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Sample records for hemolytic complement activity

  1. Direct evidence of complement activation in HELLP syndrome: A link to atypical hemolytic uremic syndrome.

    PubMed

    Vaught, Arthur J; Gavriilaki, Eleni; Hueppchen, Nancy; Blakemore, Karin; Yuan, Xuan; Seifert, Sara M; York, Sarah; Brodsky, Robert A

    2016-05-01

    HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) is a severe variant of pre-eclampsia whose pathogenesis remains unclear. Recent evidence and clinical similarities suggest a link to atypical hemolytic uremic syndrome, a disease of excessive activation of the alternative complement pathway effectively treated with a complement inhibitor, eculizumab. Therefore, we used a functional complement assay, the modified Ham test, to analyze sera of women with classic or atypical HELLP syndrome, pre-eclampsia with severe features, normal pregnancies, and healthy nonpregnant women. Sera were also evaluated using levels of the terminal product of complement activation (C5b-9). We tested the in vitro ability of eculizumab to inhibit complement activation in HELLP serum. Increased complement activation was observed in participants with classic or atypical HELLP compared with those with normal pregnancies and nonpregnant controls. Mixing HELLP serum with eculizumab-containing serum resulted in a significant decrease in cell killing compared with HELLP serum alone. We found that HELLP syndrome is associated with increased complement activation as assessed with the modified Ham test. This assay may aid in the diagnosis of HELLP syndrome and could confirm that its pathophysiology is related to that of atypical hemolytic uremic syndrome.

  2. Role of Complement in Autoimmune Hemolytic Anemia

    PubMed Central

    Berentsen, Sigbjørn

    2015-01-01

    Summary The classification of autoimmune hemolytic anemias and the complement system are reviewed. In autoimmune hemolytic anemia of the warm antibody type, complement-mediated cell lysis is clinically relevant in a proportion of the patients but is hardly essential for hemolysis in most patients. Cold antibody-mediated autoimmune hemolytic anemias (primary cold agglutinin disease, secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria) are entirely complement-mediated disorders. In cold agglutinin disease, efficient therapies have been developed in order to target the pathogenic B-cell clone, but complement modulation remains promising in some clinical situations. No established therapy exists for secondary cold agglutinin syndrome and paroxysmal cold hemoglobinuria, and the possibility of therapeutic complement inhibition is interesting. Currently, complement modulation is not clinically documented in any autoimmune hemolytic anemia. The most relevant candidate drugs and possible target levels of action are discussed. PMID:26696798

  3. Role of Complement in Autoimmune Hemolytic Anemia.

    PubMed

    Berentsen, Sigbjørn

    2015-09-01

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

  4. Shiga Toxin Promotes Podocyte Injury in Experimental Hemolytic Uremic Syndrome via Activation of the Alternative Pathway of Complement

    PubMed Central

    Locatelli, Monica; Buelli, Simona; Pezzotta, Anna; Corna, Daniela; Perico, Luca; Tomasoni, Susanna; Rottoli, Daniela; Rizzo, Paola; Conti, Debora; Thurman, Joshua M.; Remuzzi, Giuseppe; Zoja, Carlamaria

    2014-01-01

    Shiga toxin (Stx)–producing Escherichia coli is the offending agent of postdiarrhea-associated hemolytic uremic syndrome (HUS), a disorder of glomerular ischemic damage and widespread microvascular thrombosis. We previously documented that Stx induces glomerular complement activation, generating C3a responsible for microvascular thrombosis in experimental HUS. Here, we show that the presence of C3 deposits on podocytes is associated with podocyte damage and loss in HUS mice generated by the coinjection of Stx2 and LPS. Because podocyte adhesion to the glomerular basement membrane is mediated by integrins, the relevance of integrin-linked kinase (ILK) signals in podocyte dysfunction was evaluated. Podocyte expression of ILK increased after the injection of Stx2/LPS and preceded the upregulation of Snail and downregulation of nephrin and α-actinin-4. Factor B deficiency or pretreatment with an inhibitory antibody to factor B protected mice against Stx2/LPS-induced podocyte dysregulation. Similarly, pretreatment with a C3a receptor antagonist limited podocyte loss and changes in ILK, Snail, and α-actinin-4 expression. In cultured podocytes, treatment with C3a reduced α-actinin-4 expression and promoted ILK-dependent nuclear expression of Snail and cell motility. These results suggest that Stx-induced activation of the alternative pathway of complement and generation of C3a promotes ILK signaling, leading to podocyte dysfunction and loss in Stx-HUS. PMID:24578132

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

    PubMed

    Risitano, Antonio M; Marotta, Serena

    2016-06-01

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

  6. Complements Spurned: Our Experience with Atypical Hemolytic Uremic Syndrome

    PubMed Central

    Nagar, Vidya S.; Chaterjee, Rudrarpan; Sood, Ankita; Sajjan, Basavaraj; Kaushik, Aniruddha; Vyahalkar, Sameer V.

    2017-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare disorder resulting from a dysregulated activation of the alternative pathway of the complement system. It results in significant morbidity and mortality if not diagnosed and treated promptly. It lends itself to myriad renal and extrarenal manifestations, all potentially disabling. Eculizumab, a monoclonal antibody to complement C5 is now the widely accepted norm for treatment. However, in resource-limited settings, plasma exchange if instituted early may be as beneficial. We report a case of aHUS treated with extended plasma exchange with excellent results. Critical care monitoring is essential for the management of the disease in view of a tendency to develop multiple complications. Long-term immunosuppression may be successful in maintaining remission. PMID:28250608

  7. PASylated Coversin, a C5-Specific Complement Inhibitor with Extended Pharmacokinetics, Shows Enhanced Anti-Hemolytic Activity in Vitro.

    PubMed

    Kuhn, Nadine; Schmidt, Christoph Q; Schlapschy, Martin; Skerra, Arne

    2016-10-19

    The Ornithodoros moubata Complement Inhibitor (OmCI) binds complement component 5 (C5) with high affinity and, thus, selectively prevents proteolytic activation of the terminal lytic complement pathway. A recombinant version of OmCI (also known as Coversin and rEV576) has proven efficacious in several animal models of complement-mediated diseases and successfully completed a phase Ia clinical trial. Coversin is a small 17 kDa lipocalin protein which has a very short plasma half-life if not bound to C5; therefore, the drug requires frequent dosing. We have improved the pharmacokinetics of Coversin by N-terminal translational conjugation with a 600 residue polypeptide composed of Pro, Ala, and Ser (PAS) residues. To this end, PAS-Coversin as well as the unmodified Coversin were functionally expressed in the cytoplasm of E. coli and purified to homogeneity. Both versions showed identical affinity to human C5, as determined by surface plasmon resonance measurements, and revealed similar complement inhibitory activity, as measured in ELISAs with human serum. In line with the PEG-like biophysical properties, PASylation dramatically prolonged the plasma half-life of uncomplexed Coversin by a factor ≥50 in mice. In a clinically relevant in vitro model of the complement-mediated disease paroxysmal nocturnal hemoglobinuria (PNH) both versions of Coversin effectively reduced erythrocyte lysis. Unexpectedly, while the IC50 values were comparable, PAS-Coversin reached a substantially lower plateau of residual lysis at saturating inhibitor concentrations. Taken together, our data demonstrate two clinically relevant improvements of PASylated Coversin: markedly increased plasma half-life and considerably reduced background hemolysis of erythrocytes with PNH-induced phenotype.

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

    PubMed Central

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

    2008-01-01

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

  9. Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome

    PubMed Central

    de Jorge, Elena Goicoechea; Harris, Claire L.; Esparza-Gordillo, Jorge; Carreras, Luis; Arranz, Elena Aller; Garrido, Cynthia Abarrategui; López-Trascasa, Margarita; Sánchez-Corral, Pilar; Morgan, B. Paul; de Córdoba, Santiago Rodríguez

    2007-01-01

    Hemolytic uremic syndrome (HUS) is an important cause of acute renal failure in children. Mutations in one or more genes encoding complement-regulatory proteins have been reported in approximately one-third of nondiarrheal, atypical HUS (aHUS) patients, suggesting a defect in the protection of cell surfaces against complement activation in susceptible individuals. Here, we identified a subgroup of aHUS patients showing persistent activation of the complement alternative pathway and found within this subgroup two families with mutations in the gene encoding factor B (BF), a zymogen that carries the catalytic site of the complement alternative pathway convertase (C3bBb). Functional analyses demonstrated that F286L and K323E aHUS-associated BF mutations are gain-of-function mutations that result in enhanced formation of the C3bBb convertase or increased resistance to inactivation by complement regulators. These data expand our understanding of the genetic factors conferring predisposition to aHUS, demonstrate the critical role of the alternative complement pathway in the pathogenesis of aHUS, and provide support for the use of complement-inhibition therapies to prevent or reduce tissue damage caused by dysregulated complement activation. PMID:17182750

  10. Complement deposition in autoimmune hemolytic anemia is a footprint for difficult-to-detect IgM autoantibodies.

    PubMed

    Meulenbroek, Elisabeth M; de Haas, Masja; Brouwer, Conny; Folman, Claudia; Zeerleder, Sacha S; Wouters, Diana

    2015-11-01

    In autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal hemolytic anemia. Depending on whether IgG or IgM antibodies are involved, response to therapy is different. Proper identification of the isotype of the anti-erythrocyte autoantibodies is, therefore, crucial. However, detection of IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of complement C3 on a patient's erythrocytes could be used for indirect detection of anti-erythrocyte IgM. To this end, we fractionated patients' sera by size exclusion chromatography and tested which fractions yielded complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an IgM anti-erythrocyte component that could activate complement, even if no such autoantibody had been detected with any other test. This also included all tested patients with only IgG and C3 on their erythrocytes, who would previously have been classified as having an IgG-only mediated autoimmune hemolytic anemia. Depleting patients' sera of either IgG or IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in autoimmune hemolytic anemia is mostly IgM-mediated and the presence of covalent C3 on patients' erythrocytes can be taken as a footprint of the presence of anti-erythrocyte IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy.

  11. Alternative Pathway of Complement in Children with Diarrhea-Associated Hemolytic Uremic Syndrome

    PubMed Central

    Thurman, Joshua M.; Marians, Russell; Emlen, Woodruff; Wood, Susan; Smith, Christopher; Akana, Hillary; Holers, V. Michael; Lesser, Martin; Kline, Myriam; Hoffman, Cathy; Christen, Erica

    2009-01-01

    Background and objectives: Diarrhea-associated hemolytic uremic syndrome (D+HUS) is a common cause of acute kidney injury in children. Mutations in alternative pathway (AP) complement regulatory proteins have been identified in severe cases of thrombotic microangiopathy, but the role of the AP in D+HUS has not been studied. Therefore, we determined whether plasma levels of markers of activation of the AP are increased in D+HUS and are biomarkers of the severity of renal injury that predict the need for dialysis. Design, setting, participants, & measurements: Patients were randomly selected from among participants in the HUS-SYNSORB Pk trial. Plasma samples were collected on days 1, 4, 7, and 10 after enrollment and day 28 after discharge from the hospital. Levels of two complement pathway products, Bb and SC5b-9, were determined by ELISA. Results: Seventeen children (6 boys and 11 girls; age, 5.4 ± 3.5 yr) were studied. Eight (47%) required dialysis support, and two had serious extrarenal events. On the day of enrollment, plasma levels of Bb and SC5b-9 were significantly increased in all patients compared with healthy controls (P < 0.01). The elevated concentrations normalized by day 28 after discharge. Circulating levels of complement pathway fragments did not correlate with severity of renal injury or occurrence of complications. Conclusions: Patients with acute-onset D+HUS manifest activation of the AP of complement that is temporally related to the onset of disease and that resolves within 1 mo. Therapies to inhibit the AP of complement may be useful in attenuating the severity of renal injury and extrarenal complications. PMID:19820137

  12. Red Blood Cell Destruction in Autoimmune Hemolytic Anemia: Role of Complement and Potential New Targets for Therapy

    PubMed Central

    Berentsen, Sigbjørn

    2015-01-01

    Autoimmune hemolytic anemia (AIHA) is a collective term for several diseases characterized by autoantibody-initiated destruction of red blood cells (RBCs). Exact subclassification is essential. We provide a review of the respective types of AIHA with emphasis on mechanisms of RBC destruction, focusing in particular on complement involvement. Complement activation plays a definitive but limited role in warm-antibody AIHA (w-AIHA), whereas primary cold agglutinin disease (CAD), secondary cold agglutinin syndrome (CAS), and paroxysmal cold hemoglobinuria (PCH) are entirely complement-dependent disorders. The details of complement involvement differ among these subtypes. The theoretical background for therapeutic complement inhibition in selected patients is very strong in CAD, CAS, and PCH but more limited in w-AIHA. The optimal target complement component for inhibition is assumed to be important and highly dependent on the type of AIHA. Complement modulation is currently not an evidence-based therapy modality in any AIHA, but a number of experimental and preclinical studies are in progress and a few clinical observations have been reported. Clinical studies of new complement inhibitors are probably not far ahead. PMID:25705656

  13. Red blood cell destruction in autoimmune hemolytic anemia: role of complement and potential new targets for therapy.

    PubMed

    Berentsen, Sigbjørn; Sundic, Tatjana

    2015-01-01

    Autoimmune hemolytic anemia (AIHA) is a collective term for several diseases characterized by autoantibody-initiated destruction of red blood cells (RBCs). Exact subclassification is essential. We provide a review of the respective types of AIHA with emphasis on mechanisms of RBC destruction, focusing in particular on complement involvement. Complement activation plays a definitive but limited role in warm-antibody AIHA (w-AIHA), whereas primary cold agglutinin disease (CAD), secondary cold agglutinin syndrome (CAS), and paroxysmal cold hemoglobinuria (PCH) are entirely complement-dependent disorders. The details of complement involvement differ among these subtypes. The theoretical background for therapeutic complement inhibition in selected patients is very strong in CAD, CAS, and PCH but more limited in w-AIHA. The optimal target complement component for inhibition is assumed to be important and highly dependent on the type of AIHA. Complement modulation is currently not an evidence-based therapy modality in any AIHA, but a number of experimental and preclinical studies are in progress and a few clinical observations have been reported. Clinical studies of new complement inhibitors are probably not far ahead.

  14. Mount St. Helens' volcanic ash: hemolytic activity.

    PubMed

    Vallyathan, V; Mentnech, M S; Stettler, L E; Dollberg, D D; Green, F H

    1983-04-01

    Volcanic ash samples from four Mount St. Helens' volcanic eruptions were subjected to mineralogical, analytical, and hemolytic studies in order to evaluate their potential for cytotoxicity and fibrogenicity. Plagioclase minerals constituted the major component of the ash with free crystalline silica concentrations ranging from 1.5 to 7.2%. The in vitro hemolytic activity of the volcanic ash was compared to similar concentrations of cytotoxic and inert minerals. The ash was markedly hemolytic, exhibiting an activity similar to chrysotile asbestos, a known fibrogenic agent. The hemolysis of the different ash samples varied with particle size but not with crystalline silica concentration. The results of these studies taken in conjunction with the results of our animal studies indicate a fibrogenic potential of volcanic ash in heavily exposed humans.

  15. Complement Factor B Mutations in Atypical Hemolytic Uremic Syndrome—Disease-Relevant or Benign?

    PubMed Central

    Marinozzi, Maria Chiara; Vergoz, Laura; Rybkine, Tania; Ngo, Stephanie; Bettoni, Serena; Pashov, Anastas; Cayla, Mathieu; Tabarin, Fanny; Jablonski, Mathieu; Hue, Christophe; Smith, Richard J.; Noris, Marina; Halbwachs-Mecarelli, Lise; Donadelli, Roberta; Fremeaux-Bacchi, Veronique

    2014-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association. PMID:24652797

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

    PubMed

    Martinez, R D

    1978-01-01

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

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

    PubMed Central

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

    1980-01-01

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

  18. Physicochemical signatures of nanoparticle-dependent complement activation

    SciTech Connect

    Thomas, Dennis G.; Chikkagoudar, Satish; Heredia-Langner, Alejandro; Tardiff, Mark F.; Xu, Zhixiang; Hourcade, Dennis; Pham, Christine; Lanza, Gregory M.; Weinberger, Kilian Q.; Baker, Nathan A.

    2014-03-21

    Nanoparticles are potentially powerful therapeutic tools that have the capacity to target drug payloads and imaging agents. However, some nanoparticles can activate complement, a branch of the innate immune system, and cause adverse side-effects. Recently, we developed an in vitro hemolytic assay protocol for measuring the nanoparticle-dependent complement activity of serum samples and applied this protocol to several nanoparticle formulations that differed in size, surface charge, and surface chemistry; quantifying the nanoparticle-dependent complement activity using a metric called Residual Hemolytic Activity (RHA). In the present work, we have used a decision tree learning algorithm to derive the rules for estimating nanoparticle-dependent complement response based on the data generated from the hemolytic assay studies. Our results indicate that physicochemical properties of nanoparticles, namely, size, polydispersity index, zeta potential, and mole percentage of the active surface ligand of a nanoparticle, can serve as good descriptors for prediction of nanoparticle-dependent complement activation in the decision tree modeling framework. The robustness and predictability of the model can be improved by training the model with additional data points that are uniformly distributed in the RHA/physicochemical descriptor space and by incorporating instability effects on nanoparticle physicochemical properties into the model.

  19. Minor Role of Plasminogen in Complement Activation on Cell Surfaces

    PubMed Central

    Hyvärinen, Satu; Jokiranta, T. Sakari

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation. PMID:26637181

  20. Complement Activation Alters Platelet Function

    DTIC Science & Technology

    2013-10-01

    mice and mice transfused with Syk inhibitor-treated platelets . Platelet lodging was remarkably decreased in lungs of mice transfused with Syk...AD_________________ Award Number: W81XWH-12-1-0523 TITLE: Complement Activation Alters Platelet ...30September2012–29September2013 4. TITLE AND SUBTITLE Complement Activation Alters Platelet Function 5a. CONTRACT NUMBER W81XWH-12-1-0523 5b. GRANT NUMBER

  1. Hemolytic Activities of the Candida Species in Liquid Medium

    PubMed Central

    Malcok, Hilal Kuzucu; Aktas, Esin; Ayyildiz, Ahmet; Yigit, Nimet; Yazgi, Halil

    2009-01-01

    Objective The aim of this study was to evaluate the in vitro hemolytic activities of 107 Candida strains isolated from different clinical samples in liquid medium, and to examine the impact of glucose on this activity. Materials and Methods A total of 107 Candida isolates representing seven species (C. albicans, n=28; C. glabrata, n=23; C. tropicalis, n=17; C. parapsilosis, n=16; C. kefyr, n=14; C. krusei, n=5; C. guilliermondii, n=4) were included in the study. The hemolytic activities of the strains were tested on two different Sabouraud dextrose liquid media (SDB) containing 7% defibrinated human blood, one of which is supplemented with 3% glucose and the other without glucose. Cultures were evaluated at the end of a 48-hour incubation. The hemolysis in the media was detected spectrophotometrically by measuring the amount of released hemoglobin and compared with a standard hemolysate which was prepared prior to testing. The degree of hemolysis (percentage value) by an individual strain was calculated according to the following formula below: (Absorbance of supernatant media at 540 nm / Absorbance of standard hemolysate at 540 nm X 100). Results In the liquid medium without glucose, strains generally produced hemolysis at low levels. The degree of hemolysis produced by all species increased noticeably in the liquid medium with glucose. Strains of C. albicans and C.kefyr had demonstrated significant hemolytic activity, whereas others had lower activity. C. parapsilosis exerted very little hemolytic activity in the medium with glucose and showed no activity in the medium without glucose. Conclusion The hemolytic activities of most Candida species was found to be higher in the human blood-enriched SDB medium containing 3% additive glucose than in the one free from additives. This result indicates that increased blood glucose concentration may contribute to increased hemolytic activity in Candida species, and it suggests a parallel with possible pathogenesis of

  2. Structural insights on complement activation.

    PubMed

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

    2015-10-01

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

  3. Methods for Quantitative Detection of Antibody-induced Complement Activation on Red Blood Cells

    PubMed Central

    Meulenbroek, Elisabeth M.; Wouters, Diana; Zeerleder, Sacha

    2014-01-01

    Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal1. Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis1-4. However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation. PMID:24514151

  4. Legionella pneumophila lipopolysaccharide activates the classical complement pathway.

    PubMed Central

    Mintz, C S; Schultz, D R; Arnold, P I; Johnson, W

    1992-01-01

    Legionella pneumophila is a gram-negative bacterium capable of entering and growing in alveolar macrophages and monocytes. Complement and complement receptors are important in the uptake of L. pneumophila by human mononuclear phagocytes. The surface molecules of L. pneumophila that activate the complement system are unknown. To identify these factors, we investigated the effects of L. pneumophila lipopolysaccharide (LPS) on the classical and alternative complement pathways of normal human serum by functional hemolytic assays. Although incubation of LPS in normal human serum at 37 degrees C resulted in the activation of both pathways, complement activation proceeded primarily through the classical pathway. Activation of the classical pathway by LPS was dependent on natural antibodies of the immunoglobulin M class that were present in various quantities in sera from different normal individuals but were absent in an immunoglobulin-deficient serum obtained from an agammaglobulinemic patient. Additional studies using sheep erythrocytes coated with LPS suggested that the antibodies recognized antigenic sites in the carbohydrate portion of LPS. The ability of LPS to interact with the complement system suggests a role for LPS in the uptake of L. pneumophila by mononuclear phagocytes. PMID:1612744

  5. A universal method for measuring functional activity of complement in humans, laboratory, domestic, and agricultural animals, amphibians, and birds.

    PubMed

    Kuleshina, O N; Kozlov, L V; Cheremnykh, E G

    2014-06-01

    A new universal method for measuring activity of the serum complement system in humans, laboratory, domestic, agricultural animals, birds and amphibians is based on automated evaluation of the mortality of ciliate Tetrahymena pyriformis under the effect of the complement system. In contrast to the hemolytic method, measured activity of the complement shows no erroneously high results caused by reactive lysis in febrile patients. The method can be used for studies of the complement system in humans and animals without species-specific adaptation.

  6. Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans

    PubMed Central

    Narayanavari, Suneel A.; Lourdault, Kristel; Sritharan, Manjula; Haake, David A.; Matsunaga, James

    2015-01-01

    . Complementation of the mutation with the sph2 gene partially restored production of hemolytic and sphingomyelinase activities. These results indicate that the sph2 gene product contributes to the hemolytic and sphingomyelinase activities secreted by L. interrogans and most likely dominates those functions under the culture condition tested. PMID:26274394

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

    PubMed Central

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

    1985-01-01

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

  8. Streptolysin S of Streptococcus anginosus exhibits broad-range hemolytic activity.

    PubMed

    Asam, Daniela; Mauerer, Stefanie; Spellerberg, Barbara

    2015-04-01

    Streptococcus anginosus is a commensal of mucous membranes and an emerging human pathogen. Some strains, including the type strain, display a prominent β-hemolytic phenotype. A gene cluster (sag), encoding a variant of streptolysin S (SLS) has recently been identified as the genetic background for β-hemolysin production in S. anginosus. In this study, we further characterized the hemolytic and cytolytic activity of the S. anginosus hemolysin in comparison with other streptococcal hemolysins. The results indicate that SLS of S. anginosus is a broad-range hemolysin able to lyse erythrocytes of different species, including horse, bovine, rabbit and even chicken. The hemolytic activity is temperature dependent, and a down-regulation of the hemolysin expression is induced in the presence of high glucose levels. Survival assays indicate that in contrast to other streptococcal species, S. anginosus does not require SLS for survival in the presence of human granulocytes. Cross-complementation studies using the sagB and sagD genes of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis demonstrated functional similarities to the S. anginosus SLS. Nevertheless, distinct differences to other streptolysin S variants were noted and provide further insights into the molecular mechanisms of SLS pathogen host interactions.

  9. Complement Activation and Inhibition in Wound Healing

    PubMed Central

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

    2012-01-01

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

  10. Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats

    PubMed Central

    Sharp, Julia A.; Hair, Pamela S.; Pallera, Haree K.; Kumar, Parvathi S.; Mauriello, Clifford T.; Nyalwidhe, Julius O.; Phelps, Cody A.; Park, Dalnam; Thielens, Nicole M.; Pascal, Stephen M.; Chen, Waldon; Duffy, Diane M.; Lattanzio, Frank A.; Cunnion, Kenji M.; Krishna, Neel K.

    2015-01-01

    The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases. PMID:26196285

  11. Complement System Part I – Molecular Mechanisms of Activation and Regulation

    PubMed Central

    Merle, Nicolas S.; Church, Sarah Elizabeth; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T.

    2015-01-01

    Complement is a complex innate immune surveillance system, playing a key role in defense against pathogens and in host homeostasis. The complement system is initiated by conformational changes in recognition molecular complexes upon sensing danger signals. The subsequent cascade of enzymatic reactions is tightly regulated to assure that complement is activated only at specific locations requiring defense against pathogens, thus avoiding host tissue damage. Here, we discuss the recent advances describing the molecular and structural basis of activation and regulation of the complement pathways and their implication on physiology and pathology. This article will review the mechanisms of activation of alternative, classical, and lectin pathways, the formation of C3 and C5 convertases, the action of anaphylatoxins, and the membrane-attack-complex. We will also discuss the importance of structure–function relationships using the example of atypical hemolytic uremic syndrome. Lastly, we will discuss the development and benefits of therapies using complement inhibitors. PMID:26082779

  12. Properties of the Hemolytic Activities of Escherichia coli

    PubMed Central

    Short, Everett C.; Kurtz, Harold J.

    1971-01-01

    Some properties of the cell-free and cell-associated hemolysins of Escherichia coli were studied. Several strains of E. coli that were isolated from intestines of pigs with edema disease produce large quantities of cell-free hemolysin when grown in the presence of an extract of meat. The component of meat that stimulates production of cell-free hemolysin is not extracted by lipid solvents and is not dialyzable. The cell-free hemolysin is an acidic substance that occurs in two forms. It is inactivated by trypsin but not by lecithinase, lysozyme, ribonuclease, or deoxyribonuclease, shows optimum activity between pH 7 and 8, and requires calcium ion for activity. It does not appear to be an enzyme. The kinetics of the lytic reaction are most consistent with the hypothesis that one molecule of cell-free hemolysin is sufficient to lyse one erythrocyte and that it is inactivated in the lytic reaction. The cell-free hemolysin does not sufficiently damage the cell during the prelytic period to cause lysis after the hemolysin-calcium-erythrocyte complex has been disrupted. The cell-associated hemolysin was not separated from the cell by autolysis, freezing, sonic treatment, or treatment with trypsin or lysozyme. It appears to be closely associated with the metabolic status of the cell. Organisms that are highly hemolytic under usual conditions of assay immediately lose most of their hemolytic capability in the presence of sodium cyanide, streptomycin, nalidixic acid, and rifampin. PMID:16558036

  13. Properdin in Complement Activation and Tissue Injury

    PubMed Central

    Lesher, AM; B, Nilsson; Song, W-C

    2013-01-01

    The plasma protein properdin is the only known positive regulator of complement activation. Although regarded as an initiator of the alternative pathway of complement activation at the time of its discovery more than a half century ago, the role and mechanism of action of properdin in the complement cascade has undergone significant conceptual evolution since then. Despite the long history of research on properdin, however, new insight and unexpected findings on the role of properdin in complement activation, pathogen infection and host tissue injury are still being revealed by ongoing investigations. In this article, we provide a brief review on recent studies that shed new light on properdin biology, focusing on the following three topics: 1) its role as a pattern recognition molecule to direct and trigger complement activation, 2) its context-dependent requirement in complement activation on foreign and host cell surfaces, and 3) its involvement in alternative pathway complement-mediated immune disorders and considerations of properdin as a potential therapeutic target in human diseases. PMID:23816404

  14. Hemolytic activity of venom from crown-of-thorns starfish Acanthaster planci spines

    PubMed Central

    2013-01-01

    Background The crown-of-thorns starfish Acanthaster planci is a venomous species from Taiwan whose venom provokes strong hemolytic activity. To understand the hemolytic properties of A. planci venom, samples were collected from A. planci spines in the Penghu Islands, dialyzed with distilled water, and lyophilized into A. planci spine venom (ASV) powder. Results Both crude venom and ASV cause 50% hemolysis at a concentration of 20 μg/mL. The highest hemolytic activity of ASV was measured at pH 7.0-7.4; ASV-dependent hemolysis was sharply reduced when the pH was lower than 3 or greater than 8. There was almost no hemolytic activity when the Cu2+ concentration was increased to 10 mM. Furthermore, incubation at 100°C for 30 to 60 minutes sharply decreased the hemolytic activity of ASV. After treatment with the protease α-chymotrypsin, the glycoside hydrolase cellulase, and the membrane component cholesterin, the hemolytic activity of ASV was significantly inhibited. Conclusions The results of this study provide fundamental information about A. planci spine venom. The hemolytic activity was affected by pH, temperature, metal ions, EDTA, cholesterin, proteases, and glycoside hydrolases. ASV hemolysis was inhibited by Cu2+, cholesterin, α-chymotrypsin, and cellulose, factors that might prevent the hemolytic activity of venom and provide the medical treatment for sting. PMID:24063308

  15. Activation of immune complement by fly ash particles from coal combustion. [Dogs

    SciTech Connect

    Hill, J.O.; Rothenberg, S.J.; Kanapilly, G.M.; Hanson, R.L.; Scott, B.R.

    1982-06-01

    The interaction of immune complement with fly ash particles from coal combustion was studied in vitro. Fly ash from different coal combustors was incubated for 1 hr with pooled normal dog serum at 37/sup 0/C. The serum supernatants were assayed for complement by a 505 hemolytic (CH/sub 50/) endpoint method. Ash produced by burning one type of coal activated complement with up to 70% of the complement activated at 10 mg ash/ml serum. This activation was concentration dependent and a linear dose-response curve was obtained. Heat treatment and surface area measurements, as well as immunofluorescence studies, suggest that the active component(s) is volatile or heat labile, found on the surface of the particles, and removed by saline or water extraction.

  16. Complement activation by a B cell superantigen.

    PubMed

    Kozlowski, L M; Soulika, A M; Silverman, G J; Lambris, J D; Levinson, A I

    1996-08-01

    Staphylococcal protein A (SpA), acting as a B cell superantigen, binds to the Fab region of human VH3+ Igs. Using SpA abrogated of its IgG Fc binding activity (Mod SpA) as a model B cell superantigen, we determined whether such an interaction causes complement activation. Addition of Mod SpA to human serum led to complement consumption and the generation of C3a. To determine whether this complement activation 1) was due to an interaction between VH3+ Igs and the Fab binding site of SpA and 2) proceeded via the classical complement pathway, we tested a panel of monoclonal IgM proteins for the ability to hind C1q following interaction with SpA. C1q binding was restricted to SpA-reactive, VH3+ IgM proteins. To formally determine whether the binding of SpA to the reactive VH3+ IgM proteins led to complement activation, we reconstituted the serum from a hypogammaglobulinemic patient with monoclonal IgM proteins and measured complement consumption and C3a generation following the addition of Mod SpA. We observed complement consumption and C3a production only in Mod SpA-treated serum reconstituted with a VH3+, SpA-binding, IgM protein. Taken together, these results provide compelling evidence that the interaction of the Fab binding site of SpA and VH3+ Igs can lead to complement activation via the classical pathway. This novel interaction may have significant implications for the in vivo properties of a B cell superantigen.

  17. Complement activation in chronic liver disease.

    PubMed Central

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

    1982-01-01

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

  18. Inherited complement regulatory protein deficiency predisposes to human disease in acute injury and chronic inflammatory statesthe examples of vascular damage in atypical hemolytic uremic syndrome and debris accumulation in age-related macular degeneration.

    PubMed

    Richards, Anna; Kavanagh, David; Atkinson, John P

    2007-01-01

    In this chapter, we examine the role of complement regulatory activity in atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration (AMD). These diseases are representative of two distinct types of complement-mediated injury, one being acute and self-limited, the other reflecting accumulation of chronic damage. Neither condition was previously thought to have a pathologic relationship to the immune system. However, alterations in complement regulatory protein genes have now been identified as major predisposing factors for the development of both diseases. In aHUS, heterozygous mutations leading to haploinsufficiency and function-altering polymorphisms in complement regulators have been identified, while in AMD, polymorphic haplotypes in complement genes are associated with development of disease. The basic premise is that a loss of function in a plasma or membrane inhibitor of the alternative complement pathway allows for excessive activation of complement on the endothelium of the kidney in aHUS and on retinal debris in AMD. These associations have much to teach us about the host's innate immune response to acute injury and to chronic debris deposition. We all experience cellular injury and, if we live long enough, will deposit debris in blood vessel walls (atherosclerosis leading to heart attacks and strokes), the brain (amyloid proteins leading to Alzheimer's disease), and retina (lipofuscin pigments leading to AMD). These are three common causes of morbidity and mortality in the developed world. The clinical, genetic, and immunopathologic understandings derived from the two examples of aHUS and AMD may illustrate what to anticipate in related conditions. They highlight how a powerful recognition and effector system, the alternative complement pathway, reacts to altered self. A response to acute injury or chronic debris accumulation must be appropriately balanced. In either case, too much activation or too little regulation promotes

  19. Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves.

    PubMed

    Yuldasheva, L N; Carvalho, E B; Catanho, M-T J A; Krasilnikov, O V

    2005-07-01

    Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.

  20. Scanning electron microscopy in the investigation of the in vitro hemolytic activity of Trichomonas vaginalis.

    PubMed

    Rosset, Iveli; Tasca, Tiana; Tessele, Paola M; De Carli, Geraldo A

    2002-04-01

    The in vitro hemolytic activity of Trichomonas vaginalis has been previously demonstrated, but the mechanisms involved remain to be elucidated. In this work we used scanning electron microscopy to investigate the contact dependency of the hemolytic phenomenon caused by the parasites. The erythrocytes adhered to the parasites' surface and were phagocytosed. These observations suggest that the contact between T. vaginalis and erythrocytes may be an important mechanism in the injury caused to the erythrocytes. The hemolytic activity of T. vaginalis may be an efficient means of obtaining nutrients for the parasite and allow the investigation of the mechanism used by T. vaginalis to damage cellular membranes.

  1. A steryl glycoside fraction with hemolytic activity from tubers of Momordica cochinchinensis.

    PubMed

    Ng, T B; Li, W W; Yeung, H W

    1986-10-01

    A hemolytic fraction has been obtained from fresh tubers of Momordica cochinchinensis. The fraction was strongly adsorbed on DEAE-Sepharose CL6B. It did not stain with Coomassie brilliant blue in SDS-polyacrylamide gel electrophoresis and it gave no immunoprecipitin arcs in immunoelectrophoresis. The hemolytic activity of the fraction was resistant to heat and proteolytic enzymes. The behavior of the fraction in thin-layer chromatography and its positive reaction in Liebermann-Burchard test indicated that the hemolytic activity of the fraction can be attributed to a steryl glycoside(s).

  2. Hemolytic and antimicrobial activities differ among saponin-rich extracts from guar, quillaja, yucca, and soybean.

    PubMed

    Hassan, Sherif M; Byrd, James A; Cartwright, Aubry L; Bailey, Chris A

    2010-10-01

    Hemolytic and antibacterial activities of eight serial concentrations ranged from 5-666 microg/mL of saponin-rich extracts from guar meal (GM), quillaja, yucca, and soybean were tested in 96-well plates and read by enzyme-linked immunosorbent assay plate-well as 650 nm. Hemolytic assay used a 1% suspension of chicken red blood cells with water and phosphate buffered saline as positive and negative controls, respectively. Antibacterial activity against Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli were evaluated using ampicillin and bacteria without saponin-rich extract as positive and negative controls, respectively. The 100% MeOH GM and commercial quillaja saponin-rich extracts were significantly the highest in both hemolytic and antibacterial activities against all bacteria at the same concentration tested. Soybean saponin-rich extract had no antibacterial activity against any of the bacteria at the concentrations tested while yucca saponin-rich extract had no antibacterial activity against the gram-negative bacteria at the concentrations tested. GM and quillaja saponin-rich extracts were hemolytic, while yucca and soybean saponin-rich extracts were not hemolytic at the concentrations tested. No saponin-rich extract source had antibacterial activity against S. typhimurium or E. coli at the concentrations tested. Both GM and quillaja saponin-rich extracts exhibited antibacterial activity against S. aureus. Saponin-rich extracts from different plant sources have different hemolytic and antibacterial activities.

  3. Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation.

    PubMed

    Harder, Markus J; Kuhn, Nadine; Schrezenmeier, Hubert; Höchsmann, Britta; von Zabern, Inge; Weinstock, Christof; Simmet, Thomas; Ricklin, Daniel; Lambris, John D; Skerra, Arne; Anliker, Markus; Schmidt, Christoph Q

    2017-02-23

    Eculizumab inhibits the terminal, lytic pathway of complement by blocking the activation of the complement protein C5 and shows remarkable clinical benefits in certain complement-mediated diseases. However, several reports suggest that activation of C5 is not always completely suppressed in patients even under excess of eculizumab over C5, indicating that residual C5 activity may derogate the drug's therapeutic benefit under certain conditions. By using eculizumab and the tick-derived C5 inhibitor coversin, we determined conditions ex vivo in which C5 inhibition is incomplete. The degree of such residual lytic activity depended on the strength of the complement activator and the resulting surface density of the complement activation product C3b, which autoamplifies via the alternative pathway (AP) amplification loop. We show that at high C3b densities required for binding and activation of C5, both inhibitors reduce but do not abolish this interaction. The decrease of C5 binding to C3b clusters in the presence of C5 inhibitors correlated with the levels of residual hemolysis. However, by employing different C5 inhibitors simultaneously, residual hemolytic activity could be abolished. The importance of AP-produced C3b clusters for C5 activation in the presence of eculizumab was corroborated by the finding that residual hemolysis after forceful activation of the classical pathway could be reduced by blocking the AP. By providing insights into C5 activation and inhibition, our study delivers the rationale for the clinically observed phenomenon of residual terminal pathway activity under eculizumab treatment with important implications for anti-C5 therapy in general.

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

    PubMed

    Arroyave, C M; Tan, E M

    1976-01-01

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

  5. Effects of penicillinase on bactericidal and complement activities in normal human serum.

    PubMed Central

    Biggs, W H; Wunderlich, A C; Corbeil, L C; Davis, C E; Curd, J G

    1983-01-01

    During routine addition of penicillinase (beta-lactamase) to patients sera, we found that the capacity of some of these sera to kill serum-sensitive gram-negative organisms was significantly decreased. Further controlled studies showed that penicillinase decreased both the bactericidal activity of normal human sera and the total hemolytic activity (CH50) of complement in these sera. The decreased bactericidal activity correlated significantly (r = 0.57, P less than 0.05) with the reduction of CH50 in eight normal sera. These effects of penicillinase were time and temperature dependent. Measurement of individual complement component activities showed that penicillinase decreased the activity of C2, C4, and C3-C9, suggesting that the penicillinase preparation activated the classical pathway. These results cast doubts on the validity of bactericidal determinations when sera are pretreated with penicillinase. PMID:6603195

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

    PubMed

    Tichaczek-Goska, Dorota

    2012-01-01

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

  7. Characterization of serum complement activity of saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles.

    PubMed

    Merchant, Mark; Britton, Adam

    2006-04-01

    We employed a spectroscopic assay, based on the hemolysis of sheep red blood cells (SRBCs), to assess the innate immune function of saltwater and freshwater crocodiles in vitro. Incubation of serum from freshwater and saltwater crocodiles with SRBCs resulted in concentration-dependent increases in SRBC hemolysis. The hemolytic activity occurred rapidly, with detectable activity within 2 min and maximum activity at 20 min. These activities, in both crocodilian species, were heat sensitive, unaffected by 20 mM methylamine, and completely inhibited by low concentrations of EDTA, suggesting that the alternative serum complement cascade is responsible for the observed effects. The hemolytic activities of the sera were inhibited by other chelators of divalent metal ions, such as phosphate and citrate. The inhibition of SRBC hemolysis by EDTA could be completely restored by the addition of 10 mM Ca2+ or Mg2+, but not Ba2+, Cu2+ or Fe2+, indicating specificity for these metal ions. The serum complement activities of both crocodilians were temperature-dependent, with peak activities occurring at 25-30 degrees C and reduced activities below 25 degrees C and above 35 degrees C.

  8. Novel hemagglutinating, hemolytic and cytotoxic activities of the intermediate subunit of Entamoeba histolytica lectin.

    PubMed

    Kato, Kentaro; Yahata, Kazuhide; Gopal Dhoubhadel, Bhim; Fujii, Yoshito; Tachibana, Hiroshi

    2015-09-10

    Galactose and N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable lectin of Entamoeba histolytica, a common protozoan parasite, has roles in pathogenicity and induction of protective immunity in mouse models of amoebiasis. The lectin consists of heavy (Hgl), light (Lgl), and intermediate (Igl) subunits. Hgl has lectin activity and Lgl does not, but little is known about the activity of Igl. In this study, we assessed various regions of Igl for hemagglutinating activity using recombinant proteins expressed in Escherichia coli. We identified a weak hemagglutinating activity of the protein. Furthermore, we found novel hemolytic and cytotoxic activities of the lectin, which resided in the carboxy-terminal region of the protein. Antibodies against Igl inhibited the hemolytic activity of Entamoeba histolytica trophozoites. This is the first report showing hemagglutinating, hemolytic and cytotoxic activities of an amoebic molecule, Igl.

  9. Complement

    MedlinePlus

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

  10. Complement factor H, FHR-3 and FHR-1 variants associate in an extended haplotype conferring increased risk of atypical hemolytic uremic syndrome.

    PubMed

    Bernabéu-Herrero, Maria E; Jiménez-Alcázar, Miguel; Anter, Jaouad; Pinto, Sheila; Sánchez Chinchilla, Daniel; Garrido, Sofía; López-Trascasa, Margarita; Rodríguez de Córdoba, Santiago; Sánchez-Corral, Pilar

    2015-10-01

    Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy affecting the renal microvasculature and is associated with complement dysregulation caused by mutations or autoantibodies. Disease penetrance and severity is modulated by inheritance of "risk" polymorphisms in the complement genes MCP, CFH and CFHR1. We describe the prevalence of mutations, the frequency of risk polymorphisms and the occurrence of anti-FH autoantibodies in a Spanish aHUS cohort (n=367). We also report the identification of a polymorphism in CFHR3 (c.721C>T; rs379370) that is associated with increased risk of aHUS (OR=1.78; CI 1.22-2.59; p=0.002), and is most frequently included in an extended risk haplotype spanning the CFH-CFHR3-CFHR1 genes. This extended haplotype integrates polymorphisms in the promoter region of CFH and CFHR3, and is associated with poorer evolution of renal function and decreased FH levels. The CFH-CFHR3-CFHR1 aHUS-risk haplotype seems to be the same as was previously associated with protection against meningococcal infections, suggesting that the genetic variability in this region is limited to a few extended haplotypes, each with opposite effects in various human diseases. These results suggest that the combination of quantitative and qualitative variations in the complement proteins encoded by CFH, CFHR3 and CFHR1 genes is key for the association of these haplotypes with disease.

  11. Lipid selectivity in novel antimicrobial peptides: Implication on antimicrobial and hemolytic activity.

    PubMed

    Maturana, P; Martinez, M; Noguera, M E; Santos, N C; Disalvo, E A; Semorile, L; Maffia, P C; Hollmann, A

    2017-05-01

    Antimicrobial peptides (AMPs) are small cationic molecules that display antimicrobial activity against a wide range of bacteria, fungi and viruses. For an AMP to be considered as a therapeutic option, it must have not only potent antibacterial properties but also low hemolytic and cytotoxic activities [1]. Even though many studies have been conducted in order to correlate the antimicrobial activity with affinity toward model lipid membranes, the use of these membranes to explain cytotoxic effects (especially hemolysis) has been less explored. In this context, we studied lipid selectivity in two related novel AMPs, peptide 6 (P6) and peptide 6.2 (P6.2). Each peptide was designed from a previously reported AMP, and specific amino acid replacements were performed in an attempt to shift their hydrophobic moment or net charge. P6 showed no antimicrobial activity and high hemolytic activity, and P6.2 exhibited good antibacterial and low hemolytic activity. Using both peptides as a model we correlated the affinity toward membranes of different lipid composition and the antimicrobial and hemolytic activities. Our results from surface pressure and zeta potential assays showed that P6.2 exhibited a higher affinity and faster binding kinetic toward PG-containing membranes, while P6 showed this behavior for pure PC membranes. The final position and structure of P6.2 into the membrane showed an alpha-helix conversion, resulting in a parallel alignment with the Trps inserted into the membrane. On the other hand, the inability of P6 to adopt an amphipathic structure, plus its lower affinity toward PG-containing membranes seem to explain its poor antimicrobial activity. Regarding erythrocyte interactions, P6 showed the highest affinity toward erythrocyte membranes, resulting in an increased hemolytic activity. Overall, our data led us to conclude that affinity toward negatively charged lipids instead of zwitterionic ones seems to be a key factor that drives from hemolytic to

  12. Complement inhibitors to treat IgM-mediated autoimmune hemolysis

    PubMed Central

    Wouters, Diana; Zeerleder, Sacha

    2015-01-01

    Complement activation in autoimmune hemolytic anemia may exacerbate extravascular hemolysis and may occasionally result in intravascular hemolysis. IgM autoantibodies as characteristically found in cold autoantibody autoimmune hemolytic anemia, in cold agglutinin disease but also in a considerable percentage of patients with warm autoantibodies are very likely to activate complement in vivo. Therapy of IgM-mediated autoimmune hemolytic anemia mainly aims to decrease autoantibody production. However, most of these treatments require time to become effective and will not stop immediate ongoing complement-mediated hemolysis nor prevent hemolysis of transfused red blood cells. Therefore pharmacological inhibition of the complement system might be a suitable approach to halt or at least attenuate ongoing hemolysis and improve the recovery of red blood cell transfusion in autoimmune hemolytic anemia. In recent years, several complement inhibitors have become available in the clinic, some of them with proven efficacy in autoimmune hemolytic anemia. In the present review, we give a short introduction on the pathogenesis of autoimmune hemolytic anemia, followed by an overview on the complement system with a special focus on its regulation. Finally, we will discuss complement inhibitors with regard to their potential efficacy to halt or attenuate hemolysis in complement-mediated autoimmune hemolytic anemia. PMID:26521297

  13. Distribution and characterization of hemolytic activity by an oral anaerobe from the Streptococcus milleri group.

    PubMed

    Yamaguchi, T; Koreeda, H

    2004-04-01

    Some oral anaerobes from the Streptococcus milleri strain group were found to secrete human specific hemolytic toxin, which was detected when bacteria were cultured in Todd-Hewitt broth and Brain Heart Infusion broth. The toxin elicited by the Streptococcus intermedius strain was partially fractionated by ammonium sulfate precipitation. Preincubation with glutathione or cysteine showed significant inhibiting effects; however, no effects were seen with dithiothreitol or beta-mercaptoethanol, and cholesterol was a weak inhibitor. Five kinds of protease inhibitor had no effect on the hemolytic activity, and rabbit preimmune and immune sera against the bacterial cells showed weak inhibition at a similar level. Digestion with trypsin, chymotrypsin, proteinase-K, subtilisin and pronase-P brought about a rise in activity, followed by a decrease during long-term incubation. Other enzymes tested showed no effects. Further, the presence of the intermedilysin gene in the portion with hemolytic activity was not identified by polymerase chain reaction.

  14. Recognition of malondialdehyde-modified proteins by the C terminus of complement factor H is mediated via the polyanion binding site and impaired by mutations found in atypical hemolytic uremic syndrome.

    PubMed

    Hyvärinen, Satu; Uchida, Koji; Varjosalo, Markku; Jokela, Reija; Jokiranta, T Sakari

    2014-02-14

    Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy characterized by uncontrolled complement activation against endothelial and blood cells. Mutations in the C-terminal target recognition domains 19-20 of complement regulator factor H (FH) are strongly associated with aHUS, but the mechanisms triggering disease onset have remained unresolved. Here we report that several aHUS-related mutations alter the binding of FH19-20 to proteins where lysines have reacted with malondialdehyde (MDA). Although FH19-20 did not interact with MDA-modified hexylamine, lysine-containing peptides, or a proteolytically degraded protein, it bound to MDA-modified polylysine. This suggests that FH19-20 recognizes only clustered MDA adducts. Binding of MDA-modified BSA to FH19-20 was ionic by nature, depended on positive residues of FH19-20, and competed with the polyanions heparin and DNA. This could not be explained with the mainly neutral adducts known to form in MDA modification. When positive charges of lysines were eliminated by acetic anhydride instead of MDA, the acetylated BSA started to bind FH19-20. Together, these results indicate that negative charges on the modified proteins dominate the interaction with FH19-20. This is beneficial for the physiological function of FH because by binding to the negative charges of the modified target, FH could prevent excess complement activation initiated by naturally occurring antibodies recognizing MDA epitopes with multiple different structures. We propose that oxidative stress leading to formation of MDA adducts is a common feature for triggers of aHUS and that failure of FH in protecting MDA-modified surfaces from complement activation is involved in the pathogenesis of the disease.

  15. Contribution of hly homologs to the hemolytic activity of Prevotella intermedia.

    PubMed

    Suzuki, Naoko; Fukamachi, Haruka; Arimoto, Takafumi; Yamamoto, Matsuo; Igarashi, Takeshi

    2012-06-01

    Prevotella intermedia is a periodontal pathogen that requires iron for its growth. Although this organism has hemolytic activity, the precise nature of its hemolytic substances and their associated hemolytic actions are yet to be fully determined. In the present study, we identified and characterized several putative hly genes in P. intermedia ATCC25611 which appear to encode hemolysins. Six hly genes (hlyA, B, C, D, E, and hlyI) of P. intermedia were identified by comparing their nucleotide sequences to those of known hly genes of Bacteroides fragilis NCTC9343. The hlyA-E, and hlyI genes were overexpressed individually in the non-hemolytic Escherichia coli strain JW5181 and examined its contribution to the hemolytic activity on sheep blood agar plates. E. coli cells expressing the hlyA and hlyI genes exhibited hemolytic activity under anaerobic conditions. On the other hand, only E. coli cells stably expressing the hlyA gene were able to lyse the red blood cells when cultured under aerobic conditions. In addition, expression of the hlyA and hlyI genes was significantly upregulated in the presence of red blood cells. Furthermore, we found that the growth of P. intermedia was similar in an iron-limited medium supplemented with either red blood cells or heme. Taken together, our results indicate that the hlyA and hlyI genes of P. intermedia encode putative hemolysins that appear to be involved in the lysis of red blood cells, and suggest that these hemolysins might play important roles in the iron-dependent growth of this organism.

  16. Hemolytic anemia

    MedlinePlus

    Anemia - hemolytic ... bones that helps form all blood cells. Hemolytic anemia occurs when the bone marrow isn't making ... destroyed. There are several possible causes of hemolytic anemia. Red blood cells may be destroyed due to: ...

  17. Hemolytic Anemia

    MedlinePlus

    ... from the NHLBI on Twitter. What Is Hemolytic Anemia? Hemolytic anemia (HEE-moh-lit-ick uh-NEE-me-uh) ... blood cells to replace them. However, in hemolytic anemia, the bone marrow can't make red blood ...

  18. Complement activation in very early Alzheimer disease.

    PubMed

    Zanjani, H; Finch, C E; Kemper, C; Atkinson, J; McKeel, D; Morris, J C; Price, J L

    2005-01-01

    The activation of the classical complement (C)-system in early-stage Alzheimer disease (AD) and nondemented aging was examined with immunohistochemistry in subjects assessed by the Clinical Dementia Rating (CDR). Activation (staining for C3 and C4 fragments) was found in all brains with amyloid deposits, including all nondemented (CDR 0) cases, with either small numbers of diffuse plaques or with sufficient plaques and tangles to indicate preclinical AD. Staining for C3 and C4 increased in parallel with plaque density in very mild to severe clinical AD. A subset of very mild AD (CDR 0.5) cases also showed C1q (on plaques) and C5b-9 (on neuritic plaques and tangles), whereas these C-fragments were consistently found in severe AD (CDR 3). Mirror section (split-face) analysis showed that C1q, C3, and apoJ (clusterin) occurred on the same plaques. However, C-system regulators CD59, CR1, DAF, and MCP were not detected on plaques or tangles at any stage, indicating that C-activation related to AD is incompletely controlled.

  19. Can Cell Bound Complement Activation Products Predict Inherited Complement Deficiency in Systemic Lupus Erythematosus?

    PubMed Central

    Waters, Barry

    2016-01-01

    Activation of the classical pathway complement system has long been implicated in stimulating immune complex mediated tissue destruction in systemic lupus erythematosus (SLE). C3 and C4 complement levels are utilized as part of SLE diagnosis and monitoring criteria. Recently, cell bound complement activation products (CBCAPs) have shown increased sensitivity in diagnosing and monitoring lupus activity, compared to traditional markers. CBCAPs are increasingly utilized in rheumatology practice as additional serological markers in evaluating SLE patients. We report a case of a patient diagnosed with SLE that had chronically low C3 and C4, along with negative CBCAPs. We surmise that the patient has an inherited complement deficiency as the etiology of her SLE and that CBCAPs could be used to predict such deficiency. PMID:28074166

  20. Activation of the alternative complement pathway in canine normal serum by Paracoccidioides brasiliensis

    PubMed Central

    Bianchini, A.A.C.; Petroni, T.F.; Fedatto, P.F.; Bianchini, R.R.; Venancio, E.J.; Itano, E.N.; Ono, M.A.

    2009-01-01

    The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p ≤ 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis. PMID:24031350

  1. Protein ultrastructure and the nanoscience of complement activation.

    PubMed

    Vorup-Jensen, Thomas; Boesen, Thomas

    2011-09-16

    The complement system constitutes an important barrier to infection of the human body. Over more than four decades structural properties of the proteins of the complement system have been investigated with X-ray crystallography, electron microscopy, small-angle scattering, and atomic force microscopy. Here, we review the accumulated evidence that the nm-scaled dimensions and conformational changes of these proteins support functions of the complement system with regard to tissue distribution, molecular crowding effects, avidity binding, and conformational regulation of complement activation. In the targeting of complement activation to the surfaces of nanoparticulate material, such as engineered nanoparticles or fragments of the microbial cell wall, these processes play intimately together. This way the complement system is an excellent example where nanoscience may serve to unravel the molecular biology of the immune response.

  2. Investigating Biofilm Production, Coagulase and Hemolytic Activity in Candida Species Isolated From Denture Stomatitis Patients

    PubMed Central

    Yigit, Nimet; Aktas, Esin; Dagistan, Saadettin; Ayyildiz, Ahmet

    2011-01-01

    Objective: Oral candidiasis, in the form of Candida-associated denture stomatitis, represents a common disease in a large percentage of denture wearers, and Candida albicans remains the most commonly isolated species. In this study, we aimed to evaluate biofilm production, coagulase and hemolytic activity of Candida species isolated from denture stomatitis patients. Materials and Methods: This study included 70 patients (31 female, 39 male). Forty-eight of the patients were found to have a positive culture. A total of 48 Candida isolates representing five species, C. albicans (n=17), C. glabrata (n=10), C. krusei (n=9), C. kefyr (n=7) and C. parapsilosis (n=5), were tested. Their coagulase activities were evaluated by a classical tube coagulase test with rabbit plasma. A blood plate assay on 3% enriched sheep blood Sabouraud-dextrose agar (SDA) was used to determine their in vitro hemolytic activities. Biofilm production was determined by a visual tube method. Results: Twenty-one Candida isolates exhibited coagulase activity, and the coagulase activities of the C. albicans (64.7%) isolates were higher than other species. C. albicans, C. glabrata, C. kefyr and C. krusei species demonstrated beta hemolysis. C. parapsilosis strains failed to demonstrate any hemolytic activities. Fifteen (88.0%) of the C. albicans strains were biofilm positive. Six (35.2%) of these strains were strongly positive, 8 (47.0%) C. albicans strains were moderately positive and 1 (5.8%) C. albicans strain was weakly positive. Sixteen (51.6%) of the non-albicans Candida strains were biofilm positive while 15 (48.3%) did not produce biofilms. Conclusion: The results of this present study indicate coagulase, hemolytic activity and biofilm production by Candida spp. isolated from patients with denture stomatitis. Investigations of these virulence factors might be helpful in gaining information about the possible virulence of oral Candida species related to denture stomatitis. PMID:25610156

  3. Systemic complement activation in age-related macular degeneration.

    PubMed

    Scholl, Hendrik P N; Charbel Issa, Peter; Walier, Maja; Janzer, Stefanie; Pollok-Kopp, Beatrix; Börncke, Florian; Fritsche, Lars G; Chong, Ngaihang V; Fimmers, Rolf; Wienker, Thomas; Holz, Frank G; Weber, Bernhard H F; Oppermann, Martin

    2008-07-02

    Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH), factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001), were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes.This study is the first to show systemic complement activation in AMD patients. This suggests that AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were previously linked to AMD susceptibility.

  4. Trichomonas gallinae: a possible contact-dependent mechanism in the hemolytic activity.

    PubMed

    De Carli, Geraldo Attilio; Tasca, Tiana

    2002-07-02

    The in vitro hemolytic activity of Trichomonas gallinae was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of six adult animals of different species (rabbit, rat, chicken, horse, bovine, and sheep). Results showed that T. gallinae lysed all human erythrocytes groups, as well as rabbit, rat, chicken, horse, bovine and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. gallinae, or with killed organisms. The scanning electron microscopy (SEM) showed that the erythrocytes adhered to the parasite surface and were phagocytosed. These observations suggest that the contact between T. gallinae and erythrocytes may be an important mechanism in the injury caused to the erythrocytes. The hemolytic activity of T. gallinae may be an efficient means of obtaining nutrients for the parasite and allow the investigation of the mechanism used by T. gallinae to damage cellular membranes.

  5. Soluble complement receptor 1 inhibits both complement and granulocyte activation during ex vivo hemodialysis.

    PubMed

    Himmelfarb, J; McMonagle, E; Holbrook, D; Toth, C

    1995-10-01

    Hemodialysis with cellulosic membranes results in both complement and granulocyte activation. We investigated the effects of soluble complement receptor 1 (sCR1), a potent complement inhibitor, on both complement and granulocyte activation in an ex vivo model of dialysis. Measurements were made of complement activation (radioimmunoassay for C3a desArg) as well as granulocyte activation (flow cytometric measurements of reactive oxygen species production, granulocyte CD11b/CD18 (MAC-1) expression and CD62L (L-selectin) expression). sCR1 completely abolished the generation of plasma C3a desArg during ex vivo hemodialysis. Without sCR1, C3a desArg levels rose from 968 +/- 373 ng/ml to 4961 +/- 40 ng/ml by the end of the ex vivo procedure (p < 0.001). sCR1 also completely inhibited MAC-1 upregulation and L-selectin shedding from granulocytes during ex vivo hemodialysis. With sCR1 there was still a statistically significant increase in granulocyte reactive oxygen species production (from 2.42 +/- 0.1 fluorescence channels to 6.47 +/- 0.7 fluorescence channels, p < 0.01) but a 50% inhibition when compared with experiments without sCR1 (3.15 +/- 0.5 to 11.2 +/- 1.9, p < 0.01). We conclude that sCR1 completely abolishes complement activation and changes in granulocyte cell adhesion molecules during ex vivo hemodialysis with cellulosic membranes. sCR1 partially inhibits granulocyte reactive oxygen species formation.

  6. Tritrichomonas foetus: a scanning electron microscopy study of erythrocyte adhesion associated with hemolytic activity.

    PubMed

    De Carli, Geraldo Attilio; Tasca, Tiana; Pires Borges, Fernanda

    2004-01-01

    The in vitro hemolytic activity of Tritrichomonas foetus was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of nine adult animals of different species (the rabbit, rat, chicken, cat, dog, swine, horse, bovine, and sheep). The results showed that T. foetus strains (ATCC KV1, K, PAL, 5022, RJ, 90) did not present any hemolytic activity against any human erythrocyte group nor against rabbit, rat, chicken, cat, dog and swine erythrocytes. T. foetus strains, however, lysed horse, bovine, and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. foetus, nor with killed organisms. Scanning electron microscopy (SEM) showed that human erythrocytes did not adhere to the trophozoites, in contrast horse erythrocytes adhered to the surface of the parasites and were phagocytosed for up to 90 min. The parasites are able to exert their cytopathic effects through: (a) physical contact established between the two cell surfaces, (b) toxins released from parasites into the interaction media, or (c) the association of both mechanisms. Further studies are necessary to clarify the importance of the hemolytic activity in the biology of T. foetus.

  7. Comparison of the hemolytic activity between C. albicans and non-albicans Candida species.

    PubMed

    Rossoni, Rodnei Dennis; Barbosa, Júnia Oliveira; Vilela, Simone Furgeri Godinho; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos

    2013-01-01

    The ability to produce enzymes, such as hemolysins, is an important virulence factor for the genus Candida.The objective of this study was to compare the hemolytic activity between C. albicansand non-albicans Candida species. Fifty strains of Candida species, isolated from the oral cavity of patients infected with HIV were studied. The isolates included the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. dubliniensis, C. norvegensis, C. lusitaniae, and C. guilliermondii. Hemolysin production was evaluated on Sabouraud dextrose agar containing chloramphenicol, blood, and glucose. A loop-full of pure Candidaculture was spot-inoculated onto plates and incubated at 37 ºC for 24 h in a 5% CO2 atmosphere. Hemolytic activity was defined as the formation of a translucent halo around the colonies. All C. albicansstrains that were studied produced hemolysins. Among the non-albicans Candidaspecies, 86% exhibited hemolytic activity. Only C. guilliermondiiand some C. parapsilosis isolates were negative for this enzyme. In conclusion, most non-albicans Candidaspecies had a similar ability to produce hemolysins when compared to C. albicans.

  8. Complement activation promotes muscle inflammation during modified muscle use

    NASA Technical Reports Server (NTRS)

    Frenette, J.; Cai, B.; Tidball, J. G.

    2000-01-01

    Modified muscle use can result in muscle inflammation that is triggered by unidentified events. In the present investigation, we tested whether the activation of the complement system is a component of muscle inflammation that results from changes in muscle loading. Modified rat hindlimb muscle loading was achieved by removing weight-bearing from the hindlimbs for 10 days followed by reloading through normal ambulation. Experimental animals were injected with the recombinant, soluble complement receptor sCR1 to inhibit complement activation. Assays for complement C4 or factor B in sera showed that sCR1 produced large reductions in the capacity for activation of the complement system through both the classical and alternative pathways. Analysis of complement C4 concentration in serum in untreated animals showed that the classical pathway was activated during the first 2 hours of reloading. Analysis of factor B concentration in untreated animals showed activation of the alternative pathway at 6 hours of reloading. Administration of sCR1 significantly attenuated the invasion of neutrophils (-49%) and ED1(+) macrophages (-52%) that occurred in nontreated animals after 6 hours of reloading. The presence of sCR1 also reduced significantly the degree of edema by 22% as compared to untreated animals. Together, these data show that increased muscle loading activated the complement system which then briefly contributes to the early recruitment of inflammatory cells during modified muscle loading.

  9. Complement activity and pharmacological inhibition in cardiovascular disease

    PubMed Central

    Théroux, Pierre; Martel, Catherine

    2006-01-01

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

  10. Bullous pemphigoid autoantibodies directly induce blister formation without complement activation.

    PubMed

    Ujiie, Hideyuki; Sasaoka, Tetsumasa; Izumi, Kentaro; Nishie, Wataru; Shinkuma, Satoru; Natsuga, Ken; Nakamura, Hideki; Shibaki, Akihiko; Shimizu, Hiroshi

    2014-11-01

    Complement activation and subsequent recruitment of inflammatory cells at the dermal/epidermal junction are thought to be essential for blister formation in bullous pemphigoid (BP), an autoimmune blistering disease induced by autoantibodies against type XVII collagen (COL17); however, this theory does not fully explain the pathological features of BP. Recently, the involvement of complement-independent pathways has been proposed. To directly address the question of the necessity of the complement activation in blister formation, we generated C3-deficient COL17-humanized mice. First, we show that passive transfer of autoantibodies from BP patients induced blister formation in neonatal C3-deficient COL17-humanized mice without complement activation. By using newly generated human and murine mAbs against the pathogenic noncollagenous 16A domain of COL17 with high (human IgG1, murine IgG2), low (murine IgG1), or no (human IgG4) complement activation abilities, we demonstrate that the deposition of Abs, and not complements, is relevant to the induction of blister formation in neonatal and adult mice. Notably, passive transfer of BP autoantibodies reduced the amount of COL17 in lesional mice skin, as observed in cultured normal human keratinocytes treated with the same Abs. Moreover, the COL17 depletion was associated with a ubiquitin/proteasome pathway. In conclusion, the COL17 depletion induced by BP autoantibodies, and not complement activation, is essential for the blister formation under our experimental system.

  11. Pathophysiology of hemolytic transfusion reactions.

    PubMed

    Davenport, Robertson D

    2005-07-01

    Hemolytic transfusion reactions (HTR) are systemic reactions provoked by immunologic red blood cell (RBC) incompatibility. Clinical and experimental observations of such reactions indicate that they proceed through phases of humoral immune reaction, activation of phagocytes, productions of cytokine mediators, and wide-ranging cellular responses. HTR have many features in common with the systemic inflammatory response syndrome (SIRS). Knowledge of the pathophysiologic mechanisms in HTR suggest that newer biological agents that target complement intermediates or proinflammatory cytokines may be effective agents in the treatment of severe HTRs.

  12. Complement activation by antibodies to Sm in systemic lupus erythematosus.

    PubMed

    Sabharwal, U K; Fong, S; Hoch, S; Cook, R D; Vaughan, J H; Curd, J G

    1983-02-01

    An enzyme linked immunosorbent assay was developed to quantitate antibodies to Sm (anti-Sm) and to measure complement activation by anti-Sm in vitro. Anti-Sm in plasma of patients with systemic lupus erythematosus (SLE) were bound to purified Sm bound to polyvinyl chloride microtitre plates and assayed for bound IgG or IgM using enzyme linked anti-gamma or anti-mu. The activation of C4 by anti-Sm was measured by adding diluted normal human serum (complement) to the wells and quantitating the amount of C4 bound to the well surface using (Fab')2 goat anti-C4 followed by enzyme linked rabbit anti-goat IgG. The plasmas of 12 of 36 patients with SLE contained anti-Sm and all 12 activated complement (complement activating anti-Sm). Twenty-eight plasmas containing anti-Sm from 12 patients with SLE were studied. Ten of the 12 patients had anti-Sm of the IgG class whereas two had anti-Sm of both IgG and IgM classes. The amount of C4 activating anti-Sm correlated significantly with the in vivo activation of C4 measured by rocket immunoelectrophoresis for C4d and C4, suggesting that complement activation by anti-Sm is important in vivo.

  13. The Impact of Silica Nanoparticle Design on Cellular Toxicity and Hemolytic Activity

    PubMed Central

    Yu, Tian; Malugin, Alexander; Ghandehari, Hamidreza

    2011-01-01

    Understanding the toxicity of silica nanoparticles (SiO2) on the cellular level is crucial for rational design of these nanomaterials for biomedical applications. Herein, we explore the impacts of geometry, porosity and surface charge of SiO2 on cellular toxicity and hemolytic activity. Nonporous Stöber silica nanospheres (115 nm diameter), mesoporous silica nanospheres (120 nm diameter, aspect ratio 1), mesoporous silica nanorods with aspect ratio of 2, 4 and 8 (width by length 80 × 200 nm, 150 × 600 nm, 130 × 1000 nm) as well as their cationic counterparts were evaluated on macrophages, lung carcinoma cells, and human erythrocytes. It was shown that the toxicity of SiO2 is cell-type dependent and that surface charge and pore size govern cellular toxicity. Using inductively coupled plasma mass spectrometry, the cellular association of SiO2 was quantitated with the association amount increasing in the following order: mesoporous SiO2 (aspect ratio 1, 2, 4, 8) < amine-modified mesoporous SiO2 (aspect ratio 1, 2, 4, 8) < amine-modified nonporous Stöber SiO2 < nonporous Stöber SiO2. Geometry did not seem to influence the extent of SiO2 association at early or extended time points. The level of cellular association of the nanoparticles was directly linked to the extent of plasma membrane damage, suggesting a biological cause-and-effect relationship. Hemolysis assay showed that the hemolytic activity was porosity- and geometry- dependent for bare SiO2 and surface charge-dependent for amine-modified SiO2. A good correlation between hemolytic activity and cellular association was found on a similar dosage basis. These results can provide useful guidelines for the rational design of SiO2 in nanomedicine. PMID:21630682

  14. Vibrio parahaemolyticus CalR down regulates the thermostable direct hemolysin (TDH) gene transcription and thereby inhibits hemolytic activity.

    PubMed

    Zhang, Yiquan; Zhang, Ying; Gao, He; Zhang, Lingyu; Yin, Zhe; Huang, Xinxiang; Zhou, Dongsheng; Yang, Huiying; Yang, Wenhui; Wang, Li

    2017-03-04

    TDH, encoded by tdh gene, is a major virulent determinant of V. parahaemolyticus that controls various biological activities, such as hemolytic activity, cytotoxicity, and enterotoxicity. The hemolytic activity on Wagatsuma agar ascribed to TDH is called Kanagawa phenomenon (KP). All KP positive strains contain tdh1 and tdh2 genes, but tdh2 is predominantly responsible for KP. CalR is a regulatory protein that was originally identified as a repressor of swarming motility and T3SS1 gene expression in V. parahaemolyticus. In the present study, the regulation of tdh2 by CalR was investigated using a set of experiments including qRT-PCR, primer extension, LacZ fusion, hemolytic phenotype, EMSA, and DNase I footprinting assays. The results showed that His-CalR protected a single region from 224bp to 318bp upstream of tdh2 against DNase I digestion, and a transcriptional start site located at 42bp upstream of tdh2 was detected and its transcribed activity was inhibited by CalR. Moreover, the KP test results showed that the hemolytic activity of V. parahaemolyticus is also under negative control of CalR. The data demonstrated that CalR is a repressor of the tdh2 transcription and thereby inhibits the hemolytic activity of V. parahaemolyticus.

  15. Warm autoimmune hemolytic anemia.

    PubMed

    Naik, Rakhi

    2015-06-01

    Warm autoimmune hemolytic anemia (AIHA) is defined as the destruction of circulating red blood cells (RBCs) in the setting of anti-RBC autoantibodies that optimally react at 37°C. The pathophysiology of disease involves phagocytosis of autoantibody-coated RBCs in the spleen and complement-mediated hemolysis. Thus far, treatment is aimed at decreasing autoantibody production with immunosuppression or reducing phagocytosis of affected cells in the spleen. The role of complement inhibitors in warm AIHA has not been explored. This article addresses the diagnosis, etiology, and treatment of warm AIHA and highlights the role of complement in disease pathology.

  16. Investigation into the hemolytic activity of tentacle venom from jellyfish Cyanea nozakii Kishinouye

    NASA Astrophysics Data System (ADS)

    Li, Cuiping; Yu, Huahua; Li, Rongfeng; Xing, Ronge; Liu, Song; Li, Pengcheng

    2016-03-01

    Cyanea nozakii Kishinouy e ( C. nozakii), a giant cnidarian of the class Scyphomedusae, order Semaeostomeae and family Cyaneidae, is widely distributed in the East China Sea, the Yellow Sea and the Bohai Sea, and is abundant from late summer to early autumn. Venom produced by C. nozakii during mass agglomerations can contaminate seawater resulting in death of the halobios and seriously damage commercial fisheries. Swimmers and fishermen commonly suff er painful stings from this jellyfish, resulting in local edema, tingling, breathing difficulties, depressed blood pressure and even death. Such effects arise from the complex mixture of biologically active molecules that make up jellyfish venom. In the present study, the hemolytic activity of venom from tentacles of C. nozakii and factors aff ecting its activity were assayed. The HU50 ( defined as the amount of protein required to lyse 50 % of erythrocytes) of the venom against dove and chicken erythrocytes was 34 and 59 μg/mL, respectively. Carboxylmethyl chitosan and glycerol could increase hemolytic activity at concentrations greater than 0.06% and 0.2 mol/L, respectively.

  17. Cationic surfactants derived from lysine: effects of their structure and charge type on antimicrobial and hemolytic activities.

    PubMed

    Colomer, A; Pinazo, A; Manresa, M A; Vinardell, M P; Mitjans, M; Infante, M R; Pérez, L

    2011-02-24

    Three different sets of cationic surfactants from lysine have been synthesized. The first group consists of three monocatenary surfactants with one lysine as the cationic polar head with one cationic charge. The second consists of three monocatenary surfactants with two amino acids as cationic polar head with two positive charges. Finally, four gemini surfactants were synthesized in which the spacer chain and the number and type of cationic charges have been regulated. The micellization process, antimicrobial activity, and hemolytic activity were evaluated. The critical micelle concentration was dependent only on the hydrophobic character of the molecules. Nevertheless, the antimicrobial and hemolytic activities were related to the structure of the compounds as well as the type of cationic charges. The most active surfactants against the bacteria were those with a cationic charge on the trimethylated amino group, whereas all of these surfactants showed low hemolytic character.

  18. Lactobacilli Interfere with Streptococcus pyogenes Hemolytic Activity and Adherence to Host Epithelial Cells

    PubMed Central

    Saroj, Sunil D.; Maudsdotter, Lisa; Tavares, Raquel; Jonsson, Ann-Beth

    2016-01-01

    Streptococcus pyogenes [Group A streptococcus (GAS)], a frequent colonizer of the respiratory tract mucosal surface, causes a variety of human diseases, ranging from pharyngitis to the life-threatening streptococcal toxic shock-like syndrome. Lactobacilli have been demonstrated to colonize the respiratory tract. In this study, we investigated the interference of lactobacilli with the virulence phenotypes of GAS. The Lactobacillus strains L. rhamnosus Kx151A1 and L. reuteri PTA-5289, but not L. salivarius LMG9477, inhibited the hemolytic activity of S. pyogenes S165. The inhibition of hemolytic activity was attributed to a decrease in the production of streptolysin S (SLS). Conditioned medium (CM) from the growth of L. rhamnosus Kx151A1 and L. reuteri PTA-5289 was sufficient to down-regulate the expression of the sag operon, encoding SLS. The Lactobacillus strains L. rhamnosus Kx151A1, L. reuteri PTA-5289, and L. salivarius LMG9477 inhibited the initial adherence of GAS to host epithelial cells. Intriguingly, competition with a combination of Lactobacillus species reduced GAS adherence to host cells most efficiently. The data suggest that an effector molecule released from certain Lactobacillus strains attenuates the production of SLS at the transcriptional level and that combinations of Lactobacillus strains may protect the pharyngeal mucosa more efficiently from the initial colonization of GAS. The effector molecules released from Lactobacillus strains affecting the virulence phenotypes of pathogens hold potential in the development of a new generation of therapeutics. PMID:27524981

  19. Complement and contact activation in term neonates after fetal acidosis

    PubMed Central

    Sonntag, J.; Wagner, M.; Strauss, E.; Obladen, M.

    1998-01-01

    AIMS—To evaluate complement and contact activation after fetal acidosis.
METHODS—Fifteen term neonates with hypoxic-ischaemic encephalopathy after umbilical arterial pH < 7.10 were compared with 15 healthy neonates with umbilical arterial pH > 7.20. Determinations of the complement function and C1-inhibitor activity were performed as kinetic tests 22-28 hours after birth. C1q, C1-inhibitor, and factor B concentrations were determined by radial immunodiffusion and those of C3a, C5a, and factor XIIa by enzyme immunoabsorbent assay.
RESULTS—Median complement function (46 vs 73 %), C1q (4.3 vs 9.1 mg/dl), and factor B (5.2 vs 7.7 mg/dl) decreased after fetal acidosis. The activated split products C3a (260 vs 185 µg/l), C5a (5.0 vs 0.6 µg/l), and factor XIIa (3.2 vs 1.3 µg/l) increased in the neonates after fetal acidosis. No differences were found in the concentration and activity of C1-inhibitor.
CONCLUSIONS—Complement and contact activation occurred in the newborns with hypoxic-ischaemic encephalopathy. Activation of these systems generates mediators which can trigger inflammation and tissue injury.

 PMID:9577283

  20. Oxidative stress-mediated hemolytic activity of solvent exchange-prepared fullerene (C60) nanoparticles

    NASA Astrophysics Data System (ADS)

    Trpkovic, Andreja; Todorovic-Markovic, Biljana; Kleut, Duska; Misirkic, Maja; Janjetovic, Kristina; Vucicevic, Ljubica; Pantovic, Aleksandar; Jovanovic, Svetlana; Dramicanin, Miroslav; Markovic, Zoran; Trajkovic, Vladimir

    2010-09-01

    The present study investigated the hemolytic properties of fullerene (C60) nanoparticles prepared by solvent exchange using tetrahydrofuran (nC60THF), or by mechanochemically assisted complexation with macrocyclic oligosaccharide gamma-cyclodextrin (nC60CDX) or the copolymer ethylene vinyl acetate-ethylene vinyl versatate (nC60EVA-EVV). The spectrophotometrical analysis of hemoglobin release revealed that only nC60THF, but not nC60CDX or nC60EVA-EVV, was able to cause lysis of human erythrocytes in a dose- and time-dependent manner. Atomic force microscopy revealed that nC60THF-mediated hemolysis was preceded by erythrocyte shrinkage and increase in cell surface roughness. A flow cytometric analysis confirmed a decrease in erythrocyte size and demonstrated a significant increase in reactive oxygen species production in red blood cells exposed to nC60THF. The nC60THF-triggered hemolytic activity was efficiently reduced by the antioxidants N-acetylcysteine and butylated hydroxyanisole, as well as by serum albumin, the most abundant protein in human blood plasma. These data indicate that nC60THF can cause serum albumin-preventable hemolysis through oxidative stress-mediated damage of the erythrocyte membrane.

  1. Cytotoxicity and hemolytic activity of jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae) venom.

    PubMed

    Kang, Changkeun; Munawir, Al; Cha, Mijin; Sohn, Eun-Tae; Lee, Hyunkyoung; Kim, Jong-Shu; Yoon, Won Duk; Lim, Donghyun; Kim, Euikyung

    2009-07-01

    The recent bloom of a giant jellyfish Nemopilema nomurai has caused a danger to sea bathers and fishery damages in the waters of China, Korea, and Japan. The present study investigated the cytotoxic and hemolytic activities of crude venom extract of N. nomurai using a number of in vitro assays. The jellyfish venom showed a much higher cytotoxic activity in H9C2 heart myoblast than in C2C12 skeletal myoblast (LC(50)=2 microg/mL vs. 12 microg/mL, respectively), suggesting its possible in vivo selective toxicity on cardiac tissue. This result is consistent with our previous finding that cardiovascular function is a target of the venom. In order to determine the stability of N. nomurai venom, its cytotoxicity was examined under the various temperature and pH conditions. The activity was relatively well retained at low environmental temperature (or=60 degrees C). In pH stability test, the venom has abruptly lost its activity at low pH environment (pHactivity was not significantly affected even at the highest pH environment tested (pHhemolytic activity of the venom was examined using the erythrocytes of cat, dog, human, rabbit and rat. Venom concentration-dependent hemolysis could be observed from 10 microg/mL of protein equivalents or higher with variable potencies in different species, among which dog erythrocyte was the most susceptible to the venom (EC(50)=151 microg/mL). SDS-PAGE analysis of N. nomurai venom showed the molecules of 20-40 kDa and 10-15 kDa appeared to be the major protein components of the venom.

  2. Anticariogenic and Hemolytic Activity of Selected Seed Protein Extracts In vitro conditions

    PubMed Central

    Ishnava, Kalpesh B; Shah, Pankit P.

    2014-01-01

    Objective: This study aimed to assess the anticariogenic and hemolytic activity of crude plant seed protein extracts against tooth decaying bacteria. Materials and Methods: The proteins from seeds of 12 different plants were extracted and used for antimicrobial assay against six different organisms. The extraction was carried out in 10mM of sodium phosphate buffer (pH 7.0). Protein concentrations were determined as described by Bradford method. Anticariogenic activity was studied by agar well diffusion method and Minimum Inhibitory Concentration (MIC) was evaluated by the two-fold serial broth dilution method. Hemolytic activity, treatment of proteinase K and Kinetic study in Mimusops elengi crude seed protein extract. Results: The anticariogenic assay demonstrated the activity of Mimusops elengi against Staphylococcus aureus and Streptococcus pyogenes. A minor activity of Glycine wightii against Streptococcus mutans was also found. The protein content of Mimusops elengi seed protein extract was 5.84mg/ml. The MIC values for Staphylococcus aureus and Streptococcus pyogenes against Mimusops elengi seed protein extract were 364.36μg/ml and 182.19μg/ml, respectively. Kinetic study further elucidated the mode of inhibition in the presence of the Mimusops elengi plant seed protein with respect to time. The concentration of crude extract which gave 50% hemolysis compared to Triton X-100 treatment (HC50) value was 1.58 mg/ml; which is more than five times larger than that of the MIC. Treatment with proteinase K of the Mimusops elengi seed protein resulted in absence of the inhibition zone; which clearly indicates that the activity was only due to protein. Conclusion: Our results showed the prominence of Mimusops elengi plant seed protein extract as an effective herbal medication against tooth decaying bacteria. PMID:25628685

  3. Cercarial glycocalyx of Schistosoma mansoni activates human complement.

    PubMed Central

    Samuelson, J C; Caulfield, J P

    1986-01-01

    Human complement activation by cercariae and schistosomula of the human parasite Schistosoma mansoni was studied in vitro. Cercariae are composed of tails which are shed after infection of the host and bodies which transform into the larvae or schistosomula after infection. After incubation in fresh normal human serum (NHS), cercarial tails bound more anti-C3 antibodies than did cercarial bodies (CB), and the tails were rapidly lysed, while the attached CB remained intact. Complement activation by cercariae was dependent on the alternative pathway but was independent of antibody, as shown by C3 deposition by hypogammaglobulinemic human sera. By transmission microscopy, the fibrillar glycocalyx on both CB and tails was stained by NHS but not by heat-inactivated serum (HI-NHS). The glycocalyx was labeled with periodate and tritiated borohydride, and parasites were incubated in NHS and HI-NHS. After solubilization, the labeled glycocalyx on organisms incubated in NHS but not HI-NHS bound anti-C3 antibodies. Of the CB incubated with eserine sulfate to prevent transformation, 78% +/- 10% were dead after culture for 24 h in NHS. In contrast, 21% +/- 12% of the CB were dead after culture in HI-NHS. Schistosomula incubated in NHS bound 37% of the amount of anti-C3 antibodies bound by cercariae but were not killed by NHS. In conclusion, the cercarial glycocalyx activated human complement, and schistosomula were less susceptible to killing than cercariae because they had less glycocalyx and activated less complement. Images PMID:3940995

  4. Complement activation and effect of eculizumab in scleroderma renal crisis

    PubMed Central

    Devresse, Arnaud; Aydin, Selda; Le Quintrec, Moglie; Demoulin, Nathalie; Stordeur, Patrick; Lambert, Catherine; Gastoldi, Sara; Pirson, Yves; Jadoul, Michel; Morelle, Johann

    2016-01-01

    Abstract Background: Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis characterized by abrupt onset of hypertension, thrombotic microangiopathy, and kidney injury. The mechanisms of the disease remain ill-defined, but a growing body of evidence suggests that activation of the complement system may be involved. Methods: Here, we report the case of a patient presenting with severe SRC and strong evidence of complement activation, both in serum and in the kidney, in the absence of genetic defect of the complement system. Results: Immunofluorescence studies on kidney biopsy showed significant deposits of C1q and C4d in the endothelium of renal arterioles, pointing toward activation of the classical pathway. Because of the dramatic clinical and histological severity, and the lack of response to early treatment with angiotensin-converting enzyme inhibitors, calcium channel blockers and plasma exchange, the patient was treated with the specific C5 blocker eculizumab. Contrarily to conventional treatment, eculizumab efficiently blocked C5b-9 deposition ex vivo and maintained hematological remission. Unfortunately, the patient died from heart failure a few weeks later. Postmortem examination of the heart showed diffuse patchy interstitial fibrosis, the typical lesion of systemic sclerosis-related cardiomyopathy, but normal coronary arteries and myocardial microvasculature. Conclusion: SRC may lead to complement system activation through the classical pathway. Early administration of C5 inhibitor eculizumab may have therapeutic potential in patients with life-threatening SRC refractory to conventional treatment using angiotensin-converting enzyme inhibitors. PMID:27472742

  5. Hemolytic uremic syndrome

    PubMed Central

    Canpolat, Nur

    2015-01-01

    Hemolytic uremic syndrome (HUS) is a clinical syndrome characterized by the triad of thrombotic microangiopathy, thrombocytopenia, and acute kidney injury. Hemolytic uremic syndrome represents a heterogeneous group of disorders with variable etiologies that result in differences in presentation, management and outcome. In recent years, better understanding of the HUS, especially those due to genetic mutations in the alternative complement pathway have provided an update on the terminology, classification, and treatment of the disease. This review will provide the updated classification of the disease and the current diagnostic and therapeutic approaches on the complement-mediated HUS in addition to STEC-HUS which is the most common cause of the HUS in childhood. PMID:26265890

  6. Effect of Relative Arrangement of Cationic and Lipophilic Moieties on Hemolytic and Antibacterial Activities of PEGylated Polyacrylates

    PubMed Central

    Punia, Ashish; Lee, Kevin; He, Edward; Mukherjee, Sumit; Mancuso, Andrew; Banerjee, Probal; Yang, Nan-Loh

    2015-01-01

    Synthetic amphiphilic polymers have been established as potentially efficient agents to combat widespread deadly infections involving antibiotic resistant superbugs. Incorporation of poly(ethylene glycol) (PEG) side chains into amphiphilic copolymers can reduce their hemolytic activity while maintaining high antibacterial activity. Our study found that the incorporation of PEG has substantially different effects on the hemolytic and antibacterial activities of copolymers depending on structural variations in the positions of cationic centers relative to hydrophobic groups. The PEG side chains dramatically reduced the hemolytic activities in copolymers with hydrophobic hexyl and cationic groups on the same repeating unit. However, in case of terpolymers with cationic and lipophilic groups placed on separate repeating units, the presence of PEG has significantly lower effect on hemolytic activities of these copolymers. PEGylated terpolymers displayed substantially lower activity against Staphylococcus aureus (S. aureus) than Escherichia coli (E. coli) suggesting the deterring effect of S. aureus’ peptidoglycan cell wall against the penetration of PEGylated polymers. Time-kill studies confirmed the bactericidal activity of these copolymers and a 5 log reduction in E. coli colony forming units was observed within 2 h of polymer treatment. PMID:26473831

  7. Comparative hemolytic activity of undiluted organic water-miscible solvents for intravenous and intra-arterial injection.

    PubMed

    Mottu, F; Stelling, M J; Rüfenacht, D A; Doelker, E

    2001-01-01

    In humans, nonaqueous solvents are administered intravascularly in two kinds of situations. They have been used in subcutaneous or intramuscular pharmaceutical formulations to dissolve water-insoluble drugs. The need for these vehicles had increased in recent years, since the drug development process has yielded many poorly water-soluble drugs. The use of water-miscible nonaqueous solvents in therefore one of the approaches for administering these products as reference solutions useful in formulation bioequivalence studies. The intravascular use of organic solvents has also gained importance owing to a new approach for the treatment of cerebral malformations using precipitating polymers dissolved in water-miscible organic solvents. At present, the solvent most commonly used for the liquid embolics to solubilize the polymers is dimethyl sulfoxide, which exhibits some local and hemodynamic toxicities. In order to find new, less toxic vehicles for pharmaceutical formulations for the intravenous and intra-arterial routes and for embolic materials, 13 water-miscible organic solvents currently used (diluted with water) for pharmaceutical applications, were evaluated in this study. Their hemolytic activity and the morphological changes induced when mixed with blood (1:99, 5:95, 10:90 solvent:blood) were estimated in vitro. From these data, the selected organic solvents could be subdivided into four groups depending on their hemolytic activity: very highly hemolytic solvents (ethyl lactate, dimethyl sulfoxide), highly hemolytic solvents (polyethylene glycol 200, acetone), moderately hemolytic solvents (tetrahydrofurfuryl alcohol, N-methyl-2-pyrrolidone, glycerol formal, ethanol, Solketal, glycofurol) and solvents with low hemolytic activity (propylene glycol, dimethyl isosorbide, diglyme).

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

    PubMed

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

    1997-11-15

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

  9. Synthesis, characterization, in vitro anti-proliferative and hemolytic activity of hydroxyapatite

    NASA Astrophysics Data System (ADS)

    Palanivelu, R.; Ruban Kumar, A.

    2014-06-01

    Hydroxyapatite (Ca10(PO4)6(OH)2, HAP) nanoparticles are widely used in several biomedical applications due to its compositional similarities to bone mineral, excellent biocompatibility and bioactivity, osteoconductivity. In this present investigation, HAP nanoparticles synthesized by precipitation technique using calcium nitrate and di-ammonium phosphate. The crystalline nature and the functional group analysis are confirmed using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Fourier transform Raman spectroscopy (FT-Raman) respectively. The morphological observations are ascertained from field emission electron scanning electron microscope (FE-SEM) and transmission electron microscope (TEM). In vitro anti-proliferative and hemolytic activities are carried out on the synthesized HAP samples and the studies reveals that HAP have mild activity against erythrocytes.

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

    PubMed Central

    Li, Mo-fei; Li, Jun; Sun, Li

    2016-01-01

    In teleost fish, the immune functions of mannan-binding lectin (MBL) associated protein (MAP) and MBL associated serine protease (MASP) are scarcely investigated. In the present study, we examined the biological properties both MAP (CsMAP34) and MASP (CsMASP1) molecules from tongue sole (Cynoglossus semilaevis). We found that CsMAP34 and CsMASP1 expressions occurred in nine different tissues and were upregulated by bacterial challenge. CsMAP34 protein was detected in blood, especially during bacterial infection. Recombinant CsMAP34 (rCsMAP34) bound C. semilaevis MBL (rCsBML) when the latter was activated by bacteria, while recombinant CsMASP1 (rCsMASP1) bound activated rCsBML only in the presence of rCsMAP34. rCsMAP34 stimulated the hemolytic and bactericidal activities of serum complement, whereas anti-CsMAP34 antibody blocked complement activities. Knockdown of CsMASP1 in C. semilaevis resulted in significant inhibition of complement activities. Furthermore, rCsMAP34 interacted directly with peripheral blood leukocytes (PBL) and enhanced the respiratory burst, acid phosphatase activity, chemotactic activity, and gene expression of PBL. These results indicate for the first time that a teleost MAP acts one hand as a regulator that promotes the lectin pathway of complement activation via its ability to recruit MBL to MASP, and other hand as a modulator of immune cell activity. PMID:28008939

  11. Clinical grand rounds: atypical hemolytic uremic syndrome.

    PubMed

    Hodgkins, Kavita S; Bobrowski, Amy E; Lane, Jerome C; Langman, Craig B

    2012-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare, lifethreatening, chronic, genetic disease of uncontrolled alternative pathway complement activation. The understanding of the pathophysiology and genetics of this disease has expanded over recent decades and promising new developments in the management of aHUS have emerged. Regardless of the cause of aHUS, with or without a demonstrated mutation or autoantibody, blockade of terminal complement activation through C5 is of high interest as a mechanism to ameliorate the disease. Eculizumab, an existing monoclonal antibody directed against C5 with high affinity, prevents the perpetuation of the downstream activation of the complement cascade and the damage caused by generation of the anaphylotoxin C5a and the membrane attack complex C5b-9, by blocking C5 cleavage. We report the successful use of eculizumab in a patient after kidney transplantation and discuss the disease aHUS.

  12. Evaluation of antimicrobial, cytotoxic, and hemolytic activities from venom of the spider Lasiodora sp.

    PubMed

    Ferreira, Felipe Roberto Borba; da Silva, Pollyanna Michelle; Soares, Tatiana; Gonçalves Machado, Larissa; de Araújo, Larissa Cardoso Corrêa; da Silva, Teresinha Gonçalves; de Mello, Gabriela Souto Vieira; Galdino da Rocha Pitta, Maira; de Melo Rego, Moacyr Jesus Barreto; Pontual, Emmanuel Viana; Zingali, Russolina Benedeta; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes

    2016-11-01

    This study characterized the protein/peptide profile of venom isolated from the spider Lasiodora sp. (Mygalomorphae, Theraphosidae) found in northeastern Brazil and determined its antimicrobial activity, toxicity against human cells, and hemolytic activity. Protein concentration of the Lasiodora sp. venom was 4.53 ± 0.38 mg/mL. SDS-PAGE showed proteins with molecular masses up to 75 kDa, some of which contained disulfide bridges. RP-HPLC analysis separate at least 12 peaks that were identified by mass spectrometry as peptides U1-theraphotoxin-Lp1a (lasiotoxin-1), U1-theraphotoxin-Lp1c (lasiotoxin-3), U3-theraphotoxin-Lsp1a (LTx5), and ω-theraphotoxin-Asp3a as well as the proteins phospholipase A2 (PLA2) and hyaluronidase. The crude venom exhibited bactericidal effect against Aeromonas sp., Bacillus subtilis, and Micrococcus luteus and fungicidal effect against Candida parapsilosis and Candida albicans. In addition, the venom exerted bacteriostatic effect against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus and fungistatic effect against Candida tropicalis and Candida krusei. The minimum inhibitory (MIC), minimum bactericidal (MBC), and minimum fungicidal (MFC) concentrations ranged from 3.9 to 500 μg/mL. The Lasiodora sp. venom decreased the viability of human peripheral blood mononuclear cells (PBMCs) by 50%-90% at concentrations of 0.1, 1, 10, and 100 μg/mL, promoting apoptosis of these cells. On the other hand, the venom showed weak hemolytic activity against Mus musculus erythrocytes (EC50: 757 μg/mL). In conclusion, the Lasiodora sp. spider venom is a rich source of antimicrobial agents. Future studies will focus on identifying antimicrobial agents present in this venom and evaluating whether these agents contribute to its cytotoxic effects against PBMCs.

  13. Allicin from garlic neutralizes the hemolytic activity of intra- and extra-cellular pneumolysin O in vitro.

    PubMed

    Arzanlou, M; Bohlooli, S; Jannati, E; Mirzanejad-Asl, H

    2011-03-15

    Pneumolysin (PLY) is a key virulence factor contributes to the pathogenesis of Streptococcus pneumoniae. In this study we investigated the effect of allicin and aqueous garlic extracts on hemolytic activity of PLY both in prelysed and intact cells. Additionally the antimicrobial activity of allicin was tested against the bacteria. All tested materials potently inhibited the PLY hemolytic activity. Allicin neutralizes PLY in a concentration- and time-dependent manner. Twenty five minute incubation of PLY (2 HU/mL) with 0.61 μM/mL concentration of allicin, totally inhibited hemolytic activity of PLY (IC50 = 0.28 μM/mL). The inhibitory activity of old extract of garlic was similar to pure allicin (IC50 = 50.46 μL/mL; 0.31 μM/mL; P < 0.05). In contrast fresh extract of garlic inhibits the PLY hemolytic activity at lower concentrations (IC50 = 13.96 μL/mL; 0.08 μM/mL allicin). Exposure of intact cells to allicin (1.8 μM) completely inhibited hemolytic activity of PLY inside bacterial cells. The inhibitory effect of the allicin was restored by addition of reducing agent DTT at 5 mM, proposing that allicin likely inhibits the PLY by binding to cysteinyl residue in the binding site. The MIC value of allicin was determined to be 512 μg/mL (3.15 μM/mL). These results indicate that PLY is a novel target for allicin and may provide a new line of investigation on pneumococcal diseases in the future.

  14. Activated Complement Factors as Disease Markers for Sepsis

    PubMed Central

    Charchaflieh, Jean; Rushbrook, Julie; Worah, Samrat; Zhang, Ming

    2015-01-01

    Sepsis is a leading cause of death in the United States and worldwide. Early recognition and effective management are essential for improved outcome. However, early recognition is impeded by lack of clinically utilized biomarkers. Complement factors play important roles in the mechanisms leading to sepsis and can potentially serve as early markers of sepsis and of sepsis severity and outcome. This review provides a synopsis of recent animal and clinical studies of the role of complement factors in sepsis development, together with their potential as disease markers. In addition, new results from our laboratory are presented regarding the involvement of the complement factor, mannose-binding lectin, in septic shock patients. Future clinical studies are needed to obtain the complete profiles of complement factors/their activated products during the course of sepsis development. We anticipate that the results of these studies will lead to a multipanel set of sepsis biomarkers which, along with currently used laboratory tests, will facilitate earlier diagnosis, timely treatment, and improved outcome. PMID:26420913

  15. Isolation and characterization of genetic variability in bacteria with β-hemolytic and antifungal activity isolated from the rhizosphere of Medicago truncatula plants.

    PubMed

    Hernández-Salmerón, J E; Prieto-Barajas, C M; Valencia-Cantero, E; Moreno-Hagelsieb, G; Santoyo, G

    2014-07-04

    In the present study, we analyzed the frequency of hemolytic and antifungal activities in bacterial isolates from the rhizosphere of Medicago truncatula plants. Of the 2000 bacterial colonies, 96 showed β-hemolytic activities (frequency, 4.8 x 10(-2)). Hemolytic isolates were analyzed for their genetic diversity by using random amplification of polymorphic DNA, yielding 88 haplotypes. The similarity coefficient of Nei and Li showed a polymorphic diversity ranging from 0.3 to 1. Additionally, 8 of the hemolytic isolates showed antifungal activity toward plant pathogens, Diaporthe phaseolorum, Colletotrichum acutatum, Rhizoctonia solani, and Fusarium oxysporum. The 16S ribosomal sequencing analysis showed that antagonistic bacterial isolates corresponded to Bacillus subtilis (UM15, UM33, UM42, UM49, UM52, and UM91), Bacillus pumilus (UM24), and Bacillus licheniformis (UM88). The present results revealed a higher genetic diversity among hemolytic isolates compared to that of isolates with antifungal action.

  16. Critical appraisal of eculizumab for atypical hemolytic uremic syndrome.

    PubMed

    Palma, Lilian M Pereira; Langman, Craig B

    2016-01-01

    The biology of atypical hemolytic uremic syndrome has been shown to involve inability to limit activation of the alternative complement pathway, with subsequent damage to systemic endothelial beds and the vasculature, resulting in the prototypic findings of a thrombotic microangiopathy. Central to this process is the formation of the terminal membrane attack complex C5b-9. Recently, application of a monoclonal antibody that specifically binds to C5, eculizumab, became available to treat patients with atypical hemolytic uremic syndrome, replacing plasma exchange or infusion as primary therapy. This review focuses on the evidence, based on published clinical trials, case series, and case reports, on the efficacy and safety of this approach.

  17. Characterization of Antibacterial and Hemolytic Activity of Synthetic Pandinin 2 Variants and Their Inhibition against Mycobacterium tuberculosis

    PubMed Central

    Rodríguez, Alexis; Villegas, Elba; Montoya-Rosales, Alejandra; Rivas-Santiago, Bruno; Corzo, Gerardo

    2014-01-01

    The contention and treatment of Mycobacterium tuberculosis and other bacteria that cause infectious diseases require the use of new type of antibiotics. Pandinin 2 (Pin2) is a scorpion venom antimicrobial peptide highly hemolytic that has a central proline residue. This residue forms a structural “kink” linked to its pore-forming activity towards human erythrocytes. In this work, the residue Pro14 of Pin2 was both substituted and flanked using glycine residues (P14G and P14GPG) based on the low hemolytic activities of antimicrobial peptides with structural motifs Gly and GlyProGly such as magainin 2 and ponericin G1, respectively. The two Pin2 variants showed antimicrobial activity against E. coli, S. aureus, and M. tuberculosis. However, Pin2 [GPG] was less hemolytic (30%) than that of Pin2 [G] variant. In addition, based on the primary structure of Pin2 [G] and Pin2 [GPG], two short peptide variants were designed and chemically synthesized keeping attention to their physicochemical properties such as hydrophobicity and propensity to adopt alpha-helical conformations. The aim to design these two short antimicrobial peptides was to avoid the drawback cost associated to the synthesis of peptides with large sequences. The short Pin2 variants named Pin2 [14] and Pin2 [17] showed antibiotic activity against E. coli and M. tuberculosis. Besides, Pin2 [14] presented only 25% of hemolysis toward human erythrocytes at concentrations as high as 100 µM, while the peptide Pin2 [17] did not show any hemolytic effect at the same concentration. Furthermore, these short antimicrobial peptides had better activity at molar concentrations against multidrug resistance M. tuberculosis than that of the conventional antibiotics ethambutol, isoniazid and rifampicin. Therefore, Pin2 [14] and Pin2 [17] have the potential to be used as an alternative antibiotics and anti-tuberculosis agents with reduced hemolytic effects. PMID:25019413

  18. Characterization of antibacterial and hemolytic activity of synthetic pandinin 2 variants and their inhibition against Mycobacterium tuberculosis.

    PubMed

    Rodríguez, Alexis; Villegas, Elba; Montoya-Rosales, Alejandra; Rivas-Santiago, Bruno; Corzo, Gerardo

    2014-01-01

    The contention and treatment of Mycobacterium tuberculosis and other bacteria that cause infectious diseases require the use of new type of antibiotics. Pandinin 2 (Pin2) is a scorpion venom antimicrobial peptide highly hemolytic that has a central proline residue. This residue forms a structural "kink" linked to its pore-forming activity towards human erythrocytes. In this work, the residue Pro14 of Pin2 was both substituted and flanked using glycine residues (P14G and P14GPG) based on the low hemolytic activities of antimicrobial peptides with structural motifs Gly and GlyProGly such as magainin 2 and ponericin G1, respectively. The two Pin2 variants showed antimicrobial activity against E. coli, S. aureus, and M. tuberculosis. However, Pin2 [GPG] was less hemolytic (30%) than that of Pin2 [G] variant. In addition, based on the primary structure of Pin2 [G] and Pin2 [GPG], two short peptide variants were designed and chemically synthesized keeping attention to their physicochemical properties such as hydrophobicity and propensity to adopt alpha-helical conformations. The aim to design these two short antimicrobial peptides was to avoid the drawback cost associated to the synthesis of peptides with large sequences. The short Pin2 variants named Pin2 [14] and Pin2 [17] showed antibiotic activity against E. coli and M. tuberculosis. Besides, Pin2 [14] presented only 25% of hemolysis toward human erythrocytes at concentrations as high as 100 µM, while the peptide Pin2 [17] did not show any hemolytic effect at the same concentration. Furthermore, these short antimicrobial peptides had better activity at molar concentrations against multidrug resistance M. tuberculosis than that of the conventional antibiotics ethambutol, isoniazid and rifampicin. Therefore, Pin2 [14] and Pin2 [17] have the potential to be used as an alternative antibiotics and anti-tuberculosis agents with reduced hemolytic effects.

  19. Rock squirrel (Spermophilus variegatus) blood sera affects proteolytic and hemolytic activities of rattlesnake venoms.

    PubMed

    Biardi, James E; Coss, Richard G

    2011-02-01

    Rock squirrels (Spermophilus variegatus) from two sites in south central New Mexico, where prairie (Crotalus viridis viridis) and western diamondback (Crotalus atrox) rattlesnakes are common predators, were assayed for inhibition of rattlesnake venom digestive and hemostatic activities. At statistically significant levels rock squirrel blood sera reduced the metalloprotease and hemolytic activity of venoms from C. v. viridis and C. atrox more than venom from an allopatric snake species, the northern Pacific rattlesnake (Crotalus oreganus). In contrast, general proteolytic activity of venom from C. oreganus was inhibited more by S. variegatus serum defenses than activity of venom from sympatric snakes. For all three venoms, incubation with squirrel sera increased the level of fibrinolysis over venom-only treatments. These results suggest that rock squirrels (S. variegatus) can defend against metalloproteases and other proteases after envenomation from at least two of five rattlesnake predators they might encounter. However, there were statistically significant differences between general proteolytic activity and fibrinolytic activity of C. v. viridis and C. atrox venom, suggesting that rock squirrels might be differentially vulnerable to these two predators. The hypothesis that prey resistance influences snake venom evolution in a predator-prey arms race is given further support by the previously cryptic variation in venoms detected when assayed against prey defenses.

  20. Complement activation in Ghanaian children with severe Plasmodium falciparum malaria

    PubMed Central

    Helegbe, Gideon K; Goka, Bamenla Q; Kurtzhals, Joergen AL; Addae, Michael M; Ollaga, Edwin; Tetteh, John KA; Dodoo, Daniel; Ofori, Michael F; Obeng-Adjei, George; Hirayama, Kenji; Awandare, Gordon A; Akanmori, Bartholomew D

    2007-01-01

    Background Severe anaemia (SA), intravascular haemolysis (IVH) and respiratory distress (RD) are severe forms of Plasmodium falciparum malaria, with RD reported to be of prognostic importance in African children with malarial anaemia. Complement factors have been implicated in the mechanism leading to excess anaemia in acute P. falciparum infection. Methods The direct Coombs test (DCT) and flow cytometry were used to investigate the mean levels of RBC-bound complement fragments (C3d and C3bαβ) and the regulatory proteins [complement receptor 1 (CD35) and decay accelerating factor (CD55)] in children with discrete clinical forms of P. falciparum malaria. The relationship between the findings and clinical parameters including coma, haemoglobin (Hb) levels and RD were investigated. Results Of the 484 samples tested, 131(27%) were positive in DCT, out of which 115/131 (87.8%) were positive for C3d alone while 16/131 (12.2%) were positive for either IgG alone or both. 67.4% of the study population were below 5 years of age and DCT positivity was more common in this age group relative to children who were 5 years or older (Odds ratio, OR = 3.8; 95%CI, 2.2–6.7, p < 0.001). DCT correlated significantly with RD (β = -304, p = 0.006), but multiple regression analysis revealed that, Hb (β = -0.341, p = 0.012) and coma (β = -0.256, p = 0.034) were stronger predictors of RD than DCT (β = 0.228, p = 0.061). DCT was also not associated with IVH, p = 0.19, while spleen size was inversely correlated with Hb (r = -402, p = 0.001). Flow cytometry showed similar mean fluorescent intensity (MFI) values of CD35, CD55 and C3bαβ levels on the surfaces of RBC in patients and asymptomatic controls (AC). However, binding of C3bαβ correlated significantly with CD35 or CD55 (p < 0.001). Conclusion These results suggest that complement activation contributed to anaemia in acute childhood P. falciparum malaria, possibly through induction of erythrophagocytosis and haemolysis. In

  1. In vitro antibacterial and hemolytic activities of crotamine, a small basic myotoxin from rattlesnake Crotalus durissus.

    PubMed

    Oguiura, Nancy; Boni-Mitake, Malvina; Affonso, Regina; Zhang, Guolong

    2011-04-01

    Crotamine, a myotoxin from the venom of South American rattlesnake, is structurally related to β-defensins, antimicrobial peptides (AMPs) found in vertebrate animals. Here, we tested the antibacterial properties of crotamine and found that it killed several strains of Escherichia coli, with the MICs ranging from 25 to 100 μg ml⁻¹. Time-kill and bacterial membrane permeabilization assays revealed that killing of bacteria by crotamine occurred within 1 h and reached the maximum by 2 h. Additionally, the anti-E. coli activity of crotamine was completely abolished with 12.5 mM NaCl. Furthermore, the three intramolecular disulfide bonds of crotamine appeared dispensable for its antibacterial activity. The reduced form of crotamine was active against E. coli as well. However, crotamine showed no or weak activity up to 200 μg ml⁻¹ against other species of Gram-negative and Gram-positive bacteria. Crotamine showed no appreciable hemolytic activity to erythrocytes. Our studies revealed that crotamine is also an AMP that kills bacteria through membrane permeabilization. However, crotamine appears to have a narrow antibacterial spectrum, distinct from many classical β-defensins, reinforcing the notion that crotamine originated from the β-defensin gene lineage, but has undergone significant functional diversification.

  2. Types of Hemolytic Anemia

    MedlinePlus

    ... from the NHLBI on Twitter. Types of Hemolytic Anemia There are many types of hemolytic anemia. The ... the condition, but you develop it. Inherited Hemolytic Anemias With inherited hemolytic anemias, one or more of ...

  3. Biological evaluation of twenty-eight ferrocenyl tetrasubstituted olefins: cancer cell growth inhibition, ROS production and hemolytic activity.

    PubMed

    de Oliveira, Alane Cabral; Hillard, Elizabeth A; Pigeon, Pascal; Rocha, Danilo Damasceno; Rodrigues, Felipe A R; Montenegro, Raquel C; Costa-Lotufo, Letícia V; Goulart, Marilia O F; Jaouen, Gérard

    2011-09-01

    The antiproliferative effects of twenty-eight tetrasubstituted olefins bearing a ferrocenyl group, including six never-reported compounds, were evaluated against SF-295 (human glioblastoma), HCT-8 (human colon cancer), MDA-MB-435 (human melanoma) and HL-60 (human promyelocytic leukemia) using the MTT test. IC(50) values were determined for twenty-three active compounds and of these, ten compounds had IC(50) values lower than 2 μM on one or more cell lines. Of all the compounds, only two produced significant amounts of ROS on HL-60 cells, and ROS production and growth inhibition could not be correlated. The ten most antiproliferative compounds were tested for their hemolytic activity on mouse erythrocytes. Five compounds showing high antiproliferative activity and low hemolytic activity were thus identified for further study.

  4. Isolation, phylogenetic analysis and screening of marine mollusc-associated bacteria for antimicrobial, hemolytic and surface activities.

    PubMed

    Romanenko, Lyudmila A; Uchino, Masataka; Kalinovskaya, Natalia I; Mikhailov, Valery V

    2008-01-01

    This study was undertaken to survey culturable heterotrophic bacteria associated with the marine ark shell Anadara broughtoni inhabiting in the Sea of Japan, and to test isolates for their antimicrobial, hemolytic and surface activities with an emphasis on low-molecular-weight metabolites search. A total of 149 strains were isolated and identified phenotypically. A total of 27 strains were selected to be investigated phylogenetically by 165 rRNA gene sequence analysis. The most bacteria were affiliated with members of the Gammaproteobacteria and Alphaproteobacteria, and Less with Firmicutes, Actinobacteria, and Cytophaga-Flavobacterium-Bacteroides (CFB) group. The isolates capable of hemolysis were numerically abundant in the genera Pseudoalteromonas, Aeromonas and Bacillus. The six Gram-positive isolates belonging to the genera Bacillus, Paenibacillus and Saccharothrix and two Gram-negative strains related to Pseudomonas and Sphingomonas, possessed antimicrobial activity against indicator strains and to each other. Antimicrobial, hemolytic and surface activities were revealed in butanot extracts of cells or cell-free supernatant of six active strains. This points to availability of active low-molecular-weight metabolites. Substances with hemolytic and surface activities were isolated from strain Bacillus pumilus An 112 and characterized as cyclic depsipeptides with molecular masses 1021, 1035, 1049, 1063 and 1077 Da. The recovery of strains producing antimicrobial and surface-active substances suggests that microorganisms associated with the marine bivalve are potential source of bioactive metabolites.

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

    PubMed Central

    1977-01-01

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

  6. Grafting synthetic transmembrane units to the engineered low-toxicity α-hemolysin to restore its hemolytic activity.

    PubMed

    Ui, Mihoko; Harima, Kousuke; Takei, Toshiaki; Tsumoto, Kouhei; Tabata, Kazuhito V; Noji, Hiroyuki; Endo, Sumire; Akiyama, Kimio; Muraoka, Takahiro; Kinbara, Kazushi

    2014-12-01

    The chemical modification of proteins to provide desirable functions and/or structures broadens their possibilities for use in various applications. Usually, proteins can acquire new functions and characteristics, in addition to their original ones, via the introduction of synthetic functional moieties. Here, we adopted a more radical approach to protein modification, i.e., the replacement of a functional domain of proteins with alternative chemical compounds to build "cyborg proteins." As a proof of concept model, we chose staphylococcal α-hemolysin (Hla), which is a well-studied, pore-forming toxin. The hemolytic activity of Hla mutants was dramatically decreased by truncation of the stem domain, which forms a β-barrel pore in the membrane. However, the impaired hemolytic activity was significantly restored by attaching a pyrenyl-maleimide unit to the cysteine residue that was introduced in the remaining stem domain. In contrast, negatively charged fluorescein-maleimide completely abolished the remaining activity of the mutants.

  7. Vegetable Peel Waste for the Production of ZnO Nanoparticles and its Toxicological Efficiency, Antifungal, Hemolytic, and Antibacterial Activities.

    PubMed

    Surendra, T V; Roopan, Selvaraj Mohana; Al-Dhabi, Naif Abdullah; Arasu, Mariadhas Valan; Sarkar, Gargi; Suthindhiran, K

    2016-12-01

    Zinc oxide (ZnO) nanoparticles (NPs) are important materials when making different products like sun screens, textiles, and paints. In the current study, the photocatalytic effect of prepared ZnO NPs from Moringa oleifera (M. oleifera) was evaluated on degradation of crystal violet (CV) dye, which is largely released from textile industries and is harmful to the environment. Preliminarily, ZnO NP formation was confirmed using a double beam ultraviolet visible (UV-Vis) spectrophotometer; further, the NP size was estimated using XRD analysis and the functional group analysis was determined using Fourier transform infrared (FT-IR) spectroscopy. The morphology of the synthesized NPs was found to be a hexagonal shape using SEM and TEM analysis and elemental screening was analyzed using EDX. ZnO NPs were shown sized 40-45 nm and spherical in shape. The degradation percentage of ZnO NPs was calculated as 94% at 70 min and the rate of the reaction -k = 0.0282. The synthesized ZnO NPs were determined for effectiveness on biological activities such as antifungal, hemolytic, and antibacterial activity. ZnO NPs showed good antifungal activity against Alternaria saloni and Sclerrotium rolfii strains. Further, we have determined the hemolytic and antibacterial activity of ZnO NPs and we got successive results in antibacterial and hemolytic activities.

  8. Vegetable Peel Waste for the Production of ZnO Nanoparticles and its Toxicological Efficiency, Antifungal, Hemolytic, and Antibacterial Activities

    NASA Astrophysics Data System (ADS)

    Surendra, T. V.; Roopan, Selvaraj Mohana; Al-Dhabi, Naif Abdullah; Arasu, Mariadhas Valan; Sarkar, Gargi; Suthindhiran, K.

    2016-12-01

    Zinc oxide (ZnO) nanoparticles (NPs) are important materials when making different products like sun screens, textiles, and paints. In the current study, the photocatalytic effect of prepared ZnO NPs from Moringa oleifera ( M. oleifera) was evaluated on degradation of crystal violet (CV) dye, which is largely released from textile industries and is harmful to the environment. Preliminarily, ZnO NP formation was confirmed using a double beam ultraviolet visible (UV-Vis) spectrophotometer; further, the NP size was estimated using XRD analysis and the functional group analysis was determined using Fourier transform infrared (FT-IR) spectroscopy. The morphology of the synthesized NPs was found to be a hexagonal shape using SEM and TEM analysis and elemental screening was analyzed using EDX. ZnO NPs were shown sized 40-45 nm and spherical in shape. The degradation percentage of ZnO NPs was calculated as 94% at 70 min and the rate of the reaction -k = 0.0282. The synthesized ZnO NPs were determined for effectiveness on biological activities such as antifungal, hemolytic, and antibacterial activity. ZnO NPs showed good antifungal activity against Alternaria saloni and Sclerrotium rolfii strains. Further, we have determined the hemolytic and antibacterial activity of ZnO NPs and we got successive results in antibacterial and hemolytic activities.

  9. Collagenase production and hemolytic activity related to 16S rRNA variability among Parvimonas micra oral isolates.

    PubMed

    Ota-Tsuzuki, Claudia; Alves Mayer, Marcia Pinto

    2010-02-01

    Parvimonas micra are gram positive anaerobic cocci isolated from the oral cavity and frequently related to polymicrobial infections in humans. Despite reports about phenotypic differences, the genotypic variation of P. micra and its role in virulence are still not elucidated. The aim of this study was to determine the genotypic diversity of P. micra isolates obtained from the subgingival biofilm of subjects with different periodontal conditions and to correlate these findings with phenotypic traits. Three reference strains and 35 isolates of P. micra were genotyped by 16S rRNA PCR-RFLP and phenotypic traits such as collagenase production, elastolytic and hemolytic activities were evaluated. 16S rRNA PCR-RFLP showed that P. micra could be grouped into two main clusters: C1 and C2; cluster C1 harbored three genotypes (HG1259-like, HG1467-like and ICBMO583-like) while cluster C2 harbored two genotypes (ATCC33270-like and ICBMO36). A wide variability in collagenolytic activity intensities was observed among all isolates, while elastolytic activity was detected in only two isolates. There was an association between hemolytic activity in rabbit erythrocytes and cluster C2. There was an association between hemolytic activity in rabbit erythrocytes and cluster C1. Although these data suggest a possible association between P. micra genetic diversity and their pathogenic potential, further investigations are needed to confirm this hypothesis.

  10. Activation of the Complement Classical Pathway (C1q Binding) by Mesophilic Aeromonas hydrophila Outer Membrane Protein

    PubMed Central

    Merino, Susana; Nogueras, Maria Mercedes; Aguilar, Alicia; Rubires, Xavier; Albertí, Sebastian; Benedí, Vicente Javier; Tomás, Juan M.

    1998-01-01

    The mechanism of killing of Aeromonas hydrophila serum-sensitive strains in nonimmune serum by the complement classical pathway has been studied. The bacterial cell surface component that binds C1q more efficiently was identified as a major outer membrane protein of 39 kDa, presumably the porin II described by D. Jeanteur, N. Gletsu, F. Pattus, and J. T. Buckley (Mol. Microbiol. 6:3355–3363, 1992), of these microorganisms. We have demonstrated that the purified form of porin II binds C1q and activates the classical pathway in an antibody-independent manner, with the subsequent consumption of C4 and reduction of the serum total hemolytic activity. Activation of the classical pathway has been observed in human nonimmune serum and agammaglobulinemic serum (both depleted of factor D). Binding of C1q to other components of the bacterial outer membrane, in particular to rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the outer membrane protein. The strains possessing O-antigen lipopolysaccharide bind less C1q than the serum-sensitive strains, because the outer membrane protein is less accessible, and are resistant to complement-mediated killing. Finally, a similar or identical outer membrane protein (presumably porin II) that binds C1q was shown to be present in strains from the most common mesophilic Aeromonas O serogroups. PMID:9673268

  11. Red wines and flavonoids diminish Staphylococcus aureus virulence with anti-biofilm and anti-hemolytic activities.

    PubMed

    Cho, Hyun Seob; Lee, Jin-Hyung; Cho, Moo Hwan; Lee, Jintae

    2015-01-01

    The emergence of antibiotic resistant Staphylococcus aureus presents a worldwide problem that requires non-antibiotic strategies. This study investigated the anti-biofilm and anti-hemolytic activities of four red wines and two white wines against three S. aureus strains. All red wines at 0.5-2% significantly inhibited S. aureus biofilm formation and hemolysis by S. aureus, whereas the two white wines had no effect. Furthermore, at these concentrations, red wines did not affect bacterial growth. Analyses of hemolysis and active component identification in red wines revealed that the anti-biofilm compounds and anti-hemolytic compounds largely responsible were tannic acid, trans-resveratrol, and several flavonoids. In addition, red wines attenuated S. aureus virulence in vivo in the nematode Caenorhabditis elegans, which is killed by S. aureus. These findings show that red wines and their compounds warrant further attention in antivirulence strategies against persistent S. aureus infection.

  12. Hemolytic and proteolytic activities of Aeromonas hydrophila and Aeromonas veronii biovar sobria in broth and salmon extract at different temperatures.

    PubMed

    González-Rodríguez, María-Nieves; Santos, Jesús A; Otero, Andrés; García-López, Maria-Luisa

    2004-02-01

    Expression of hemolytic and proteolytic activities throughout the growth cycle was investigated with two enterotoxic aeromonad strains assigned to the species Aeromonas hydrophila and Aeromonas veronii biovar sobria. Although growth kinetic data were dependent on strain, temperature, and substrate, maximum populations attained were higher than 9 log CFU/ml in aerated tryptone soya broth plus yeast extract (TSBYE) and salmon extract within the range 4 to 28 degrees C. For both strains in TSBYE, variable amounts of hemolytic activity were first detected at any temperature when aeromonad counts were over 9 log CFU/ml. Afterwards, this activity increased up to similar levels (109 to 112 hemolytic units per ml) without a significant increase in populations. Salmon extract supported hemolysin synthesis at 28 but not 4 degrees C. Proteolytic activity of the A. hydrophila strain was only expressed in salmon extract at 28 degrees C, whereas A. veronii biovar sobria did at 28 degrees C in both substrates and at 10 degrees C in TSBYE.

  13. Complement factor B activation in patients with preeclampsia.

    PubMed

    Velickovic, Ivan; Dalloul, Mudar; Wong, Karen A; Bakare, Olufunke; Schweis, Franz; Garala, Maya; Alam, Amit; Medranda, Giorgio; Lekovic, Jovana; Shuaib, Waqas; Tedjasukmana, Andreas; Little, Perry; Hanono, Daniel; Wijetilaka, Ruvini; Weedon, Jeremy; Lin, Jun; Toledano, Roulhac d'Arby; Zhang, Ming

    2015-06-01

    Preeclampsia is a leading cause of maternal and fetal morbidity and mortality. Bb, the active fragment of complement factor B (fB), has been reported to be a predictor of preeclampsia. However, conflicting results have been found by some investigators. We hypothesized that the disagreement in findings may be due to the racial/ethnic differences among various study groups, and that fB activation is significant in women of an ethnic minority with preeclampsia. We investigated the maternal and fetal levels of Bb (the activated fB fragment) in pregnant women of an ethnic minority with or without preeclampsia. We enrolled 291 pregnant women (96% of an ethnic minority, including 78% African-American). Thirteen percent of these were diagnosed with preeclampsia. Maternal venous blood was collected from all participants together with fetal umbilical cord blood samples from 154 deliveries in the 291 women. The results were analyzed using the Mann-Whitney U test and multivariate analyses. Maternal Bb levels were significantly higher in the preeclamptic group than in the nonpreeclamptic group. Levels of Bb in fetal cord blood were similar in both groups. Subgroup analyses of African-American patients' results confirmed the study hypothesis that there would be a significant increase in Bb in the maternal blood of the preeclamptic group and no increase in Bb in the fetal cord blood of this group. These results suggest that a maternal immune response through complement fB might play a role in the development of preeclampsia, particularly in African-American patients.

  14. Expression of activated molecules on CD5(+)B lymphocytes in autoimmune hemolytic anemia.

    PubMed

    Zhu, Hongli; Xu, Wenyan; Liu, Hong; Wang, Huaquan; Fu, Rong; Wu, Yuhong; Qu, Wen; Wang, Guojin; Guan, Jing; Song, Jia; Xing, Limin; Shao, Zonghong

    2016-05-01

    To investigate the expression of activation molecules on CD5(+)B lymphocytes in peripheral blood of autoimmune hemolytic anemia (AIHA)/Evans patients. The expression of CD80, CD86, and CD69 on CD5(+)B lymphocytes was detected using flow cytometry in 30 AIHA/Evans patients, 18 normal controls (NC) and nine chronic lymphocytic leukemia (CLL) patients. CD80 on CD5(+)B lymphocytes in untreated patients was higher than that in remission patients (P < 0.05), NC (P < 0.01) and CLL patients (P < 0.01). CD80 on CD5(+)B lymphocytes was higher than that on CD5(-)B lymphocytes in untreated patients (P > 0.05), but lower than those of CD5(-)B lymphocytes in remission patients and NC (P < 0.05). CD86 on CD5(+)B lymphocytes of untreated patients was higher than that of remission patients (P < 0.05), NC (P < 0.01). CD86 on CD5(+)B lymphocytes of CLL was higher than that of NC, remission (P < 0.05), and untreated patients (P > 0.05). CD80 and CD86 on CD5(+)B lymphocytes was negatively correlated with hemoglobin (HB), C3, C4 (P < 0.05) and positively correlated with reticulocyte (Ret) (P < 0.05). CD69 on CD5(+) and CD5(-)B lymphocytes of CLL was higher than those of AIHA/Evans patients and NC (P < 0.05). The active molecules on CD5(+)B lymphocytes in peripheral blood of AIHA/Evans patients differ from those on CD5(-) and clonal CD5(+)B lymphocytes.

  15. Biological effects of short-term, high-concentration exposure to methyl isocyanate. VI. In vitro and in vivo complement activation studies.

    PubMed Central

    Kolb, W P; Savary, J R; Troup, C M; Dodd, D E; Tamerius, J D

    1987-01-01

    The ability of MIC to induce complement activation in vitro and in vivo was investigated. For the in vitro studies, both human and guinea pig serum or EDTA-plasma samples were exposed to 1167 to 1260 ppm MIC vapor for 15 min at room temperature. The human serum samples exposed to MIC showed significant reductions in Factor B, C2, C4, C3, C5, and total hemolytic complement CH50 activity levels. C6 functional activity was unaffected. The C3, C5, and CH50 functional activities in guinea pig serum (the only functional tests conducted on these samples) were more sensitive to MIC-mediated reduction than the corresponding activity reductions observed in the human serum samples. The human and single guinea pig EDTA-plasma samples exposed to MIC vapor showed no evidence of C3 consumption but did show significant reductions in CH50 levels. Thus, MIC vapor was able to activate, and thereby reduce serum complement C3 activity in vitro by a complement-dependent process. However, the data suggest at least one complement component other than C3 was inactivated in EDTA-plasma by a complement-independent mechanism. For the in vivo studies, five pairs of guinea pigs were exposed to 644 to 702 ppm MIC vapor until one of the pair died (11-15 min). MIC exposure was then discontinued, the surviving guinea pig was sacrificed, and EDTA-plasma was obtained from both animals and analyzed for complement consumption.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3622434

  16. Glycoproteins, antigens, and regulation of complement activation on the surface of the protozoan parasite Trypanosoma lewisi: implications for immune evasion

    SciTech Connect

    Sturtevant, J.E.

    1985-01-01

    The surface antigens and glycoproteins of the rat parasitic protozoan, Trypanosoma lewisi were characterized. Radioiodination with /sup 125/I identified 10 out of more 40 polypeptides separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis. All of these components were identified as glycoproteins by peroxidase-conjugated Conconavalin A (HR-Con A) lectin affinoblotting. This analysis detected that quantitative but not qualitative changes occurred during infection. Localization of most of the reactive determinants was indicated by immunoblotting extracts of radioiodinated T. lewisi. Changes in the antigenicity as related to survival in the host are discussed. The presence of IgG and IgM on the surface of T. lewisi isolated from intact and ..gamma..-irradiated rats (irr.) and that determinants bind Ig from uninfected rat sera (NRS) was indicated by flow cytometric analysis. Immunoblotting identified the major NRS IgG binding component as the 74 kd surface glycoprotein. Complement component C3 deposition during infection was indicated by flow cytometric analysis and immunoblotting. Incubation of intact T. lewisi with normal human sera indicated that C3, C5, and factor B deposition was Mg/sup 2 +/ dependent, Ca/sup 2 +/ independent and deposited C3 was rapidly processed to hemolytically inactive fragments. Radioiodination of intact and protease T. lewisi after cultivation identified three components which correlate with resistance to lysis. This suggests that surface moieties on intact T. lewisi modulate host complement activity by restricting C3/C5 convertase activity.

  17. Acute kidney injury in a patient with hemolytic anemia and thrombocytopenia.

    PubMed

    Breunig, Michael; Lalama, Miguel; Rivard, Gabrielle; Kashiwagi, Deanne; Cornell, Lynn

    2016-11-01

    Atypical hemolytic uremic syndrome (HUS) is clinically difficult to distinguish from HUS and thrombotic thrombocytopenic purpura. Atypical HUS results from dysregulation of complement activation causing thrombotic microangiopathy affecting multiple organ systems. Atypical HUS is associated with high morbidity and mortality, making early recognition and appropriate therapy necessary to improve patient outcomes.

  18. Chemical analysis and hemolytic activity of the fava bean aglycon divicine.

    PubMed

    McMillan, D C; Schey, K L; Meier, G P; Jollow, D J

    1993-01-01

    Divicine is an unstable aglycon metabolite of the fava bean pyrimidine beta-glucoside vicine. Divicine has long been thought to be a mediator of an acute hemolytic crisis, known as favism, in susceptible individuals who ingest fava beans (Vicia faba). However, a recent report has questioned the chemical identity of the divicine that was used in most of the studies on divicine hemotoxicity. The present study was undertaken to examine the hemolytic potential of synthetic divicine. Divicine was synthesized and its identity and purity were confirmed by HPLC, mass spectrometry, and NMR spectroscopy. The stability and redox behavior of divicine, under physiological conditions, were examined by HPLC and cyclic voltammetry. The data indicate that divicine is readily oxidized under aerobic conditions; however, it was sufficiently stable at pH 7.4 to permit its experimental manipulation. When 51Cr-labeled rat erythrocytes were exposed in vitro to the parent glucoside, vicine (5 mM), and then readministered to rats, no decrease in erythrocyte survival was observed. In contrast, erythrocyte survival was dramatically reduced by in vitro exposure to divicine (1.5 mM). These data demonstrate that divicine is a direct-acting hemolytic agent and thus may be a mediator of the hemolytic crisis induced by fava bean ingestion.

  19. Elastase and metalloproteinase activities regulate soluble complement receptor 1 release.

    PubMed

    Sadallah, S; Hess, C; Miot, S; Spertini, O; Lutz, H; Schifferli, J A

    1999-11-01

    Complement receptor 1 (CR1) is cleaved from the surface of polymorphonuclear cells (PMN) in the membrane-proximal region to yield a soluble fragment (sCR1) that contains the functional domains. The enzymes involved in this cleavage are produced by the PMN itself, since in vitro stimulation of purified PMN is followed by sCR1 release. Purified human neutrophil elastase (HNE) cleaved CR1 from erythrocytes and urinary vesicles originating from podocytes and enhanced tenfold the cleavage of CR1 from activated PMN. The largest fragment released from PMN by HNE was identical in size to CR1 shed spontaneously. The CR1 fragments cleaved from erythrocytes were functional. The shedding of sCR1 by activated PMN was inhibited by phenylmethylsulfonyl fluoride (80 +/- 10%), alpha1-antiprotease (50 +/- 5%) and elafin (60 +/- 5%). Furthermore the cleavage was blocked by the metalloprotease inhibitor 1,10-phenanthroline (70 +/- 6 %) as well as by a monoclonal antibody against human neutrophil collagenase MMP8 (40 +/- 10%). Maximal inhibition of sCR1 shedding was obtained by a combination of 1,10-phenanthroline with elafin (86 +/- 6%). These inhibitors had no effect on L-selectin shedding, indicating that the cleavage of CR1 was specific. In conclusion, elastase or elastase-like activity may be responsible for the shedding of functional sCR1 in vivo, and this activity is controlled by the local release of PMN metalloproteases and alpha1antiprotease.

  20. Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A.

    PubMed Central

    Carroll, M C; Fathallah, D M; Bergamaschini, L; Alicot, E M; Isenman, D E

    1990-01-01

    The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)--namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the alpha chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed (ester vs. amide). Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not "catalytic" as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to "select" binding sites on potential acceptor molecules. Images PMID:2395880

  1. The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

    PubMed

    Shewell, Lucy K; Harvey, Richard M; Higgins, Melanie A; Day, Christopher J; Hartley-Tassell, Lauren E; Chen, Austen Y; Gillen, Christine M; James, David B A; Alonzo, Francis; Torres, Victor J; Walker, Mark J; Paton, Adrienne W; Paton, James C; Jennings, Michael P

    2014-12-09

    The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

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

    PubMed Central

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

    2015-01-01

    Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1–5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3–1 deletion (CFHR3–1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype–phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3–1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN. PMID:25205734

  3. A novel anti-inflammatory activity of lysozyme: modulation of serum complement activation.

    PubMed Central

    Ogundele, M O

    1998-01-01

    Lysozyme is an ubiquitous enzyme found in most biological secretions and leukocytes. This study was aimed at investigating its interaction with other inflammatory mediators on mucosa surfaces, particularly the complement system. Lysozyme has been shown in our present study, to inhibit the haemolytic activity of serum complement in a dose-dependent fashion, when tested within the levels present in normal and inflamed breast-milk samples, and other mucosal secretions. This represents a new anti-inflammatory action of lysozyme in relation to the serum complement, and the exact mode of the interaction need further studies. PMID:9883972

  4. Calcium-chelating alizarin and other anthraquinones inhibit biofilm formation and the hemolytic activity of Staphylococcus aureus

    PubMed Central

    Lee, Jin-Hyung; Kim, Yong-Guy; Yong Ryu, Shi; Lee, Jintae

    2016-01-01

    Staphylococcal biofilms are problematic and play a critical role in the persistence of chronic infections because of their abilities to tolerate antimicrobial agents. Thus, the inhibitions of biofilm formation and/or toxin production are viewed as alternative means of controlling Staphylococcus aureus infections. Here, the antibiofilm activities of 560 purified phytochemicals were examined. Alizarin at 10 μg/ml was found to efficiently inhibit biofilm formation by three S. aureus strains and a Staphylococcus epidermidis strain. In addition, two other anthraquinones purpurin and quinalizarin were found to have antibiofilm activity. Binding of Ca2+ by alizarin decreased S. aureus biofilm formation and a calcium-specific chelating agent suppressed the effect of calcium. These three anthraquinones also markedly inhibited the hemolytic activity of S. aureus, and in-line with their antibiofilm activities, increased cell aggregation. A chemical structure-activity relationship study revealed that two hydroxyl units at the C-1 and C-2 positions of anthraquinone play important roles in antibiofilm and anti-hemolytic activities. Transcriptional analyses showed that alizarin repressed the α-hemolysin hla gene, biofilm-related genes (psmα, rbf, and spa), and modulated the expressions of cid/lrg genes (the holin/antiholin system). These findings suggest anthraquinones, especially alizarin, are potentially useful for controlling biofilm formation and the virulence of S. aureus. PMID:26763935

  5. An effective treatment of atypical hemolytic uremic syndrome with plasma exchange and eculizumab: A case report.

    PubMed

    Sengul Samanci, Nilay; Ayer, Mesut; Ergen, Abdulkadir; Ozturk, Savas

    2015-06-01

    Atypical hemolytic uremic syndrome is a rare thrombotic microangiopathy caused by chronic defective regulation of the complement activation. This activation results in systemic endothelial damage leading to renal failure. Eculizumab, an anti-C5 antibody, is effective in limiting complement activation in patients with aHUS and has recently came out as a therapeutic option for aHUS. Here we present a case showing that first-line eculizumab treatment successfully prevents the induction of the terminal complement cascade and blocked the progression of thrombotic microangiopathy in aHUS.

  6. [Ligand spectrum of hemoglobin activity of methemoglobin-reductase and hemolytic resistance of erythrocytes during chronic exposure to nitrates].

    PubMed

    Kiiza, D A; Artiukh, V P; Starodub, N F; Khmel'nitskiĭ, G A

    1992-01-01

    It is found that nitrite-ions formed as a result of biotransformation during long term feeding of calves with sodium and potassium nitrates induce changes in some biochemical parameters of blood, including HS-glutathione content in erythrocytes, acid hemolytic resistance of erythrocytes, activity of NAD-dependent methemoglobin-reductase, correlation of ligand forms of hemoglobin and its total content. It is supposed that the observed changes are of an adaptational character and, as a whole, provide for the optimization of both quantitative and qualitative composition of population of erythroid cells at the expense of erythropoiesis intensification.

  7. High Antimicrobial Effectiveness with Low Hemolytic and Cytotoxic Activity for PEG/Quaternary Copolyoxetanes

    PubMed Central

    2015-01-01

    The alkyl chain length of quaternary ammonium/PEG copolyoxetanes has been varied to discern effects on solution antimicrobial efficacy, hemolytic activity and cytotoxicity. Monomers 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) were used to prepare precursor P[(BBOx)(ME2Ox)-50:50–4 kDa] copolyoxetane via cationic ring opening polymerization. The 1:1 copolymer composition and Mn (4 kDa) were confirmed by 1H NMR spectroscopy. After C–Br substitution by a series of tertiary amines, ionic liquid Cx-50 copolyoxetanes were obtained, where 50 is the mole percent of quaternary repeat units and “x” is quaternary alkyl chain length (2, 6, 8, 10, 12, 14, or 16 carbons). Modulated differential scanning calorimetry (MDSC) studies showed Tgs between −40 and −60 °C and melting endotherms for C14–50 and C16–50. Minimum inhibitory concentrations (MIC) were determined for Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. A systematic dependence of MIC on alkyl chain length was found. The most effective antimicrobials were in the C6–50 to C12–50 range. C8–50 had better overall performance with MICs of 4 μg/mL, E. coli; 2 μg/mL, S. aureus; and 24 μg/mL, P. aeruginosa. At 5 × MIC, C8–50 effected >99% kill in 1 h against S. aureus, E. coli, and P. aeruginosa challenges of 108 cfu/mL; log reductions (1 h) were 7, 3, and 5, respectively. To provide additional insight into polycation interactions with bacterial membranes, a geometric model based on the dimensions of E. coli is described that provides an estimate of the maximum number of polycations that can chemisorb. Chain dimensions were estimated for polycation C8–50 with a molecular weight of 5 kDa. Considering the approximations for polycation chemisorption (PCC), it is surprising that a calculation based on geometric considerations gives a C8–50 concentration within a factor of 2 of the MIC, 4.0 (±1.2) μg/mL for

  8. Inhibitory role of acyl homoserine lactones in hemolytic activity and viability of Streptococcus pyogenes M6 S165

    PubMed Central

    Saroj, Sunil D.; Holmer, Linda; Berengueras, Júlia M.; Jonsson, Ann-Beth

    2017-01-01

    Streptococcus pyogenes an adapted human pathogen asymptomatically colonizes the nasopharynx, among other polymicrobial communities. However, information on the events leading to the colonization and expression of virulence markers subject to interspecies and host-bacteria interactions are limited. The interference of acyl homoserine lactones (AHLs) with the hemolytic activity and viability of S. pyogenes M6 S165 was examined. AHLs, with fatty acid side chains ≥12 carbon atoms, inhibited hemolytic activity by downregulating the expression of the sag operon involved in the production of streptolysin S. Inhibitory AHLs upregulated the expression of transcriptional regulator LuxR. Electrophoretic mobility shift assays revealed the interaction of LuxR with the region upstream of sagA. AHL-mediated bactericidal activity observed at higher concentrations (mM range) was an energy-dependent process, constrained by the requirement of glucose and iron. Ferrichrome transporter FtsABCD facilitated transport of AHLs across the streptococcal membrane. The study demonstrates a previously unreported role for AHLs in S. pyogenes virulence. PMID:28303956

  9. Relationship between heat-induced fibrillogenicity and hemolytic activity of thermostable direct hemolysin and a related hemolysin of Vibrio parahaemolyticus.

    PubMed

    Ohnishi, Kiyouhisa; Nakahira, Kumiko; Unzai, Satoru; Mayanagi, Kouta; Hashimoto, Hiroshi; Shiraki, Kentaro; Honda, Takeshi; Yanagihara, Itaru

    2011-05-01

    The formation of nonspecific ion channels by small oligomeric amyloid intermediates is toxic to the host's cellular membranes. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of Vibrio parahaemolyticus. We have previously reported the crystal structure of TDH tetramer with the central channel. We have also identified the molecular mechanism underlying the paradoxical responses to heat treatment of TDH, known as the Arrhenius effect, which is the reversible amyloidogenic property. In the present report, we describe the biophysical properties of TRH, which displays 67% amino acid similarity with TDH. Molecular modeling provided a good fit of the overall structure of TDH and TRH. Size-exclusion chromatography, ultracentrifugation, and transmission electron microscopy revealed that TRH formed tetramer in solution. These toxins showed similar hemolytic activity on red blood cells. However, TRH had less amyloid-like structure than TDH analyzed by thioflavin T-binding assay and far-UV circular dichroism spectra. These data indicated that amyloidogenicity upon heating is not essential for the membrane disruption of erythrocytes, but the maintenance of tetrameric structure is indispensable for the hemolytic activity of the TDH and TRH.

  10. Complement activity is associated with disease severity in multifocal motor neuropathy

    PubMed Central

    Vlam, Lotte; Cats, Elisabeth A.; Harschnitz, Oliver; Jansen, Marc D.; Piepers, Sanne; Veldink, Jan Herman; Franssen, Hessel; Stork, Abraham C.J.; Heezius, Erik; Rooijakkers, Suzan H.M.; Herpers, Bjorn L.; van Strijp, Jos A.; van den Berg, Leonard H.

    2015-01-01

    Objective: To investigate whether high innate activity of the classical and lectin pathways of complement is associated with multifocal motor neuropathy (MMN) and whether levels of innate complement activity or the potential of anti-GM1 antibodies to activate the complement system correlate with disease severity. Methods: We performed a case-control study including 79 patients with MMN and 79 matched healthy controls. Muscle weakness was documented with Medical Research Council scale sum score and axonal loss with nerve conduction studies. Activity of the classical and lectin pathways of complement was assessed by ELISA. We also determined serum mannose-binding lectin (MBL) concentrations and polymorphisms in the MBL gene (MBL2) and quantified complement-activating properties of anti-GM1 IgM antibodies by ELISA. Results: Activity of the classical and lectin pathways, MBL2 genotypes, and serum MBL concentrations did not differ between patients and controls. Complement activation by anti-GM1 IgM antibodies was exclusively mediated through the classical pathway and correlated with antibody titers (p < 0.001). Logistic regression analysis showed that both high innate activity of the classical pathway of complement and high complement-activating capacity of anti-GM1 IgM antibodies were significantly associated with more severe muscle weakness and axonal loss. Conclusion: High innate activity of the classical pathway of complement and efficient complement-activating properties of anti-GM1 IgM antibodies are determinants of disease severity in patients with MMN. These findings underline the importance of anti-GM1 antibody–mediated complement activation in the pathogenesis and clinical course of MMN. PMID:26161430

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

    PubMed

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

    2015-08-01

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

  12. The bacteria binding glycoprotein salivary agglutinin (SAG/gp340) activates complement via the lectin pathway.

    PubMed

    Leito, Jelani T D; Ligtenberg, Antoon J M; van Houdt, Michel; van den Berg, Timo K; Wouters, Diana

    2011-10-01

    Salivary agglutinin (SAG), also known as gp-340 and Deleted in Malignant Brain Tumours 1, is a glycoprotein that is present in tears, lung fluid and mucosal surfaces along the gastrointestinal tract. It is encoded by the Deleted in Malignant Brain Tumours 1 gene, a member of the Scavenger Receptor Cysteine Rich group B protein superfamily. SAG aggregates bacteria thus promoting their clearance from the oral cavity and activates the complement system. Complement proteins may enter the oral cavity in case of serum leakage, which occurs after mucosal damage. The purpose of this study was to investigate the mode of complement activation. We showed a dose-dependent C4 deposition on SAG-coated microplates showing that either the classical or lectin pathway of complement was activated. Antibodies against mannose binding lectin inhibited C4 deposition and SAG induced no C4 deposition in MBL deficient sera showing SAG activated complement through the MBL pathway. Periodate treatment of SAG abolished MBL pathway activation consistent with an involvement of SAG glycans in complement activation. This provides the first evidence for a role of SAG in complement activation through the MBL pathway and suggests a potential role of SAG as a complement activating factor at the mucosal epithelia.

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

    PubMed

    Lamichhane, D; Weinstein, A

    2016-08-01

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

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

    PubMed

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

    2014-01-15

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

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

    PubMed

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

    2013-11-01

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

  16. Different hydroxyapatite magnetic nanoparticles for medical imaging: Its effects on hemostatic, hemolytic activity and cellular cytotoxicity.

    PubMed

    Laranjeira, Marta S; Moço, Ana; Ferreira, Jorge; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Ferreira, Paulo J; Monteiro, Fernando J

    2016-10-01

    Magnetic nanoparticles (MNPs) should be highly biocompatible, stable and safely eliminated from the body, and can therefore be successfully used in modern medicine. Synthetic hydroxyapatite (HAP) has well established biocompatible and non-inflammatory properties, as well as a highly stable and flexible structure that allows for an easy incorporation of magnetic ions. This study characterized and compared the in vitro cytotoxicity and hemocompatibility of hydroxyapatite MNPs doped with different ions (Gd(3+/)Fe(2+)/Fe(3+)/Co(2+)). HAP doped with 10% of Gd and Fe(III) presented the highest magnetic moments. Our results showed that Gd doped HAP nanoparticles are non-cytotoxic, hemocompatible, non-hemolytic and non-thrombogenic, in contrast with Fe(III) doped HAP that can be considered thrombogenic. For these reasons we propose that, Gd doped HAP nanoparticles have the most potential for application as a MRI contrast agents. However, use of Fe (III) doped HAP as MRI contrast agents should be further investigated.

  17. The effects of soluble recombinant complement receptor 1 on complement-mediated experimental glomerulonephritis.

    PubMed

    Couser, W G; Johnson, R J; Young, B A; Yeh, C G; Toth, C A; Rudolph, A R

    1995-05-01

    Complement is a major mediator of tissue injury in several types of glomerulonephritis. However, no therapeutic agents that inhibit complement activation are available for human use. sCR1 (TP10, BRL 55736) is a recombinant, soluble human complement receptor 1 (CR1) molecule lacking transmembrane and cytoplasmic domains that inhibits C3 and C5 convertase activity by preferentially binding C4b and C3b. To test the efficacy of sCR1 on complement-mediated glomerulonephritis, rats were pretreated with sCR1 (60 mg/kg per day) before and during the induction of three models of complement-dependent glomerulonephritis (concanavalin A and antithymocyte serum models of proliferative glomerulonephritis, passive Heyman nephritis). Daily sCR1 and complement hemolytic activity levels were measured, and renal histology and urine protein excretion were examined. Mean serum sCR1 levels of 100 to 200 micrograms/mL were maintained with a reduction in complement hemolytic activity to less than 15% in most animals. In the antithymocyte serum model, sCR1-treated animals had significant reductions in mesangiolysis, glomerular platelet and macrophage infiltrates, and proteinuria at 48 h. In the concanavalin A model, sCR1 significantly reduced glomerular C3 and fibrin deposits, platelet infiltrates, and proteinuria at 48 h. In passive Heymann nephritis, proteinuria was also significantly reduced (199 +/- 8.5 versus 125 +/- 16 mg/day, P < 0.002) at 5 days. It was concluded that sCR1 significantly reduces both morphologic and functional consequences of several different types of complement-mediated glomerulonephritis and deserves evaluation as a potential therapeutic agent in complement-mediated immune glomerular disease in humans.

  18. Critical appraisal of eculizumab for atypical hemolytic uremic syndrome

    PubMed Central

    Palma, Lilian M Pereira; Langman, Craig B

    2016-01-01

    The biology of atypical hemolytic uremic syndrome has been shown to involve inability to limit activation of the alternative complement pathway, with subsequent damage to systemic endothelial beds and the vasculature, resulting in the prototypic findings of a thrombotic microangiopathy. Central to this process is the formation of the terminal membrane attack complex C5b-9. Recently, application of a monoclonal antibody that specifically binds to C5, eculizumab, became available to treat patients with atypical hemolytic uremic syndrome, replacing plasma exchange or infusion as primary therapy. This review focuses on the evidence, based on published clinical trials, case series, and case reports, on the efficacy and safety of this approach. PMID:27110144

  19. Activation of complement pathways after contusion-induced spinal cord injury.

    PubMed

    Anderson, Aileen J; Robert, Stephanie; Huang, Wencheng; Young, Wise; Cotman, Carl W

    2004-12-01

    Previous studies have shown that a cellular inflammatory response is initiated, and inflammatory cytokines are synthesized, following experimental spinal cord injury (SCI). In the present study, we tested the hypothesis that the complement cascade, a major component of both the innate and adaptive immune response, is also activated following experimental SCI. We investigated the pathways, cellular localization, timecourse, and degree of complement activation in rat spinal cord following acute contusion-induced SCI using the New York University (NYU) weight drop impactor. Mild and severe injuries (12.5 and 50 mm drop heights) at 1, 7, and 42 days post injury time points were evaluated. Classical (C1q and C4), alternative (Factor B) and terminal (C5b-9) complement pathways were strongly activated within 1 day of SCI. Complement protein immunoreactivity was predominantly found in cell types vulnerable to degeneration, neurons and oligodendrocytes, and was not generally observed in inflammatory or astroglial cells. Surprisingly, immunoreactivity for complement proteins was also evident 6 weeks after injury, and complement activation was observed as far as 20 mm rostral to the site of injury. Axonal staining by C1q and Factor B was also observed, suggesting a potential role for the complement cascade in demyelination or axonal degeneration. These data support the hypothesis that complement activation plays a role in SCI.

  20. Flavonoids from the leaves of Litsea japonica and their anti-complement activity.

    PubMed

    Lee, Sun-Young; Min, Byung-Sun; Kim, Jung-Hee; Lee, Joongku; Kim, Tae-Jin; Kim, Chan-Soo; Kim, Young-Ho; Lee, Hyeong-Kyu

    2005-04-01

    Four flavonoids, epicatechin (1), afzelin (2), quercitrin (3), and tiliroside (4), were isolated from the leaves of Litsea japonica (Thunb.) Jussieu (Lauraceae). The structures of compounds were identified by comparing their chemical and spectral data with those previously reported. The flavonoids (1-4) were tested for their anti-complement activity against classical pathway of complement system. Compounds 2-4 showed inhibitory activity against complement system with IC50 values of 258, 440, and 101 microm, respectively, whereas 1 was inactive. For the evaluation of the structure-activity relationship of 5,7-dihydroxyflavones, myricitrin (5) from Juglans mandshurica also tested for it's anti-complement activity and is inactive in this assay system. Furthermore, compounds 2, 3, and 5 were hydrolyzed with naringinase to give kaempferol (2a), quercetin (3a), and myricetin (5a), and these were also tested for their activity. Of the three aglycones, 2a exhibited anti-complement activity with an IC50 value of 730 microM, while 3a and 5a were inactive. The inhibitory potencies of 2, 2a, 3, 3a, 5, and 5a against complement activity increased in inverse proportion to number of free hydroxyls on B-ring of 5,7-dihydroxyflavone. Of the compounds tested, 4 showed the most potent inhibitory activity against the complement system.

  1. Antibacterial and hemolytic activities of linenscin OC2, a hydrophobic substance produced by Brevibacterium linens OC2.

    PubMed

    Boucabeille, C; Mengin-Lecreulx, D; Henckes, G; Simonet, J M; van Heijenoort, J

    1997-08-15

    Linenscin OC2 is an antibacterial substance produced by the orange cheese coryneform bacterium Brevibacterium linens OC2. It inhibits the growth of Gram-positive bacteria but it is inactive against Gram-negative bacteria. The intact outer membrane of Gram-negative bacteria was shown to be an effective permeability barrier against linenscin OC2. At high dosage the effect of linenscin OC2 was bacteriolytic on Listeria innocua. Bacteriostasis was observed at low dosage and peptidoglycan biosynthesis was affected at an early step upstream of the UDP-N-acetylglucosamine. Hemolytic activity of this substance on sheep erythrocytes suggested a common mode of action on prokaryotic and eukaryotic cells. It also suggested that the cytoplasmic membrane might be the primary target of linenscin OC2.

  2. Inactivation of complement by Loxosceles reclusa spider venom.

    PubMed

    Gebel, H M; Finke, J H; Elgert, K D; Cambell, B J; Barrett, J T

    1979-07-01

    Zymosan depletion of serum complement in guinea pigs rendered them highly resistant to lesion by Loxosceles reclusa spider venom. Guinea pigs deficient in C4 of the complement system are as sensitive to the venom as normal guinea pigs. The injection of 35 micrograms of whole recluse venom intradermally into guinea pigs lowered their complement level by 35.7%. Brown recluse spider venom in concentrations as slight as 0.02 micrograms protein/ml can totally inactivate one CH50 of guinea pig complement in vitro. Bee, scorpion, and other spider venoms had no influence on the hemolytic titer of complement. Fractionation of recluse spider venom by Sephadex G-200 filtration separated the complement-inactivating property of the venom into three major regions which could be distinguished on the basis of heat stability as well as size. None was neutralized by antivenom. Polyacrylamide gel electrophoresis of venom resolved the complement inactivators into five fractions. Complement inactivated by whole venom or the Sephadex fractions could be restored to hemolytic activity by supplements of fresh serum but not by heat-inactivated serum, pure C3, pure C5, or C3 and C5 in combination.

  3. Recombinant Complement Receptor 2 Radiolabeled with [99mTc(CO)3]+ : A Potential New Radiopharmaceutical for Imaging Activated Complement

    PubMed Central

    McDonnell, James M.; Yahya, Norhakim; Thakor, David; Razavi, Reza; Smith, Richard; Sacks, Steven; Mullen, Gregory E. D.

    2011-01-01

    We describe the design and synthesis of a new Tc-99m labeled bioconjugate for imaging activated complement, based on Short Consensus Repeats 1 and 2 of Complement Receptor 2 (CR2), the binding domain for C3d. To avoid non specific modification of CR2 and the potential for modifying lysine residues critical to the CR2/C3d contact surface, we engineered a new protein, recombinant CR2 (rCR2), to include the C-terminal sequence VFPLECHHHHHH, a hexahistidine tag (for site-specific radiolabeling with [99mTc(CO)3(OH2)3]+). The protein was characterized by N-terminal sequencing, SDS-PAGE and size exclusion chromatography. To test the function of the recombinant CR2, binding to C3d was confirmed by enzyme-linked immunosorbent assay (ELISA). The function was further confirmed by binding of rCR2 to C3d+ red blood cells (RBC) which were generated by deposition of human or rat C3d and analyzed by fluorescence microscopy and flow cytometry. The affinity of rCR2 for C3d+, in presence of 150 mM NaCl, was measured using surface plasma resonance giving rise to a KD≈500 nM. Radiolabeling of rCR2 or an inactive mutant of rCR2 (K41E CR2) or an unrelated protein of a similar size (C2A) with [99mTc(CO)3(OH2)3]+ at gave radiochemical yields >95%. Site-specifically radiolabeled rCR2 bound to C3d to C3d+ RBC. Binding of radiolabeled rCR2 to C3d was inhibited by anti-C3d and the radiolabeled inactive mutant K41E CR2 and C2A did not bind to C3d+ RBCs. We conclude that rCR2-Tc99m has excellent radiolabeling, stability and C3d binding characteristics and warrants in vivo evaluation as an activated complement imaging agent. PMID:21494666

  4. High Fc Density Particles Result in Binary Complement Activation but Tunable Macrophage Phagocytosis

    NASA Astrophysics Data System (ADS)

    Sulchek, Todd; Pacheco, Patricia; White, David

    2014-03-01

    Macrophage phagocytosis and complement system activation represent two key components of the immune system and both can be activated through the presentation of multiple Fc domains of IgG antibodies. We have created functionalized micro- and nanoparticles with various densities of Fc domains to understand the modulation of the immune system for eventual use as a novel immunomodulation platform. Phagocytosis assays were carried out by adding functionalized particles to macrophage cells and quantitatively determined using fluorescent microscopy and flow cytometry. Complement system activation by the functionalized particles in human serum was quantified with an enzyme immunoassay. Our phagocytosis assay revealed a strong dependence on particle size and Fc density. For small particles, as the Fc density increased, the number of particles phagocytosed also increased. Large particles were phagocytosed at significantly lower levels and showed no dependency on Fc density. Complement was successfully activated at levels comparable to positive controls for small particles at high Fc densities. However at low Fc densities, there is a significant decrease in complement activation. This result suggests a binary response for complement system activation with a threshold density for successful activation. Therefore, varying the Fc density on micro/nanoparticles resulted in a tunable response in macrophage phagocytosis while a more binary response for complement activation.

  5. Classical and lectin complement pathway activity in polyneuropathy associated with IgM monoclonal gammopathy.

    PubMed

    Stork, Abraham C J; Cats, Elisabeth A; Vlam, Lotte; Heezius, Erik; Rooijakkers, Suzan; Herpers, Bjorn; de Jong, Ben A W; Rijkers, Ger; van Strijp, Jos; Notermans, Nicolette C; van den Berg, Leonard H; van der Pol, W-Ludo

    2016-01-15

    Polyneuropathy associated with IgM monoclonal gammopathy (IgM-PNP) is a slowly progressive, sensorimotor neuropathy. It is assumed that complement activation contributes to IgM-PNP pathogenesis. We investigated whether innate differences in complement activity of the classical and mannose binding lectin (MBL) pathways are associated with IgM-PNP or its severity. We measured complement activity using ELISA and determined MBL serumc oncentrations and MBL gene polymorphisms in 83 patients and 83 healthy controls. We did not observe differences between IgM-PNP patients and healthy controls nor associations with different disease severities. Differences in innate complement activity are not likely to explain susceptibility to or severity of IgM-PNP.

  6. 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

  7. The Structure-Activity Relationship between Marine Algae Polysaccharides and Anti-Complement Activity

    PubMed Central

    Jin, Weihua; Zhang, Wenjing; Liang, Hongze; Zhang, Quanbin

    2015-01-01

    In this study, 33 different polysaccharides were prepared to investigate the structure-activity relationships between the polysaccharides, mainly from marine algae, and anti-complement activity in the classical pathway. Factors considered included extraction methods, fractionations, molecular weight, molar ratio of galactose to fucose, sulfate, uronic acid (UA) content, linkage, branching, and the type of monosaccharide. It was shown that the larger the molecular weights, the better the activities. The molar ratio of galactose (Gal) to fucose (Fuc) was a positive factor at a concentration lower than 10 µg/mL, while it had no effect at a concentration more than 10 µg/mL. In addition, sulfate was necessary; however, the sulfate content, the sulfate pattern, linkage and branching had no effect at a concentration of more than 10 µg/mL. Moreover, the type of monosaccharide had no effect. Laminaran and UA fractions had no activity; however, they could reduce the activity by decreasing the effective concentration of the active composition when they were mixed with the active compositions. The effect of the extraction methods could not be determined. Finally, it was observed that sulfated galactofucan showed good anti-complement activity after separation. PMID:26712768

  8. The Structure-Activity Relationship between Marine Algae Polysaccharides and Anti-Complement Activity.

    PubMed

    Jin, Weihua; Zhang, Wenjing; Liang, Hongze; Zhang, Quanbin

    2015-12-25

    In this study, 33 different polysaccharides were prepared to investigate the structure-activity relationships between the polysaccharides, mainly from marine algae, and anti-complement activity in the classical pathway. Factors considered included extraction methods, fractionations, molecular weight, molar ratio of galactose to fucose, sulfate, uronic acid (UA) content, linkage, branching, and the type of monosaccharide. It was shown that the larger the molecular weights, the better the activities. The molar ratio of galactose (Gal) to fucose (Fuc) was a positive factor at a concentration lower than 10 µg/mL, while it had no effect at a concentration more than 10 µg/mL. In addition, sulfate was necessary; however, the sulfate content, the sulfate pattern, linkage and branching had no effect at a concentration of more than 10 µg/mL. Moreover, the type of monosaccharide had no effect. Laminaran and UA fractions had no activity; however, they could reduce the activity by decreasing the effective concentration of the active composition when they were mixed with the active compositions. The effect of the extraction methods could not be determined. Finally, it was observed that sulfated galactofucan showed good anti-complement activity after separation.

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

    PubMed Central

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

    2015-01-01

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

  10. New Insights in the Pathogenesis of Autoimmune Hemolytic Anemia

    PubMed Central

    Barcellini, Wilma

    2015-01-01

    Summary Autoimmune hemolytic anemia (AIHA) is caused by the increased destruction of red blood cells (RBCs) by anti-RBC autoantibodies with or without complement activation. RBC destruction may occur both by a direct lysis through the sequential activation of the final components of the complement cascade (membrane attack complex), or by antibody-dependent cell-mediated cytotoxicity (ADCC). The pathogenic role of autoantibodies depends on their class (the most frequent are IgG and IgM), subclass, thermal amplitude (warm and cold forms),as well as affinity and efficiency in activating complement. Several cytokines and cytotoxic mechanisms (CD8+ T and natural killer cells) are further involved in RBC destruction. Moreover, activated macrophages carrying Fc receptors may recognize and phagocyte erythrocytes opsonized by autoantibodies and complement. Direct complement-mediated lysis takes place mainly in the circulations and liver, whereas ADCC, cytotoxicity, and phagocytosis occur preferentially in the spleen and lymphoid organs. The degree of intravascular hemolysis is 10-fold greater than extravascular one. Finally, the efficacy of the erythroblastic compensatory response can greatly influence the clinical picture of AIHA. The interplay and relative burden of all these pathogenic mechanisms give reason for the great clinical heterogeneity of AIHAs, from fully compensated to rapidly evolving fatal cases. PMID:26696796

  11. 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.

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

    PubMed Central

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

    2015-01-01

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

  13. Interrelation Between Oxidative Stress and Complement Activation in Models of Age-Related Macular Degeneration.

    PubMed

    Pujol-Lereis, Luciana M; Schäfer, Nicole; Kuhn, Laura B; Rohrer, Bärbel; Pauly, Diana

    2016-01-01

    Millions of individuals older than 50-years suffer from age-related macular degeneration (AMD). Associated with this multifactorial disease are polymorphisms of complement factor genes and a main environmental risk factor-oxidative stress. Until now the linkage between these risk factors for AMD has not been fully understood. Recent studies, integrating results on oxidative stress, complement activation, epidemiology and ocular pathology suggested the following sequence in AMD-etiology: initially, chronic oxidative stress results in modification of proteins and lipids in the posterior of the eye; these tissue alterations trigger chronic inflammation, involving the complement system; and finally, invasive immune cells facilitate pathology in the retina. Here, we summarize the results for animal studies which aim to elucidate this molecular interplay of oxidative events and tissue-specific complement activation in the eye.

  14. Evidence for intrathecal synthesis of alternative pathway complement activation proteins in experimental meningitis.

    PubMed Central

    Stahel, P. F.; Frei, K.; Fontana, A.; Eugster, H. P.; Ault, B. H.; Barnum, S. R.

    1997-01-01

    Complement has been shown to contribute to intrathecal inflammation in bacterial meningitis. However, the cellular source of complement in the infected central nervous system has not been determined. In this study, we analyzed protein and mRNA expression of two alternative pathway complement activation proteins, C3 and factor B, in the brains of mice with Listeria monocytogenes meningitis. Complement protein levels were found elevated in the cerebrospinal fluid of infected mice, compared with mock-infected animals. In the course of the disease, enhanced C3 and factor B mRNA expression was detected on pyramidal neurons and Purkinje cells within 6 hours, peaking at 12 hours and then gradually decreasing by 72 hours after infection. In addition, leukocytes infiltrating the subarachnoid space, within 12 to 24 hours, expressed mRNA for C3 and factor B. The cellular infiltration increased dramatically up to 72 hours. Intraperitoneal injection of tumor necrosis factor (TNF)-alpha up-regulated C3 and factor B mRNA expression on neurons in normal mice, suggesting that TNF-alpha may represent one cytokine regulating complement expression in this model of bacterial meningitis. However, additional mediators may be involved in regulation of intrathecal complement expression, as infected mice deficient of TNF/lymphotoxin-alpha genes did not demonstrate attenuated complement expression in the brain. Images Figure 1 Figure 2 Figure 3 PMID:9327721

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

    PubMed

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

    1990-05-04

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

  16. Effects of L-arginine immobilization on the anticoagulant activity and hemolytic property of polyethylene terephthalate films

    NASA Astrophysics Data System (ADS)

    Liu, Yun; Yang, Yun; Wu, Feng

    2010-04-01

    Surface modification of polyethylene terephthalate (PET) films was performed with L-arginine ( L-Arg) to gain an improved anticoagulant surface. The surface chemistry changes of modified films were characterized by X-ray photoelectron spectroscopy (XPS) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. The in vitro anticoagulant activities of the surface-modified PET films were evaluated by blood clotting test, hemolytic test, and the measurement of clotting time including plasma recalcification time (PRT), activated partial thromboplastin time (APTT), and prothrombin time (PT). The data of blood coagulation index (BCI) for L-arginine modified PET films (PET-Arg) was larger than that for PET at the same blood-sample contact time. The hemolysis ratio for PET-Arg was less than that for PET and within the accepted standard for biomaterials. The PRT and APTT for PET-Arg were significantly prolonged by 189 s and 25 s, respectively, compared to those for the unmodified PET. All results suggested that the currently described modification method could be a possible candidate to create antithrombogenic PET surfaces which would be useful for further medical applications.

  17. Healing of complement activating Ti implants compared with non-activating Ti in rat tibia.

    PubMed

    Harmankaya, N; Igawa, K; Stenlund, P; Palmquist, A; Tengvall, P

    2012-09-01

    Recent studies have revealed that ozone ultraviolet (UVO) illumination of titanium (Ti) implants improves bone-implant anchorage by altering the physico-chemical and immune activating properties of the titanium dioxide (TiO(2)) layer. In the present rat tibia model, the authors compared the early events of inflammation and bone formation around UVO-treated Ti and complement activating immunoglobin g (IgG)-coated Ti. Machined Ti and machined Ti coated with a physical vapour-deposited Ti layer were used as references. Screw-shaped test and reference implants were implanted into rat tibia and harvested after 1, 7 and 28 days. Messenger RNA expression of implant adhered cells and peri-implant tissue ~250 μm from the surface were subsequently analysed with regard to IL-1β, TNF-α, osteocalcin, cathepsin K, BMP-2 and PDGF. Separate implants were retrieved after 7 and 28 days for removal torque measurements, and histological staining and histomorphometric analysis of bone area and bone-to-implant contact. While enhanced expression of inflammatory markers, TNF-α and IL-1β, was observed on IgG-coated surfaces throughout the observation time, UVO-treated surfaces indicated a significantly lower early inflammatory response. In the early phases (1 and 7 days), the UVO-treated surfaces displayed a significantly higher expression of osteoblast markers BMP-2 and osteocalcin. In summary, complement activating Ti implants elicited a stronger inflammatory response than UVO-treated Ti, with low complement activation during the first week of healing. In spite of this, the UVO-treated Ti induced only marginally more bone growth outside the implants.

  18. Isolation and characterization of a complement-activating lipid extracted from human atherosclerotic lesions

    PubMed Central

    1990-01-01

    The major characteristics of human atherosclerotic lesions are similar to those of a chronic inflammatory reaction, namely fibrosis, mesenchymal cell proliferation, the presence of resident macrophages, and cell necrosis. Atherosclerosis exhibits in addition the feature of lipid (mainly cholesterol) accumulation. The results of the present report demonstrate that a specific cholesterol-containing lipid particle present in human atherosclerotic lesions activates the complement system to completion. Thus, lipid could represent a stimulatory factor for the inflammatory reaction, whose underlying mechanistic basis may be, at least in part, complement activation. The complement-activating lipid was purified from saline extracts of aortic atherosclerotic lesions by sucrose density gradient centrifugation followed by molecular sieve chromatography on Sepharose 2B. It contained little protein other than albumin, was 100-500 nm in size, exhibited an unesterified to total cholesterol ratio of 0.58 and an unesterified cholesterol to phospholipid ratio of 1.2. The lipid, termed lesion lipid complement (LCA), activated the alternative pathway of complement in a dose-dependent manner. Lesion-extracted low density lipoprotein (LDL) obtained during the purification procedure failed to activate complement. Specific generation of C3a desArg and C5b-9 by LCA indicated C3/C5 convertase formation with activation proceeding to completion. Biochemical and electron microscopic evaluations revealed that much of the C5b-9 present in atherosclerotic lesions is membraneous, rather than fluid phase SC5b-9. The observations reported herein establish a link between lipid insudation and inflammation in atherosclerotic lesions via the mechanism of complement activation. PMID:2373993

  19. Clinical Practice Guidelines for the Management of Atypical Hemolytic Uremic Syndrome in Korea

    PubMed Central

    2016-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare syndrome characterized by micro-angiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. The major pathogenesis of aHUS involves dysregulation of the complement system. Eculizumab, which blocks complement C5 activation, has recently been proven as an effective agent. Delayed diagnosis and treatment of aHUS can cause death or end-stage renal disease. Therefore, a diagnosis that differentiates aHUS from other forms of thrombotic microangiopathy is very important for appropriate management. These guidelines aim to offer recommendations for the diagnosis and treatment of patients with aHUS in Korea. The guidelines have largely been adopted from the current guidelines due to the lack of evidence concerning the Korean population. PMID:27550478

  20. Effect of Complement on HIV-2 Plasma Antiviral Activity Is Intratype Specific and Potent

    PubMed Central

    Özkaya Şahin, Gülşen; Holmgren, Birgitta; Sheik-Khalil, Enas; da Silva, Zacarias; Nielsen, Jens; Nowroozalizadeh, Salma; Månsson, Fredrik; Norrgren, Hans; Aaby, Peter; Fenyö, Eva Maria

    2013-01-01

    Human immunodeficiency virus type 2 (HIV-2)-infected individuals develop immunodeficiency with a considerable delay and transmit the virus at rates lower than HIV-1-infected persons. Conceivably, comparative studies on the immune responsiveness of HIV-1- and HIV-2-infected hosts may help to explain the differences in pathogenesis and transmission between the two types of infection. Previous studies have shown that the neutralizing antibody response is more potent and broader in HIV-2 than in HIV-1 infection. In the present study, we have examined further the function of the humoral immune response and studied the effect of complement on the antiviral activity of plasma from singly HIV-1- or HIV-2-infected individuals, as well as HIV-1/HIV-2 dually infected individuals. The neutralization and antibody-dependent complement-mediated inactivation of HIV-1 and HIV-2 isolates were tested in a plaque reduction assay using U87.CD4.CCR5 cells. The results showed that the addition of complement increased intratype antiviral activities of both HIV-1 and HIV-2 plasma samples, although the complement effect was more pronounced with HIV-2 than HIV-1 plasma. Using an area-under-the-curve (AUC)-based readout, multivariate statistical analysis confirmed that the type of HIV infection was independently associated with the magnitude of the complement effect. The analyses carried out with purified IgG indicated that the complement effect was largely exerted through the classical complement pathway involving IgG in both HIV-1 and HIV-2 infections. In summary, these findings suggest that antibody binding to HIV-2 structures facilitates the efficient use of complement and thereby may be one factor contributing to a strong antiviral activity present in HIV-2 infection. PMID:23077299

  1. SALSA: A Regulator of the Early Steps of Complement Activation on Mucosal Surfaces.

    PubMed

    Reichhardt, Martin Parnov; Meri, Seppo

    2016-01-01

    Complement is present mainly in blood. However, following mechanical damage or inflammation, serous exudates enter the mucosal surfaces. Here, the complement proteins interact with other endogenous molecules to keep microbes from entering the parenteral tissues. One of the mucosal proteins known to interact with the early complement components of both the classical and the lectin pathway is the salivary scavenger and agglutinin (SALSA). SALSA is also known as deleted in malignant brain tumors 1 and gp340. It is found both attached to the epithelium and secreted into the surrounding fluids of most mucosal surfaces. SALSA has been shown to bind directly to C1q, mannose-binding lectin, and the ficolins. Through these interactions SALSA regulates activation of the complement system. In addition, SALSA interacts with surfactant proteins A and D, secretory IgA, and lactoferrin. Ulcerative colitis and Crohn's disease are examples of diseases, where complement activation in mucosal tissues may occur. This review describes the latest advances in our understanding of how the early complement components interact with the SALSA molecule. Furthermore, we discuss how these interactions may affect disease propagation on mucosal surfaces in immunological and inflammatory diseases.

  2. Systemic Administration of Induced Neural Stem Cells Regulates Complement Activation in Mouse Closed Head Injury Models

    PubMed Central

    Gao, Mou; Dong, Qin; Yao, Hui; Lu, Yingzhou; Ji, Xinchao; Zou, Mingming; Yang, Zhijun; Xu, Minhui; Xu, Ruxiang

    2017-01-01

    Complement activation plays important roles in the pathogenesis of central nervous system (CNS) diseases. Patients face neurological disorders due to the development of complement activation, which contributes to cell apoptosis, brain edema, blood-brain barrier dysfunction and inflammatory infiltration. We previously reported that induced neural stem cells (iNSCs) can promote neurological functional recovery in closed head injury (CHI) animals. Remarkably, we discovered that local iNSC grafts have the potential to modulate CNS inflammation post-CHI. In this study, we aimed to explore the role of systemically delivered iNSCs in complement activation following CNS injury. Our data showed that iNSC grafts decreased the levels of sera C3a and C5a and down-regulated the expression of C3d, C9, active Caspase-3 and Bax in the brain, kidney and lung tissues of CHI mice. Furthermore, iNSC grafts decreased the levels of C3d+/NeuN+, C5b-9+/NeuN+, C3d+/Map2+ and C5b-9+/Map2+ neurons in the injured cortices of CHI mice. Subsequently, we explored the mechanisms underlying these effects. With flow cytometry analysis, we observed a dramatic increase in complement receptor type 1-related protein y (Crry) expression in iNSCs after CHI mouse serum treatment. Moreover, both in vitro and in vivo loss-of-function studies revealed that iNSCs could modulate complement activation via Crry expression. PMID:28383046

  3. 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

  4. Atypical Hemolytic Uremic Syndrome and Chronic Ulcerative Colitis Treated with Eculizumab

    PubMed Central

    Webb, Tennille N.; Griffiths, Heidi; Miyashita, Yosuke; Bhatt, Riha; Jaffe, Ronald; Moritz, Michael; Hofer, Johannes; Swiatecka-Urban, Agnieszka

    2016-01-01

    Background Hemolytic-uremic syndrome (HUS) presents with hemolytic anemia, thrombocytopenia, and thrombotic microangiopathy of the kidney and usually results from Shiga-toxin induced activation of the alternative complement pathway. Gastroenteritis is a common feature of the Shiga-toxin producing Escherichia coli HUS, referred to as STEC-HUS. An inherited or acquired complement dysregulation may lead to HUS referred to as non-STEC or atypical (a)HUS. Although gastroenteritis is not a common presentation of aHUS, some patients develop ischemic colitis and may be misdiagnosed as acute appendicitis or acute ulcerative colitis (UC). Case Diagnosis –Treatment We present a patient with low circulating complement (C) 3 levels who developed aHUS in the course of chronic active UC. Resolution of renal and gastrointestinal manifestations in response to treatment with eculizumab, a humanized monoclonal antibody against terminal C5 protein suggests the role of alternative complement in the pathogenesis of both, aHUS and UC. Conclusion This case illustrates that dysregulation of the alternative complement pathway may manifest in other organs besides the kidney and that the circulating C3 levels do not correlate with the disease activity or the clinical response to eculizumab. PMID:27135055

  5. Functional Contributions of Positive Charges in the Pore-Lining Helix 3 of the Bordetella pertussis CyaA-Hemolysin to Hemolytic Activity and Ion-Channel Opening

    PubMed Central

    Kurehong, Chattip; Kanchanawarin, Chalermpol; Powthongchin, Busaba; Prangkio, Panchika; Katzenmeier, Gerd; Angsuthanasombat, Chanan

    2017-01-01

    The Bordetella pertussis CyaA-hemolysin (CyaA-Hly) domain was previously demonstrated to be an important determinant for hemolysis against target erythrocytes and ion-channel formation in planar lipid bilayers (PLBs). Here, net-charge variations in the pore-lining helix of thirteen related RTX cytolysins including CyaA-Hly were revealed by amino acid sequence alignments, reflecting their different degrees of hemolytic activity. To analyze possible functional effects of net-charge alterations on hemolytic activity and channel formation of CyaA-Hly, specific mutations were made at Gln574 or Glu581 in its pore-lining α3 of which both residues are highly conserved Lys in the three highly active RTX cytolysins (i.e., Escherichia coli α-hemolysin, Actinobacillus pleuropneumoniae toxin, and Aggregatibacter actinomycetemcomitans leukotoxin). All six constructed CyaA-Hly mutants that were over-expressed in E. coli as 126 kDa His-tagged soluble proteins were successfully purified via immobilized Ni2+-affinity chromatography. Both positive-charge substitutions (Q574K, Q574R, E581K, E581R) and negative-charge elimination (E581Q) appeared to increase the kinetics of toxin-induced hemolysis while the substitution with a negatively-charged side-chain (Q574E) completely abolished its hemolytic activity. When incorporated into PLBs under symmetrical conditions (1.0 M KCl, pH 7.4), all five mutant toxins with the increased hemolytic activity produced clearly-resolved single channels with higher open probability and longer lifetime than the wild-type toxin, albeit with a half decrease in their maximum conductance. Molecular dynamics simulations for 50 ns of a trimeric CyaA-Hly pore model comprising three α2-loop-α3 transmembrane hairpins revealed a significant role of the positive charge at both target positions in the structural stability and enlarged diameter of the simulated pore. Altogether, our present data have disclosed functional contributions of positively-charged side

  6. Functional Contributions of Positive Charges in the Pore-Lining Helix 3 of the Bordetella pertussis CyaA-Hemolysin to Hemolytic Activity and Ion-Channel Opening.

    PubMed

    Kurehong, Chattip; Kanchanawarin, Chalermpol; Powthongchin, Busaba; Prangkio, Panchika; Katzenmeier, Gerd; Angsuthanasombat, Chanan

    2017-03-16

    The Bordetella pertussis CyaA-hemolysin (CyaA-Hly) domain was previously demonstrated to be an important determinant for hemolysis against target erythrocytes and ion-channel formation in planar lipid bilayers (PLBs). Here, net-charge variations in the pore-lining helix of thirteen related RTX cytolysins including CyaA-Hly were revealed by amino acid sequence alignments, reflecting their different degrees of hemolytic activity. To analyze possible functional effects of net-charge alterations on hemolytic activity and channel formation of CyaA-Hly, specific mutations were made at Gln(574) or Glu(581) in its pore-lining α3 of which both residues are highly conserved Lys in the three highly active RTX cytolysins (i.e., Escherichia coli α-hemolysin, Actinobacillus pleuropneumoniae toxin, and Aggregatibacter actinomycetemcomitans leukotoxin). All six constructed CyaA-Hly mutants that were over-expressed in E. coli as 126 kDa His-tagged soluble proteins were successfully purified via immobilized Ni(2+)-affinity chromatography. Both positive-charge substitutions (Q574K, Q574R, E581K, E581R) and negative-charge elimination (E581Q) appeared to increase the kinetics of toxin-induced hemolysis while the substitution with a negatively-charged side-chain (Q574E) completely abolished its hemolytic activity. When incorporated into PLBs under symmetrical conditions (1.0 M KCl, pH 7.4), all five mutant toxins with the increased hemolytic activity produced clearly-resolved single channels with higher open probability and longer lifetime than the wild-type toxin, albeit with a half decrease in their maximum conductance. Molecular dynamics simulations for 50 ns of a trimeric CyaA-Hly pore model comprising three α2-loop-α3 transmembrane hairpins revealed a significant role of the positive charge at both target positions in the structural stability and enlarged diameter of the simulated pore. Altogether, our present data have disclosed functional contributions of positively

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

    ERIC Educational Resources Information Center

    Dengler, Mary

    2008-01-01

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

  8. Enhanced complement activation is part of the unfavourable cardiovascular risk profile in South Asians

    PubMed Central

    Siezenga, M A; Chandie Shaw, P K; van der Geest, R N; Mollnes, T E; Daha, M R; Rabelink, T J; Berger, S P

    2009-01-01

    South Asian immigrants in western societies exhibit a high burden of diabetes and subsequent vascular complications. Diabetic vascular complications are associated with vascular inflammation. We hypothesize that enhanced complement activation is involved. Therefore, levels of complement C3 and SC5b-9 – the soluble end product of complement activation – in a group of 200 South Asians were compared with an age- and sex-matched control group of native Caucasians. In addition, the association between complement levels and albuminuria, an indicator of renal damage and a cardiovascular risk marker, was assessed in the diabetic South Asian group. Compared with native Caucasians, South Asians had significantly higher levels of both serum C3 and plasma SC5b-9, even when only non-diabetic South Asians were considered. Diabetic South Asians had significantly higher C3 levels compared with non-diabetic South Asians. In diabetic South Asians, higher levels of SC5b-9 were associated with an increased prevalence of albuminuria (odds ratio 5·4, 95% confidence interval 1·8–15·8). These results suggest that enhanced complement activation is part of the unfavourable cardiovascular risk profile in South Asians. PMID:19659775

  9. Demonstration of alternative and classical complement pathway activity in colostrum from buffalo (Bubalus bubalis).

    PubMed

    Matheswaran, K; Dhinakar Raj, G; Nachimuthu, K

    2003-09-01

    Buffalo colostrum caused lysis of unsensitized red blood cells (RBC) from sheep, goats, rabbits and chickens. RBC from cattle and buffalo were resistant to lysis. That lysis was due to the presence of natural antibodies to these RBC was ruled out since there was no reduction in haemolytic titres even after adsorption with the respective RBC. The addition of EGTA to the diluent had no effect on the haemolytic activity. These findings indicate the presence of alternative complement pathway (ACP) activity in buffalo colostrum. The haemolytic activity of buffalo complement for unsensitized rabbit RBC was reduced to very low levels by heating at 50 degrees C for 45 min. Treatment with zymosan also inhibited the haemolytic activity, while inulin had no effect. The maximum activity of ACP occurred in the presence of 4 mmol/L Mg(2+) in the diluent. The range of ACP activities in colostrum from buffaloes varied from 4.06 to 8.48 CH50 units/ml. Using a standard system for titrating the classical complement pathway and rabbit red blood cells sensitized with goat haemolysin, the range of complement activity in buffalo colostrum was 4.81-6.77 CH50/ml.

  10. Complement System Part II: Role in Immunity

    PubMed Central

    Merle, Nicolas S.; Noe, Remi; Halbwachs-Mecarelli, Lise; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T.

    2015-01-01

    The complement system has been considered for a long time as a simple lytic cascade, aimed to kill bacteria infecting the host organism. Nowadays, this vision has changed and it is well accepted that complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. This review discusses recent advances in the understanding of the role of complement in physiology and pathology. It starts with a description of complement contribution to the normal physiology (homeostasis) of a healthy organism, including the silent clearance of apoptotic cells and maintenance of cell survival. In pathology, complement can be a friend or a foe. It acts as a friend in the defense against pathogens, by inducing opsonization and a direct killing by C5b–9 membrane attack complex and by triggering inflammatory responses with the anaphylatoxins C3a and C5a. Opsonization plays also a major role in the mounting of an adaptive immune response, involving antigen presenting cells, T-, and B-lymphocytes. Nevertheless, it can be also an enemy, when pathogens hijack complement regulators to protect themselves from the immune system. Inadequate complement activation becomes a disease cause, as in atypical hemolytic uremic syndrome, C3 glomerulopathies, and systemic lupus erythematosus. Age-related macular degeneration and cancer will be described as examples showing that complement contributes to a large variety of conditions, far exceeding the classical examples of diseases associated with complement deficiencies. Finally, we discuss complement as a therapeutic target. PMID:26074922

  11. Carboxyhemoglobin elevation due to hemolytic anemia.

    PubMed

    Hampson, Neil B

    2007-07-01

    A critically ill man with drug-induced hemolytic anemia and hepatic failure was hospitalized at a private academic medical center in Seattle, Washington. Intravascular hemolysis with associated endogenous carbon monoxide (CO) production resulted in elevation of the patient's carboxyhemoglobin (COHb) level to as high as 9.7%. Serial measurements of the patient's COHb level were obtained and compared with other conventional measures of hemolytic activity. With the availability of new non-invasive measurement technology to detect COHb elevations, emergency clinicians are likely to see COHb elevation as a manifestation of hemolytic anemia.

  12. Effect of calcium on the hemolytic activity of Stichodactyla helianthus toxin sticholysin II on human erythrocytes.

    PubMed

    Celedón, Gloria; González, Gustavo; Lissi, Eduardo; Cerda, Tania; Martinez, Diana; Soto, Carmen; Pupo, Mario; Pazos, Fabiola; Lanio, Maria E; Alvarez, Carlos

    2009-11-01

    Sticholysin II (St II) is a toxin from the sea anemona Stichodactyla helianthus that produces erythrocytes lysis at low concentration and its activity depends on the presence of calcium. Calcium may act modifying toxin interaction with erythrocyte membranes or activating cellular processes which may result in a modified St II lytic action. In this study we are reporting that, in the presence of external K(+), extracellular calcium decreased St II activity on erythrocytes. On the other hand an increase of intracellular calcium promotes Sty II lytic activity. The effect of intracellular calcium was specifically studied in relation to membrane lipid translocation elicited by scramblases and how this action influence St II lytic activity on erythrocytes. We used 0.5 mmol/L calcium and 10 mmol/L A23187, as calcium ionophore, for scramblases activation and found increased St II activity associated to increase of intracellular calcium. N-ethyl maleimide (activator) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (inhibitor) were used as scramblases modulators in the assays which produced an increase and a decrease of the calcium effect, respectively. Results reported suggest an improved St II membrane pore-forming capacity promoted by intracellular calcium associated to membrane phospholipids translocation.

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

    PubMed

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

    2013-07-01

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

  14. Soluble human complement receptor type 1 inhibits complement-mediated host defense.

    PubMed

    Swift, A J; Collins, T S; Bugelski, P; Winkelstein, J A

    1994-09-01

    Soluble complement receptor type 1 (sCR1) is a powerful inhibitor of complement activation. Because of this ability, sCR1 may prove to be an important therapeutic agent that can be used to block the immunopathologic effects of uncontrolled complement activation in a variety of clinically significant disorders. Although several previous studies have examined the ability of sCR1 to inhibit complemented-mediated immunopathologic damage, there is no information on its ability to interfere with the host's defense against infection. In the current experiments sCR1 exerted a concentration-dependent inhibitory effect on the phagocytosis of Streptococcus pneumoniae by human polymorphonuclear leukocytes in vitro. Not only di sCR1 inhibit complement-dependent opsonization of the pneumococcus but at higher concentrations it also inhibited the ingestion of bacteria which had been previously opsonized. Furthermore, when rats were injected with sCR1, it inhibited both their serum hemolytic activity and serum opsonic activity in a dose-dependent fashion. Finally, for rats treated with sCR1, the 50% lethal dose was S. pneumoniae and Pseudomonas aeruginosa. These data demonstrate that sCR1 significantly inhibits complement-mediated host against bacterial infection.

  15. Association of Vibrio parahaemolyticus thermostable direct hemolysin with lipid rafts is essential for cytotoxicity but not hemolytic activity.

    PubMed

    Matsuda, Shigeaki; Kodama, Toshio; Okada, Natsumi; Okayama, Kanna; Honda, Takeshi; Iida, Tetsuya

    2010-02-01

    Thermostable direct hemolysin (TDH), a major virulence factor of Vibrio parahaemolyticus, induces cytotoxicity in cultured cells. However, the mechanism of TDH's cytotoxic effect including its target molecules on the plasma membrane of eukaryotic cells remains unclear. In this study, we identified the role of lipid rafts, cholesterol- and sphingolipid-enriched microdomains, in TDH cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (MbetaCD), a raft-disrupting agent, inhibited TDH cytotoxicity. TDH was associated with detergent-resistant membranes (DRMs), and MbetaCD eliminated this association. In contrast, there was no such association between a nontoxic TDH mutant and DRMs. The disruption of lipid rafts neither affected hemolysis nor inhibited Ca(2+) influx into HeLa cells induced by TDH. These findings indicate that the cytotoxicity but not the hemolytic activity of TDH is dependent on lipid rafts. The exogenous and endogenous depletion of cellular sphingomyelin also prevented TDH cytotoxicity, but a direct interaction between TDH and sphingomyelin was not detected with either a lipid overlay assay or a liposome absorption test. Treatment with sphingomyelinase (SMase) at 100 mU/ml disrupted the association of TDH with DRMs but did not affect the localization of lipid raft marker proteins (caveolin-1 and flotillin-1) with DRMs. These results suggest that sphingomyelin is important for the association of TDH with lipid rafts but is not a molecular target of TDH. We hypothesize that TDH may target a certain group of rafts that are sensitive to SMase at a certain concentration, which does not affect other types of rafts.

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

    PubMed

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

    2009-01-02

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

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

    PubMed Central

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

    1993-01-01

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

  18. Annexin A2 Enhances Complement Activation by Inhibiting Factor H1

    PubMed Central

    Renner, Brandon; Tong, Hua Hua; Laskowski, Jennifer; Jonscher, Karen; Goetz, Lindsey; Woolaver, Rachel; Hannan, Jonathan; Li, Yong Xing; Hourcade, Dennis; Pickering, Matthew C.; Holers, V. Michael; Thurman, Joshua M.

    2015-01-01

    Factor H is a circulating protein that regulates activation of the alternative pathway (AP) of complement. Mutations and genetic variations of factor H are associated with several AP-mediated diseases, highlighting the critical role of factor H in AP regulation. AP-mediated inflammation is typically triggered by illness or tissue injury, however, and tissue injury can trigger AP activation in individuals with fully functional factor H. This suggests that factor H function is affected by local conditions within tissues. We hypothesized that inducible proteins impair the ability of factor H to locally control the AP, thereby increasing AP activation. We used purified murine factor H to immunoprecipitate binding partners from mouse kidneys. Using immunoaffinity liquid chromatography-mass spectrometry we then identified annexin A2 as a factor H binding partner. Further experiments showed that annexin A2 reduces the binding of factor H to cell surfaces. Recombinant annexin A2 impaired complement regulation by factor H, and increased complement activation on renal cell surfaces in vitro and in vivo. In a murine model of acute pneumococcal otitis media the administration of annexin A2 increased AP-mediated bacterial opsonization and clearance. In conclusion, the local production of annexin A2 within tissues suppresses regulation of the AP by factor H. Annexin A2 can contribute to AP-mediated tissue inflammation by locally impairing factor H function, but annexin A2 can also improve complement-mediated bacterial clearance. PMID:26729803

  19. Complement activation by polyethoxylated pharmaceutical surfactants: Cremophor-EL, Tween-80 and Tween-20.

    PubMed

    Weiszhár, Zsóka; Czúcz, Judit; Révész, Csaba; Rosivall, László; Szebeni, János; Rozsnyay, Zoltán

    2012-03-12

    Immunosafety analysis of pharmaceutical surfactants is an important step in understanding the complex mechanisms by which they induce side effects in susceptible patients. This paper provides experimental evidences that polyethoxylated surfactants, Cremophor-EL and Tween-80, also known as Polysorbate-80, activate the complement system in vitro, in normal human serum and plasma. They appeared to be more efficient reactogens than their structural homolog, Tween-20. Cremophor-EL and Tween-80 promoted the generation of biologically active complement products, C3a, C5a and C5b-9. Consistently, Paclitaxel and Taxotere (Docetaxel), pharmaceuticals formulated in Cremophor-EL and Tween-80, activated the complement system in similar extent. Moreover, comparison of serum reactivity against the drug-loaded and drug-free formulations exhibited a significant linear correlation. Taken together, these results are consistent with the hypothesis that therapeutic side effects, such as acute hypersensitivity and systemic immunostimulation, caused by intravenous nanomedicines containing polyethoxylated detergents such as Cremophor-EL and Tween-80, can be attributed to complement activation-derived inflammatory mediators.

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

    PubMed

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

    2008-12-01

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

  1. Antibodies to glycolipids activate complement and promote proteinuria in passive Heymann nephritis.

    PubMed

    Susani, M; Schulze, M; Exner, M; Kerjaschki, D

    1994-04-01

    Passive Heymann nephritis is an experimental rat model of human membranous nephropathy induced by injection of antisera against crude renal cortical fractions such as Fx1A or rat tubular microvilli. This results in the formation of subepithelial immune deposits, the activation of the C5b-9 membrane attack complex of complement, and severe proteinuria. While the formation of immune deposits is attributed to in situ immune complex formation with antibodies specific for the gp330-Heymann nephritis antigenic complex (HNAC), activation of complement and proteinuria appear to be caused by at least one additional antibody species present in anti-Fx1A sera. We have separated by affinity absorption polyspecific antisera against Fx1A and rat microvilli into one IgG fraction directed specifically against microvillar proteins (anti-Fx1A-prot) and another IgG fraction specific for glycolipids (ant-Fx1A-lip) of tubular microvilli. When injected into rats, the anti-Fx1A-prot fraction induced immune deposits but failed to activate complement or produce proteinuria, similar to results obtained with affinity-purified anti-gp330 IgG. When the antibodies of the anti-Fx1A-lip fraction were injected alone they did not bind to glomeruli. By contrast, when the IgGs specific for the Fx1A-prot fraction (or for gp330-HNAC) were combined with those directed against the Fx1A-lip glycolipid preparation, immune deposits were formed, in situ complement activation was observed, and also proteinuria was induced. It is concluded that within anti-Fx1A and anti-microvillar sera there are at least two IgG fractions of relevance for the development of PHN: one directed against the gp330-HNAC complex which is responsible for the development of immune deposits, and a second specific for glycolipid antigen(s) which activate(s) the complement cascade.

  2. 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

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

    PubMed Central

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

    2008-01-01

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

  4. Targeting complement activation in brain-dead donors improves renal function after transplantation.

    PubMed

    Damman, Jeffrey; Hoeger, Simone; Boneschansker, Leo; Theruvath, Ashok; Waldherr, Ruediger; Leuvenink, Henri G; Ploeg, Rutger J; Yard, Benito A; Seelen, Marc A

    2011-05-01

    Kidneys recovered from brain-dead donors have inferior outcomes after transplantation compared to kidneys from living donors. Since complement activation plays an important role in renal transplant related injury, targeting complement activation in brain-dead donors might improve renal function after transplantation. Brain death (BD) was induced in Fisher rats by inflation of an epidurally placed balloon catheter and ventilated for 6h. BD animals were treated with soluble complement receptor 1 (sCR1) 1h before or 1h after BD. Kidney transplantation was performed and 7 days after transplantation animals were sacrificed. Plasma creatinine and urea were measured at days 0, 1, 3, 5 and 7 after transplantation. Renal function was significantly better at day 1 after transplantation in recipients receiving a sCR1 pre-treated donor kidney compared to recipients of a non-treated donor graft. Also treatment with sCR1, 1h after the diagnosis of BD, resulted in a better renal function after transplantation. Gene expression of IL-6, IL-1beta and TGF-beta were significantly lower in renal allografts recovered from treated donors. This study shows that targeting complement activation, during BD in the donor, leads to an improved renal function after transplantation in the recipient.

  5. Assessment of hemolytic activity, enzyme production and bacteriocin characterization of Bacillus subtilis LR1 isolated from the gastrointestinal tract of fish.

    PubMed

    Banerjee, Goutam; Nandi, Ankita; Ray, Arun Kumar

    2017-01-01

    In the present investigation, probiotic potential (antagonistic activity, enzyme production, hemolytic activity, biosafety, antibiotic sensitivity and bile tolerance level) of Bacillus subtilis LR1 was evaluated. Bacteriocin produced by the bacterial strain B. subtilis LR1 isolated from the gastrointestinal tract of Labeo rohita was purified and characterized. The molecular weight of the purified bacteriocin was ~50 kDa in 12 % Native PAGE and showed inhibitory activity against four fish pathogens such as Bacillus mycoides, Aeromonas salmonicida, Pseudomonas fluorescens and Aeromonas hydrophila. The purified bacteriocin was maximally active at temperature 40 °C and pH 7.0, while none of the tested surfactants affect the bacteriocin activity. Extracellular enzyme activity of the selected bacterial strain was also evaluated. Amylase activity was estimated to be highest (38.23 ± 1.15 µg of maltose liberated mg(-1) protein ml(-1) of culture filtrate) followed by cellulase and protease activity. The selected bacterium was sensitive to most of the antibiotics used in this experiment, can tolerate 0.25 % bile salt and non-hemolytic in nature. Finally, the efficiency of the proposed probiotic candidate was evaluated in in vivo condition. It was detected that the bacterial strain can effectively reduce bacterial pathogenicity in Indian major carps.

  6. Complement activation on poly(ethylene oxide)-like RFGD-deposited surfaces

    PubMed Central

    Szott, Luisa Mayorga; Stein, M. Jeanette; Ratner, Buddy D.; Horbett, Thomas A.

    2010-01-01

    Non-specific protein adsorption, particularly fibrinogen (Fg), is thought to be an initiating step in the foreign body response (FBR) to biomaterials by promoting phagocyte attachment. In previous studies, we therefore prepared radio frequency glow discharge (RFGD) polyethylene oxide (PEO)-like tetraglyme coatings (CH3O(CH2CH2O)4CH3) adsorbing less than 10 ng/cm2 Fg and showed that they had the expected low monocyte adhesion in vitro. However, when these were implanted in vivo, many adherent inflammatory cells and a fibrous capsule were found, suggesting the role of alternative proteins, such as activated complement proteins, in the FBR to these materials. We therefore investigated complement interactions with the tetraglyme surfaces. First, because of its well known role in complement C3 activation, we measured the hydroxyl group (-OH) content of tetraglyme, but found it to be very low. Second, we measured C3 adsorption to tetraglyme from plasma. Low amounts of C3 adsorbed on tetraglyme, though it displayed higher binding strength than the control surfaces. Finally, complement activation was determined by measuring C3a and SC5b-9 levels in serum after incubating with tetraglyme, as well as other surfaces that served as positive and negative controls, namely poly(vinyl alcohol) hydrogels, Silastic sheeting, and poly(ethylene glycol) self-assembled monolayers with different end groups. Despite displaying low hydroxyl group concentration, relatively high C3a and SC5b-9 levels were found in serum exposed to tetraglyme, similar to the values due to our positive control, PVA. Our results support the conclusion that complement activation by tetraglyme is a possible mechanism involved in the FBR to these biomaterials. PMID:21105163

  7. Complement activation by candidate biomaterials of an implantable microfabricated medical device.

    PubMed

    Sokolov, Andrey; Hellerud, Bernt C; Pharo, Anne; Johannessen, Erik A; Mollnes, Tom E

    2011-08-01

    Implantable devices realized by microfabrication have introduced a new class of potential biomaterials whose properties would need to be assessed. Such devices include sensors for measuring biological substances like glucose. Thus, 14 different candidate materials intended for design of such a device were investigated with respect to their complement activation potential in human serum. The fluid-phase activation was measured by the products C4d, Bb, C3bc, and the terminal complement complex (TCC), whereas solid-phase activation was measured by deposition of TCC on the material surfaces. No fluid-phase activation was found for materials related to the capsule, carrier, or sealing. Fluid-phase activation was, however, triggered to a various extent in three of the four nanoporous membranes (cellulose, polyamide, and aluminium oxide), whereas polycarbonate was rendered inactive. Solid-phase activation discriminated more sensitively between all the materials, revealing that the capsule candidate polydimethylsiloxane and sealing candidate silicone 3140 were highly compatible, showing significantly lower TCC deposition than the negative control (p < 0.01). Three of the candidate materials were indifferent, whereas the remaining nine showed significantly higher deposition of TCC than the negative control (p < 0.01). In conclusion, complement activation, in particular when examined on the solid phase, discriminated well between the different candidate materials tested and could be used as a guide for the selection of the best-suited materials for further investigation and development of the device.

  8. Fcγ and Complement Receptors and Complement Proteins in Neutrophil Activation in Rheumatoid Arthritis: Contribution to Pathogenesis and Progression and Modulation by Natural Products

    PubMed Central

    Paoliello-Paschoalato, Adriana Balbina; Marchi, Larissa Fávaro; de Andrade, Micássio Fernandes; Kabeya, Luciana Mariko; Donadi, Eduardo Antônio; Lucisano-Valim, Yara Maria

    2015-01-01

    Rheumatoid arthritis (RA) is a highly disabling disease that affects all structures of the joint and significantly impacts on morbidity and mortality in RA patients. RA is characterized by persistent inflammation of the synovial membrane lining the joint associated with infiltration of immune cells. Eighty to 90% of the leukocytes infiltrating the synovia are neutrophils. The specific role that neutrophils play in the onset of RA is not clear, but recent studies have evidenced that they have an important participation in joint damage and disease progression through the release of proteolytic enzymes, reactive oxygen species (ROS), cytokines, and neutrophil extracellular traps, in particular during frustrated phagocytosis of immune complexes (ICs). In addition, the local and systemic activation of the complement system contributes to the pathogenesis of RA and other IC-mediated diseases. This review discusses (i) the participation of Fcγ and complement receptors in mediating the effector functions of neutrophils in RA; (ii) the contribution of the complement system and ROS-dependent and ROS-independent mechanisms to joint damage in RA; and (iii) the use of plant extracts, dietary compounds, and isolated natural compounds in the treatment of RA, focusing on modulation of the effector functions of neutrophils and the complement system activity and/or activation. PMID:26346244

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

    PubMed Central

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

    1972-01-01

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

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

    SciTech Connect

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

    1987-08-01

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

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

    PubMed

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

    2013-12-12

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

  12. [Hemolytic anemias in adults].

    PubMed

    Müller, A; Zimmermann, R; Krause, S W

    2011-11-01

    The erythrocyte lifespan in haemolytic anemia is shortened while erythropoesis is increased. Important labaratory findings are increased reticulocytes, LDH, indirect bilirubin and a decreased haptoglobin level. The most important diagnostic tool for further work up of hemolytic anemia is the direct antiglobulin test (DAT, Coombs test) to differentiate autoimmune hemolytic anemia (AIHA) from other causes. Another important group are fragmentation syndroms (hemolytic uremic syndrome and thrombotic thrombocytopenic purpura). In these forms of haemolytic anemia fragmented red blood cells can be found in the blood smear together with thrombocytopenia. A severe problem in paroxysmal nocturnal hematuria is the incidence of thrombosis. The following review describes the most important forms of hemolytic anemia in the adult and the diagnostic and therapeutic strategies.

  13. Bioactive Lysophospholipids Generated by Hepatic Lipase Degradation of Lipoproteins Lead to Complement Activation via the Classical Pathway

    PubMed Central

    Ma, Wanchao; Paik, David C.; Barile, Gaetano R.

    2014-01-01

    Purpose. We determined bioactivity of lysophospholipids generated by degradation of the low-density (LDL), very low-density (VLDL), and high-density (HDL) lipoproteins with hepatic lipase (HL), cholesterol esterase (CE), and lipoprotein-associated phospholipase A2 (Lp-PLA2). Methods. The LDL, VLDL, and HDL were treated with HL, CE, and Lp-PLA2 after immobilization on plates, and complement activation studies were performed with diluted human serum. Complement component 3 (C3) fixation, a marker for complement activation, was determined with a monoclonal anti-human C3d antibody. Enzymatic properties of HL and CE were assayed with triglyceride and phosphatidylcholine substrates for triglyceride hydrolase and phospholipase A activities. The ARPE-19 cells were used for viability studies. Results. The HL degradation of human lipoproteins LDL, VLDL, or HDL results in the formation of modified lipoproteins that can activate the complement pathway. Complement activation is dose- and time-dependent upon HL and occurs via the classical pathway. Enzymatic studies suggest that the phospholipase A1 activity of HL generates complement-activating lysophospholipids. C-reactive protein (CRP), known to simultaneously interact with complement C1 and complement factor H (CFH), further enhances HL-induced complement activation. The lysophospholipids, 1-Palmitoyl-sn-glycero-3-phosphocholine and 1-Oleoyl-sn-glycero-3-phosphocholine, can be directly cytotoxic to ARPE-19 cells. Conclusions. The HL degradation of lipoproteins, known to accumulate in the outer retina and in drusen, can lead to the formation of bioactive lysophospholipids that can trigger complement activation and induce RPE cellular dysfunction. Given the known risk associations for age-related macular degeneration (AMD) with HL, CRP, and CFH, this study elucidates a possible damage pathway for age-related macular degeneration (AMD) in genetically predisposed individuals, that HL activity may lead to accumulation of

  14. The Carbohydrate-linked Phosphorylcholine of the Parasitic Nematode Product ES-62 Modulates Complement Activation*

    PubMed Central

    Ahmed, Umul Kulthum; Maller, N. Claire; Iqbal, Asif J.; Al-Riyami, Lamyaa; Harnett, William; Raynes, John G.

    2016-01-01

    Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications. PMID:27044740

  15. The Carbohydrate-linked Phosphorylcholine of the Parasitic Nematode Product ES-62 Modulates Complement Activation.

    PubMed

    Ahmed, Umul Kulthum; Maller, N Claire; Iqbal, Asif J; Al-Riyami, Lamyaa; Harnett, William; Raynes, John G

    2016-05-27

    Parasitic nematodes manufacture various carbohydrate-linked phosphorylcholine (PCh)-containing molecules, including ES-62, a protein with an N-linked glycan terminally substituted with PCh. The PCh component is biologically important because it is required for immunomodulatory effects. We showed that most ES-62 was bound to a single protein, C-reactive protein (CRP), in normal human serum, displaying a calcium-dependent, high-avidity interaction and ability to form large complexes. Unexpectedly, CRP binding to ES-62 failed to efficiently activate complement as far as the C3 convertase stage in comparison with PCh-BSA and PCh-containing Streptococcus pneumoniae cell wall polysaccharide. C1q capture assays demonstrated an ES-62-CRP-C1q interaction in serum. The three ligands all activated C1 and generated C4b to similar extents. However, a C2a active site was not generated following ES-62 binding to CRP, demonstrating that C2 cleavage was far less efficient for ES-62-containing complexes. We proposed that failure of C2 cleavage was due to the flexible nature of carbohydrate-bound PCh and that reduced proximity of the C1 complex was the reason that C2 was poorly cleaved. This was confirmed using synthetic analogues that were similar to ES-62 only in respect of having a flexible PCh. Furthermore, ES-62 was shown to deplete early complement components, such as the rate-limiting C4, following CRP interaction and thereby inhibit classical pathway activation. Thus, flexible PCh-glycan represents a novel mechanism for subversion of complement activation. These data illustrate the importance of the rate-limiting C4/C2 stage of complement activation and reveal a new addition to the repertoire of ES-62 immunomodulatory mechanisms with possible therapeutic applications.

  16. Roles of the valine clusters in domain 3 of the hemolytic lectin CEL-III in its oligomerization and hemolytic abilities.

    PubMed

    Hisamatsu, Keigo; Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2009-01-01

    The hemolytic lectin CEL-III and its site-directed mutants were expressed in Escherichia coli cells. Replacement of the valine clusters in domain 3 with alanine residues led to increased self-oligomerization in solution and higher hemolytic activity. The results suggest the involvement of these valine clusters in CEL-III oligomerization and hemolytic activity.

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

    PubMed

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

    2015-08-15

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

  18. Effect of functionalization of carbon nanotubes with psychosine on complement activation and protein adsorption.

    PubMed

    Rybak-Smith, Malgorzata J; Tripisciano, Carla; Borowiak-Palen, Ewa; Lamprecht, Constanze; Sim, Robert B

    2011-12-01

    Carbon nanotubes possess interesting physicochemical properties which make them potentially usable in medicine. Single-walled carbon nanotubes and multi-walled carbon nanotubes, for example, may carry and deliver anticancer drugs, such as cisplatin. Magnetic nanoparticles, like iron filled MWCNT, can be used in hyperthermia therapy. However, their hydrophobic character is a major difficulty, as preparation of stable dispersions of carbon nanotubes in biological buffers is an essential step towards biomedical applications. Recently, a novel treatment using the glycolipid, Galactosyl-beta1-sphingosine (psychosine), was employed to make stable suspensions of psychosine-functionalized carbon nanotubes in biological buffers. In this paper, the interactions of psychosine-functionalized carbon nanotubes with a part of the human immune system, complement, is presented. To investigate if human serum complement proteins can interact with psychosine-functionalized carbon nanotubes, complement consumption (depletion) assays were conducted. Moreover, direct protein binding studies, to analyze the interaction of plasma proteins with the psychosine-functionalized carbon nanotubes, using affinity chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis techniques, were applied. The psychosine-functionalized carbon nanotubes activate human complement via the classical pathway. Interestingly, as the hydrophilic part of the glycolipid may bind to ficolins, the lectin pathway could also be involved. Binding of human plasma proteins is very selective as only very few proteins adsorb to the psychosine-functionalized carbon nanotube surface, when placed in contact with human plasma. Bovine serum albumin-coated carbon nanotubes were used as a standard to find the differences in complement activation and protein adsorption patterns, caused by various non-covalent coatings of carbon nanotubes.

  19. Potassium humate inhibits complement activation and the production of inflammatory cytokines in vitro

    SciTech Connect

    van Rensburg, C.E.J.; Naude, P.J.

    2009-08-15

    The effects of brown coal derived potassium humate on lymphocyte proliferation, cytokine production and complement activation were investigated in vitro. Potassium humate increased lymphocyte proliferation of phytohaemaglutinin A (PHA) and pokeweed mitogen (PWM) stimulated mononuclear lymphocytes (MNL) in vitro from concentrations of 20 to 80 {mu} g/ml, in a dose dependant manner. On the other hand potassium humate, at 40 {mu} g/ml, significantly inhibited the release of TNF-alpha, IL-1 beta, IL-6 and IL-10 by PHA stimulated MNL. Regarding complement activation it was found that potassium humate inhibits the activation of both the alternative and classical pathways without affecting the stability of the red blood cell membranes. These results indicate that the anti-inflammatory potential of potassium humate could be partially due to the inhibition of pro-inflammatory cytokines responsible for the initiation of these reactions as well as inhibition of complement activation. The increased lymphocyte proliferation observed, might be due to increased IL-2 production as previously been documented.

  20. Detection of complement activation using monoclonal antibodies against C3d

    PubMed Central

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

    2013-01-01

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

  1. Synergistic hemolytic reactions between staphylococci and Micrococcus lylae.

    PubMed

    Lämmler, C; Brückler, J

    1989-06-01

    The primary culture of a clinical specimen obtained from a dog with an acute squamous eczema revealed three different bacterial species which demonstrated synergistic hemolytic activities on sheep blood agar plates. The three cultures were identified as beta-hemolytic Staphylococcus intermedius, as a coagulase-negative staphylococcal species, producing a delta-like hemolysin and as non-hemolytic Micrococcus lylae. The coagulase-negative staphylococcal species as well as M. lylae produced synergistically with beta-hemolytic S. intermedius zones of complete hemolysis. The occurrence of three different synergistically active bacterial species from one clinical specimen might be of clinical significance.

  2. Complement activation and liver impairment in trichloroethylene-sensitized BALB/c mice.

    PubMed

    Zhang, Jiaxiang; Zha, Wansheng; Wang, Feng; Jiang, Tao; Xu, Shuhai; Yu, Junfeng; Zhou, Chengfan; Shen, Tong; Wu, Changhao; Zhu, Qixing

    2013-01-01

    Our recent studies have shown that trichloroethylene (TCE) was able to induce multisystem injuries in the form of occupational medicamentosa-like dermatitis, including skin, kidney, and liver damages. However, the role of complement activation in the immune-mediated liver injury is not known. This study examined the role of complement activation in the liver injury in a mouse model of TCE-induced sensitization. Treatment of female BALB/c mice with TCE under specific dosing protocols resulted in skin inflammation and sensitization. Skin edema and erythema occurred in TCE-sensitized groups. Trichloroethylene sensitization produced liver histopathological lesions, increased serum alanine aminotransferase, aspartate transaminase activities, and the relative liver weight. The concentrations of serum complement components C3a-desArg, C5a-desArg, and C5b-9 were significantly increased in 24-hour, 48-hour, and 72-hour sensitization-positive groups treated with TCE and peaked in the 72-hour sensitization-positive group. Depositions of C3a, C5a, and C5b-9 into the liver tissue were also revealed by immunohistochemistry. Immunofluorescence further verified high C5b-9 expression in 24-hour, 48-hour, and 72-hour sensitization-positive groups in response to TCE treatment. Reverse transcription-polymerase chain reaction detected C3 messenger RNA expression in the liver, and this was significantly increased in 24-hour and 48-hour sensitization-positive groups with a transient reduction at 72 hours. These results provide the first experimental evidence that complement activation may play a key role in the generation and progression of immune-mediated hepatic injury by exposure to TCE.

  3. Differential activity of candidate microbicides against early steps of HIV-1 infection upon complement virus opsonization

    PubMed Central

    2010-01-01

    Background HIV-1 in genital secretions may be opsonized by several molecules including complement components. Opsonized HIV-1 by complement enhances the infection of various mucosal target cells, such as dendritic cells (DC) and epithelial cells. Results We herein evaluated the effect of HIV-1 complement opsonization on microbicide candidates' activity, by using three in vitro mucosal models: CCR5-tropic HIV-1JR-CSF transcytosis through epithelial cells, HIV-1JR-CSF attachment on immature monocyte-derived dendritic cells (iMDDC), and infectivity of iMDDC by CCR5-tropic HIV-1BaL and CXCR4-tropic HIV-1NDK. A panel of 10 microbicide candidates [T20, CADA, lectines HHA & GNA, PVAS, human lactoferrin, and monoclonal antibodies IgG1B12, 12G5, 2G12 and 2F5], were investigated using cell-free unopsonized or opsonized HIV-1 by complements. Only HHA and PVAS were able to inhibit HIV trancytosis. Upon opsonization, transcytosis was affected only by HHA, HIV-1 adsorption on iMDDC by four molecules (lactoferrin, IgG1B12, IgG2G5, IgG2G12), and replication in iMDDC of HIV-1BaL by five molecules (lactoferrin, CADA, T20, IgG1B12, IgG2F5) and of HIV-1NDK by two molecules (lactoferrin, IgG12G5). Conclusion These observations demonstrate that HIV-1 opsonization by complements may modulate in vitro the efficiency of candidate microbicides to inhibit HIV-1 infection of mucosal target cells, as well as its crossing through mucosa. PMID:20546571

  4. Anti-complement activity of essential oils from red and black rice bran.

    PubMed

    Chung, Ill-Min; Yeo, Min-A; Kim, Sun-Jin; Moon, Hyung-In

    2011-05-01

    The volatile essential oils from red and black rice bran were obtained by hydrodistillation using a clevenger-type apparatus, and the components of that oil were analyzed by capillary gas chromatography-mass spectroscopy (GC-MS). The present study involved characterizing the chemical compositions, their amounts and the anti-complement activities of red and black rice bran. The red rice bran essential oils yield was 0.031%, and GC-MS analysis revealed that its major constituents were (E)-β-ocimene (3.12%), nonanal (11.32%), (2E, 4E)-decadienal (2.54%), myristic acid (41.32%), geranyactone (2.41%) and methyl oleate (2.46%). The black rice bran essential oils yield was 0.053%, and GC-MS analysis revealed that its major constituents were nonanal (8.31%), acrylic acid (3.21%), 2-hydroxy-6-methylbenzaldehyde (2.81%), pelargonic acid (4.21%) and myrisitc acid (28.07%). The essential oils showed inhibitory activity against complement system with 50% inhibitory concentrations (IC(50)) values of 246 ppm (red rice bran) and 193 ppm (black rice bran). Also, myristic acid, nonanal, (E)-β-ocimene and pelargonic acid were tested against complement system. Pelargonic acid was shown to moderate activity (50% inhibitory concentration = 132 μM).

  5. Antibodies to glycolipids activate complement and promote proteinuria in passive Heymann nephritis.

    PubMed Central

    Susani, M.; Schulze, M.; Exner, M.; Kerjaschki, D.

    1994-01-01

    Passive Heymann nephritis is an experimental rat model of human membranous nephropathy induced by injection of antisera against crude renal cortical fractions such as Fx1A or rat tubular microvilli. This results in the formation of subepithelial immune deposits, the activation of the C5b-9 membrane attack complex of complement, and severe proteinuria. While the formation of immune deposits is attributed to in situ immune complex formation with antibodies specific for the gp330-Heymann nephritis antigenic complex (HNAC), activation of complement and proteinuria appear to be caused by at least one additional antibody species present in anti-Fx1A sera. We have separated by affinity absorption polyspecific antisera against Fx1A and rat microvilli into one IgG fraction directed specifically against microvillar proteins (anti-Fx1A-prot) and another IgG fraction specific for glycolipids (ant-Fx1A-lip) of tubular microvilli. When injected into rats, the anti-Fx1A-prot fraction induced immune deposits but failed to activate complement or produce proteinuria, similar to results obtained with affinity-purified anti-gp330 IgG. When the antibodies of the anti-Fx1A-lip fraction were injected alone they did not bind to glomeruli. By contrast, when the IgGs specific for the Fx1A-prot fraction (or for gp330-HNAC) were combined with those directed against the Fx1A-lip glycolipid preparation, immune deposits were formed, in situ complement activation was observed, and also proteinuria was induced. It is concluded that within anti-Fx1A and anti-microvillar sera there are at least two IgG fractions of relevance for the development of PHN: one directed against the gp330-HNAC complex which is responsible for the development of immune deposits, and a second specific for glycolipid antigen(s) which activate(s) the complement cascade. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:8160779

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

    PubMed

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

    2006-07-01

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

  7. Molluskan Hemocyanins Activate the Classical Pathway of the Human Complement System through Natural Antibodies

    PubMed Central

    Pizarro-Bauerle, Javier; Maldonado, Ismael; Sosoniuk-Roche, Eduardo; Vallejos, Gerardo; López, Mercedes N.; Salazar-Onfray, Flavio; Aguilar-Guzmán, Lorena; Valck, Carolina; Ferreira, Arturo; Becker, María Inés

    2017-01-01

    Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a Th1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropod Megathura crenulata [keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities: Fisurella latimarginata hemocyanin (FLH) and Concholepas concholepas hemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting

  8. Disseminated intravascular coagulation due to IgM-mediated autoimmune hemolytic anemia.

    PubMed

    Bleakly, N Teresa; Fontaine, Magali J; Pate, Lisa L; Sutherland, Scott M; Jeng, Michael

    2011-08-01

    Disseminated intravascular coagulation (DIC) due to red cell hemolysis has been previously attributed to transfusion-related hemolytic reactions, but not to autoimmune hemolytic anemia. We report a case of DIC in a child with complement-fixing IgM-mediated cold-agglutinin autoimmune hemolysis, which resulted in arterial thrombosis and gangrene of the upper and lower extremities.

  9. A teleost complement factor Ba possesses antimicrobial activity and inhibits bacterial infection in fish.

    PubMed

    Li, Xue-Peng; Sun, Li

    2017-01-24

    Complement factor B (Bf) is a component of the complement system. Following activation of the alternative pathway of the complement system, factor B is cleaved into Ba and Bb fragments. In fish, the Bf of rainbow trout is known to act as a C3 convertase, but the function of the Ba fragment is essentially unknown. In this study, we examined the expression patterns of tongue sole Cynoglossus semilaevis Bf (named CsBf) and the biological activity of the Ba fragment of CsBf (named CsBa). CsBf possesses the conserved domains of Bf and shares 39.9%-56.4% sequence identities with other fish Bf. CsBf expression was high in liver, muscle, and heart, and low in intestine, blood, and kidney. Bacterial infection significantly induced CsBf expression in kidney, spleen, and liver in a time-dependent manner. Recombinant CsBa (rCsBa) exhibited apparent binding capacities to bacteria and tongue sole peripheral blood leukocytes, and binding of rCsBa to bacteria inhibited bacterial growth. When overexpressed in tongue sole, CsBa significantly reduced bacterial dissemination in fish tissues. Together these results indicate for the first time that a fish Ba possesses antibacterial effect as well as immune cell-binding capacity, and thus probably plays a role in host immune defense against bacterial infection.

  10. Complement activation and choriocapillaris loss in early AMD: Implications for pathophysiology and therapy

    PubMed Central

    Whitmore, S.Scott; Sohn, Elliott H.; Chirco, Kathleen R.; Drack, Arlene V.; Stone, Edwin M.; Tucker, Budd A.; Mullins, Robert F.

    2015-01-01

    Age-related macular degeneration (AMD) is a common and devastating disease that can result in severe visual dysfunction. Over the last decade, great progress has been made in identifying genetic variants that contribute to AMD, many of which lie in genes involved in the complement cascade. In this review we discuss the significance of complement activation in AMD, particularly with respect to the formation of the membrane attack complex in the aging choriocapillaris. We review the clinical, histological and biochemical data that indicate that vascular loss in the choroid occurs very early in the pathogenesis of AMD, and discuss the potential impact of vascular dropout on the retinal pigment epithelium, Bruch's membrane and the photoreceptor cells. Finally, we present a hypothesis for the pathogenesis of early AMD and consider the implications of this model on the development of new therapies. PMID:25486088

  11. Recessive mutations in DGKE cause atypical hemolytic-uremic syndrome

    PubMed Central

    Lemaire, Mathieu; Frémeaux-Bacchi, Véronique; Schaefer, Franz; Choi, Murim; Tang, Wai Ho; Le Quintrec, Moglie; Fakhouri, Fadi; Taque, Sophie; Nobili, François; Martinez, Frank; Ji, Weizhen; Overton, John D.; Mane, Shrikant M.; Nürnberg, Gudrun; Altmüller, Janine; Thiele, Holger; Morin, Denis; Deschenes, Georges; Baudouin, Véronique; Llanas, Brigitte; Collard, Laure; Majid, Mohammed A.; Simkova, Eva; Nürnberg, Peter; Rioux-Leclerc, Nathalie; Moeckel, Gilbert W.; Gubler, Marie Claire; Hwa, John; Loirat, Chantal; Lifton, Richard P.

    2013-01-01

    Pathologic thrombosis is a major cause of mortality. Hemolytic-uremic syndrome (HUS) features episodes of small vessel thrombosis resulting in microangiopathic hemolytic anemia, thrombocytopenia and renal failure1. Atypical HUS (aHUS) can result from genetic or autoimmune factors2 that lead to pathologic complement cascade activation3. By exome sequencing we identify recessive mutations in DGKE (diacylglycerol kinase epsilon) that co-segregate with aHUS in 9 unrelated kindreds, defining a distinctive Mendelian disease. Affected patients present with aHUS before age 1, have persistent hypertension, hematuria and proteinuria (sometimes nephrotic range), and develop chronic kidney disease with age. DGKE is found in endothelium, platelets, and podocytes. Arachidonic acid-containing diacylglycerols (DAG) activate protein kinase C, which promotes thrombosis. DGKE normally inactivates DAG signaling. We infer that loss of DGKE function results in a pro-thrombotic state. These findings identify a new mechanism of pathologic thrombosis and kidney failure and have immediate implications for treatment of aHUS patients. PMID:23542698

  12. Complement factor I from flatfish half-smooth tongue (Cynoglossus semilaevis) exhibited anti-microbial activities.

    PubMed

    Xiang, Jinsong; Li, Xihong; Chen, Yadong; Lu, Yang; Yu, Mengjun; Chen, Xuejie; Zhang, Wenting; Zeng, Yan; Sun, Luming; Chen, Songlin; Sha, Zhenxia

    2015-11-01

    Complement factor I (Cfi) is a soluble serine protease which plays a crucial role in the modulation of complement cascades. In the presence of substrate modulating cofactors (such as complement factor H, C4bp, CR1, etc), Cfi cleaves and inactivates C3b and C4b, thereby controlling the complement-mediated processes. In this study, we sequenced and characterized Cfi gene from Cynoglossus Semilaevis (designated as CsCfi) for the first time. The full-length cDNA of CsCfi was 2230 bp in length, including a 98 bp 5'-untranslated region (UTR), a 164 bp 3'-UTR and a 1968 bp open reading frame (ORF). It encoded a polypeptide of 656 amino acids, with a molecular mass of 72.28 kDa and an isoelectric point of 7.71. A signal peptide was defined at N-terminus, resulting in a 626-residue mature protein. Multiple sequence alignment revealed that Cfi proteins were well conserved with the typical modular architecture and identical active sites throughout the vertebrates, which suggested the conserved function of Cfi. Phylogenetic analysis indicated that CsCfi and the homologous Cfi sequences from teleosts clustered into a clade, separating from another clade from the cartilaginous fish and other vertebrates. Tissue expression profile analysis by quantitative real-time PCR (qRT-PCR) showed that CsCfi mRNA constitutively expressed in all tested tissues, with the predominant expression in liver and the lowest in stomach. Temporal expression levels of CsCfi after challenging with Vibrio anguillarum showed different expression patterns in intestine, spleen, skin, blood, head kidney and liver. The recombinant CsCfi (rCsCfi) protein showed broad-spectrum antimicrobial activities against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and Shewanella putrefaciens. The research revealed that CsCfi plays an important role in C. Semilaevis immunity.

  13. Recombinant glycoproteins that inhibit complement activation and also bind the selectin adhesion molecules.

    PubMed

    Rittershaus, C W; Thomas, L J; Miller, D P; Picard, M D; Geoghegan-Barek, K M; Scesney, S M; Henry, L D; Sen, A C; Bertino, A M; Hannig, G; Adari, H; Mealey, R A; Gosselin, M L; Couto, M; Hayman, E G; Levin, J L; Reinhold, V N; Marsh, H C

    1999-04-16

    Soluble human complement receptor type 1 (sCR1, TP10) has been expressed in Chinese hamster ovary (CHO) DUKX-B11 cells and shown to inhibit the classical and alternative complement pathways in vitro and in vivo. A truncated version of sCR1 lacking the long homologous repeat-A domain (LHR-A) containing the C4b binding site has similarly been expressed and designated sCR1[desLHR-A]. sCR1[desLHR-A] was shown to be a selective inhibitor of the alternative complement pathway in vitro and to function in vivo. In this study, sCR1 and sCR1[desLHR-A] were expressed in CHO LEC11 cells with an active alpha(1,3)-fucosyltransferase, which makes possible the biosynthesis of the sialyl-Lewisx (sLex) tetrasaccharide (NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc) during post-translational glycosylation. The resulting glycoproteins, designated sCR1sLex and sCR1[desLHR-A]sLex, respectively, retained the complement regulatory activities of their DUKX B11 counterparts, which lack alpha(1-3)-fucose. Carbohydrate analysis of purified sCR1sLex and sCR1[desLHR-A]sLex indicated an average incorporation of 10 and 8 mol of sLex/mol of glycoprotein, respectively. sLex is a carbohydrate ligand for the selectin adhesion molecules. sCR1sLex was shown to specifically bind CHO cells expressing cell surface E-selectin. sCR1[desLHR-A]sLex inhibited the binding of the monocytic cell line U937 to human aortic endothelial cells, which had been activated with tumor necrosis factor-alpha to up-regulate the expression of E-selectin. sCR1sLex inhibited the binding of U937 cells to surface-adsorbed P-selectin-IgG. sCR1sLex and sCR1[desLHR-A]sLex have thus demonstrated both complement regulatory activity and the capacity to bind selectins and to inhibit selectin-mediated cell adhesion in vitro.

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

    PubMed

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

    2016-12-01

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

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

    ERIC Educational Resources Information Center

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

    2008-01-01

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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed Central

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

    2008-01-01

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

  18. Congenital Hemolytic Anemia.

    PubMed

    Haley, Kristina

    2017-03-01

    Red blood cell (RBC) destruction can be secondary to intrinsic disorders of the RBC or to extrinsic causes. In the congenital hemolytic anemias, intrinsic RBC enzyme, RBC membrane, and hemoglobin disorders result in hemolysis. The typical clinical presentation is a patient with pallor, anemia, jaundice, and often splenomegaly. The laboratory features include anemia, hyperbilirubinemia, and reticulocytosis. For some congenital hemolytic anemias, splenectomy is curative. However, in other diseases, avoidance of drugs and toxins is the best therapy. Supportive care with transfusions are also mainstays of therapy. Chronic hemolysis often results in the formation of gallstones, and cholecystectomy is often indicated.

  19. A pathogenic role of complement in arterial hypertension and hypertensive end organ damage.

    PubMed

    Wenzel, Ulrich O; Bode, Marlies; Köhl, Jörg; Ehmke, Heimo

    2017-03-01

    The self-amplifying cascade of messenger and effector molecules of the complement system serves as a powerful danger-sensing system that protects the host from a hostile microbial environment, while maintaining proper tissue and organ function through effective clearance of altered or dying cells. As an important effector arm of innate immunity, it also plays important roles in the regulation of adaptive immunity. Innate and adaptive immune responses have been identified as crucial players in the pathogenesis of arterial hypertension and hypertensive end organ damage. In line with this view, complement activation may drive the pathology of hypertension and hypertensive injury through its impact on innate and adaptive immune responses. It is well known that complement activation can cause tissue inflammation and injury and complement-inhibitory drugs are effective treatments for several inflammatory diseases. In addition to these proinflammatory properties, complement cleavage fragments of C3 and C5 can exert anti-inflammatory effects that dampen the inflammatory response to injury. Recent experimental data strongly support a role for complement in arterial hypertension. The remarkably similar clinical and histopathological features of malignant nephrosclerosis and atypical hemolytic uremic syndrome, which is driven by complement activation, suggest a role for complement also in the development of malignant nephrosclerosis. Herein, we will review canonical and noncanonical pathways of complement activation as the framework to understand the multiple roles of complement in arterial hypertension and hypertensive end organ damage.

  20. [Autoimmune hemolytic anemia in children].

    PubMed

    Becheur, M; Bouslama, B; Slama, H; Toumi, N E H

    2015-01-01

    Autoimmune hemolytic anemia is a rare condition in children which differs from the adult form. It is defined by immune-mediated destruction of red blood cells caused by autoantibodies. Characteristics of the autoantibodies are responsible for the various clinical entities. Classifications of autoimmune hemolytic anemia include warm autoimmune hemolytic anemia, cold autoimmune hemolytic anemia, and paroxysmal cold hemoglobinuria. For each classification, this review discusses the epidemiology, etiology, clinical presentation, laboratory evaluation, and treatment options.

  1. THE CONVERSION OF HEMOLYTIC STREPTOCOCCI TO NON-HEMOLYTIC FORMS

    PubMed Central

    Todd, E. W.

    1928-01-01

    From one strain of hemolytic streptococcus three forms were isolated, which produced three different degrees of hemolysis on the surface of blood agar in the presence of oxygen. The original form was moderately hemolytic; the glossy variant was more hemolytic than the original form; and the third form, obtained by passing the original culture through mice, was non-hemolytic. Under anaerobic conditions all three forms were hemolytic. The non-hemolytic passage culture, in the presence of an ample supply of oxygen, not only destroyed its own hemolysin, which only appeared under anaerobic conditions, but was also able to destroy the hemolysin of other cultures of hemolytic streptococci. It is possible that these observations may throw some light on experiments reported by a number of workers showing that Streptococcus hæmolyticus can be transmuted to Streptococcus viridans by animal passage. PMID:19869500

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

    PubMed

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

    2015-12-01

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

  3. Autoimmune Hemolytic Anemia.

    PubMed

    Liebman, Howard A; Weitz, Ilene C

    2017-03-01

    Autoimmune hemolytic anemia is an acquired autoimmune disorder resulting in the production of antibodies directed against red blood cell antigens causing shortened erythrocyte survival. The disorders can present as a primary disorder (idiopathic) or secondary to other autoimmune disorders, malignancies, or infections. Treatment involves immune modulation with corticosteroids and other agents.

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

    PubMed

    Kunchithapautham, Kannan; Atkinson, Carl; Rohrer, Bärbel

    2014-05-23

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

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

    PubMed

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

    2012-10-01

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

  6. Activation of the lectin complement pathway in post-streptococcal acute glomerulonephritis.

    PubMed

    Hisano, Satoshi; Matsushita, Misao; Fujita, Teizo; Takeshita, Morishige; Iwasaki, Hiroshi

    2007-06-01

    The aim of the present study was to elucidate the correlation between complement pathways and clinicopathological findings in post-streptococcal acute glomerulonephritis (PSAGN). Immunohistological staining was performed on renal specimens obtained from 18 patients with PSAGN and 20 controls, using antibodies against IgG, IgA, IgM, C1q, C3c, C4, fibrinogen, factor B, C4-binding protein (C4-bp), C5b-9, CD59, mannose-binding lectin (MBL) and MBL-associated serine protease-1 (MASP-1). Controls showed no deposition of any antibody. In seven patients, glomerular deposits of C3c, C4, factor B, C4-bp, C5b-9, CD59, MBL and MASP-1 were found. In the remaining 11 patients, glomerular deposits of neither C4 nor MBL/MASP-1 were found, and glomerular deposits of C3c, factor B, C5b-9 and CD59 were evident. C4-bp was detected in seven of these 11 patients. Glomerular deposits of fibrinogen were detected in five of seven patients with MBL/MASP-1 deposits and in only two of 11 patients without MBL/MASP-1 deposits. Hematuria was prolonged in three of seven patients with MBL/MASP-1 deposits through follow up, whereas urinalysis was normal in all patients without MBL/MASP-1 deposits. However, the histological indicators were not different between the two groups. To the authors' knowledge this is the first report to show that complement activation through both the alternative and lectin pathways is evident in some patients with PSAGN. Complement activation is promoted in situ in the glomerulus.

  7. Case report of atypical hemolytic uremic syndrome with retinal arterial and venous occlusion treated with eculizumab

    PubMed Central

    Greenwood, Gregory T

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare disease caused by chronic, uncontrolled activation of the alternative complement pathway, leading to thrombotic microangiopathy. Renal impairment and progression to end-stage renal disease are common in untreated patients with aHUS, and extrarenal manifestations are being increasingly characterized in the literature. Ocular involvement remains rare in aHUS. This report describes a patient with aHUS with bilateral central retinal artery and vein occlusion, vitreous hemorrhage, and blindness in addition to renal impairment. The patient’s hematologic and renal parameters and ocular manifestation improved following initiation of eculizumab therapy. PMID:26508891

  8. Complement requirement for virus neutralization by antibody and reduced serum complement levels associated with experimental equine herpesvirus 1 infection.

    PubMed Central

    Snyder, D B; Myrup, A C; Dutta, S K

    1981-01-01

    Pony foals, negative for detectable serum-neutralizing antibody to equine herpesvirus 1 by the standard tube-culture virus neutralization test, were experimentally infected with equine herpesvirus 1. Complement-requiring (CR) and non-complement-requiring (NCR) serum-neutralizing antibodies were evaluated in preinfection and postinfection sera by means of a complement-enhanced plaque reduction assay. Low levels of CR antibodies were found in the preinfection sera of only group II ponies. Upon infection, CR antibodies were detected by day 2 postinfection and reached peak titers between 7 and 14 days postinfection in the antisera of all ponies. NCR antibodies were detected later than CR antibodies and at levels approximately 40 to 150 times lower than the latter. CR/NCR ratios indicated that complement requirement was greatest early in the acute stages of disease and that this requirement decreased during the convalescent phase. Fractionation of 1-week and 2-week postinfection antisera of group I ponies indicated the CR antibody activity resided in both the 7S and 19S fractions. Total serum complement levels of the ponies were quantified throughout the infection with an equine anti-goat erythrocyte hemolytic system. In vivo, complement levels were depressed for all ponies during the first 2 weeks of infection. A decline in complement levels was seen as early as day 2, and they decreased to an average of 35% of preinfection levels on day 10 postinfection for all ponies. PMID:6260672

  9. Novel twin streptolysin S-like peptides encoded in the sag operon homologue of beta-hemolytic Streptococcus anginosus.

    PubMed

    Tabata, Atsushi; Nakano, Kota; Ohkura, Kazuto; Tomoyasu, Toshifumi; Kikuchi, Ken; Whiley, Robert A; Nagamune, Hideaki

    2013-03-01

    Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci.

  10. Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

    PubMed Central

    Tabata, Atsushi; Nakano, Kota; Ohkura, Kazuto; Tomoyasu, Toshifumi; Kikuchi, Ken; Whiley, Robert A.

    2013-01-01

    Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci. PMID:23292771

  11. 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,…

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

    PubMed Central

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

    2016-01-01

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

  13. Complement activation and kidney injury molecule-1-associated proximal tubule injury in severe preeclampsia.

    PubMed

    Burwick, Richard M; Easter, Sarah Rae; Dawood, Hassan Y; Yamamoto, Hidemi S; Fichorova, Raina N; Feinberg, Bruce B

    2014-10-01

    Kidney injury with proteinuria is a characteristic feature of preeclampsia, yet the nature of injury in specific regions of the nephron is incompletely understood. Our study aimed to use existing urinary biomarkers to describe the pattern of kidney injury and proteinuria in pregnancies affected by severe preeclampsia. We performed a case-control study of pregnant women from Brigham and Women's Hospital from 2012 to 2013. We matched cases of severe preeclampsia (n=25) 1:1 by parity and gestational age to 2 control groups with and without chronic hypertension. Urinary levels of kidney injury molecule-1 and complement components (C3a, C5a, and C5b-9) were measured by enzyme-linked immunosorbent assay, and other markers (albumin, β2 microglobulin, cystatin C, epithelial growth factor, neutrophil gelatinase-associated lipocalin, osteopontin, and uromodulin) were measured simultaneously with a multiplex electrochemiluminescence assay. Median values between groups were compared with the Wilcoxon signed-rank test and correlations with Spearman correlation coefficient. Analysis of urinary markers revealed higher excretion of albumin and kidney injury molecule-1 and lower excretion of neutrophil gelatinase-associated lipocalin and epithelial growth factor in severe preeclampsia compared with chronic hypertension and healthy controls. Among subjects with severe preeclampsia, urinary excretion of complement activation products correlated most closely with kidney injury molecule-1, a specific marker of proximal tubule injury (C5a: r=0.60; P=0.001; and C5b-9: r=0.75; P<0.0001). Taken together, we describe a pattern of kidney injury in severe preeclampsia that is characterized by glomerular impairment and complement-mediated inflammation and injury, possibly localized to the proximal tubule in association with kidney injury molecule-1.

  14. Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase

    PubMed Central

    Spath, Brigitte; Fischer, Cornelia; Stolz, Moritz; Ayuk, Francis A.; Kröger, Nicolaus; Bokemeyer, Carsten; Ruf, Wolfram

    2013-01-01

    Lymphocyte depletion with antithymocyte globulin (ATG) can be complicated by systemic coagulation activation. We found that ATG activated tissue factor procoagulant activity (TF PCA) on monocytic cells more potently than other stimuli that decrypt TF, including cell disruption, TF pathway inhibitor inhibition, and calcium ionophore treatment. Induction of TF PCA by ATG was dependent on lipid raft integrity and complement activation. We showed that ATG-mediated TF activation required complement activation until assembly of the C5b-7 membrane insertion complex, but not lytic pore formation by the membrane attack complex C5b-9. Consistently, induction of TF PCA by ATG did not require maximal phosphatidylserine membrane exposure and was not correlated with the magnitude of complement-induced lytic cell injury. Blockade of free thiols, an inhibitory monoclonal antibody to protein disulfide isomerase (PDI), and the small-molecule PDI antagonist quercetin-3-rutinoside prevented ATG-mediated TF activation, and C5 complement activation resulted in oxidation of cell surface PDI. This rapid and potent mechanism of cellular TF activation represents a novel connection between the complement system and cell surface PDI-mediated thiol-disulfide exchange. Delineation of this clinically relevant mechanism of activation of the extrinsic coagulation pathway during immunosuppressive therapy with ATG may have broader implications for vascular thrombosis associated with inflammatory disorders. PMID:23315166

  15. The terminal pathway of the complement system is activated in focal penetrating but not in mild diffuse traumatic brain injury.

    PubMed

    Rostami, Elham; Davidsson, Johan; Gyorgy, Andrea; Agoston, Denes V; Risling, Mårten; Bellander, Bo-Michael

    2013-12-01

    The complement system plays an important role in the inflammatory response activated by many central nervous system disorders. However, its significance in traumatic diffuse traumatic axonal injury (TAI) is not fully known. Here we analyze the complement activity in two rat models of traumatic brain injury (TBI); a focal penetration injury (pen-TBI) and a rotational acceleration injury (rot-TBI) that leads to a mild TAI. We used in situ hybridization to examine the distribution of mRNA for C1q and C3 and immunohistochemistry to examine the presence of the C3 protein and C5b-9 complex at 1-5 days after injury. We found a time-dependent complement activity in both models. However, the responses caused by the two models were different. We detected C5b-9 surrounding the cavity in pen-TBI, but C5b-9 was not found in the rot-TBI. Our findings suggest that the terminal complement pathway is progressed to the formation of the C5b-9 membrane attack complex only in the penetrating TBI but not in isolated TAI model. This indicates that the complement activation does not lead to membrane-damaging effects and a subsequent secondary axotomy in TAI by the terminal complex C5b-9. The role of complement activation in TAI is unclear, but might indicate an alternative function following rot-TBI, such as opsonizing the synapses for elimination.

  16. Complement cascade and kidney transplantation: The rediscovery of an ancient enemy

    PubMed Central

    Mella, Alberto; Messina, Maria; Lavacca, Antonio; Biancone, Luigi

    2014-01-01

    The identification of complement activity in serum and immunohistochemical samples represents a core element of nephropathology. On the basis of this observation, different experimental models and molecular studies have shown the role of this cascade in glomerular disease etiology, but the absence of inhibiting drugs have limited its importance. Since 2006, the availability of target-therapies re-defined this ancient pathway, and its blockage, as the new challenging frontier in renal disease treatment. In the graft, the complement cascade is able to initiate and propagate the damage in ischemia-reperfusion injury, C3 glomerulopathy, acute and chronic rejection, atypical hemolytic uremic syndrome and, probably, in many other conditions. The importance of complement-focused research is revealed by the evidence that eculizumab, the first complement-targeting drug, is now considered a valid option in atypical hemolytic uremic syndrome treatment but it is also under investigation in all the aforementioned conditions. In this review we evaluate the importance of complement cascade in renal transplantation diseases, focusing on available treatments, and we propose a speculative identification of areas where complement inhibition may be a promising strategy. PMID:25346889

  17. Alternative Pathway Dysregulation and the Conundrum of Complement Activation by IgG4 Immune Complexes in Membranous Nephropathy

    PubMed Central

    Borza, Dorin-Bogdan

    2016-01-01

    Membranous nephropathy (MN), a major cause of nephrotic syndrome, is a non-inflammatory immune kidney disease mediated by IgG antibodies that form glomerular subepithelial immune complexes. In primary MN, autoantibodies target proteins expressed on the podocyte surface, often phospholipase A2 receptor (PLA2R1). Pathology is driven by complement activation, leading to podocyte injury and proteinuria. This article overviews the mechanisms of complement activation and regulation in MN, addressing the paradox that anti-PLA2R1 and other antibodies causing primary MN are predominantly (but not exclusively) IgG4, an IgG subclass that does not fix complement. Besides immune complexes, alterations of the glomerular basement membrane (GBM) in MN may lead to impaired regulation of the alternative pathway (AP). The AP amplifies complement activation on surfaces insufficiently protected by complement regulatory proteins. Whereas podocytes are protected by cell-bound regulators, the GBM must recruit plasma factor H, which inhibits the AP on host surfaces carrying certain polyanions, such as heparan sulfate (HS) chains. Because HS chains present in the normal GBM are lost in MN, we posit that the local complement regulation by factor H may be impaired as a result. Thus, the loss of GBM HS in MN creates a micro-environment that promotes local amplification of complement activation, which in turn may be initiated via the classical or lectin pathways by subsets of IgG in immune complexes. A detailed understanding of the mechanisms of complement activation and dysregulation in MN is important for designing more effective therapies. PMID:27199983

  18. Studies on the phenylethanoid glycosides with anti-complement activity from Paulownia tomentosa var. tomentosa wood.

    PubMed

    Si, Chuan-Ling; Deng, Xiao-Juan; Liu, Zhong; Kim, Jin-Kyu; Bae, Young-Soo

    2008-01-01

    Four epimeric phenylethanoid glycosides, including a new one, R,S-beta-ethoxy-beta-(3,4-dihydroxyphenyl)-ethyl-O-alpha-L-rhamnopyranosyl(1-->3)-beta-D-(6-O-E-caffeoyl)-glucopyranoside named isoilicifolioside A (1), and three known compounds, ilicifolioside A (2), campneoside II (3), and isocampneoside II (4), were isolated from Paulownia tomentosa var. tomentosa wood. The structures of the four compounds were elucidated by the interpretation of 1D and 2D NMR and MS spectra. This is the first report of the chemical profile of this tree. Compounds 1-4 exhibited excellent anti-complement activity with IC(50) values less than 74 microM, compared with tiliroside (IC(50) = 104 microM) and rosmarinic acid (IC(50) = 182 microM) that were used as positive controls.

  19. Antibacterial activity of peptides derived from the C-terminal region of a hemolytic lectin, CEL-III, from the marine invertebrate Cucumaria echinata.

    PubMed

    Hatakeyama, Tomomitsu; Suenaga, Tomoko; Eto, Seiichiro; Niidome, Takuro; Aoyagi, Haruhiko

    2004-01-01

    Several synthetic peptides derived from the C-terminal domain sequence of a hemolytic lectin, CEL-III, were examined as to their action on bacteria and artificial lipid membranes. Peptide P332 (KGVIFAKASVSVKVTASLSK-NH(2)), corresponding to the sequence from residue 332, exhibited strong antibacterial activity toward Gram-positive bacteria. Replacement of each Lys in P332 by Ala markedly decreased the activity. However, when all Lys were replaced by Arg, the antibacterial activity increased, indicating the importance of positively charged residues at these positions. Replacement of Val by Leu also led to higher antibacterial activity, especially toward Gram-negative bacteria. The antibacterial activity of these peptides was correlated with their membrane-permeabilizing activity toward the bacterial inner membrane and artificial lipid vesicles, indicating that the antibacterial action is due to perturbation of bacterial cell membranes, leading to enhancement of their permeability. These results also suggest that the hydrophobic region of CEL-III, from which P332 and its analogs were derived, may play some role in the interaction with target cell membranes to trigger hemolysis.

  20. 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

  1. Prevention of complement-mediated immune hemolysis by a small molecule compound.

    PubMed

    Mqadmi, Amina; Zheng, Xiaoying; Song, Jinmei; Abramowitz, Steven; Giclas, Patricia; Yazdanbakhsh, Karina

    2004-12-24

    Complement sensitization of red blood cells (RBCs) can result in transfusion reactions and hemolytic anemias. We hypothesized that manipulating the complement system using small organic molecules might prevent RBC destruction, thereby prolonging RBC survival in patients. Using a simple, rapid, large-scale hemolytic assay, we screened a 10,000 compound library, enriched in anti-inflammatory compounds at a final concentration of 25 microM, and identified a 549Da compound (C(34)H(24)N(6)O(2)) with a symmetrical structure containing two benzimidazole rings that, as compared to a known anti-complement molecule FUT-175, was more effective in reducing hemolysis by the classical pathway and had comparable anti-hemolytic activity against the alternative pathway. Furthermore, in a xenotransfusion mouse model, treatment of mice with 1.2mg/kg of the compound significantly prolonged the survival of transfused RBCs, reducing C3 deposition, but not the deposition of control IgG or IgM, for the first hour post-transfusion. These data suggest that further studies are warranted to determine if this compound has usefulness in a transfusion setting.

  2. Contribution of Chondroitin Sulfate A to the Binding of Complement Proteins to Activated Platelets

    PubMed Central

    Lasaosa, Maria; Ricklin, Daniel; Lambris, John D.; Nilsson, Bo; Nilsson Ekdahl, Kristina

    2010-01-01

    Background Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of the complement recognition molecule C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets. Principal Findings Human blood serum was passed over Sepharose conjugated with CS-A, and CS-A-specific binding proteins were identified by Western blotting and mass spectrometric analysis. C1q was shown to be the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and then not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were also shown to bind to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on activated platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets. Conclusions This study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q also seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases. PMID:20886107

  3. The paralogous salivary anti-complement proteins IRAC I and IRAC II encoded by Ixodes ricinus ticks have broad and complementary inhibitory activities against the complement of different host species.

    PubMed

    Schroeder, Hélène; Daix, Virginie; Gillet, Laurent; Renauld, Jean-Christophe; Vanderplasschen, Alain

    2007-02-01

    Several observations suggest that inhibition of the host complement alternative pathway by Ixodes tick saliva is crucial to achieve blood feeding. We recently described two paralogous anti-complement proteins called Ixodes ricinus anti-complement (IRAC) proteins I and II co-expressed in I. ricinus salivary glands. Phylogenetic analyses suggested that these sequences were diversifying by a process of positive Darwinian selection, possibly leading to molecules with different biological properties. In the present study, we tested the hypothesis that each paralogue may have different inhibitory activities against the complement of different natural host species, thereby contributing to broaden the host range of I. ricinus ticks. IRAC I and IRAC II were tested against the complement of eight I. ricinus natural host species (six mammals and two birds). The results demonstrate that IRAC I and IRAC II have broad and complementary inhibition activities against the complement of different host species. This report is the first description of paralogous anti-complement molecules encoded by a pathogen with broad and complementary inhibitory activities against the complement of different host species.

  4. Near-planar Solution Structures of Mannose-binding Lectin Oligomers Provide Insight on Activation of Lectin Pathway of Complement

    PubMed Central

    Miller, Ami; Phillips, Anna; Gor, Jayesh; Wallis, Russell; Perkins, Stephen J.

    2012-01-01

    The complement system is a fundamental component of innate immunity that orchestrates complex immunological and inflammatory processes. Complement comprises over 30 proteins that eliminate invading microorganisms while maintaining host cell integrity. Protein-carbohydrate interactions play critical roles in both the activation and regulation of complement. Mannose-binding lectin (MBL) activates the lectin pathway of complement via the recognition of sugar arrays on pathogenic surfaces. To determine the solution structure of MBL, synchrotron x-ray scattering and analytical ultracentrifugation experiments showed that the carbohydrate-recognition domains in the MBL dimer, trimer, and tetramer are positioned close to each other in near-planar fan-like structures. These data were subjected to constrained modeling fits. A bent structure for the MBL monomer was identified starting from two crystal structures for its carbohydrate-recognition domain and its triple helical region. The MBL monomer structure was used to identify 10–12 near-planar solution structures for each of the MBL dimers, trimers, and tetramers starting from 900 to 6,859 randomized structures for each. These near-planar fan-like solution structures joined at an N-terminal hub clarified how the carbohydrate-recognition domain of MBL binds to pathogenic surfaces. They also provided insight on how MBL presents a structural template for the binding and auto-activation of the MBL-associated serine proteases to initiate the lectin pathway of complement activation. PMID:22167201

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2000-04-01

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

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

    PubMed Central

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

    2017-01-01

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

  8. 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.

  9. Mechanisms of action of (meth)acrylates in hemolytic activity, in vivo toxicity and dipalmitoylphosphatidylcholine (DPPC) liposomes determined using NMR spectroscopy.

    PubMed

    Fujisawa, Seiichiro; Kadoma, Yoshinori

    2012-01-01

    We investigated the quantitative structure-activity relationships between hemolytic activity (log 1/H(50)) or in vivo mouse intraperitoneal (ip) LD(50) using reported data for α,β-unsaturated carbonyl compounds such as (meth)acrylate monomers and their (13)C-NMR β-carbon chemical shift (δ). The log 1/H(50) value for methacrylates was linearly correlated with the δC(β) value. That for (meth)acrylates was linearly correlated with log P, an index of lipophilicity. The ipLD(50) for (meth)acrylates was linearly correlated with δC(β) but not with log P. For (meth)acrylates, the δC(β) value, which is dependent on the π-electron density on the β-carbon, was linearly correlated with PM3-based theoretical parameters (chemical hardness, η; electronegativity, χ; electrophilicity, ω), whereas log P was linearly correlated with heat of formation (HF). Also, the interaction between (meth)acrylates and DPPC liposomes in cell membrane molecular models was investigated using (1)H-NMR spectroscopy and differential scanning calorimetry (DSC). The log 1/H(50) value was related to the difference in chemical shift (ΔδHa) (Ha: H (trans) attached to the β-carbon) between the free monomer and the DPPC liposome-bound monomer. Monomer-induced DSC phase transition properties were related to HF for monomers. NMR chemical shifts may represent a valuable parameter for investigating the biological mechanisms of action of (meth)acrylates.

  10. 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/C3adesarg 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.

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

    PubMed Central

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

    2015-01-01

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

  12. Activity Recognition Using Community Data to Complement Small Amounts of Labeled Instances †

    PubMed Central

    Garcia-Ceja, Enrique; Brena, Ramon F.

    2016-01-01

    Human Activity Recognition (HAR) is an important part of ambient intelligence systems since it can provide user-context information, thus allowing a greater personalization of services. One of the problems with HAR systems is that the labeling process for the training data is costly, which has hindered its practical application. A common approach is to train a general model with the aggregated data from all users. The problem is that for a new target user, this model can perform poorly because it is biased towards the majority type of users and does not take into account the particular characteristics of the target user. To overcome this limitation, a user-dependent model can be trained with data only from the target user that will be optimal for this particular user; however, this requires a considerable amount of labeled data, which is cumbersome to obtain. In this work, we propose a method to build a personalized model for a given target user that does not require large amounts of labeled data. Our method uses data already labeled by a community of users to complement the scarce labeled data of the target user. Our results showed that the personalized model outperformed the general and the user-dependent models when labeled data is scarce. PMID:27314355

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

    PubMed

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

    1984-12-20

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

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

    PubMed Central

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

    2014-01-01

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

  15. A Serine Protease Isolated from the Bristles of the Amazonic Caterpillar, Premolis semirufa, Is a Potent Complement System Activator

    PubMed Central

    Villas Boas, Isadora Maria; Pidde-Queiroz, Giselle; Magnoli, Fabio Carlos; Gonçalves-de-Andrade, Rute M.; van den Berg, Carmen W.; Tambourgi, Denise V.

    2015-01-01

    Background The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called “Pararamose”, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa’s bristles extract could interfere with the human complement system. Results The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly

  16. The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

    PubMed Central

    Scambi, Cinzia; Ugolini, Sara; Jokiranta, T. Sakari; De Franceschi, Lucia; Bortolami, Oscar; La Verde, Valentina; Guarini, Patrizia; Caramaschi, Paola; Ravagnani, Viviana; Martignoni, Guido; Colato, Chiara; Pedron, Serena; Benedetti, Fabio; Sorio, Marco; Poli, Fabio; Biasi, Domenico

    2015-01-01

    Objective The role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region. Methods C5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method. Results Even though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases. Conclusions Our results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation. PMID:25658605

  17. Interaction of complement and leukocytes in severe acute pancreatitis: potential for therapeutic intervention.

    PubMed

    Hartwig, Werner; Klafs, Martina; Kirschfink, Michael; Hackert, Thilo; Schneider, Lutz; Gebhard, Martha-Maria; Büchler, Markus W; Werner, Jens

    2006-11-01

    In acute pancreatitis, local as well as systemic organ complications are mediated by the activation of various inflammatory cascades. The role of complement in this setting is unclear. The aim of the present study was to determine the level of complement activation in experimental pancreatitis, to evaluate the interaction of complement and leukocyte-endothelium activation, and to assess the effects of complement inhibition by soluble complement receptor 1 (sCR1) in this setting. Necrotizing pancreatitis was induced in Wistar rats by the combination of intravenous cerulein and retrograde infusion of glycodeoxycholic acid into the biliopancreatic duct; edematous pancreatitis was induced by intravenous cerulein only. In control animals, a sham operation (midline laparotomy) was performed. Complement activation, leukocyte sequestration, and pancreatic as well as pulmonary injury were assessed in the presence/absence of sCR1. Increased levels of C3a were found in necrotizing but not in edematous pancreatitis. When complement activation in necrotizing pancreatitis was blocked by sCR1, levels of C3a and total hemolytic activity (CH50) were decreased. Leukocyte-endothelial interaction, as assessed by intravital microscopy, and pancreatic as well as pulmonary organ injury (wet-to-dry weight ratio, MPO activity, and histology) were ameliorated by sCR1. As a result of the present study, necrotizing but not edematous pancreatitis is characterized by significant and early complement activation. Based on the interaction of complement and leukocytes, complement inhibition by sCR1 may be a valuable option in the treatment of leukocyte-associated organ injury in severe pancreatitis.

  18. Immune Response to Snake Envenoming and Treatment with Antivenom; Complement Activation, Cytokine Production and Mast Cell Degranulation

    PubMed Central

    Stone, Shelley F.; Isbister, Geoffrey K.; Shahmy, Seyed; Mohamed, Fahim; Abeysinghe, Chandana; Karunathilake, Harendra; Ariaratnam, Ariaranee; Jacoby-Alner, Tamara E.; Cotterell, Claire L.; Brown, Simon G. A.

    2013-01-01

    Background Snake bite is one of the most neglected public health issues in poor rural communities worldwide. In addition to the clinical effects of envenoming, treatment with antivenom frequently causes serious adverse reactions, including hypersensitivity reactions (including anaphylaxis) and pyrogenic reactions. We aimed to investigate the immune responses to Sri Lankan snake envenoming (predominantly by Russell's viper) and antivenom treatment. Methodology/Principal Findings Plasma concentrations of Interleukin (IL)-6, IL-10, tumor necrosis factor α (TNFα), soluble TNF receptor I (sTNFRI), anaphylatoxins (C3a, C4a, C5a; markers of complement activation), mast cell tryptase (MCT), and histamine were measured in 120 Sri Lankan snakebite victims, both before and after treatment with antivenom. Immune mediator concentrations were correlated with envenoming features and the severity of antivenom-induced reactions including anaphylaxis. Envenoming was associated with complement activation and increased cytokine concentrations prior to antivenom administration, which correlated with non-specific systemic symptoms of envenoming but not with coagulopathy or neurotoxicity. Typical hypersensitivity reactions to antivenom occurred in 77/120 patients (64%), satisfying criteria for a diagnosis of anaphylaxis in 57/120 (48%). Pyrogenic reactions were observed in 32/120 patients (27%). All patients had further elevations in cytokine concentrations, but not complement activation, after the administration of antivenom, whether a reaction was noted to occur or not. Patients with anaphylaxis had significantly elevated concentrations of MCT and histamine. Conclusions/Significance We have demonstrated that Sri Lankan snake envenoming is characterized by significant complement activation and release of inflammatory mediators. Antivenom treatment further enhances the release of inflammatory mediators in all patients, with anaphylactic reactions characterised by high levels of mast

  19. Complement Activation by Giardia duodenalis Parasites through the Lectin Pathway Contributes to Mast Cell Responses and Parasite Control

    PubMed Central

    Li, Erqiu; Tako, Ernest A.

    2016-01-01

    Infection with Giardia duodenalis is one of the most common causes of diarrheal disease in the world. While numerous studies have identified important contributions of adaptive immune responses to parasite control, much less work has examined innate immunity and its connections to the adaptive response during this infection. We explored the role of complement in immunity to Giardia using mice deficient in mannose-binding lectin (Mbl2) or complement factor 3a receptor (C3aR). Both strains exhibited delayed clearance of parasites and a reduced ability to recruit mast cells in the intestinal submucosa. C3aR-deficient mice had normal production of antiparasite IgA, but ex vivo T cell recall responses were impaired. These data suggest that complement is a key factor in the innate recognition of Giardia and that recruitment of mast cells and activation of T cell immunity through C3a are important for parasite control. PMID:26831470

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

    PubMed Central

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

    2017-01-01

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

  1. Lectin Pathway of Complement Activation Is Associated with Vulnerability of Atherosclerotic Plaques

    PubMed Central

    Fumagalli, Stefano; Perego, Carlo; Zangari, Rosalia; De Blasio, Daiana; Oggioni, Marco; De Nigris, Francesca; Snider, Francesco; Garred, Peter; Ferrante, Angela M. R.; De Simoni, Maria-Grazia

    2017-01-01

    Inflammatory mechanisms may be involved in atherosclerotic plaque rupture. By using a novel histology-based method to quantify plaque instability here, we assess whether lectin pathway (LP) of complement activation, a major inflammation arm, could represent an index of plaque instability. Plaques from 42 consecutive patients undergoing carotid endarterectomy were stained with hematoxylin-eosin and the lipid core, cholesterol clefts, hemorrhagic content, thickness of tunica media, and intima, including or not infiltration of cellular debris and cholesterol, were determined. The presence of ficolin-1, -2, and -3 and mannose-binding lectin (MBL), LP initiators, was assessed in the plaques by immunofluorescence and in plasma by ELISA. LP activation was assessed in plasma by functional in vitro assays. Patients presenting low stenosis (≤75%) had higher hemorrhagic content than those with high stenosis (>75%), indicating increased erosion. Increased hemorrhagic content and tunica media thickness, as well as decreased lipid core and infiltrated content were associated with vulnerable plaques and therefore used to establish a plaque vulnerability score that allowed to classify patients according to plaque vulnerability. Ficolins and MBL were found both in plaques’ necrotic core and tunica media. Patients with vulnerable plaques showed decreased plasma levels and intraplaque deposition of ficolin-2. Symptomatic patients experiencing a transient ischemic attack had lower plasma levels of ficolin-1. We show that the LP initiators are present within the plaques and their circulating levels change in atherosclerotic patients. In particular, we show that decreased ficolin-2 levels are associated with rupture-prone vulnerable plaques, indicating its potential use as marker for cardiovascular risk assessment in atherosclerotic patients. PMID:28360913

  2. Disease-causing mutations in genes of the complement system.

    PubMed

    Degn, Søren E; Jensenius, Jens C; Thiel, Steffen

    2011-06-10

    Recent studies have revealed profound developmental consequences of mutations in genes encoding proteins of the lectin pathway of complement activation, a central component of the innate immune system. Apart from impairment of immunity against microorganisms, it is known that hereditary deficiencies of this system predispose one to autoimmune conditions. Polymorphisms in complement genes are linked to, for example, atypical hemolytic uremia and age-dependent macular degeneration. The complement system comprises three convergent pathways of activation: the classical, the alternative, and the lectin pathway. The recently discovered lectin pathway is less studied, but polymorphisms in the plasma pattern-recognition molecule mannan-binding lectin (MBL) are known to impact its level, and polymorphisms in the MBL-associated serine protease-2 (MASP-2) result in defects of complement activation. Recent studies have described roles outside complement and immunity of another MBL-associated serine protease, MASP-3, in the etiology of 3MC syndrome, an autosomal-recessive disorder involving a spectrum of developmental features, including characteristic facial dysmorphism. Syndrome-causing mutations were identified in MASP1, encoding MASP-3 and two additional proteins, MASP-1 and MAp44. Furthermore, an association was discovered between 3MC syndrome and mutations in COLEC11, encoding CL-K1, another molecule of the lectin pathway. The findings were confirmed in zebrafish, indicating that MASP-3 and CL-K1 underlie an evolutionarily conserved pathway of embryonic development. Along with the discovery of a role of C1q in pruning synapses in mice, these recent advances point toward a broader role of complement in development. Here, we compare the functional immunologic consequences of "conventional" complement deficiencies with these newly described developmental roles.

  3. Disease-Causing Mutations in Genes of the Complement System

    PubMed Central

    Degn, Søren E.; Jensenius, Jens C.; Thiel, Steffen

    2011-01-01

    Recent studies have revealed profound developmental consequences of mutations in genes encoding proteins of the lectin pathway of complement activation, a central component of the innate immune system. Apart from impairment of immunity against microorganisms, it is known that hereditary deficiencies of this system predispose one to autoimmune conditions. Polymorphisms in complement genes are linked to, for example, atypical hemolytic uremia and age-dependent macular degeneration. The complement system comprises three convergent pathways of activation: the classical, the alternative, and the lectin pathway. The recently discovered lectin pathway is less studied, but polymorphisms in the plasma pattern-recognition molecule mannan-binding lectin (MBL) are known to impact its level, and polymorphisms in the MBL-associated serine protease-2 (MASP-2) result in defects of complement activation. Recent studies have described roles outside complement and immunity of another MBL-associated serine protease, MASP-3, in the etiology of 3MC syndrome, an autosomal-recessive disorder involving a spectrum of developmental features, including characteristic facial dysmorphism. Syndrome-causing mutations were identified in MASP1, encoding MASP-3 and two additional proteins, MASP-1 and MAp44. Furthermore, an association was discovered between 3MC syndrome and mutations in COLEC11, encoding CL-K1, another molecule of the lectin pathway. The findings were confirmed in zebrafish, indicating that MASP-3 and CL-K1 underlie an evolutionarily conserved pathway of embryonic development. Along with the discovery of a role of C1q in pruning synapses in mice, these recent advances point toward a broader role of complement in development. Here, we compare the functional immunologic consequences of “conventional” complement deficiencies with these newly described developmental roles. PMID:21664996

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

    PubMed Central

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

    2016-01-01

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

  5. Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement.

    PubMed

    Choy, Henry A

    2012-01-01

    Microbial pathogens acquire the immediate imperative to avoid or counteract the formidable defense of innate immunity as soon as they overcome the initial physical barriers of the host. Many have adopted the strategy of directly disrupting the complement system through the capture of its components, using proteins on the pathogen's surface. In leptospirosis, pathogenic Leptospira spp. are resistant to complement-mediated killing, in contrast to the highly vulnerable non-pathogenic strains. Pathogenic L. interrogans uses LenA/LfhA and LcpA to respectively sequester and commandeer the function of two regulators, factor H and C4BP, which in turn bind C3b or C4b to interrupt the alternative or classical pathways of complement activation. LigB, another surface-proximal protein originally characterized as an adhesin binding multiple host proteins, has other activities suggesting its importance early in infection, including binding extracellular matrix, plasma, and cutaneous repair proteins and inhibiting hemostasis. In this study, we used a recent model of ectopic expression of LigB in the saprophyte, L. biflexa, to test the hypothesis that LigB also interacts with complement proteins C3b and C4b to promote the virulence of L. interrogans. The surface expression of LigB partially rescued the non-pathogen from killing by 5% normal human serum, showing 1.3- to 48-fold greater survival 4 to 6 d following exposure to complement than cultures of the non-expressing parental strain. Recombinant LigB7'-12 comprising the LigB-specific immunoglobulin repeats binds directly to human complement proteins, C3b and C4b, with respective K(d)s of 43±26 nM and 69±18 nM. Repeats 9 to 11, previously shown to contain the binding domain for fibronectin and fibrinogen, are also important in LigB-complement interactions, which interfere with the alternative and classical pathways measured by complement-mediated hemolysis of erythrocytes. Thus, LigB is an adaptable interface for L. interrogans

  6. Antibacterial Activity of the Contact and Complement Systems Is Blocked by SIC, a Protein Secreted by Streptococcus pyogenes*

    PubMed Central

    Frick, Inga-Maria; Shannon, Oonagh; Åkesson, Per; Mörgelin, Matthias; Collin, Mattias; Schmidtchen, Artur; Björck, Lars

    2011-01-01

    Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides. Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein. Cases of invasive and life-threatening S. pyogenes infections are commonly associated with isolates of the M1 serotype, and in contrast to the large majority of M serotypes, M1 isolates all secrete the SIC protein. Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation. This effect promotes the growth of S. pyogenes in human plasma, and in a mouse model of S. pyogenes sepsis, SIC enhances bacterial dissemination, results which help explain the high frequency of severe S. pyogenes infections caused by isolates of the M1 serotype. PMID:21068386

  7. Anti-microbial activity and anti-complement activity of extracts obtained from selected Hawaiian medicinal plants.

    PubMed

    Locher, C P; Burch, M T; Mower, H F; Berestecky, J; Davis, H; Van Poel, B; Lasure, A; Vanden Berghe, D A; Vlietinck, A J

    1995-11-17

    Selected plants having a history of use in Polynesian traditional medicine for the treatment of infectious disease were investigated for anti-viral, anti-fungal and anti-bacterial activity in vitro. Extracts from Scaevola sericea, Psychotria hawaiiensis, Pipturus albidus and Eugenia malaccensis showed selective anti-viral activity against Herpes Simplex Virus-1 and 2 and Vesicular Stomatitis Virus. Aleurites moluccana extracts showed anti-bacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, while Pipturus albidus and Eugenia malaccensis extracts showed growth inhibition of Staphylococcus aureus and Streptococcus pyogenes. Psychotria hawaiiensis and Solanum niger inhibited growth of the fungi Microsporum canis, Trichophyton rubrum and Epidermophyton floccosum, while Ipomoea sp., Pipturus albidus, Scaevola sericea, Eugenia malaccensis, Piper methysticum, Barringtonia asiatica and Adansonia digitata extracts showed anti-fungal activity to a lesser extent. Eugenia malaccensis was also found to inhibit the classical pathway of complement suggesting that an immunological basis for its in vivo activity was identified. This study has confirmed some of the ethnobotanical reports of Hawaiian medicinal plants having curative properties against infections using biological assays in vitro.

  8. 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.

  9. Endothelial targeting with C1-inhibitor reduces complement activation in vitro and during ex vivo reperfusion of pig liver

    PubMed Central

    Bergamaschini, L; Gobbo, G; Gatti, S; Caccamo, L; Prato, P; Maggioni, M; Braidotti, P; Di Stefano, R; Fassati, L R

    2001-01-01

    Tissue damage during cold storage and reperfusion remains a major obstacle to wider use of transplantation. Vascular endothelial cells and complement activation are thought to be involved in the inflammatory reactions following reperfusion, so endothelial targeting of complement inhibitors is of great interest. Using an in vitro model of human umbilical vein endothelial cells (HUVEC) cold storage and an animal model of ex vivo liver reperfusion after cold ischaemia, we assessed the effect of C1-INH on cell functions and liver damage. We found that in vitro C1-INH bound to HUVEC in a manner depending on the duration of cold storage. Cell-bound C1-INH was functionally active since retained the ability to inhibit exogenous C1s. To assess the ability of cell-bound C1-INH to prevent complement activation during organ reperfusion, we added C1-INH to the preservation solution in an animal model of extracorporeal liver reperfusion. Ex vivo liver reperfusion after 8 h of cold ischaemia resulted in plasma C3 activation and reduction of total serum haemolytic activity, and at tissue level deposition of C3 associated with variable level of inflammatory cell infiltration and tissue damage. These findings were reduced when livers were stored in preservation solution containing C1-INH. Immunohistochemical analysis of C1-INH-treated livers showed immunoreactivity localized on the sinusoidal pole of the liver trabeculae, linked to sinusoidal endothelium, so it is likely that the protective effect was due to C1-INH retained by the livers. These results suggest that adding C1-INH to the preservation solution may be useful to reduce complement activation and tissue injury during the reperfusion of an ischaemic liver. PMID:11737055

  10. Endothelial targeting with C1-inhibitor reduces complement activation in vitro and during ex vivo reperfusion of pig liver.

    PubMed

    Bergamaschini, L; Gobbo, G; Gatti, S; Caccamo, L; Prato, P; Maggioni, M; Braidotti, P; Di Stefano, R; Fassati, L R

    2001-12-01

    Tissue damage during cold storage and reperfusion remains a major obstacle to wider use of transplantation. Vascular endothelial cells and complement activation are thought to be involved in the inflammatory reactions following reperfusion, so endothelial targeting of complement inhibitors is of great interest. Using an in vitro model of human umbilical vein endothelial cells (HUVEC) cold storage and an animal model of ex vivo liver reperfusion after cold ischaemia, we assessed the effect of C1-INH on cell functions and liver damage. We found that in vitro C1-INH bound to HUVEC in a manner depending on the duration of cold storage. Cell-bound C1-INH was functionally active since retained the ability to inhibit exogenous C1s. To assess the ability of cell-bound C1-INH to prevent complement activation during organ reperfusion, we added C1-INH to the preservation solution in an animal model of extracorporeal liver reperfusion. Ex vivo liver reperfusion after 8 h of cold ischaemia resulted in plasma C3 activation and reduction of total serum haemolytic activity, and at tissue level deposition of C3 associated with variable level of inflammatory cell infiltration and tissue damage. These findings were reduced when livers were stored in preservation solution containing C1-INH. Immunohistochemical analysis of C1-INH-treated livers showed immunoreactivity localized on the sinusoidal pole of the liver trabeculae, linked to sinusoidal endothelium, so it is likely that the protective effect was due to C1-INH retained by the livers. These results suggest that adding C1-INH to the preservation solution may be useful to reduce complement activation and tissue injury during the reperfusion of an ischaemic liver.

  11. A Web Server and Mobile App for Computing Hemolytic Potency of Peptides

    PubMed Central

    Chaudhary, Kumardeep; Kumar, Ritesh; Singh, Sandeep; Tuknait, Abhishek; Gautam, Ankur; Mathur, Deepika; Anand, Priya; Varshney, Grish C.; Raghava, Gajendra P. S.

    2016-01-01

    Numerous therapeutic peptides do not enter the clinical trials just because of their high hemolytic activity. Recently, we developed a database, Hemolytik, for maintaining experimentally validated hemolytic and non-hemolytic peptides. The present study describes a web server and mobile app developed for predicting, and screening of peptides having hemolytic potency. Firstly, we generated a dataset HemoPI-1 that contains 552 hemolytic peptides extracted from Hemolytik database and 552 random non-hemolytic peptides (from Swiss-Prot). The sequence analysis of these peptides revealed that certain residues (e.g., L, K, F, W) and motifs (e.g., “FKK”, “LKL”, “KKLL”, “KWK”, “VLK”, “CYCR”, “CRR”, “RFC”, “RRR”, “LKKL”) are more abundant in hemolytic peptides. Therefore, we developed models for discriminating hemolytic and non-hemolytic peptides using various machine learning techniques and achieved more than 95% accuracy. We also developed models for discriminating peptides having high and low hemolytic potential on different datasets called HemoPI-2 and HemoPI-3. In order to serve the scientific community, we developed a web server, mobile app and JAVA-based standalone software (http://crdd.osdd.net/raghava/hemopi/). PMID:26953092

  12. A Web Server and Mobile App for Computing Hemolytic Potency of Peptides

    NASA Astrophysics Data System (ADS)

    Chaudhary, Kumardeep; Kumar, Ritesh; Singh, Sandeep; Tuknait, Abhishek; Gautam, Ankur; Mathur, Deepika; Anand, Priya; Varshney, Grish C.; Raghava, Gajendra P. S.

    2016-03-01

    Numerous therapeutic peptides do not enter the clinical trials just because of their high hemolytic activity. Recently, we developed a database, Hemolytik, for maintaining experimentally validated hemolytic and non-hemolytic peptides. The present study describes a web server and mobile app developed for predicting, and screening of peptides having hemolytic potency. Firstly, we generated a dataset HemoPI-1 that contains 552 hemolytic peptides extracted from Hemolytik database and 552 random non-hemolytic peptides (from Swiss-Prot). The sequence analysis of these peptides revealed that certain residues (e.g., L, K, F, W) and motifs (e.g., “FKK”, “LKL”, “KKLL”, “KWK”, “VLK”, “CYCR”, “CRR”, “RFC”, “RRR”, “LKKL”) are more abundant in hemolytic peptides. Therefore, we developed models for discriminating hemolytic and non-hemolytic peptides using various machine learning techniques and achieved more than 95% accuracy. We also developed models for discriminating peptides having high and low hemolytic potential on different datasets called HemoPI-2 and HemoPI-3. In order to serve the scientific community, we developed a web server, mobile app and JAVA-based standalone software (http://crdd.osdd.net/raghava/hemopi/).

  13. A Web Server and Mobile App for Computing Hemolytic Potency of Peptides.

    PubMed

    Chaudhary, Kumardeep; Kumar, Ritesh; Singh, Sandeep; Tuknait, Abhishek; Gautam, Ankur; Mathur, Deepika; Anand, Priya; Varshney, Grish C; Raghava, Gajendra P S

    2016-03-08

    Numerous therapeutic peptides do not enter the clinical trials just because of their high hemolytic activity. Recently, we developed a database, Hemolytik, for maintaining experimentally validated hemolytic and non-hemolytic peptides. The present study describes a web server and mobile app developed for predicting, and screening of peptides having hemolytic potency. Firstly, we generated a dataset HemoPI-1 that contains 552 hemolytic peptides extracted from Hemolytik database and 552 random non-hemolytic peptides (from Swiss-Prot). The sequence analysis of these peptides revealed that certain residues (e.g., L, K, F, W) and motifs (e.g., "FKK", "LKL", "KKLL", "KWK", "VLK", "CYCR", "CRR", "RFC", "RRR", "LKKL") are more abundant in hemolytic peptides. Therefore, we developed models for discriminating hemolytic and non-hemolytic peptides using various machine learning techniques and achieved more than 95% accuracy. We also developed models for discriminating peptides having high and low hemolytic potential on different datasets called HemoPI-2 and HemoPI-3. In order to serve the scientific community, we developed a web server, mobile app and JAVA-based standalone software (http://crdd.osdd.net/raghava/hemopi/).

  14. A Gammaherpesvirus Complement Regulatory Protein Promotes Initiation of Infection by Activation of Protein Kinase Akt/PKB

    PubMed Central

    Steer, Beatrix; Adler, Barbara; Jonjic, Stipan; Stewart, James P.; Adler, Heiko

    2010-01-01

    Background Viruses have evolved to evade the host's complement system. The open reading frames 4 (ORF4) of gammaherpesviruses encode homologs of regulators of complement activation (RCA) proteins, which inhibit complement activation at the level of C3 and C4 deposition. Besides complement regulation, these proteins are involved in heparan sulfate and glycosaminoglycan binding, and in case of MHV-68, also in viral DNA synthesis in macrophages. Methodology/Principal Findings Here, we made use of MHV-68 to study the role of ORF4 during infection of fibroblasts. While attachment and penetration of virions lacking the RCA protein were not affected, we observed a delayed delivery of the viral genome to the nucleus of infected cells. Analysis of the phosphorylation status of a variety of kinases revealed a significant reduction in phosphorylation of the protein kinase Akt in cells infected with ORF4 mutant virus, when compared to cells infected with wt virus. Consistent with a role of Akt activation in initial stages of infection, inhibition of Akt signaling in wt virus infected cells resulted in a phenotype resembling the phenotype of the ORF4 mutant virus, and activation of Akt by addition of insulin partially reversed the phenotype of the ORF4 mutant virus. Importantly, the homologous ORF4 of KSHV was able to rescue the phenotype of the MHV-68 ORF4 mutant, indicating that ORF4 is functionally conserved and that ORF4 of KSHV might have a similar function in infection initiation. Conclusions/Significance In summary, our studies demonstrate that ORF4 contributes to efficient infection by activation of the protein kinase Akt and thus reveal a novel function of a gammaherpesvirus RCA protein. PMID:20657771

  15. A Novel Interaction between Complement Inhibitor C4b-binding Protein and Plasminogen That Enhances Plasminogen Activation*

    PubMed Central

    Agarwal, Vaibhav; Talens, Simone; Grandits, Alexander M.; Blom, Anna M.

    2015-01-01

    The complement, coagulation, and fibrinolytic systems are crucial for the maintenance of tissue homeostasis. To date numerous interactions and cross-talks have been identified between these cascades. In line with this, here we propose a novel, hitherto unknown interaction between the complement inhibitor C4b-binding protein (C4BP) and plasminogen of the fibrinolytic pathway. Binding of C4BP to Streptococcus pneumoniae is a known virulence mechanism of this pathogen and it was increased in the presence of plasminogen. Interestingly, the acute phase variant of C4BP lacking the β-chain and protein S binds plasminogen much stronger than the main isoform containing the β-chain and protein S. Indeed, the complement control protein (CCP) 8 domain of C4BP, which would otherwise be sterically hindered by the β-chain, primarily mediates this interaction. Moreover, the lysine-binding sites in plasminogen kringle domains facilitate the C4BP-plasminogen interaction. Furthermore, C4BP readily forms complexes with plasminogen in fluid phase and such complexes are present in human serum and plasma. Importantly, whereas the presence of plasminogen did not affect the factor I cofactor activity of C4BP, the activation of plasminogen by urokinase-type plasminogen activator to active plasmin was significantly augmented in the presence of C4BP. Taken together, our data demonstrate a novel interaction between two proteins of the complement and fibrinolytic system. Most complexes might be formed during the acute phase of inflammation and have an effect on the homeostasis at the site of injury or acute inflammation. PMID:26067271

  16. Secondary Cell Wall Polymers of Enterococcus faecalis Are Critical for Resistance to Complement Activation via Mannose-binding Lectin*

    PubMed Central

    Geiss-Liebisch, Stefan; Rooijakkers, Suzan H. M.; Beczala, Agnieszka; Sanchez-Carballo, Patricia; Kruszynska, Karolina; Repp, Christian; Sakinc, Tuerkan; Vinogradov, Evgeny; Holst, Otto; Huebner, Johannes; Theilacker, Christian

    2012-01-01

    The complement system is part of our first line of defense against invading pathogens. The strategies used by Enterococcus faecalis to evade recognition by human complement are incompletely understood. In this study, we identified an insertional mutant of the wall teichoic acid (WTA) synthesis gene tagB in E. faecalis V583 that exhibited an increased susceptibility to complement-mediated killing by neutrophils. Further analysis revealed that increased killing of the mutant was due to a higher rate of phagocytosis by neutrophils, which correlated with higher C3b deposition on the bacterial surface. Our studies indicated that complement activation via the lectin pathway was much stronger on the tagB mutant compared with wild type. In concordance, we found an increased binding of the key lectin pathway components mannose-binding lectin and mannose-binding lectin-associated serine protease-2 (MASP-2) on the mutant. To understand the mechanism of lectin pathway inhibition by E. faecalis, we purified and characterized cell wall carbohydrates of E. faecalis wild type and V583ΔtagB. NMR analysis revealed that the mutant strain lacked two WTAs with a repeating unit of →6)[α-l-Rhap-(1→3)]β-d-GalpNAc-(1→5)-Rbo-1-P and →6) β-d-Glcp-(1→3) [α-d-Glcp-(1→4)]-β-d-GalpNAc-(1→5)-Rbo-1-P→, respectively (Rbo, ribitol). In addition, compositional changes in the enterococcal rhamnopolysaccharide were noticed. Our study indicates that in E. faecalis, modification of peptidoglycan by secondary cell wall polymers is critical to evade recognition by the complement system. PMID:22908219

  17. Activated signal transducers and activators of transcription 3 signaling induces CD46 expression and protects human cancer cells from complement-dependent cytotoxicity.

    PubMed

    Buettner, Ralf; Huang, Mei; Gritsko, Tanya; Karras, Jim; Enkemann, Steve; Mesa, Tania; Nam, Sangkil; Yu, Hua; Jove, Richard

    2007-08-01

    CD46 is one of the complement-regulatory proteins expressed on the surface of normal and tumor cells for protection against complement-dependent cytotoxicity. Cancer cells need to access the blood circulation for continued growth and metastasis, thus exposing themselves to destruction by complement system components. Previous studies have established that the signal transducers and activators of transcription 3 (STAT3) transcription factor is persistently activated in a wide variety of human cancer cells and primary tumor tissues compared with their normal counterparts. Using microarray gene expression profiling, we identified the CD46 gene as a target for activated STAT3 signaling in human breast and prostate cancer cells. The CD46 promoter contains two binding sites for activated STAT3 and mutations introduced into the major site abolished STAT3 binding. Chromatin immunoprecipitation confirms binding of STAT3 to the CD46 promoter. CD46 promoter activity is induced by activation of STAT3 and blocked by a dominant-negative form of STAT3 in luciferase reporter assays. CD46 mRNA expression is induced by interleukin-6 and by transient transfection of normal human epithelial cells with a persistently active mutant construct of STAT3, STAT3C. Furthermore, we show that inhibition of STAT3-mediated CD46 cell surface expression sensitizes DU145 prostate cancer cells to cytotoxicity in an in vitro complement lysis assay using rabbit anti-DU145 antiserum and rabbit complement. These results show that activated STAT3 signaling induces the CD46 promoter and protects human cancer cells from complement-dependent cytotoxicity, suggesting a potential mechanism whereby oncogenic signaling contributes to tumor cell evasion of antibody-mediated immunity.

  18. Moss-Produced, Glycosylation-Optimized Human Factor H for Therapeutic Application in Complement Disorders.

    PubMed

    Michelfelder, Stefan; Parsons, Juliana; Bohlender, Lennard L; Hoernstein, Sebastian N W; Niederkrüger, Holger; Busch, Andreas; Krieghoff, Nicola; Koch, Jonas; Fode, Benjamin; Schaaf, Andreas; Frischmuth, Thomas; Pohl, Martin; Zipfel, Peter F; Reski, Ralf; Decker, Eva L; Häffner, Karsten

    2016-12-08

    Genetic defects in complement regulatory proteins can lead to severe renal diseases, including atypical hemolytic uremic syndrome and C3 glomerulopathies, and age-related macular degeneration. The majority of the mutations found in patients with these diseases affect the glycoprotein complement factor H, the main regulator of the alternative pathway of complement activation. Therapeutic options are limited, and novel treatments, specifically those targeting alternative pathway activation, are highly desirable. Substitution with biologically active factor H could potentially treat a variety of diseases that involve increased alternative pathway activation, but no therapeutic factor H is commercially available. We recently reported the expression of full-length recombinant factor H in moss (Physcomitrella patens). Here, we present the production of an improved moss-derived recombinant human factor H devoid of potentially immunogenic plant-specific sugar residues on protein N-glycans, yielding approximately 1 mg purified moss-derived human factor H per liter of initial P. patens culture after a multistep purification process. This glycosylation-optimized factor H showed full in vitro complement regulatory activity similar to that of plasma-derived factor H and efficiently blocked LPS-induced alternative pathway activation and hemolysis induced by sera from patients with atypical hemolytic uremic syndrome. Furthermore, injection of moss-derived factor H reduced C3 deposition and increased serum C3 levels in a murine model of C3 glomerulopathy. Thus, we consider moss-produced recombinant human factor H a promising pharmaceutical product for therapeutic intervention in patients suffering from complement dysregulation.

  19. C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities.

    PubMed

    Zhang, Jihua; Markiewicz, Matthew J; Mowery, Brendan P; Weisblum, Bernard; Stahl, Shannon S; Gellman, Samuel H

    2012-02-13

    Nylon-3 polymers contain β-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete, we introduce a new β-lactam, approximately 1 equiv relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new β-lactam should become attached at this position. If the terminating β-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials.

  20. Structural characterization of a homogalacturonan from Capparis spinosa L. fruits and anti-complement activity of its sulfated derivative.

    PubMed

    Wang, Huijun; Wang, Hongwei; Shi, Songshan; Duan, Jinyou; Wang, Shunchun

    2012-08-01

    A water-soluble polysaccharide CSPS-2B-2 with a molecular mass of 8.8 kDa, was obtained from the fruits of Capparis spinosa L. Chemical and NMR spectral analysis verified CSPS-2B-2 was a linear poly-(1-4)-α-D-galactopyranosyluronic acid in which 12.9±0.4% of carboxyl groups existed as methyl ester and 2.6±0.1% of D-GalpA residues were acetylated. A sulfated derivative Sul-2B-2 with a sulfation degree of 0.88±0.02 was prepared via the substitution of C-2 and/or C-3 of GalpA residues in CSPS-2B-2. Bioassay on the complement and coagulation system demonstrated that Sul-2B-2 (CH(50): 3.5±0.2 μg/mL) had a stronger inhibitory effect on the activation of complement system through the classic pathway than that of heparin (CH(50): 8.9±0.3 μg/mL). Interestingly, Sul-2B-2 at low dose even middle dose (for example 52 μg/mL) had no effect on coagulation system, which was totally different from heparin. Thus, our observation indicated that Sul-2B-2 was more efficient than heparin in inhibiting the activation of the complement system through classical pathway and exhibiting a relatively less anti-coagulant activity. These results suggested that the sulfated derivative Sul-2B-2 prepared from the homogalacturonan in the fruits of Capparis spinosa L, might be a promising drug candidate in case of necessary therapeutic complement inhibition.

  1. Overcoming technical challenges when treating atypical hemolytic uremic syndrome with therapeutic plasma exchange.

    PubMed

    Zimbudzi, Edward

    2013-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a very rare, life-threatening, progressive disease that frequently has a genetic component and in most cases is triggered by an uncontrolled activation of the complement system. Successful treatment of aHUS with plasma infusions and therapeutic plasma exchange (TPE) is well reported. TPE has been the treatment of choice in most adult patients with aHUS. However, due to severe hemolysis, which is common among aHUS patients, there are some technical challenges that can affect TPE treatment such as the continuous activation of the blood leak alarm due to hemolysis. Our experience shows that such patients can be managed better on a centrifuge based TPE machine compared to a membrane based TPE machine.

  2. Targeting complement in therapy.

    PubMed

    Kirschfink, M

    2001-04-01

    With increasing evidence that complement activation significantly contributes to the pathogenesis of a large number of inflammatory diseases, strategies that interfere with its deleterious action have become a major focus in pharmacological research. Endogenous soluble complement inhibitors (C1 inhibitor, recombinant soluble complement receptor 1, antibodies) blocking key proteins of the cascade reaction, neutralizing the action of the complement-derived anaphylatoxin C5a, or interfering with complement receptor 3 (CR3, CD18/11b)-mediated adhesion of inflammatory cells to the vascular endothelium have successfully been tested in various animal models over the past years. Promising results consequently led to clinical trials. Furthermore, incorporation of membrane-bound complement regulators (decay-accelerating factor (CD55), membrane co-factor protein (CD46), CD59) in transgenic animals has provided a major step forward in protecting xenografts from hyperacute rejection. At the same time, the poor contribution of complement to the antitumor response, which is caused by multiple resistance mechanisms that hamper the efficacy of antibody-based tumor therapy, is increasingly recognized and requires pharmacologic intervention. First attempts have now been made to interfere with the resistance mechanisms, thereby improving complement-mediated tumor cell destruction.

  3. Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease

    PubMed Central

    Lian, Hong; Litvinchuk, Alexandra; Chiang, Angie C.-A.; Aithmitti, Nadia; Jankowsky, Joanna L.

    2016-01-01

    Increasing evidence supports a role of neuroinflammation in the pathogenesis of Alzheimer's disease (AD). Previously, we identified a neuron–glia signaling pathway whereby Aβ acts as an upstream activator of astroglial nuclear factor kappa B (NF-κB), leading to the release of complement C3, which acts on the neuronal C3a receptor (C3aR) to influence dendritic morphology and cognitive function. Here we report that astrocytic complement activation also regulates Aβ dynamics in vitro and amyloid pathology in AD mouse models through microglial C3aR. We show that in primary microglial cultures, acute C3 or C3a activation promotes, whereas chronic C3/C3a treatment attenuates, microglial phagocytosis and that the effect of chronic C3 exposure can be blocked by cotreatment with a C3aR antagonist and by genetic deletion of C3aR. We further demonstrate that Aβ pathology and neuroinflammation in amyloid precursor protein (APP) transgenic mice are worsened by astroglial NF-κB hyperactivation and resulting C3 elevation, whereas treatment with the C3aR antagonist (C3aRA) ameliorates plaque load and microgliosis. Our studies define a complement-dependent intercellular cross talk in which neuronal overproduction of Aβ activates astroglial NF-κB to elicit extracellular release of C3. This promotes a pathogenic cycle by which C3 in turn interacts with neuronal and microglial C3aR to alter cognitive function and impair Aβ phagocytosis. This feedforward loop can be effectively blocked by C3aR inhibition, supporting the therapeutic potential of C3aR antagonists under chronic neuroinflammation conditions. SIGNIFICANCE STATEMENT The complement pathway is activated in Alzheimer's disease. Here we show that the central complement factor C3 secreted from astrocytes interacts with microglial C3a receptor (C3aR) to mediate β-amyloid pathology and neuroinflammation in AD mouse models. Our study provides support for targeting C3aR as a potential therapy for Alzheimer's disease. PMID

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

    PubMed

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

    2014-11-01

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

  5. Almost total protection from age-related macular degeneration by haplotypes of the Regulators of Complement Activation.

    PubMed

    Williamson, Joseph F; McLure, Craig A; Guymer, Robyn H; Baird, Paul N; Millman, John; Cantsilieris, Stuart; Dawkins, Roger L

    2011-12-01

    Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. It has been proposed that the polymorphism encoding Y402H (T1277C) in the complement factor H gene (CFH) is one of the main determinants of disease. We genotyped the polymorphism at a number of loci in the region encompassing the Regulators of Complement Activation (RCA) on chromosome 1, including T1277C SNP, in 187 patients and 146 controls. Haplotypes have been classified as protective (P) or susceptible (S) with respect to AMD. This included the identification of an S haplotype with a T at 1277. The results show that no single locus should be assumed to be directly responsible for AMD, but rather argue for the existence of RCA haplotypes, which can be assigned meaningful predictive values for AMD. We conclude that the critical sequences are within a region 450 kb centromeric to 128 kb telomeric of CFH.

  6. Structural features and complement-fixing activity of pectin from three Brassica oleracea varieties: white cabbage, kale, and red kale.

    PubMed

    Samuelsen, Anne Berit; Westereng, Bjørge; Yousif, Osman; Holtekjølen, Ann Katrin; Michaelsen, Terje E; Knutsen, Svein H

    2007-02-01

    Leaves of different cabbage species are used both as food and as wound healing remedies in traditional medicine. This supposed wound healing activity might be connected to presence of immunomodulating water soluble polysaccharides. To study this, three different cabbage varieties, white cabbage (W), kale (K), and red kale (RK), were pretreated with 80% ethanol and then extracted with water at 50 degrees C and 100 degrees C for isolation of polysaccharide-containing fractions. The fractions were analyzed for monosaccharide composition, glycosidic linkages, Mw distribution, protein content, and phenolic compounds and then tested for complement-fixing activity. All fractions contained pectin type polysaccharides with linkages corresponding to homogalacturonan and hairy regions. Those extracted at 50 degrees C contained higher amounts of neutral side chains and were more active in the complement-fixation test than those extracted at 100 degrees C. The fractions can be ranged by decreasing activity: K-50 > RK-50 > W-50 approximately = K-100 > RK100 approximately = W-100. Studies on structure-activity relationships (SAR) employing multivariate statistical analysis strongly suggest that the magnitude of the measured activity is influenced by the content of certain side chains in the polymers. High activity correlates to large neutral side chains with high amounts of (1-->6)- and (1-->3,6)-linked Gal and low amounts of (1-->4)-linked GalA but not on molecular weight distribution of the polymers.

  7. Blood-brain barrier disruption and complement activation in the brain following rapid correction of chronic hyponatremia.

    PubMed

    Baker, E A; Tian, Y; Adler, S; Verbalis, J G

    2000-10-01

    In previous studies we developed a rat model in which demyelination is reproducibly produced following rapid correction of chronic hyponatremia and demonstrated that the development of demyelination in this model is strongly associated with NMR indices of blood-brain barrier (BBB) disruption. Because complement is toxic to oligodendrocytes, we evaluated the hypothesis that BBB disruption precipitated by correction of hypoosmolality is followed by an influx of complement into the brain, which then contributes to the demyelination that occurs under these conditions. We studied four groups of rats with immunocytochemical analysis using primary antibodies to IgG and the C3d split-fragment of activated complement: (1) normal rats; (2) rats in which hyponatremia was maintained for 7 days; (3) chronically hyponatremic rats in which the plasma [Na(+)] was rapidly corrected with hypertonic saline administration 20 h prior to perfusion; and (4) chronically hyponatremic rats in which the plasma [Na(+)] was rapidly corrected with hypertonic saline administration 5 days prior to perfusion. In normonatremic and uncorrected hyponatremic rats only background staining was observed in areas lacking a BBB and in blood vessel walls, whereas marked increases in IgG and C3d staining were seen in the brains of rats both 20 h and 5 days after rapid correction of hyponatremia. The staining intensity was significantly correlated with the degree of neurological impairment. These results provide evidence for functional BBB disruption following rapid correction of hyponatremia and support the hypothesis that complement activation may be involved in the pathogenesis of osmotic demyelination.

  8. 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

  9. Chimeric CD46/DAF molecules reveal a cryptic functional role for SCR1 of DAF in regulating complement activation.

    PubMed

    Christiansen, D; Loveland, B; Kyriakou, P; Lanteri, M; Rubinstein, E; Gerlier, D

    2000-01-01

    Chimeric proteins using membrane cofactor (CD46) and decay accelerating factor (DAF or CD55) were generated to further investigate the functional domains involved in the regulation of human serum complement. Following activation of the classical pathway, the isolated substitution of CD46 SCR III (x3DAF) exhibited a modest regulatory activity comparable to that of CD46. The isolated substitution of CD46 SCR IV (x4DAF), and the combined CD46 SCR III+IV substitutions (x3/4DAF) were essentially as efficient as DAF. No regulation of C3b deposition was observed with the combined CD46 SCR I+II substitutions (x1/2DAF). When tested after activation of the alternative pathway, both the x3DAF and x3/4DAF chimeras failed to regulate C3b deposition, while the x4DAF chimera still displayed some activity. In contrast to that observed following classical pathway activation, the x1/2DAF chimera exhibited a similar efficiency to wild type CD46 and DAF in controlling C3b deposition. Using SCR specific antibodies, the regulatory activity of the x1/2DAF chimera against the alternative pathway was mapped to the first three distal SCR (i.e. DAF 1, DAF 2 and CD46 III). These data demonstrate that several combinations of SCR domains from two related complement regulators can result in functional molecules, and reveal a novel and cryptic functional role for DAF SCR1.

  10. Influence of surface modulations by enzymes and monoclonal antibodies on alternative complement pathway activation by Yersinia enterocolitica.

    PubMed Central

    Wachter, E; Brade, V

    1989-01-01

    Effector mechanisms resulting from alternative complement pathway (ACP) activation cannot act efficiently against Yersinia enterocolitica serotype O3, as indicated by poor C3 to C9 consumption and by survival in EGTA (ethyleneglycoldiaminetetraacetic acid) Mg-serum. These results were not influenced by the lack or presence of plasmid-encoded outer membrane proteins or lipopolysaccharides (LPS) with different amounts of side chains or by treatment of the bacteria with pronase or neuraminidase. Surface modulation of Y. enterocolitica with polyclonal immunoglobulin G or the immunoglobulin G fragments F(ab')2 and Fab always converted Y. enterocolitica to a high ACP activator, with strong C3 to C9 consumption and surface deposition of activated C3. Killing of Y. enterocolitica as a result of antibody-mediated ACP activation was observed only with bacteria grown at 22 degrees C but not with bacteria from 37 degrees C cultures. The expression of complement resistance in Y. enterocolitica grown at 37 degrees C was not influenced by the presence or absence of plasmids. Using different monoclonal antibodies (MAb), we found that MAb with LPS specificity mediated ACP activation, whereas MAb specific for different plasmid-encoded outer membrane proteins were ineffective, despite surface binding. These results suggest a major inhibitory role of LPS on ACP activation which was neutralized by LPS-specific antibodies. PMID:2731980

  11. Drug-induced immune hemolytic anemia

    MedlinePlus

    Immune hemolytic anemia secondary to drugs; Anemia - immune hemolytic - secondary to drugs ... In some cases, a drug can cause the immune system to mistake your own red blood cells for foreign substances. The body responds by making ...

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

    PubMed

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

    2010-02-01

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

  13. Creating functional sophistication from simple protein building blocks, exemplified by factor H and the regulators of complement activation.

    PubMed

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

    2015-10-01

    Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a β-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins.

  14. Deletion of wboA Enhances Activation of the Lectin Pathway of Complement in Brucella abortus and Brucella melitensis

    PubMed Central

    Fernandez-Prada, Carmen M.; Nikolich, Mikeljon; Vemulapalli, Ramesh; Sriranganathan, Nammalwar; Boyle, Stephen M.; Schurig, Gerhardt G.; Hadfield, Ted L.; Hoover, David L.

    2001-01-01

    Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae. PMID:11401980

  15. [Diagnosis and treatment of hemolytic anemia].

    PubMed

    Kamesaki, Toyomi

    2015-10-01

    Hemolytic anemia is defined as anemia due to a reduction of the RBC lifespan to less than the normal range of approximately 120 days. Patients with anemia and jaundice are often suspected to have hemolysis. Herein, different causes of hemolysis and the diagnostic algorithm are reviewed. Currently, there is no generic treatment for hemolytic anemia. Appropriate management of a patient with hemolytic anemia requires determination of the underlying cause. Treatments for the different causes of hemolytic anemia are also reviewed.

  16. Severe autoimmune hemolytic anemia with renal neoplasm.

    PubMed

    Rhodes, Emily C; Parikh, Sahil P; Bhattacharyya, Nishith

    2014-02-01

    Autoimmune hemolytic anemia is a type of hemolytic anemia characterized by autoantibodies directed against red blood cells shortening their survival. When autoimmune hemolytic anemia is secondary to a paraneoplastic process, severe anemia can occur leading to significant morbidity and even mortality. Here we discuss the literature and present the case of a child with autoimmune hemolytic anemia from a paraneoplastic syndrome secondary to a renal tumor.

  17. New insights into Shiga toxin-mediated endothelial dysfunction in hemolytic uremic syndrome

    PubMed Central

    Petruzziello-Pellegrini, Tania N; Moslemi-Naeini, Mozhgan; Marsden, Philip A

    2013-01-01

    Shiga toxin-producing E. coli represents a significant global health concern, especially as hypervirulent pathogens surface amidst outbreaks of hemolytic uremic syndrome (HUS). Shiga toxin (Stx) is key in the microangiopathic events underlying the disease and its central role is underscored by the unprecedented HUS outbreak in Germany in 2011. The mechanisms of Stx-mediated endothelial dysfunction have been a major focus of research that has contributed to the current understanding of the pathogenic changes in endothelial phenotype leading to HUS. Among the newer concepts are Stx-mediated gene regulation in the absence of protein synthesis inhibition, a potential role for complement activation, and accumulating evidence for detectable serum markers before the onset of the classic clinical features of HUS. Further investigation of newer therapeutic targets and potential prognostic markers is essential to assess their utility in mitigating disease and/or predicting outcomes and will provide an improved overall understanding of HUS pathogenesis. PMID:23955166

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

    PubMed Central

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

    2008-01-01

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

  19. Membrane-controlled depletion of complement activity by spin-label-specific IgM

    PubMed Central

    Humphries, Gillian M. K.; McConnell, Harden M.

    1977-01-01

    Complement depletion mediated by high molecular weight (IgM) rabbit antibodies specifically bound to spin-label lipid haptens dispersed in model membranes is controlled by various physical attributes of those membranes other than the total number of exposed determinants that they provide. Carrier lipids used at 32° were (i) a “fluid” phosphatidylcholine (PC), (ii) a “solid” PC, and (iii) a cholesterol/PC mixture. The concentration of hapten in the plane of the membranes (two-dimensional concentration) was varied while the overall hapten molarity (three-dimensional concentration) was kept constant. Both specific binding and the efficiency of depletion by IgM are markedly enhanced by systematically decreasing the average distance between haptens (∞ → 26 A). Heterogeneous distribution was found to be more favorable than a random homogeneous distribution of the same number of haptens in the same total quantity of lipids. IgM efficiency is also markedly increased by the inclusion of cholesterol in PC membranes, an effect thought to result from enhanced projection of the determinant from the surface of the membrane and hence increased accessibility to the antibody-binding site. Furthermore, the efficiency of IgM was increased by using haptens dispersed in fluid rather than in solid PC membranes. The results are consistent with the hypothesis that IgM molecules must be bound to a critical multiple of antigenic determinants at a membrane surface in order to induce complement-mediated attack and that subtle variation of the physical state of membrane antigens can be the crucial factor in determining the outcome of this type of efferent immune response. PMID:198789

  20. Seroprevalence of Antibody-Mediated, Complement-Dependent Opsonophagocytic Activity against Neisseria meningitidis Serogroup B in England.

    PubMed

    Humphries, Holly E; Brookes, Charlotte; Allen, Lauren; Kuisma, Eeva; Gorringe, Andrew; Taylor, Stephen

    2015-05-01

    The correlate of protection for the licensure of meningococcal vaccines is serum bactericidal activity. However, evidence indicates that a complex situation and other mechanisms, such as antibody-mediated, complement-dependent opsonophagocytosis (OP), may play a role in protection and should be investigated in order to understand immunity to this disease. In this study, a high-throughput flow cytometric opsonophagocytic assay (OPA) was optimized. The assay measures the presence of killed fluorescently labeled Neisseria meningitidis within human granulocytes (differentiated HL60 cells) by flow cytometry, using IgG-depleted pooled human plasma as an exogenous source of complement. This method was found to be reliable and correlated with the results of an opsonophagocytic killing assay. The OPA was used to measure OP activity in 1,878 serum samples from individuals ranging from 0 to 99 years of age against N. meningitidis strain NZ98/254 (B:4:P1.7-2,4). The levels of OP activity in individual serum samples varied greatly. OP activity showed an initial peak in the 6- to 12-month age group corresponding to a peak in disease incidence. The OP activity dropped in childhood until the late teenage years, although there was still a higher percentage of individuals with OP activity than with protective bactericidal antibody titers. OP activity reached a peak in the 30- to 39-year age group and then declined. This later peak in OP activity did not coincide with the young adults in whom peak serum bactericidal activity and disease incidence occurred. The demonstration of OP activity when disease incidence is low and when protective bactericidal antibody titers are not detected may indicate a role for OP in protection from meningococcal disease in these age groups.

  1. Seroprevalence of Antibody-Mediated, Complement-Dependent Opsonophagocytic Activity against Neisseria meningitidis Serogroup B in England

    PubMed Central

    Brookes, Charlotte; Allen, Lauren; Kuisma, Eeva; Gorringe, Andrew; Taylor, Stephen

    2015-01-01

    The correlate of protection for the licensure of meningococcal vaccines is serum bactericidal activity. However, evidence indicates that a complex situation and other mechanisms, such as antibody-mediated, complement-dependent opsonophagocytosis (OP), may play a role in protection and should be investigated in order to understand immunity to this disease. In this study, a high-throughput flow cytometric opsonophagocytic assay (OPA) was optimized. The assay measures the presence of killed fluorescently labeled Neisseria meningitidis within human granulocytes (differentiated HL60 cells) by flow cytometry, using IgG-depleted pooled human plasma as an exogenous source of complement. This method was found to be reliable and correlated with the results of an opsonophagocytic killing assay. The OPA was used to measure OP activity in 1,878 serum samples from individuals ranging from 0 to 99 years of age against N. meningitidis strain NZ98/254 (B:4:P1.7-2,4). The levels of OP activity in individual serum samples varied greatly. OP activity showed an initial peak in the 6- to 12-month age group corresponding to a peak in disease incidence. The OP activity dropped in childhood until the late teenage years, although there was still a higher percentage of individuals with OP activity than with protective bactericidal antibody titers. OP activity reached a peak in the 30- to 39-year age group and then declined. This later peak in OP activity did not coincide with the young adults in whom peak serum bactericidal activity and disease incidence occurred. The demonstration of OP activity when disease incidence is low and when protective bactericidal antibody titers are not detected may indicate a role for OP in protection from meningococcal disease in these age groups. PMID:25739917

  2. Interaction of Leptospira elongation factor Tu with plasminogen and complement factor H: a metabolic leptospiral protein with moonlighting activities.

    PubMed

    Wolff, Danielly G; Castiblanco-Valencia, Mónica M; 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.

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

    PubMed

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

    2015-04-14

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

  4. Dissociable effects of Sry and sex chromosome complement on activity, feeding and anxiety-related behaviours in mice.

    PubMed

    Kopsida, Eleni; Lynn, Phoebe M; Humby, Trevor; Wilkinson, Lawrence S; Davies, William

    2013-01-01

    Whilst gonadal hormones can substantially influence sexual differentiation of the brain, recent findings have suggested that sex-linked genes may also directly influence neurodevelopment. Here we used the well-established murine 'four core genotype' (FCG) model on a gonadally-intact, outbred genetic background to characterise the contribution of Sry-dependent effects (i.e. those arising from the expression of the Y-linked Sry gene in the brain, or from hormonal sequelae of gonadal Sry expression) and direct effects of sex-linked genes other than Sry ('sex chromosome complement' effects) to sexually dimorphic mouse behavioural phenotypes. Over a 24 hour period, XX and XY gonadally female mice (lacking Sry) exhibited greater horizontal locomotor activity and reduced food consumption per unit bodyweight than XX and XY gonadally male mice (possessing Sry); in two behavioural tests (the elevated plus and zero mazes) XX and XY gonadally female mice showed evidence for increased anxiety-related behaviours relative to XX and XY gonadally male mice. Exploratory correlational analyses indicated that these Sry-dependent effects could not be simply explained by brain expression of the gene, nor by circulating testosterone levels. We also noted a sex chromosome complement effect on food (but not water) consumption whereby XY mice consumed more over a 24hr period than XX mice, and a sex chromosome complement effect in a third test of anxiety-related behaviour, the light-dark box. The present data suggest that: i) the male-specific factor Sry may influence activity and feeding behaviours in mice, and ii) dissociable feeding and anxiety-related murine phenotypes may be differentially modulated by Sry and by other sex-linked genes. Our results may have relevance for understanding the molecular underpinnings of sexually dimorphic behavioural phenotypes in healthy men and women, and in individuals with abnormal sex chromosome constitutions.

  5. Response gene to complement 32 protein promotes macrophage phagocytosis via activation of protein kinase C pathway.

    PubMed

    Tang, Rui; Zhang, Gui; Chen, Shi-You

    2014-08-15

    Macrophage phagocytosis plays an important role in host defense. The molecular mechanism, especially factors regulating the phagocytosis, however, is not completely understood. In the present study, we found that response gene to complement 32 (RGC-32) is an important regulator of phagocytosis. Although RGC-32 is induced and abundantly expressed in macrophage during monocyte-macrophage differentiation, RGC-32 appears not to be important for this process because RGC-32-deficient bone marrow progenitor can normally differentiate to macrophage. However, both peritoneal macrophages and bone marrow-derived macrophages with RGC-32 deficiency exhibit significant defects in phagocytosis, whereas RGC-32-overexpressed macrophages show increased phagocytosis. Mechanistically, RGC-32 is recruited to macrophage membrane where it promotes F-actin assembly and the formation of phagocytic cups. RGC-32 knock-out impairs F-actin assembly. RGC-32 appears to interact with PKC to regulate PKC-induced phosphorylation of F-actin cross-linking protein myristoylated alanine-rich protein kinase C substrate. Taken together, our results demonstrate for the first time that RGC-32 is a novel membrane regulator for macrophage phagocytosis.

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

    PubMed Central

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

    2010-01-01

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

  7. [Hemolytic anemias and vitamin B12 deficieny].

    PubMed

    Dietzfelbinger, Hermann; Hubmann, Max

    2015-08-01

    Hemolytic anemias consist of corpuscular, immun-hemolytic and toxic hemolytic anemias. Within the group of corpuscular hemolytic anemias, except for the paroxysmal nocturnal hemoglobinuria (PNH), all symptoms are caused by underlying heredetiary disorders within the red blood cell membran (hereditary spherocytosis), deficiencies of red cell enzymes (G6PDH- and pyrovatkinase deficiency) or disorders in the hemoglobin molecule (thalassaemia and sickle cell disease). Immune-hemolytic anemias are acquired hemolytic anemias and hemolysis is caused by auto- or allo-antibodies which are directed against red blood cell antigens. They are classified as warm, cold, mixed type or drug-induced hemolytic anemia. Therapy consists of glucocorticoids and other immunsuppressive drugs. Pernicious anemia is the most important vitamin B12 deficiency disorder. Diagnosis relies on cobalamin deficiency and antibodies to intrinsic factor. The management should focus on a possibly life-long replacement treatment with cobalamin.

  8. Insights into hemolytic uremic syndrome: segregation of three independent predisposition factors in a large, multiple affected pedigree.

    PubMed

    Esparza-Gordillo, Jorge; Jorge, Elena Goicoechea de; Garrido, Cynthia Abarrategui; Carreras, Luis; López-Trascasa, Margarita; Sánchez-Corral, Pilar; de Córdoba, Santiago Rodríguez

    2006-04-01

    Mutations in the complement regulators factor H, membrane cofactor protein (MCP), and factor I are associated with atypical hemolytic uremic syndrome (aHUS, MIM 235400), suggesting that the disease develops as a consequence of the inefficient protection of the renal endothelium from damage by the complement system. Incomplete penetrance of the disease in individuals carrying these mutations is, however, relatively frequent. Here, we report the identification of a large, multiple affected aHUS pedigree in which there is independent segregation of three different aHUS risk factors: a MCP missense mutation (c.-598C>T; Pro165Ser) that decreases MCP expression on the cell surface, a dinucleotide insertion in the coding sequence of factor I (c.-1610insAT) that introduces a premature stop codon in the factor I protein, and the MCPggaac SNP haplotype block that was previously shown to decrease the transcription activity from the MCP promoter. Interestingly, individuals affected by aHUS in the pedigree are only those who have inherited the three aHUS risk factors. These data show an additive effect for mutations in MCP and factor I and provide definitive support to the conclusion that aHUS results from a defective protection of cellular surfaces from complement activation. Furthermore, they help to explain the incomplete penetrance of the disease, illustrating that concurrence of multiple hits in complement regulatory proteins may be necessary to significantly impair host tissue protection and to confer susceptibility to aHUS.

  9. Plasma-derived human C1-esterase inhibitor does not prevent mechanical ventilation-induced pulmonary complement activation in a rat model of Streptococcus pneumoniae pneumonia.

    PubMed

    de Beer, F M; Aslami, H; Hoeksma, J; van Mierlo, G; Wouters, D; Zeerleder, S; Roelofs, J J T H; Juffermans, N P; Schultz, M J; Lagrand, W K

    2014-11-01

    Mechanical ventilation has the potential to cause lung injury, and the role of complement activation herein is uncertain. We hypothesized that inhibition of the complement cascade by administration of plasma-derived human C1-esterase inhibitor (C1-INH) prevents ventilation-induced pulmonary complement activation, and as such attenuates lung inflammation and lung injury in a rat model of Streptococcus pneumoniae pneumonia. Forty hours after intratracheal challenge with S. pneumoniae causing pneumonia rats were subjected to ventilation with lower tidal volumes and positive end-expiratory pressure (PEEP) or high tidal volumes without PEEP, after an intravenous bolus of C1-INH (200 U/kg) or placebo (saline). After 4 h of ventilation blood, broncho-alveolar lavage fluid and lung tissue were collected. Non-ventilated rats with S. pneumoniae pneumonia served as controls. While ventilation with lower tidal volumes and PEEP slightly amplified pneumonia-induced complement activation in the lungs, ventilation with higher tidal volumes without PEEP augmented local complement activation more strongly. Systemic pre-treatment with C1-INH, however, failed to alter ventilation-induced complement activation with both ventilation strategies. In accordance, lung inflammation and lung injury were not affected by pre-treatment with C1-INH, neither in rats ventilated with lower tidal volumes and PEEP, nor rats ventilated with high tidal volumes without PEEP. Ventilation augments pulmonary complement activation in a rat model of S. pneumoniae pneumonia. Systemic administration of C1-INH, however, does not attenuate ventilation-induced complement activation, lung inflammation, and lung injury.

  10. Complement inhibition in cancer therapy.

    PubMed

    Pio, Ruben; Ajona, Daniel; Lambris, John D

    2013-02-01

    For decades, complement has been recognized as an effector arm of the immune system that contributes to the destruction of tumor cells. In fact, many therapeutic strategies have been proposed that are based on the intensification of complement-mediated responses against tumors. However, recent studies have challenged this paradigm by demonstrating a tumor-promoting role for complement. Cancer cells seem to be able to establish a convenient balance between complement activation and inhibition, taking advantage of complement initiation without suffering its deleterious effects. Complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and activate cancer-related signaling pathways. In this context, inhibition of complement activation would be a therapeutic option for treating cancer. This concept is relatively new and deserves closer attention. In this article, we summarize the mechanisms of complement activation on cancer cells, the cancer-promoting effect of complement initiation, and the rationale behind the use of complement inhibition as a therapeutic strategy against cancer.

  11. Tsetse GmmSRPN10 Has Anti-complement Activity and Is Important for Successful Establishment of Trypanosome Infections in the Fly Midgut

    PubMed Central

    Ooi, Cher-Pheng; Haines, Lee R.; Southern, Daniel M.; Lehane, Michael J.; Acosta-Serrano, Alvaro

    2015-01-01

    The complement cascade in mammalian blood can damage the alimentary tract of haematophagous arthropods. As such, these animals have evolved their own repertoire of complement-inactivating factors, which are inadvertently exploited by blood-borne pathogens to escape complement lysis. Unlike the bloodstream stages, the procyclic (insect) stage of Trypanosoma brucei is highly susceptible to complement killing, which is puzzling considering that a tsetse takes a bloodmeal every 2–4 days. In this study, we identified four tsetse (Glossina morsitans morsitans) serine protease inhibitors (serpins) from a midgut expressed sequence tag (EST) library (GmmSRPN3, GmmSRPN5, GmmSRPN9 and GmmSRPN10) and investigated their role in modulating the establishment of a T. brucei infection in the midgut. Although not having evolved in a common blood-feeding ancestor, all four serpins have an active site sharing remarkable homology with the human complement C1-inhibitor serpin, SerpinG1. RNAi knockdown of individual GmmSRPN9 and GmmSRPN10 genes resulted in a significant decreased rate of infection by procyclic form T. brucei. Furthermore, recombinant GmmSRPN10 was both able to inhibit the activity of human complement-cascade serine proteases, C1s and Factor D, and to protect the in vitro killing of procyclic trypanosomes when incubated with complement-activated human serum. Thus, the secretion of serpins, which may be part of a bloodmeal complement inactivation system in tsetse, is used by procyclic trypanosomes to evade an influx of fresh trypanolytic complement with each bloodmeal. This highlights another facet of the complicated relationship between T. brucei and its tsetse vector, where the parasite takes advantage of tsetse physiology to further its chances of propagation and transmission. PMID:25569180

  12. Complement (C5)-derived chemotactic activity accounts for accumulation of polymorphonuclear leukocytes in cerebrospinal fluid of rabbits with pneumococcal meningitis.

    PubMed Central

    Ernst, J D; Hartiala, K T; Goldstein, I M; Sande, M A

    1984-01-01

    Experiments were performed to identify the chemoattractant for polymorphonuclear leukocytes that appears in the cerebrospinal fluid of rabbits with experimental pneumococcal meningitis. Meningitis was induced in anesthetized New Zealand white rabbits by injecting 10(4) cells of stationary-phase Streptococcus pneumoniae type III intracisternally. Before bacteria were injected, cerebrospinal fluid contained neither polymorphonuclear leukocytes nor chemotactic activity. Significant chemotactic activity for rabbit polymorphonuclear leukocytes was detected 12 h after inoculation with bacteria and was maximal after 18 to 20 h. Chemotactic activity appeared in cerebrospinal fluid while concentrations of pneumococci and total protein were increasing but before there was any accumulation of polymorphonuclear leukocytes. The chemotactic activity in cerebrospinal fluid was heat stable (56 degrees C for 30 min), eluted from Sephadex G-75 with a profile identical to that of the chemotactic activity in zymosan-activated rabbit serum, and was inhibited by treatment with antibodies to native human C5. In addition, preincubation of polymorphonuclear leukocytes with partially purified rabbit C5a selectively inhibited their subsequent chemotactic responses to cerebrospinal fluid. These data indicate that complement (C5)-derived chemotactic activity appears in cerebrospinal fluid during the course of experimental pneumococcal meningitis in rabbits and suggest that this activity accounts for the accumulation of polymorphonuclear leukocytes observed in this infection. PMID:6480117

  13. Complement activating antibodies to myelin oligodendrocyte glycoprotein in neuromyelitis optica and related disorders

    PubMed Central

    2011-01-01

    Background Serum autoantibodies against the water channel aquaporin-4 (AQP4) are important diagnostic biomarkers and pathogenic factors for neuromyelitis optica (NMO). However, AQP4-IgG are absent in 5-40% of all NMO patients and the target of the autoimmune response in these patients is unknown. Since recent studies indicate that autoimmune responses to myelin oligodendrocyte glycoprotein (MOG) can induce an NMO-like disease in experimental animal models, we speculate that MOG might be an autoantigen in AQP4-IgG seronegative NMO. Although high-titer autoantibodies to human native MOG were mainly detected in a subgroup of pediatric acute disseminated encephalomyelitis (ADEM) and multiple sclerosis (MS) patients, their role in NMO and High-risk NMO (HR-NMO; recurrent optic neuritis-rON or longitudinally extensive transverse myelitis-LETM) remains unresolved. Results We analyzed patients with definite NMO (n = 45), HR-NMO (n = 53), ADEM (n = 33), clinically isolated syndromes presenting with myelitis or optic neuritis (CIS, n = 32), MS (n = 71) and controls (n = 101; 24 other neurological diseases-OND, 27 systemic lupus erythematosus-SLE and 50 healthy subjects) for serum IgG to MOG and AQP4. Furthermore, we investigated whether these antibodies can mediate complement dependent cytotoxicity (CDC). AQP4-IgG was found in patients with NMO (n = 43, 96%), HR-NMO (n = 32, 60%) and in one CIS patient (3%), but was absent in ADEM, MS and controls. High-titer MOG-IgG was found in patients with ADEM (n = 14, 42%), NMO (n = 3, 7%), HR-NMO (n = 7, 13%, 5 rON and 2 LETM), CIS (n = 2, 6%), MS (n = 2, 3%) and controls (n = 3, 3%, two SLE and one OND). Two of the three MOG-IgG positive NMO patients and all seven MOG-IgG positive HR-NMO patients were negative for AQP4-IgG. Thus, MOG-IgG were found in both AQP4-IgG seronegative NMO patients and seven of 21 (33%) AQP4-IgG negative HR-NMO patients. Antibodies to MOG and AQP4 were predominantly of the IgG1 subtype, and were able to

  14. Complement C5 Activation during Influenza A Infection in Mice Contributes to Neutrophil Recruitment and Lung Injury

    PubMed Central

    Garcia, Cristiana C.; Weston-Davies, Wynne; Russo, Remo C.; Tavares, Luciana P.; Rachid, Milene A.; Alves-Filho, José C.; Machado, Alexandre V.; Ryffel, Bernhard; Nunn, Miles A.; Teixeira, Mauro M.

    2013-01-01

    Influenza virus A (IAV) causes annual epidemics and intermittent pandemics that affect millions of people worldwide. Potent inflammatory responses are commonly associated with severe cases of IAV infection. The complement system, an important mechanism of innate and humoral immune responses to infections, is activated during primary IAV infection and mediates, in association with natural IgM, viral neutralization by virion aggregation and coating of viral hemmagglutinin. Increased levels of the anaphylatoxin C5a were found in patients fatally infected with the most recent H1N1 pandemic virus. In this study, our aim was to evaluate whether targeting C5 activation alters inflammatory lung injury and viral load in a murine model of IAV infection. To address this question C57Bl/6j mice were infected intranasally with 104 PFU of the mouse adapted Influenza A virus A/WSN/33 (H1N1) or inoculated with PBS (Mock). We demonstrated that C5a is increased in bronchoalveolar lavage fluid (BALF) upon experimental IAV infection. To evaluate the role of C5, we used OmCI, a potent arthropod-derived inhibitor of C5 activation that binds to C5 and prevents release of C5a by complement. OmCI was given daily by intraperitoneal injection from the day of IAV infection until day 5. Treatment with OmCI only partially reduced C5a levels in BALF. However, there was significant inhibition of neutrophil and macrophage infiltration in the airways, Neutrophil Extracellular Traps (NETs) formation, death of leukocytes, lung epithelial injury and overall lung damage induced by the infection. There was no effect on viral load. Taken together, these data suggest that targeting C5 activation with OmCI during IAV infection could be a promising approach to reduce excessive inflammatory reactions associated with the severe forms of IAV infections. PMID:23696894

  15. The role of complement, platelet-activating factor and leukotriene B4 in a reversed passive Arthus reaction.

    PubMed Central

    Rossi, A. G.; Norman, K. E.; Donigi-Gale, D.; Shoupe, T. S.; Edwards, R.; Williams, T. J.

    1992-01-01

    1. The mechanisms underlying oedema formation induced in a reversed passive Arthus (RPA) reaction and, for comparison, in response to zymosan in rabbit skin were investigated. 2. Oedema formation at skin sites was quantified by the accumulation of intravenously-injected 125I-labelled human serum albumin. 3. Recombinant soluble complement receptor type 1 (sCR1), administered locally in rabbit skin, suppressed oedema formation induced in the RPA reaction and by zymosan. 4. The platelet-activating factor (PAF) antagonists, WEB 2086 and PF10040 administered locally, inhibited oedema formation induced in the RPA reaction and by PAF but not by zymosan. 5. A locally administered leukotriene B4 (LTB4) antagonist, LY-255283, inhibited oedema formation induced by LTB4 but did not inhibit oedema responses to PAF, zymosan or the RPA reaction. 6. The results demonstrate a role for complement in oedema formation in both the RPA reaction and in response to zymosan. An important contribution by PAF is indicated in the RPA reaction but not in response to zymosan whereas no evidence was obtained to suggest a role for LTB4 in either inflammatory response. PMID:1330163

  16. Meningococcal surface fibril (Msf) binds to activated vitronectin and inhibits the terminal complement pathway to increase serum resistance.

    PubMed

    Griffiths, Natalie J; Hill, Darryl J; Borodina, Elena; Sessions, Richard B; Devos, Nathalie I; Feron, Christiane M; Poolman, Jan T; Virji, Mumtaz

    2011-12-01

    Complement evasion is an important survival strategy of Neisseria meningitidis (Nm) during colonization and infection. Previously, we have shown that Nm Opc binds to serum vitronectin to inhibit complement-mediated killing. In this study, we demonstrate meningococcal interactions with vitronectin via a novel adhesin, Msf (meningococcal surface fibril, previously NhhA or Hsf). As with Opc, Msf binds preferentially to activated vitronectin (aVn), engaging at its N-terminal region but the C-terminal heparin binding domain may also participate. However, unlike Opc, the latter binding is not heparin-mediated. By binding to aVn, Msf or Opc can impart serum resistance, which is further increased in coexpressers, a phenomenon dependent on serum aVn concentrations. The survival fitness of aVn-binding derivatives was evident from mixed population studies, in which msf/opc mutants were preferentially depleted. In addition, using vitronectin peptides to block Msf-aVn interactions, aVn-induced inhibition of lytic C5b-9 formation and of serum killing could be reversed. As Msf-encoding gene is ubiquitous in the meningococcal strains examined and is expressed in vivo, serum resistance via Msf may be of significance to meningococcal pathogenesis. The data imply that vitronectin binding may be an important strategy for the in vivo survival of Nm for which the bacterium has evolved redundant mechanisms.

  17. MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked

    PubMed Central

    Dobó, József; Szakács, Dávid; Oroszlán, Gábor; Kortvely, Elod; Kiss, Bence; Boros, Eszter; Szász, Róbert; Závodszky, Péter; Gál, Péter; Pál, Gábor

    2016-01-01

    MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways. PMID:27535802

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  19. Remarkable Activation of the Complement System and Aberrant Neuronal Localization of the Membrane Attack Complex in the Brain Tissues of Scrapie-Infected Rodents.

    PubMed

    Lv, Yan; Chen, Cao; Zhang, Bao-Yun; Xiao, Kang; Wang, Jing; Chen, Li-Na; Sun, Jing; Gao, Chen; Shi, Qi; Dong, Xiao-Ping

    2015-12-01

    As an integral part of the innate immunity, the complement system has been reported to involve in the pathogenesis of prion diseases (PrD). However, the states of expression and activity of complement proteins in experimental models of scrapie infection are still not fully understood. Herein, the state of complement activation, the presence, and distribution as well as localization of C3 and membrane attack complex (MAC) in the brains of several scrapie-infected rodents were comparatively assessed through various methodologies. Our data illustrated a significant increase in the total complement activity (CH50, U/ml) in several scrapie-infected rodent brains at the terminal stage and a time-dependent upregulation of C1q in 263K-infected hamsters during the incubation period, intimating the sustained and progressive activation of the classical pathway during PrD progression. Confocal microscopy revealed robust activation of C3 and its localization to various central nervous system (CNS) cells with differential morphology in the brain tissues of both 263K-infected hamsters and 139A-infected C57BL/6 mice at disease end stages. Dynamic analyses of MAC in the brains of 263K-infected hamsters and 139A-infected C57BL/6 mice demonstrated remarkably time-dependent deposition during the incubation period, which may highlight a persistently activated terminal complement components. Moreover, immunofluorescent assays (IFAs) showed that MAC-specific signals appeared to overlap with morphologically abnormal neurons rather than proliferative astrocytes or activated microglia throughout the CNS of both 263K-infected hamsters and 139A-infected C57BL/6 mice. Overall, these results indicate that the activation of the complement system and the subsequent localization of the complement components to neurons may be a hallmark during prion infection, which ultimately contribute to the neurodegeneration in PrD.

  20. Sub-lethal concentrations of activated complement increase rat lymphocyte glutamine utilization and oxidation while lethal concentrations cause death by a mechanism involving ATP depletion.

    PubMed

    Bacurau, R F P; O'Toole, C E; Newsholme, P; Costa Rosa, L F B P

    2002-09-01

    Nucleated cells are more resistant to complement-mediated cell death than anucleated cells such as erythrocytes. There are few reports concerning the metabolic response of nucleated cells subjected to sub-lethal complement attack. It is possible that the rate of utilization of specific metabolic fuels by the cell is increased to enhance cell defence. We have measured the maximum activity of hexokinase, citrate synthase, glucose 6-phosphate dehydrogenase and glutaminase in rat mesenteric lymphocytes exposed to sub-lethal concentrations of activated complement (present in zymosan-activated serum, ZAS). These enzymes were carefully selected as they indicate changes of flux in glycolysis, TCA cycle, pentose phosphate pathway and glutaminolysis, respectively. The only enzyme activity to change on exposure of lymphocytes to ZAS was glutaminase, which was enhanced approximately by two-fold. Although rates of both glutamine and glucose utilization were enhanced by exposure to ZAS, only the rate of oxidation of glutamine was increased. Complement kills anucleated cells by simple osmotic lysis. However, it is likely that some nucleated cells will display characteristics of an ordered death mechanism and we have demonstrated that the concentration of lymphocyte ATP is dramatically decreased by activated complement. Nevertheless, the extent of cell death could be significantly reduced by the addition of inhibitors of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). We conclude that glutamine metabolism is not only important for lymphocyte proliferative responses but is also important for cell defence from sub-lethal concentrations of activated complement. The rapid rate of complement-induced lymphocyte death reported here is suggested to be a consequence of over-activation of the nuclear enzyme PARP and ATP depletion.

  1. [Autoimmune hemolytic anemia: diagnosis and management].

    PubMed

    Philippe, Pierre

    2007-12-01

    Autoimmune hemolytic anemia (AIHA) is diagnosed in the presence of anemia, usually macrocytic and of variable intensity, reticulocytosis, and a positive direct and/or indirect antiglobulin test, after ruling out other types of hemolytic anemia. A positive direct antiglobulin test alone is not sufficient to diagnose AIHA and may be positive in many patients without anemia or negative in some patients with AIHA. AIHA may be classified into two major categories according to the optimal temperature of antibody activity: warm-reacting autoantibodies (usually IgG) optimal around 37 degrees C and cold-reacting autoantibodies, optimal at 4 degrees C (usually IgM). This classification guides the selection of tests and treatment. AIHA is widely reported to be associated with a variety of other diseases, although these associations are often fortuitous. A minimal set of useful investigations is appropriate since AIHA may be secondary to viral infections, lymphoid malignancies, or autoimmune disorders such as lupus. Transfusion should remain rare in AHAI, but close contact with the transfusion service is necessary if it is to succeed. As for many autoimmune and/or systemic diseases, numerous types of treatment have been proposed but have not been validated in controlled multicenter studies. These are necessary to improve the management of these rare disorders.

  2. Hemolytic uremic syndrome associated with paraquat intoxication.

    PubMed

    Jang, Ha Nee; Bae, Eun Jin; Hwang, Kyungo; Kang, Yeojin; Yun, Seongeun; Cho, Hyun Seop; Chang, Se-Ho; Park, Dong Jun

    2014-06-01

    We report a case of a 66-year-old patient with paraquat intoxication resulting in the requirement for hemoperfusion, hemodialysis, and plasma exchange. His initial serum paraquat level was 0.24 µg/mL (0.0-0.1 µg/mL). Activated charcoal (50 g) was administered orally, and high-dose N-acetylcysteine (150 mg/kg) was administered intravenously. In addition, immediate 4 h hemoperfusion was also performed for three consecutive days after admission. Hemodialysis was started on the 4th day after admission because of uremia. On the 9th day after admission, laboratory findings demonstrated hemolytic uremic syndrome (HUS): microangiopathic hemolytic anemia (MAHA), thrombocytopenia, elevated reticulocyte count, and lactate dehydrogenase (LDH). Plasma exchange was performed three times consecutively. Anemia and thrombocytopenia were improved, and LDH was normalized after plasma exchange. Urine output increased to 2240 mL/day on the 18th day after admission, and hemodialysis was discontinued. He is currently being observed at our follow-up clinic without renal impairment or pulmonary dysfunction for 1.5 years since discharge. We should suspect paraquat-associated HUS when thrombocytopenia and anemia are maintained for a long time after paraquat intoxication.

  3. Inhibition of the activation of Hageman factor (factor XII) by complement subcomponent C1q.

    PubMed

    Rehmus, E H; Greene, B M; Everson, B A; Ratnoff, O D

    1987-08-01

    Hageman factor (HF, Factor XII) is activated by glass, collagen, and ellagic acid, and initiates blood coagulation via the intrinsic pathway. C1q inhibits collagen-induced platelet aggregation and adherence of platelets to glass, effects attributable to the collagen-like region of C1q. We examined the actions of C1q on HF activation. Incubation of C1q with HF before addition of HF-deficient plasma extended the activated partial thromboplastin time. Similarly, when glass tubes were coated with C1q before testing, the partial thromboplastin time of normal plasma was increased. C1q reduced the activation of HF by ellagic acid, as measured by the release of p-nitroaniline from the synthetic substrate H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride, an effect inhibited by monoclonal anti-human C1q murine IgG and by digestion of C1q by collagenase. Thus, C1q inhibits activation of HF in vitro in clot-promoting and amidolytic assays and suggests a regulatory mechanism for the inhibition of coagulation.

  4. Inhibition of the activation of Hageman factor (factor XII) by complement subcomponent C1q.

    PubMed Central

    Rehmus, E H; Greene, B M; Everson, B A; Ratnoff, O D

    1987-01-01

    Hageman factor (HF, Factor XII) is activated by glass, collagen, and ellagic acid, and initiates blood coagulation via the intrinsic pathway. C1q inhibits collagen-induced platelet aggregation and adherence of platelets to glass, effects attributable to the collagen-like region of C1q. We examined the actions of C1q on HF activation. Incubation of C1q with HF before addition of HF-deficient plasma extended the activated partial thromboplastin time. Similarly, when glass tubes were coated with C1q before testing, the partial thromboplastin time of normal plasma was increased. C1q reduced the activation of HF by ellagic acid, as measured by the release of p-nitroaniline from the synthetic substrate H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide dihydrochloride, an effect inhibited by monoclonal anti-human C1q murine IgG and by digestion of C1q by collagenase. Thus, C1q inhibits activation of HF in vitro in clot-promoting and amidolytic assays and suggests a regulatory mechanism for the inhibition of coagulation. PMID:3038961

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

    SciTech Connect

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu . E-mail: dxliu001@yahoo.com

    2007-07-13

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

  6. Inhibition of Myeloperoxidase Activity in Cystic Fibrosis Sputum by Peptide Inhibitor of Complement C1 (PIC1)

    PubMed Central

    Hair, Pamela S.; Sass, Laura A.; Krishna, Neel K.

    2017-01-01

    Myeloperoxidase is the major peroxidase enzyme in neutrophil granules and implicated in contributing to inflammatory lung damage in cystic fibrosis. Free myeloperoxidase is present in cystic fibrosis lung fluid and generates hypochlorous acid. Here we report a new inhibitor of myeloperoxidase activity, Peptide Inhibitor of Complement C1 (PIC1). Using TMB as the oxidizing substrate, PIC1 inhibited myeloperoxidase activity in cystic fibrosis sputum soluble fractions by an average of a 3.4-fold decrease (P = 0.02). PIC1 also dose-dependently inhibited myeloperoxidase activity in a neutrophil lysate or purified myeloperoxidase by up to 28-fold (P < 0.001). PIC1 inhibited myeloperoxidase activity similarly, on a molar basis, as the specific myeloperoxidase inhibitor 4-Aminobenzoic acid hydrazide (ABAH) for various oxidizing substrates. PIC1 was able to protect the heme ring of myeloperoxidase from destruction by NaOCl, assayed by spectral analysis. PIC1 incubated with oxidized TMB reversed the oxidation state of TMB, as measured by absorbance at 450 nm, with a 20-fold reduction in oxidized TMB (P = 0.02). This result was consistent with an antioxidant mechanism for PIC1. In summary, PIC1 inhibits the peroxidase activity of myeloperoxidase in CF sputum likely via an antioxidant mechanism. PMID:28135312

  7. Amblyomma americanum tick calreticulin binds C1q but does not inhibit activation of the classical complement cascade

    PubMed Central

    Kim, Tae Kwon; Ibelli, Adriana Mércia Guaratini; Mulenga, Albert

    2014-01-01

    In this study we characterized Amblyomma americanum (Aam) tick calreticulin (CRT) homolog in tick feeding physiology. In nature, different tick species can be found feeding on the same animal host. This suggests that different tick species found feeding on the same host can modulate the same host anti-tick defense pathways to successfully feed. From this perspective it’s plausible that different tick species can utilize universally conserved proteins such as CRT to regulate and facilitate feeding. CRT is a multi-functional protein found in most taxa that is injected into the vertebrate host during tick feeding. Apart from it’s current use as a biomarker for human tick bites, role(s) of this protein in tick feeding physiology have not been elucidated. Here we show that annotated functional CRT amino acid motifs are well conserved in tick CRT. However our data show that despite high amino acid identity levels to functionally characterized CRT homologs in other organisms, AamCRT is apparently functionally different. Pichia pastoris expressed recombinant (r) AamCRT bound C1q, the first component of the classical complement system, but it did not inhibit activation of this pathway. This contrast with reports of other parasite CRT that inhibited activation of the classical complement pathway through sequestration of C1q. Furthermore rAamCRT did not bind factor Xa in contrast to reports of parasite CRT binding factor Xa, an important protease in the blood clotting system. Consistent with this observation, rAamCRT did not affect plasma clotting or platelet aggregation aggregation. We discuss our findings in the context of tick feeding physiology. PMID:25454607

  8. A peptide derived from the parasite receptor, complement C2 receptor inhibitor trispanning, suppresses immune complex-mediated inflammation in mice.

    PubMed

    Inal, Jameel M; Schneider, Brigitte; Armanini, Marta; Schifferli, Jürg A

    2003-04-15

    Complement C2 receptor inhibitor trispanning (CRIT) is a Schistosoma protein that binds the human complement protein, C2. We recently showed that peptides based on the ligand binding region of CRIT inhibit the classical pathway (CP) of complement activation in human serum, using hemolytic assays and so speculated that on the parasite surface CRIT has the function of evading human complement. We now show that in vitro the C2-binding 11-aa C terminus of the first extracellular domain of CRIT, a 1.3-kDa peptide termed CRIT-H17, inhibits CP activation in a species-specific manner, inhibiting mouse and rat complement but not that from guinea pig. Hitherto, the ability of CRIT to regulate complement in vivo has not been assessed. In this study we show that by inhibiting the CP, CRIT-H17 is able to reduce immune complex-mediated inflammation (dermal reversed passive Arthus reaction) in BALB/c mice. Upon intradermal injection of CRIT-H17, and similarly with recombinant soluble complement receptor type 1, there was a 41% reduction in edema and hemorrhage, a 72% reduction in neutrophil influx, and a reduced C3 deposition. Furthermore, when H17 was administered i.v. at a 1 mg/kg dose, inflammation was reduced by 31%. We propose that CRIT-H17 is a potential therapeutic agent against CP complement-mediated inflammatory tissue destruction.

  9. Complement Inhibition for Prevention and Treatment of Antibody-Mediated Rejection in Renal Allograft Recipients.

    PubMed

    Jordan, S C; Choi, J; Kahwaji, J; Vo, A

    2016-04-01

    Therapeutic interventions aimed at the human complement system are recognized as potentially important strategies for the treatment of inflammatory and autoimmune diseases because there is often evidence of complement-mediated injury according to pathologic assessments. In addition, there are a large number of potential targets, both soluble and cell bound, that might offer potential for new drug development, but progress in this area has met with significant challenges. Currently, 2 drugs are approved aimed at inhibition of complement activation. The first option is eculizumab (anti-C5), which is approved for the treatment of paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Eculizumab has also been studied in human transplantation for the treatment and prevention of antibody-mediated rejection (ABMR). Initial data from uncontrolled studies suggested a significant benefit of eculizumab for the prevention of ABMR in highly HLA-sensitized patients, but a subsequent randomized, placebo-controlled trial failed to meet its primary endpoint. Anecdotal data, primarily from case studies, showed benefits in treating complement-mediated ABMR. A second approved complement-inhibiting therapy is C1 esterase inhibitor (C1-INH), which is approved for use in patients with hereditary angioedema, a condition caused by mutations in the gene that codes for C1-INH. A recent placebo-controlled trial of C1-INH for prevention of ABMR in HLA-sensitized patients found that the drug was safe, with evidence for inhibition of systemic complement activation and complement-activating donor-specific antibodies. Other drugs are now under development.

  10. The Holozoan Capsaspora owczarzaki Possesses a Diverse Complement of Active Transposable Element Families

    PubMed Central

    Carr, Martin; Suga, Hiroshi

    2014-01-01

    Capsaspora owczarzaki, a protistan symbiont of the pulmonate snail Biomphalaria glabrata, is the centre of much interest in evolutionary biology due to its close relationship to Metazoa. The whole genome sequence of this protist has revealed new insights into the ancestral genome composition of Metazoa, in particular with regard to gene families involved in the evolution of multicellularity. The draft genome revealed the presence of 23 families of transposable element, made up from DNA transposon as well as long terminal repeat (LTR) and non-LTR retrotransposon families. The phylogenetic analyses presented here show that all of the transposable elements identified in the C. owczarzaki genome have orthologous families in Metazoa, indicating that the ancestral metazoan also had a rich diversity of elements. Molecular evolutionary analyses also show that the majority of families has recently been active within the Capsaspora genome. One family now appears to be inactive and a further five families show no evidence of current transposition. Most individual element copies are evolutionarily young; however, a small proportion of inserts appear to have persisted for longer in the genome. The families present in the genome show contrasting population histories and appear to be in different stages of their life cycles. Transcriptome data have been analyzed from multiple stages in the C. owczarzaki life cycle. Expression levels vary greatly both between families and between different stages of the life cycle, suggesting an unexpectedly complex level of transposable element regulation in a single celled organism. PMID:24696401

  11. Peptide redesign for inhibition of the complement system: Targeting age-related macular degeneration

    PubMed Central

    Mohan, Rohith R.; Cabrera, Andrea P.; Harrison, Reed E. S.; Gorham, Ronald D.; Johnson, Lincoln V.; Ghosh, Kaustabh

    2016-01-01

    Purpose To redesign a complement-inhibiting peptide with the potential to become a therapeutic for dry and wet age-related macular degeneration (AMD). Methods We present a new potent peptide (Peptide 2) of the compstatin family. The peptide is developed by rational design, based on a mechanistic binding hypothesis, and structural and physicochemical properties derived from molecular dynamics (MD) simulation. The inhibitory activity, efficacy, and solubility of Peptide 2 are evaluated using a hemolytic assay, a human RPE cell–based assay, and ultraviolet (UV) absorption properties, respectively, and compared to the respective properties of its parent peptide (Peptide 1). Results The sequence of Peptide 2 contains an arginine-serine N-terminal extension (a characteristic of parent Peptide 1) and a novel 8-polyethylene glycol (PEG) block C-terminal extension. Peptide 2 has significantly improved aqueous solubility compared to Peptide 1 and comparable complement inhibitory activity. In addition, Peptide 2 is more efficacious in inhibiting complement activation in a cell-based model that mimics the pathobiology of dry AMD. Conclusions We have designed a new peptide analog of compstatin that combines N-terminal polar amino acid extensions and C-terminal PEGylation extensions. This peptide demonstrates significantly improved aqueous solubility and complement inhibitory efficacy, compared to the parent peptide. The new peptide overcomes the aggregation limitation for clinical translation of previous compstatin analogs and is a candidate to become a therapeutic for the treatment of AMD. PMID:27829783

  12. Role of membrane depolarization and extracellular calcium in increased complement receptor expression during neutrophil (PMN) activation

    SciTech Connect

    Berger, M.; Wetzler, E.; Birx, D.L.

    1986-03-05

    During PMN activation the surface expression of receptors (R) for C3b and C3bi increases rapidly. This is necessary for optimal cell adhesion, migration, and phagocytosis. Following stimulation with fMLP or LTB-4, the increased expression of C3bR depends only on the Ca/sup + +/ released from intracellular stores and is not inhibited by 5mM EDTA, while the increase in C3biR also requires extracellular Ca/sup + +/. CR expression also increases when the PMN are depolarized with 140 mM K/sup +/, but with this stimulus, EDTA inhibits C3bR by 67% and C3biR 100%, suggesting that intracellular Ca/sup + +/ stores may not be released. Pertussis toxin caused dose-dependent inhibition of both CR responses to fMLP and also inhibited the increases in both CR induced by K/sup +/. Membrane depolarization (monitored by di-O-C5 fluorescence) due to fMLP was similarly inhibited by toxin but the depolarization due to K/sup +/ was not. The dose of phorbol myristate acetate that maximally increased CR expression, 0.1 ng/ml, did not depolarize the membrane. These results suggest that membrane depolarization is neither necessary nor sufficient for increased CR expression. A Ca/sup + +/ and GTP binding protein-dependent enzyme such as phospholipase C is necessary to the amplify initial signals generated either by release of Ca/sup + +/ stores or by opening voltage dependent Ca/sup + +/ channels following membrane depolarization.

  13. Pregnant Woman with Atypical Hemolytic Uremic Syndrome Delivered a Healthy Newborn under Eculizumab Treatment

    PubMed Central

    Demir, Erol; Yazici, Halil; Ozluk, Yasemin; Kilicaslan, Isin; Turkmen, Aydin

    2016-01-01

    Pregnancy-associated thrombotic microangiopathy is a very rare condition; however, it significantly increases fetal or maternal morbidity and mortality. Pregnancy may trigger atypical hemolytic uremic syndrome (aHUS) or thrombotic thrombocytopenic purpura. The risk for pregnancy-associated aHUS is highest during the second pregnancy. The outcome is usually poor with 50–60% mortality; renal dysfunction and hypertension are the rule in those who survive the acute episode. After the development of complement regulation mechanisms and aHUS pathogenesis, eculizumab has been widely used as a first-line treatment in aHUS. Eculizumab has been produced to minimize immunogenicity and Fc-mediated functions, including recruitment of inflammatory cells and complement activation, and using eculizumab in first-line treatment improves kidney function. Recent studies showed that early diagnosis and rapid use of eculizumab in first-line treatment improve outcomes. We demonstrate a case with pregnancy-triggered aHUS occurring in the second trimester, who was successfully treated and delivered a healthy baby under eculizumab treatment. PMID:28101502

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

    PubMed

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

    2013-05-01

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

  15. Genetic Complementation Screen Identifies a Mitogen-activated Protein Kinase Phosphatase, MKP3, as a Regulator of Dopamine Transporter Trafficking

    PubMed Central

    Larsen, Mads Breum; Prasad, Balakrishna M.; Amara, Susan G.

    2008-01-01

    The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C–induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis. PMID:18434601

  16. Autoimmune hemolytic anaemia in Hodgkin's lymphoma.

    PubMed

    Shah, Mihir B; Nanjapp, Veena; Devaraj, H S; Sindhu, K S

    2013-07-01

    Autoimmune hemolytic anaemia is a rare presentation of Hodgkin's lymphoma though its association with Non- Hodgkin's lymphoma is well known. It is usually detected at the time of diagnosis when it accompanies Hodgkin's and rarely precedes it. It is a warm immune hemolytic anemia which is responsive to steroids and rituximab. We hereby report a case of advanced Hodgkin's disease who presented as AIHA.

  17. Topical Application of PPADS Inhibits Complement Activation and Choroidal Neovascularization in a Model of Age-Related Macular Degeneration

    PubMed Central

    Birke, Kerstin; Lipo, Erion; Birke, Marco T.; Kumar-Singh, Rajendra

    2013-01-01

    Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly. AMD patients have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and retinal pigment epithelium (RPE). MAC forms pores in cell membranes. Low levels of MAC result in an elevation of cytokine release such as vascular endothelial growth factor (VEGF) that promotes the formation of choroidal neovascularization (CNV). High levels of MAC result in cell lysis and RPE degeneration is a hallmark of advanced AMD. The current standard of care for CNV associated with wet AMD is intravitreal injection of anti-VEGF molecules every 4 to 12 weeks. Such injections have significant side effects. Recently, it has been found that membrane pore-forming proteins such as α-haemolysin can mediate their toxic effects through auto- and paracrine signaling and that complement-induced lysis is amplified through ATP release followed by P2X receptor activation. We hypothesized that attenuation of P2X receptor activation may lead to a reduction in MAC deposition and consequent formation of CNV. Hence, in this study we investigated topical application of the purinergic P2X antagonist Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) as a potential treatment for AMD. We found that 4.17 µM PPADS inhibited formation of HUVEC master junctions and master segments by 74.7%. In a human complement mediated cell lysis assay, 104 µM PPADS enabled almost complete protection of Hepa1c1c7 cells from 1% normal human serum mediated cell lysis. Daily topical application of 4.17 mM PPADS for 3 days attenuated the progression of laser induced CNV in mice by 41.8% and attenuated the deposition of MAC at the site of the laser injury by 19.7%. Our data have implications for the future treatment of AMD and potentially other ocular disorders involving CNV such as angioid streaks, choroidal rupture and high myopia. PMID:24130789

  18. Atypical Hemolytic Uremic Syndrome: Differential Diagnosis from TTP/HUS and Management.

    PubMed

    Yenerel, Mustafa N

    2014-09-05

    Atypical hemolytic uremic syndrome (aHUS) is a rare form of thrombotic microangiopathy (TMA). It has an unfavorable outcome with death rates as high as 25% during the acute phase and up to 50% of cases progressing to end-stage renal failure. Uncontrolled complement activation through the alternative pathway is thought to be the main underlying pathopysiology of aHUS and corresponds to all the deleterious findings of the disease. Thrombotic thrombocytopenic purpura (TTP) and Shiga toxin-associated HUS are the 2 other important TMA diseases. Although differentiating HUS from TTP is relatively easy in children with a preceding diarrheal illness or invasive S. pneumoniae, differentiating aHUS from TTP or other microangiopathic disorders can present a major diagnostic challenge in adults. ADAMTS13 analysis is currently the most informative diagnostic test for differentiating TTP, congenital TTP, and aHUS. Today empiric plasma therapy still is recommended by expert opinion to be used as early as possible in any patient with symptoms of aHUS. The overall treatment goal remains restoration of a physiological balance between activation and control of the alternative complement pathway. So it is a reasonable approach to block the terminal complement complex with eculizumab in order to prevent further organ injury and increase the likelihood organ recovery. Persistence of hemolysis or lack of improvement of renal function after 3-5 daily plasmaphereses have to be regarded as the major criteria for uncontrolled TMA even if platelet count has normalized and as an indication to switch the treatment to eculizumab. Eculizumab has changed the future perspectives of patients with aHUS and both the FDA and the EMA have approved it as life-long treatment. However, there are still some unresolved issues about the follow-up such as the optimal duration of eculizumab treatment and whether it can be stopped or how to stop the therapy.

  19. Atypical Hemolytic Uremic Syndrome: Differential Diagnosis from TTP/HUS and Management

    PubMed Central

    Yenerel, Mustafa N.

    2014-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare form of thrombotic microangiopathy (TMA). It has an unfavorable outcome with death rates as high as 25% during the acute phase and up to 50% of cases progressing to end-stage renal failure. Uncontrolled complement activation through the alternative pathway is thought to be the main underlying pathopysiology of aHUS and corresponds to all the deleterious findings of the disease. Thrombotic thrombocytopenic purpura (TTP) and Shiga toxin-associated HUS are the 2 other important TMA diseases. Although differentiating HUS from TTP is relatively easy in children with a preceding diarrheal illness or invasive S. pneumoniae, differentiating aHUS from TTP or other microangiopathic disorders can present a major diagnostic challenge in adults. ADAMTS13 analysis is currently the most informative diagnostic test for differentiating TTP, congenital TTP, and aHUS. Today empiric plasma therapy still is recommended by expert opinion to be used as early as possible in any patient with symptoms of aHUS. The overall treatment goal remains restoration of a physiological balance between activation and control of the alternative complement pathway. So it is a reasonable approach to block the terminal complement complex with eculizumab in order to prevent further organ injury and increase the likelihood organ recovery. Persistence of hemolysis or lack of improvement of renal function after 3-5 daily plasmaphereses have to be regarded as the major criteria for uncontrolled TMA even if platelet count has normalized and as an indication to switch the treatment to eculizumab. Eculizumab has changed the future perspectives of patients with aHUS and both the FDA and the EMA have approved it as life-long treatment. However, there are still some unresolved issues about the follow-up such as the optimal duration of eculizumab treatment and whether it can be stopped or how to stop the therapy. PMID:25319590

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

    PubMed

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

    2017-01-01

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

  1. Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B.

    PubMed Central

    Williams, S C; Hinshelwood, J; Perkins, S J; Sim, R B

    1999-01-01

    Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system. It binds to other complement proteins C3b and properdin, and is activated by the protease factor D. The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A). A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys(267)-->Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of (125)I-labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain. The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains. Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b. PMID:10477273

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

    PubMed

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

    2016-04-15

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

  3. Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes

    SciTech Connect

    Venema, J.; van Hoffen, A.; Karcagi, V.; Natarajan, A.T.; van Zeeland, A.A.; Mullenders, L.H. )

    1991-08-01

    The authors have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5{prime} part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3{prime} part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

  4. Fosb gene products contribute to excitotoxic microglial activation by regulating the expression of complement C5a receptors in microglia

    PubMed Central

    Nomaru, Hiroko; Sakumi, Kunihiko; Katogi, Atsuhisa; Ohnishi, Yoshinori N; Kajitani, Kosuke; Tsuchimoto, Daisuke; Nestler, Eric J.; Nakabeppu, Yusaku

    2014-01-01

    The Fosb gene encodes subunits of the activator protein-1 transcription factor complex. Two mature mRNAs, Fosb and ΔFosb, encoding full-length FOSB and ΔFOSB proteins respectively, are formed by alternative splicing of Fosb mRNA. Fosb products are expressed in several brain regions. Moreover, Fosb-null mice exhibit depressive-like behaviors and adult-onset spontaneous epilepsy, demonstrating important roles in neurological and psychiatric disorders. Study of Fosb products has focused almost exclusively on neurons; their function in glial cells remains to be explored. In this study, we found that microglia express equivalent levels of Fosb and ΔFosb mRNAs to hippocampal neurons and, using microarray analysis, we identified six microglial genes whose expression is dependent on Fosb products. Of these genes, we focused on C5ar1 and C5ar2, which encode receptors for complement C5a. In isolated Fosb-null microglia, chemotactic responsiveness toward the truncated form of C5a was significantly lower than that in wild-type cells. Fosb-null mice were significantly resistant to kainate-induced seizures compared with wild-type mice. C5ar1 mRNA levels and C5aR1 immunoreactivity were increased in wild-type hippocampus 24 hours after kainate administration; however, such induction was significantly reduced in Fosb-null hippocampus. Furthermore, microglial activation after kainate administration was significantly diminished in Fosb-null hippocampus, as shown by significant reductions in CD68 immunoreactivity, morphological change and reduced levels of Il6 and Tnf mRNAs, although no change in the number of Iba-1-positive cells was observed. These findings demonstrate that, under excitotoxicity, Fosb products contribute to a neuroinflammatory response in the hippocampus through regulation of microglial C5ar1 and C5ar2 expression. PMID:24771617

  5. Isolation and in vitro partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish Stomolophus meleagris.

    PubMed

    Li, Rongfeng; Yu, Huahua; Xing, Ronge; Liu, Song; Qing, Yukun; Li, Kecheng; Li, Bing; Meng, Xiangtao; Cui, Jinhui; Li, Pengcheng

    2013-09-01

    Jellyfish venom contains various toxins and can cause itching, edema, muscle aches, shortness of breath, blood pressure depression, shock or even death after being stung. Hemolytic protein is one of the most hazardous components in the venom. The present study investigated the hemolytic activity of the nematocyst venom from jellyfish Stomolophus meleagris. Anion exchange chromatography, DEAE Sepharose Fast Flow, and gel filtration chromatography, Superdex200 had been employed to isolate hemolytic proteins from the nematocyst venom of jellyfish S. meleagris. Hemolysis of chicken red blood cells was used to quantify hemolytic potency of crude nematocyst venom and chromatography fractions during the purification process. Native-PAGE profile displayed one protein band in the purified hemolytic protein (SmTX); however, two protein bands with apparent molecular weights of ≈ 45 kDa and 52 kDa were observed in the reducing SDS-PAGE analysis. Approximately 70 μg/mL of SmTX caused 50% hemolysis (HU50) of the erythrocyte suspension. The hemolytic activity of SmTX was shown to be temperature and pH dependent, with the optimum temperature and pH being 37°C and pH 5.0. The present study is the first report of isolation and partial characterization of hemolytic proteins from the nematocyst venom of the jellyfish S. meleagris. The mechanism of the hemolytic activity of SmTX is not clear and deserves further investigation.

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

    PubMed Central

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

    2013-01-01

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

  7. Effects of Ca2+ on refolding of the recombinant hemolytic lectin CEL-III.

    PubMed

    Hisamatsu, Keigo; Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2009-05-01

    CEL-III is a hemolytic lectin isolated from Cucumaria echinata. Although recombinant CEL-III (rCEL-III) expressed in Escherichia coli showed very weak hemolytic activity compared with native protein, it was considerably enhanced by refolding in the presence of Ca(2+). This suggests that Ca(2+) supported correct folding of the carbohydrate-binding domains of rCEL-III, leading to effective binding to the cell surface and subsequent self-oligomerization.

  8. 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

  9. Complement regulates conventional DC-mediated NK-cell activation by inducing TGF-β1 in Gr-1+ myeloid cells.

    PubMed

    Qing, Xiaoping; Koo, Gloria C; Salmon, Jane E

    2012-07-01

    Complement activation modulates DC-mediated T-cell activation, but whether complement affects DC-mediated priming of NK cells is unknown. Here, we demonstrated that conventional DCs (cDCs) from C3(-/-) and C5aR(-/-) mice are hyperresponsive to polyI:C, a TLR3 ligand, leading to enhanced NK-cell activation. We found that cDCs lack C5a receptor (C5aR) and do not respond to C5a directly. Depletion of Gr-1(+) myeloid cells augments polyI:C-induced cDC activation in WT but not in C3(-/-) or C5aR(-/-) mice, indicating that the effect of complement activation on cDCs is indirectly mediated through C5aR-expressing Gr-1(+) myeloid cells. We further demonstrated that the mechanism by which Gr-1(+) myeloid cells regulate the activity of cDCs involves C5a-dependent TGF-β1 production in Gr-1(+) myeloid cells. C5a enhances and blocking C5aR decreases TGF-β1 production in cultured bone marrow Gr-1(+) CD11b(+) cells. C5aR deficiency is associated with reduced circulating TGF-β1 levels, while depleting Gr-1(+) myeloid cells abrogates this difference between WT and C5aR(-/-) mice. Lastly, we showed that enhanced cDC-NK-cell activity in C3(-/-) mice led to delayed melanoma tumor growth. Thus, complement activation indirectly regulates cDC-NK-cell activation in response to inflammatory stimuli such as TLR3 by promoting TGF-β1 production in Gr-1(+) myeloid cells at steady state.

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

    PubMed Central

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

    2015-01-01

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

  11. The Avise Lupus Test and Cell-bound Complement Activation Products Aid the Diagnosis of Systemic Lupus Erythematosus

    PubMed Central

    Mossell, James; Goldman, John A.; Barken, Derren; Alexander, Roberta Vezza

    2016-01-01

    Background: Systemic lupus erythematosus (SLE) is a multifaceted disease, and its diagnosis may be challenging. A blood test for the diagnosis of SLE, the Avise Lupus test, has been recently commercialized and validated in clinical studies. Objectives: To evaluate the use of the Avise Lupus test by community rheumatologists. Methods: The study is a longitudinal, case-control, retrospective review of medical charts. Cases had a positive test result, and controls had a negative result; all patients were anti-nuclear antibodies (ANA) positive but negative for SLE-specific autoantibodies. Features of SLE, diagnosis, and medications at two time points were recorded. Results: Twenty of the 23 cases (87%) and 4 of the 23 controls (17%) were diagnosed with SLE (sensitivity=83%; specificity=86%). More cases than controls (43% vs. 17%) fulfilled 4 American College of Rheumatology (ACR) classification criteria of SLE. Sensitivity of the test was significantly higher than the ACR score (83% vs. 42%, p=0.006). A higher percentage of patients who met the classification criteria had elevated cell-bound complement activation products (CB-CAPs) compared to patients who did not. Anti-rheumatic medications were used in a higher percentage of cases than controls (83% vs. 35% at baseline, p=0.002), suggesting that cases were treated more aggressively early on. Conclusion: A positive Avise Lupus test result aids in formulating a SLE diagnosis when diagnosis based on standard-of-care tests and clinical features may be challenging, and impacts patient management. Prospective studies will be performed to better evaluate the clinical utility of the test and of CB-CAPs as biomarkers of SLE. PMID:27867431

  12. A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast.

    PubMed

    Pérez-Castiñeira, José R; Hernández, Agustín; Drake, Rocío; Serrano, Aurelio

    2011-07-15

    V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.

  13. Complement in health and disease.

    PubMed

    Carroll, Maria V; Sim, Robert B

    2011-09-16

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

  14. Complement Evasion by Pathogenic Leptospira.

    PubMed

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

    2016-01-01

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

  15. Complement Evasion by Pathogenic Leptospira

    PubMed Central

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

    2016-01-01

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

  16. Hemolytic anemia caused by chemicals and toxins

    MedlinePlus

    ... Rho immune globulin (WinRho) Ribavirin Snake bites (some snake venom contains hemolytic toxins ) Sulfonamides Sulfones This list is not all-inclusive. Alternative Names ... Review Date 2/12/2016 Updated ...

  17. Deficiency of human complement factor I associated with lowered factor H.

    PubMed

    Naked, G M; Florido, M P; Ferreira de Paula, P; Vinet, A M; Inostroza, J S; Isaac, L

    2000-08-01

    Deficiencies of factor I and/or factor H result in an increased consumption of C3 and higher susceptibility to recurrent infections. Here we describe a case of human factor I deficiency and lowered factor H levels. C3 concentration was 50% lower than normal, the classical pathway-dependent hemolytic activity was reduced to almost 30% of normal, and alternative pathway-dependent activity was completely absent. The killing by peripheral leukocytes of Candida albicans treated with deficient serum and the production of complement-dependent chemotactic factors were reduced in the proband's serum when compared with normal serum. Finally, we observed that C3 antigen present in the proband's serum has a different electrophoretic mobility than native C3 (most likely C3b), confirming the deregulation of complement activation due to the lack of regulatory proteins factors I and H. The impaired complement system described in this case, the first of its kind described in a Chile, explains the higher susceptibility to infections found in the proband.

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

    PubMed Central

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

    1986-01-01

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

  19. Human IgG1 monoclonal antibody against human collagen 17 noncollagenous 16A domain induces blisters via complement activation in experimental bullous pemphigoid model.

    PubMed

    Li, Qiang; Ujiie, Hideyuki; Shibaki, Akihiko; Wang, Gang; Moriuchi, Reine; Qiao, Hong-jiang; Morioka, Hiroshi; Shinkuma, Satoru; Natsuga, Ken; Long, Heather A; Nishie, Wataru; Shimizu, Hiroshi

    2010-12-15

    Bullous pemphigoid (BP) is an autoimmune blistering disease caused by IgG autoantibodies targeting the noncollagenous 16A (NC16A) domain of human collagen 17 (hCOL17), which triggers blister formation via complement activation. Previous in vitro analysis demonstrated that IgG1 autoantibodies showed much stronger pathogenic activity than IgG4 autoantibodies; however, the exact pathogenic role of IgG1 autoantibodies has not been fully demonstrated in vivo. We constructed a recombinant IgG1 mAb against hCOL17 NC16A from BP patients. In COL17-humanized mice, this mAb effectively reproduced a BP phenotype that included subepidermal blisters, deposition of IgG1, C1q and C3, neutrophil infiltration, and mast cell degranulation. Subsequently, alanine substitutions at various C1q binding sites were separately introduced to the Fc region of the IgG1 mAb. Among these mutated mAbs, the one that was mutated at the P331 residue completely failed to activate the complement in vitro and drastically lost pathogenic activity in COL17-humanized mice. These findings indicate that P331 is a key residue required for complement activation and that IgG1-dependent complement activation is essential for blister formation in BP. This study is, to our knowledge, the first direct evidence that IgG1 Abs to hCOL17 NC16A can induce blister formation in vivo, and it raises the possibility that IgG1 mAbs with Fc modification may be used to block pathogenic epitopes in autoimmune diseases.

  20. Factor H and factor H-related protein 1 bind to human neutrophils via complement receptor 3, mediate attachment to Candida albicans, and enhance neutrophil antimicrobial activity.

    PubMed

    Losse, Josephine; Zipfel, Peter F; Józsi, Mihály

    2010-01-15

    The host complement system plays an important role in protection against infections. Several human-pathogenic microbes were shown to acquire host complement regulators, such as factor H (CFH), that downregulate complement activation at the microbial surface and protect the pathogens from the opsonic and lytic effects of complement. Because CFH can also bind to host cells, we addressed the role of CFH and CFH-related proteins as adhesion ligands in host-pathogen interactions. We show that the CFH family proteins CFH, CFH-like protein 1 (CFHL1), CFH-related protein (CFHR) 1, and CFHR4 long isoform bind to human neutrophil granulocytes and to the opportunistic human-pathogenic yeast Candida albicans. Two major binding sites, one within the N-terminus and one in the C-terminus of CFH, were found to mediate binding to neutrophils. Complement receptor 3 (CD11b/CD18; alpha(M)beta2 integrin) was identified as the major cellular receptor on neutrophils for CFH, CFHL1, and CFHR1, but not for CFHR4 long isoform. CFH and CFHR1 supported cell migration. Furthermore, CFH, CFHL1, and CFHR1 increased attachment of neutrophils to C. albicans. Adhesion of neutrophils to plasma-opsonized yeasts was reduced when CFH binding was inhibited by specific Abs or when using CFH-depleted plasma. Yeast-bound CFH and CFHR1 enhanced the generation of reactive oxygen species and the release of the antimicrobial protein lactoferrin by human neutrophils, and resulted in a more efficient killing of the pathogen. Thus, CFH and CFHR1, when bound on the surface of C. albicans, enhance antimicrobial activity of human neutrophils.

  1. Tumor-Derived Tissue Factor Aberrantly Activates Complement and Facilitates Lung Tumor Progression via Recruitment of Myeloid-Derived Suppressor Cells

    PubMed Central

    Han, Xiao; Zha, Haoran; Yang, Fei; Guo, Bo; Zhu, Bo

    2017-01-01

    The initiator of extrinsic coagulation, tissue factor (TF), and its non-coagulant isoform alternatively spliced TF (asTF) are closely associated with tumor development. In the tumor microenvironment, the role of TF-induced coagulation in tumor progression remains to be fully elucidated. Using TF-knockdown lung tumor cells, we showed that TF is the dominant component of procoagulant activity but is dispensable in the cellular biology of tumor cells. In a xenograft model, using immunohistochemical analysis and flow cytometry analysis of the tumor microenvironment, we demonstrated that TF-induced fibrin deposition, which is correlated with complement activation and myeloid-derived suppressor cell (MDSC) recruitment, is positively associated with tumor progression. C5aR antagonism blunted the effect of TF on tumor progression and decreased MDSC recruitment. In conclusion, our data suggested that in tumor microenvironment, TF-induced coagulation activated the complement system and subsequently recruited myeloid-derived suppressor cells to promote tumor growth, which brings new insights into the coagulation-induced complement activation within the tumor microenvironment during tumor progression. PMID:28106852

  2. Tumor-Derived Tissue Factor Aberrantly Activates Complement and Facilitates Lung Tumor Progression via Recruitment of Myeloid-Derived Suppressor Cells.

    PubMed

    Han, Xiao; Zha, Haoran; Yang, Fei; Guo, Bo; Zhu, Bo

    2017-01-19

    The initiator of extrinsic coagulation, tissue factor (TF), and its non-coagulant isoform alternatively spliced TF (asTF) are closely associated with tumor development. In the tumor microenvironment, the role of TF-induced coagulation in tumor progression remains to be fully elucidated. Using TF-knockdown lung tumor cells, we showed that TF is the dominant component of procoagulant activity but is dispensable in the cellular biology of tumor cells. In a xenograft model, using immunohistochemical analysis and flow cytometry analysis of the tumor microenvironment, we demonstrated that TF-induced fibrin deposition, which is correlated with complement activation and myeloid-derived suppressor cell (MDSC) recruitment, is positively associated with tumor progression. C5aR antagonism blunted the effect of TF on tumor progression and decreased MDSC recruitment. In conclusion, our data suggested that in tumor microenvironment, TF-induced coagulation activated the complement system and subsequently recruited myeloid-derived suppressor cells to promote tumor growth, which brings new insights into the coagulation-induced complement activation within the tumor microenvironment during tumor progression.

  3. Treatment of autoimmune hemolytic anemias.

    PubMed

    Zanella, Alberto; Barcellini, Wilma

    2014-10-01

    Autoimmune hemolytic anemia (AIHA) is a relatively uncommon disorder caused by autoantibodies directed against self red blood cells. It can be idiopathic or secondary, and classified as warm, cold (cold hemagglutinin disease (CAD) and paroxysmal cold hemoglobinuria) or mixed, according to the thermal range of the autoantibody. AIHA may develop gradually, or have a fulminant onset with life-threatening anemia. The treatment of AIHA is still not evidence-based. The first-line therapy for warm AIHA are corticosteroids, which are effective in 70-85% of patients and should be slowly tapered over a time period of 6-12 months. For refractory/relapsed cases, the current sequence of second-line therapy is splenectomy (effective approx. in 2 out of 3 cases but with a presumed cure rate of up to 20%), rituximab (effective in approx. 80-90% of cases), and thereafter any of the immunosuppressive drugs (azathioprine, cyclophosphamide, cyclosporin, mycophenolate mofetil). Additional therapies are intravenous immunoglobulins, danazol, plasma-exchange, and alemtuzumab and high-dose cyclophosphamide as last resort option. As the experience with rituximab evolves, it is likely that this drug will be located at an earlier point in therapy of warm AIHA, before more toxic immunosuppressants, and in place of splenectomy in some cases. In CAD, rituximab is now recommended as first-line treatment.

  4. A Case of Atypical Hemolytic Uremic Syndrome Successfully Treated with Eculizumab

    PubMed Central

    Thajudeen, B.; Sussman, A.; Bracamonte, E.

    2013-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare thrombotic microangiopathy (TMA) characterized by the triad of hemolytic anemia, thrombocytopenia, and acute renal failure. Eculizumab, a monoclonal complement C5 antibody which prevents the induction of the terminal complement cascade, has recently emerged as a therapeutic option for aHUS. We report a case of aHUS successfully treated with eculizumab. A 51-year-old male was admitted to the hospital following a mechanical fall. His past medical history was significant for rheumatic valve disease and mitral valve replacement; he was on warfarin for anticoagulation. A computed tomography scan of the head revealed a right-sided subdural hematoma due to coagulopathy resulting from a supratherapeutic international normalized ratio (INR). Following treatment with prothrombin complex concentrate to reverse the INR, urine output dropped and his serum creatinine subsequently increased to 247.52 μmol/l from the admission value of 70.72 μmol/l. Laboratory evaluation was remarkable for hemolytic anemia, thrombocytopenia, elevated lactate dehydrogenase (LDH), low haptoglobin, and low complement C3. A renal biopsy was consistent with TMA, favoring a diagnosis of aHUS. Treatment with eculizumab was initiated which resulted in the stabilization of his hemoglobin, platelets, and LDH. Hemodialysis was terminated after 2.5 months due to improvement in urine output and solute clearance. The interaction between thrombin and complement pathway might be responsible for the pathogenesis of aHUS in this case. Eculizumab is an effective therapeutic agent in the treatment of aHUS. Early targeting of the complement system may modify disease progression and thus treat aHUS more effectively. PMID:24570684

  5. Extravascular hemolysis and complement consumption in Paroxysmal Nocturnal Hemoglobinuria patients undergoing eculizumab treatment.

    PubMed

    Subías Hidalgo, Marta; Martin Merinero, Hector; López, Alicia; Anter, Jaouad; García, Sheila Pinto; Ataúlfo Gonzalez-Fernández, Fernando; Forés, Rafael; Lopez-Trascasa, Margarita; Villegas, Ana; Ojeda, Emilio; Rodríguez de Córdoba, Santiago

    2017-02-01

    Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis that is effectively treated with eculizumab. However, treatment responses are reported heterogeneous with some patients presenting residual hemolysis and requiring RBC transfusions. Recent reports have shown that both extravascular hemolysis and incomplete C5 blockade can explain these suboptimal hematological responses. Here we have tested our eculizumab-treated PNH patients (n=12) for signs of hemolysis and assessed complement biomarkers. Patients were also genotyped for complement receptor 1 (CR1, CD35) and C5 polymorphisms and evaluated for free eculizumab in plasma. We report that 10 patients (83%) present parameters suggesting persistent hemolysis, although they did not require additional transfusions. Seven of them (58%) become direct Coombs-test positive as a consequence of treatment, including all patients carrying the low-expression CR1-L allele. CH50 and sC5b-9 assays demonstrate that the persistent low-level hemolysis identified in our treated patients is not a consequence of incomplete C5 blockade, supporting that this hemolysis, as has been suggested previously, results from the extravascular removal of C3 opsonized PNH erythrocytes. We also show that continuous alternative pathway activation in eculizumab-treated individuals carrying the CR1-L allele results in abnormally decreased levels of C3 in plasma that could, potentially, increase their susceptibility to bacterial infections. Finally, we encourage a routine evaluation of free eculizumab levels and terminal pathway activity to personalize eculizumab administration.

  6. Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

    SciTech Connect

    Sjoewall, Christopher; Askendal, Agneta; Almroth, Gunnel

    2007-01-05

    C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fc{gamma} receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficiently down-regulated at CRP levels >150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.

  7. Complement receptor 1 inhibitors for prevention of immune-mediated red cell destruction: potential use in transfusion therapy.

    PubMed

    Yazdanbakhsh, Karina; Kang, Stanley; Tamasauskas, Daniel; Sung, Dorothy; Scaradavou, Andromachi

    2003-06-15

    Activation of complement cascade via the antibody-mediated classical pathway can initiate red blood cell (RBC) destruction, causing transfusion reactions and hemolytic anemia. In the present study, we have assessed the ability of a human recombinant soluble form of complement receptor 1 (sCR1) to inhibit complement-mediated RBC destruction in vitro and in vivo. Using an in vitro alloimmune incompatibility model, sCR1 inhibited complement activation and prevented hemolysis. Following transfusion of human group O RBCs into mice lacking detectable pre-existing antibodies against the transfused RBCs, systemic coadministration of 10 mg/kg sCR1, a dose well tolerated in human subjects for prevention of tissue injury, completely inhibited the in vivo clearance of the transfused RBCs and surface C3 deposition in the first hour after transfusion, correlating with the half-life of sCR1 in the circulation. Treatment with sCR1 increased the survival of transfused human group A RBCs in the circulation of mice with pre-existing anti-A for 2 hours after transfusion by 50%, reduced intravascular hemolysis, and lowered the levels of complement deposition (C3 and C4), but not immunoglobulin G (IgG) or IgM, on the transfused cells by 100-fold. We further identified potential functional domains in CR1 that can act to limit complement-mediated RBC destruction in vitro and in vivo. Collectively, our data highlight a potential use of CR1-based inhibitors for prevention of complement-dependent immune hemolysis.

  8. Activation of bovine monocytes and neutrophils by the Bb fragment of complement factor B: demonstration by the uptake of 3H-deoxyglucose.

    PubMed Central

    Sethi, M S; Tabel, H; Misra, V

    1990-01-01

    The Bb fragment is the enzymatically active split product of bovine complement factor B. The Bb fragment was obtained after zymosan treatment of fresh bovine serum and fractionation of the treated serum, first over diethylaminoethyl-Sephacel and then over an affinity column made up of monoclonal antibody to bovine Bb, coupled to cyanogen-bromide-activated Sepharose. Purified Bb has a molecular weight of 64,000, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The ability of purified Bb to activate phagocytes was assessed. The activation assay was based on the principle that the primary source of energy for the phagocytes is obtained from glucose. 3H-deoxyglucose, a nonmetabolizable analogue of glucose, was used to obtain the quantitative measurement of the activation process. The activation by Bb was shown by the uptake of the labelled deoxyglucose in the phagocytic cells and was comparable to the activation caused by phorbol myristate acetate and N-formyl-L-methionyl-L-leucyl-L-phenylalanine, run in parallel. These data showed that fragment Bb activates bovine monocytes and neutrophils and also suggested that, when generated after complement activation, Bb may stimulate monocytes and neutrophils for enhanced phagocytosis. Images Fig. 1. Fig. 2. PMID:2306658

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

    PubMed Central

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

    2010-01-01

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

  10. Genetics of the complement system.

    PubMed Central

    Lachmann, P

    1975-01-01

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

  11. The Serine Protease Pic From Enteroaggregative Escherichia coli Mediates Immune Evasion by the Direct Cleavage of Complement Proteins.

    PubMed

    Abreu, Afonso G; Fraga, Tatiana R; Granados Martínez, Adriana P; Kondo, Marcia Y; Juliano, Maria A; Juliano, Luiz; Navarro-Garcia, Fernando; Isaac, Lourdes; Barbosa, Angela S; Elias, Waldir P

    2015-07-01

    Enteroaggregative and uropathogenic Escherichia coli, Shigella flexneri 2a, and the hybrid enteroaggregative/Shiga toxin-producing E. coli strain (O104:H4) are important pathogens responsible for intestinal and urinary tract infections, as well as sepsis and hemolytic uremic syndrome. They have in common the production of a serine protease called Pic. Several biological roles for Pic have been described, including protection of E. coli DH5α from complement-mediated killing. Hereby we showed that Pic significantly reduces complement activation by all 3 pathways. Pic cleaves purified C3/C3b and other proteins from the classic and lectin pathways, such as C4 and C2. Cleavage fragments of C3, C4, and C2 were also observed with HB101(pPic1) culture supernatants, and C3 cleavage sites were mapped by fluorescence resonance energy transfer peptides. Experiments using human serum as a source of complement proteins confirmed Pic proteolytic activity on these proteins. Furthermore, Pic works synergistically with the human complement regulators factor I and factor H, promoting inactivation of C3b. In the presence of both regulators, further degradation of C3 α' chain was observed. Therefore, Pic may contribute to immune evasion of E. coli and S. flexneri, favoring invasiveness and increasing the severity of the disorders caused by these pathogens.

  12. Specifically binding of L-ficolin to N-glycans of HCV envelope glycoproteins E1 and E2 leads to complement activation.

    PubMed

    Liu, Jun; Ali, Mohammed A M; Shi, Yinghua; Zhao, Yinglan; Luo, Fenglin; Yu, Jin; Xiang, Tian; Tang, Jie; Li, Dongqing; Hu, Quan; Ho, Wenzhe; Zhang, Xiaolian

    2009-08-01

    L-ficolin, one of lectin families, is a recently identified complement factor that initiates lectin pathway of complement. Little is known about its role in viral hepatitis. In the present study, we found that L-ficolin in serum from 103 patients with hepatitis C virus (HCV), were significantly higher than that in 150 healthy controls. We further found that L-ficolin expressions were significantly increased in vitro study by HCV JFH-1 infected human hepatocyte cell line Huh7.5.1. Investigation of the mechanisms of the L-ficolin action on HCV demonstrated that L-ficolin protein could recognize and bind to envelope glycoproteins E1 and E2 of HCV, activating the lectin complement pathway-mediated cytolytic activity in HCV-infected hepatocyte. This interaction between L-ficolin and HCV E1 and E2 glycoproteins was attributed to the N-glycans of E1 and E2. These findings provide new insights into the biological functions of L-ficolin in clinically important hepatic viral diseases.

  13. Genetic diagnosis for congenital hemolytic anemia.

    PubMed

    Ohga, Shouichi

    Congenital hemolytic anemia is a group of monogenic diseases presenting with anemia due to increased destruction of circulating erythrocytes. The etiology of inherited anemia accounts for germline mutations of the responsible genes coding for the structural components of erythrocytes and extra-erythrocytes. The erythrocyte abnormalities are classified into three major disorders of red cell membrane defects, hemoglobinopathies, and red cell enzymopathies. The extra-erythrocyte abnormalities, typified by consumption coagulopathy and intravascular hemolysis, include Upshaw-Schulman syndrome and atypical hemolytic uremic syndrome. The clinical manifestations of congenital hemolytic anemia are anemia, jaundice, cholelithiasis and splenomegaly, while the onset mode and severity are both variable. Genetic overlapping of red cell membrane protein disorders, and distinct frequency and mutation spectra differing among races make it difficult to understand this disease entity. On the other hand, genetic modifiers for the phenotype of β-globin diseases provide useful information for selecting the optimal treatment and for long-term management. Recently, next generation sequencing techniques have enabled us to determine the novel causative genes in patients with undiagnosed hemolytic anemias. We herein review the concept and strategy for genetic diagnosis of inherited hemolytic anemias.

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

    PubMed Central

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

    1995-01-01

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

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

    PubMed

    Franchi, Nicola; Ballarin, Loriano

    2014-10-01

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

  16. A Saccharomyces Cerevisiae Rad52 Allele Expressing a C-Terminal Truncation Protein: Activities and Intragenic Complementation of Missense Mutations

    PubMed Central

    Boundy-Mills, K. L.; Livingston, D. M.

    1993-01-01

    A nonsense allele of the yeast RAD52 gene, rad52-327, which expresses the N-terminal 65% of the protein was compared to two missense alleles, rad52-1 and rad52-2, and to a deletion allele. While the rad52-1 and the deletion mutants have severe defects in DNA repair, recombination and sporulation, the rad52-327 and rad52-2 mutants retain either partial or complete capabilities in repair and recombination. These two mutants behave similarly in most tests of repair and recombination during mitotic growth. One difference between these two alleles is that a homozygous rad52-2 diploid fails to sporulate, whereas the homozygous rad52-327 diploid sporulates weakly. The low level of sporulation by the rad52-327 diploid is accompanied by a low percentage of spore viability. Among these viable spores the frequency of crossing over for markers along chromosome VII is the same as that found in wild-type spores. rad52-327 complements rad52-2 for repair and sporulation. Weaker intragenic complementation occurs between rad52-327 and rad52-1. PMID:8417987

  17. Irregular antibodies in no hemolytic autoimmune diseases are able to induce erythrophagocytosis.

    PubMed

    López-Díaz, Paola Ester; Ruiz-Olivera, María Del Rocío; Hernández-Osorio, Luis Alberto; Vargas-Arzola, Jaime; Valle-Jiménez, Xareni; Aguilar-Ruiz, Sergio Roberto; Torres-Aguilar, Honorio

    2016-08-25

    Irregular antibodies are produced by alloimmunization because of pregnancies or blood transfusions. They are called "irregular" due to target erythrocyte antigens from "rare blood systems," those different from the ABO system. Irregular antibodies have been widely investigated in immunohematology since their presence in blood donors may lead to difficulties in blood typing and in blood cross-matching, or to induce hemolytic transfusion reactions. Nevertheless, their incidence and participation in the physiopathology of autoimmune diseases have not been thoroughly studied. In this work, we analyzed the presence and pro-hemolytic capabilities of irregular antibodies in patients with different autoimmune diseases lacking signs of hemolytic anemia, in comparison with healthy multiparous women. Five of 141 autoimmune patients (3.5 %) and two of 77 multiparous women (2.6 %) were positive. Although frequency was relatively low and similar in both populations, the targeted antigens were Kell (k, Kp(b), Js(b)) and Luth (Lu(b)) in multiparous women, and the same plus Duffy (Fy(a)), Kidd (Jk(a)) and MNS (M, s) in autoimmune patients. Irregular antibodies from autoimmune patients did not induce complement-mediated hemolysis (intravascular), but they were able to induce macrophages-mediated phagocytosis (extravascular hemolysis) in vitro. It is the first approach exploring the presence of irregular antibodies associated with the loss of immune tolerance and demonstrating their hemolytic potential in autoimmune patients without hemolytic manifestations. The presence of irregular antibodies targeted to Duffy (Fya), Kidd (Jka) and MNS (M, s) antigens only in autoimmune patients suggests a loss of immune tolerance to these erythrocyte antigens.

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

    PubMed

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

    2015-12-10

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

  19. Stevia rebaudiana Bertoni effect on the hemolytic potential of Listeria monocytogenes.

    PubMed

    Sansano, S; Rivas, A; Pina-Pérez, M C; Martinez, A; Rodrigo, D

    2017-03-16

    The effect of Stevia rebaudiana Bertoni on the hemolytic potential of Listeria monocytogenes was studied by means of the assessment of the Listeriolysin O (LLO) production. The three factors under study, stevia concentration in the range [0-2.5] % (w/v), incubation temperature (10 and 37°C), and exposure time (0-65h) significantly affected (p≤0.05) the hemolytic activity of L. monocytogenes. Results showed that at the lower incubation temperature the hemolytic potential of the bacterium was significantly reduced, from 100% at 37°C to 8% at 10°C (after 65h of incubation) in unsupplemented substrate (0% stevia). Irrespective of the temperature, 10 or 37°C, supplementation of the medium with stevia at 2.5 % (w/v) reduced the bacterium's hemolytic activity by a maximum of 100%. Furthermore, the time of exposure to 2.5 % (w/v) stevia concentration was also a significant factor reducing the hemolytic capability of L. monocytogenes. The possibility of reducing the pathogenic potential of L. monocytogenes (hemolysis) by exposure to stevia should be confirmed in real food matrices, opening a research niche with a valuable future impact on food safety.

  20. Activation of the complement system and accumulation of hemoglobin-haptoglobin complexes in plasma during an adverse reaction to penicillin treatment.

    PubMed

    Brandslund, I; Svehag, S E; Teisner, B; Hyltoft Petersen, P

    1983-01-01

    A patient treated with penicillin intravenously developed a serum sickness-like reaction. Classical pathway complement (C) activation was indicated by quantitation of the split products C3c and C3d as well as demonstration of C4 conversion. Circulating immune complexes could, however, not be detected by the solid-phase Clq and PEG-precipitation methods. A concomitant accumulation of circulating hemoglobin-haptoglobin complexes and a marked fall in serum-fibronectin concentrations suggested saturation of the reticuloendothelial system. The plasma became a deep-red color, and a diffuse intravascular coagulation followed. The patient recovered completely upon discontinuation of penicillin administration.

  1. Inducible ASABF-type antimicrobial peptide from the sponge Suberites domuncula: microbicidal and hemolytic activity in vitro and toxic effect on molluscs in vivo.

    PubMed

    Wiens, Matthias; Schröder, Heinz C; Korzhev, Michael; Wang, Xiao-Hong; Batel, Renato; Müller, Werner E G

    2011-01-01

    Since sponges, as typical filter-feeders, are exposed to a high load of attacking prokaryotic and eukaryotic organisms, they are armed with a wide arsenal of antimicrobial/cytostatic low-molecular-weight, non-proteinaceous bioactive compounds. Here we present the first sponge agent belonging to the group of ASABF-type antimicrobial peptides. The ASABF gene was identified and cloned from the demosponge Suberites domuncula. The mature peptide, with a length of 64 aa residues has a predicted pI of 9.24, and comprises the characteristic CSα β structural motif. Consequently, the S. domuncula ASABF shares high similarity with the nematode ASABFs; it is distantly related to the defensins. The recombinant peptide was found to display besides microbicidal activity, anti-fungal activity. In addition, the peptide lyses human erythrocytes. The expression of ASABF is upregulated after exposure to the apoptosis-inducing agent 2,2'-dipyridyl. During the process of apoptosis of surface tissue of S. domuncula, grazing gastropods (Bittium sp.) are attracted by quinolinic acid which is synthesized through the kynurenine pathway by the enzyme 3-hydroxyanthranilate 3,4-dioxygenase (HAD). Finally, the gastropods are repelled from the sponge tissue by the ASABF. It is shown that the effector peptide ASABF is sequentially expressed after the induction of the HAD gene and a caspase, as a central enzyme executing apoptosis.

  2. [Biermer's disease and autoimmune hemolytic anemia].

    PubMed

    Nafil, Hatim; Tazi, Illias; Mahmal, Lahoucine

    2012-01-01

    Biermer's disease is an autoimmune atrophic gastritis of the fundus predominantly responsible for a malabsorption of vitamin B12. Despite its association with several autoimmune disorders, few observations have reported an association with autoimmune hemolytic anemia (AIHA). We report a case of Biermer's disease associated with AIHA in a patient of 66 years old.

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

    PubMed Central

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

    1999-01-01

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

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

    PubMed

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

    1999-03-01

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

  5. Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice

    PubMed Central

    Husain, Mainul; Wu, Dongmei; Saber, Anne T.; Decan, Nathalie; Jacobsen, Nicklas R.; Williams, Andrew; Yauk, Carole L.; Wallin, Hakan; Vogel, Ulla; Halappanavar, Sabina

    2015-01-01

    Abstract An estimated 1% or less of nanoparticles (NPs) deposited in the lungs translocate to systemic circulation and enter other organs; however, this estimation may not be accurate given the low sensitivity of existing in vivo NP detection methods. Moreover, the biological effects of such low levels of translocation are unclear. We employed a nano-scale hyperspectral microscope to spatially observe and spectrally profile NPs in tissues and blood following pulmonary deposition in mice. In addition, we characterized effects occurring in blood, liver and heart at the mRNA and protein level following translocation from the lungs. Adult female C57BL/6 mice were exposed via intratracheal instillation to 18 or 162 µg of industrially relevant titanium dioxide nanoparticles (nano-TiO2) alongside vehicle controls. Using the nano-scale hyperspectral microscope, translocation to heart and liver was confirmed at both doses, and to blood at the highest dose, in mice analyzed 24 h post-exposure. Global gene expression profiling and ELISA analysis revealed activation of complement cascade and inflammatory processes in heart and specific activation of complement factor 3 in blood, suggesting activation of an early innate immune response essential for particle opsonisation and clearance. The liver showed a subtle response with changes in the expression of genes associated with acute phase response. This study characterizes the subtle systemic effects that occur in liver and heart tissues following pulmonary exposure and low levels of translocation of nano-TiO2 from lungs. PMID:25993494

  6. Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis.

    PubMed

    Ansar, Waliza; Mukhopadhyay, Sumi; Habib, S K Hasan; Basu, Shyamasree; Saha, Bibhuti; Sen, Asish Kumar; Mandal, C N; Mandal, Chitra

    2009-12-01

    Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7-8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (10(4)-10(5) M(-1)) and number of binding sites (10(6)-10(7)sites/erythrocyte) for CRP on patients' erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10 microg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two diseases.

  7. A species-specific nucleotide sequence of Mycobacterium tuberculosis encodes a protein that exhibits hemolytic activity when expressed in Escherichia coli.

    PubMed Central

    Leão, S C; Rocha, C L; Murillo, L A; Parra, C A; Patarroyo, M E

    1995-01-01

    Species-specific proteins may be implicated in the unique pathogenic mechanisms characteristic of Mycobacterium tuberculosis. In previous studies, a 3.0-kb species-specific DNA fragment of M. tuberculosis was identified (C. A. Parra, L. P. Londoño, P. del Portillo, and M. E. Patarroyo, Immun. 59:3411-3417, 1991). The nucleotide sequence of this 3.0-kb fragment has been obtained. This sequence was shown to contain two open reading frames (ORFs) whose putative gene products share 68.9% identity between each other. The major ORF shows 57.8% similarity with PLC-N and 53.2% similarity with PLC-H, two phospholipase C enzymes from Pseudomonas aeruginosa. The major ORF was amplified by PCR and cloned into the pGEX-5T expression vector. Cell extracts of Escherichia coli overexpressing this glutathione S-transferase fusion protein were shown to produce beta-hemolysis suggestive of phospholipase activity. Since phospholipase C enzymes have been reported as virulence factors of P. aeruginosa and also of the intracellular pathogen Listeria monocytogenes, it is possible that the proteins identified in this study could also play a role in sustaining tuberculosis infection in humans. PMID:7591062

  8. Serum complement and immunoconglutinin in malnutrition.

    PubMed Central

    Chandra, R K

    1975-01-01

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

  9. The mechanisms of complement activation in normal bovine serum and normal horse serum against Yersinia enterocolitica O:9 strains with different outer membrane proteins content.

    PubMed

    Miętka, K; Brzostek, K; Guz-Regner, K; Bugla-Płoskońska, G

    2016-01-01

    Yersinia enterocolitica is a common zoonotic pathogen and facultative intracellular bacterium which can survive within blood cells. Cattle and horses are considered a reservoir of Y. enterocolitica which often causes several serious syndromes associated with yersiniosis such as abortions, premature births or infertility. The aim of our investigation was to determine the vitality of Y. enterocolitica O:9 strains (Ye9) in bovine and horse sera (NBS and NHrS) and explain the role of outer membrane proteins (OMPs) in serum resistance of these bacteria. Our previous studies demonstrated moderate human serum (NHS) resistance of the wild type Ye9 strain, whereas mutants lacking YadA, Ail or OmpC remained sensitive to the bactericidal activity of NHS. The present study showed that the wild type of Ye9 strain was resistant to the bactericidal activity of both NHrS and NBS, while Ye9 mutants lacking the YadA, Ail and OmpC proteins were sensitive to NHrS and NBS as well as to NHS. The mechanisms of complement activation against Ye9 strains lacking Ail and YadA were distinguished, i.e. activation of the classical/lectin pathways decisive in the bactericidal mechanism of complement activation of NBS, parallel activation of the classical/lectin and alternative pathways of NHrS. In this research the mechanism of independent activation of the classical/lectin or the alternative pathway of NBS and NHrS against Ye9 lacking OmpC porin was also established. The results indicate that serum resistance of Ye9 is multifactorial, in which extracellular structures, i.e. outer membrane proteins (OMPs) such as Ail, OmpC or YadA, play the main role.

  10. Effects of amino acid mutations in the pore-forming domain of the hemolytic lectin CEL-III.

    PubMed

    Nagao, Tomonao; Masaki, Risa; Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2016-10-01

    The hemolytic lectin CEL-III forms transmembrane pores in the membranes of target cells. A study on the effect of site-directed mutation at Lys405 in domain 3 of CEL-III indicated that replacements of this residue by relatively smaller residues lead to a marked increase in hemolytic activity, suggesting that moderately destabilizing domain 3 facilitates formation of transmembrane pores through conformational changes.

  11. A high-resolution map of the regulator of the complement activation gene cluster on 1q32 that integrates new genes and markers.

    PubMed

    Heine-Suñer, D; Díaz-Guillén, M A; de Villena, F P; Robledo, M; Benítez, J; Rodríguez de Córdoba, S

    1997-01-01

    Sixteen microsatellite markers, including two described here, were used to construct a high-resolution map of the 1q32 region encompassing the regulator of the complement activation (RCA) gene cluster. The RCA genes are a group of related genes coding for plasma and membrane associated proteins that collectively control activation of the complement component C3. We provide here the location of two new genes within the RCA gene cluster. These genes are PFKFB2 that maps 15 kilobases (kb) upstream of the C4BPB gene, and a gene located 4 kb downstream of C4BPA, which seems to code for the 72 000 Mr component of the signal recognition particle (SRP72). Neither of these two genes is related structurally or functionally to the RCA genes. In addition, our map shows the centromere-telomere orientation of the C4BPB/MCP linkage group, which is: centromere-PFKFB2-C4BPB-C4BPA-SRP72-C4BPAL1++ +-C4BPAL2-telomere, and outlines an interval with a significant female-male recombination difference which suggests the presence of a female-specific hotspot(s) of recombination.

  12. Complement-mediated killing of Vibrio species by the humoral fluids of amphioxus Branchiostoma belcheri: implications for a dual role of O-antigens in the resistance to bactericidal activity.

    PubMed

    Li, Zhimin; Zhang, Shicui; Wang, Changfa; Pang, Qiuxiang

    2008-02-01

    The functional properties of complement in invertebrate deuterostomes are rather ill-defined. Here we showed that the humoral fluids from amphioxus Branchiostoma belcheri were capable of causing lysis of some Vibrio species including Vibrio alginolyticus HW284, Vibrio parahaemolyticus HW458 and Vibrio harvey SF-1, the first such data in the invertebrate deuterostomes. The fluid bacteriolytic activity was abolished by pre-incubation with heat-inactivated rabbit anti-human C3 serum, heating at 45 degrees C for 30 min, and repeated thawing and freezing. Additionally, the bacteriolytic activity was Mg(2+)-dependent and Ca(2+)-independent, and selective activation of the alternative pathway by zymosan A induced a loss of bacteriolytic activity. This strongly suggests that activation of the alternative complement pathway is responsible for the fluid bacteriolytic activity. It was also shown that some Vibrio species like Vibrio cincinnatiensis HW287 appeared resistant to the complement-mediated lysis. The LPS profiling revealed that the fluid-resistant V. cincinnatiensis HW287 had an LPS profile with a ladder of both high-molecular-weight (HMW) and low-molecular-weight (LMW) O-antigen bands, whereas the fluid-sensitive V. alginolyticus HW284 had few HMW O-antigen bands, suggesting a positive correlation between O-antigen size and humoral fluid resistance. Moreover, complement consumption assays demonstrated that both V. alginolyticus HW284 and V. cincinnatiensis HW287 consumed complement, with the former consuming significantly higher complement than the latter. Overall, it is suggested that HMW O-antigens may protect the fluid-resistant Vibrio species by a dual act of avoiding initiating complement activation as well as sterically hindering complement from gaining access to and damaging the cell membrane.

  13. Infectious diseases associated with complement deficiencies.

    PubMed Central

    Figueroa, J E; Densen, P

    1991-01-01

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

  14. Case of cytomegalovirus-associated direct anti-globulin test-negative autoimmune hemolytic anemia.

    PubMed

    Kaneko, Saeko; Sato, Masanori; Sasaki, Goro; Eguchi, Hiroyuki; Oishi, Tsutomu; Kamesaki, Toyomi; Kawaguchi, Hiroyuki

    2013-12-01

    A 1-year-old boy developed autoimmune hemolytic anemia after a negative direct anti-globulin test. The concentration of erythrocyte membrane-associated immunoglobulin G, determined using an immunoradiometric assay, correlated with disease activity. He was positive for cytomegalovirus (CMV) both serologically and by quantitative real-time polymerase chain reaction, indicating that his autoimmune hemolytic anemia was directly caused by CMV infection. Since anti-CMV immunoglobulin G was not absorbed by the patient's erythrocytes, cross-reaction between erythrocyte antigens and CMV was not likely a causative factor for hemolysis.

  15. Influence of the electric charge of the antigen and the immune complex (IC) lattice on the IC activation of human complement

    PubMed Central

    Michelin, M A; Crott, L S P; Assis-pandochi, A I; Coimbra, T M; Teixeira, J E; Barbosa, J E

    2002-01-01

    In order to understand the mechanism of complement (C) activation by immune complexes (ICs), the anti-complementary effect of ICs containing cationized antigens was compared in vitro to that using ICs formed by native antigens. ICs were prepared with affinity-purified rabbit polyclonal IgG antibovine serum albumin (BSA) antibody and either native BSA (isoelectric point 4.2) or BSA rendered cationic by treatment with ethylenediamine (isoelectric point 9.4). Native and cationized antigens were characterized by isoelectric focusing. ICs containing anti-BSA IgG or F(ab′)2, formed either at equivalence or in excess of native or cationized antigen, were submitted to ultracentrifugation in a sucrose gradient for mesh size determination. The anti-complementary effect of ICs was evaluated by kinetic determination of haemolytic activity of human serum on haemolysin-sensitized sheep red blood cells. In conditions of antigen excess, the ICs formed by cationized BSA were significantly more efficient in activating human complement than those formed by native antigen. This higher activity was dependent on cationized antigen complexed with complete antibody molecules, as non-complexed cationized BSA or ICs prepared with F(ab′)2 fragments were inactive under the same experimental conditions. Furthermore, this difference did not depend on the mesh size of the immune complexes. Our results suggest that the balance between antigen, antibody and C may be of importance in vivo for the onset and course of infections and other pathological processes involving IC formation. ICs containing cationized antigens should be proven of value in experimental models for studies on the regulation of C activation. PMID:12084047

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

    PubMed Central

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

    1991-01-01

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

  17. Complement membrane attack complexes activate noncanonical NF-κB by forming an Akt+NIK+ signalosome on Rab5+ endosomes

    PubMed Central

    Jane-wit, Dan; Surovtseva, Yulia V.; Qin, Lingfeng; Li, Guangxin; Liu, Rebecca; Clark, Pamela; Manes, Thomas D.; Wang, Chen; Kashgarian, Michael; Kirkiles-Smith, Nancy C.; Tellides, George; Pober, Jordan S.

    2015-01-01

    Complement membrane attack complexes (MACs) promote inflammatory functions in endothelial cells (ECs) by stabilizing NF-κB–inducing kinase (NIK) and activating noncanonical NF-κB signaling. Here we report a novel endosome-based signaling complex induced by MACs to stabilize NIK. We found that, in contrast to cytokine-mediated activation, NIK stabilization by MACs did not involve cIAP2 or TRAF3. Informed by a genome-wide siRNA screen, instead this response required internalization of MACs in a clathrin-, AP2-, and dynamin-dependent manner into Rab5+endosomes, which recruited activated Akt, stabilized NIK, and led to phosphorylation of IκB kinase (IKK)-α. Active Rab5 was required for recruitment of activated Akt to MAC+ endosomes, but not for MAC internalization or for Akt activation. Consistent with these in vitro observations, MAC internalization occurred in human coronary ECs in vivo and was similarly required for NIK stabilization and EC activation. We conclude that MACs activate noncanonical NF-κB by forming a novel Akt+NIK+ signalosome on Rab5+ endosomes. PMID:26195760

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

    PubMed Central

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

    2014-01-01

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

  19. Immunization with LytB protein of Streptococcus pneumoniae activates complement-mediated phagocytosis and induces protection against pneumonia and sepsis.

    PubMed

    Corsini, Bruno; Aguinagalde, Leire; Ruiz, Susana; Domenech, Mirian; Antequera, María Luisa; Fenoll, Asunción; García, Pedro; García, Ernesto; Yuste, Jose

    2016-12-07

    The cell wall glucosaminidase LytB of Streptococcus pneumoniae is a surface exposed protein involved in daughter cell separation, biofilm formation and contributes to different aspects of the pathogenesis process. In this study we have characterized the antibody responses after immunization of mice with LytB in the presence of alhydrogel as an adjuvant. Enzyme-linked immunosorbent assays measuring different subclasses of immunoglobulin G, demonstrated that the antibody responses to LytB were predominantly IgG1 and IgG2b, followed by IgG3 and IgG2a subclasses. Complement-mediated immunity against two different pneumococcal serotypes was investigated using sera from immunized mice. Immunization with LytB increased the recognition of S. pneumoniae by complement components C1q and C3b demonstrating that anti-LytB antibodies trigger activation of the classical pathway. Phagocytosis assays showed that serum containing antibodies to LytB stimulates neutrophil-mediated phagocytosis against S. pneumoniae. Animal models of infection including invasive pneumonia and sepsis were performed with two different clinical isolates. Vaccination with LytB increased bacterial clearance and induced protection demonstrating that LytB might be a good candidate to be considered in a future protein-based vaccine against S. pneumoniae.

  20. Role of complement in xenotransplantation.

    PubMed

    Mollnes, Tom Eirik; Fiane, A E

    2002-01-01

    The xenotransplantation research is driven by the increasing gap between the number of patients with end-stage organ failure on waiting lists for transplantation and the supply of allografts. The lack of success in developing suitable artificial organs for permanent treatment of organ failure has further strengthened the need for xenotransplantation research. Pigs are now generally accepted to be the source animal of choice. Transplantation of pig organs to humans faces several barriers which have to be overcome before it comes to clinical application: (1) anatomical and physiological conditions; (2) immunological rejection mechanisms; (3) molecular compatibility between signal molecules of the two species; (4) risk of transmission of microorganisms, particularly pig endogenous retroviruses; and (5) legal and ethical aspects both with respect to the animal and the recipient. Here we will focus on the role of the complement system in the rejection of immediately vascularized pig-to-primate xenografts. The hyperacute rejection occurring within minutes after transplantation is mediated by binding of natural antibodies to the Galalpha(l-3)Gal epitope on the endothelial cells with subsequent complement activation. Whereas inhibition of complement activation protects against hyperacute rejection, the role of complement in the later rejection phases is less clarified.

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

    PubMed

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

    2000-04-14

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

  2. Fatal autoimmune hemolytic anemia due to immunoglobulin g autoantibody exacerbated by epstein-barr virus.

    PubMed

    Fadeyi, Emmanuel A; Simmons, Julie H; Jones, Mary Rose; Palavecino, Elizabeth L; Pomper, Gregory J

    2015-01-01

    Most cases of autoimmune hemolytic anemia (AIHA) are caused by the production of an autoantibody that targets determinants on red blood cells (RBCs). This autoantibody can be immunoglobulin (Ig) G, IgM, or IgA. Some autoantibodies react optimally at 0° to 4°C (ie, cold agglutinin) and usually are clinically insignificant. High-titer cold agglutinins are associated with IgM autoantibody and complement fixation induced by infectious agents, including the Epstein-Barr virus (EBV). This case report describes a 31-year-old man who had jaundice, a hemoglobin of 6.0 gdL, and was diagnosed with a hemolytic crisis of AIHA. He received a total of 11 RBC transfusions during a 15-hour period without sustained response and later died. The direct antiglobulin test results for this patient were positive, whereas the cold-agglutinin-testing results were negative. We detected EBV DNA in blood via polymerase chain reaction (PCR). We report a rare case of AIHA associated with an IgG autoantibody and exacerbated by EBV infection, causing a fatal hemolytic anemia.

  3. In vitro inhibition of the classical pathway of human complement by a natural microbial product, colistin sulphate.

    PubMed

    Asghar, S S; de Koster, A; van der Helm, H J

    1986-09-01

    Colistin sulphate was found to be an inhibitor of the classical pathway of the complement system. The main sites of inhibition were the interaction of EAC14 with C2 and EAC142 with C3. It also inhibited EAC14 formation from EA and C2-deficient serum, EAC1-7 formation from EAC1-3, C5, C6 and C7 and the interaction of EAC1-7 with C8 and C9, though less efficiently. It did not inhibit formation of C3/C5 convertase of the alternative pathway. The inhibition of the classical pathway was reversible since hemolytic activity was completely restored after dialysis.

  4. Anti-Mouse Properdin TSR 5/6 Monoclonal Antibodies Block Complement Alternative Pathway-dependent Pathogenesis

    PubMed Central

    Bertram, Paula; Akk, Antonina M.; Zhou, Hui-fang; Mitchell, Lynne M.; Pham, Christine T.N.

    2015-01-01

    The complement alternative pathway (AP) is a major contributor to a broad and growing spectrum of diseases that includes age-related macular degeneration, atypical hemolytic uremic syndrome, and preeclampsia. As a result, there is much interest in the therapeutic disruption of AP activity. Properdin, the only positive regulator of the AP, is a particularly promising AP target. Several issues need to be clarified before the potential for properdin-directed therapy can be realized. In this report we use a portion of the mouse properdin protein, expressed in a bacterial system, to raise rabbit polyclonal and hamster monoclonal antibodies that block properdin-dependent pathogenesis. These antibodies, when employed with AP-dependent mouse disease models, can help evaluate the feasibility of properdin-directed therapy. PMID:25723276

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

    DTIC Science & Technology

    1977-01-12

    alternative activation pathway appears possible which involves the reaction of certain polymeric m i- tiators (inulin, endotoxin, or zyinosan) with non ...displayed the ability to consume C’3, and anti-C’2 did not interfere with this activity. These findings suggested that there was an alternate ( non ...included using 5 g dextrose per liter veronal buffered diluent (VBD) . VBD prepared with the stock buffer containing Mg~~ and Ca++ was designated VBD

  6. Anticomplement activity of polyacetylenes from leaves of Dendropanax morbifera Leveille.

    PubMed

    Chung, Ill-Min; Song, Hong-Keun; Kim, Sun-Jin; Moon, Hyung-In

    2011-05-01

    The present study evaluated the anticomplement effect of polyacetylenes from Dendropanax morbifera (Araliaceae) in the classical pathway complement system. The leaves of D. morbifera were evaluated with regard to its anticomplement activity, and its active principles identified following activity-guided isolation. An aqueous CCl(4) fraction of the leaves of D. morbifera exhibited significant anticomplement activity on the classical pathway complement system, which was expressed as total hemolytic activity. Three polyacetylenes isolated from the leaves of D. morbifera, namely (3S)-falcarinol (1), (3S,8S)-falcarindiol (2) and (3S)-diynene (3). Compounds 1, 2 and 3 showed inhibitory activity against complement system with 50% inhibitory concentrations (IC(50)) values of 87.3 µM, 15.2 µM and 39.8 µM. Among the compounds tested, 2 showed the most potent anticomplement activity (IC(50), 15.2 µM).

  7. The N- and C-terminal autolytic fragments of CAPN3/p94/calpain-3 restore proteolytic activity by intermolecular complementation

    PubMed Central

    Ono, Yasuko; Shindo, Mayumi; Doi, Naoko; Kitamura, Fujiko; Gregorio, Carol C.

    2014-01-01

    CAPN3/p94/calpain-3, a calpain protease family member predominantly expressed in skeletal muscle, possesses unusually rapid and exhaustive autolytic activity. Mutations in the human CAPN3 gene impairing its protease functions cause limb-girdle muscular dystrophy type 2A (LGMD2A); yet, the connection between CAPN3’s autolytic activity and the enzyme’s function in vivo remain unclear. Here, we demonstrated that CAPN3 protease activity was reconstituted by intermolecular complementation (iMOC) between its two autolytic fragments. Furthermore, the activity of full-length CAPN3 active-site mutants was surprisingly rescued through iMOC with autolytic fragments containing WT amino acid sequences. These results provide evidence that WT CAPN3 can be formed by the iMOC of two different complementary CAPN3 mutants. The finding of iMOC-mediated restoration of calpain activity indicates a novel mechanism for the genotype–phenotype links in LGMD2A. PMID:25512505

  8. Complement related kidney diseases: Recurrence after transplantation.

    PubMed

    Salvadori, Maurizio; Bertoni, Elisabetta

    2016-12-24

    The recurrence of renal disease after renal transplantation is becoming one of the main causes of graft loss after kidney transplantation. This principally concerns some of the original diseases as the atypical hemolytic uremic syndrome (HUS), the membranoproliferative glomerulonephritis (MPGN), in particular the MPGN now called C3 glomerulopathy. Both this groups of renal diseases are characterized by congenital (genetic) or acquired (auto-antibodies) modifications of the alternative pathway of complement. These abnormalities often remain after transplantation because they are constitutional and poorly influenced by the immunosuppression. This fact justifies the high recurrence rate of these diseases. Early diagnosis of recurrence is essential for an optimal therapeutically approach, whenever possible. Patients affected by end stage renal disease due to C3 glomerulopathies or to atypical HUS, may be transplanted with extreme caution. Living donor donation from relatives is not recommended because members of the same family may be affected by the same gene mutation. Different therapeutically approaches have been attempted either for recurrence prevention and treatment. The most promising approach is represented by complement inhibitors. Eculizumab, a monoclonal antibody against C5 convertase is the most promising drug, even if to date is not known how long the therapy should be continued and which are the best dosing. These facts face the high costs of the treatment. Eculizumab resistant patients have been described. They could benefit by a C3 convertase inhibitor, but this class of drugs is by now the object of randomized controlled trials.

  9. Complement related kidney diseases: Recurrence after transplantation

    PubMed Central

    Salvadori, Maurizio; Bertoni, Elisabetta

    2016-01-01

    The recurrence of renal disease after renal transplantation is becoming one of the main causes of graft loss after kidney transplantation. This principally concerns some of the original diseases as the atypical hemolytic uremic syndrome (HUS), the membranoproliferative glomerulonephritis (MPGN), in particular the MPGN now called C3 glomerulopathy. Both this groups of renal diseases are characterized by congenital (genetic) or acquired (auto-antibodies) modifications of the alternative pathway of complement. These abnormalities often remain after transplantation because they are constitutional and poorly influenced by the immunosuppression. This fact justifies the high recurrence rate of these diseases. Early diagnosis of recurrence is essential for an optimal therapeutically approach, whenever possible. Patients affected by end stage renal disease due to C3 glomerulopathies or to atypical HUS, may be transplanted with extreme caution. Living donor donation from relatives is not recommended because members of the same family may be affected by the same gene mutation. Different therapeutically approaches have been attempted either for recurrence prevention and treatment. The most promising approach is represented by complement inhibitors. Eculizumab, a monoclonal antibody against C5 convertase is the most promising drug, even if to date is not known how long the therapy should be continued and which are the best dosing. These facts face the high costs of the treatment. Eculizumab resistant patients have been described. They could benefit by a C3 convertase inhibitor, but this class of drugs is by now the object of randomized controlled trials. PMID:28058212

  10. Cobalt-doped nanohydroxyapatite: synthesis, characterization, antimicrobial and hemolytic studies

    NASA Astrophysics Data System (ADS)

    Tank, Kashmira P.; Chudasama, Kiran S.; Thaker, Vrinda S.; Joshi, Mihir J.

    2013-05-01

    Hydroxyapatite (Ca10(PO4)6(OH)2; HAP) is a major mineral component of the calcified tissues, and it has various applications in medicine and dentistry. In the present investigation, cobalt-doped hydroxyapatite (Co-HAP) nanoparticles were synthesized by surfactant-mediated approach and characterized by different techniques. The EDAX was carried out to estimate the amount of doping in Co-HAP. The transmission electron microscopy result suggested the transformation of morphology from needle shaped to spherical type on increasing the doping concentration. The powder XRD study indicated the formation of a new phase of brushite for higher concentration of cobalt. The average particle size and strain were calculated using Williamson-Hall analysis. The average particle size was found to be 30-60 nm. The FTIR study confirmed the presence of various functional groups in the samples. The antimicrobial activity was evaluated against four organisms Pseudomonas aeruginosa and Shigella flexneri as Gram negative as well as Micrococcus luteus and Staphylococcus aureus as Gram positive. The hemolytic test result suggested that all samples were non-hemolytic. The photoluminescence study was carried out to identify its possible applicability as a fluorescent probe.

  11. The C5 convertase is not required for activation of the terminal complement pathway in murine experimental cerebral malaria.

    PubMed

    Ramos, Theresa N; Darley, Meghan M; Weckbach, Sebastian; Stahel, Philip F; Tomlinson, Stephen; Barnum, Scott R

    2012-07-13

    Cerebral malaria (CM) is the most severe manifestation of clinical malaria syndromes and has a high fatality rate especially in the developing world. Recent studies demonstrated that C5(-/-) mice are resistant to experimental CM (ECM) and that protection was due to the inability to form the membrane attack complex. Unexpectedly, we observed that C4(-/-) and factor B(-/-) mice were fully susceptible to disease, indicating that activation of the classical or alternative pathways is not required for ECM. C3(-/-) mice were also susceptible to ECM, indicating that the canonical C5 convertases are not required for ECM development and progression. Abrogation of ECM by treatment with anti-C9 antibody and detection of C5a in serum of C3(-/-) mice confirmed that C5 activation occurs in ECM independent of C5 convertases. Our data indicate that activation of C5 in ECM likely occurs via coagulation enzymes of the extrinsic protease pathway.

  12. T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4⁺ T cells.

    PubMed

    Arbore, Giuseppina; West, Erin E; Spolski, Rosanne; Robertson, Avril A B; Klos, Andreas; Rheinheimer, Claudia; Dutow, Pavel; Woodruff, Trent M; Yu, Zu Xi; O'Neill, Luke A; Coll, Rebecca C; Sher, Alan; Leonard, Warren J; Köhl, Jörg; Monk, Pete; Cooper, Matthew A; Arno, Matthew; Afzali, Behdad; Lachmann, Helen J; Cope, Andrew P; Mayer-Barber, Katrin D; Kemper, Claudia

    2016-06-17

    The NLRP3 inflammasome controls interleukin-1β maturation in antigen-presenting cells, but a direct role for NLRP3 in human adaptive immune cells has not been described. We found that the NLRP3 inflammasome assembles in human CD4(+) T cells and initiates caspase-1-dependent interleukin-1β secretion, thereby promoting interferon-γ production and T helper 1 (T(H)1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in T cells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to "innate immune cells" but is an integral component of normal adaptive T(H)1 responses.

  13. Interplay between base excision repair activity and toxicity of 3-methyladenine DNA glycosylases in an E. coli complementation system.

    PubMed

    Troll, Christopher J; Adhikary, Suraj; Cueff, Marie; Mitra, Ileena; Eichman, Brandt F; Camps, Manel

    2014-01-01

    DNA glycosylases carry out the first step of base excision repair by removing damaged bases from DNA. The N3-methyladenine (3MeA) DNA glycosylases specialize in alkylation repair and are either constitutively expressed or induced by exposure to alkylating agents. To study the functional and evolutionary significance of constitutive versus inducible expression, we expressed two closely related yeast 3MeA DNA glycosylases - inducible Saccharomyces cerevisiae MAG and constitutive S. pombe Mag1 - in a glycosylase-deficient Escherichia coli strain. In both cases, constitutive expression conferred resistance to alkylating agent exposure. However, in the absence of exogenous alkylation, high levels of expression of both glycosylases were deleterious. We attribute this toxicity to excessive glycosylase activity, since suppressing spMag1 expression correlated with improved growth in liquid culture, and spMag1 mutants exhibiting decreased glycosylase activity showed improved growth and viability. Selection of a random spMag1 mutant library for increased survival in the presence of exogenous alkylation resulted in the selection of hypomorphic mutants, providing evidence for the presence of a genetic barrier to the evolution of enhanced glycosylase activity when constitutively expressed. We also show that low levels of 3MeA glycosylase expression improve fitness in our glycosylase-deficient host, implying that 3MeA glycosylase activity is likely necessary for repair of endogenous lesions. These findings suggest that 3MeA glycosylase activity is evolutionarily conserved for repair of endogenously produced alkyl lesions, and that inducible expression represents a common strategy to rectify deleterious effects of excessive 3MeA activity in the absence of exogenous alkylation challenge.

  14. Exploiting the nephrotoxic effects of venom from the sea anemone, Phyllodiscus semoni, to create a hemolytic uremic syndrome model in the rat.

    PubMed

    Mizuno, Masashi; Ito, Yasuhiko; Morgan, B Paul

    2012-07-01

    In the natural world, there are many creatures with venoms that have interesting and varied activities. Although the sea anemone, a member of the phylum Coelenterata, has venom that it uses to capture and immobilise small fishes and shrimp and for protection from predators, most sea anemones are harmless to man. However, a few species are highly toxic; some have venoms containing neurotoxins, recently suggested as potential immune-modulators for therapeutic application in immune diseases. Phyllodiscus semoni is a highly toxic sea anemone; the venom has multiple effects, including lethality, hemolysis and renal injuries. We previously reported that venom extracted from Phyllodiscus semoni induced acute glomerular endothelial injuries in rats resembling hemolytic uremic syndrome (HUS), accompanied with complement dysregulation in glomeruli and suggested that the model might be useful for analyses of pathology and development of therapeutic approaches in HUS. In this mini-review, we describe in detail the venom-induced acute renal injuries in rat and summarize how the venom of Phyllodiscus semoni could have potential as a tool for analyses of complement activation and therapeutic interventions in HUS.

  15. Exploiting the Nephrotoxic Effects of Venom from the Sea Anemone, Phyllodiscus semoni, to Create a Hemolytic Uremic Syndrome Model in the Rat

    PubMed Central

    Mizuno, Masashi; Ito, Yasuhiko; Morgan, B. Paul

    2012-01-01

    In the natural world, there are many creatures with venoms that have interesting and varied activities. Although the sea anemone, a member of the phylum Coelenterata, has venom that it uses to capture and immobilise small fishes and shrimp and for protection from predators, most sea anemones are harmless to man. However, a few species are highly toxic; some have venoms containing neurotoxins, recently suggested as potential immune-modulators for therapeutic application in immune diseases. Phyllodiscus semoni is a highly toxic sea anemone; the venom has multiple effects, including lethality, hemolysis and renal injuries. We previously reported that venom extracted from Phyllodiscus semoni induced acute glomerular endothelial injuries in rats resembling hemolytic uremic syndrome (HUS), accompanied with complement dysregulation in glomeruli and suggested that the model might be useful for analyses of pathology and development of therapeutic approaches in HUS. In this mini-review, we describe in detail the venom-induced acute renal injuries in rat and summarize how the venom of Phyllodiscus semoni could have potential as a tool for analyses of complement activation and therapeutic interventions in HUS. PMID:22851928

  16. Efficacy and safety of eculizumab in atypical hemolytic uremic syndrome from 2-year extensions of phase 2 studies.

    PubMed

    Licht, Christoph; Greenbaum, Larry A; Muus, Petra; Babu, Sunil; Bedrosian, Camille L; Cohen, David J; Delmas, Yahsou; Douglas, Kenneth; Furman, Richard R; Gaber, Osama A; Goodship, Timothy; Herthelius, Maria; Hourmant, Maryvonne; Legendre, Christophe M; Remuzzi, Giuseppe; Sheerin, Neil; Trivelli, Antonella; Loirat, Chantal

    2015-05-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare, possibly life-threatening disease characterized by platelet activation, hemolysis and thrombotic microangiopathy (TMA) leading to renal and other end-organ damage. We originally conducted two phase 2 studies (26 weeks and 1 year) evaluating eculizumab, a terminal complement inhibitor, in patients with progressing TMA (trial 1) and those with long duration of aHUS and chronic kidney disease (trial 2). The current analysis assessed outcomes after 2 years (median eculizumab exposure 100 and 114 weeks, respectively). At all scheduled time points, eculizumab inhibited terminal complement activity. In trial 1 with 17 patients, the platelet count was significantly improved from baseline, and hematologic normalization was achieved in 13 patients at week 26, and in 15 patients at both 1 and 2 years. The estimated glomerular filtration rate (eGFR) was significantly improved compared with baseline and year 1. In trial 2 with 20 patients, TMA event-free status was achieved by 16 patients at week 26, 17 patients at year 1, and 19 patients at year 2. Criteria for hematologic normalization were met by 18 patients at each time point. Improvement of 15 ml/min per 1.73 m(2) or more in eGFR was achieved by 1 patient at week 26, 3 patients at 1 year, and 8 patients at 2 years. The mean change in eGFR was not significant compared with baseline, week 26, or year 1. Eculizumab was well tolerated, with no new safety concerns or meningococcal infections. Thus, a 2-year analysis found that the earlier clinical benefits achieved by eculizumab treatment of aHUS were maintained at 2 years of follow-up.

  17. The C5 Convertase Is Not Required for Activation of the Terminal Complement Pathway in Murine Experimental Cerebral Malaria*

    PubMed Central

    Ramos, Theresa N.; Darley, Meghan M.; Weckbach, Sebastian; Stahel, Philip F.; Tomlinson, Stephen; Barnum, Scott R.

    2012-01-01

    Cerebral malaria (CM) is the most severe manifestation of clinical malaria syndromes and has a high fatality rate especially in the developing world. Recent studies demonstrated that C5−/− mice are resistant to experimental CM (ECM) and that protection was due to the inability to form the membrane attack complex. Unexpectedly, we observed that C4−/− and factor B−/− mice were fully susceptible to disease, indicating that activation of the classical or alternative pathways is not required for ECM. C3−/− mice were also susceptible to ECM, indicating that the canonical C5 convertases are not required for ECM development and progression. Abrogation of ECM by treatment with anti-C9 antibody and detection of C5a in serum of C3−/− mice confirmed that C5 activation occurs in ECM independent of C5 convertases. Our data indicate that activation of C5 in ECM likely occurs via coagulation enzymes of the extrinsic protease pathway. PMID:22689574

  18. Correlation among Antioxidant, Antimicrobial, Hemolytic, and Antiproliferative Properties of Leiothrix spiralis Leaves Extract

    PubMed Central

    de Freitas Araújo, Marcelo Gonzaga; Hilário, Felipe; Vilegas, Wagner; dos Santos, Lourdes Campaner; Brunetti, Iguatemy Lourenço; Sotomayor, Claudia Elena; Bauab, Tais Maria

    2012-01-01

    The biological activities of a plant extract depend on a complex sum of individual properties including the antioxidant activity. Several biological activities protect against the harmful action of reactive oxygen species (ROS), and here we focused our attention on the relationship between the biological activities tested and the antioxidant properties. In this study, the total flavonoid content as well as the antioxidant, antimicrobial, hemolytic and cytotoxicity activities of the methanolic extract of Leitothrix spiralis leaves were evaluated. The extract showed a total flavonoid content of 19.26% and the chemical characterization by HPLC-PAD confirmed the presence of flavonoids as the major secondary metabolite compounds. Significant antioxidant activity (IC50 = 1.743 μg/mL ± 0.063) was demonstrated and was effective against Gram-negative organisms and all Candida strains tested, and showed an ability to inhibit hyphal formation. Non-hemolytic and antiproliferative activity could be demonstrated. PMID:22942765

  19. IgG autoantibodies to C1q do not detectably influence complement activation in vivo and in vitro in systemic lupus erythematosus.

    PubMed

    Siegert, C E; Daha, M R; Lobatto, S; van der Voort, E A; Breedveld, F C

    1992-01-01

    The influence of IgG antibodies to C1q (C1qAb) on activation of the classical pathway of the complement system was investigated in patients with systemic lupus erythematosus (SLE). In in vivo experiments, a prototype for immune complexes was administered intravenously to 14 patients and 9 healthy controls. Eight SLE patients had increased C1qAb titers. The increase of C3a levels, which was measured as a parameter of C1 activation, was significantly lower in SLE patients than in the healthy controls (p = 0.01). No correlation was found between C3a increases and C1qAb titers. In in vitro experiments the influence on C1 activation of monomeric IgG isolated from serum of 11 SLE patients, 7 of whom had increased C1qAb titers, was measured in a C4 consumption assay. The presence of C1qAb did not influence C4 consumption. The results demonstrate that C1qAb do not influence C1 activation by immune complexes in SLE patients.

  20. The role of complement in AMD.

    PubMed

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

    2010-01-01

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

  1. Familial atypical hemolytic uremic syndrome: a review of its genetic and clinical aspects.

    PubMed

    Bu, Fengxiao; Borsa, Nicolo; Gianluigi, Ardissino; Smith, Richard J H

    2012-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare renal disease (two per one million in the USA) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Both sporadic (80% of cases) and familial (20% of cases) forms are recognized. The study of familial aHUS has implicated genetic variation in multiple genes in the complement system in disease pathogenesis, helping to define the mechanism whereby complement dysregulation at the cell surface level leads to both sporadic and familial disease. This understanding has culminated in the use of Eculizumab as first-line therapy in disease treatment, significantly changing the care and prognosis of affected patients. However, even with this bright outlook, major challenges remain to understand the complexity of aHUS at the genetic level. It is possible that a more detailed picture of aHUS can be translated to an improved understanding of disease penetrance, which is highly variable, and response to therapy, both in the short and long terms.

  2. Clinical Applications of Hemolytic Markers in the Differential Diagnosis and Management of Hemolytic Anemia.

    PubMed

    Barcellini, W; Fattizzo, B

    2015-01-01

    Several hemolytic markers are available to guide the differential diagnosis and to monitor treatment of hemolytic conditions. They include increased reticulocytes, an indicator of marrow compensatory response, elevated lactate dehydrogenase, a marker of intravascular hemolysis, reduced haptoglobin, and unconjugated hyperbilirubinemia. The direct antiglobulin test is the cornerstone of autoimmune forms, and blood smear examination is fundamental in the diagnosis of congenital membrane defects and thrombotic microangiopathies. Marked increase of lactate dehydrogenase and hemosiderinuria are typical of intravascular hemolysis, as observed in paroxysmal nocturnal hemoglobinuria, and hyperferritinemia is associated with chronic hemolysis. Prosthetic valve replacement and stenting are also associated with intravascular and chronic hemolysis. Compensatory reticulocytosis may be inadequate/absent in case of marrow involvement, iron/vitamin deficiency, infections, or autoimmune reaction against bone marrow-precursors. Reticulocytopenia occurs in 20-40% of autoimmune hemolytic anemia cases and is a poor prognostic factor. Increased reticulocytes, lactate dehydrogenase, and bilirubin, as well as reduced haptoglobin, are observed in conditions other than hemolysis that may confound the clinical picture. Hemoglobin defines the clinical severity of hemolysis, and thrombocytopenia suggests a possible thrombotic microangiopathy or Evans' syndrome. A comprehensive clinical and laboratory evaluation is advisable for a correct diagnostic and therapeutic workup of the different hemolytic conditions.

  3. [Autoimmune hemolytic anemia associated with mesenteric teratoma].

    PubMed

    Ntanishyan, K I; Sabirov, K R; Shcherbakova, O V; Vybornykh, D E; Shupletsova, I A; Tsvetaeva, N V

    2017-01-01

    The paper describes a case of autoimmune hemolytic anemia (AIHA) in a 27-year-old woman whose examination revealed mesenteric teratoma. AIHA was characterized by a hypertensive crisis and a temporary response to corticosteroid therapy that was complicated by the development of somatogenic psychosis and discontinued. A relapse of hemolysis developed 6 months later. The patient underwent laparoscopic splenectomy and removal of mesenteric root teratoma. Immediately after surgery, a hematological response was obtained as relief of hemolysis and restoration of a normal hemoglobin level. There is a sustained remission of AIHA for the next 16 months.

  4. The Clinical Pictures of Autoimmune Hemolytic Anemia

    PubMed Central

    Packman, Charles H.

    2015-01-01

    Summary Autoimmune hemolytic anemia is characterized by shortened red blood cell survival and a positive Coombs test. The responsible autoantibodies may be either warm reactive or cold reactive. The rate of hemolysis and the severity of the anemia may vary from mild to severe and life-threatening. Diagnosis is made in the laboratory by the findings of anemia, reticulocytosis, a positive Coombs test, and specific serologic tests. The prognosis is generally good but renal failure and death sometimes occur, especially in cases mediated by drugs. PMID:26696800

  5. Diagnosis and classification of autoimmune hemolytic anemia.

    PubMed

    Bass, Garrett F; Tuscano, Emily T; Tuscano, Joseph M

    2014-01-01

    Uncompensated autoantibody-mediated red blood cell (RBC) consumption is the hallmark of autoimmune hemolytic anemia (AIHA). Classification of AIHA is pathophysiologically based and divides AIHA into warm, mixed or cold-reactive subtypes. This thermal-based classification is based on the optimal autoantibody-RBC reactivity temperatures. AIHA is further subcategorized into idiopathic and secondary with the later being associated with a number of underlying infectious, neoplastic and autoimmune disorders. In most cases AIHA is confirmed by a positive direct antiglobulin test (DAT). The standard therapeutic approaches to treatment of AIHA include corticosteroids, splenectomy, immunosuppressive agents and monoclonal antibodies.

  6. Complement associated pathogenic mechanisms in myasthenia gravis.

    PubMed

    Tüzün, Erdem; Christadoss, Premkumar

    2013-07-01

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

  7. Progress and Trends in Complement Therapeutics

    PubMed Central

    Ricklin, Daniel; Lambris, John D.

    2012-01-01

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

  8. Antiglycopeptide Mouse Monoclonal Antibody LpMab-21 Exerts Antitumor Activity Against Human Podoplanin Through Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

    PubMed

    Kato, Yukinari; Kunita, Akiko; Fukayama, Masashi; Abe, Shinji; Nishioka, Yasuhiko; Uchida, Hiroaki; Tahara, Hideaki; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Fujii, Yuki; Honma, Ryusuke; Takagi, Michiaki; Ogasawara, Satoshi; Murata, Takeshi; Kaneko, Mika K

    2017-02-01

    The interaction between podoplanin (PDPN) and C-type lectin-like receptor 2 (CLEC-2) is involved in tumor malignancy. We have established many monoclonal antibodies (mAbs) against human podoplanin using the cancer-specific mAb (CasMab) technology. LpMab-21, one of the mouse antipodoplanin mAbs, is of the IgG2a subclass, and its minimum epitope was determined to be Thr76-Arg79 of the human podoplanin. Importantly, sialic acid is linked to Thr76; therefore, LpMab-21 is an antiglycopeptide mAb (GpMab). In this study, we investigated whether LpMab-21 shows antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against human podoplanin-expressing cancer cell lines in vitro and also studied its antitumor activities using a xenograft model. LpMab-21 showed high ADCC and CDC activities against not only podoplanin-expressing Chinese hamster ovary cells but also LN319 glioblastoma cells and PC-10 lung cancer cells, both of which endogenously express podoplanin. Furthermore, LpMab-21 decreased tumor growth in vivo, indicating that LpMab-21 could be useful for antibody therapy against human podoplanin-expressing cancers.

  9. Complement and thrombosis in the antiphospholipid syndrome.

    PubMed

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

    2016-10-01

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

  10. Investigation of whether the acute hemolysis associated with Rho(D) immune globulin intravenous (human) administration for treatment of immune thrombocytopenic purpura is consistent with the acute hemolytic transfusion reaction model

    PubMed Central

    Gaines, Ann Reed; Lee-Stroka, Hallie; Byrne, Karen; Scott, Dorothy E.; Uhl, Lynne; Lazarus, Ellen; Stroncek, David F.

    2012-01-01

    BACKGROUND Immune thrombocytopenic purpura and secondary thrombocytopenia patients treated with Rho(D) immune globulin intravenous (human; anti-D IGIV) have experienced acute hemolysis, which is inconsistent with the typical presentation of extravascular hemolysis—the presumed mechanism of action of anti-D IGIV. Although the mechanism of anti-D-IGIV–associated acute hemolysis has not been established, the onset, signs/symptoms, and complications appear consistent with the intravascular hemolysis of acute hemolytic transfusion reactions (AHTRs). In transfusion medicine, the red blood cell (RBC) antigen-antibody incompatibility(-ies) that precipitate AHTRs can be detected in vitro with compatibility testing. Under the premise that anti-D-IGIV–associated acute hemolysis results from RBC antigen-antibody–mediated complement activation, this study evaluated whether the incompatibility(-ies) could be detected in vitro with a hemolysin assay, which would support the AHTR model as the hemolytic mechanism. STUDY DESIGN AND METHODS Seven anti-D IGIV lots were tested to determine the RBC antibody identities in those lots, including four lots that had been implicated in acute hemolytic episodes. Hemolysin assays were performed that tested each of 73 RBC specimens against each lot, including the RBCs of one patient who had experienced acute hemolysis after anti-D IGIV administration. RESULTS Only two anti-D IGIV lots contained RBC antibodies beyond those expected. No hemolysis endpoint was observed in any of the hemolysin assays. CONCLUSION Although the findings did not support the AHTR model, the results are reported to contribute knowledge about the mechanism of anti-D-IGIV–associated acute hemolysis and to prompt continued investigation into cause(s), prediction, and prevention of this potentially serious adverse event. PMID:19220820

  11. Age-related macular degeneration: Complement in action.

    PubMed

    van Lookeren Campagne, Menno; Strauss, Erich C; Yaspan, Brian L

    2016-06-01

    The complement system plays a key role in host-defense against common pathogens but must be tightly controlled to avoid inflammation and tissue damage. Polymorphisms in genes encoding two important negative regulators of the alternative complement pathway, complement factor H (CFH) and complement factor I (CFI), are associated with the risk for Age-Related Macular Degeneration (AMD), a leading cause of vision impairment in the ageing population. In this review, we will discuss the genetic basis of AMD and the potential impact of complement de-regulation on disease pathogenesis. Finally, we will highlight recent therapeutic approaches aimed at controlling complement activation in patients with AMD.

  12. Hemolytic anemia caused by glucose-6-phosphate dehydrogenase deficiency.

    PubMed

    Olivares, N; Medina, C; Sánchez-Corona, J; Rivas, F; Rivera, H; Hernández, A; Delgado, J L; Ibarra, B; Cantú, J M; Vaca, G; Martínez, C

    1979-01-01

    Results are reported concerning quantitation of glucose -6- phosphate dehydrogenase (G6PD) enzyme activity where in one of the members of a family a clinical diagnosis of acute hemolytic anemia due to G6PD deficiency had been established. In the propositus, G6PD levels were found to be less than 10 per cent thus confirming diagnosis; the same enzymatic deficiency was identified in one of the siblings without a history of hematologic pathology and in a maternal cousin with a history of neonatal jaundice as well as two obliged carriers. Electrophoretical enzyme phenotype was similar to A variant in three affected males. Advantages of prevention and medical care possible with early diagnosis of G6PD deficiency are discussed.

  13. Outline of Hungarian Complementation.

    ERIC Educational Resources Information Center

    Szamosi, Michael

    This study presents a preliminary analysis of Hungarian complement constructions and the syntactic operations needed to account for them. The expository framework (and the implicit framework of the research itself) is based upon that of Rosenbaum (1967). The aim of the paper is to arrive at a rough picture of the kinds of structures and syntactic…

  14. 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…

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

    PubMed

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

    2016-02-01

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

  16. Growth, serum biochemistry, complement activity, and liver gene expression responses of Pekin ducklings to graded levels of cultured aflatoxin B1.

    PubMed

    Chen, X; Horn, N; Cotter, P F; Applegate, T J

    2014-08-01

    A 14-d study was conducted to evaluate the effects of cultured aflatoxin B1 (AFB1) on performance, serum biochemistry, serum natural antibody and complement activity, and hepatic gene expression parameters in Pekin ducklings. A total of 144 male Pekin ducklings were weighed, tagged, and randomly allotted to 4 dietary treatments containing 4 concentrations of AFB1 (0, 0.11, 0.14, and 0.21 mg/kg) from 0 to 14 d of age (6 cages per diet; 6 ducklings per cage). Compared with the control group, there was a 10.9, 31.7, and 47.4% (P < 0.05) decrease in cumulative BW gain with 0.11, 0.14, and 0.21 mg of AFB1/kg of diet, respectively, but feed efficiency was not affected. Increasing concentrations of AFB1 reduced cumulative BW gain and feed intake both linearly and quadratically, and regression equations were developed with r(2) ≥0.73. Feeding 0.11 to 0.21 mg of AFB1/kg reduced serum glucose, creatinine, albumin, total protein, globulin, Ca, P, and creatine phosphokinase linearly, whereas serum urea N, Cl, alkaline phosphatase, and aspartate amino transferase concentrations increased linearly with increasing AFB1 (P < 0.05). Additionally, 0.11 to 0.21 mg of AFB1/kg diets impaired classical and alternative complement pathways in the duckling serum when tested by lysis of rabbit, human type O, and horse erythrocytes, and decreased rabbit and horse agglutinins (P < 0.05). Liver peroxisome proliferator activated receptor α (PPARα) expression was linearly downregulated by AFB1 (P < 0.01). Results from this study indicate that for every 0.10 mg/kg increase in dietary AFB1, cumulative feed intake and BW gain decrease approximately 230 and 169 g per duckling from hatch to 14 d; and that AFB1 at very low concentrations can significantly impair liver function and gene expression, and innate immune dynamics in Pekin ducklings.

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

    PubMed

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

    2015-10-01

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

  18. Primary Sjögren syndrome presenting with hemolytic anemia and pure red cell aplasia following delivery due to Coombs-negative autoimmune hemolytic anemia and hemophagocytosis.

    PubMed

    Komaru, Yohei; Higuchi, Takakazu; Koyamada, Ryosuke; Haji, Youichiro; Okada, Masato; Kamesaki, Toyomi; Okada, Sadamu

    2013-01-01

    A 36-year-old woman presented with hemolytic anemia without a reticulocyte response 38 days after delivery. A marked reduction in erythroid cells and an increase in macrophages with active hemophagocytosis were noted in the bone marrow. While conventional Coombs' tests were negative, the level of red blood cell (RBC)-bound immunoglobulin G (IgG) was increased. The patient was diagnosed with primary Sjögren syndrome (pSS) based on her symptoms, positive anti-SS-A antibodies, Coombs-negative autoimmune hemolytic anemia and pure red cell aplasia associated with RBC-bound IgG and hemophagocytosis. The unique presentation was considered to be a consequence of immunological derangement associated with pSS, pregnancy and delivery.

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

    PubMed

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

    1991-11-01

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

  20. Complement Activation Alters Platelet Function

    DTIC Science & Technology

    2014-10-01

    Negative T cells than B6.lpr mice. This suggests that the absence of PF4 alleviates some tissue damage in the lupus prone mice. 6...mice with PF4-/- mice may alleviate multi organ dysfunction in Lupus prone mice. Reportable Outcomes Nothing to report Conclusions We have...dysfunction in lupus models. We have evaluated the relationship between Syk and platelets and have thus far identified a role for Syk in platelet lodging in