tyrosine-sulfated proteins is an important first step toward understanding the role of ECM in retinal health and diseases. These secreted proteins are members of the retinal ECM. Tyrosine sulfation was shown to be necessary for the development of proper retinal structure and function. The importance of tyrosine sulfation is further demonstrated by the evolutionary presence of tyrosylprotein sulfotransferases, enzymes that catalyze proteins’ tyrosine sulfation, and the compensatory abilities of these enzymes. Research has identified four tyrosine-sulfated retinal proteins: fibulin 2, vitronectin, complement factor H (CFH), and opticin. Vitronectin and CFH regulate the activation of the complement system and are involved in the etiology of some cases of age-related macular degeneration. Analysis of the role of tyrosine sulfation in fibulin function showed that sulfation influences the protein's ability to regulate growth and migration. Although opticin was recently shown to exhibit anti-angiogenic properties, it is not yet determined what role sulfation plays in that function. Future studies focusing on identifying all of the tyrosine-sulfated retinal proteins would be instrumental in determining the impact of sulfation on retinal protein function in retinal homeostasis and diseases. PMID:25819460

Modulation of cytotoxic T lymphocyte, natural killer cell, antibody-dependent cellular cytotoxicity, and antibody-dependent complement-mediated cytotoxicity by Vernonia cinerea L. and vernolide-A in BALB/c mice via enhanced production of cytokines IL-2 and IFN-γ.

PubMed

Pratheeshkumar, Poyil; Kuttan, Girija

2012-02-01

Effect of Vernonia cinerea L. and vernolide-A on cell-mediated immune (CMI) response was studied in normal as well as tumor-bearing BALB/c mice. Administration of V. cinerea and vernolide-A significantly enhanced natural killer (NK) cell activity in both normal as well as tumor-bearing animals, and the activity was observed earlier than in tumor-bearing control animals. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were also enhanced significantly in both normal as well as tumor-bearing animals after V. cinerea and vernolide-A administration compared with untreated control tumor-bearing animals. Extract and vernolide-A showed a significant increase in cytotoxic T lymphocyte (CTL) production in both the in vivo and in vitro models. The level of cytokines such as interleukin (IL)-2 and interferon (IFN)-γ were also enhanced by the treatment of V. cinerea and vernolide-A in both normal as well as tumor-bearing animals. This study demonstrated that V. cinerea extract and vernolide-A stimulate the CTL, NK cell, ADCC, and ADCC through enhanced secretion of IL-2 and IFN-γ.

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

PubMed Central

Wang, Li; Carnegie, Graeme K.

2013-01-01

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction. PMID:23979513

Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction.

PubMed

Wang, Li; Carnegie, Graeme K

2013-08-15

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Biocompatibility of nanoporous alumina membranes for immunoisolation

PubMed Central

La Flamme, Kristen E.; Popat, Ketul C.; Leoni, Lara; Markiewicz, Erica; LaTempa, Thomas J.; Roman, Brian B.; Grimes, Craig A.; Desai, Tejal A.

2011-01-01

Cellular immunoisolation using semi-permeable barriers has been investigated over the past several decades as a promising treatment approach for diseases such as Parkinson’s, Alzheimer’s, and Type 1 diabetes. Typically, polymeric membranes are used for immunoisolation applications; however, recent advances in technology have led to the development of more robust membranes that are able to more completely meet the requirements for a successful immunoisolation device, including well controlled pore size, chemical and mechanical stability, non-biodegradability, and biocompatibility with both the graft tissue as well as the host. It has been shown previously that nanoporous alumina biocapsules can act effectively as immunoisolation devices, and support the viability and functionality of encapsulated β cells. The aim of this investigation was to assess the biocompatibility of the material with host tissue. The cytotoxicity of the capsule, as well as its ability to activate complement and inflammation was studied. Further, the effects of PEG-modification on the tissue response to implanted capsules were studied. Our results have shown that the device is non-toxic and does not induce significant complement activation. Further, in vivo work has demonstrated that implantation of these capsules into the peritoneal cavity of rats induces a transient inflammatory response, and that PEG is useful in minimizing the host response to the material. PMID:17335895

Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA

PubMed Central

Shell, Steven M.; Hawkins, Edward K.; Tsai, Miaw-Sheue; Hlaing, Aye Su; Rizzo, Carmelo J.; Chazin, Walter J.

2013-01-01

The xeroderma pigmentosum complementation group C protein (XPC) serves as the primary initiating factor in the global genome nucleotide excision repair pathway (GG-NER). Recent reports suggest XPC also stimulates repair of oxidative lesions by base excision repair. However, whether XPC distinguishes among various types of DNA lesions remains unclear. Although the DNA binding properties of XPC have been studied by several groups, there is a lack of consensus over whether XPC discriminates between DNA damaged by lesions associated with NER activity versus those that are not. In this study we report a high-throughput fluorescence anisotropy assay used to measure the DNA binding affinity of XPC for a panel of DNA substrates containing a range of chemical lesions in a common sequence. Our results demonstrate that while XPC displays a preference for binding damaged DNA, the identity of the lesion has little effect on the binding affinity of XPC. Moreover, XPC was equally capable of binding to DNA substrates containing lesions not repaired by GG-NER. Our results support an indirect read-out model for sensing the presence of lesions by human XPC and suggest XPC may act as a general sensor of damaged DNA capable of recognizing DNA containing lesions not repaired by NER. PMID:24051049

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

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

Interaction of factor H-binding protein of Streptococcus suis with globotriaosylceramide promotes the development of meningitis

PubMed Central

Kong, Decong; Chen, Zhe; Wang, Junping; Lv, Qingyu; Jiang, Hua; Zheng, Yuling; Xu, Maokai; Zhou, Xuyu; Hao, Huaijie; Jiang, Yongqiang

2017-01-01

ABSTRACT Streptococcus suis is an important emerging zoonotic agent that causes acute bacterial meningitis in humans with high mortality and morbidity. Our previous work showed that factor H-binding protein (Fhb) contributed to virulence of S. suis, but the role of Fhb in the development of S. suis meningitis remained unclear. In this study, we demonstrated for the first time that Fhb contributed to the traversal of S. suis across the human blood–brain barrier by allelic-exchange mutagenesis, complementation and specific antibody blocking studies. We also showed that globotriaosylceramide (Gb3), the receptor of Fhb, was involved in this process and affected S. suis infection-induced activation of myosin light chain 2 through Rho/ROCK signaling in hCMEC/D3 cells. Using a murine model of S. suis meningitis, we further demonstrated that Gb3-deficiency prevented the mice from developing severe brain inflammation or injury. Our results demonstrate that the Fhb-Gb3 interaction plays an important role in the development of S. suis meningitis and might be a potential therapeutic target against S. suis infection. PMID:28402705

Mannose-binding lectin and its associated proteases (MASPs) mediate coagulation and its deficiency is a risk factor in developing complications from infection, including disseminated intravascular coagulation

PubMed Central

Takahashi, Kazue; Chang, Wei-Chuan; Takahashi, Minoru; Pavlov, Vasile; Ishida, Yumi; La Bonte, Laura; Shi, Lei; Fujita, Teizo; Stahl, Gregory L.; Van Cott, Elizabeth M.

2010-01-01

The first line of host defense is the innate immune system that includes coagulation factors and pattern recognition molecules, one of which is mannose-binding lectin (MBL). Previous studies have demonstrated that MBL deficiency increases susceptibility to infection. Several mechanisms are associated with increased susceptibility to infection, including reduced opsonophagocytic killing and reduced lectin complement pathway activation. In this study, we demonstrate that MBL and MBL-associated serine protease (MASP)-1/3 together mediate coagulation factor-like activities, including thrombin-like activity. MBL and/or MASP-1/3 deficient hosts demonstrate in vivo evidence that MBL and MASP-1/3 are involved with hemostasis following injury. Staphylococcus aureus infected MBL null mice developed disseminated intravascular coagulation (DIC), which was associated with elevated blood IL-6 levels (but not TNF-α and multi-organ inflammatory responses). Infected MBL null mice also develop liver injury. These findings suggest that MBL deficiency may manifest into DIC and organ failure during infectious diseases. PMID:20399528

The acyl desaturase CER17 is involved in producing wax unsaturated primary alcohols and cutin monomers

USDA-ARS?s Scientific Manuscript database

We report here n-6 mono-unsaturated primary alcohols (the C26:1, C28:1, and C30:1 homologues) in the cuticular waxes of Arabidopsis inflorescence stem, a class of wax compound not previously reported in Arabidopsis. Further, we used mutation and transgenic complementation analyses to demonstrate tha...

"Mouse Clone Model" for evaluating the immunogenicity and tumorigenicity of pluripotent stem cells.

PubMed

Zhang, Gang; Zhang, Yi

2015-12-18

To investigate the immune-rejection and tumor-formation potentials of induced pluripotent stem cells and other stem cells, we devised a model-designated the "Mouse Clone Model"-which combined the theory of somatic animal cloning, tetraploid complementation, and induced pluripotent stem cells to demonstrate the applicability of stem cells for transplantation therapy.

Two Wrongs Make a Right: Using Pseudoscience and Reasoning Fallacies to Complement Primary Literature

ERIC Educational Resources Information Center

Stover, Shawn

2016-01-01

Undergraduate science students benefit greatly by learning to read and interpret primary research articles. However, once they obtain a level of competence in analyzing primary literature and develop a better understanding of the nature of science, they may become frustrated by the lack of scientific literacy and objectivity demonstrated by the…

Nuclear Bragg scattering studies in [sup 57]Fe with synchrotron radiation

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

Haustein, P.E.

1993-01-01

Studies of nuclear Bragg x-ray scattering of synchrotron radiation, using crystals of [alpha]-[sup 57]Fe[sub 2]O[sub 3], have been carried out at the NSLS at Brookhaven National Laboratory and at the Cornell University CHESS facility. These studies have demonstrated that nuclear resonance states can be used to produce filtered x-ray beams which have extremely narrow bandwidth, small angular divergence and unique polarization and temporal properties. this combination of characteristics, unobtainable with radioactive sources, makes synchrotron-based Moessbauer spectroscopy feasible and is an important complement to existing methods. A review of the experimental methodology is presented. As well as come suggestions for fullermore » exploitation of this new technique.« less

Nuclear Bragg scattering studies in {sup 57}Fe with synchrotron radiation

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

Haustein, P.E.

1993-03-01

Studies of nuclear Bragg x-ray scattering of synchrotron radiation, using crystals of {alpha}-{sup 57}Fe{sub 2}O{sub 3}, have been carried out at the NSLS at Brookhaven National Laboratory and at the Cornell University CHESS facility. These studies have demonstrated that nuclear resonance states can be used to produce filtered x-ray beams which have extremely narrow bandwidth, small angular divergence and unique polarization and temporal properties. this combination of characteristics, unobtainable with radioactive sources, makes synchrotron-based Moessbauer spectroscopy feasible and is an important complement to existing methods. A review of the experimental methodology is presented. As well as come suggestions for fullermore » exploitation of this new technique.« less

Language and False Belief: Evidence for General, Not Specific, Effects in Cantonese-Speaking Preschoolers

ERIC Educational Resources Information Center

Tardif, Twila; So, Catherine Wing-Chee; Kaciroti, Niko

2007-01-01

Two studies were conducted with Cantonese-speaking preschoolers examining J. de Villiers's (1995) hypothesis that syntactic complements play a unique role in the acquisition of false belief (FB). In Study 1, the authors found a positive correlation between FB and syntactic complements in 72 four- to six-year-old Cantonese-speaking preschoolers.…

Detection of plum pox potyviral protein-protein interactions in planta using an optimized mRFP-based bimolecular fluorescence complementation system.

PubMed

Zilian, Eva; Maiss, Edgar

2011-12-01

In previous studies, protein interaction maps of different potyviruses have been generated using yeast two-hybrid (YTH) systems, and these maps have demonstrated a high diversity of interactions of potyviral proteins. Using an optimized bimolecular fluorescence complementation (BiFC) system, a complete interaction matrix for proteins of a potyvirus was developed for the first time under in planta conditions with ten proteins from plum pox virus (PPV). In total, 52 of 100 possible interactions were detected, including the self-interactions of CI, 6K2, VPg, NIa-Pro, NIb and CP, which is more interactions than have ever been detected for any other potyvirus in a YTH approach. Moreover, the BiFC system was shown to be able to localize the protein interactions, which was typified for the protein self-interactions indicated above. Additionally, experiments were carried out with the P3N-PIPO protein, revealing an interaction with CI but not with CP and supporting the involvement of P3N-PIPO in the cell-to-cell movement of potyviruses. No self-interaction of the PPV helper component-proteinase (HC-Pro) was detected using BiFC in planta. Therefore, additional experiments with turnip mosaic virus (TuMV) HC-Pro, PPV_HC-Pro and their mutants were conducted. The self-interaction of TuMV_HCpro, as recently demonstrated, and the self-interaction of the TuMV_ and PPV_HC-Pro mutants were shown by BiFC in planta, indicating that HC-Pro self-interactions may be species-specific. BiFC is a very useful and reliable method for the detection and localization of protein interactions in planta, thus enabling investigations under more natural conditions than studies in yeast cells.

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

A comprehensive strategy for the subtyping of patients with Fanconi anaemia: conclusions from the Spanish Fanconi Anemia Research Network.

PubMed

Antonio Casado, José; Callén, Elsa; Jacome, Ariana; Río, Paula; Castella, Maria; Lobitz, Stephan; Ferro, Teresa; Muñoz, Arturo; Sevilla, Julián; Cantalejo, Angeles; Cela, Elena; Cervera, José; Sánchez-Calero, Jesús; Badell, Isabel; Estella, Jesús; Dasí, Angeles; Olivé, Teresa; José Ortega, Juan; Rodriguez-Villa, Antonia; Tapia, María; Molinés, Antonio; Madero, Luis; Segovia, José C; Neveling, Kornelia; Kalb, Reinhard; Schindler, Detlev; Hanenberg, Helmut; Surrallés, Jordi; Bueren, Juan A

2007-04-01

Fanconi anaemia is a heterogeneous genetic disease, where 12 complementation groups have been already described. Identifying the complementation group in patients with Fanconi anaemia constitutes a direct procedure to confirm the diagnosis of the disease and is required for the recruitment of these patients in gene therapy trials. To determine the subtype of Fanconi anaemia patients in Spain, a Mediterranean country with a relatively high population (23%) of Fanconi anaemia patients belonging to the gypsy race. Most patients could be subtyped by retroviral complementation approaches in peripheral blood T cells, although some mosaic patients were subtyped in cultured skin fibroblasts. Other approaches, mainly based on western blot analysis and generation of nuclear RAD51 and FANCJ foci, were required for the subtyping of a minor number of patients. From a total of 125 patients included in the Registry of Fanconi Anaemia, samples from 102 patients were available for subtyping analyses. In 89 cases the subtype could be determined and in 8 cases exclusions of common complementation groups were made. Compared with other international studies, a skewed distribution of complementation groups was observed in Spain, where 80% of the families belonged to the Fanconi anaemia group A (FA-A) complementation group. The high proportion of gypsy patients, all of them FA-A, and the absence of patients with FA-C account for this characteristic distribution of complementation groups.

[Construction of enterohemorrhagic Escherichia coli O157:H7 strains with espF gene deletion and complementation].

PubMed

Hua, Ying; Sun, Qi; Wang, Xiangyu; DU, Yanli; Shao, Na; Zhang, Qiwei; Zhao, Wei; Wan, Chengsong

2015-11-01

To construct enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains with delection espF gene and its nucleotide fragment and with espF gene complementation. A pair of homologous arm primers was designed to amplify the gene fragment of kanamycin resistance, which was transformed into EHEC O157:H7 EDL933w strain via the PKD46 plasmid by electroporation. The replacement of the espF gene by kanamycin resistance gene through the PKD46-mediated red recombination system was confirmed by PCR and sequencing. The entire coding region of espF along with its nucleotide fragment was amplified by PCR and cloned into pBAD33 plasmid, which was transformed into a mutant strain to construct the strain with espF complementation. RT-PCR was used to verify the transcription of espF and its nucleotide fragment in the complemented mutant strain. We established EHEC O157:H7 EDL933w strains with espF gene deletion and with espF gene complementation. Both espF and its nucleotide fragment were transcribed in the complemented mutant strain. The two strains provide a basis for further study of the regulatory mechanism of espF.

A randomized, first-in-human, healthy volunteer trial of BIVV009, a humanized antibody for the specific inhibition of the classical complement pathway.

PubMed

Bartko, Johann; Schoergenhofer, Christian; Schwameis, Michael; Firbas, Christa; Beliveau, Martin; Chang, Colin; Marier, Jean-Francois; Nix, Darrell; Gilbert, James C; Panicker, Sandip; Jilma, Bernd

2018-05-08

Aberrant activation of the classical complement pathway is the common underlying pathophysiology of orphan diseases such as bullous pemphigoid, antibody-mediated rejection of organ transplants, cold agglutinin disease and warm autoimmune haemolytic anaemia. Therapeutic options for these complement-mediated disorders are limited and BIVV009, a humanized monoclonal antibody directed against complement factor C1s, may be potentially useful for inhibition of the classical complement pathway. A phase-1, first-in-human, double-blind, randomized, placebo-controlled, dose-escalation trial of single and multiple doses of BIVV009 or placebo was conducted in 64 volunteers to evaluate safety, tolerability, pharmacokinetic, and pharmacodynamic profiles. Single and multiple infusions of BIVV009 were well tolerated without any safety concerns. BIVV009 exhibited a steep concentration-effect relationship with a Hill coefficient of 2.4, and an IC90 of 15.5 µg/mL. This study establishes the foundation for using BIVV009 as a highly selective inhibitor of the classical complement pathway in different diseases. This article is protected by copyright. All rights reserved. © 2018 American Society for Clinical Pharmacology and Therapeutics.

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

PubMed

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

2016-10-01

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

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.

A selection that reports on protein–protein interactions within a thermophilic bacterium

PubMed Central

Nguyen, Peter Q.; Silberg, Jonathan J.

2010-01-01

Many proteins can be split into fragments that exhibit enhanced function upon fusion to interacting proteins. While this strategy has been widely used to create protein-fragment complementation assays (PCAs) for discovering protein–protein interactions within mesophilic organisms, similar assays have not yet been developed for studying natural and engineered protein complexes at the temperatures where thermophilic microbes grow. We describe the development of a selection for protein–protein interactions within Thermus thermophilus that is based upon growth complementation by fragments of Thermotoga neapolitana adenylate kinase (AKTn). Complementation studies with an engineered thermophile (PQN1) that is not viable above 75°C because its adk gene has been replaced by a Geobacillus stearothermophilus ortholog revealed that growth could be restored at 78°C by a vector that coexpresses polypeptides corresponding to residues 1–79 and 80–220 of AKTn. In contrast, PQN1 growth was not complemented by AKTn fragments harboring a C156A mutation within the zinc-binding tetracysteine motif unless these fragments were fused to Thermotoga maritima chemotaxis proteins that heterodimerize (CheA and CheY) or homodimerize (CheX). This enhanced complementation is interpreted as arising from chemotaxis protein–protein interactions, since AKTn-C156A fragments having only one polypeptide fused to a chemotaxis protein did not complement PQN1 to the same extent. This selection increases the maximum temperature where a PCA can be used to engineer thermostable protein complexes and to map protein–protein interactions. PMID:20418388

A selection that reports on protein-protein interactions within a thermophilic bacterium.

PubMed

Nguyen, Peter Q; Silberg, Jonathan J

2010-07-01

Many proteins can be split into fragments that exhibit enhanced function upon fusion to interacting proteins. While this strategy has been widely used to create protein-fragment complementation assays (PCAs) for discovering protein-protein interactions within mesophilic organisms, similar assays have not yet been developed for studying natural and engineered protein complexes at the temperatures where thermophilic microbes grow. We describe the development of a selection for protein-protein interactions within Thermus thermophilus that is based upon growth complementation by fragments of Thermotoga neapolitana adenylate kinase (AK(Tn)). Complementation studies with an engineered thermophile (PQN1) that is not viable above 75 degrees C because its adk gene has been replaced by a Geobacillus stearothermophilus ortholog revealed that growth could be restored at 78 degrees C by a vector that coexpresses polypeptides corresponding to residues 1-79 and 80-220 of AK(Tn). In contrast, PQN1 growth was not complemented by AK(Tn) fragments harboring a C156A mutation within the zinc-binding tetracysteine motif unless these fragments were fused to Thermotoga maritima chemotaxis proteins that heterodimerize (CheA and CheY) or homodimerize (CheX). This enhanced complementation is interpreted as arising from chemotaxis protein-protein interactions, since AK(Tn)-C156A fragments having only one polypeptide fused to a chemotaxis protein did not complement PQN1 to the same extent. This selection increases the maximum temperature where a PCA can be used to engineer thermostable protein complexes and to map protein-protein interactions.

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.

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.

Complement receptor 1 variants confer protection from severe malaria in Odisha, India.

PubMed

Panda, Aditya K; Panda, Madhumita; Tripathy, Rina; Pattanaik, Sarit S; Ravindran, Balachandran; Das, Bidyut K

2012-01-01

In Plasmodium falciparum infection, complement receptor-1 (CR1) on erythrocyte's surface and ABO blood group play important roles in formation of rosettes which are presumed to be contributory in the pathogenesis of severe malaria. Although several studies have attempted to determine the association of CR1 polymorphisms with severe malaria, observations remain inconsistent. Therefore, a case control study and meta-analysis was performed to address this issue. Common CR1 polymorphisms (intron 27 and exon 22) and blood group were typed in 353 cases of severe malaria (SM) [97 cerebral malaria (CM), 129 multi-organ dysfunction (MOD), 127 non-cerebral severe malaria (NCSM)], 141 un-complicated malaria and 100 healthy controls from an endemic region of Odisha, India. Relevant publications for meta-analysis were searched from the database. The homozygous polymorphisms of CR1 intron 27 and exon 22 (TT and GG) and alleles (T and G) that are associated with low expression of CR1 on red blood cells, conferred significant protection against CM, MOD and malaria deaths. Combined analysis showed significant association of blood group B/intron 27-AA/exon 22-AA with susceptibility to SM (CM and MOD). Meta-analysis revealed that the CR1 exon 22 low expression polymorphism is significantly associated with protection against severe malaria. The results of the present study demonstrate that common CR1 variants significantly protect against severe malaria in an endemic area.

Risk factors for age-related maculopathy.

PubMed

Connell, Paul P; Keane, Pearse A; O'Neill, Evelyn C; Altaie, Rasha W; Loane, Edward; Neelam, Kumari; Nolan, John M; Beatty, Stephen

2009-01-01

Age-related maculopathy (ARM) is the leading cause of blindness in the elderly. Although beneficial therapeutic strategies have recently begun to emerge, much remains unclear regarding the etiopathogenesis of this disorder. Epidemiologic studies have enhanced our understanding of ARM, but the data, often conflicting, has led to difficulties with drawing firm conclusions with respect to risk for this condition. As a consequence, we saw a need to assimilate the published findings with respect to risk factors for ARM, through a review of the literature appraising results from published cross-sectional studies, prospective cohort studies, case series, and case control studies investigating risk for this condition. Our review shows that, to date, and across a spectrum of epidemiologic study designs, only age, cigarette smoking, and family history of ARM have been consistently demonstrated to represent risk for this condition. In addition, genetic studies have recently implicated many genes in the pathogenesis of age-related maculopathy, including Complement Factor H, PLEKHA 1, and LOC387715/HTRA1, demonstrating that environmental and genetic factors are important for the development of ARM suggesting that gene-environment interaction plays an important role in the pathogenesis of this condition.

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

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

Systematic mutagenesis of genes encoding predicted autotransported proteins of Burkholderia pseudomallei identifies factors mediating virulence in mice, net intracellular replication and a novel protein conferring serum resistance.

PubMed

Lazar Adler, Natalie R; Stevens, Mark P; Dean, Rachel E; Saint, Richard J; Pankhania, Depesh; Prior, Joann L; Atkins, Timothy P; Kessler, Bianca; Nithichanon, Arnone; Lertmemongkolchai, Ganjana; Galyov, Edouard E

2015-01-01

Burkholderia pseudomallei is the causative agent of the severe tropical disease melioidosis, which commonly presents as sepsis. The B. pseudomallei K96243 genome encodes eleven predicted autotransporters, a diverse family of secreted and outer membrane proteins often associated with virulence. In a systematic study of these autotransporters, we constructed insertion mutants in each gene predicted to encode an autotransporter and assessed them for three pathogenesis-associated phenotypes: virulence in the BALB/c intra-peritoneal mouse melioidosis model, net intracellular replication in J774.2 murine macrophage-like cells and survival in 45% (v/v) normal human serum. From the complete repertoire of eleven autotransporter mutants, we identified eight mutants which exhibited an increase in median lethal dose of 1 to 2-log10 compared to the isogenic parent strain (bcaA, boaA, boaB, bpaA, bpaC, bpaE, bpaF and bimA). Four mutants, all demonstrating attenuation for virulence, exhibited reduced net intracellular replication in J774.2 macrophage-like cells (bimA, boaB, bpaC and bpaE). A single mutant (bpaC) was identified that exhibited significantly reduced serum survival compared to wild-type. The bpaC mutant, which demonstrated attenuation for virulence and net intracellular replication, was sensitive to complement-mediated killing via the classical and/or lectin pathway. Serum resistance was rescued by in trans complementation. Subsequently, we expressed recombinant proteins of the passenger domain of four predicted autotransporters representing each of the phenotypic groups identified: those attenuated for virulence (BcaA), those attenuated for virulence and net intracellular replication (BpaE), the BpaC mutant with defects in virulence, net intracellular replication and serum resistance and those displaying wild-type phenotypes (BatA). Only BcaA and BpaE elicited a strong IFN-γ response in a restimulation assay using whole blood from seropositive donors and were recognised by seropositive human sera from the endemic area. To conclude, several predicted autotransporters contribute to B. pseudomallei virulence and BpaC may do so by conferring resistance against complement-mediated killing.

Depression of in vivo clearance function of hepatic macrophage complement receptors following thermal injury.

PubMed

Cuddy, B G; Loegering, D J; Blumenstock, F A

1984-09-01

Previous studies have implicated a role for impaired hepatic macrophage blood clearance function in the increased susceptibility to infection caused by experimental thermal injury. The present study evaluated in vivo hepatic macrophage complement receptor clearance function as a possible factor contributing to impaired hepatic clearance after thermal injury. Rat erythrocytes treated with anti-erythrocyte serum (EA) were used as the test particle in rats. EA were rapidly removed from the circulation primarily by the liver and hepatic uptake of EA was greatly depressed in animals rendered C3 deficient by treatment with cobra venom factor. Thermal injury caused a large depression in the hepatic uptake of EA. It was shown that the depression in the binding of EA to hepatic macrophages was not due to decreased hepatic blood flow, decreased serum complement levels, or increased fluid phase C3b. Also, the depression of the hepatic uptake of EA incubated with serum prior to injection (EAC) was not different from that of EA after thermal injury. On this basis it was concluded that the impairment in binding of EA to the macrophages was at the cellular level and represented a depression in complement receptor clearance function. Additional studies showed that the injection of erythrocyte stroma, as a model of intravascular hemolysis, also depressed in vivo hepatic macrophage complement receptor clearance function. This latter finding suggests that the intravascular hemolysis caused by thermal injury may contribute to the depression of macrophage receptor function. The depression of hepatic macrophage complement receptor clearance function may contribute to the impaired bacterial clearance and increased susceptibility to infection following experimental thermal injury.

A hybrid two-component system protein from Azospirillum brasilense Sp7 was involved in chemotaxis.

PubMed

Cui, Yanhua; Tu, Ran; Wu, Lixian; Hong, Yuanyuan; Chen, Sanfeng

2011-09-20

We here report the sequence and functional analysis of org35 of Azospirillum brasilense Sp7, which was originally identified to be able to interact with NifA in yeast-two-hybrid system. The org35 encodes a hybrid two-component system protein, including N-terminal PAS domains, a histidine kinase (HPK) domain and a response regulator (RR) domain in C-terminal. To determine the function of the Org35, a deletion-insertion mutant in PAS domain [named Sp7353] and a complemental strain Sp7353C were constructed. The mutant had reduced chemotaxis ability compared to that of wild-type, and the complemental strain was similar to the wild-type strain. These data suggested that the A. brasilense org35 played a key role in chemotaxis. Variants containing different domains of the org35 were expressed, and the functions of these domains were studied in vitro. Phosphorylation assays in vitro demonstrated that the HPK domain of Org35 possessed the autokinase activity and that the phosphorylated HPK was able to transfer phosphate groups to the RR domain. The result indicated Org35 was a phosphorylation-communicating protein. Copyright © 2010 Elsevier GmbH. All rights reserved.

Expression of complement and pentraxin proteins in acute phase response elicited by tumor photodynamic therapy: the engagement of adrenal hormones.

PubMed

Merchant, Soroush; Huang, Naiyan; Korbelik, Mladen

2010-12-01

Treatment of solid tumors by photodynamic therapy (PDT) was recently shown to trigger a strong acute phase response. Using the mouse Lewis lung carcinoma (LLC) model, the present study examined complement and pentraxin proteins as PDT-induced acute phase reactants. The results show a distinct pattern of changes in the expression of genes encoding these proteins in the tumor, as well as host liver and spleen, following PDT mediated by photosensitizer Photofrin™. These changes were influenced by glucocorticoid hormones, as evidenced by transcriptional activation of glucocorticoid receptor and the upregulation of gene encoding this receptor. The expression of gene for glucocorticoid-induced zipper (GILZ) protein, whose activity is particularly susceptible to glucocorticoid regulation, was also changed in PDT-treated tumors. A direct demonstration that tumor PDT induces glucocorticoid hormone upregulation is provided by documenting elevated levels of serum corticosterone in mice bearing PDT-treated LLC tumors. Tumor response to PDT was negatively affected by blocking glucocorticoid receptor activity, which suggests that glucocorticoid hormones have a positive impact on the therapeutic outcome with this therapy. Copyright © 2010 Elsevier B.V. All rights reserved.

Host Immune Evasion by Lyme and Relapsing Fever Borreliae: Findings to Lead Future Studies for Borrelia miyamotoi

PubMed Central

Stone, Brandee L.; Brissette, Catherine A.

2017-01-01

The emerging pathogen, Borrelia miyamotoi, is a relapsing fever spirochete vectored by the same species of Ixodes ticks that carry the causative agents of Lyme disease in the US, Europe, and Asia. Symptoms caused by infection with B. miyamotoi are similar to a relapsing fever infection. However, B. miyamotoi has adapted to different vectors and reservoirs, which could result in unique physiology, including immune evasion mechanisms. Lyme Borrelia utilize a combination of Ixodes-produced inhibitors and native proteins [i.e., factor H-binding proteins (FHBPs)/complement regulator-acquiring surface proteins, p43, BBK32, BGA66, BGA71, CD59-like protein] to inhibit complement, while some relapsing fever spirochetes use C4b-binding protein and likely Ornithodoros-produced inhibitors. To evade the humoral response, Borrelia utilize antigenic variation of either outer surface proteins (Osps) and the Vmp-like sequences (Vls) system (Lyme borreliae) or variable membrane proteins (Vmps, relapsing fever borreliae). B. miyamotoi possesses putative FHBPs and antigenic variation of Vmps has been demonstrated. This review summarizes and compares the common mechanisms utilized by Lyme and relapsing fever spirochetes, as well as the current state of understanding immune evasion by B. miyamotoi. PMID:28154563

Host Immune Evasion by Lyme and Relapsing Fever Borreliae: Findings to Lead Future Studies for Borrelia miyamotoi.

PubMed

Stone, Brandee L; Brissette, Catherine A

2017-01-01

The emerging pathogen, Borrelia miyamotoi , is a relapsing fever spirochete vectored by the same species of Ixodes ticks that carry the causative agents of Lyme disease in the US, Europe, and Asia. Symptoms caused by infection with B. miyamotoi are similar to a relapsing fever infection. However, B. miyamotoi has adapted to different vectors and reservoirs, which could result in unique physiology, including immune evasion mechanisms. Lyme Borrelia utilize a combination of Ixodes -produced inhibitors and native proteins [i.e., factor H-binding proteins (FHBPs)/complement regulator-acquiring surface proteins, p43, BBK32, BGA66, BGA71, CD59-like protein] to inhibit complement, while some relapsing fever spirochetes use C4b-binding protein and likely Ornithodoros -produced inhibitors. To evade the humoral response, Borrelia utilize antigenic variation of either outer surface proteins (Osps) and the Vmp-like sequences (Vls) system (Lyme borreliae) or variable membrane proteins (Vmps, relapsing fever borreliae). B. miyamotoi possesses putative FHBPs and antigenic variation of Vmps has been demonstrated. This review summarizes and compares the common mechanisms utilized by Lyme and relapsing fever spirochetes, as well as the current state of understanding immune evasion by B. miyamotoi .

Kidney lesions in baboons infected with Schistosoma mansoni.

PubMed Central

Houba, V; Sturrock, R F; Butterworth, A E

1977-01-01

Glomerular lesions in baboons (Papio anubis) infected with different dosage regimes of Schistosoma mansoni were studied by immunofluorescence and light microscopy on kidney sections and by countercurrent immunoelectrophoresis on kidney homogenates and tissue eluates. Mild lesions, characterized by focal and segmental deposits of immune complexes, developed in sixty-two out of 103 baboons, irrespective of the intensity and duration of the infection. Severe, diffuse lesions developed in six baboons after prolonged and heavy infections. Adult worm and soluble egg antigens, together with IgM, IgG and C3, were detected in most of the severe lesions and in some of the mild lesions. In some animals, antigens were detected in most of the severe lesions and in some of the mild lesions. In some animals, antigens were detected in acid homogenates and eluates of kidneys which showed no deposits of immunoglobulins or complement. These observations indicate that renal lesions in S. mansoni infections may be attributable to the deposition of immune complexes pre-formed in the circulation. However, the demonstration of antigens alone in some animals may suggest an alternative possibility, namely that antigens are deposited first with a subsequent binding of antibody and complement. PMID:414868

Genetic Complementation of the Obligate Marine Actinobacterium Salinispora tropica with the Large Mechanosensitive Channel Gene mscL Rescues Cells from Osmotic Downshock

PubMed Central

Bucarey, Sergio A.; Penn, Kevin; Paul, Lauren; Fenical, William

2012-01-01

Marine actinomycetes in the genus Salinispora fail to grow when seawater is replaced with deionized (DI) water in complex growth media. While bioinformatic analyses have led to the identification of a number of candidate marine adaptation genes, there is currently no experimental evidence to support the genetic basis for the osmotic requirements associated with this taxon. One hypothesis is that the lineage-specific loss of mscL is responsible for the failure of strains to grow in media prepared with DI water. The mscL gene encodes a conserved transmembrane protein that reduces turgor pressure under conditions of acute osmotic downshock. In the present study, the mscL gene from a Micromonospora strain capable of growth on media prepared with DI water was transformed into S. tropica strain CNB-440. The single-copy, chromosomal genetic complementation yielded a recombinant Salinispora mscL+ strain that demonstrated an increased capacity to survive osmotic downshock. The enhanced survival of the S. tropica transformant provides experimental evidence that the loss of mscL is associated with the failure of Salinispora spp. to grow in low-osmotic-strength media. PMID:22492446

Topical application of probiotics in skin: adhesion, antimicrobial and antibiofilm in vitro assays.

PubMed

Lopes, E G; Moreira, D A; Gullón, P; Gullón, B; Cardelle-Cobas, A; Tavaria, F K

2017-02-01

When skin dysbiosis occurs as a result of skin disorders, probiotics can act as modulators, restoring microbial balance. Several properties of selected probiotics were evaluated so that their topical application could be considered. Adhesion, antimicrobial, quorum sensing and antibiofilm assays were carried out with several probiotic strains and tested against selected skin pathogens. All tested strains displayed significant adhesion to keratin. All lactobacilli with the exception of Lactobacillus delbrueckii, showed antimicrobial activity against skin pathogens, mainly due to organic acid production. Most of them also prevented biofilm formation, but only Propioniferax innocua was able to break down mature biofilms. This study demonstrates that although all tested probiotics adhered to human keratin, they showed limited ability to prevent adhesion of some potential skin pathogens. Most of the tested probiotics successfully prevented biofilm formation, suggesting that they may be successfully used in the future as a complement to conventional therapies in the treatment of a range of skin disorders. The topically used probiotics may be a natural, targeted treatment approach to several skin disorders and a complement to conventional therapies which present many undesirable side effects. © 2016 The Society for Applied Microbiology.

Concerted Action of Sphingomyelinase and Non-Hemolytic Enterotoxin in Pathogenic Bacillus cereus

PubMed Central

Doll, Viktoria M.

2013-01-01

Bacillus cereus causes food poisoning and serious non-gastrointestinal-tract infections. Non-hemolytic enterotoxin (Nhe), which is present in most B. cereus strains, is considered to be one of the main virulence factors. However, a B. cereus ΔnheBC mutant strain lacking Nhe is still cytotoxic to intestinal epithelial cells. In a screen for additional cytotoxic factors using an in vitro model for polarized colon epithelial cells we identified B. cereus sphingomyelinase (SMase) as a strong inducer of epithelial cell death. Using single and double deletion mutants of sph, the gene encoding for SMase, and nheBC in B. cereus we demonstrated that SMase is an important factor for B. cereus cytotoxicity in vitro and pathogenicity in vivo. SMase substantially complemented Nhe induced cytotoxicity in vitro. In addition, SMase but not Nhe contributed significantly to the mortality rate of larvae in vivo in the insect model Galleria mellonella. Our study suggests that the role of B. cereus SMase as a secreted virulence factor for in vivo pathogenesis has been underestimated and that Nhe and SMase complement each other significantly to cause full B. cereus virulence hence disease formation. PMID:23613846

A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites

PubMed Central

Sajid, Mohammed; Chevalley-Maurel, Séverine; Ramesar, Jai; Klop, Onny; Franke-Fayard, Blandine M. D.; Janse, Chris J.; Khan, Shahid M.

2011-01-01

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research. PMID:22216235

Defining the Complement Biomarker Profile of C3 Glomerulopathy

PubMed Central

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

2014-01-01

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

Synchronization of chaotic and nonchaotic oscillators: Application to bipolar disorder

NASA Astrophysics Data System (ADS)

Nono Dueyou Buckjohn, C.; Siewe Siewe, M.; Tchawoua, C.; Kofane, T. C.

2010-08-01

In this Letter, we use a synchronization scheme on two bipolar disorder models consisting of a strong nonlinear system with multiplicative excitation and a nonlinear oscillator without parametric harmonic forcing. The stability condition following our control function is analytically demonstrated using the Lyapunov theory and Routh-Hurwitz criteria, we then have the condition for the existence of a feedback gain matrix. A convenient demonstration of the accuracy of the method is complemented by the numerical simulations from which we illustrate the synchronized dynamics between the two non-identical bipolar disorder patients.

Complement-Related Regulates Autophagy in Neighboring Cells.

PubMed

Lin, Lin; Rodrigues, Frederico S L M; Kary, Christina; Contet, Alicia; Logan, Mary; Baxter, Richard H G; Wood, Will; Baehrecke, Eric H

2017-06-29

Autophagy degrades cytoplasmic components and is important for development and human health. Although autophagy is known to be influenced by systemic intercellular signals, the proteins that control autophagy are largely thought to function within individual cells. Here, we report that Drosophila macroglobulin complement-related (Mcr), a complement ortholog, plays an essential role during developmental cell death and inflammation by influencing autophagy in neighboring cells. This function of Mcr involves the immune receptor Draper, suggesting a relationship between autophagy and the control of inflammation. Interestingly, Mcr function in epithelial cells is required for macrophage autophagy and migration to epithelial wounds, a Draper-dependent process. This study reveals, unexpectedly, that complement-related from one cell regulates autophagy in neighboring cells via an ancient immune signaling program. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

Evaluation of serum levels of C3 and C4 complement factors in patients with beta thalassemia major in Khuzestan Province, Southwest Iran.

PubMed

Ghafourian, Mehri; Esmaeili, Mehrnosh; Dashti-Gerdabi, Nader; Sadeghi, Alireza; Malekei Naseri, Ali; Kazemi, Akhtar

2017-01-01

Thalassemia syndrome is the most common genetic disorder in the world and infection is the second cause of death in these patients. Measurement of serum C3 and C4 complement factors in serum was done in 60 patients with beta thalassemia major in comparison with 30 healthy subjects as control group. The serum level of C3 and C4 complement factors in 60 patients with beta thalassemia major who were randomly selected from among the patients referred to Shafa Hospital of Ahvaz was evaluated and compared with 30 samples from healthy individuals with no history of recent infectious or autoimmune diseases. It should be noted that single-radial-immunodiffusion assay was used in this study. This study has shown a significant reduction in serum levels of C3 and C4 in patients compared to controls (P value < 0.05). Decreased synthesis or increased consumption of complement factors in patients receiving multiple blood transfusions might lead to continuous contact between the immune system and various antigens, causing nonstop use of complement factors, recurrent infections, changes in parameters of the immune system due to iron overload as well as exposure to infectious factors such as HBV, HCV, HIV, and HTLV through blood transfusion.

Sundanese Complementation

ERIC Educational Resources Information Center

Kurniawan, Eri

2013-01-01

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

Complement Evasion Strategies of Viruses: An Overview

PubMed Central

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

2017-01-01

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

Increased HDL cholesterol levels in mice with XX versus XY sex chromosomes

PubMed Central

Link, Jenny C.; Chen, Xuqi; Prien, Christopher; Borja, Mark S.; Hammerson, Bradley; Oda, Michael N.; Arnold, Arthur P.; Reue, Karen

2015-01-01

Objective The molecular mechanisms underlying sex differences in dyslipidemia are poorly understood. We aimed to distinguish genetic and hormonal regulators of sex differences in plasma lipid levels. Approach and Results We assessed the role of gonadal hormones and sex chromosome complement on lipid levels using the Four Core Genotypes mouse model (XX females, XX males, XY females, and XY males). In gonadally intact mice fed a chow diet, lipid levels were influenced by both male–female gonadal sex and XX–XY chromosome complement. Gonadectomy of adult mice revealed that the male–female differences are dependent on acute effects of gonadal hormones. In both intact and gonadectomized animals, XX mice had higher HDL cholesterol (HDL-C) levels than XY mice, regardless of male–female sex. Feeding a cholesterol-enriched diet produced distinct patterns of sex differences in lipid levels compared to a chow diet, revealing the interaction of gonadal and chromosomal sex with diet. Notably, under all dietary and gonadal conditions, HDL-C levels were higher in mice with two X chromosomes compared to mice with an X and Y chromosome. By generating mice with XX, XY and XXY chromosome complements, we determined that the presence of two X chromosomes, and not the absence of the Y chromosome, influences HDL-C concentration. Conclusions We demonstrate that having two X chromosomes versus an X and Y chromosome complement drives sex differences in HDL-C. It is conceivable that increased expression of genes escaping X-inactivation in XX mice regulates downstream processes to establish sexual dimorphism in plasma lipid levels. PMID:26112012

Increased high-density lipoprotein cholesterol levels in mice with XX versus XY sex chromosomes.

PubMed

Link, Jenny C; Chen, Xuqi; Prien, Christopher; Borja, Mark S; Hammerson, Bradley; Oda, Michael N; Arnold, Arthur P; Reue, Karen

2015-08-01

The molecular mechanisms underlying sex differences in dyslipidemia are poorly understood. We aimed to distinguish genetic and hormonal regulators of sex differences in plasma lipid levels. We assessed the role of gonadal hormones and sex chromosome complement on lipid levels using the four core genotypes mouse model (XX females, XX males, XY females, and XY males). In gonadally intact mice fed a chow diet, lipid levels were influenced by both male-female gonadal sex and XX-XY chromosome complement. Gonadectomy of adult mice revealed that the male-female differences are dependent on acute effects of gonadal hormones. In both intact and gonadectomized animals, XX mice had higher HDL cholesterol (HDL-C) levels than XY mice, regardless of male-female sex. Feeding a cholesterol-enriched diet produced distinct patterns of sex differences in lipid levels compared with a chow diet, revealing the interaction of gonadal and chromosomal sex with diet. Notably, under all dietary and gonadal conditions, HDL-C levels were higher in mice with 2 X chromosomes compared with mice with an X and Y chromosome. By generating mice with XX, XY, and XXY chromosome complements, we determined that the presence of 2 X chromosomes, and not the absence of the Y chromosome, influences HDL-C concentration. We demonstrate that having 2 X chromosomes versus an X and Y chromosome complement drives sex differences in HDL-C. It is conceivable that increased expression of genes escaping X-inactivation in XX mice regulates downstream processes to establish sexual dimorphism in plasma lipid levels. © 2015 American Heart Association, Inc.

The Importance of Being a Complement: CED Effects Revisited

ERIC Educational Resources Information Center

Jurka, Johannes

2010-01-01

This dissertation revisits subject island effects (Ross 1967, Chomsky 1973) cross-linguistically. Controlled acceptability judgment studies in German, English, Japanese and Serbian show that extraction out of specifiers is consistently degraded compared to extraction out of complements, indicating that the Condition on Extraction domains (CED,…

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

PubMed

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

2016-12-01

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

[Complement 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.

Spatially resolved density and ionization measurements of shocked foams using x-ray fluorescence

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

MacDonald, M. J.; Keiter, P. A.; Montgomery, D. S.

2016-09-22

We present experiments at the Trident laser facility demonstrating the use of x-ray fluorescence (XRF) to simultaneously measure density, ionization state populations, and electron temperature in shocked foams. An imaging x-ray spectrometer was used to obtain spatially-resolved measurements of Ti K-more » $$\\alpha$$ emission. Density profiles were measured from K-$$\\alpha$$ intensity. Ti ionization state distributions and electron temperatures were inferred by fitting K-$$\\alpha$$ spectra to spectra from CRETIN simulations. This study shows that XRF provides a powerful tool to complement other diagnostics to make equation of state measurements of shocked materials containing a suitable tracer element.« less

Treating ADHD With Suggestion: Neurofeedback and Placebo Therapeutics.

PubMed

Thibault, Robert T; Veissière, Samuel; Olson, Jay A; Raz, Amir

2018-06-01

We propose that clinicians can use suggestion to help treat conditions such as ADHD. We use EEG neurofeedback as a case study, alongside evidence from a recent pilot experiment utilizing a sham MRI scanner to highlight the therapeutic potential of suggestion-based treatments. The medical literature demonstrates that many practitioners already prescribe treatments that hardly outperform placebo comparators. Moreover, the sham MRI experiment showed that, even with full disclosure of the procedure, suggestion alone can reduce the symptomatology of ADHD. Non-deceptive suggestion-based treatments, especially those drawing on accessories from neuroscience, may offer a safe complement and potential alternative to current standard of care for individuals with ADHD.

Regulation of Macrophage Recognition through the Interplay of Nanoparticle Surface Functionality and Protein Corona.

PubMed

Saha, Krishnendu; Rahimi, Mehran; Yazdani, Mahdieh; Kim, Sung Tae; Moyano, Daniel F; Hou, Singyuk; Das, Ridhha; Mout, Rubul; Rezaee, Farhad; Mahmoudi, Morteza; Rotello, Vincent M

2016-04-26

Using a family of cationic gold nanoparticles (NPs) with similar size and charge, we demonstrate that proper surface engineering can control the nature and identity of protein corona in physiological serum conditions. The protein coronas were highly dependent on the hydrophobicity and arrangement of chemical motifs on NP surface. The NPs were uptaken in macrophages in a corona-dependent manner, predominantly through recognition of specific complement proteins in the NP corona. Taken together, this study shows that surface functionality can be used to tune the protein corona formed on NP surface, dictating the interaction of NPs with macrophages.

Designing a serious game for historical heritage: a case study of Heerlen Roman bathhouse

NASA Astrophysics Data System (ADS)

Qi, Wen

2014-01-01

The advances of computer games have shown their potentials for developing edutainment content and services. Current cultural heritages often make use of games in order to complement existing presentations and to create a memorable exhibition. It offers opportunities to reorganize and conceptualize historical, cultural and technological information about the exhibits. To demonstrate the benefits of serious games in terms of facilitating the learning activities in a constructive and meaningful way, we designed a video game about the Heerlen bathhouse heritage. This paper explains the design considerations of this Roman bathhouse game, with a particular focus on the link between game play and learning.

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.

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.

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.

Complementary Curves of Descent

DTIC Science & Technology

2012-11-16

a lemniscate of Bernoulli . Alternatively, the wires can be tracks down which round objects undergo a rolling race. The level of presentation is...A common mechanics demonstration consists of racing cars or balls down tracks of various shapes and qualitatively or quantitatively measuring the...problem), which is self complementary. A striking example is a straight wire whose complement is a lemniscate of Bernoulli . Alternatively the wires can

Demonstration of Lenz's law: Analysis of a magnet falling through a conducting tube

NASA Astrophysics Data System (ADS)

Roy, M. K.; Harbola, Manoj K.; Verma, H. C.

2007-08-01

We revisit a recent analysis of the time of fall of a magnet as it slows down while passing through a conducting tube. We complement recent work by considering the effect of the thickness of the tube on the time of fall. The resulting expression gives a more accurate expression for the time of fall.

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

A comprehensive strategy for the subtyping of patients with Fanconi anaemia: conclusions from the Spanish Fanconi Anemia Research Network

PubMed Central

Casado, José Antonio; Callén, Elsa; Jacome, Ariana; Río, Paula; Castella, Maria; Lobitz, Stephan; Ferro, Teresa; Muñoz, Arturo; Sevilla, Julián; Cantalejo, Ángeles; Cela, Elena; Cervera, José; Sánchez‐Calero, Jesús; Badell, Isabel; Estella, Jesús; Dasí, Ángeles; Olivé, Teresa; Ortega, Juan José; Rodriguez‐Villa, Antonia; Tapia, María; Molinés, Antonio; Madero, Luis; Segovia, José C; Neveling, Kornelia; Kalb, Reinhard; Schindler, Detlev; Hanenberg, Helmut; Surrallés, Jordi; Bueren, Juan A

2007-01-01

Background Fanconi anaemia is a heterogeneous genetic disease, where 12 complementation groups have been already described. Identifying the complementation group in patients with Fanconi anaemia constitutes a direct procedure to confirm the diagnosis of the disease and is required for the recruitment of these patients in gene therapy trials. Objective To determine the subtype of Fanconi anaemia patients in Spain, a Mediterranean country with a relatively high population (23%) of Fanconi anaemia patients belonging to the gypsy race. Methods Most patients could be subtyped by retroviral complementation approaches in peripheral blood T cells, although some mosaic patients were subtyped in cultured skin fibroblasts. Other approaches, mainly based on western blot analysis and generation of nuclear RAD51 and FANCJ foci, were required for the subtyping of a minor number of patients. Results and conclusions From a total of 125 patients included in the Registry of Fanconi Anaemia, samples from 102 patients were available for subtyping analyses. In 89 cases the subtype could be determined and in 8 cases exclusions of common complementation groups were made. Compared with other international studies, a skewed distribution of complementation groups was observed in Spain, where 80% of the families belonged to the Fanconi anaemia group A (FA‐A) complementation group. The high proportion of gypsy patients, all of them FA‐A, and the absence of patients with FA‐C account for this characteristic distribution of complementation groups. PMID:17105750

Genomic resources for gene discovery, functional genome annotation, and evolutionary studies of maize and its close relatives.

PubMed

Wang, Chao; Shi, Xue; Liu, Lin; Li, Haiyan; Ammiraju, Jetty S S; Kudrna, David A; Xiong, Wentao; Wang, Hao; Dai, Zhaozhao; Zheng, Yonglian; Lai, Jinsheng; Jin, Weiwei; Messing, Joachim; Bennetzen, Jeffrey L; Wing, Rod A; Luo, Meizhong

2013-11-01

Maize is one of the most important food crops and a key model for genetics and developmental biology. A genetically anchored and high-quality draft genome sequence of maize inbred B73 has been obtained to serve as a reference sequence. To facilitate evolutionary studies in maize and its close relatives, much like the Oryza Map Alignment Project (OMAP) (www.OMAP.org) bacterial artificial chromosome (BAC) resource did for the rice community, we constructed BAC libraries for maize inbred lines Zheng58, Chang7-2, and Mo17 and maize wild relatives Zea mays ssp. parviglumis and Tripsacum dactyloides. Furthermore, to extend functional genomic studies to maize and sorghum, we also constructed binary BAC (BIBAC) libraries for the maize inbred B73 and the sorghum landrace Nengsi-1. The BAC/BIBAC vectors facilitate transfer of large intact DNA inserts from BAC clones to the BIBAC vector and functional complementation of large DNA fragments. These seven Zea Map Alignment Project (ZMAP) BAC/BIBAC libraries have average insert sizes ranging from 92 to 148 kb, organellar DNA from 0.17 to 2.3%, empty vector rates between 0.35 and 5.56%, and genome equivalents of 4.7- to 8.4-fold. The usefulness of the Parviglumis and Tripsacum BAC libraries was demonstrated by mapping clones to the reference genome. Novel genes and alleles present in these ZMAP libraries can now be used for functional complementation studies and positional or homology-based cloning of genes for translational genomics.

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

Language and False-Belief Task Performance in Children With Autism Spectrum Disorder.

PubMed

Jeffrey Farrar, M; Seung, Hye Kyeung; Lee, Hyeonjin

2017-07-12

Language is related to false-belief (FB) understanding in both typically developing children and children with autism spectrum disorder (ASD). The current study examined the role of complementation and general language in FB understanding. Of interest was whether language plays similar or different roles in the groups' FB performance. Participants were 16 typically developing children (mean age = 5.0 years; mental age = 6.7) and 18 with ASD (mean age = 7.3 years; mental age = 8.3). Children were administered FB and language tasks (say- and think-complements), receptive and expressive vocabulary tests, and relative clauses. When mental age and receptive and expressive vocabulary were used as separate covariates, the typical control group outperformed the children with ASD in FB task performance. Chi-square analyses indicated that passing both complementation tasks was linked to the FB understanding of children with ASD. Children with ASD who passed FB tasks all passed say- and think-complement tasks. However, some children in the control group were able to pass the FB tasks, even if they failed the say- and think-complement tasks. The results indicate that children with ASD relied more on complement understanding to pass FB than typically developing children. Results are discussed regarding the developmental pathways for FB understanding.

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.

Let’s Tie the Knot: Marriage of Complement and Adaptive Immunity in Pathogen Evasion, for Better or Worse

PubMed Central

Bennett, Kaila M.; Rooijakkers, Suzan H. M.; Gorham, Ronald D.

2017-01-01

The complement system is typically regarded as an effector arm of innate immunity, leading to recognition and killing of microbial invaders in body fluids. Consequently, pathogens have engaged in an arms race, evolving molecules that can interfere with proper complement responses. However, complement is no longer viewed as an isolated system, and links with other immune mechanisms are continually being discovered. Complement forms an important bridge between innate and adaptive immunity. While its roles in innate immunity are well-documented, its function in adaptive immunity is less characterized. Therefore, it is no surprise that the field of pathogenic complement evasion has focused on blockade of innate effector functions, while potential inhibition of adaptive immune responses (via complement) has been overlooked to a certain extent. In this review, we highlight past and recent developments on the involvement of complement in the adaptive immune response. We discuss the mechanisms by which complement aids in lymphocyte stimulation and regulation, as well as in antigen presentation. In addition, we discuss microbial complement evasion strategies, and highlight specific examples in the context of adaptive immune responses. These emerging ties between complement and adaptive immunity provide a catalyst for future discovery in not only the field of adaptive immune evasion but in elucidating new roles of complement. PMID:28197139

Let's Tie the Knot: Marriage of Complement and Adaptive Immunity in Pathogen Evasion, for Better or Worse.

PubMed

Bennett, Kaila M; Rooijakkers, Suzan H M; Gorham, Ronald D

2017-01-01

The complement system is typically regarded as an effector arm of innate immunity, leading to recognition and killing of microbial invaders in body fluids. Consequently, pathogens have engaged in an arms race, evolving molecules that can interfere with proper complement responses. However, complement is no longer viewed as an isolated system, and links with other immune mechanisms are continually being discovered. Complement forms an important bridge between innate and adaptive immunity. While its roles in innate immunity are well-documented, its function in adaptive immunity is less characterized. Therefore, it is no surprise that the field of pathogenic complement evasion has focused on blockade of innate effector functions, while potential inhibition of adaptive immune responses (via complement) has been overlooked to a certain extent. In this review, we highlight past and recent developments on the involvement of complement in the adaptive immune response. We discuss the mechanisms by which complement aids in lymphocyte stimulation and regulation, as well as in antigen presentation. In addition, we discuss microbial complement evasion strategies, and highlight specific examples in the context of adaptive immune responses. These emerging ties between complement and adaptive immunity provide a catalyst for future discovery in not only the field of adaptive immune evasion but in elucidating new roles of complement.

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.

Juvenile Justice and a Strengths Perspective: Complement or Clash?

ERIC Educational Resources Information Center

Clark, Michael D.

2009-01-01

Does the new realm of positive psychology and strength-based strategies complement or clash with the remedial discipline of social control traditionally practiced in juvenile justice programs? Many welcome the balance of positive psychology, the strengths perspective, and coping and resilience studies. Although emerging from different disciplines,…

Cognitive State Verbs and Complement Clauses in Children with SLI and Their Typically Developing Peers

ERIC Educational Resources Information Center

Van Horne, Amanda J. Owen; Lin, Shanju

2011-01-01

This study investigated the use of cognitive state verbs (CSVs) and complement clauses in children with specific language impairment (SLI) and their typically developing (TD) peers. In Study 1, conversational samples from 23 children with SLI (M = 6;2), 24 age-matched TD children (M = 6;2) and 21 vocabulary-matched TD children (M = 4;9) were…

Low-loss electron energy loss spectroscopy: An atomic-resolution complement to optical spectroscopies and application to graphene

DOE PAGES

Kapetanakis, Myron; Zhou, Wu; Oxley, Mark P.; ...

2015-09-25

Photon-based spectroscopies have played a central role in exploring the electronic properties of crystalline solids and thin films. They are a powerful tool for probing the electronic properties of nanostructures, but they are limited by lack of spatial resolution. On the other hand, electron-based spectroscopies, e.g., electron energy loss spectroscopy (EELS), are now capable of subangstrom spatial resolution. Core-loss EELS, a spatially resolved analog of x-ray absorption, has been used extensively in the study of inhomogeneous complex systems. In this paper, we demonstrate that low-loss EELS in an aberration-corrected scanning transmission electron microscope, which probes low-energy excitations, combined with amore » theoretical framework for simulating and analyzing the spectra, is a powerful tool to probe low-energy electron excitations with atomic-scale resolution. The theoretical component of the method combines density functional theory–based calculations of the excitations with dynamical scattering theory for the electron beam. We apply the method to monolayer graphene in order to demonstrate that atomic-scale contrast is inherent in low-loss EELS even in a perfectly periodic structure. The method is a complement to optical spectroscopy as it probes transitions entailing momentum transfer. The theoretical analysis identifies the spatial and orbital origins of excitations, holding the promise of ultimately becoming a powerful probe of the structure and electronic properties of individual point and extended defects in both crystals and inhomogeneous complex nanostructures. The method can be extended to probe magnetic and vibrational properties with atomic resolution.« less

Mechanistic Explanations for Restricted Evolutionary Paths That Emerge from Gene Regulatory Networks

PubMed Central

Cotterell, James; Sharpe, James

2013-01-01

The extent and the nature of the constraints to evolutionary trajectories are central issues in biology. Constraints can be the result of systems dynamics causing a non-linear mapping between genotype and phenotype. How prevalent are these developmental constraints and what is their mechanistic basis? Although this has been extensively explored at the level of epistatic interactions between nucleotides within a gene, or amino acids within a protein, selection acts at the level of the whole organism, and therefore epistasis between disparate genes in the genome is expected due to their functional interactions within gene regulatory networks (GRNs) which are responsible for many aspects of organismal phenotype. Here we explore epistasis within GRNs capable of performing a common developmental function – converting a continuous morphogen input into discrete spatial domains. By exploring the full complement of GRN wiring designs that are able to perform this function, we analyzed all possible mutational routes between functional GRNs. Through this study we demonstrate that mechanistic constraints are common for GRNs that perform even a simple function. We demonstrate a common mechanistic cause for such a constraint involving complementation between counter-balanced gene-gene interactions. Furthermore we show how such constraints can be bypassed by means of “permissive” mutations that buffer changes in a direct route between two GRN topologies that would normally be unviable. We show that such bypasses are common and thus we suggest that unlike what was observed in protein sequence-function relationships, the “tape of life” is less reproducible when one considers higher levels of biological organization. PMID:23613807

Applications of Computational Methods for Dynamic Stability and Control Derivatives

NASA Technical Reports Server (NTRS)

Green, Lawrence L.; Spence, Angela M.

2004-01-01

Initial steps in the application o f a low-order panel method computational fluid dynamic (CFD) code to the calculation of aircraft dynamic stability and control (S&C) derivatives are documented. Several capabilities, unique to CFD but not unique to this particular demonstration, are identified and demonstrated in this paper. These unique capabilities complement conventional S&C techniques and they include the ability to: 1) perform maneuvers without the flow-kinematic restrictions and support interference commonly associated with experimental S&C facilities, 2) easily simulate advanced S&C testing techniques, 3) compute exact S&C derivatives with uncertainty propagation bounds, and 4) alter the flow physics associated with a particular testing technique from those observed in a wind or water tunnel test in order to isolate effects. Also presented are discussions about some computational issues associated with the simulation of S&C tests and selected results from numerous surface grid resolution studies performed during the course of the study.

Practical barriers to the implementation of early goal directed therapy in the UK: trainee skills and awareness.

PubMed

McNally, S J; MacKinnon, M; Hawkins, M

2009-08-01

The Surviving Sepsis Campaign (SSC) recommends Early Goal Directed Therapy (EGDT) in the treatment of septic shock, which requires key critical care skills and knowledge. This study evaluates the availability of these skills in Specialist Registrars in acute hospital specialities in the UK. A questionnaire was sent to Specialist Registrars in Anaesthetics, General Surgery and General Medicine throughout Scotland. One hundred and eighty five responses were obtained. One hundred percent of anaesthetists, 70% of surgeons and 51% of physicians were aware of EGDT Only 62 trainees (6% of surgeons, 79% of anaesthetists, 19% of physicians) had the full complement of skills and knowledge to implement EGDT. This study demonstrates that non-anaesthetic registrars in the UK lack both knowledge and skills required to provide EGDT. The main deficit was in awareness, demonstrating that knowledge of EGDT is not penetrating into specialities beyond anaesthesia. It is now time for the SSC to specifically target non-anaesthetic specialities.

Studying light-harvesting models with superconducting circuits.

PubMed

Potočnik, Anton; Bargerbos, Arno; Schröder, Florian A Y N; Khan, Saeed A; Collodo, Michele C; Gasparinetti, Simone; Salathé, Yves; Creatore, Celestino; Eichler, Christopher; Türeci, Hakan E; Chin, Alex W; Wallraff, Andreas

2018-03-02

The process of photosynthesis, the main source of energy in the living world, converts sunlight into chemical energy. The high efficiency of this process is believed to be enabled by an interplay between the quantum nature of molecular structures in photosynthetic complexes and their interaction with the environment. Investigating these effects in biological samples is challenging due to their complex and disordered structure. Here we experimentally demonstrate a technique for studying photosynthetic models based on superconducting quantum circuits, which complements existing experimental, theoretical, and computational approaches. We demonstrate a high degree of freedom in design and experimental control of our approach based on a simplified three-site model of a pigment protein complex with realistic parameters scaled down in energy by a factor of 10 5 . We show that the excitation transport between quantum-coherent sites disordered in energy can be enabled through the interaction with environmental noise. We also show that the efficiency of the process is maximized for structured noise resembling intramolecular phononic environments found in photosynthetic complexes.

[Comparative data on the formation of complement-binding and hemagglutinating antibodies to penicillin].

PubMed

Sluvko, A L

1976-10-01

Comparative data on production of complement-binding and hemagglutinating antibodies in the process of the antigenic effect of benzylpenicillin under experimental conditions are presented. 30 rabbit antisera and 3 sera of intact animals were studied. The hemagglutinating antibodies were determined in 19 antisera, high and reliable titers of the antipenicillin hemagglutinating antibodies being found only in 8 antisera. The antipenicillin complement-binding antibodies using complex antibiotic antibodies were also found in 19 antisera. The process of antibody production was more pronounced in the complement-binding reaction (CBR). Both types of the antibodies were detected simultaneously in 14 antisera. It is concluded that the CBR with the use of the penicillin complex antigenes on the stroma of the erythrocytes and in combination with the blood serum is a rather sensitive reaction for detection of antipenicillin antibodies.

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

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

Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter, OSTalpha-OSTbeta.

PubMed

Seward, David J; Koh, Albert S; Boyer, James L; Ballatori, Nazzareno

2003-07-25

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.

The effect of heparin rinse on the biocompatibility of continuous veno-venous hemodiafiltration.

PubMed

Opatrný, K; Polanská, K; Krouzecký, A; Vít, L; Novák, I; Kasal, E

2002-06-01

The aims of our cross-over randomized study were (1) to assess hemostasis in patients with acute renal failure (ARF) and (2) to determine whether or not the generally recommended heparin rinse of the extracorporeal circuit (ECC) prior to the procedure affects thrombogenicity, complement activation, and leukocyte count in blood during continuous venovenous hemodiafiltration (CVVHDF). Eleven critically ill ARF patients were treated, in random order, using CVVHDF in postdilution setup following ECC rinse with saline (A) with heparin at a concentration of 2,000 IU/L (10 procedures), (B) with heparin at a concentration of 10,000 IU/L (7 procedures), and (C) without heparin (9 procedures). Except for the rinse, anticoagulation therapy did not differ in individual patients during the procedures. Blood was withdrawn before, and at minutes 15, 60, and 360 invariably at diafilter inlet and outlet. Compared with healthy individuals, patients showed lower blood thrombocyte counts (153 vs 233*10(9)/L, p<0.01, arithmetic means, Student's t test), longer aPTT (44 vs 36 s, p<0.05), higher plasma levels of heparin (0.1 vs 0.0 U/mL, p<0.05), D-dimer (1129 vs 36 ng/mL, p<0.001) and beta-thromboglobulin (BTG) (159 vs 37 U/mL, p<0.001) prior to CVVHDF. The comparison of procedures with different rinsing technique did not reveal any significant difference in their effects on blood thrombocyte and leukocyte counts, aPTT, plasma levels of heparin, BTG, thrombin-antithrombin III complexes, D-dimer, or the C5a complement component. (1) Patients indicated for CVVHDF show impaired hemostasis involving thrombocytes, coagulation, and fibrinolysis, (2) no beneficial effect of heparin rinse on CVVHDF ECC thrombogenicity, complement activation or blood leukocyte counts was demonstrated.

Genetic susceptibility to chronic wasting disease in free-ranging white-tailed deer: complement component C1q and Prnp polymorphisms

USGS Publications Warehouse

Blanchong, Julie A.; Heisey, Dennis M.; Scribner, Kim T.; Libants, Scot V.; Johnson, Chad; Aiken, Judd M.; Langenberg, Julia A.; Samuel, Michael D.

2009-01-01

The genetic basis of susceptibility to chronic wasting disease (CWD) in free-ranging cervids is of great interest. Association studies of disease susceptibility in free-ranging populations, however, face considerable challenges including: the need for large sample sizes when disease is rare, animals of unknown pedigree create a risk of spurious results due to population admixture, and the inability to control disease exposure or dose. We used an innovative matched case–control design and conditional logistic regression to evaluate associations between polymorphisms of complement C1q and prion protein (Prnp) genes and CWD infection in white-tailed deer from the CWD endemic area in south-central Wisconsin. To reduce problems due to admixture or disease-risk confounding, we used neutral genetic (microsatellite) data to identify closely related CWD-positive (n = 68) and CWD-negative (n = 91) female deer to serve as matched cases and controls. Cases and controls were also matched on factors (sex, location, age) previously demonstrated to affect CWD infection risk. For Prnp, deer with at least one Serine (S) at amino acid 96 were significantly less likely to be CWD-positive relative to deer homozygous for Glycine (G). This is the first characterization of genes associated with the complement system in white-tailed deer. No tests for association between any C1q polymorphism and CWD infection were significant at p < 0.05. After controlling for Prnp, we found weak support for an elevated risk of CWD infection in deer with at least one Glycine (G) at amino acid 56 of the C1qC gene. While we documented numerous amino acid polymorphisms in C1q genes none appear to be strongly associated with CWD susceptibility.

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

PubMed

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

2015-01-01

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

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

Complement factor H-related proteins in IgA nephropathy-sometimes a gentle nudge does the trick.

PubMed

Thurman, Joshua M; Laskowski, Jennifer

2017-10-01

Complement activation probably contributes to glomerular inflammation and damage in IgA nephropathy. In this issue, 2 groups report that levels of factor H-related protein 1 are elevated in patients with IgA nephropathy and correlate with disease progression. These studies provide new evidence that the complement cascade is important to the pathogenesis of this disease. These results also suggest that factor H-related protein 1 levels may be useful for identifying those patients at high risk of disease progression. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Endotoxin-associated protein: a potent stimulus for human granulocytopoietic activity which may be accessory cell independent.

PubMed Central

Bjornson, B H; Agura, E; Harvey, J M; Johns, M; Andrews, R G; McCabe, W R

1988-01-01

Proteins coextracted with endotoxin, termed endotoxin-associated protein (EAP), have been shown to exert interleukin 1-like activities. The present studies demonstrate that EAP also exerts potent granulopoietic colony-stimulating activity (CSA) on human peripheral blood and bone marrow progenitor cells, comparable to that seen with various types of conditioned media. The CSA observed with EAP appeared to be heat (100 degrees C, 30 min) and trypsin resistant and partially pronase resistant. Similar resistance was observed with the porin proteins of the outer membrane of gram-negative bacteria, and similar CSA activity was observed with a purified porin preparation of Neisseria gonorrhoeae. The CSA of EAP could be demonstrated in human peripheral blood and bone marrow leukocytes rigorously depleted of monocytes, T lymphocytes, and B lymphocytes by treatment with specific monoclonal antibodies and complement. PMID:2836311

Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry.

PubMed

Ejsing, Christer S; Sampaio, Julio L; Surendranath, Vineeth; Duchoslav, Eva; Ekroos, Kim; Klemm, Robin W; Simons, Kai; Shevchenko, Andrej

2009-02-17

Although the transcriptome, proteome, and interactome of several eukaryotic model organisms have been described in detail, lipidomes remain relatively uncharacterized. Using Saccharomyces cerevisiae as an example, we demonstrate that automated shotgun lipidomics analysis enabled lipidome-wide absolute quantification of individual molecular lipid species by streamlined processing of a single sample of only 2 million yeast cells. By comparative lipidomics, we achieved the absolute quantification of 250 molecular lipid species covering 21 major lipid classes. This analysis provided approximately 95% coverage of the yeast lipidome achieved with 125-fold improvement in sensitivity compared with previous approaches. Comparative lipidomics demonstrated that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes, and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches.

The industrial anaerobe Clostridium acetobutylicum uses polyketides to regulate cellular differentiation.

PubMed

Herman, Nicolaus A; Kim, Seong Jong; Li, Jeffrey S; Cai, Wenlong; Koshino, Hiroyuki; Zhang, Wenjun

2017-11-15

Polyketides are an important class of bioactive small molecules valued not only for their diverse therapeutic applications, but also for their role in controlling interesting biological phenotypes in their producing organisms. While numerous polyketides are known to be derived from aerobic organisms, only a single family of polyketides has been identified from anaerobic organisms. Here we uncover a family of polyketides native to the anaerobic bacterium Clostridium acetobutylicum, an organism well-known for its historical use as an industrial producer of the organic solvents acetone, butanol, and ethanol. Through mutational analysis and chemical complementation assays, we demonstrate that these polyketides act as chemical triggers of sporulation and granulose accumulation in this strain. This study represents a significant addition to the body of work demonstrating the existence and importance of polyketides in anaerobes, and showcases a strategy of manipulating the secondary metabolism of an organism to improve traits relevant for industrial applications.

Strong Broadband Terahertz Optical Activity through Control of the Blaschke Phase with Chiral Metasurfaces

NASA Astrophysics Data System (ADS)

Cole, Michael A.; Chen, Wen-chen; Liu, Mingkai; Kruk, Sergey S.; Padilla, Willie J.; Shadrivov, Ilya V.; Powell, David A.

2017-07-01

We demonstrate terahertz chiral metamaterials that achieve resonant transmission and strong optical activity. This response is realized in a metasurface coupled to its Babinet complement, with additional twist. Uniquely, the optical activity achieved in this type of metamaterial is weakly dispersive around the resonant transmission maxima, but it can be highly dispersive around the transmission minima. It has recently been shown that this unique optical activity response is closely related to zeros in the transmission spectra of circular polarizations through the Kramers-Kronig relations and strong resonant features in the optical activity spectrum corresponding to the Blaschke phase terms. Here we demonstrate how modifying the meta-atom geometry greatly affects the location and magnitude of these Blaschke phase terms. We study three different meta-atoms, which are variations on the simple cross structure. Their responses are measured using terahertz time-domain spectroscopy and analyzed via numerical simulations.

Identification of the HrpS binding site in the hrpL promoter and effect of the RpoN binding site of HrpS on the regulation of the type III secretion system in Erwinia amylovora.

PubMed

Lee, Jae Hoon; Sundin, George W; Zhao, Youfu

2016-06-01

The type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by an RpoN-HrpL sigma factor cascade, which is activated by the bacterial alarmone (p)ppGpp. In this study, the binding site of HrpS, an enhancer binding protein, was identified for the first time in plant-pathogenic bacteria. Complementation of the hrpL mutant with promoter deletion constructs of the hrpL gene and promoter activity analyses using various lengths of the hrpL promoter fused to a promoter-less green fluorescent protein (gfp) reporter gene delineated the upstream region for HrpS binding. Sequence analysis revealed a dyad symmetry sequence between -138 and -125 nucleotides (TGCAA-N4-TTGCA) as the potential HrpS binding site, which is conserved in the promoter of the hrpL gene among plant enterobacterial pathogens. Results of quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and electrophoresis mobility shift assay coupled with site-directed mutagenesis (SDM) analysis showed that the intact dyad symmetry sequence was essential for HrpS binding, full activation of T3SS gene expression and virulence. In addition, the role of the GAYTGA motif (RpoN binding site) of HrpS in the regulation of T3SS gene expression in E. amylovora was characterized by complementation of the hrpS mutant using mutant variants generated by SDM. Results showed that a Y100F substitution of HrpS complemented the hrpS mutant, whereas Y100A and Y101A substitutions did not. These results suggest that tyrosine (Y) and phenylalanine (F) function interchangeably in the conserved GAYTGA motif of HrpS in E. amylovora. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Modification of the plasma complement protein profile by exogenous estrogens is indicative of a compromised immune competence in marine medaka (Oryzias melastigma).

PubMed

Dong, Miao; Seemann, Frauke; Humble, Joseph L; Liang, Yimin; Peterson, Drew R; Ye, Rui; Ren, Honglin; Kim, Hui-Su; Lee, Jae-Seong; Au, Doris W T; Lam, Yun Wah

2017-11-01

Growing evidence suggests that the immune system of teleost is vulnerable to xenoestrogens, which are ubiquitous in the marine environment. This study detected and identified the major circulatory immune proteins deregulated by 17α-ethinylestradiol (EE2), which may be linked to fish susceptibility to pathogens in the marine medaka, Oryzias melastigma. Fish immune competence was determined using a host resistance assay to pathogenic bacteria Edwardsiella tarda. Females were consistently more susceptible to infection-induced mortality than males. Exposure to EE2 could narrow the sex gap of mortality by increasing infection-induced death in male fish. Proteomic analysis revealed that the major plasma immune proteins of adult fish were highly sexually dimorphic. EE2 induced pronounced sex-specific changes in the plasma proteome, with the male plasma composition clearly becoming "feminised". Male plasma was found to contain a higher level of fibrinogens, WAP63 and ependymin-2-like protein, which are involved in coagulation, inflammation and regeneration. For the first time, we demonstrated that expression of C1q subunit B (C1Q), an initiating factor of the classical complement pathway, was higher in males and was suppressed in both sexes in response to EE2 and bacterial challenge. Moreover, cleavage and post-translational modification of C3, the central component of the complement system, could be altered by EE2 treatment in males (C3dg down; C3g up). Multiple regression analysis indicated that C1Q is possibly an indicator of fish survival, which warrants further confirmation. The findings support the potential application of plasma immune proteins for prognosis/diagnosis of fish immune competence. Moreover, this study provides the first biochemical basis of the sex-differences in fish immunity and how these differences might be modified by xenoestrogens. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Complement Constructions in English: Fairly Difficult for EFL Language Learners

ERIC Educational Resources Information Center

Fazeli, Fatemeh; Shokrpour, Nasrin

2012-01-01

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

An 'instant gene bank' method for gene cloning by mutant complementation.

PubMed

Gems, D; Aleksenko, A; Belenky, L; Robertson, S; Ramsden, M; Vinetski, Y; Clutterbuck, A J

1994-02-01

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid'). Transformant colonies appear as the result of the joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this "instant gene bank" technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.

Human SAP is a novel peptidoglycan recognition protein that induces complement- independent phagocytosis of Staphylococcus aureus

PubMed Central

An, Jang-Hyun; Kurokawa, Kenji; Jung, Dong-Jun; Kim, Min-Jung; Kim, Chan-Hee; Fujimoto, Yukari; Fukase, Koichi; Coggeshall, K. Mark; Lee, Bok Luel

2014-01-01

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin (MBL) binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer, a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an Fcγ receptor-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain (NOD)-like receptors. PMID:23966633

Complement fixation test to C burnetii

MedlinePlus

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

Fanconi Anemia complementation group C protein in metabolic disorders.

PubMed

Nepal, Manoj; Ma, Chi; Xie, Guoxiang; Jia, Wei; Fei, Peiwen

2018-06-21

Given importance of 22-Fanconi Anemia (FA) proteins together to act in a signaling pathway in preventing deleterious clinical symptoms, e.g. severe bone marrow failure, congenital defects, an early onset of aging and cancer, studies on each FA protein become increasingly attractive. However, an unbiased and systematic investigation of cellular effects resulting from each FA protein is missing. Here, we report roles of FA complementation C group protein (FANCC) in the protection from metabolic disorders. This study was prompted by the diabetes-prone feature displayed in FANCC knockout mice, which is not typically shown in patients with FA. We found that in cells expressing FANCC at different levels, there are representative alterations in metabolites associated with aging (glycine, citrulline, ornithine, L-asparagine, L-tyrosine, L-arginine, L-glutamine, L-leucine, L-isoleucine, L-valine, L-proline and L-alanine), Diabetes Mellitus (DM) (carbon monoxide, collagens, fatty acids, D-glucose, fumaric acid, 2-oxoglutaric acid, C3), inflammation (inosine, L-arginine, L-isoleucine, L-leucine, L-lysine, L-phenylalanine, hypoxanthine, L-methionine), and cancer ( L-methionine, sphingomyelin, acetyl-L-carnitine, L-aspartic acid, L-glutamic acid, niacinamide, phospho-rylethanolamine). We also found that FANCC can act in an FA-pathway-independent manner in tumor suppression. Collectively, featured-metabolic alterations are readouts of functional mechanisms underlying reduced tumorigenicity driven by FANCC, demonstrating close links among cancer, aging, inflammation and DM.

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

PubMed Central

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

2011-01-01

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

Study of concentration-dependent cobalt ion doping of TiO2 and TiO(2-x)Nx at the nanoscale.

PubMed

Gole, James L; Prokes, Sharka M; Glembocki, O J; Wang, Junwei; Qiu, Xiaofeng; Burda, Clemens

2010-07-01

Experiments with a porous sol-gel generated TiO(2) nanocolloid and its corresponding oxynitride TiO(2-x)N(x) are carried out to evaluate those transformations which accompany additional doping with transition metals. In this study, doping with cobalt (Co(ii)) ions is evaluated using a combination of core level and VB-photoelectron and optical spectroscopy, complementing data obtained from Raman spectroscopy. Raman spectroscopy suggests that cobalt doping of porous sol-gel generated anatase TiO(2) and nitridated TiO(2-x)N(x) introduces a spinel-like structure into the TiO(2) and TiO(2-x)N(x) lattices. TEM and XPS data complemented by valence band-photoelectron spectra demonstrate that metallic cobalt clusters are not formed even at high doping levels. As evidenced by Raman spectroscopy, the creation of a spinel-like structure is commensurate with the room temperature conversion of the oxide and its oxynitride from the anatase to the rutile form. The onset of this kinetically driven process correlates with the formation of spinel sites within the TiO(2) and TiO(2-x)N(x) particles. Despite their visible light absorption, the photocatalytic activity of these cobalt seeded systems is diminished relative to the oxynitride TiO(2-x)N(x).

Treatment of 9L gliosarcoma in rats by ferrociphenol-loaded lipid nanocapsules based on a passive targeting strategy via the EPR effect.

PubMed

Huynh, Ngoc Trinh; Morille, Marie; Bejaud, Jerome; Legras, Pierre; Vessieres, Anne; Jaouen, Gerard; Benoit, Jean-Pierre; Passirani, Catherine

2011-12-01

To study a passive targeting strategy, via the enhanced permeability and retention effect following systemic administration of lipid nanocapsules (LNCs) loaded with ferrociphenol, FcdiOH. Long chains of polyethylene glycol (DSPE-mPEG2000) were incorporated onto the surface of LNCs by post-insertion technique. Stealth properties of LNCs were investigated by in vitro complement consumption and macrophage uptake, and in vivo pharmacokinetics in healthy rats. Antitumour effect of FcdiOH-loaded LNCs was evaluated in subcutaneous and intracranial 9L gliosarcoma rat models. LNCs and DSPE-mPEG2000-LNCs presented low complement activation and weak macrophage uptake. DSPE-mPEG2000-LNCs exhibited prolonged half-life and extended area under the curve in healthy rats. In a subcutaneous gliosarcoma model, a single intravenous injection of FcdiOH-LNCs (400 μL, 2.4 mg/rat) considerably inhibited tumour growth when compared to the control. DSPE-mPEG2000-FcdiOH-LNCs exhibited a strong antitumour effect by nearly eradicating the tumour by the end of the study. In intracranial gliosarcoma model, treatment with DSPE-mPEG2000-FcdiOH-LNCs and FcdiOH-LNCs statistically improved median survival time (28 and 27.5 days, respectively) compared to the control (25 days). These results demonstrate the interesting perspectives for the systemic treatment of glioma thanks to bio-organometallic chemotherapy via lipid nanocapsules.

Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes

PubMed Central

Fernie-King, Barbara A; Seilly, David J; Willers, Christine; Würzner, Reinhard; Davies, Alexandra; Lachmann, Peter J

2001-01-01

Streptococcal inhibitor of complement (SIC) was first described in 1996 as a putative inhibitor of the membrane attack complex of complement (MAC). SIC is a 31 000 MW protein secreted in large quantities by the virulent Streptococcus pyogenes strains M1 and M57, and is encoded by a gene which is extremely variable. In order to study further the interactions of SIC with the MAC, we have made a recombinant form of SIC (rSIC) in Escherichia coli and purified native M1 SIC which was used to raise a polyclonal antibody. SIC prevented reactive lysis of guinea pig erythrocytes by the MAC at a stage prior to C5b67 complexes binding to cell membranes, presumably by blocking the transiently expressed membrane insertion site on C7. The ability of SIC and clusterin (another putative fluid phase complement inhibitor) to inhibit complement lysis was compared, and found to be equally efficient. In parallel, by enzyme-linked immunosorbent assay both SIC and rSIC bound strongly to C5b67 and C5b678 complexes and to a lesser extent C5b-9, but only weakly to individual complement components. The implications of these data for virulence of SIC-positive streptococci are discussed, in light of the fact that Gram-positive organisms are already protected against complement lysis by the presence of their peptidoglycan cell walls. We speculate that MAC inhibition may not be the sole function of SIC. PMID:11454069

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

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