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Sample records for hemolytic complement activity

  1. Complement in hemolytic anemia.

    PubMed

    Brodsky, Robert A

    2015-11-26

    Complement is increasingly being recognized as an important driver of human disease, including many hemolytic anemias. Paroxysmal nocturnal hemoglobinuria (PNH) cells are susceptible to hemolysis because of a loss of the complement regulatory proteins CD59 and CD55. Patients with atypical hemolytic uremic syndrome (aHUS) develop a thrombotic microangiopathy (TMA) that in most cases is attributable to mutations that lead to activation of the alternative pathway of complement. For optimal therapy, it is critical, but often difficult, to distinguish aHUS from other TMAs, such as thrombotic thrombocytopenic purpura; however, novel bioassays are being developed. In cold agglutinin disease (CAD), immunoglobulin M autoantibodies fix complement on the surface of red cells, resulting in extravascular hemolysis by the reticuloendothelial system. Drugs that inhibit complement activation are increasingly being used to treat these diseases. This article discusses the pathophysiology, diagnosis, and therapy for PNH, aHUS, and CAD. PMID:26582375

  2. Complement in hemolytic anemia.

    PubMed

    Brodsky, Robert A

    2015-01-01

    Complement is increasingly being recognized as an important driver of human disease, including many hemolytic anemias. Paroxysmal nocturnal hemoglobinuria (PNH) cells are susceptible to hemolysis because of a loss of the complement regulatory proteins CD59 and CD55. Patients with atypical hemolytic uremic syndrome (aHUS) develop a thrombotic microangiopathy (TMA) that in most cases is attributable to mutations that lead to activation of the alternative pathway of complement. For optimal therapy, it is critical, but often difficult, to distinguish aHUS from other TMAs, such as thrombotic thrombocytopenic purpura; however, novel bioassays are being developed. In cold agglutinin disease (CAD), immunoglobulin M autoantibodies fix complement on the surface of red cells, resulting in extravascular hemolysis by the reticuloendothelial system. Drugs that inhibit complement activation are increasingly being used to treat these diseases. This article discusses the pathophysiology, diagnosis, and therapy for PNH, aHUS, and CAD. PMID:26637747

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Khoa, D V A; Wimmers, K

    2015-09-01

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

  5. Initiation and Regulation of Complement during Hemolytic Transfusion Reactions

    PubMed Central

    Stowell, Sean R.; Winkler, Anne M.; Maier, Cheryl L.; Arthur, C. Maridith; Smith, Nicole H.; Girard-Pierce, Kathryn R.; Cummings, Richard D.; Zimring, James C.; Hendrickson, Jeanne E.

    2012-01-01

    Hemolytic transfusion reactions represent one of the most common causes of transfusion-related mortality. Although many factors influence hemolytic transfusion reactions, complement activation represents one of the most common features associated with fatality. In this paper we will focus on the role of complement in initiating and regulating hemolytic transfusion reactions and will discuss potential strategies aimed at mitigating or favorably modulating complement during incompatible red blood cell transfusions. PMID:23118779

  6. 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. PMID:26696798

  7. 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

  8. Hemolytic complement activity and concentrations of its third component during maturation of the immune response in colostrum-deprived foals.

    PubMed

    Bernoco, M M; Liu, I K; Willits, N H

    1994-07-01

    Six foals were deprived of colostrum for the first 36 hours after birth and, instead, received reconstituted powdered milk. Five control foals suckled their dams naturally. Blood samples were obtained from all the foals after birth and at approximately weekly intervals until at least 5.5 months of age. Sera were analyzed for hemolytic complement activity, complement component C3, and correlating IgG concentration. Hemolytic complement (P = 0.0145) and C3 (P = 0.0002) values were significantly higher in colostrum-deprived foals (CDF) than in naturally nursed foals at 2 to 5 days of age. In addition, significantly (P = 0.0149) higher IgG concentration was found in CDF than in naturally nursed foals between 3 and 5.5 months of age. It was concluded that the observed high complement activity in CDF within 2 to 5 days of age may provide an alternative in immune defense for IgG-deprived foals after failure of colostral transfer. PMID:7978631

  9. 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

  10. Intravenous and standard immune serum globulin preparations interfere with uptake of /sup 125/I-C3 onto sensitized erythrocytes and inhibit hemolytic complement activity

    SciTech Connect

    Berger, M.; Rosenkranz, P.; Brown, C.Y.

    1985-02-01

    Antibody-sensitized sheep erythrocytes were used as a model to determine the effects of therapeutic immune serum globulin (ISG) preparations on the ability of this particulate activator to fix C3 and initiate hemolysis. Both standard and intravenous forms of ISG inhibit uptake of /sup 125/I-C3, presumably by competing for the deposition of ''nascent'' C3b molecules onto the erythrocytes. Both forms of ISG also inhibit hemolytic activity of whole serum or purified complement components. The inhibition appears to be a specific property of IgG itself, since similar inhibition was not caused by equivalent concentrations of human serum albumin, and was not affected by the buffer in which the ISG was dissolved. Interference with C3 uptake onto antibody-sensitized platelets and/or inhibition of hemolytic complement activity could contribute to the efficacy of high dose intravenous ISG in idiopathic thrombocytopenic purpura.

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

    PubMed

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

    2016-06-01

    The regulators of complement activation cluster at chromosome 1q32 contains the complement factor H (CFH) and five complement factor H-related (CFHR) genes. This area of the genome arose from several large genomic duplications, and these low-copy repeats can cause genome instability in this region. Genomic disorders affecting these genes have been described in atypical hemolytic uremic syndrome, arising commonly through nonallelic homologous recombination. We describe a novel CFH/CFHR3 hybrid gene secondary to a de novo 6.3-kb deletion that arose through microhomology-mediated end joining rather than nonallelic homologous recombination. We confirmed a transcript from this hybrid gene and showed a secreted protein product that lacks the recognition domain of factor H and exhibits impaired cell surface complement regulation. The fact that the formation of this hybrid gene arose as a de novo event suggests that this cluster is a dynamic area of the genome in which additional genomic disorders may arise. PMID:26490391

  12. Hemolytic activity of Borrelia burgdorferi.

    PubMed Central

    Williams, L R; Austin, F E

    1992-01-01

    Zones of beta-hemolysis occurred around colonies of Borrelia burgdorferi grown on Barbour-Stoenner-Kelly medium containing agarose and horse blood. Blood plates were inoculated with either the infective strain Sh-2-82 or noninfective strain B-31 in an overlay and incubated in a candle jar. Both strains of B. burgdorferi displayed beta-hemolysis after 1 to 2 weeks of incubation. The hemolytic activity diffused out from the borrelial colonies, eventually resulting in lysis of the entire blood plate. Hemolysis was most pronounced with horse blood and was less intense with bovine, sheep, and rabbit blood. Hemolysis was enhanced by hot-cold incubation, which is typical of phospholipase-like activities in other bacteria. Further characterization of the borrelial hemolysin by using a spectrophotometric assay revealed its presence in the supernatant fluids of stationary-phase cultures. Detection of the borrelial hemolytic activity was dependent on activation of the hemolysin by the reducing agent cysteine. This study provides the first evidence of hemolytic activity associated with B. burgdorferi. Images PMID:1639493

  13. Postoperative atypical hemolytic uremic syndrome associated with complement c3 mutation.

    PubMed

    Matsukuma, Eiji; Imamura, Atsushi; Iwata, Yusuke; Takeuchi, Takamasa; Yoshida, Yoko; Fujimura, Yoshihiro; Fan, Xinping; Miyata, Toshiyuki; Kuwahara, Takashi

    2014-01-01

    Atypical hemolytic uremic syndrome (aHUS) can be distinguished from typical or Shiga-like toxin-induced HUS. The clinical outcome is unfavorable; up to 50% of affected patients progress to end-stage renal failure and 25% die during the acute phase. Multiple conditions have been associated with aHUS, including infections, drugs, autoimmune conditions, transplantation, pregnancy, and metabolic conditions. aHUS in the nontransplant postsurgical period, however, is rare. An 8-month-old boy underwent surgical repair of tetralogy of Fallot. Neurological disturbances, acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia developed 25 days later, and aHUS was diagnosed. Further evaluation revealed that his complement factor H (CFH) level was normal and that anti-FH antibodies were not detected in his plasma. Sequencing of his CFH, complement factor I, membrane cofactor protein, complement factor B, and thrombomodulin genes was normal. His ADAMTS-13 (a disintegrin-like and metalloprotease with thrombospondin-1 repeats 13) activity was also normal. However, he had a potentially causative mutation (R425C) in complement component C3. Restriction fragment length polymorphism analysis revealed that his father and aunt also had this mutation; however, they had no symptoms of aHUS. We herein report a case of aHUS that developed after cardiovascular surgery and was caused by a complement C3 mutation. PMID:25431709

  14. 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

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

    PubMed Central

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

    2015-01-01

    In autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal hemolytic anemia. Depending on whether IgG or IgM antibodies are involved, response to therapy is different. Proper identification of the isotype of the anti-erythrocyte autoantibodies is, therefore, crucial. However, detection of IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of complement C3 on a patient’s erythrocytes could be used for indirect detection of anti-erythrocyte IgM. To this end, we fractionated patients’ sera by size exclusion chromatography and tested which fractions yielded complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an IgM anti-erythrocyte component that could activate complement, even if no such autoantibody had been detected with any other test. This also included all tested patients with only IgG and C3 on their erythrocytes, who would previously have been classified as having an IgG-only mediated autoimmune hemolytic anemia. Depleting patients’ sera of either IgG or IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in autoimmune hemolytic anemia is mostly IgM-mediated and the presence of covalent C3 on patients’ erythrocytes can be taken as a footprint of the presence of anti-erythrocyte IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy. PMID:26354757

  16. Complement activation induced by rabbit rheumatoid factor.

    PubMed Central

    Meyer, R R; Brown, J C

    1980-01-01

    Rabbit rheumatoid factor produced in animals by hyperimmunized with group C streptococcal vaccine activated guinea pig complement. Anti-streptococcal serum was fractionated by Sephacryl S-200 chromatography into excluded (19S) and included (7S) material and examined for hemolytic activity in a sensitive homologous hemolytic assay system. In the presence of complement, both 19S and 7S antistreptococcal serum fractions induced lysis of bovine (ox) erythrocytes coated with mildly reduced and carboxymethylated rabbit anti-erythrocyte immunoglobulin G. That rabbit rheumatoid factor was responsible for the observed hemolytic activity was substantiated by hemolytic inhibition assays. Significant inhibition of hemolysis was effected when antistreptococcal serum fractions were incubated in the presence of human immunoglobulin G, rabbit immunoglobulin G, and Fc, whereas, no inhibition was detected when the same fractions were tested in the presence of rabbit Fab or F(ab')2 fragments. Deaggregation of inhibitor preparations revealed a preferential reactivity of rheumatoid factor for rabbit immunoglobulin G. In addition to the rheumatoid factor-dependent hemolytic activity observed in humoral preparations, immunoglobulin G-specific antibody-forming cells in spleen and peripheral blood lymphocyte isolates were enumerated by plaque-forming cell assay. PMID:7399707

  17. 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

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

    PubMed

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

    2013-08-01

    Enterohemorrhagic Escherichia coli (EHEC) produce ribosome-inactivating Shiga toxins (Stx1, Stx2) responsible for development of hemolytic uremic syndrome (HUS) and acute kidney injury (AKI). Some patients show complement activation during EHEC infection, raising the possibility of therapeutic targeting of complement for relief. Our juvenile nonhuman primate (Papio baboons) models of endotoxin-free Stx challenge exhibit full spectrum HUS, including thrombocytopenia, hemolytic anemia, and AKI with glomerular thrombotic microangiopathy. There were no significant increases in soluble terminal complement complex (C5b-9) levels after challenge with lethal Stx1 (n = 6) or Stx2 (n = 5) in plasma samples from T0 to euthanasia at 49.5 to 128 hours post-challenge. d-dimer and cell injury markers (HMGB1, histones) confirmed coagulopathy and cell injury. Thus, complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these preclinical models, and benefits or risks of complement inhibition should be studied further for this infection. PMID:23733336

  19. 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. PMID:6832120

  20. 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. PMID:25705656

  1. 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

  2. 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

  3. The Alternative Pathway of Complement and the Evolving Clinical-Pathophysiological Spectrum of Atypical Hemolytic Uremic Syndrome.

    PubMed

    Berger, Bruce E

    2016-08-01

    Complement-mediated atypical hemolytic uremic syndrome (aHUS) comprises approximately 90% of cases of aHUS, and results from dysregulation of endothelial-anchored complement activation with resultant endothelial damage. The discovery of biomarker ADAMTS13 has enabled a more accurate diagnosis of thrombotic thrombocytopenic purpura (TTP) and an appreciation of overlapping clinical features of TTP and aHUS. Given our present understanding of the pathogenic pathways involved in aHUS, it is unlikely that a specific test will be developed. Rather the use of biomarker data, complement functional analyses, genomic analyses and clinical presentation will be required to diagnose aHUS. This approach would serve to clarify whether a thrombotic microangiopathy present in a complement-amplifying condition arises from the unmasking of a genetically driven aHUS versus a time-limited complement storm-mediated aHUS due to direct endothelial damage in which no genetic predisposition is present. Although both scenarios result in the phenotypic expression of aHUS and involve the alternate pathway of complement activation, long-term management would differ. PMID:27524217

  4. Physicochemical signatures of nanoparticle-dependent complement activation

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

    Nanoparticles are potentially powerful therapeutic tools that have the capacity to target drug payloads and imaging agents. However, some nanoparticles can activate complement, a branch of the innate immune system, and cause adverse side-effects. Recently, we employed an in vitro hemolysis assay to measure the serum complement activity of perfluorocarbon nanoparticles that differed by size, surface charge, and surface chemistry, quantifying the nanoparticle-dependent complement activity using a metric called Residual Hemolytic Activity (RHA). In the present work, we have used a decision tree learning algorithm to derive the rules for estimating nanoparticle-dependent complement response based on the data generated from the hemolytic assay studies. Our results indicate that physicochemical properties of nanoparticles, namely, size, polydispersity index, zeta potential, and mole percentage of the active surface ligand of a nanoparticle, can serve as good descriptors for prediction of nanoparticle-dependent complement activation in the decision tree modeling framework.

  5. The molecular and structural bases for the association of complement C3 mutations with atypical hemolytic uremic syndrome

    PubMed Central

    Martínez-Barricarte, Rubén; Heurich, Meike; López-Perrote, Andrés; Tortajada, Agustin; Pinto, Sheila; López-Trascasa, Margarita; Sánchez-Corral, Pilar; Morgan, B. Paul; Llorca, Oscar; Harris, Claire L.; Rodríguez de Córdoba, Santiago

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) associates with complement dysregulation caused by mutations and polymorphisms in complement activators and regulators. However, the reasons why some mutations in complement proteins predispose to aHUS are poorly understood. Here, we have investigated the functional consequences of three aHUS-associated mutations in C3, R592W, R161W and I1157T. First, we provide evidence that penetrance and disease severity for these mutations is modulated by inheritance of documented “risk” haplotypes as has been observed with mutations in other complement genes. Next, we show that all three mutations markedly reduce the efficiency of factor I-mediated C3b cleavage when catalyzed by membrane cofactor protein (MCP), but not when catalyzed by factor H. Biacore analysis showed that each mutant C3b bound sMCP (recombinant soluble MCP; CD46) at reduced affinity, providing a molecular basis for its reduced cofactor activity. Lastly, we show by electron microscopy structural analysis a displacement of the TED domain from the MG ring in C3b in two of the C3 mutants that explains these defects in regulation. As a whole our data suggest that aHUS-associated mutations in C3 selectively affect regulation of complement on surfaces and provide a structural framework to predict the functional consequences of the C3 genetic variants found in patients. PMID:25879158

  6. 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

  7. Efficacy of plasma therapy in atypical hemolytic uremic syndrome with complement factor H mutations.

    PubMed

    Lapeyraque, Anne-Laure; Wagner, Eric; Phan, Véronique; Clermont, Marie-José; Merouani, Aïcha; Frémeaux-Bacchi, Véronique; Goodship, Timothy H J; Robitaille, Pierre

    2008-08-01

    Atypical hemolytic uremic syndrome (aHUS) frequently results in end-stage renal failure and can be lethal. Several studies have established an association between quantitative or qualitative abnormalities in complement factor H and aHUS. Although plasma infusion and exchange are often advocated, guidelines have yet to be established. Long-term outcome for patients under treatment is still unknown. We describe a patient who, at 7 months of age, presented with aHUS associated with combined de novo complement factor H mutations (S1191L and V1197A) on the same allele. Laboratory investigations showed normal levels of complements C4, C3 and factor H. Plasma exchanges and large-dose infusion therapy resulted in a resolution of hemolysis and recovery of renal function. Three recurrences were successfully treated by intensification of the plasma infusion treatment to intervals of 2 or 3 days. This patient showed good response to large doses of plasma infusions and her condition remained stable for 30 months with weekly plasma infusions (30 ml/kg). Long-term tolerance and efficacy of such intensive plasma therapy are still unknown. Reported secondary failure of plasma therapy in factor H deficiency warrants the search for alternative therapeutic approaches. PMID:18425537

  8. Beta-hemolytic activity of Trichomonas vaginalis correlates with virulence.

    PubMed Central

    Krieger, J N; Poisson, M A; Rein, M F

    1983-01-01

    The reasons that some women develop symptomatic trichomonal vaginitis, whereas many other infected women remain asymptomatic, are unclear, but it has been suggested that Trichomonas vaginalis strains vary in their intrinsic virulence. We describe beta-hemolytic activity in T. vaginalis which correlates with virulence in patients as well as in an animal model and in tissue culture. Fresh T. vaginalis isolates from four women with severe, symptomatic trichomoniasis had high-level (86.3 +/- 6.6%) hemolytic activity, whereas isolates from three completely asymptomatic women had low-level (45.3 +/- 8.4%) hemolytic activity (P less than 0.001). Hemolytic activity also correlated with the production of subcutaneous abscesses in mice (r = 0.74) and with destruction of CHO cell monolayers (r = 0.94). All of the 20 clinical isolates of T. vaginalis tested possessed hemolytic activity. The beta-hemolysin may be a virulence factor for T. vaginalis. Images PMID:6604026

  9. 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.

  10. Anti-Legionella activity of staphylococcal hemolytic peptides.

    PubMed

    Marchand, A; Verdon, J; Lacombe, C; Crapart, S; Héchard, Y; Berjeaud, J M

    2011-05-01

    A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic. PMID:21291938

  11. Disturbed sialic acid recognition on endothelial cells and platelets in complement attack causes atypical hemolytic uremic syndrome.

    PubMed

    Hyvärinen, Satu; Meri, Seppo; Jokiranta, T Sakari

    2016-06-01

    Uncontrolled activation of the complement system against endothelial and blood cells is central to the pathogenesis of atypical hemolytic uremic syndrome (aHUS). aHUS patients frequently carry mutations in the inhibitory complement regulator factor H (FH). Mutations cluster in domains 19 and 20 (FH19-20), which are critical for recognizing self surfaces. On endothelial cells, binding of FH is generally attributed to heparan sulfate. This theory, however, is questioned by the puzzling observation that some aHUS-associated mutations markedly enhance FH binding to heparin and endothelial cells. In this article, we show that, instead of disturbed heparin interactions, the impaired ability of C-terminal mutant FH molecules to recognize sialic acid in the context of surface-bound C3b explains their pathogenicity. By using recombinant FH19-20 as a competitor for FH and measuring erythrocyte lysis and deposition of complement C3b and C5b-9 on endothelial cells and platelets, we now show that several aHUS-associated mutations, which have been predicted to impair FH19-20 binding to sialic acid, prevent FH19-20 from antagonizing FH function on cells. When sialic acid was removed, the wild-type FH19-20 also lost its ability to interfere with FH function on cells. These results indicate that sialic acid is critical for FH-mediated complement regulation on erythrocytes, endothelial cells, and platelets. The inability of C-terminal mutant FH molecules to simultaneously bind sialic acid and C3b on cells provides a unifying explanation for their association with aHUS. Proper formation of FH-sialic acid-C3b complexes on surfaces exposed to plasma is essential for preventing cell damage and thrombogenesis characteristic of aHUS. PMID:27006390

  12. Combined Complement Gene Mutations in Atypical Hemolytic Uremic Syndrome Influence Clinical Phenotype

    PubMed Central

    Bresin, Elena; Rurali, Erica; Caprioli, Jessica; Sanchez-Corral, Pilar; Fremeaux-Bacchi, Veronique; Rodriguez de Cordoba, Santiago; Pinto, Sheila; Goodship, Timothy H.J.; Alberti, Marta; Ribes, David; Valoti, Elisabetta; Remuzzi, Giuseppe

    2013-01-01

    Several abnormalities in complement genes reportedly contribute to atypical hemolytic uremic syndrome (aHUS), but incomplete penetrance suggests that additional factors are necessary for the disease to manifest. Here, we sought to describe genotype–phenotype correlations among patients with combined mutations, defined as mutations in more than one complement gene. We screened 795 patients with aHUS and identified single mutations in 41% and combined mutations in 3%. Only 8%–10% of patients with mutations in CFH, C3, or CFB had combined mutations, whereas approximately 25% of patients with mutations in MCP or CFI had combined mutations. The concomitant presence of CFH and MCP risk haplotypes significantly increased disease penetrance in combined mutated carriers, with 73% penetrance among carriers with two risk haplotypes compared with 36% penetrance among carriers with zero or one risk haplotype. Among patients with CFH or CFI mutations, the presence of mutations in other genes did not modify prognosis; in contrast, 50% of patients with combined MCP mutation developed end stage renal failure within 3 years from onset compared with 19% of patients with an isolated MCP mutation. Patients with combined mutations achieved remission with plasma treatment similar to patients with single mutations. Kidney transplant outcomes were worse, however, for patients with combined MCP mutation compared with an isolated MCP mutation. In summary, these data suggest that genotyping for the risk haplotypes in CFH and MCP may help predict the risk of developing aHUS in unaffected carriers of mutations. Furthermore, screening patients with aHUS for all known disease-associated genes may inform decisions about kidney transplantation. PMID:23431077

  13. 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

  14. Complement, complement activation and anaphylatoxins in human ovarian follicular fluid.

    PubMed Central

    Perricone, R; de Carolis, C; Moretti, C; Santuari, E; de Sanctis, G; Fontana, L

    1990-01-01

    Functionally active complement was sought and detected in human follicular fluids obtained during the pre-ovulatory period. All the functional complement activities tested, including total haemolytic complement, classical pathway activity and alternative pathway activity were present in nine fluids from four different donors with values within the normal serum range. The immunochemical analysis demonstrated the presence of complement factors from C1 to C9, of B and of C1 INH, H, I. Complement anaphylatoxins were found employing RIA techniques in amounts significantly higher than in human plasma, thus demonstrating that follicular fluid complement, at least during the pre-ovulatory period, is partially activated. A possible role for urokinase-like substances in such an activation was indicated by further in vitro experiments. The presence of active complement in follicular fluid can be relevant for the function of the enzymatic multi-factorial mechanism of ovulation. PMID:2242616

  15. Complement

    MedlinePlus

    ... the suspected disease are done first. C3 and C4 are the complement components measured most often. A ... normal levels of the complement proteins C3 and C4 . Complement activity varies throughout the body. For example, ...

  16. Variation in hemolytic activity of Brachyspira hyodysenteriae strains from pigs.

    PubMed

    Mahu, Maxime; De Pauw, Nele; Vande Maele, Lien; Verlinden, Marc; Boyen, Filip; Ducatelle, Richard; Haesebrouck, Freddy; Martel, An; Pasmans, Frank

    2016-01-01

    Brachyspira hyodysenteriae is the primary cause of swine dysentery, which is responsible for major economic losses to the pig industry worldwide. The hemolytic activity of 10 B. hyodysenteriae strains isolated from stools of pigs with mild to mucohemorrhagic diarrhea was compared and seven hemolysis associated genes were sequenced. Hemolysis induced by these strains varied from strong to near absent. One weakly hemolytic B. hyodysenteriae strain showed sequence changes in five hemolysis associated genes (tlyA, tlyB, hemolysin III, hemolysin activation protein and hemolysin III channel protein) resulting in amino acid substitutions. The occurrence of weakly hemolytic strains identifiable as B. hyodysenteriae should be taken into account in swine dysentery diagnostics. The presence of these strains may affect herd dysentery status, with great impact on a farms trading opportunities. PMID:27338265

  17. Examining coagulation-complement crosstalk: complement activation and thrombosis.

    PubMed

    Foley, Jonathan H

    2016-05-01

    The coagulation and complement systems are ancestrally related enzymatic cascades of the blood. Although their primary purposes have diverged over the past few hundred million years, they remain inextricably connected. Both complement and coagulation systems limit infection by pathogens through innate immune mechanisms. Recently, it has been shown that hyperactive complement (in particular, elevated C5a/C5b-9) is involved in the pathogenesis (including thrombosis) of diseases such as paroxysmal nocturnal hemoglobinuria, atypical haemolytic uremic syndrome, antiphospholipid syndrome and bacteremia. Although these diseases together account for many thrombosis cases, there are many more where complement activation is not considered a causative factor leading to thrombosis. To better understand what role complement may play in the pathogenesis of thrombosis a better understanding of the mechanisms that cause over-active complement in thrombotic disease is required. PMID:27207425

  18. Cationic amphiphilic non-hemolytic polyacrylates with superior antibacterial activity.

    PubMed

    Punia, Ashish; He, Edward; Lee, Kevin; Banerjee, Probal; Yang, Nan-Loh

    2014-07-01

    Acrylic copolymers with appropriate compositions of counits having cationic charge with 2-carbon and 6-carbon spacer arms can show superior antibacterial activities with concomitant very low hemolytic effect. These amphiphilic copolymers represent one of the most promising synthetic polymer antibacterial systems reported. PMID:24854366

  19. Hemolytic activity of dermatophytes species isolated from clinical specimens.

    PubMed

    Aktas, E; Yıgıt, N

    2015-03-01

    Hemolytic activity was recently reported for several pathogenic fungal species, such as Aspergillus, Candida, Trichophyton, Penicillium and Fusarium. Based on a number of mechanistic and characterization studies, several fungal hemolysins have been proposed as virulence factors. Hemolysins lyse red blood cells resulting in the release of iron, an important growth factor for microbes especially during infection. The requirement of iron in fungal growth is necessary for metabolic processes and as a catalyst for various biochemical processes. Expression of a hemolytic protein with capabilities to lyse red blood cells has also been suggested to provide a survival strategy for fungi during opportunistic infections. The aims of this study were to investigate the hemolytic activities of dermatophytes species isolated from patients with dermatophytosis. Hair, skin and nail samples of patients were examined with direct microscopy using potassium hydroxide and cultivated on Mycobiotic agar and Sabouraud's dextrose agar. To determine hemolytic activities of dermatophytes species, they were subcultured on Columbia Agar with 5% sheep blood and incubated for 7-14 days at 25°C in aerobic conditions. Media which displayed hemolysis were further incubated for 1-5 days at 37°C to increase hemolytic activity. In this study, 66 dermatophytes strains were isolated from clinical specimens and were identified by six different species: 43 (65.1%) Trichophyton rubrum, 7 (10.7%) Trichophyton mentagrophytes, 5 (7.6%) Microsporum canis, 5 (7.6%) Trichophyton tonsurans, 4 (6.0%) Epidermophyton floccosum and 2 (3.0%) Trichophyton violaceum. Twenty-one T. rubrum strains showed incomplete (alpha) hemolysis and nine T. rubrum strains showed complete (beta) hemolysis, whereas hemolysis was absent in 13 T. rubrum strains. Four T. mentagrophytes strains showed complete hemolysis and three T. tonsurans strains showed incomplete hemolysis. However, M. canis, E. floccosum and T. violaceum species had

  20. Methods for quantitative detection of antibody-induced complement activation on red blood cells.

    PubMed

    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 - fatal(1). 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 lysis(1-4). 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

  1. 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

  2. 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

  3. Complement Activation in Placental Malaria

    PubMed Central

    McDonald, Chloe R.; Tran, Vanessa; Kain, Kevin C.

    2015-01-01

    Sixty percent of all pregnancies worldwide occur in malaria endemic regions. Pregnant women are at greater risk of malaria infection than their non-pregnant counterparts and have a higher risk of adverse birth outcomes including low birth weight resulting from intrauterine growth restriction and/or preterm birth. The complement system plays an essential role in placental and fetal development as well as the host innate immune response to malaria infection. Excessive or dysregulated complement activation has been associated with the pathobiology of severe malaria and with poor pregnancy outcomes, dependent and independent of infection. Here we review the role of complement in malaria and pregnancy and discuss its part in mediating altered placental angiogenesis, malaria-induced adverse birth outcomes, and disruptions to the in utero environment with possible consequences on fetal neurodevelopment. A detailed understanding of the mechanisms underlying adverse birth outcomes, and the impact of maternal malaria infection on fetal neurodevelopment, may lead to biomarkers to identify at-risk pregnancies and novel therapeutic interventions to prevent these complications. PMID:26733992

  4. 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. PMID:25381594

  5. ATYPICAL HEMOLYTIC UREMIC SYNDROME AND GENETIC ABERRATIONS IN THE COMPLEMENT FACTOR H RELATED 5 GENE

    PubMed Central

    Westra, Dineke; Vernon, Katherine A.; Volokhina, Elena B.; Pickering, Matthew C.; van de Kar, Nicole C.A.J.; van den Heuvel, Lambert P.

    2012-01-01

    Atypical HUS (aHUS) is a severe renal disorder that is associated with mutations in the genes encoding proteins of the complement alternative pathway. Previously, we identified pathogenic variations in genes encoding complement regulators (CFH, CFI, and MCP) in our aHUS cohort. In this study, we screened for mutations in the alternative pathway regulator CFHR5 in 65 aHUS patients by means of PCR on genomic DNA and sequence analysis. Potential pathogenicity of genetic alterations was determined by published data on CFHR5 variants, evolutionary conservation, and in silico mutation prediction programs. Detection of serum CFHR5 was performed by western blot analysis and ELISA. A potentially pathogenic sequence variation was found in CFHR5 in three patients (4.6%). All variations were located in SCRs that might be involved in binding to C3b, heparin, or CRP. The identified CFHR5 mutations require functional studies to determine their relevance to aHUS, but they might be candidates for an altered genetic profile predisposing to the disease. PMID:22622361

  6. Hemolytic activity in enterotoxigenic and non-enterotoxigenic strains of Escherichia coli.

    PubMed Central

    DeBoy, J M; Wachsmuth, I K; Davis, B R

    1980-01-01

    We screened 223 strains of Escherichia coli belonging to serotypes previously associated with the production of enterotoxin for hemolytic activity, using horse erythrocytes in liquid and in agar media. Thirty-eight were hemolytic. They belonged to nine different serotypes; most (65.8%) belonged to one serotype, O6: H-. Additionally, all 38 strains were specifically assayed for a filterable, heat-labile hemolytic activity previously associated with a hemolysin plasmid. A comparison of hemolytic activity and enterotoxicity showed that none of 32 strains hemolytic in both media was enterotoxigenic; 28 of the 32 expressed heat-labile hemolytic activity. Four of the six strains hemolytic in only one of the media were enterotoxigenic; none of these six expressed heat-labile hemolytic activity. Of 223 strains, 176 that were of human origin and isolated in the United States were further assayed for three traditionally plasmid-mediated characteristics: heat-labile enterotoxin, heat-stable enterotoxin, and colonization factors. The interrelationships of these characteristics, including hemolytic activity, may reflect varying degrees of plasmid compatibility. PMID:7014606

  7. Recent approaches for reducing hemolytic activity of chemotherapeutic agents.

    PubMed

    Jeswani, Gunjan; Alexander, Amit; Saraf, Shailendra; Saraf, Swarnlata; Qureshi, Azra; Ajazuddin

    2015-08-10

    Drug induced hemolysis is a frequent complication associated with chemotherapy. It results from interaction of drug with erythrocyte membrane and leads to cell lysis. In recent past, various approaches were made to reduce drug-induced hemolysis, which includes drug polymer conjugation, drug delivery via colloidal carriers and hydrogels, co-administration of botanical agents and modification in molecular chemistry of drug molecules. The basic concept behind these strategies is to protect the red blood cells from membrane damaging effects of drugs. There are several examples of drug polymer conjugate that either are approved by Food and Drug Administration or are under clinical trial for delivering drugs with reduced toxicities. Likewise, colloidal carriers are also used successfully nowadays for the delivery of various chemotherapeutic agents like gemcitabine and amphotericin B with remarkable decrease in their hemolytic activity. Similarly, co-administration of botanical agents with drugs works as secondary system proving protection and strength to erythrocyte membranes. In addition to the above statement, interaction hindrance between RBC and drug molecule by molecular modification plays an important role in reducing hemolysis. This review predominantly describes the above recent approaches explored to achieve the reduced hemolytic activity of drugs especially chemotherapeutic agents. PMID:26047758

  8. 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

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

    PubMed

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

    2015-01-01

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

  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. 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

  12. 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

  13. 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). PMID:3821135

  14. Zinc Supplementation Inhibits Complement Activation in Age-Related Macular Degeneration

    PubMed Central

    Blom, Anna M.; Mohlin, Frida C.; den Hollander, Anneke I.; van de Ven, Johannes P. H.; van Huet, Ramon A. C.; Groenewoud, Joannes M. M.; Tian, Yuan; Berendschot, Tos T. J. M.; Lechanteur, Yara T. E.; Fauser, Sascha; de Bruijn, Chris; Daha, Mohamed R.; van der Wilt, Gert Jan; Hoyng, Carel B.; Klevering, B. Jeroen

    2014-01-01

    Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. AMD is a multifactorial disorder but complement-mediated inflammation at the level of the retina plays a pivotal role. Oral zinc supplementation can reduce the progression of AMD but the precise mechanism of this protective effect is as yet unclear. We investigated whether zinc supplementation directly affects the degree of complement activation in AMD and whether there is a relation between serum complement catabolism during zinc administration and the complement factor H (CFH) gene or the Age-Related Maculopathy susceptibility 2 (ARMS2) genotype. In this open-label clinical study, 72 randomly selected AMD patients in various stages of AMD received a daily supplement of 50 mg zinc sulphate and 1 mg cupric sulphate for three months. Serum complement catabolism–defined as the C3d/C3 ratio–was measured at baseline, throughout the three months of supplementation and after discontinuation of zinc administration. Additionally, downstream inhibition of complement catabolism was evaluated by measurement of anaphylatoxin C5a. Furthermore, we investigated the effect of zinc on complement activation in vitro. AMD patients with high levels of complement catabolism at baseline exhibited a steeper decline in serum complement activation (p<0.001) during the three month zinc supplementation period compared to patients with low complement levels. There was no significant association of change in complement catabolism and CFH and ARMS2 genotype. In vitro zinc sulphate directly inhibits complement catabolism in hemolytic assays and membrane attack complex (MAC) deposition on RPE cells. This study provides evidence that daily administration of 50 mg zinc sulphate can inhibit complement catabolism in AMD patients with increased complement activation. This could explain part of the mechanism by which zinc slows AMD progression. Trial Registration The Netherlands National Trial Register

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

    PubMed

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

    2001-02-01

    Complement is an important mediator of vascular injury following oxidative stress. We recently demonstrated that complement activation following endothelial oxidative stress is mediated by mannose-binding lectin (MBL) and activation of the lectin complement pathway. Here, we investigated whether nine plant lectins which have a binding profile similar to that of MBL competitively inhibit MBL deposition and subsequent complement activation following human umbilical vein endothelial cell (HUVEC) oxidative stress. HUVEC oxidative stress (1% O(2), 24 hr) significantly increased Ulex europaeus agglutinin II (UEA-II) binding by 72 +/- 9% compared to normoxic cells. UEA-II inhibited MBL binding to HUVEC in a concentration-dependent manner following oxidative stress. Further, MBL inhibited UEA-II binding to HUVEC in a concentration-dependent manner following oxidative stress, suggesting a common ligand. UEA-II (< or = 100 micromol/L) did not attenuate the hemolytic activity, nor did it inhibit C3a des Arg formation from alternative or classical complement pathway-specific hemolytic assays. C3 deposition (measured by ELISA) following HUVEC oxidative stress was inhibited by UEA-II in a concentration-dependent manner (IC(50) = 10 pmol/L). UEA-II inhibited C3 and MBL co-localization (confocal microscopy) in a concentration-dependent manner on HUVEC following oxidative stress (IC(50) approximately 1 pmol/L). Finally, UEA-II significantly inhibited complement-dependent neutrophil chemotaxis, but failed to inhibit fMLP-mediated chemotaxis, following endothelial oxidative stress. These data demonstrate that UEA-II is a novel, potent inhibitor of human MBL deposition and complement activation following human endothelial oxidative stress. PMID:11266613

  16. Eculizumab reduces complement activation, inflammation, endothelial damage, thrombosis, and renal injury markers in aHUS

    PubMed Central

    Cofiell, Roxanne; Kukreja, Anjli; Bedard, Krystin; Yan, Yan; Mickle, Angela P.; Ogawa, Masayo; Bedrosian, Camille L.

    2015-01-01

    Atypical hemolytic uremic syndrome (aHUS) is a genetic, life-threatening disease characterized by uncontrolled complement activation, systemic thrombotic microangiopathy (TMA), and vital organ damage. We evaluated the effect of terminal complement blockade with the anti-C5 monoclonal antibody eculizumab on biomarkers of cellular processes involved in TMA in patients with aHUS longitudinally, during up to 1 year of treatment, compared with in healthy volunteers. Biomarker levels were elevated at baseline in most patients, regardless of mutational status, plasma exchange/infusion use, platelet count, or lactate dehydrogenase or haptoglobin levels. Eculizumab reduced terminal complement activation (C5a and sC5b-9) and renal injury markers (clusterin, cystatin-C, β2-microglobulin, and liver fatty acid binding protein-1) to healthy volunteer levels and reduced inflammation (soluble tumor necrosis factor receptor-1), coagulation (prothrombin fragment F1+2 and d-dimer), and endothelial damage (thrombomodulin) markers to near-normal levels. Alternative pathway activation (Ba) and endothelial activation markers (soluble vascular cell adhesion molecule-1) decreased but remained elevated, reflecting ongoing complement activation in aHUS despite complete terminal complement blockade. These results highlight links between terminal complement activation and inflammation, endothelial damage, thrombosis, and renal injury and underscore ongoing risk for systemic TMA and progression to organ damage. Further research regarding underlying complement dysregulation is warranted. This trial was registered at www.clinicaltrials.gov as #NCT01194973. PMID:25833956

  17. Complement activation in discordant hepatic xenotransplantation.

    PubMed

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

    1998-11-01

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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26354528

  19. Characteristics of hemolytic activity induced by skin secretions of the frog Kaloula pulchra hainana

    PubMed Central

    2013-01-01

    Background The hemolytic activity of skin secretions obtained by stimulating the frog Kaloula pulchra hainana with diethyl ether was tested using human, cattle, rabbit, and chicken erythrocytes. The skin secretions had a significant concentration-dependent hemolytic effect on erythrocytes. The hemolytic activity of the skin secretions was studied in the presence of osmotic protectants (polyethylene glycols and carbohydrates), cations (Mg2+, Ca2+, Ba2+, Cu2+, and K+), or antioxidants (ascorbic acid, reduced glutathione, and cysteine). Results Depending on their molecular mass, osmotic protectants effectively inhibited hemolysis. The inhibition of skin hemolysis was observed after treatment with polyethylene glycols (1000, 3400, and 6000 Da). Among divalent cations, only 1 mM Cu2+ markedly inhibited hemolytic activity. Antioxidant compounds slightly reduced the hemolytic activity. Conclusions The results suggested that skin secretions of K. pulchra hainana induce a pore-forming mechanism to form pores with a diameter of 1.36-2.0 nm rather than causing oxidative damage to the erythrocyte membrane. PMID:24499077

  20. Carbamazepine-induced hemolytic and aplastic crises associated with reduced glutathione peroxidase activity of erythrocytes.

    PubMed

    Yamamoto, Masaki; Suzuki, Nobuhiro; Hatakeyama, Naoki; Kubo, Noriaki; Tachi, Nobutada; Kanno, Hitoshi; Fujii, Hisaichi; Tsutsumi, Hiroyuki

    2007-11-01

    Although pure red cell aplasia is a well-known side effect of carbamazepine treatment, intravascular hemolytic anemia is rare. We describe a 5-year-old boy who developed concurrent intravascular hemolytic anemia and erythroblastopenia, probably due to carbamazepine. Carbamazepine treatment was subsequently discontinued, and the patient was treated with red blood cell transfusions, haptoglobin, and methylprednisolone. His hematologic abnormalities were almost fully recovered within 2 weeks. Examination of the patient's and mother's erythrocyte enzyme activities revealed mildly decreased erythrocyte glutathione peroxidase (GSH-Px) activity. We speculate that patients with reduced GSH-Px activity are at a high risk of developing carbamazepine-induced hemolytic crisis and/or aplastic crisis. PMID:18055338

  1. Hemolytic activity of plasma and urine from rabbits experimentally infected with Legionella pneumophila.

    PubMed

    Baine, W B; Rasheed, J K; Maca, H W; Kaufmann, A F

    1979-01-01

    Rabbits were infected with Legionella pneumophila by intravenous administration of allantoic fluid from eggs infected with this organism. Heated plasma from animals with severe illness caused by L. pneumophila lysed erythrocytes from guinea pigs in a radial hemolysis assay. Plasma from control rabbits did not lyse guinea pig erythrocytes in parallel assays. Urine from two of the infected animals also showed hemolytic activity. Attempts to induce illness in rabbits by intranasal administration of L. pneumohpila were less successful. Allantoic fluid from embrynated hen eggs developed hemolytic activity when maintained eithr in vitro at room temperature or in eggs whose embryos were killed by refrigeration. Hemolytic activity in filtrates of allantoic fluid from eggs infected with L. pneumophila, as previously reported, may not be due to the presence of bacterial hemolysins in the fluid. PMID:399383

  2. Comparison of Hemagglutination and Hemolytic Activity of Various Bacterial Clinical Isolates Against Different Human Blood Groups

    PubMed Central

    HRV, Rajkumar; Devaki, Ramakrishna

    2016-01-01

    Among the various pathogenic determinants shown by microorganisms hemagglutination and hemolysin production assume greater significance in terms of laboratory identification. This study evaluated the hemagglutination and hemolytic activity of various bacterial isolates against different blood groups. One hundred and fifty bacterial strains, isolated from clinical specimens like urine, pus, blood, and other body fluids were tested for their hemagglutinating and hemolytic activity against human A, B, AB, and O group red blood cells. Among the 150 isolates 81 were Escherichia coli, 18 were Klebsiella pneumoniae, 19 were Pseudomonas aeruginosa, 10 were Pseudomonas spp, six were Proteus mirabilis, and the rest 16 were Staphylococcus aureus. Nearly 85% of the isolates agglutinated A group cells followed by B and AB group (59.3% and 60.6% respectively). Least number of isolates agglutinated O group cells (38.0%). When the hemolytic activity was tested, out of these 150 isolates 79 (52.6%) hemolyzed A group cells, 61 (40.6%) hemolyzed AB group cells, 46 (30.6%) hemolyzed B group cells, and 57 (38.6%) isolates hemolyzed O group cells. Forty-six percent of the isolates exhibited both hemagglutinating and hemolytic property against A group cells, followed by B and AB group cells (28.6% and 21.3% respectively). Least number of isolates i.e., 32 (21.3%) showed both the properties against O group cells. The isolates showed wide variation in their hemagglutination and hemolytic properties against different combinations of human blood group cells. The study highlights the importance of selection of the type of cells especially when human RBCs are used for studying the hemagglutination and hemolytic activity of bacterial isolates because these two properties are considered as characteristic of pathogenic strains. PMID:27014523

  3. Comparison of Hemagglutination and Hemolytic Activity of Various Bacterial Clinical Isolates Against Different Human Blood Groups.

    PubMed

    Hrv, Rajkumar; Devaki, Ramakrishna; Kandi, Venkataramana

    2016-01-01

    Among the various pathogenic determinants shown by microorganisms hemagglutination and hemolysin production assume greater significance in terms of laboratory identification. This study evaluated the hemagglutination and hemolytic activity of various bacterial isolates against different blood groups. One hundred and fifty bacterial strains, isolated from clinical specimens like urine, pus, blood, and other body fluids were tested for their hemagglutinating and hemolytic activity against human A, B, AB, and O group red blood cells. Among the 150 isolates 81 were Escherichia coli, 18 were Klebsiella pneumoniae, 19 were Pseudomonas aeruginosa, 10 were Pseudomonas spp, six were Proteus mirabilis, and the rest 16 were Staphylococcus aureus. Nearly 85% of the isolates agglutinated A group cells followed by B and AB group (59.3% and 60.6% respectively). Least number of isolates agglutinated O group cells (38.0%). When the hemolytic activity was tested, out of these 150 isolates 79 (52.6%) hemolyzed A group cells, 61 (40.6%) hemolyzed AB group cells, 46 (30.6%) hemolyzed B group cells, and 57 (38.6%) isolates hemolyzed O group cells. Forty-six percent of the isolates exhibited both hemagglutinating and hemolytic property against A group cells, followed by B and AB group cells (28.6% and 21.3% respectively). Least number of isolates i.e., 32 (21.3%) showed both the properties against O group cells. The isolates showed wide variation in their hemagglutination and hemolytic properties against different combinations of human blood group cells. The study highlights the importance of selection of the type of cells especially when human RBCs are used for studying the hemagglutination and hemolytic activity of bacterial isolates because these two properties are considered as characteristic of pathogenic strains. PMID:27014523

  4. 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: ...

  5. 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 ...

  6. Hemolytic anemia

    MedlinePlus

    Anemia - hemolytic ... Hemolytic anemia occurs when the bone marrow is unable to replace the red blood cells that are being destroyed. Immune hemolytic anemia occurs when the immune system mistakenly sees your ...

  7. Complement inhibitors to treat IgM-mediated autoimmune hemolysis.

    PubMed

    Wouters, Diana; Zeerleder, Sacha

    2015-11-01

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

  8. 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

  9. Membrane-associated CD93 regulates leukocyte migration and C1q-hemolytic activity during murine peritonitis1

    PubMed Central

    Greenlee-Wacker, Mallary C.; Briseño, Carlos; Galvan, Manuel; Moriel, Gabriela; Velázquez, Peter; Bohlson, Suzanne S.

    2011-01-01

    CD93 is emerging as a novel regulator of inflammation; however, its molecular function is unknown. CD93 exists as a membrane-associated glycoprotein on the surface of cells involved in the inflammatory cascade, including endothelial and myeloid cells. A soluble form (sCD93) is detectable in blood and is elevated with inflammation. Here we demonstrate heightened susceptibility to thioglycollate-induced peritonitis in CD93−/− mice. CD93−/− mice showed a 1.6 to 1.8-fold increase in leukocyte infiltration during thioglycollate-induced peritonitis between 3 and 24 hours that returned to wildtype levels by 96 hours. Impaired vascular integrity in CD93−/− mice during peritonitis was demonstrated using fluorescence multi-photon intravital microscopy; however, no differences in cytokine or chemokine levels were detected by Luminex Multiplex or ELISA analysis. C1q-hemolytic activity in CD93−/− mice was decreased by 22% at time zero and by 46% 3 hours post thioglycollate injection suggesting a defect in the classical complement pathway. Leukocyte recruitment and C1q-hemolytic activity was restored to wildtype levels when CD93 was expressed on either hematopoietic cells or non-hematopoietic cells in bone marrow chimeric mice. However, elevated levels of sCD93 in inflammatory fluid were observed only when CD93 was expressed on non-hematopoietic cells. Since cell-associated CD93 was sufficient to restore a normal inflammatory response, these data suggest that cell-associated CD93, and not sCD93, regulates leukocyte recruitment and complement activation during murine peritonitis. PMID:21849679

  10. Membrane-associated CD93 regulates leukocyte migration and C1q-hemolytic activity during murine peritonitis.

    PubMed

    Greenlee-Wacker, Mallary C; Briseño, Carlos; Galvan, Manuel; Moriel, Gabriela; Velázquez, Peter; Bohlson, Suzanne S

    2011-09-15

    CD93 is emerging as a novel regulator of inflammation; however, its molecular function is unknown. CD93 exists as a membrane-associated glycoprotein on the surface of cells involved in the inflammatory cascade, including endothelial and myeloid cells. A soluble form (sCD93) is detectable in blood and is elevated with inflammation. In this study, we demonstrate heightened susceptibility to thioglycollate-induced peritonitis in CD93(-/-) mice. CD93(-/-) mice showed a 1.6-1.8-fold increase in leukocyte infiltration during thioglycollate-induced peritonitis between 3 and 24 h that returned to wild type levels by 96 h. Impaired vascular integrity in CD93(-/-) mice during peritonitis was demonstrated using fluorescence multiphoton intravital microscopy; however, no differences in cytokine or chemokine levels were detected with Luminex Multiplex or ELISA analysis. C1q-hemolytic activity in CD93(-/-) mice was decreased by 22% at time zero and by 46% 3 h after thioglycollate injection, suggesting a defect in the classical complement pathway. Leukocyte recruitment and C1q-hemolytic activity was restored to wild type levels when CD93 was expressed on either hematopoietic cells or nonhematopoietic cells in bone marrow chimeric mice. However, elevated levels of sCD93 in inflammatory fluid were observed only when CD93 was expressed on nonhematopoietic cells. Because cell-associated CD93 was sufficient to restore a normal inflammatory response, these data suggest that cell-associated CD93, and not sCD93, regulates leukocyte recruitment and complement activation during murine peritonitis. PMID:21849679

  11. Typical Hus: Evidence of Acute Phase Complement Activation from a Daycare Outbreak

    PubMed Central

    Brady, Tammy M; Pruette, Cozumel; Loeffler, Lauren F; Weidemann, Darcy; Strouse, John J; Gavriilaki, Eleni; Brodsky, Robert A

    2016-01-01

    The clinical manifestations of typical hemolytic uremic syndrome (HUS) encompass a wide spectrum. Despite the potentially severe sequelae from this syndrome, treatment approaches remain supportive. We present the clinical course of a child who contracted Shiga toxin-positive E. coli (STEC) from a daycare center during an outbreak. Utilizing the modified Ham test which is a rapid, serum-based functional assay used to detect activation of the alternative pathway of complement as observed in atypical HUS, patient sera revealed evidence of increased complement activation in the acute phase of the syndrome but not after resolution. Further, this complement activation was attenuated by eculizumab in vitro, an effect that was replicated in vitro utilizing Shiga toxin as a stimulus of complement activation in normal serum. Our report suggests that complement blockade may be effective in the treatment of STEC-HUS when initiated early in the disease. Given the epidemic nature of the disease that limits the feasibility of randomized clinical trials, further studies are needed to determine the value of early eculizumab treatment in STEC-HUS. PMID:27413789

  12. Synthesis, cytotoxicity, and hemolytic activity of 6'-O-substituted dioscin derivatives.

    PubMed

    Li, Wei; Qiu, Zaozao; Wang, Yibing; Zhang, Yichun; Li, Ming; Yu, Jia; Zhang, Lihong; Zhu, Ziyan; Yu, Biao

    2007-12-28

    Dioscin derivatives (1-12) with a variety of substitutions at the 6'-OH of the chacotriosyl residue and the 3',6'-anhydrosaponin derivatives (26, 30, and 32) were synthesized. All these derivatives showed much lower cytotoxicity than that of the parent dioscin, while their hemolytic activities were partially retained depending on the various 6'-O-substitutions. PMID:17945208

  13. Sigma E Regulators Control Hemolytic Activity and Virulence in a Shrimp Pathogenic Vibrio harveyi

    PubMed Central

    Rattanama, Pimonsri; Thompson, Janelle R.; Kongkerd, Natthawan; Srinitiwarawong, Kanchana; Vuddhakul, Varaporn; Mekalanos, John J.

    2012-01-01

    Members of the genus Vibrio are important marine and aquaculture pathogens. Hemolytic activity has been identified as a virulence factor in many pathogenic vibrios including V. cholerae, V. parahaemolyticus, V. alginolyticus, V. harveyi and V. vulnificus. We have used transposon mutagenesis to identify genes involved in the hemolytic activity of shrimp-pathogenic V. harveyi strain PSU3316. Out of 1,764 mutants screened, five mutants showed reduced hemolytic activity on sheep blood agar and exhibited virulence attenuation in shrimp (Litopenaeus vannamei). Mutants were identified by comparing transposon junction sequences to a draft of assembly of the PSU3316 genome. Surprisingly none of the disrupted open reading frames or gene neighborhoods contained genes annotated as hemolysins. The gene encoding RseB, a negative regulator of the sigma factor (σE), was interrupted in 2 out of 5 transposon mutants, in addition, the transcription factor CytR, a threonine synthetase, and an efflux-associated cytoplasmic protein were also identified. Knockout mutations introduced into the rpoE operon at the rseB gene exhibited low hemolytic activity in sheep blood agar, and were 3-to 7-fold attenuated for colonization in shrimp. Comparison of whole cell extracted proteins in the rseB mutant (PSU4030) to the wild-type by 2-D gel electrophoresis revealed 6 differentially expressed proteins, including two down-regulated porins (OmpC-like and OmpN) and an upregulated protease (DegQ) which have been associated with σE in other organisms. Our study is the first report linking hemolytic activity to the σE regulators in pathogenic Vibrio species and suggests expression of this virulence-linked phenotype is governed by multiple regulatory pathways within the V. harveyi. PMID:22384269

  14. Human Astrovirus Coat Protein Inhibits Serum Complement Activation via C1, the First Component of the Classical Pathway▿

    PubMed Central

    Bonaparte, Rheba S.; Hair, Pamela S.; Banthia, Deepa; Marshall, Dawn M.; Cunnion, Kenji M.; Krishna, Neel K.

    2008-01-01

    Human astroviruses (HAstVs) belong to a family of nonenveloped, icosahedral RNA viruses that cause noninflammatory gastroenteritis, predominantly in infants. Eight HAstV serotypes have been identified, with a worldwide distribution. While the HAstVs represent a significant public health concern, very little is known about the pathogenesis of and host immune response to these viruses. Here we demonstrate that HAstV type 1 (HAstV-1) virions, specifically the viral coat protein (CP), suppress the complement system, a fundamental component of the innate immune response in vertebrates. HAstV-1 virions and purified CP both suppress hemolytic complement activity. Hemolytic assays utilizing sera depleted of individual complement factors as well as adding back purified factors demonstrated that HAstV CP suppresses classical pathway activation at the first component, C1. HAstV-1 CP bound the A chain of C1q and inhibited serum complement activation, resulting in decreased C4b, iC3b, and terminal C5b-9 formation. Inhibition of complement activation was also demonstrated for HAstV serotypes 2 to 4, suggesting that this phenomenon is a general feature of these human pathogens. Since complement is a major contributor to the initiation and amplification of inflammation, the observed CP-mediated inhibition of complement activity may contribute to the lack of inflammation associated with astrovirus-induced gastroenteritis. Although diverse mechanisms of inhibition of complement activation have been described for many enveloped animal viruses, this is the first report of a nonenveloped icosahedral virus CP inhibiting classical pathway activation at C1. PMID:17959658

  15. Hemolytic activity and solubilizing capacity of raffinose and melezitose fatty acid monoesters prepared by enzymatic synthesis.

    PubMed

    Carvalho, Luis; Morales, Juan C; Pérez-Victoria, José M; Pérez-Victoria, Ignacio

    2015-05-01

    The hemolytic activity and solubilizing capacity of two families of non-reducing trisaccharide fatty acid monoesters have been studied to assess their usefulness as surfactants for pharmaceutical applications. The carbohydrate-based surfactants investigated included homologous series of raffinose and melezitose monoesters bearing C10 to C18 acyl chains prepared by lipase-catalyzed synthesis in organic media. The hemolytic activity was determined in vitro using a static method based on the addition of the surfactants to an erythrocyte suspension and subsequent spectrophotometric determination of the released hemoglobin. The effect of the carbohydrate head group, the acyl chain length and the regioisomeric purity was investigated. In all cases, the carbohydrate monoester surfactants decreased their hemolytic activity (with respect to their critical micelle concentration) when increasing the length of the acyl chain. A very similar behaviour was observed either the carbohydrate head-group (raffinose and melezitose) or regardless of the regioisomeric purity. Interestingly, decanoyl (C10) and lauroyl (C12) monoesters were just marginally hemolytic at their critical micelle concentrations while the longer palmitoyl (C16) and (C18) stearoyl monoesters become hemolytic at concentrations much higher than their respective cmc. The palmitoyl and stearoyl monoesters also displayed higher solubilization capacity than the shorter acyl chain monoesters in a solubilization assay of a hydrophobic dye as a model drug mimic. These results suggest that raffinose and melezitose monoesters with long-chain fatty acids (C16 to C18) are promising surfactants for pharmaceutical applications and could be an alternative to the use of current commercial nonionic polyoxyethylene-based surfactants in parenteral formulations. PMID:25753196

  16. Antioxidant, hemolytic and cytotoxic activities of Senecio species used in traditional medicine of northwestern Argentina.

    PubMed

    Lizarraga, Emilio; Castro, Felipe; Fernández, Francisco; de Lampasona, Marina P; Catalán, César A N

    2012-05-01

    Senecio nutans Sch. Bip., S. viridis var. viridis Phill. and S. spegazzinii Cabrera are native species used in traditional medicine of northwestern Argentina. The total phenolics, flavonoids and caffeoylquinic acids contents, as well as radical scavenging, antioxidant, hemolytic and cytotoxic activities of aqueous extracts (infusion and decoction) of all three species were determined. S. nutans was the most active. The extracts did not show antibacterial activity. Alkaloids were not detected in any of the aqueous extracts of the three studied species. PMID:22799087

  17. Infliximab treatment reduces complement activation in patients with rheumatoid arthritis

    PubMed Central

    Familian, A; Voskuyl, A; van Mierlo, G J; Heijst, H; Twisk, J; Dijkmans, B; Hack, C

    2005-01-01

    Background: Tumour necrosis factor (TNF) blocking agents decrease C reactive protein (CRP) levels in rheumatoid arthritis (RA). It has been shown that CRP may contribute to complement activation in RA. Objective: To assess the effect of intravenous infliximab treatment on complement activation, especially that mediated by CRP, in RA. Methods: 35 patients with active RA (28 joint count Disease Activity Score (DAS28) >4.4) were treated with intravenous injections of infliximab (3 mg/kg, at weeks 0, 2, 6, 14, and 22). Clinical response and plasma levels of complement activation products, of CRP and of CRP-complement complexes, which are specific markers for CRP mediated complement activation, were assessed at the indicated time points up to 22 weeks. The relationship between CRP and CRP-complement complexes was analysed by paired t test between two time points and by generalised estimated equation, to test differences of variables over time. Results: At 2 weeks after the first dose, infliximab significantly reduced overall C3 and C4 activation and plasma levels of CRP and CRP-complement complexes were also significantly reduced at this time point. The effects of infliximab on CRP and complement continued throughout the observation period and were more pronounced in patients with a good response to infliximab treatment. Conclusion: Treatment with infliximab decreases plasma levels of CRP and CRP dependent complement activation products and concomitantly may reduce complement activation in RA. Complement activation may be among the effector mechanisms of TNF in RA. PMID:15958758

  18. The cytolytic C5b-9 complement complex: feedback inhibition of complement activation.

    PubMed Central

    Bhakdi, S; Maillet, F; Muhly, M; Kazatchkine, M D

    1988-01-01

    We describe a regulatory function of the terminal cytolytic C5b-9 complex [C5b-9(m)] of human complement. Purified C5b-9(m) complexes isolated from target membranes, whether in solution or bound to liposomes, inhibited lysis of sensitized sheep erythrocytes by whole human serum in a dose-dependent manner. C9 was not required for the inhibitory function since C5b-7 and C5b-8 complexes isolated from membranes were also effective. No effect was found with the cytolytically inactive, fluid-phase SC5b-9 complex. However, tryptic modification of SC5b-9 conferred an inhibitory capacity to the complex, due probably to partial removal of the S protein. Experiments using purified components demonstrated that C5b-9(m) exerts a regulatory effect on the formation of the classical- and alternative-pathway C3 convertases and on the utilization of C5 by cell-bound C5 convertases. C5b-9(m) complexes were unable to inhibit the lysis of cells bearing C5b-7(m) by C8 and C9. Addition of C5b-9(m) to whole human serum abolished its bactericidal effect on the serum-sensitive Escherichia coli K-12 strain W 3110 and suppressed its hemolytic function on antibody-sensitized, autologous erythrocytes. Feedback inhibition by C5b-9(m) represents a biologically relevant mechanism through which complement may autoregulate its effector functions. Images PMID:3162317

  19. Disease Recurrence After Early Discontinuation of Eculizumab in a Patient With Atypical Hemolytic Uremic Syndrome With Complement C3 I1157T Mutation.

    PubMed

    Toyoda, Hidemi; Wada, Hideo; Miyata, Toshiyuki; Amano, Keishiro; Kihira, Kentaro; Iwamoto, Shotaro; Hirayama, Masahiro; Komada, Yoshihiro

    2016-04-01

    Eculizumab, terminal complement inhibitor, has become the frontline treatment for atypical hemolytic uremic syndrome (aHUS). However, the optimal treatment schedule has not yet been established. We describe here an aHUS patient with a mutation of C3 I1157T who achieved remission with eculizumab and suffered a recurrence after eculizumab discontinuation, a clinical situation that has not been previously described in patients with C3 mutation. A 9-year-old male experienced an onset of aHUS after viral gastroenteritis and was treated with hemodialysis. At 13 years of age he developed bacterial enterocolitis due to Campylobacter jejuni and experienced a recurrence of aHUS. Eculizumab was initiated on day 4 after disease onset resulting in recovering laboratory parameters. The patient received eculizumab for 5 months before its discontinuation. Second relapse induced by bacterial pharyngitis was confirmed 4 months after eculizumab discontinuation and prompt eculizumab reinitiation resulted in rapid remission. The patients carrying mutations in CFH or C3 have a high frequency of relapse and worse prognosis. More than 50% of aHUS relapses occurred during the first year after the onset. Therefore, long-term treatment with eculizumab is appropriate in patients with aHUS who have experienced a relapse or have mutations associated with poor prognosis. PMID:26840081

  20. Hemolytic crisis

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003270.htm Hemolytic crisis To use the sharing features on this page, please enable JavaScript. Hemolytic crisis occurs when large numbers of red blood cells ...

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

    PubMed

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

    2009-04-01

    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

  2. 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

  3. Relationship of bacteriocin-like inhibitor production to the pigmentation and hemolytic activity of mutans streptococci.

    PubMed

    Crooks, M; James, S M; Tagg, J R

    1987-03-01

    An inhibitor production typing (P-typing) scheme originally devised for hemolytic streptococci of Lancefield groups A-G has been successfully applied to 35 mutans streptococcus isolates recovered from plaque cultures of 60 Dunedin schoolchildren. Thirteen different P-type designations were identified. Although 11 (31%) of the isolates failed to produce detectable inhibitory activity on the conventional blood agar medium used for P-typing, four of these isolates were inhibitor-positive on Trypticase Soy agar supplemented with 2% yeast extract and 0.5% calcium carbonate (TSYCa). Four mutans strains displayed strong beta-hemolysis on Columbia agar base containing human blood when incubated in a 5% CO2 in air atmosphere. Three of these also produced weak beta-hemolysis on sheep blood-supplemented medium and were further distinctive in that they were the only inhibitor P-type 767 strains to be detected in the present study. Five mutans isolates were pigment producers and this property seemed to occur independently of both the beta-hemolytic activity and the P-type designation. Upon testing an additional collection of 18 mutans strains of various serotypes, only seven (39%) were inhibitor-positive. However, three of the four serotype c strains were inhibitor producers. Two strains of serotype d and one of serotype g were more hemolytic on sheep than on human blood agar medium. In general, it seems that the most common human mutans streptococci (serotype c strains) are more likely than are other mutans strains to produce bacteriocin-like inhibitory activity and to be hemolytic for human rather than sheep erythrocytes. PMID:3604497

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

    PubMed Central

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

    2015-01-01

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

  5. Vitronectin-binding staphylococci enhance surface-associated complement activation.

    PubMed Central

    Lundberg, F; Lea, T; Ljungh, A

    1997-01-01

    Coagulase-negative staphylococci are well recognized in medical device-associated infections. Complement activation is known to occur at the biomaterial surface, resulting in unspecific inflammation around the biomaterial. The human serum protein vitronectin (Vn), a potent inhibitor of complement activation by formation of an inactive terminal complement complex, adsorbs to biomaterial surfaces in contact with blood. In this report, we discuss the possibility that surface-immobilized Vn inhibits complement activation and the effect of Vn-binding staphylococci on complement activation on surfaces precoated with Vn. The extent of complement activation was measured with a rabbit anti-human C3c antibody and a mouse anti-human C9 antibody, raised against the neoepitope of C9. Our data show that Vn immobilized on a biomaterial surface retains its ability to inhibit complement activation. The additive complement activation-inhibitory effect of Vn on a heparinized surface is very small. In the presence of Vn-binding strain, Staphylococcus hemolyticus SM131, complement activation on a surface precoated with Vn occurred as it did in the absence of Vn precoating. For S. epidermidis 3380, which does not express binding of Vn, complement activation on a Vn-precoated surface was significantly decreased. The results could be repeated on heparinized surfaces. These data suggest that Vn adsorbed to a biomaterial surface may serve to protect against surface-associated complement activation. Furthermore, Vn-binding staphylococcal cells may enhance surface-associated complement activation by blocking the inhibitory effect of preadsorbed Vn. PMID:9038294

  6. [Hemolytic activity of venoms from snakes of the genera Bothrop, Lachesis, Crotalus, and Micrurus (Serpentes: Viperidae and Elapidae].

    PubMed

    Martínez Cadillo, E; Bonilla Ferreyra, C; Zvealeta, A

    1991-11-01

    Hemolytic activity of eight Peruvian snake venoms from the families Viperidae and Elapidae (Bothrops atrox, B. pictus, B. hyoprorus, B. bilineatus, B. neuwedii, Lachesis m. muta, Crotalus d. terrificus, Micrurus tschudi), and three Brazilian viperids (B. jararacussu, B. alternatus and C. d. collilineatus) is described. None of the venoms caused direct lysis on washed human erythrocytes. However, all of them caused indirect hemolysis provided that the incubation medium contains an exogenous source of lecithin. Venom of Micrurus tschudi was the most hemolytic (HD50 2.8 ug/ml) while that of B. bilineatus was the least (HD50 681.3 ug/ml). Only six of eleven venoms showed parallel curves of hemolytic activity, and the HD50 varied from 198 to 681 ug/ml and the following decreasing order of hemolytic activity was obtained: L. muta, C. d. terrificus, C. d. collilineatus, B. hyoprorus, B. bilineatus, B. alternatus. PMID:1844159

  7. [Resistance to antimicrobial agents, hemolytic activity and plasmids in Aeromonas species].

    PubMed

    Morita, K; Watanabe, N; Kanamori, M

    1990-06-01

    A total of 174 Aeromonas isolates consisting of 100 strains from patients with diarrhea being mainly overseas travellers nd healthy subjects, and 74 strains from environmental sources including foods, fish, fresh water, sea water and river soil collected in the area of Tokyo Metropolis and Kanagawa Prefecture was examined for the antimicrobial resistance, presence of plasmids and hemolytic activity. Almost all the isolates (99.4%) were resistant to aminobenzyl penicillin. The isolation frequency of chloramphenicol- or tetracycline-resistant strain was low. Most environmental isolates of A. hydrophila were resistant to multiple antimicrobial agents. Thirty-seven percent of environmental isolates and 39% of human fecal ones carried plasmids. In environmental isolates, seven A. hydrophila and three A. sobria strains carried 63- to 150-kilobase pair (kb) conjugative R plasmids. Two A. hydrophila strains from both the healthy subject and domestic case with diarrhea carried 58- to 90-kb conjugative R plasmids, respectively. None of the isolates from the feces of overseas traveller's diarrhea carried the plasmid. Irrespective of the sources. A. hydrophila showed the highest hemolytic activity among three Aeromonas species. Eighty percent or more of A. hydrophila isolates were of hemolysin positive. The hemolytic titer of A. hydrophila strains from human feces was higher than that of the strains from environmental sources. PMID:2401817

  8. Chronic Low Level Complement Activation within the Eye Is Controlled by Intraocular Complement Regulatory Proteins

    PubMed Central

    Sohn, Jeong-Hyeon; Kaplan, Henry J.; Suk, Hye-Jung; Bora, Puran S.; Bora, Nalini S.

    2007-01-01

    Purpose To explore the role of the complement system and complement regulatory proteins in an immune-privileged organ, the eye. Methods Eyes of normal Lewis rats were analyzed for the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay-acceleration factor (DAF), membrane inhibitor of reactive lysis (MIRL, CD59), and cell surface regulator of complement (Crry), using immunohistochemistry, Western blot analysis, and reverse transcription–polymerase chain reaction (RT-PCR). Zymosan, a known activator of the alternative pathway of complement system was injected into the anterior chamber of the eye of Lewis rats. Animals were also injected intracamerally with 5 μl (25 μg) of neutralizing monoclonal antibody (mAb) against rat Crry (5I2) or CD59 (6D1) in an attempt to develop antibody induced anterior uveitis; control animals received 5 μl of sterile phosphate-buffered saline (PBS), OX-18 (25 μg), G-16-510E3 (25 μg), or MOPC-21 (25 μg). The role of complement system in antibody-induced uveitis was explored by intraperitoneal injection of 35 U cobra venom factor (CVF), 24 hours before antibody injection. Immunohistochemical staining and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with Western blot analysis were used to detect the presence of membrane attack complex (MAC) and C3 activation products, respectively, in normal and antibody-injected rat eyes. Results Complement activation product MAC was present in the normal rat eye, and intraocular injection of zymosan induced severe anterior uveitis. The complement regulatory proteins, MCP, DAF, CD59, and Crry, were identified in the normal rat eye. Soluble forms of Crry and CD59 were also detected in normal rat aqueous humor. Severe anterior uveitis developed in Lewis rats injected with a neutralizing mAb against Crry, with increased formation of C3 split products. Systemic complement depletion by CVF prevented the induction of anterior uveitis by anti

  9. Alternative complement pathway and factor B activities in rats with altered blood levels of thyroid hormone

    PubMed Central

    Bitencourt, C.S.; Duarte, C.G.; Azzolini, A.E.C.S.; Assis-Pandochi, A.I.

    2012-01-01

    Evaluating the activity of the complement system under conditions of altered thyroid hormone levels might help elucidate the role of complement in triggering autoimmune processes. Here, we investigated alternative pathway (AP) activity in male Wistar rats (180 ± 10 g) after altering their thyroid hormone levels by treatment with triiodothyronine (T3), propylthiouracil (PTU) or thyroidectomy. T3 and thyroxine (T4) levels were determined by chemiluminescence assays. Hemolytic assays were performed to evaluate the lytic activity of the AP. Factor B activity was evaluated using factor B-deficient serum. An anti-human factor B antibody was used to measure factor B levels in serum by radial immunodiffusion. T3 measurements in thyroidectomized animals or animals treated with PTU demonstrated a significant reduction in hormone levels compared to control. The results showed a reduction in AP lytic activity in rats treated with increasing amounts of T3 (1, 10, or 50 µg). Factor B activity was also decreased in the sera of hyperthyroid rats treated with 1 to 50 µg T3. Additionally, treating rats with 25 µg T3 significantly increased factor B levels in their sera (P < 0.01). In contrast, increased factor B concentration and activity (32%) were observed in hypothyroid rats. We conclude that alterations in thyroid hormone levels affect the activity of the AP and factor B, which may in turn affect the roles of AP and factor B in antibody production. PMID:22370704

  10. Structural Analysis and Anti-Complement Activity of Polysaccharides from Kjellmaniella crsaaifolia

    PubMed Central

    Zhang, Wenjing; Jin, Weihua; Sun, Delin; Zhao, Luyu; Wang, Jing; Duan, Delin; Zhang, Quanbin

    2015-01-01

    Two polysaccharides, named KCA and KCW, were extracted from Kjellmaniella crassifolia using dilute hydrochloric acid and water, respectively. Composition analysis showed that these polysaccharides predominantly consisted of fucose, with galactose, mannose and glucuronic acid as minor components. After degradation and partial desulfation, electrospray ionization mass spectrometry (ESI-MS) was performed, which showed that the polysaccharides consisted of sulfated fucooligosaccharides, sulfated galactofucooligosaccharides and methyl glycosides of mono-sulfated/multi-sulfated fucooligosaccharides. The structures of the oligomeric fragments were further characterized by electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS2 and ESI-CID-MS3). Moreover, the activity of KCA and KCW against the hemolytic activity of both the classical and alternative complement pathways was determined. The activity of KCA was found to be similar to KCW, suggesting that the method of extraction did not influence the activity. In addition, the degraded polysaccharides (DKCA and DKCW) displayed lower activity levels than the crude polysaccharides (KCA and KCW), indicating that molecular weight had an effect on activity. Moreover, the desulfated fractions (ds-DKCA and ds-DKCW) showed less or no activity, which confirmed that sulfate was important for activity. In conclusion, polysaccharides from K. crassifolia may be good candidates for the treatment of diseases involving the complement pathway. PMID:25786064

  11. Poly-lysine peptidomimetics having potent antimicrobial activity without hemolytic activity.

    PubMed

    Ahn, Mija; Jacob, Binu; Gunasekaran, Pethaiah; Murugan, Ravichandran N; Ryu, Eun Kyoung; Lee, Ga-hyang; Hyun, Jae-Kyung; Cheong, Chaejoon; Kim, Nam-Hyung; Shin, Song Yub; Bang, Jeong Kyu

    2014-09-01

    Diversity of sequence and structure in naturally occurring antimicrobial peptides (AMPs) limits their intensive structure-activity relationship (SAR) study. In contrast, peptidomimetics have several advantages compared to naturally occurring peptide in terms of simple structure, convenient to analog synthesis, rapid elucidation of optimal physiochemical properties and low-cost synthesis. In search of short antimicrobial peptides using peptidomimetics, which provide facile access to identify the key factors involving in the destruction of pathogens through SAR study, a series of simple and short peptidomimetics consisting of multi-Lys residues and lipophilic moiety have been prepared and found to be active against several Gram-negative and Gram-positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) without hemolytic activity. Based on the SAR studies, we found that hydrophobicity, +5 charges of multiple Lys residues, hydrocarbon tail lengths and cyclohexyl group were crucial for antimicrobial activity. Furthermore, membrane depolarization, dye leakage, inner membrane permeability and time-killing kinetics revealed that bacterial-killing mechanism of our peptidomimetics is different from the membrane-targeting AMPs (e. g. melittin and SMAP-29) and implied our peptidomimetics might kill bacteria via the intracellular-targeting mechanism as done by buforin-2. PMID:24961649

  12. Atypical hemolytic uremic syndrome

    PubMed Central

    2011-01-01

    Hemolytic uremic syndrome (HUS) is defined by the triad of mechanical hemolytic anemia, thrombocytopenia and renal impairment. Atypical HUS (aHUS) defines non Shiga-toxin-HUS and even if some authors include secondary aHUS due to Streptococcus pneumoniae or other causes, aHUS designates a primary disease due to a disorder in complement alternative pathway regulation. Atypical HUS represents 5 -10% of HUS in children, but the majority of HUS in adults. The incidence of complement-aHUS is not known precisely. However, more than 1000 aHUS patients investigated for complement abnormalities have been reported. Onset is from the neonatal period to the adult age. Most patients present with hemolytic anemia, thrombocytopenia and renal failure and 20% have extra renal manifestations. Two to 10% die and one third progress to end-stage renal failure at first episode. Half of patients have relapses. Mutations in the genes encoding complement regulatory proteins factor H, membrane cofactor protein (MCP), factor I or thrombomodulin have been demonstrated in 20-30%, 5-15%, 4-10% and 3-5% of patients respectively, and mutations in the genes of C3 convertase proteins, C3 and factor B, in 2-10% and 1-4%. In addition, 6-10% of patients have anti-factor H antibodies. Diagnosis of aHUS relies on 1) No associated disease 2) No criteria for Shigatoxin-HUS (stool culture and PCR for Shiga-toxins; serology for anti-lipopolysaccharides antibodies) 3) No criteria for thrombotic thrombocytopenic purpura (serum ADAMTS 13 activity > 10%). Investigation of the complement system is required (C3, C4, factor H and factor I plasma concentration, MCP expression on leukocytes and anti-factor H antibodies; genetic screening to identify risk factors). The disease is familial in approximately 20% of pedigrees, with an autosomal recessive or dominant mode of transmission. As penetrance of the disease is 50%, genetic counseling is difficult. Plasmatherapy has been first line treatment until presently

  13. Complement Regulatory Activity of Normal Human Intraocular Fluid Is Mediated by MCP, DAF, and CD59

    PubMed Central

    Sohn, Jeong-Hyeon; Kaplan, Henry J.; Suk, Hye-Jung; Bora, Puran S.; Bora, Nalini S.

    2007-01-01

    Purpose To identify the molecules in normal human intraocular fluid (aqueous humor and vitreous) that inhibit the functional activity of the complement system. Methods Aqueous humor and vitreous were obtained from patients with noninflammatory ocular disease at the time of surgery. Samples were incubated with normal human serum (NHS), and the mixture assayed for inhibition of the classical and alternative complement pathways using standard CH50 and AH50 hemolytic assays, respectively. Both aqueous humor and vitreous were fractionated by microconcentrators and size exclusion column chromatography. The inhibitory molecules were identified by immunoblotting as well as by studying the effect of depletion of membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59 on inhibitory activity. Results Both aqueous humor and vitreous inhibited the activity of the classical pathway (CH50). Microcentrifugation revealed the major inhibitory activity resided in the fraction with an Mr ≥ 3 kDa. Chromatography on an S-100-HR column demonstrated that the most potent inhibition was associated with the high-molecular-weight fractions (≥ 19.5 kDa). In contrast to unfractionated aqueous and vitreous, fractions with an Mr ≥ 3 kDa also had an inhibitory effect on the alternative pathway activity (AH50). The complement regulatory activity in normal human intraocular fluid was partially blocked by monoclonal antibodies against MCP, DAF, and CD59. Immunoblot analysis confirmed the presence of these three molecules in normal intraocular fluid. Conclusions Our results demonstrate that normal human intraocular fluid (aqueous humor and vitreous) contains complement inhibitory factors. Furthermore, the high-molecular-weight factors appear to be the soluble forms of MCP, DAF, and CD59. PMID:11095615

  14. Phenolic compounds in drumstick peel for the evaluation of antibacterial, hemolytic and photocatalytic activities.

    PubMed

    Surendra, T V; Roopan, Selvaraj Mohana; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Sridharan, Makuteswaran

    2016-08-01

    Most of the wastes emitted from the food processing industries are not utilized for any further purpose. The economic value of the food waste is very less when compared to the collection or reuse or discard. To increase the economic value we have to design the food waste as useful product or applicable in most of the current field. Nothing is waste in this world with this concept we have investigated the phytochemical analysis of drumstick peel (Moringa oleifera). The result supports the presence of phenols, alkaloids, flavanoids, glycosides and tannins. Since various functional groups containing molecules are present in the extract; it has been further subjected to antibacterial and hemolytic activities. To analysis the antibacterial studies we have employed human pathogenic Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) bacterium. The result of antibacterial activity clearly shows that it possesses significant activity on both bacterial cultures. The hemolytic activity was performed on red blood cells (RBCs). From this result we observed that drumstick peel extract has been considered as non-toxic on RBCs. Malachite green was selected to perform photocatalytic activity. The results stated that the drumstick peel extract possessed good behaviour towards photocatalytic investigation. The malachite green was degraded upto 99.7% using drumstick peel extract. PMID:27318603

  15. Complement-activating ability of leucocytes from patients with complement factor I deficiency.

    PubMed Central

    Marquart, H V; Rasmussen, J M; Leslie, R G

    1997-01-01

    Previous studies from this laboratory have shown that normal peripheral blood B cells are capable of activating complement via the alternative pathway (AP), that the activation is associated with complement receptor type 2 (CR2) expression, and that erythrocytes at normal blood levels partially inhibit the activation. The purpose of the present study was to investigate whether factor I (FI) deficiency, which leads to continued formation of the AP convertase (C3bBb) resulting in the consumption of factor B and C3 and large scale generation of C3b fragments, affects the phenotype and/or function of the patients' B cells. Using flow cytometry, peripheral blood leucocytes (PBL) from two FI-deficient patients were investigated for expression of complement receptors and complement regulatory proteins, in vivo-deposited C3 fragments and in vitro complement-activating ability. CR1 levels on B cells were significantly lower in FI-deficient patients than in normal individuals, whereas CR2 levels were found to be reduced, although not to a significant extent. CR1 levels on monocytes and polymorphonuclear leucocytes (PMN) were found to be normal or slightly raised. All leucocyte subpopulations were found to be covered in vivo with C3b fragments. AP activation on B cells from FI-deficient patients in homologous serum was significantly reduced compared with that for normal individuals, whereas no in vitro activation was seen in autologous serum. In addition, the in vivo-bound C3b fragments were degraded to C3d,g when the patients' PBL were incubated in homologous serum containing EDTA. Finally, the patients, erythrocytes failed to exert any inhibition on AP activation in homologous serum. PMID:9301541

  16. Heme: Modulator of Plasma Systems in Hemolytic Diseases.

    PubMed

    Roumenina, Lubka T; Rayes, Julie; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D

    2016-03-01

    Hemolytic diseases such as sickle-cell disease, β-thalassemia, malaria, and autoimmune hemolytic anemia continue to present serious clinical hurdles. In these diseases, lysis of erythrocytes causes the release of hemoglobin and heme into plasma. Extracellular heme has strong proinflammatory potential and activates immune cells and endothelium, thus contributing to disease pathogenesis. Recent studies have revealed that heme can interfere with the function of plasma effector systems such as the coagulation and complement cascades, in addition to the activity of immunoglobulins. Any perturbation in such functions may have severe pathological consequences. In this review we analyze heme interactions with coagulation, complement, and immunoglobulins. Deciphering such interactions to better understand the complex pathogenesis of hemolytic diseases is pivotal. PMID:26875449

  17. 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.

  18. 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.

  19. Involvement of Antilipoarabinomannan Antibodies in Classical Complement Activation in Tuberculosis

    PubMed Central

    Hetland, Geir; Wiker, Harald G.; Høgåsen, Kolbjørn; Hamasur, Beston; Svenson, Stefan B.; Harboe, Morten

    1998-01-01

    We examined alternative and classical complement activation induced by whole bacilli of Mycobacterium bovis BCG and Mycobacterium tuberculosis products. After exposure to BCG, there were higher levels of the terminal complement complex in sera from Indian tuberculosis patients than in sera from healthy controls. The addition of BCG with or without EGTA to these sera indicated that approximately 70 to 85% of the total levels of the terminal complement complex was formed by classical activation. Sera from Indian tuberculosis patients contained more antibody to lipoarabinomannan (LAM) than sera from healthy Indians. Levels of anti-LAM immunoglobulin G2 (IgG2), but not anti-LAM IgM, correlated positively with classical activation induced by BCG in the sera. By flow cytometry, deposition of C3 and terminal complement complex on bacilli incubated with normal human serum was demonstrated. The anticomplement staining was significantly reduced in the presence of EGTA and EDTA. Flow cytometry also revealed the binding of complement to BCG incubated with rabbit anti-LAM and then with factor B-depleted serum. This indicates that classical activation plays a major role in complement activation induced by mycobacteria and that anti-LAM IgG on the bacilli can mediate this response. Classical complement activation may be important for the extent of phagocytosis of M. tuberculosis by mononuclear phagocytes, which may influence the course after infection. PMID:9521145

  20. Complement Activation in Trauma Patients Alters Platelet Function.

    PubMed

    Atefi, Gelareh; Aisiku, Omozuanvbo; Shapiro, Nathan; Hauser, Carl; Dalle Lucca, Jurandir; Flaumenhaft, Robert; Tsokos, George C

    2016-09-01

    Trauma remains the main cause of death for both civilians and those in uniform. Trauma-associated coagulopathy is a complex process involving inflammation, coagulation, and platelet dysfunction. It is unknown whether activation of complement, which occurs invariably in trauma patients, is involved in the expression of trauma-associated coagulopathy. We designed a prospective study in which we enrolled 40 trauma patients and 30 healthy donors upon arrival to the emergency department of BIDMC. Platelets from healthy individuals were incubated with sera from trauma patients and their responsiveness to a thrombin receptor-activating peptide was measured using aggregometry. Complement deposition on platelets from trauma patients was measured by flow cytometry. Normal platelets displayed hypoactivity after incubation with trauma sera even though exposure to trauma sera resulted in increased agonist-induced calcium flux. Depletion of complement from sera further blocked activation of hypoactive platelets. Conversely, complement activation increased aggregation of platelets. Platelets from trauma patients were found to have significantly higher amounts of C3a and C4d on their surface compared with platelets from controls. Depletion of complement (C4d, C3a) reversed the ability of trauma sera to augment agonist-induced calcium flux in donor platelets. Our data indicate that complement enhances platelet aggregation. Despite its complement content, trauma sera render platelets hypoactive and complement depletion further blocks activation of hypoactive platelets. The defect in platelet activation induced by trauma sera is distal to receptor activation since agonist-induced Ca2+ flux is elevated in the presence of trauma sera owing to complement deposition. PMID:27355402

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

    PubMed

    Shih, Angela R; Murali, Mandakolathur R

    2015-12-01

    The complement pathway is a cascade of proteases that is involved in immune surveillance and innate immunity, as well as adaptive immunity. Dysfunction of the complement cascade may be mediated by aberrations in the pathways of activation, complement regulatory proteins, or complement deficiencies, and has been linked to a number of hematologic disorders, including paroxysmal noctural hemoglobinuria (PNH), hereditary angioedema (HAE), and atypical hemolytic-uremic syndrome (aHUS). Here, current laboratory tests for disorders of the complement pathway are reviewed, and their utility and limitations in hematologic disorders and systemic diseases are discussed. Current therapeutic advances targeting the complement pathway in treatment of complement-mediated hematologic disorders are also reviewed. PMID:26437749

  2. Purification, characterization and activities of two hemolytic and antibacterial proteins from coelomic fluid of the annelid Eisenia fetida andrei.

    PubMed

    Milochau, A; Lassègues, M; Valembois, P

    1997-01-01

    The coelomic fluid of the earthworm Eisenia fetida andrei exhibits antibacterial, hemolytic and hemagglutinating activities. These activities are mainly mediated by two proteins, named fetidins, of apparent molecular mass 40 kDa and 45 kDa, respectively. For the first time, the two proteins have been purified to homogeneity from dialysed coelomic fluid by means of anion-exchange chromatography. Three peaks had hemolytic activity. The first fraction was found to correspond to the 40 kDa fetidin, the second to mixed 40 and 45 kDa fetidins, the last one to the 45 kDa fetidin. Both purified proteins still exhibited their hemolytic and antibacterial activities as dialysed coelomic fluid did. In this study, the amino-acid sequence of purified proteins is compared to the amino-acid sequence predicted by cDNA. This cDNA was isolated by screening an expression cDNA library from earthworm total tissues (unpublished data). PMID:9003444

  3. Role of Complement Activation in Obliterative Bronchiolitis Post Lung Transplantation

    PubMed Central

    Suzuki, Hidemi; Lasbury, Mark E.; Fan, Lin; Vittal, Ragini; Mickler, Elizabeth A.; Benson, Heather L.; Shilling, Rebecca; Wu, Qiang; Weber, Daniel J.; Wagner, Sarah R.; Lasaro, Melissa; Devore, Denise; Wang, Yi; Sandusky, George E.; Lipking, Kelsey; Pandya, Pankita; Reynolds, John; Love, Robert; Wozniak, Thomas; Gu, Hongmei; Brown, Krista M.; Wilkes, David S.

    2013-01-01

    Obliterative bronchiolitis (OB) post lung transplantation involves IL-17 regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB are unknown. The current study examines the role of complement activation in OB. Complement regulatory protein (CRP) (CD55, CD46, Crry/CD46) expression was down regulated in human and murine OB; and C3a, a marker of complement activation, was up regulated locally. IL-17 differentially suppressed Crry expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen or autoantigen (type V collagen) reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17 mediated down regulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed forward loop that may enhance CRP down regulation, suggesting that complement blockade could be a therapeutic strategy for OB. PMID:24043901

  4. Involvement of a phospholipase C in the hemolytic activity of a clinical strain of Pseudomonas fluorescens

    PubMed Central

    Rossignol, Gaelle; Merieau, Annabelle; Guerillon, Josette; Veron, Wilfried; Lesouhaitier, Olivier; Feuilloley, Marc GJ; Orange, Nicole

    2008-01-01

    Background Pseudomonas fluorescens is a ubiquitous Gram-negative bacterium frequently encountered in hospitals as a contaminant of injectable material and surfaces. This psychrotrophic bacterium, commonly described as unable to grow at temperatures above 32°C, is now considered non pathogenic. We studied a recently identified clinical strain of P. fluorescens biovar I, MFN1032, which is considered to cause human lung infection and can grow at 37°C in laboratory conditions. Results We found that MFN1032 secreted extracellular factors with a lytic potential at least as high as that of MF37, a psychrotrophic strain of P. fluorescens or the mesophilic opportunistic pathogen, Pseudomonas aeruginosa PAO1. We demonstrated the direct, and indirect – through increases in biosurfactant release – involvement of a phospholipase C in the hemolytic activity of this bacterium. Sequence analysis assigned this phospholipase C to a new group of phospholipases C different from those produced by P. aeruginosa. We show that changes in PlcC production have pleiotropic effects and that plcC overexpression and plcC extinction increase MFN1032 toxicity and colonization, respectively. Conclusion This study provides the first demonstration that a PLC is involved in the secreted hemolytic activity of a clinical strain of Pseudomonas fluorescens. Moreover, this phospholipase C seems to belong to a complex biological network associated with the biosurfactant production. PMID:18973676

  5. 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

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

    PubMed

    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

  7. Antifungal activity of the local complement system in cerebral aspergillosis.

    PubMed

    Rambach, Günter; Hagleitner, Magdalena; Mohsenipour, Iradj; Lass-Flörl, Cornelia; Maier, Hans; Würzner, Reinhard; Dierich, Manfred P; Speth, Cornelia

    2005-10-01

    Dissemination of aspergillosis into the central nervous system is associated with nearly 100% mortality. To study the reasons for the antifungal immune failure we analyzed the efficacy of cerebral complement to combat the fungus Aspergillus. Incubation of Aspergillus in non-inflammatory cerebrospinal fluid (CSF) revealed that complement levels were sufficient to obtain a deposition on the surface, but opsonization was much weaker than in serum. Consequently complement deposition from normal CSF on fungal surface stimulated a very low phagocytic activity of microglia, granulocytes, monocytes and macrophages compared to stimulation by conidia opsonized in serum. Similarly, opsonization of Aspergillus by CSF was not sufficient to induce an oxidative burst in infiltrating granulocytes, whereas conidia opsonized in serum induced a clear respiratory signal. Thus, granulocytes were capable of considerably reducing the viability of serum-opsonized Aspergillus conidia, but not of conidia opsonized in CSF. The limited efficacy of antifungal attack by cerebral complement can be partly compensated by enhanced synthesis, leading to elevated complement concentrations in CSF derived from a patient with cerebral aspergillosis. This inflammatory CSF was able to induce (i) a higher complement deposition on the Aspergillus surface than non-inflammatory CSF, (ii) an accumulation of complement activation products and (iii) an increase in phagocytic and killing activity of infiltrating granulocytes. However, levels and efficacy of the serum-derived complement were not reached. These data indicate that low local complement synthesis and activation may represent a central reason for the insufficient antifungal defense in the brain and the high mortality rate of cerebral aspergillosis. PMID:16027023

  8. Leishmania species: mechanisms of complement activation by five strains of promastigotes.

    PubMed

    Mosser, D M; Burke, S K; Coutavas, E E; Wedgwood, J F; Edelson, P J

    1986-12-01

    The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade

  9. 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.

  10. Evaluation of the antibacterial and hemolytic activities of Latvian herbal preparation.

    PubMed

    Atroshi, F; Ali-Vehmas, T; Westermarck, T; Rizzo, A; Selga, G; Baltais, A; Linars, J; Saulite, V; Daukste, A

    2000-12-01

    Three extracts originating from a combination of various Latvian plant species were tested for their antibacterial activities by evaluating growth delays using a fully automated microturbidimetric method. Ten different human and bovine strains of the genera Staphylococcus and Micrococcus were used as test microorganisms. The inhibitory effect in vitro was defined as the difference between the growth rate without herbs and the growth rate in the presence of an extract. Among the tested strains, Staphylococcus aureus was found sensitive to all 3 extracts. However, extract I was the most effective in slowing the growth of all strains tested. Using appropriate tester strains it should be possible to set up a broad-range microtubidimetry assay for individual herb screening in vitro. The hemolytic effects of the individual extracts on human erythrocytes were also studied at different concentrations. Two of the herbal extracts had minimal lytic effects on eurocaryotic cells. An additional hemolysis test was conducted in the presence of coenzyme Q10 (CoQ10) as a free radical scavenger: CoQ10 had no effect on the hemolytic reaction. PMID:11111939

  11. Modulation of Backbone Flexibility for Effective Dissociation of Antibacterial and Hemolytic Activity in Cyclic Peptides.

    PubMed

    Oddo, Alberto; Thomsen, Thomas T; Britt, Hannah M; Løbner-Olesen, Anders; Thulstrup, Peter W; Sanderson, John M; Hansen, Paul R

    2016-08-11

    Bacterial resistance to antibiotic therapy is on the rise and threatens to evolve into a worldwide emergency: alternative solutions to current therapies are urgently needed. Cationic amphipathic peptides are potent membrane-active agents that hold promise as the next-generation therapy for multidrug-resistant infections. The peptides' behavior upon encountering the bacterial cell wall is crucial, and much effort has been dedicated to the investigation and optimization of this amphipathicity-driven interaction. In this study we examined the interaction of a novel series of nine-membered flexible cyclic AMPs with liposomes mimicking the characteristics of bacterial membranes. Employed techniques included circular dichroism and marker release assays, as well as microbiological experiments. Our analysis was aimed at correlating ring flexibility with their antimicrobial, hemolytic, and membrane activity. By doing so, we obtained useful insights to guide the optimization of cyclic antimicrobial peptides via modulation of their backbone flexibility without loss of activity. PMID:27563396

  12. 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

  13. 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

  14. 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

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

    PubMed

    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

  16. 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). PMID:11212416

  17. 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.

  18. Influence of antimicrobial subinhibitory concentrations on hemolytic activity and bacteriocin-like substances in oral Fusobacterium nucleatum.

    PubMed

    Okamoto, A C; Gaetti-Jardim, E; Cai, S; Avila-Campos, M J

    2000-04-01

    Fusobacterium nucleatum is considered for its role in colonization of initial and late microorganisms in dental plaque and for its coaggregation with other bacterial species. It is known that action of different antimicrobial substances may interfere with either virulence factors or with host-bacteria interaction. The goal of this study was to examine the influence of subinhibitory concentrations of chlorhexidine, triclosan, penicillin G and metronidazole on hemolytic activity and bacteriocin-like substance production of oral F. nucleatum. A high resistance to penicillin G was observed and 63% of the isolates were beta-lactamase positive. All the tested isolates were susceptible to metronidazole. F. nucleatum isolates grown with or without antimicrobials were alpha-hemolytics. Bacteriocin-like substance production was increased in isolates grown with penicillin G. Impaired production of hemolytic or antagonic substances can suggest a role in the regulation of oral microbiota. PMID:10872683

  19. Complement Activation in Patients with Focal Segmental Glomerulosclerosis

    PubMed Central

    Thurman, Joshua M.; Wong, Maria; Renner, Brandon; Frazer-Abel, Ashley; Giclas, Patricia C.; Joy, Melanie S.; Jalal, Diana; Radeva, Milena K.; Gassman, Jennifer; Gipson, Debbie S.; Kaskel, Frederick; Friedman, Aaron; Trachtman, Howard

    2015-01-01

    Background Recent pre-clinical studies have shown that complement activation contributes to glomerular and tubular injury in experimental FSGS. Although complement proteins are detected in the glomeruli of some patients with FSGS, it is not known whether this is due to complement activation or whether the proteins are simply trapped in sclerotic glomeruli. We measured complement activation fragments in the plasma and urine of patients with primary FSGS to determine whether complement activation is part of the disease process. Study Design Plasma and urine samples from patients with biopsy-proven FSGS who participated in the FSGS Clinical Trial were analyzed. Setting and Participants We identified 19 patients for whom samples were available from weeks 0, 26, 52 and 78. The results for these FSGS patients were compared to results in samples from 10 healthy controls, 10 patients with chronic kidney disease (CKD), 20 patients with vasculitis, and 23 patients with lupus nephritis. Outcomes Longitudinal control of proteinuria and estimated glomerular filtration rate (eGFR). Measurements Levels of the complement fragments Ba, Bb, C4a, and sC5b-9 in plasma and urine. Results Plasma and urine Ba, C4a, sC5b-9 were significantly higher in FSGS patients at the time of diagnosis than in the control groups. Plasma Ba levels inversely correlated with the eGFR at the time of diagnosis and at the end of the study. Plasma and urine Ba levels at the end of the study positively correlated with the level of proteinuria, the primary outcome of the study. Limitations Limited number of patients with samples from all time-points. Conclusions The complement system is activated in patients with primary FSGS, and elevated levels of plasma Ba correlate with more severe disease. Measurement of complement fragments may identify a subset of patients in whom the complement system is activated. Further investigations are needed to confirm our findings and to determine the prognostic significance of

  20. LPS induces pulp progenitor cell recruitment via complement activation.

    PubMed

    Chmilewsky, F; Jeanneau, C; Laurent, P; About, I

    2015-01-01

    Complement system, a major component of the natural immunity, has been recently identified as an important mediator of the dentin-pulp regeneration process through STRO-1 pulp cell recruitment by the C5a active fragment. Moreover, it has been shown recently that under stimulation with lipoteichoic acid, a complex component of the Gram-positive bacteria cell wall, human pulp fibroblasts are able to synthesize all proteins required for complement activation. However, Gram-negative bacteria, which are also involved in tooth decay, are known as powerful activators of complement system and inflammation. Here, we investigated the role of Gram-negative bacteria-induced complement activation on the pulp progenitor cell recruitment using lipopolysaccharide (LPS), a major component of all Gram-negative bacteria. Our results show that incubating pulp fibroblasts with LPS induced membrane attack complex formation and C5a release in serum-free fibroblast cultures. The produced C5a binds to the pulp progenitor cells' membrane and induces their migration toward the LPS stimulation chamber, as revealed by the dynamic transwell migration assays. The inhibition of this migration by the C5aR-specific antagonist W54011 indicates that the pulp progenitor migration is mediated by the interaction between C5a and C5aR. Our findings demonstrate, for the first time, a direct interaction between the recruitment of progenitor pulp cells and the activation of complement system generated by pulp fibroblast stimulation with LPS. PMID:25359783

  1. Degradation of Complement 3 by Streptococcal Pyrogenic Exotoxin B Inhibits Complement Activation and Neutrophil Opsonophagocytosis▿

    PubMed Central

    Kuo, Chih-Feng; Lin, Yee-Shin; Chuang, Woei-Jer; Wu, Jiunn-Jong; Tsao, Nina

    2008-01-01

    Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcus (GAS) infection. The inhibition of phagocytic activity by SPE B may help prevent bacteria from being ingested. In this study, we examined the mechanism SPE B uses to enable bacteria to resist opsonophagocytosis. Using an enzyme-linked immunosorbent assay, we found that SPE B-treated serum impaired the activation of the classical, the lectin, and the alternative complement pathways. In contrast, C192S, a SPE B mutant lacking protease activity, had no effect on complement activation. Further study showed that cleavage of serum C3 by SPE B, but not C192S, blocked zymosan-induced production of reactive oxygen species in neutrophils as a result of decreased deposition of C3 fragments on the zymosan surface. Reconstitution of C3 into SPE B-treated serum unblocked zymosan-mediated neutrophil activation dose dependently. SPE B-treated, but not C192S-treated, serum also impaired opsonization of C3 fragments on the surface of GAS strain A20. Moreover, the amount of C3 fragments on the A20 cell surface, a SPE B-producing strain, was less than that on its isogenic mutant strain, SW507, after opsonization with normal serum. A20 opsonized with SPE B-treated serum was more resistant to neutrophil killing than A20 opsonized with normal serum, and SPE B-mediated resistance was C3 dependent. These results suggest a novel SPE B mechanism, one which degrades serum C3 and enables GAS to resist complement damage and opsonophagocytosis. PMID:18174338

  2. 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

    2014-01-01

    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. PMID:25062484

  3. 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

  4. Hypersensitivity reactions to radiocontrast media: the role of complement activation.

    PubMed

    Szebeni, Janos

    2004-01-01

    Although intravenous use of radiocontrast media (RCM) for a variety of radiographic procedures is generally safe, clinically significant acute hypersensitivity reactions still occur in a significant percentage of patients. The mechanism of these anaphylactoid, or "pseudoallergic," reactions is complex, involving complement activation, direct degranulation of mast cells and basophils, and modulation of enzymes and proteolytic cascades in plasma. In this review, basic information on different RCMs and their reactogenicity is summarized and updated, and the prevalence, pathomechanism, prediction, prevention, treatment, and economic impact of hypersensitivity reactions are discussed. Particular attention is paid to the in vitro and in vivo evidence supporting complement activation as an underlying cause of RCM reactions. PMID:14680617

  5. Complement-Coagulation Cross-Talk: A Potential Mediator of the Physiological Activation of Complement by Low pH

    PubMed Central

    Kenawy, Hany Ibrahim; Boral, Ismet; Bevington, Alan

    2015-01-01

    The complement system is a major constituent of the innate immune system. It not only bridges innate and adaptive arms of the immune system but also links the immune system with the coagulation system. Current understanding of the role of complement has extended far beyond fighting of infections, and now encompasses maintenance of homeostasis, tissue regeneration, and pathophysiology of multiple diseases. It has been known for many years that complement activation is strongly pH sensitive, but only relatively recently has the physiological significance of this been appreciated. Most complement assays are carried out at the physiological pH 7.4. However, pH in some extracellular compartments, for example, renal tubular fluid in parts of the tubule, and extracellular fluid at inflammation loci, is sufficiently acidic to activate complement. The exact molecular mechanism of this activation is still unclear, but possible cross-talk between the contact system (intrinsic pathway) and complement may exist at low pH with subsequent complement activation. The current article reviews the published data on the effect of pH on the contact system and complement activity, the nature of the pH sensor molecules, and the clinical implications of these effects. Of particular interest is chronic kidney disease (CKD) accompanied by metabolic acidosis, in which therapeutic alkalinization of urine has been shown significantly to reduce tubular complement activation products, an effect, which may have important implications for slowing progression of CKD. PMID:25999953

  6. Complement activation in leprosy: a retrospective study shows elevated circulating terminal complement complex in reactional leprosy.

    PubMed

    Bahia El Idrissi, N; Hakobyan, S; Ramaglia, V; Geluk, A; Morgan, B Paul; Das, P Kumar; Baas, F

    2016-06-01

    Mycobacterium leprae infection gives rise to the immunologically and histopathologically classified spectrum of leprosy. At present, several tools for the stratification of patients are based on acquired immunity markers. However, the role of innate immunity, particularly the complement system, is largely unexplored. The present retrospective study was undertaken to explore whether the systemic levels of complement activation components and regulators can stratify leprosy patients, particularly in reference to the reactional state of the disease. Serum samples from two cohorts were analysed. The cohort from Bangladesh included multi-bacillary (MB) patients with (n = 12) or without (n = 46) reaction (R) at intake and endemic controls (n = 20). The cohort from Ethiopia included pauci-bacillary (PB) (n = 7) and MB (n = 23) patients without reaction and MB (n = 15) patients with reaction. The results showed that the activation products terminal complement complex (TCC) (P ≤ 0·01), C4d (P ≤ 0·05) and iC3b (P ≤ 0·05) were specifically elevated in Bangladeshi patients with reaction at intake compared to endemic controls. In addition, levels of the regulator clusterin (P ≤ 0·001 without R; P < 0·05 with R) were also elevated in MB patients, irrespective of a reaction. Similar analysis of the Ethiopian cohort confirmed that, irrespective of a reaction, serum TCC levels were increased significantly in patients with reactions compared to patients without reactions (P ≤ 0·05). Our findings suggests that serum TCC levels may prove to be a valuable tool in diagnosing patients at risk of developing reactions. PMID:26749503

  7. Factor H–Related Protein 5 Interacts with Pentraxin 3 and the Extracellular Matrix and Modulates Complement Activation

    PubMed Central

    Csincsi, Ádám I.; Kopp, Anne; Zöldi, Miklós; Bánlaki, Zsófia; Uzonyi, Barbara; Hebecker, Mario; Caesar, Joseph J. E.; Pickering, Matthew C.; Daigo, Kenji; Hamakubo, Takao; Lea, Susan M.; Goicoechea de Jorge, Elena

    2015-01-01

    The physiological roles of the factor H (FH)-related proteins are controversial and poorly understood. Based on genetic studies, FH-related protein 5 (CFHR5) is implicated in glomerular diseases, such as atypical hemolytic uremic syndrome, dense deposit disease, and CFHR5 nephropathy. CFHR5 was also identified in glomerular immune deposits at the protein level. For CFHR5, weak complement regulatory activity and competition for C3b binding with the plasma complement inhibitor FH have been reported, but its function remains elusive. In this study, we identify pentraxin 3 (PTX3) as a novel ligand of CFHR5. Binding of native CFHR5 to PTX3 was detected in human plasma and the interaction was characterized using recombinant proteins. The binding of PTX3 to CFHR5 is of ∼2-fold higher affinity compared with that of FH. CFHR5 dose-dependently inhibited FH binding to PTX3 and also to the monomeric, denatured form of the short pentraxin C–reactive protein. Binding of PTX3 to CFHR5 resulted in increased C1q binding. Additionally, CFHR5 bound to extracellular matrix in vitro in a dose-dependent manner and competed with FH for binding. Altogether, CFHR5 reduced FH binding and its cofactor activity on pentraxins and the extracellular matrix, while at the same time allowed for enhanced C1q binding. Furthermore, CFHR5 allowed formation of the alternative pathway C3 convertase and supported complement activation. Thus, CFHR5 may locally enhance complement activation via interference with the complement-inhibiting function of FH, by enhancement of C1q binding, and by activating complement, thereby contributing to glomerular disease. PMID:25855355

  8. Peptide inhibitor of complement c1, a novel suppressor of classical pathway activation: mechanistic studies and clinical potential.

    PubMed

    Sharp, Julia A; Whitley, Pamela H; Cunnion, Kenji M; Krishna, Neel K

    2014-01-01

    The classical pathway of complement plays multiple physiological roles including modulating immunological effectors initiated by adaptive immune responses and an essential homeostatic role in the clearance of damaged self-antigens. However, dysregulated classical pathway activation is associated with antibody-initiated, inflammatory diseases processes like cold agglutinin disease, acute intravascular hemolytic transfusion reaction (AIHTR), and acute/hyperacute transplantation rejection. To date, only one putative classical pathway inhibitor, C1 esterase inhibitor (C1-INH), is currently commercially available and its only approved indication is for replacement treatment in hereditary angioedema, which is predominantly a kinin pathway disease. Given the variety of disease conditions in which the classical pathway is implicated, development of therapeutics that specifically inhibits complement initiation represents a major unmet medical need. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement. In vitro studies have demonstrated that these peptide inhibitors of complement C1 (PIC1) bind to the collagen-like region of the initiator molecule of the classical pathway, C1q. PIC1 binding to C1q blocks activation of the associated serine proteases (C1s-C1r-C1r-C1s) and subsequent downstream complement activation. Rational design optimization of PIC1 has resulted in the generation of a highly potent derivative of 15 amino acids. PIC1 inhibits classical pathway mediated complement activation in ABO incompatibility in vitro and inhibiting classical pathway activation in vivo in rats. This review will focus on the pre-clinical development of PIC1 and discuss its potential as a therapeutic in antibody-mediated classical pathway disease, specifically AIHTR. PMID:25202312

  9. Peptide Inhibitor of Complement C1, a Novel Suppressor of Classical Pathway Activation: Mechanistic Studies and Clinical Potential

    PubMed Central

    Sharp, Julia A.; Whitley, Pamela H.; Cunnion, Kenji M.; Krishna, Neel K.

    2014-01-01

    The classical pathway of complement plays multiple physiological roles including modulating immunological effectors initiated by adaptive immune responses and an essential homeostatic role in the clearance of damaged self-antigens. However, dysregulated classical pathway activation is associated with antibody-initiated, inflammatory diseases processes like cold agglutinin disease, acute intravascular hemolytic transfusion reaction (AIHTR), and acute/hyperacute transplantation rejection. To date, only one putative classical pathway inhibitor, C1 esterase inhibitor (C1-INH), is currently commercially available and its only approved indication is for replacement treatment in hereditary angioedema, which is predominantly a kinin pathway disease. Given the variety of disease conditions in which the classical pathway is implicated, development of therapeutics that specifically inhibits complement initiation represents a major unmet medical need. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement. In vitro studies have demonstrated that these peptide inhibitors of complement C1 (PIC1) bind to the collagen-like region of the initiator molecule of the classical pathway, C1q. PIC1 binding to C1q blocks activation of the associated serine proteases (C1s–C1r–C1r–C1s) and subsequent downstream complement activation. Rational design optimization of PIC1 has resulted in the generation of a highly potent derivative of 15 amino acids. PIC1 inhibits classical pathway mediated complement activation in ABO incompatibility in vitro and inhibiting classical pathway activation in vivo in rats. This review will focus on the pre-clinical development of PIC1 and discuss its potential as a therapeutic in antibody-mediated classical pathway disease, specifically AIHTR. PMID:25202312

  10. Mesenchymal stem cells inhibit complement activation by secreting factor H.

    PubMed

    Tu, Zhidan; Li, Qing; Bu, Hong; Lin, Feng

    2010-11-01

    Mesenchymal stem cells (MSCs) possess potent and broad immunosuppressive capabilities, and have shown promise in clinical trials treating many inflammatory diseases. Previous studies have found that MSCs inhibit dendritic cell, T-cell, and B-cell activities in the adaptive immunity; however, whether MSCs inhibit complement in the innate immunity, and if so, by which mechanism, have not been established. In this report, we found that MSCs constitutively secrete factor H, which potently inhibits complement activation. Depletion of factor H in the MSC-conditioned serum-free media abolishes their complement inhibitory activities. In addition, production of factor H by MSCs is augmented by inflammatory cytokines TNF-α and interferon-γ (IFN-γ) in dose- and time-dependent manners, while IL-6 does not have a significant effect. Furthermore, the factor H production from MSCs is significantly suppressed by the prostaglandin E2 (PGE2) synthesis inhibitor indomethacin and the indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl-d-tryptophan (1-MT), both of which inhibitors are known to efficiently dampen MSCs immunosuppressive activity. These results indicate that MSCs inhibit complement activation by producing factor H, which could be another mechanism underlying MSCs broad immunosuppressive capabilities. PMID:20163251

  11. 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 ...

  12. Guanylated polymethacrylates: a class of potent antimicrobial polymers with low hemolytic activity.

    PubMed

    Locock, Katherine E S; Michl, Thomas D; Valentin, Jules D P; Vasilev, Krasimir; Hayball, John D; Qu, Yue; Traven, Ana; Griesser, Hans J; Meagher, Laurence; Haeussler, Matthias

    2013-11-11

    We have synthesized a series of copolymers containing both positively charged (amine, guanidine) and hydrophobic side chains (amphiphilic antimicrobial peptide mimics). To investigate the structure-activity relationships of these polymers, low polydispersity polymethacrylates of varying but uniform molecular weight and composition were synthesized, using a reversible addition-fragmentation chain transfer (RAFT) approach. In a facile second reaction, pendant amine groups were converted to guanidines, allowing for direct comparison of cation structure on activity and toxicity. The guanidine copolymers were much more active against Staphylococcus epidermidis and Candida albicans compared to the amine analogues. Activity against Staphylococcus epidermidis in the presence of fetal bovine serum was only maintained for guanidine copolymers. Selectivity for bacterial over mammalian cells was assessed using hemolytic and hemagglutination toxicity assays. Guanidine copolymers of low to moderate molecular weight and hydrophobicity had high antimicrobial activity with low toxicity. Optimum properties appear to be a balance between charge density, hydrophobic character, and polymer chain length. In conclusion, a suite of guanidine copolymers has been identified that represent a new class of antimicrobial polymers with high potency and low toxicity. PMID:24099527

  13. Complement profile and activation mechanisms by different LDL apheresis systems.

    PubMed

    Hovland, Anders; Hardersen, Randolf; Nielsen, Erik Waage; Enebakk, Terje; Christiansen, Dorte; Ludviksen, Judith Krey; Mollnes, Tom Eirik; Lappegård, Knut Tore

    2012-07-01

    Extracorporeal removal of low-density lipoprotein (LDL) cholesterol by means of selective LDL apheresis is indicated in otherwise uncontrolled familial hypercholesterolemia. During blood-biomaterial interaction other constituents than the LDL particles are affected, including the complement system. We set up an ex vivo model in which human whole blood was passed through an LDL apheresis system with one of three different apheresis columns: whole blood adsorption, plasma adsorption and plasma filtration. The concentrations of complement activation products revealed distinctly different patterns of activation and adsorption by the different systems. Evaluated as the final common terminal complement complex (TCC) the whole blood system was inert, in contrast to the plasma systems, which generated substantial and equal amounts of TCC. Initial classical pathway activation was revealed equally for both plasma systems as increases in the C1rs-C1inh complex and C4d. Alternative pathway activation (Bb) was most pronounced for the plasma adsorption system. Although the anaphylatoxins (C3a and C5a) were equally generated by the two plasma separation systems, they were efficiently adsorbed to the plasma adsorption column before the "outlet", whereas they were left free in the plasma in the filtration system. Consequently, during blood-biomaterial interaction in LDL apheresis the complement system is modulated in different manners depending on the device composition. PMID:22373816

  14. TNT003, an inhibitor of the serine protease C1s, prevents complement activation induced by cold agglutinins.

    PubMed

    Shi, Ju; Rose, Eileen L; Singh, Andrew; Hussain, Sami; Stagliano, Nancy E; Parry, Graham C; Panicker, Sandip

    2014-06-26

    Activation of the classical pathway (CP) of complement is often associated with autoimmune disorders in which disease pathology is linked to the presence of an autoantibody. One such disorder is cold agglutinin disease (CAD), an autoimmune hemolytic anemia in which autoantibodies (cold agglutinins) bind to red blood cells (RBCs) at low temperatures. Anemia occurs as a result of autoantibody-mediated CP activation on the surface of the erythrocyte, leading to the deposition of complement opsonins that drive extravascular hemolysis in the liver. Here we test the effects of TNT003, a mouse monoclonal antibody targeting the CP-specific serine protease C1s, on CP activity induced by cold agglutinins on human RBCs. We collected 40 individual CAD patient samples and showed that TNT003 prevented cold agglutinin-mediated deposition of complement opsonins that promote phagocytosis of RBCs. Furthermore, we show that by preventing CP activation, TNT003 also prevents cold agglutinin-driven generation of anaphylatoxins. Finally, we provide evidence that CP activity in CAD patients terminates prior to activation of the terminal cascade, supporting the hypothesis that the primary route of RBC destruction in these patients occurs via extravascular hemolysis. Our results support the development of a CP inhibitor for the treatment of CAD. PMID:24695853

  15. Intracellular sensing of complement C3 activates cell autonomous immunity.

    PubMed

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

    2014-09-01

    Pathogens traverse multiple barriers during infection, including cell membranes. We found that during this transition, pathogens carried covalently attached complement C3 into the cell, triggering immediate signaling and effector responses. Sensing of C3 in the cytosol activated mitochondrial antiviral signaling (MAVS)-dependent signaling cascades and induced proinflammatory cytokine secretion. C3 also flagged viruses for rapid proteasomal degradation, preventing their replication. This system could detect both viral and bacterial pathogens but was antagonized by enteroviruses, such as rhinovirus and poliovirus, which cleave C3 using their 3C protease. The antiviral rupintrivir inhibited 3C protease and prevented C3 cleavage, rendering enteroviruses susceptible to intracellular complement sensing. Thus, complement C3 allows cells to detect and disable pathogens that have invaded the cytosol. PMID:25190799

  16. Intracellular sensing of complement C3 activates cell autonomous immunity

    PubMed Central

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

    2014-01-01

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

  17. RNA Helicase Important for Listeria monocytogenes Hemolytic Activity and Virulence Factor Expression

    PubMed Central

    Netterling, Sakura; Bäreclev, Caroline; Vaitkevicius, Karolis

    2015-01-01

    RNA helicases have been shown to be important for the function of RNA molecules at several levels, although their putative involvement in microbial pathogenesis has remained elusive. We have previously shown that Listeria monocytogenes DExD-box RNA helicases are important for bacterial growth, motility, ribosomal maturation, and rRNA processing. We assessed the importance of the RNA helicase Lmo0866 (here named CshA) for expression of virulence traits. We observed a reduction in hemolytic activity in a strain lacking CshA compared to the wild type. This phenomenon was less evident in strains lacking other RNA helicases. The reduced hemolysis was accompanied by lower expression of major listerial virulence factors in the ΔcshA strain, mainly listeriolysin O, but also to some degree the actin polymerizing factor ActA. Reduced expression of these virulence factors in the strain lacking CshA did not, however, correlate with a decreased level of the virulence regulator PrfA. When combining the ΔcshA knockout with a mutation creating a constitutively active PrfA protein (PrfA*), the effect of the ΔcshA knockout on LLO expression was negated. These data suggest a role for the RNA helicase CshA in posttranslational activation of PrfA. Surprisingly, although the expression of several virulence factors was reduced, the ΔcshA strain did not demonstrate any reduced ability to infect nonphagocytic cells compared to the wild-type strain. PMID:26483402

  18. Streptomyces-derived actinomycin D inhibits biofilm formation by Staphylococcus aureus and its hemolytic activity.

    PubMed

    Lee, Jin-Hyung; Kim, Yong-Guy; Lee, Kayeon; Kim, Chang-Jin; Park, Dong-Jin; Ju, Yoonjung; Lee, Jae-Chan; Wood, Thomas K; Lee, Jintae

    2016-01-01

    Staphylococcus aureus is a versatile human pathogen that produces diverse virulence factors, and its biofilm cells are difficult to eradicate due to their inherent ability to tolerate antibiotics. The anti-biofilm activities of the spent media of 252 diverse endophytic microorganisms were investigated using three S. aureus strains. An attempt was made to identify anti-biofilm compounds in active spent media and to assess their anti-hemolytic activities and hydrophobicities in order to investigate action mechanisms. Unlike other antibiotics, actinomycin D (0.5 μg ml(-1)) from Streptomyces parvulus significantly inhibited biofilm formation by all three S. aureus strains. Actinomycin D inhibited slime production in S. aureus and it inhibited hemolysis by S. aureus and caused S. aureus cells to become less hydrophobic, thus supporting its anti-biofilm effect. In addition, surface coatings containing actinomycin D prevented S. aureus biofilm formation on glass surfaces. Given these results, FDA-approved actinomycin D warrants further attention as a potential antivirulence agent against S. aureus infections. PMID:26785934

  19. Complement fixation by rheumatoid factor.

    PubMed Central

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

    1975-01-01

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

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

    PubMed

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

    2015-05-01

    Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients. PMID:25772973

  1. Pulp Fibroblasts Control Nerve Regeneration through Complement Activation.

    PubMed

    Chmilewsky, F; About, I; Chung, S-H

    2016-07-01

    Dentin-pulp regeneration is closely linked to the presence of nerve fibers in the pulp and to the healing mechanism by sprouting of the nerve fiber's terminal branches beneath the carious injury site. However, little is known about the initial mechanisms regulating this process in carious teeth. It has been recently demonstrated that the complement system activation, which is one of the first immune responses, contributes to tissue regeneration through the local production of anaphylatoxins such as C5a. While few pulp fibroblasts in intact teeth and in untreated fibroblast cultures express the C5a receptor (C5aR), here we show that all dental pulp fibroblasts, localized beneath the carious injury site, do express this receptor. This observation is consistent with our in vitro results, which showed expression of C5aR in lipoteichoic acid-stimulated pulp fibroblasts. The interaction of C5a, produced after complement synthesis and activation from pulp fibroblasts, with the C5aR of these cells mediated the local brain-derived neurotropic factor (BDNF) secretion. Overall, this activation guided the neuronal growth toward the lipoteichoic acid-stimulated fibroblasts. Thus, our findings highlight a new mechanism in one of the initial steps of the dentin-pulp regeneration process, linking pulp fibroblasts to the nerve sprouting through the complement system activation. This may provide a useful future therapeutic tool in targeting the fibroblasts in the dentin-pulp regeneration process. PMID:27053117

  2. [Atypical hemolytic uremic syndrome].

    PubMed

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

    2015-11-20

    The hemolytic uremic syndrome (HUS) is a clinical entity characterized by thrombocytopenia, non-immune hemolytic anemia and renal impairment. Kidney pathology shows thrombotic microangiopathy (TMA) with endothelial cell injury leading to thrombotic occlusion of arterioles and capillaries. Traditionally, HUS was classified in 2 forms: Typical HUS, most frequently occurring in children and caused by Shiga-toxin-producing bacteria, and atypical HUS (aHUS). aHUS is associated with mutations in complement genes in 50-60% of patients and has worse prognosis, with the majority of patients developing end stage renal disease. After kidney transplantation HUS may develop as a recurrence of aHUS or as de novo disease. Over the last years, many studies have demonstrated that complement dysregulation underlies the endothelial damage that triggers the development of TMA in most of these patients. Advances in our understanding of the pathogenic mechanisms of aHUS, together with the availability of novel therapeutic options, will enable better strategies for the early diagnosis and etiological treatment, which are changing the natural history of aHUS. This review summarizes the aHUS clinical entity and describes the role of complement dysregulation in the pathogenesis of aHUS. Finally, we review the differential diagnosis and the therapeutic options available to patients with aHUS. PMID:25433773

  3. Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities.

    PubMed Central

    Chu, L; Ebersole, J L; Kurzban, G P; Holt, S C

    1997-01-01

    A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule. PMID:9234780

  4. Can Pulp Fibroblasts Kill Cariogenic Bacteria? Role of Complement Activation.

    PubMed

    Jeanneau, C; Rufas, P; Rombouts, C; Giraud, T; Dejou, J; About, I

    2015-12-01

    Complement system activation has been shown to be involved in inflammation and regeneration processes that can be observed within the dental pulp after moderate carious decay. Studies simulating carious injuries in vitro have shown that when human pulp fibroblasts are stimulated by lipoteichoic acid (LTA), they synthetize all complement components. Complement activation leads to the formation of the membrane attack complex (MAC), which is known for its bacterial lytic effect. This work was designed to find out whether human pulp fibroblasts can kill Streptococcus mutans and Streptococcus sanguinis via complement activation. First, histological staining of carious tooth sections showed that the presence of S. mutans correlated with an intense MAC staining. Next, to simulate bacterial infection in vitro, human pulp fibroblasts were incubated in serum-free medium with LTA. Quantification by an enzymatic assay showed a significant increase of MAC formation on bacteria grown in this LTA-conditioned medium. To determine whether the MAC produced by pulp fibroblasts was functional, bacteria sensitivity to LTA-conditioned medium was evaluated using agar well diffusion assay and succinyl dehydrogenase (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide [MTT]) assay. Both assays showed that S. mutans and S. sanguinis were sensitive to LTA-conditioned medium. Finally, to evaluate whether MAC formation on cariogenic bacteria, by pulp fibroblasts, can be directly induced by the presence of these bacteria, a specific coculture model of human pulp fibroblasts and bacteria was developed. Immunofluorescence revealed an intense MAC labeling on bacteria after direct contact with pulp fibroblasts. The observed MAC formation and its lethal effects were significantly reduced when CD59, an inhibitor of MAC formation, was added. Our findings demonstrate that the MAC produced by LTA-stimulated pulp fibroblasts is functional and can kill S. mutans and S. sanguinis. Taken together

  5. 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. PMID:26968550

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

    SciTech Connect

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

    1987-06-01

    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 reduction in Factor B, C2, C4, C3, C5, and total hemolytic complement CH/sub 50/ activity levels. The C3, C5, and CH/sub 50/ functional activities in guinea pig serum 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 CH/sub 50/ levels. Thus, MIC vapor was able to active, and thereby reduce serum complement C3 activity in vitro by a complement-dependent process. 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. Clear evidence was obtained to indicate that complement activation had occurred in these animals exposed to MIC for 11 to 15 min. In addition, the complement activation profile observed in these guinea pigs was qualitatively similar to that seen in the guinea pig serum samples exposed to MIC vapor in vitro. The total protein concentration present in plasma samples obtained from guinea pigs that had died from MIC exposure was elevated significantly. The possible contribution of complement activation to the fatal reaction(s) observed in these MIC-treated animals is discussed.

  7. Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype*

    PubMed Central

    Radu, Roxana A.; Hu, Jane; Jiang, Zhichun; Bok, Dean

    2014-01-01

    Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4−/− OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4−/− or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4−/− OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease. PMID:24550392

  8. 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. PMID:8374040

  9. C1q binding and activation of the complement classical pathway by Klebsiella pneumoniae outer membrane proteins.

    PubMed Central

    Albertí, S; Marqués, G; Camprubí, S; Merino, S; Tomás, J M; Vivanco, F; Benedí, V J

    1993-01-01

    The mechanisms of killing of Klebsiella pneumoniae serum-sensitive strains in nonimmune serum by the complement classical pathway have been studied. The bacterial cell surface components that bind C1q more efficiently were identified as two major outer membrane proteins, presumably the porins of this bacterial species. These two outer membrane proteins were isolated from a representative serum-sensitive strain. We have demonstrated that in their purified form, they bind C1q and activate 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 in factor D). Binding of C1q to other components of the bacterial outer membrane, in particular the rough lipopolysaccharide, could not be demonstrated. Activation of the classical pathway by this lipopolysaccharide was also much less efficient than activation by the two outer membrane proteins. The antibody-independent binding of C1q to serum-sensitive strains was independent of the presence of capsular polysaccharide, while strains possessing lipopolysaccharide O antigen bind less C1q and are resistant to complement-mediated killing. Images PMID:8432605

  10. Autoimmune hemolytic anemia.

    PubMed

    Dacie, J V

    1975-10-01

    Warm-type autoantibodies of autoimmune hemolytic anemia (AIHA) are usually IgG but may be IgM or IgA. They are usual Rh specific. Cold-type antibodies are IgM or IgG (Donath-Landsteiner [DL] antibody). IgM antibodies are usually anit-l (occasionally anti-i) and DL antibodies anti-P. The warm IgG antibodies do not fix complement (C); they cause red blood cell (RBC) destruction predominantly in the spleen as the result of interaction between fixing; they cause RBC destruction either by intravascular lysis (complement sequence completed) or by interaction between C3-coated RBCs and phagocytes in liver and spleen. Gentic factors, immunoglobulin deficiency, somatic mutation, viral infections and drugs, and failure of T-lymphocyte function, all probably play a part in breaking immunological tolerance and the development of AIHA. PMID:1164110

  11. TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells.

    PubMed

    Sartain, Sarah E; Turner, Nancy A; Moake, Joel L

    2016-01-15

    Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1β or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1β had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC-mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure. PMID:26673143

  12. Structure-function relationship of the saponins from the roots of Platycodon grandiflorum for hemolytic and adjuvant activity.

    PubMed

    Sun, Hongxiang; Chen, Liqing; Wang, Juanjuan; Wang, Kuiwu; Zhou, Jiyong

    2011-12-01

    To assess the contribution of the aglycone and sugar chain to the biological activity of saponins from Platycodon grandiflorum, seven structurally consecutive saponins, platycodin D (PD), D2 (PD2), D3 (PD3), platycoside A (PA), E (PE), deapioplatycoside E (DPE), and polygalacin D2 (PGD) were compared for their hemolytic activities and adjuvant potentials on the immune responses to Newcastle disease virus-based recombinant avian influenza vaccine (rL-H5) in mice. Among seven compounds, the order of the hemolytic activity was PGD ≈ PD > PD2 > PA > PD3 > PE > DPE. PD, PD2, PA, and PGD significantly not only promoted concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-induced splenocyte proliferation, but enhanced the NK cell activity in mice immunized with rL-H5. PD and PD2 increased the antigen specific IgG, IgG1, IgG2a, and IgG2b antibody titers, while PA and PGD only induce the IgG and IgG1 antibody responses in the immunized mice. However, the other three saponins were not observed for adjuvant activity. The results suggested that the sugar chains attached to C-3, the glycidic moiety at C-28 of aglycone, as well as aglycone affect their biological activities. Interestingly, their hemolytic and adjuvant activities increased with the retention time by reverse phase HPLC analysis. The retention time may be useful for primary estimation of fundamental adjuvanticity of saponin with the same aglycone. PMID:21945665

  13. Complement activation associated with polysorbate 80 in beagle dogs.

    PubMed

    Qiu, Shidong; Liu, Zhaohua; Hou, Li; Li, Yuanyuan; Wang, Jiao; Wang, Hong; Du, Wu; Wang, Wenfang; Qin, Yizhuo; Liu, Zhaoping

    2013-01-01

    Polysorbate 80 (Tween® 80) is the most extensively used surfactant in parenteral drug formulation. Its application as an adjunct for intravenous drug administration is approved by the Food and Drug Administration. However, severe hypersensitive reactions, which are typical non-immune anaphylactic reactions (pseudoallergy) characterized by the release of histamine and unvaried IgE antibodies, have been associated with Tween® 80. In order to explore the non-immune anaphylactic mechanisms of Tween® 80, we performed in vivo experiments to assess the changes in physiological and hematologic indicators after intravenous injection of Tween® 80 into dogs. Tween® 80 induced the release of histamine, and a 2-fold increase in SC5b-9, 2.5-fold increase in C4d, 1.3-fold increase in Bb, while IgE remained unchanged. It also produced changes in pulmonary pressure, systemic pressure and ECG. In in vitro experiments, Tween® 80 was incubated with dog serum in the presence of an inhibitor of complement activation (EGTA/Mg(2+)). Under these conditions, Tween® 80 increased the contents of C4d and Bb. The results of this study reveal that Tween® 80 can cause cardiopulmonary distress in dogs and activate the complement system through classical and alternative pathways as indicated in both in vivo and in vitro preparations. Moreover, they demonstrate the utility of the beagle dog as an animal model for the study of complement activation-related pseudoallergy. These findings raise concerns with regard to the indiscriminate use of Tween® 80 in clinical applications. PMID:23159336

  14. Complement activating factor(s) of Trypanosoma lewisi: some physiochemical characteristics of the active components.

    PubMed Central

    Nielsen, K; Sheppard, J; Tizard, I; Holmes, W

    1978-01-01

    Of the complement activating factors present in Trypanosoma lewisi, the major component, a carbohydrate containing substance was further investigated. This component was found to have a lag time of complete activation of 2 CH50 units of bovine complement of approximately 15 minutes while 1% trypsin (a known activator of complement, used as a control system) was capable of instant consumption of a similar quantity of complement. In addition, the complement activating factor of trypanosomes was observed to be stable at 100 degrees C for 15 minutes and over a pH range of 3.0 to 11.0. Thin layer chromatography studies suggested that at least part of the active component contained lipid, perhaps indicating that it may be glycolipid in nature. PMID:25701

  15. The alternative pathway of complement and the thrombotic microangiopathies.

    PubMed

    Teoh, Chia Wei; Riedl, Magdalena; Licht, Christoph

    2016-04-01

    Thrombotic microangiopathies (TMA) are disorders defined by microangiopathic hemolytic anemia, non-immune thrombocytopenia and have multi-organ involvement including the kidneys, brain, gastrointestinal, respiratory tract and skin. Emerging evidence points to the central role of complement dysregulation in leading to microvascular endothelial injury which is crucial for the development of TMAs. This key insight has led to the development of complement-targeted therapy. Eculizumab is an anti-C5 monoclonal antibody, which has revolutionized the treatment of atypical hemolytic uremic syndrome. Several other anti-complement therapeutic agents are currently in development, offering a potential armamentarium of therapies available to treat complement-mediated TMAs. The development of sensitive, reliable and easy to perform assays to monitor complement activity and therapeutic efficacy will be key to devising an individualized treatment regime with the potential of safely weaning or discontinuing treatment in the appropriate clinical setting. PMID:27160864

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

    PubMed Central

    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.

    2014-01-01

    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 Kd 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. PMID:25422425

  17. The relative merits of therapies being developed to tackle inappropriate ('self'-directed) complement activation.

    PubMed

    Antwi-Baffour, Samuel; Kyeremeh, Ransford; Adjei, Jonathan Kofi; Aryeh, Claudia; Kpentey, George

    2016-12-01

    The complement system is an enzyme cascade that helps defend against infection. Many complement proteins occur in serum as inactive enzyme precursors or reside on cell surfaces. Complement components have many biologic functions and their activation can eventually damage the plasma membranes of cells and some bacteria. Although a direct link between complement activation and autoimmune diseases has not been found, there is increasing evidence that complement activation significantly contributes to the pathogenesis of a large number of inflammatory diseases that may have autoimmune linkage. The inhibition of complement may therefore be very important in a variety of autoimmune diseases since their activation may be detrimental to the individual involved. However, a complete and long-term inhibition of complement may have some contra side effects such as increased susceptibility to infection. The site of complement activation will, however, determine the type of inhibitor to be used, its route of application and dosage level. Compared with conventional drugs, complement inhibitors may be the best option for treatment of autoimmune diseases. The review takes a critical look at the relative merits of therapies being developed to tackle inappropriate complement activation that are likely to result in sporadic autoimmune diseases or worsen already existing one. It covers the complement system, general aspects of complement inhibition therapy, therapeutic strategies and examples of complement inhibitors. It concludes by highlighting on the possibility that a better inhibitor of complement activation when found will help provide a formidable treatment for autoimmune diseases as well as preventing one. PMID:26935316

  18. 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

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

    PubMed

    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 Ca(2+) 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

  20. Complement Alternative Pathway Activation in Human Nonalcoholic Steatohepatitis

    PubMed Central

    Segers, Filip M.; Verdam, Froukje J.; de Jonge, Charlotte; Boonen, Bas; Driessen, Ann; Shiri-Sverdlov, Ronit; Bouvy, Nicole D.; Greve, Jan Willem M.; Buurman, Wim A.; Rensen, Sander S.

    2014-01-01

    The innate immune system plays a major role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Recently we reported complement activation in human NASH. However, it remained unclear whether the alternative pathway of complement, which amplifies C3 activation and which is frequently associated with pathological complement activation leading to disease, was involved. Here, alternative pathway components were investigated in liver biopsies of obese subjects with healthy livers (n = 10) or with NASH (n = 12) using quantitative PCR, Western blotting, and immunofluorescence staining. Properdin accumulated in areas where neutrophils surrounded steatotic hepatocytes, and colocalized with the C3 activation product C3c. C3 activation status as expressed by the C3c/native C3 ratio was 2.6-fold higher (p<0.01) in subjects with NASH despite reduced native C3 concentrations (0.94±0.12 vs. 0.57±0.09; p<0.01). Hepatic properdin levels positively correlated with levels of C3c (rs = 0.69; p<0.05) and C3c/C3 activation ratio (rs = 0.59; p<0.05). C3c, C3 activation status (C3c/C3 ratio) and properdin levels increased with higher lobular inflammation scores as determined according to the Kleiner classification (C3c: p<0.01, C3c/C3 ratio: p<0.05, properdin: p<0.05). Hepatic mRNA expression of factor B and factor D did not differ between subjects with healthy livers and subjects with NASH (factor B: 1.00±0.19 vs. 0.71±0.07, p = 0.26; factor D: 1.00±0.21 vs. 0.66±0.14, p = 0.29;). Hepatic mRNA and protein levels of Decay Accelerating Factor tended to be increased in subjects with NASH (mRNA: 1.00±0.14 vs. 2.37±0.72; p = 0.22; protein: 0.51±0.11 vs. 1.97±0.67; p = 0.28). In contrast, factor H mRNA was downregulated in patients with NASH (1.00±0.09 vs. 0.71±0.06; p<0.05) and a similar trend was observed with hepatic protein levels (1.12±0.16 vs. 0.78±0.07; p = 0.08). Collectively, these data suggest a role for alternative pathway

  1. 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

  2. High antimicrobial effectiveness with low hemolytic and cytotoxic activity for PEG/quaternary copolyoxetanes.

    PubMed

    King, Allison; Chakrabarty, Souvik; Zhang, Wei; Zeng, Xiaomei; Ohman, Dennis E; Wood, Lynn F; Abraham, Sheena; Rao, Raj; Wynne, Kenneth J

    2014-02-10

    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 (1)H 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 10(8) 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 E. coli . Cx-50

  3. Non-linear dynamics of the complement system activation.

    PubMed

    Korotaevskiy, Andrey A; Hanin, Leonid G; Khanin, Mikhail A

    2009-12-01

    The complement system (CS) plays a prominent role in the immune defense. The goal of this work is to study the dynamics of activation of the classic and alternative CS pathways based on the method of mathematical modeling. The principal difficulty that hinders modeling effort is the absence of the measured values of kinetic constants of many biochemical reactions forming the CS. To surmount this difficulty, an optimization procedure consisting of constrained minimization of the total protein consumption by the CS was designed. The constraints made use of published data on the in vitro kinetics of elimination of the Borrelia burgdorferi bacteria by the CS. Special features of the problem at hand called for a significant modification of the general constrained optimization procedure to include a mathematical model of the bactericidal effect of the CS in the iterative setting. Determination of the unknown kinetic constants of biochemical reactions forming the CS led to a fully specified mathematical model of the dynamics of cell killing induced by the CS. On the basis of the model, effects of the initial concentrations of complements and their inhibitors on the bactericidal action of the CS were studied. Proteins playing a critical role in the regulation of the bactericidal action of the CS were identified. Results obtained in this work serve as an important stepping stone for the study of functioning of the CS as a whole as well as for developing methods for control of pathogenic processes. PMID:19854207

  4. Persistent complement activation on tumor cells in breast cancer.

    PubMed Central

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

    1992-01-01

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

  5. Antibody-independent activation of the classical pathway of human serum complement by lipid A is restricted to re-chemotype lipopolysaccharide and purified lipid A.

    PubMed Central

    Vukajlovich, S W

    1986-01-01

    Incubation of most bacterial lipopolysaccharides (LPS) with normal human sera at 37 degrees C activates the serum complement system, resulting in decreased levels of hemolytic complement. A panel of R-chemotype LPS preparations isolated from Salmonella minnesota rough mutant strains, as well as smooth wild-type LPS from S. minnesota, Escherichia coli O55-B5, Serratia marcescens, and Yersinia enterolitica, were used to examine the effect of LPS polysaccharide chain length on LPS lipid (lipid A)-dependent activation of the classical pathway of complement (CPC). To examine specific lipid A-dependent activation of the CPC, sera deficient in alternative pathway of complement activity were prepared by the removal of factor D. Absorption of normal human sera with formalinized rabbit erythrocytes was found to remove natural antibodies, factors capable of forming LPS complexes which activate the CPC, or both. By using such factor D-depleted formalinized rabbit erythrocyte-absorbed normal human sera, only isolated lipid A and Re-chemotype LPS (R595 LPS) were found to activate the CPC. Thus, the presence of the additional monosaccharide L-glycero-D-mannoheptose in the Rd2 LPS oligosaccharide chain compared with the L-glycero-D-mannoheptose-deficient Re-chemotype LPS structure is sufficient to block lipid A-dependent activation of the CPC by LPS. PMID:3744547

  6. 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.

  7. Free Radical Scavenging, Cytotoxic and Hemolytic Activities from Leaves of Acacia nilotica (L.) Wild. ex. Delile subsp. indica (Benth.) Brenan

    PubMed Central

    Kalaivani, T.; Rajasekaran, C.; Suthindhiran, K.; Mathew, Lazar

    2011-01-01

    Dietary intake of phytochemicals having antioxidant activity is associated with a lower risk of mortality from many diseases. Therefore, the aim of this study was to determine the free radical scavenging, cytotoxic and hemolytic activities of leaves of Acacia nilotica by using various methods. The results of the present study revealed that ethanol extract was the most effective and IC50 value was found to be 53.6 μg mL−1 for Vero cell lines and 28.9 μg mL−1 for Hela cell lines in cytotoxicity assays. The zone of color retention was 14.2 mm in β-carotene bleaching assay, which was as significant as positive control, butylated hydroxy toluene. None of the tested extracts possessed any hemolytic activity against rat and human erythrocytes revealing their cytotoxic mechanism and non-toxicity. Thus, only the ethanol extract could be considered as a potential source of anticancer and antioxidant compounds. Further phytochemical studies will be performed for specification of the biologically active principles. PMID:21799676

  8. Complement Factor H-Related 5-Hybrid Proteins Anchor Properdin and Activate Complement at Self-Surfaces.

    PubMed

    Chen, Qian; Manzke, Melanie; Hartmann, Andrea; Büttner, Maike; Amann, Kerstin; Pauly, Diana; Wiesener, Michael; Skerka, Christine; Zipfel, Peter F

    2016-05-01

    C3 glomerulopathy (C3G) is a severe kidney disease for which no specific therapy exists. The causes of C3G are heterogeneous, and defective complement regulation is often linked to C3G pathogenesis. Copy number variations in the complement factor H-related (CFHR) gene cluster on chromosome 1q32 and CFHR5 mutant proteins associate with this disease. Here, we identified CFHR5 as a pattern recognition protein that binds to damaged human endothelial cell surfaces and to properdin, the human complement activator. We found the two N-terminal short consensus repeat domains of CFHR5 contact properdin and mediate dimer formation. These properdin-binding segments are duplicated in two mutant CFHR5 proteins, CFHR2-CFHR5Hyb from German patients with C3G and CFHR5Dup from Cypriot patients with C3G. Each of these mutated proteins assembled into large multimeric complexes and, compared to CFHR5, bound damaged human cell surfaces and properdin with greater intensity and exacerbated local complement activation. This enhanced surface binding and properdin recruitment was further evidenced in the mesangia of a transplanted and explanted kidney from a German patient with a CFHR2-CFHR5Hyb protein. Enhanced properdin staining correlated with local complement activation with C3b and C5b-9 deposition on the mesangial cell surface in vitro This gain of function in complement activation for two disease-associated CFHR5 mutants describes a new disease mechanism of C3G, which is relevant for defining appropriate treatment options for this disorder. PMID:26432903

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

    PubMed Central

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

    2013-01-01

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

  10. Current treatment of atypical hemolytic uremic syndrome

    PubMed Central

    Kaplan, Bernard S.; Ruebner, Rebecca L.; Spinale, Joann M.; Copelovitch, Lawrence

    2014-01-01

    Summary Tremendous advances have been made in understanding the pathogenesis of atypical Hemolytic Uremic Syndrome (aHUS), an extremely rare disease. Insights into the molecular biology of aHUS resulted in rapid advances in treatment with eculizumab (Soliris®, Alexion Pharmaceuticals Inc.). Historically, aHUS was associated with very high rates of mortality and morbidity. Prior therapies included plasma therapy and/or liver transplantation. Although often life saving, these were imperfect and had many complications. We review the conditions included under the rubric of aHUS: S. pneumoniae HUS (SpHUS), inborn errors of metabolism, and disorders of complement regulation, emphasizing their differences and similarities. We focus on the clinical features, diagnosis, and pathogenesis, and treatment of aHUS that results from mutations in genes encoding alternative complement regulators, SpHUS and HUS associated with inborn errors of metabolism. Mutations in complement genes, or antibodies to their protein products, result in unregulated activity of the alternate complement pathway, endothelial injury, and thrombotic microangiopathy (TMA). Eculizumab is a humanized monoclonal antibody that inhibits the production of the terminal complement components C5a and the membrane attack complex (C5b-9) by binding to complement protein C5a. This blocks the proinflammatory and cytolytic effects of terminal complement activation. Eculizumab use has been reported in many case reports, and retrospective and prospective clinical trials in aHUS. There have been few serious side effects and no reports of tachphylaxis or drug resistance. The results are very encouraging and eculizumab is now recognized as the treatment of choice for aHUS. PMID:25343125

  11. Measuring initiator caspase activation by bimolecular fluorescence complementation.

    PubMed

    Parsons, Melissa J; Bouchier-Hayes, Lisa

    2015-01-01

    Initiator caspases, including caspase-2, -8, and -9, are activated by the proximity-driven dimerization that occurs after their recruitment to activation platforms. Here we describe the use of caspase bimolecular fluorescence complementation (caspase BiFC) to measure this induced proximity. BiFC assays rely on the use of a split fluorescent protein to identify protein-protein interactions in cells. When fused to interacting proteins, the fragments of the split fluorescent protein (which do not fluoresce on their own) can associate and fluoresce. In this protocol, we use the fluorescent protein Venus, a brighter and more photostable variant of yellow fluorescent protein (YFP), to detect the induced proximity of caspase-2. Plasmids encoding two fusion products (caspase-2 fused to either the amino- or carboxy-terminal halves of Venus) are transfected into cells. The cells are then treated with an activating (death) stimulus. The induced proximity (and subsequent activation) of caspase-2 in the cells is visualized as Venus fluorescence. The proportion of Venus-positive cells at a single time point can be determined using fluorescence microscopy. Alternatively, the increase in fluorescence intensity over time can be evaluated by time-lapse confocal microscopy. The caspase BiFC strategy described here should also work for other initiator caspases, such as caspase-8 or -9, as long as the correct controls are used. PMID:25561623

  12. Methylation of the phosphate oxygen moiety of phospholipid-methoxy(polyethylene glycol) conjugate prevents PEGylated liposome-mediated complement activation and anaphylatoxin production.

    PubMed

    Moghimi, S Moein; Hamad, Islam; Andresen, Thomas L; Jørgensen, Kent; Szebeni, Janos

    2006-12-01

    Methoxy(polyethylene glycol), mPEG, -grafted liposomes are known to exhibit prolonged circulation time in the blood, but their infusion into a substantial percentage of human subjects triggers immediate non-IgE-mediated hypersensitivity reactions. These reactions are strongly believed to arise from anaphylatoxin production through complement activation. Despite the general view that vesicle surface camouflaging with mPEG should dramatically suppress complement activation, here we show that bilayer enrichment of noncomplement activating liposomes [dipalmitoylphosphatidylcholine (DPPC) vesicles] with phospholipid-mPEG conjugate induces complement activation resulting in vesicle recognition by macrophage complement receptors. The extent of vesicle uptake, however, is dependent on surface mPEG density. We have delineated the likely structural features of phospholipid-mPEG conjugate responsible for PEGylated liposome-induced complement activation in normal as well as C1q-deficient human sera, using DPPC vesicles bearing the classical as well as newly synthesized lipid-mPEG conjugates. With PEGylated DPPC vesicles, the net anionic charge on the phosphate moiety of phospholipid-mPEG conjugate played a key role in activation of both classical and alternative pathways of complement and anaphylatoxin production (reflected in significant rises in SC5b-9, C4d, and C3a-desarg levels in normal human sera as well as SC5b-9 in EGTA-chelated/Mg2+ supplemented serum), since methylation of the phosphate oxygen of phospholipid-mPEG conjugate, and hence the removal of the negative charge, totally prevented complement activation. To further corroborate on the role of the negative charge in complement activation, vesicles bearing anionic phospholipid-mPEG conjugates, but not the methylated phospholipid-mPEG, were shown to significantly decrease serum hemolytic activity and increase plasma thromboxane B2 levels in rats. In contrast to liposomes, phospholipid-mPEG micelles had no effect on

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

    PubMed

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

    2015-05-01

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

  14. Systemic complement activation, lung injury, and products of lipid peroxidation.

    PubMed Central

    Ward, P A; Till, G O; Hatherill, J R; Annesley, T M; Kunkel, R G

    1985-01-01

    Previously we have demonstrated that systemic activation of the complement system after intravenous injection of cobra venom factor (CVF) results in acute lung injury as reflected by increases in the vascular permeability of the lung as well as by morphologic evidence of damage to lung vascular endothelial cells. In using the vascular permeability of the lung as the reference, the current studies show a quantitative correlation between lung injury and the appearance in plasma of lipid peroxidation products (conjugated dienes) as well as increased concentrations of lactic dehydrogenase (LDH) and one of its isoenzymes (LDH-4). After injection of CVF, extracts of lungs also showed elevated levels of conjugated dienes, whereas no elevations were found in extracts of liver, kidney, and spleen. There was no evidence in CVF-injected rats of renal or hepatic injury as reflected by the lack of development of proteinuria and the failure to detect increased serum levels of liver-related enzymes. Other peroxidation products identified in plasma of CVF-injected rats involved hydroperoxides and fluorescent compounds with features of Schiff bases. Not surprisingly, malondialdehyde was not found to be a reliable plasma indicator of lipid peroxidation associated with oxygen radical-mediated lung vascular injury. In using a model of oxygen radical-independent lung injury induced by oleic acid, although large amounts of LDH and LDH-4 were found in the plasma, no increases in plasma levels of conjugated dienes were detected. In CVF-injected animals treated with interventions protective against lung injury (neutrophil depletion, catalase, hydroxyl radical scavengers, or iron chelators), there were striking reductions in the plasma levels of conjugated dienes, hydroperoxides, and fluorochromic products. Morphometric analysis of lung sections revealed that the protective interventions did not interfere with the accumulation of neutrophils in lung interstitial capillaries after systemic

  15. Fatal carboplatin-induced immune hemolytic anemia in a child with a brain tumor

    PubMed Central

    Haley, Kristina M; Russell, Thomas B; Boshkov, Lynn; Leger, Regina M; Garratty, George; Recht, Michael; Nazemi, Kellie J

    2014-01-01

    Drug-induced immune hemolytic anemia (DIIHA) is an uncommon side effect of pharmacologic intervention. A rare mediator of DIIHA, carboplatin is an agent used to treat many pediatric cancers. We describe here, the first case of fatal carboplatin induced DIIHA in a pediatric patient and a brief review of the literature. Our patient developed acute onset of multi-organ failure with evidence of complement activation, secondary to a drug induced red cell antibody. Early recognition of the systemic insult associated with carboplatin induced hemolytic anemia may allow for future affected patients to receive plasmapheresis, a potentially effective therapy. PMID:24868179

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

    PubMed Central

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

    2015-01-01

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

  17. Antibody-independent activation of the classical pathway of complement by Epstein-Barr virus.

    PubMed

    Martin, H; McConnell, I; Gorick, B; Hughes-Jones, N C

    1987-03-01

    A purified preparation of Epstein-Barr virus (EBV) has been shown to activate the classical complement pathway by direct interaction with the first component of complement, C1, without the intervention of antibody. No evidence was found for activation of the alternative pathway. Following classical pathway activation the specific affinity of EBV for B cells can be presumed to be lost since the virus will become opsonized for clearance by phagocytic cells bearing complement receptors, CR1 and CR3. This activation is further evidence that complement plays a role in defence mechanisms independently of antibody activity. PMID:3038440

  18. Atypical Hemolytic-Uremic Syndrome: A Clinical Review.

    PubMed

    Nayer, Ali; Asif, Arif

    2016-01-01

    Atypical hemolytic-uremic syndrome (HUS) is a rare life-threatening disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and ischemic injury to organs, especially the kidneys. Microvascular injury and thrombosis are the dominant histologic findings. Complement activation through the alternative pathway plays a critical role in the pathogenesis of atypical HUS. Genetic abnormalities involving complement regulatory proteins and complement components form the molecular basis for complement activation. Endothelial cell dysfunction, probably because of the effects of complement activation, is an intermediate stage in the pathophysiologic cascade. Atypical HUS has a grave prognosis. Although mortality approaches 25% during the acute phase, end-stage renal disease develops in nearly half of patients within a year. Atypical HUS has a high recurrence rate after renal transplantation, and recurrent disease often leads to graft loss. Plasma therapy in the form of plasma exchange or infusion has remained the standard treatment for atypical HUS. However, many patients do not respond to plasma therapy and some require prolonged treatment. Approved by the Food and Drug Administration in the treatment of atypical HUS, eculizumab is a humanized monoclonal antibody that blocks cleavage of complement C5 into biologically active mediators of inflammation and cytolysis. Although case reports have shown the efficacy of eculizumab, randomized clinical trials are lacking. Therapeutic strategies targeting endothelial cells have demonstrated promising results in experimental settings. Therefore, inhibitors of angiotensin-converting enzyme, HMG-CoA reductase, and xanthine oxidase as well as antioxidants, such as ascorbic acid, may have salutary effects in patients with atypical HUS. PMID:24681522

  19. 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

  20. [Separation of lysozyme from sera with complement or antibody activities (author's transl)].

    PubMed

    Müller, F; Oetting, C; Beck, E

    1975-10-01

    A gel-filtration method for separating lysozyme from sera with haemolytic complement activity and/or antibody activity is described. It is shown that the gel-filtration has only a small effect, whereas bentonite absorption results in a known lost of haemolytic complement activity. PMID:129979

  1. 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

  2. Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway

    PubMed Central

    2014-01-01

    Background Therapeutic hypothermia is a treatment modality that is increasingly used to improve clinical neurological outcomes for ischemia-reperfusion injury-mediated diseases. Antibody-initiated classical complement pathway activation has been shown to contribute to ischemia-reperfusion injury in multiple disease processes. However, how therapeutic hypothermia affects complement activation is unknown. Our goal was to measure the independent effect of temperature on complement activation, and more specifically, examine the relationship between clinical hypothermia temperatures (31–33°C), and complement activation. Methods Antibody-sensitized erythrocytes were used to assay complement activation at temperatures ranging from 0-41°C. Individual complement pathway components were assayed by ELISA, Western blot, and quantitative dot blot. Peptide Inhibitor of complement C1 (PIC1) was used to specifically inhibit activation of C1. Results Antibody-initiated complement activation resulting in eukaryotic cell lysis was increased by 2-fold at 31°C compared with 37°C. Antibody-initiated complement activation in human serum increased as temperature decreased from 37°C until dramatically decreasing at 13°C. Quantitation of individual complement components showed significantly increased activation of C4, C3, and C5 at clinical hypothermia temperatures. In contrast, C1s activation by heat-aggregated IgG decreased at therapeutic hypothermia temperatures consistent with decreased enzymatic activity at lower temperatures. However, C1q binding to antibody-coated erythrocytes increased at lower temperatures, suggesting that increased classical complement pathway activation is mediated by increased C1 binding at therapeutic hypothermia temperatures. PIC1 inhibited hypothermia-enhanced complement-mediated cell lysis at 31°C by up to 60% (P = 0.001) in a dose dependent manner. Conclusions In summary, therapeutic hypothermia temperatures increased antibody

  3. 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. PMID:27388965

  4. Complement-mediated 'bystander' damage initiates host NLRP3 inflammasome activation.

    PubMed

    Suresh, Rahul; Chandrasekaran, Prabha; Sutterwala, Fayyaz S; Mosser, David M

    2016-05-01

    Complement activation has long been associated with inflammation, primarily due to the elaboration of the complement anaphylotoxins C5a and C3a. In this work, we demonstrate that the phagocytosis of complement-opsonized particles promotes host inflammatory responses by a new mechanism that depends on the terminal complement components (C5b-C9). We demonstrate that during the phagocytosis of complement-opsonized particles, the membrane attack complex (MAC) of complement can be transferred from the activating particle to the macrophage plasma membrane by a 'bystander' mechanism. This MAC-mediated bystander damage initiates NLRP3 inflammasome activation, resulting in caspase-1 activation and IL-1β and IL-18 secretion. Inflammasome activation is not induced when macrophages phagocytize unopsonized particles or particles opsonized with serum deficient in one of the terminal complement components. The secretion of IL-1β and IL-18 by macrophages depends on NLRP3, ASC (also known as PYCARD) and caspase-1, as macrophages deficient in any one of these components fail to secrete these cytokines following phagocytosis. The phagocytosis of complement-opsonized particles increases leukocyte recruitment and promotes T helper 17 cell (TH17) biasing. These findings reveal a new mechanism by which complement promotes inflammation and regulates innate and adaptive immunity. PMID:27006116

  5. 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. PMID:26119290

  6. 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. PMID:26911153

  7. A Novel Quantitative Hemolytic Assay Coupled with Restriction Fragment Length Polymorphisms Analysis Enabled Early Diagnosis of Atypical Hemolytic Uremic Syndrome and Identified Unique Predisposing Mutations in Japan

    PubMed Central

    Yoshida, Yoko; Miyata, Toshiyuki; Matsumoto, Masanori; Shirotani-Ikejima, Hiroko; Uchida, Yumiko; Ohyama, Yoshifumi; Kokubo, Tetsuro; Fujimura, Yoshihiro

    2015-01-01

    For thrombotic microangiopathies (TMAs), the diagnosis of atypical hemolytic uremic syndrome (aHUS) is made by ruling out Shiga toxin-producing Escherichia coli (STEC)-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP), often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs) and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH) monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45) had moderate-to-severe (≥50%) hemolysis, whereas the remaining 76% (34/45) patients had mild or no hemolysis (<50%). The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34), which were identified by restriction fragment length polymorphism (RFLP) analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45) of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients. PMID:25951460

  8. The role of complement activation in atherogenesis: the first 40 years.

    PubMed

    Vlaicu, Sonia I; Tatomir, Alexandru; Rus, Violeta; Mekala, Armugam P; Mircea, Petru A; Niculescu, Florin; Rus, Horea

    2016-02-01

    The pathogenesis of atherosclerotic inflammation is a multi-step process defined by the interweaving of excess modified lipid particles, monocyte-macrophages populations, and innate immune and adaptive immunity effectors. A part of innate immunity, the complement system, is an important player in the induction and progression of atherosclerosis. The accumulation of either oxidized or enzymatically modified LDL-bound to C-reactive protein or not-prompts complement activation leading to the assembly of the terminal complement C5b-9 complex in the atherosclerotic lesion. The sublytic C5b-9 assembly leads to the activation and proliferation of smooth muscle and endothelial cells, accompanied by the release of various chemotactic, pro-adhesion, and procoagulant cytokines from these cells. Response gene to complement (RGC)-32, an essential effector of the terminal complement complex C5b-9, also affects atherogenesis, propelling vascular smooth muscle cell proliferation and migration, stimulating endothelial proliferation, and promoting vascular lesion formation. A substantial amount of experimental work has suggested a role for the complement system activation during atherosclerotic plaque formation, with the proximal classical complement pathway seemingly having a protective effect and terminal complement contributing to accelerated atherogenesis. All these data suggest that complement plays an important role in atherogenesis. PMID:26091721

  9. In vitro and in vivo changes in human complement caused by silage.

    PubMed Central

    Olenchock, S A; May, J J; Pratt, D S; Lewis, D M; Mull, J C; Stallones, L

    1986-01-01

    Aqueous extracts of silage samples from four farms in up-state New York were reacted in vitro with normal human serum. Hemolytic levels of complement component C3 were consumed in a dose-dependent fashion, and the four extracts differed in their relative activity rankings. Studies with chelated serum indicate that the alternative complement pathway is involved in the activation, and the active fragment C3b was demonstrated. Serum levels of hemolytic C3 and C4 in vivo were quantified before and after farmers performed their normal silo unloading operations. Although the study groups were small, suggestive evidence of in vivo complement consumption was found. IgE-related allergy did not appear to be of significance to the study groups. Complement activation may be an initiator of or contributor to adverse reactions in farmers who are exposed to airborne silage dusts. Images FIGURE 2. FIGURE 3. PMID:3709488

  10. Regulation of complement and modulation of its activity in monoclonal antibody therapy of cancer

    PubMed Central

    Meyer, Saskia; Leusen, Jeanette HW; Boross, Peter

    2014-01-01

    The complement system is a powerful tool of the innate immune system to eradicate pathogens. Both in vitro and in vivo evidence indicates that therapeutic anti-tumor monoclonal antibodies (mAbs) can activate the complement system by the classical pathway. However, the contribution of complement to the efficacy of mAbs is still debated, mainly due to the lack of convincing data in patients. A beneficial role for complement during mAb therapy is supported by the fact that cancer cells often upregulate complement-regulatory proteins (CRPs). Polymorphisms in various CRPs were previously associated with complement-mediated disorders. In this review the role of complement in anti-tumor mAb therapy will be discussed with special emphasis on strategies aiming at modifying complement activity. In the future, clinical efficacy of mAbs with enhanced effector functions together with comprehensive analysis of polymorphisms in CRPs in mAb-treated patients will further clarify the role of complement in mAb therapy. PMID:25517299

  11. Partial ADAMTS13 deficiency in atypical hemolytic uremic syndrome

    PubMed Central

    Feng, Shuju; Eyler, Stephen J.; Zhang, Yuzhou; Maga, Tara; Nester, Carla M.; Kroll, Michael H.

    2013-01-01

    Complement dysregulation leads to atypical hemolytic uremic syndrome (aHUS), while ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura. We investigated whether genetic variations in the ADAMTS13 gene partially explain the reduced activity known to occur in some patients with aHUS. We measured complement activity and ADAMTS13 function, and completed mutation screening of multiple complement genes and ADAMTS13 in a large cohort of aHUS patients. In over 50% of patients we identified complement gene mutations. Surprisingly, 80% of patients also carried at least 1 nonsynonymous change in ADAMTS13, and in 38% of patients, multiple ADAMTS13 variations were found. Six of the 9 amino acid substitutions in ADAMTS13 were common single nucleotide polymorphisms; however, 3 variants—A747V, V832M, and R1096H— were rare, with minor allele frequencies of 0.0094%, 0.5%, and 0.32%, respectively. Reduced complement and ADAMTS13 activity (<60% of normal activity) were found in over 60% and 50% of patients, respectively. We concluded that partial ADAMTS13 deficiency is a common finding in aHUS patients and that genetic screening and functional tests of ADAMTS13 should be considered in these patients. PMID:23847193

  12. 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.

  13. [Atypical HUS caused by complement-related abnormalities].

    PubMed

    Yoshida, Yoko; Matsumoto, Masanori

    2015-02-01

    Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. The term aHUS was historically used to distinguish this disorder from Shiga-toxin producing Escherichia coli (STEC)-HUS. Many aHUS cases (approximately 70%) are reportedly caused by uncontrolled complement activation due to genetic mutations in the alternative pathway, including complement factor H (CFH), complement factor I (CFI), membrane cofactor protein (MCP), thrombomodulin (THBD), complement component C3 (C3), and complement factor B (CFB). Mutations in the coagulation pathway, such as diacylglycerol kinase ε (DGKE) and plasminogen, are also reported to be causes of aHUS. In this review, we have focused on aHUS due to complement dysfunction. aHUS is suspected based on plasma ADAMTS13 activity of 10% or more, and being negative for STEC-HUS, in addition to the aforementioned triad. Complement genetic studies provide a more specific diagnosis of aHUS. Plasma therapy is the first-line treatment for patients with aHUS and should be initiated as soon as the diagnosis is suspected. Recently, eculizumab, a humanized monoclonal antibody against C5, was shown to be an effective treatment for aHUS. Therefore, early diagnosis and identification of the underlying pathogenic mechanism is important for improving the outcome of aHUS. PMID:25765799

  14. Extra-Renal Manifestations of Complement-Mediated Thrombotic Microangiopathies

    PubMed Central

    Hofer, Johannes; Rosales, Alejandra; Fischer, Caroline; Giner, Thomas

    2014-01-01

    Thrombotic microangiopathies (TMA) are rare but severe disorders, characterized by endothelial cell activation and thrombus formation leading to hemolytic anemia, thrombocytopenia, and organ failure. Complement over activation in combination with defects in its regulation is described in an increasing number of TMA and if primary for the disease denominated as atypical hemolytic-uremic syndrome. Although TMA predominantly affects the renal microvasculature, extra-renal manifestations are observed in 20% of patients including involvement of the central nerve system, cardiovascular system, lungs, skin, skeletal muscle, and gastrointestinal tract. Prompt diagnosis and treatment initiation are therefore crucial for the prognosis of disease acute phase and the long-term outcome. This review summarizes the available evidence on extra-renal TMA manifestations and discusses the role of acute and chronic complement activation by highlighting its complex interaction with inflammation, coagulation, and endothelial homeostasis. PMID:25250305

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

    PubMed

    Cheong, Hae Il; Jo, Sang Kyung; Yoon, Sung Soo; Cho, Heeyeon; Kim, Jin Seok; Kim, Young Ok; Koo, Ja Ryong; Park, Yong; Park, Young Seo; Shin, Jae Il; Yoo, Kee Hwan; Oh, Doyeun

    2016-10-01

    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

  16. Cold agglutinin-mediated autoimmune hemolytic anemia.

    PubMed

    Berentsen, Sigbjørn; Randen, Ulla; Tjønnfjord, Geir E

    2015-06-01

    Cold antibody types account for about 25% of autoimmune hemolytic anemias. Primary chronic cold agglutinin disease (CAD) is characterized by a clonal lymphoproliferative disorder. Secondary cold agglutinin syndrome (CAS) complicates specific infections and malignancies. Hemolysis in CAD and CAS is mediated by the classical complement pathway and is predominantly extravascular. Not all patients require treatment. Successful CAD therapy targets the pathogenic B-cell clone. Complement modulation seems promising in both CAD and CAS. Further development and documentation are necessary before clinical use. We review options for possible complement-directed therapy. PMID:26043385

  17. 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.

  18. Candida tropicalis from veterinary and human sources shows similar in vitro hemolytic activity, antifungal biofilm susceptibility and pathogenesis against Caenorhabditis elegans.

    PubMed

    Brilhante, Raimunda Sâmia Nogueira; Oliveira, Jonathas Sales de; Evangelista, Antônio José de Jesus; Serpa, Rosana; Silva, Aline Lobão da; Aguiar, Felipe Rodrigues Magalhães de; Pereira, Vandbergue Santos; Castelo-Branco, Débora de Souza Collares Maia; Pereira-Neto, Waldemiro Aquino; Cordeiro, Rossana de Aguiar; Sidrim, José Júlio Costa; Rocha, Marcos Fábio Gadelha

    2016-08-30

    The aim of this study was to evaluate the in vitro hemolytic activity and biofilm antifungal susceptibility of veterinary and human Candida tropicalis strains, as well as their pathogenesis against Caenorhabditis elegans. Twenty veterinary isolates and 20 human clinical isolates of C. tropicalis were used. The strains were evaluated for their hemolytic activity and biofilm production. Biofilm susceptibility to itraconazole, fluconazole, voriconazole, amphotericin B and caspofungin was assessed using broth microdilution assay. The in vivo evaluation of strain pathogenicity was investigated using the nematode C. elegans. Hemolytic factor was observed in 95% of the strains and 97.5% of the isolates showed ability to form biofilm. Caspofungin and amphotericin B showed better results than azole antifungals against mature biofilms. Paradoxical effect on mature biofilm metabolic activity was observed at elevated concentrations of caspofungin (8-64μg/mL). Azole antifungals were not able to inhibit mature C. tropicalis biofilms, even at the higher tested concentrations. High mortality rates of C. elegans were observed when the worms were exposed to with C. tropicalis strains, reaching up to 96%, 96h after exposure of the worms to C. tropicalis strains. These results reinforce the high pathogenicity of C. tropicalis from veterinary and human sources and show the effectiveness of caspofungin and amphotericin B against mature biofilms of this species. PMID:27527785

  19. 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. PMID:26711574

  20. Targeting mechanisms at sites of complement activation for imaging and therapy.

    PubMed

    Holers, V Michael

    2016-06-01

    The complement system plays a key role in many acute injury states as well as chronic autoimmune and inflammatory diseases. Localized complement activation and alternative pathway-mediated amplification on diverse target surfaces promote local recruitment of pro-inflammatory cells and elaboration of other mediators. Despite a general understanding of the architecture of the system, though, many of the mechanisms that underlie site-specific complement activation and amplification in vivo are incompletely understood. In addition, there is no capability yet to measure the level of local tissue site-specific complement activation in patients without performing biopsies to detect products using immunohistochemical techniques. Herein is reviewed emerging evidence obtained through clinical research studies of human rheumatoid arthritis along with translational studies of its disease models which demonstrate that several parallel mechanisms are involved in site-specific amplification of activation of the complement system in vivo. Among these processes are de-regulation of the alternative pathway, effector pathway-catalyzed amplification of proximal complement activation, recognition of injury-associated ligands by components of the lectin pathway, and engagement of pathogenic natural antibodies that recognize a limited set of injury-associated neoepitopes. Studies suggest that each of these inter-related processes can play key roles in amplification of complement-dependent injury on self-tissues in vivo. These findings, in addition to development of an imaging strategy described herein designed to quantitatively measure local complement C3 fixation, have relevance to therapeutic and diagnostic strategies targeting the complement system. PMID:25979851

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

    PubMed

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

    2016-01-01

    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

  2. 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

  3. Complement regulation: physiology and disease relevance

    PubMed Central

    2015-01-01

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

  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. Complement Test

    MedlinePlus

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

  6. C1q binding and complement activation by prions and amyloids.

    PubMed

    Sim, Robert B; Kishore, Uday; Villiers, Christian L; Marche, Patrice N; Mitchell, Daniel A

    2007-01-01

    C1q binds to many non-self and altered-self-materials. These include microorganisms, immune complexes, apoptotic and necrotic cells and their breakdown products, and amyloids. C1q binding to amyloid fibrils found as extracellular deposits in tissues, and subsequent complement activation are involved in the pathology of several amyloid diseases, such as Alzheimer's disease. Prion diseases, such as scrapie also involve formation of amyloid by polymerization of the host prion protein (PrP). Complement activation is likely to contribute to neuronal damage in the end stages of prion diseases, but is also thought to participate in the initial infection, dissemination and replication stages. Infectious prion particles are likely to bind C1q and activate the complement system. Bound complement proteins may then influence the uptake and transport of prion particles by dendritic cells (DCs) and their subsequent proliferation at sites such as follicular DCs. PMID:17544820

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

    PubMed

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

    2016-01-01

    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

  8. Characterization of a Novel Hemolytic Activity of Human IgG Fractions Arising from Diversity in Protein and Oligosaccharide Components

    PubMed Central

    Zhang, Yueling; Ye, Xiangqun; Zhong, Mingqi; Cao, Jinsong; Zou, Haiying; Chen, Jiehui

    2014-01-01

    Human IgG is a well-established multifunctional antigen specific immunoglobulin molecule of the adaptive immune system. However, an antigen nonspecific immunological function of human IgG has never been reported. In this study, human IgG was isolated using ammonium sulfate fractional precipitation and diethylaminoethanol (DEAE) cellulose 52 ion exchange chromatography, from which h-IgG and hs-IgG fractions were purified on the basis of their differential binding to rabbit anti-shrimp hemocyanin antibody (h) and rabbit anti-shrimp hemocyanin's small subunit antibody (hs), respectively. We found that h-IgG had a higher hemolytic activity than hs-IgG against erythrocytes from humans, rabbits, mice and chickens, whereas the control IgG showed negligible activity. h-IgG could interact directly with erythrocyte membranes, and this interaction was suppressed by high molecular weight osmoprotectants, showing that it may follow a colloid-osmotic mechanism. In comparative proteomics and glycomics studies, h-IgG and hs-IgG yielded 20 and 5 significantly altered protein spots, respectively, on a 2-D gel. The mean carbohydrate content of h-IgG and hs-IgG was approximately 3.6- and 2-fold higher than that of IgG, respectively, and the α-d-mannose/α-d-glucose content was in the order of h-IgG>hs-IgG>IgG. In this study, a novel antigen nonspecific immune property of human IgG was investigated, and the diversity in the protein constituents and glycosylation levels may have functional signficance. PMID:24465658

  9. A novel method for direct measurement of complement convertases activity in human serum.

    PubMed

    Blom, A M; Volokhina, E B; Fransson, V; Strömberg, P; Berghard, L; Viktorelius, M; Mollnes, T E; López-Trascasa, M; van den Heuvel, L P; Goodship, T H; Marchbank, K J; Okroj, M

    2014-10-01

    Complement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function leads directly or indirectly to pathological conditions, including higher infection rate, kidney diseases, autoimmune- or neurodegenerative diseases and ischaemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and, thus, convertase formation occurs under non-physiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. The use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of haemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong the half-life of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay. PMID:24853370

  10. [L forms of Staphylococcus aureus. Behavior of coagulase, hemolytic and desoxyribonuclease activities and antibiotic sensitivity].

    PubMed

    Loschiavo, F; Giarrizzo, S

    1977-01-01

    L Forms derived from strains of coagulase positive Staphylococcus aureus, have, on the whole, preserved their DNAsic, haemolitic and coagulastic activities. L. forms showed high resistence to antibiotics acting on the bacterial cell-wall. The sensibility to other antibiotics was, roughly, analogous for the L forms as well as for the bacterial strains ones, with the exception of the clortetraciclin and the diidrostreptomicin, ehich proved to be comparatively more active on the L forms. PMID:614141

  11. Inhibition of aberrant complement activation by a dimer of acetylsalicylic acid.

    PubMed

    Lee, Moonhee; Wathier, Matthew; Love, Jennifer A; McGeer, Edith; McGeer, Patrick L

    2015-10-01

    We here report synthesis for the first time of the acetyl salicylic acid dimer 5,5'-methylenebis(2-acetoxybenzoic acid) (DAS). DAS inhibits aberrant complement activation by selectively blocking factor D of the alternative complement pathway and C9 of the membrane attack complex. We have previously identified aurin tricarboxylic and its oligomers as promising agents in this regard. DAS is much more potent, inhibiting erythrocyte hemolysis by complement-activated serum with an IC50 in the 100-170 nanomolar range. There are numerous conditions where self-damage from the complement system has been implicated in the pathology, including such chronic degenerative diseases of aging as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and age-related macular degeneration. Consequently, there is a high priority for the discovery and development of agents that can successfully treat such conditions. DAS holds considerable promise for being such an agent. PMID:26248865

  12. Strategies for Surveillance of Pediatric Hemolytic Uremic Syndrome: Foodborne Diseases Active Surveillance Network (FoodNet), 2000–2007

    PubMed Central

    Ong, Kanyin L.; Apostal, Mirasol; Comstock, Nicole; Hurd, Sharon; Webb, Tameka Hayes; Mickelson, Stephanie; Scheftel, Joni; Smith, Glenda; Shiferaw, Beletshachew; Boothe, Effie

    2012-01-01

    Background. Postdiarrheal hemolytic uremic syndrome (HUS) is the most common cause of acute kidney failure among US children. The Foodborne Diseases Active Surveillance Network (FoodNet) conducts population-based surveillance of pediatric HUS to measure the incidence of disease and to validate surveillance trends in associated Shiga toxin–producing Escherichia coli (STEC) O157 infection. Methods. We report the incidence of pediatric HUS, which is defined as HUS in children <18 years. We compare the results from provider-based surveillance and hospital discharge data review and examine the impact of different case definitions on the findings of the surveillance system. Results. During 2000–2007, 627 pediatric HUS cases were reported. Fifty-two percent of cases were classified as confirmed (diarrhea, anemia, microangiopathic changes, low platelet count, and acute renal impairment). The average annual crude incidence rate for all reported cases of pediatric HUS was 0.78 per 100 000 children <18 years. Regardless of the case definition used, the year-to-year pattern of incidence appeared similar. More cases were captured by provider-based surveillance (76%) than by hospital discharge data review (68%); only 49% were identified by both methods. Conclusions. The overall incidence of pediatric HUS was affected by key characteristics of the surveillance system, including the method of ascertainment and the case definitions. However, year-to-year patterns were similar for all methods examined, suggesting that several approaches to HUS surveillance can be used to track trends. PMID:22572665

  13. Molecules Great and Small: The Complement System.

    PubMed

    Mathern, Douglas R; Heeger, Peter S

    2015-09-01

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

  14. Antifungal Imidazole-Decorated Cationic Amphiphiles with Markedly Low Hemolytic Activity.

    PubMed

    Benhamou, Raphael I; Steinbuch, Kfir B; Fridman, Micha

    2016-08-01

    Herein we report that an imidazole-decorated cationic amphiphile derived from the pseudo-disaccharide nebramine has potent antifungal activity against strains of Candida glabrata pathogens. In combination with the natural bis-benzylisoquinoline alkaloid tetrandrine the reported antifungal cationic amphiphile demonstrated synergistic antifungal activity against Candida albicans pathogens. This unique membrane disruptor caused no detectible mammalian red blood cell hemolysis at concentrations up to more than two orders of magnitude greater than its minimal inhibitory concentrations against the tested C. glabrata strains. We provide evidence that potency against C. glabrata may be associated with differences between the drug efflux pumps of C. albicans and C. glabrata. Imidazole decorated-cationic amphiphiles show promise for the development of less toxic membrane-disrupting antifungal drugs and drug combinations. PMID:27258738

  15. Soluble complement complex C5b-9 promotes microglia activation.

    PubMed

    Yang, Chao; Yang, Li; Liu, Yong

    2014-02-15

    Soluble C5b-9 has been described as a pro-inflammatory mediator that triggers cell activation rather than inducing cell death. Microglia is the most important immune cell involved in inflammatory response in the CNS. Although microglia activation induced by various stimuli has been well characterized, the role of C5b-9 in microglia has not been well studied. In the current experiment, we utilized assembled functional C5b-9 to treat microglia and analyzed the function. We found that soluble C5b-9 could promote microglia activation by up-regulation of costimulatory molecules and increase cytokine secretion. Our results suggested that soluble C5b-9 possessed immunoregulatory potential on microglia. PMID:24434076

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

    PubMed Central

    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. PMID:27014265

  17. A novel peptide inhibitor of classical and lectin complement activation including ABO incompatibility

    PubMed Central

    Mauriello, Clifford T.; Pallera, Haree K.; Sharp, Julia A.; Woltmann, Jon L.; Qian, Shizhi; Hair, Pamela S.; van der Pol, Pieter; van Kooten, Cees; Thielens, Nicole M.; Lattanzio, Frank A.; Cunnion, Kenji M.; Krishna, Neel K.

    2012-01-01

    Previous experiments from our laboratories have identified peptides derived from the human astrovirus coat protein (CP) that bind C1q and mannose binding lectin (MBL) inhibiting activation of the classical and lectin pathways of complement, respectively. The purpose of this study was to evaluate the function of these coat protein peptides (CPPs) in an in vitro model of complement-mediated disease (ABO incompatibility), preliminarily assess their in vivo complement suppression profile and develop more highly potent derivatives of these molecules. E23A, a 30 amino acid CPP derivative previously demonstrated to inhibit classical pathway activation was able to dose-dependently inhibit lysis of AB erythrocytes treated with mismatched human O serum. Additionally, when injected into rats, E23A inhibited the animals’ serum from lysing antibody-sensitized erythrocytes, providing preliminary in vivo functional evidence that this CPP can cross the species barrier to inhibit serum complement activity in rodents. A rational drug design approach was implemented to identify more potent CPP derivatives, resulting in the identification and characterization of a 15 residue peptide (Polar Assortant (PA)), which demonstrated both superior inhibition of classical complement pathway activation and robust binding to C1q collagen-like tails. PA also inhibited ABO incompatibility in vitro and demonstrated in vivo complement suppression up to 24 hours post-injection. CPP’s ability to inhibit ABO incompatibility in vitro, proof of concept in vivo inhibitory activity in rats and the development of the highly potent PA derivative set the stage for preclinical testing of this molecule in small animal models of complement-mediated disease. PMID:22906481

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

    PubMed

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

    2015-11-01

    Colitis-associated colorectal cancer (CAC) is the most serious complication of inflammatory bowel disease (IBD). Excessive complement activation has been shown to be involved in the pathogenesis of IBD. However, its role in the development of CAC is largely unknown. Here, using a CAC model induced by combined administration of azoxymethane (AOM) and dextran sulfate sodium (DSS), we demonstrated that complement activation was required for CAC pathogenesis. Deficiency in key components of complement (e.g., C3, C5, or C5a receptor) rendered tumor repression in mice subjected to AOM/DSS. Mechanistic investigation revealed that complement ablation dramatically reduced proinflammatory cytokine interleukin (IL)-1β levels in the colonic tissues that was mainly produced by infiltrating neutrophils. IL-1β promoted colon carcinogenesis by eliciting IL-17 response in intestinal myeloid cells. Furthermore, complement-activation product C5a represented a potent inducer for IL-1β in neutrophil, accounting for downregulation of IL-1β levels in the employed complement-deficient mice. Overall, our study proposes a protumorigenic role of complement in inflammation-related colorectal cancer and that the therapeutic strategies targeting complement may be beneficial for the treatment of CAC in clinic. PMID:25736459

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

    PubMed

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

    2014-01-01

    Activity-guided fractionation for complement inhibitors led to the isolation of 23 known compounds from Houttuynia cordata Thunb. Seven flavonoids, two alkaloids, one coumarin and two phenols showed anti-complementary activity. Preliminary inhibitory mechanism of four flavonoids, including quercitrin, afzelin, isoquercitrin and quercetin in the complement activation cascade were examined for the first time. The results indicated that the target components of flavonols are different from those of flavonosides, and the glycoside moieties may be necessary to block C3 and C4 components. PMID:24423008

  20. Deficient activity of the alternative pathway of complement in beta thalassemia major.

    PubMed

    Corry, J M; Marshall, W C; Guthrie, L A; Peerless, A G; Johnston, R B

    1981-06-01

    Patients with thalassemia major suffer frequent and serious infections, especially after splenectomy. To explore the basis for this susceptibility, we examined activity of the complement system in sera from 24 patients. All sera had normal or increased activity of the classic complement pathway. However, six of the 24 (three with and three without splenectomy) had abnormal alternative pathway function, and mean alternative pathway activity was significantly decreased in both splenectomized and nonsplenectomized patients. Mean concentrations of C3, factor B, properdin, and immunoglobulins were normal. Defective alternative pathway function, especially in conjunction with asplenia, could contribute to the propensity to infection that exists in thalassemia. PMID:6908998

  1. Polyphosphate suppresses complement via the terminal pathway

    PubMed Central

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

    2014-01-01

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

  2. Structure-activity relationships for substrate-based inhibitors of human complement factor B.

    PubMed

    Ruiz-Gómez, Gloria; Lim, Junxian; Halili, Maria A; Le, Giang T; Madala, Praveen K; Abbenante, Giovanni; Fairlie, David P

    2009-10-01

    Human complement is a cascading network of plasma proteins important in immune defense, cooperatively effecting recognition, opsonization, destruction, and removal of pathogens and infected/damaged cells. Overstimulated or unregulated complement activation can result in immunoinflammatory diseases. Key serine proteases in this cascade are difficult to study due to their multiprotein composition, short lifetimes, formation on membranes, or serum circulation as inactive zymogens. Factor B is inactive at pH 7, but a catalytically active serine protease under alkaline conditions, enabling structure-activity relationship studies for 63 substrate-based peptide inhibitors with 4-7 residues and a C-terminal aldehyde. A potent factor B inhibitor was hexpeptide Ac-RLTbaLAR-H (IC(50) 250 nM, pH 9.5), which at pH 7 also blocked formation of membrane attack complex via the "alternative pathway" of complement activation and inhibited human complement mediated lysis of rabbit erythrocytes. Inhibitors of factor B may be valuable probes and drug leads for complement mediated immunity and disease. PMID:19743866

  3. Mild hypothermia inhibits systemic and cerebral complement activation in a swine model of cardiac arrest

    PubMed Central

    Gong, Ping; Zhao, Hong; Hua, Rong; Zhang, Mingyue; Tang, Ziren; Mei, Xue; Cui, Juan; Li, Chunsheng

    2015-01-01

    Complement activation has been implicated in ischemia/reperfusion injury. This study aimed to determine whether mild hypothermia (HT) inhibits systemic and cerebral complement activation after resuscitation from cardiac arrest. Sixteen minipigs resuscitated from 8 minutes of untreated ventricular fibrillation were randomized into two groups: HT group (n=8), treated with HT (33°C) for 12 hours; and normothermia group (n=8), treated similarly as HT group except for cooling. Blood samples were collected at baseline and 0.5, 6, 12, and 24 hours after return of spontaneous circulation (ROSC). The brain cortex was harvested 24 hours after ROSC. Complement and pro-inflammatory markers were detected using enzyme-linked immunosorbent assay. Neurologic deficit scores were evaluated 24 hours after ROSC. C1q, Bb, mannose-binding lectin (MBL), C3b, C3a, C5a, interleukin-6, and tumor necrosis factor-α levels were significantly increased under normothermia within 24 hours after ROSC. However, these increases were significantly reduced by HT. Hypothermia decreased brain C1q, MBL, C3b, and C5a contents 24 hours after ROSC. Hypothermic pigs had a better neurologic outcome than normothermic pigs. In conclusion, complement is activated through classic, alternative, and MBL pathways after ROSC. Hypothermia inhibits systemic and cerebral complement activation, which may provide an additional mechanism of cerebral protection. PMID:25757755

  4. Noninvasive Imaging of Activated Complement in Ischemia-Reperfusion Injury Post–Cardiac Transplant

    PubMed Central

    Sharif-Paghaleh, E; Yap, M L; Meader, L L; Chuamsaamarkkee, K; Kampmeier, F; Badar, A; Smith, R A; Sacks, S; Mullen, G E

    2015-01-01

    Ischemia-reperfusion injury (IRI) is inevitable in solid organ transplantation, due to the transplanted organ being ischemic for prolonged periods prior to transplantation followed by reperfusion. The complement molecule C3 is present in the circulation and is also synthesized by tissue parenchyma in early response to IRI and the final stable fragment of activated C3, C3d, can be detected on injured tissue for several days post-IRI. Complement activation post-IRI was monitored noninvasively by single photon emission computed tomography (SPECT) and CT using 99mTc-recombinant complement receptor 2 (99mTc-rCR2) in murine models of cardiac transplantation following the induction of IRI and compared to 99mTc-rCR2 in C3−/− mice or with the irrelevant protein 99mTc-prostate–specific membrane antigen antibody fragment (PSMA). Significant uptake with 99mTc-rCR2 was observed as compared to C3−/− or 99mTc-PSMA. In addition, the transplanted heart to muscle ratio of 99mTc-rCR2 was significantly higher than 99mTc-PSMA or C3−/−. The results were confirmed by histology and autoradiography. 99mTc-rCR2 can be used for noninvasive detection of activated complement and in future may be used to quantify the severity of transplant damage due to complement activation postreperfusion. PMID:25906673

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

    PubMed

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

    2016-06-15

    Herpes simplex encephalitis (HSE) is characterized by a pronounced inflammatory activity in the central nervous system (CNS). Here, we investigated the acute and prolonged complement system activity in HSE patients, by using enzyme-linked immunosorbent assays (ELISAs) for numerous complement components (C). We found increased cerebrospinal fluid concentrations of C3a, C3b, C5 and C5a in HSE patients compared with healthy controls. C3a and C5a concentrations remained increased also compared with patient controls. Our results conclude that the complement system is activated in CNS during HSE in the acute phase, and interestingly also in later stages supporting previous reports of prolonged inflammation. PMID:27235358

  6. Application of a hemolysis assay for analysis of complement activation by perfluorocarbon nanoparticles

    PubMed Central

    Pham, Christine T.N.; Thomas, Dennis G.; Beiser, Julia; Mitchell, Lynne M.; Huang, Jennifer L.; Senpan, Angana; Hu, Grace; Gordon, Mae; Baker, Nathan A.; Pan, Dipanjan; Lanza, Gregory M.; Hourcade, Dennis E.

    2013-01-01

    Nanoparticles offer new options for medical diagnosis and therapeutics with their capacity to specifically target cells and tissues with imaging agents and/or drug payloads. The unique physical aspects of nanoparticles present new challenges for this promising technology. Studies indicate that nanoparticles often elicit moderate to severe complement activation. Using human in vitro assays that corroborated the mouse in vivo results we previously presented mechanistic studies that define the pathway and key components involved in modulating complement interactions with several gadolinium-functionalized perfluorocarbon nanoparticles (PFOB). Here we employ a modified in vitro hemolysis-based assay developed in conjunction with the mouse in vivo model to broaden our analysis to include PFOBs of varying size, charge and surface chemistry and examine the variations in nanoparticle-mediated complement activity between individuals. This approach may provide the tools for an in-depth structure-activity relationship study that will guide the eventual development of biocompatible nanoparticles. PMID:24211337

  7. Chemotaxigenesis and activation of the alternative complement pathway by encapsulated and non-encapsulated Cryptococcus neoformans.

    PubMed Central

    Laxalt, K A; Kozel, T R

    1979-01-01

    In the presence of serum, whole cells of encapsulated and non-encapsulated Cryptococcus neoformans generated a chemotactic response by neutrophils. Heat inactivation of serum ablated all chemotactic activity. Cryptococcal polysaccharide was not chemotaxigenic. Assays for alternative complement pathway activation such as depletion of alternative complement pathway factor B or electrophoretic conversion of factor B closely paralleled chemotaxis assays. Cells of encapsulated and non-encapsulated C. neoformans activated the alternative complement pathway, whereas cryptococcal polysaccharide was inactive. Failure of the capsular material to activate the alternative pathway was not due to serotype specificity because polysaccharide of several serotypes failed to achieve activation. The results suggest that chemotaxigenesis and alternative complement pathway activation are functions of the yeast cell wall. The results support our proposal that the cryptococcal capsul does not prevent potential opsonins from reaching binding and activation sites at the yeast cell wall or the release of biologically active soluble cleavage products into the surrounding medium; however, cell wall-bound cleavage products remain bound to the cell wall beneath the capsule. Therefore, they are unable to participate as opsonins in phagocytosis. PMID:397927

  8. 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…

  9. 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

  10. Monomeric C-reactive protein inhibits renal cell-directed complement activation mediated by properdin.

    PubMed

    O'Flynn, Joseph; van der Pol, Pieter; Dixon, Karen O; Prohászka, Zoltán; Daha, Mohamed R; van Kooten, Cees

    2016-06-01

    Previous studies have shown that complement activation on renal tubular cells is involved in the induction of interstitial fibrosis and cellular injury. Evidence suggests that the tubular cell damage is initiated by the alternative pathway (AP) of complement with properdin having an instrumental role. Properdin is a positive regulator of the AP, which can bind necrotic cells as well as viable proximal tubular epithelial cells (PTECs), inducing complement activation. Various studies have indicated that in the circulation there is an unidentified inhibitor of properdin. We investigated the ability of C-reactive protein (CRP), both in its monomeric (mCRP) and pentameric (pCRP) form, to inhibit AP activation and injury in vitro on renal tubular cells by fluorescent microscopy, ELISA, and flow cytometry. We demonstrated that preincubation of properdin with normal human serum inhibits properdin binding to viable PTECs. We identified mCRP as a factor able to bind to properdin in solution, thereby inhibiting its binding to PTECs. In contrast, pCRP exhibited no such binding and inhibitory effect. Furthermore, mCRP was able to inhibit properdin-directed C3 and C5b-9 deposition on viable PTECs. The inhibitory ability of mCRP was not unique for viable cells but also demonstrated for binding to necrotic Jurkat cells, a target for properdin binding and complement activation. In summary, mCRP is an inhibitor of properdin in both binding to necrotic cells and viable renal cells, regulating complement activation on the cell surface. We propose that mCRP limits amplification of tissue injury by controlling properdin-directed complement activation by damaged tissue and cells. PMID:26984957

  11. Bactericidal/permeability-increasing protein promotes complement activation for neutrophil-mediated phagocytosis on bacterial surface

    PubMed Central

    Nishimura, H; Gogami, A; Miyagawa, Y; Nanbo, A; Murakami, Y; Baba, T; Nagasawa, S

    2001-01-01

    The neutrophil bactericidal/permeability-increasing protein (BPI) has both bactericidal and lipopolysaccharide-neutralizing activities. The present study suggests that BPI also plays an important role in phagocytosis of Escherichia coli by neutrophils through promotion of complement activation on the bacterial surface. Flow cytometric analysis indicated that fluorescein-labelled E. coli treated with BPI were phagocytosed in the presence of serum at two- to five-fold higher levels than phagocytosis of the bacteria without the treatment. In contrast, phagocytosis of the fluoresceined bacteria with or without treatment by BPI did not occur at all in the absence of serum. The phagocytosis stimulated by BPI and serum was dose-dependent. The effect of BPI on phagocytosis in the presence of serum was not observed on Gram-positive bacteria (Staphylococcus aureus). Interestingly, the complement C3b/iC3b fragments were deposited onto the bacterial surface also as a function of the BPI concentration under conditions similar to those for phagocytosis. Furthermore, the BPI-promoted phagocytosis was blocked completely by anti-C3 F(ab′)2 and partially by anti-complement receptor (CR) type 1 and/or anti-CR type 3. These findings suggest that BPI accelerates complement activation to opsonize bacteria with complement-derived fragments, leading to stimulation of phagocytosis by neutrophils via CR(s). PMID:11529944

  12. Assessment of phytochemicals, antioxidant, anti-lipid peroxidation and anti-hemolytic activity of extract and various fractions of Maytenus royleanus leaves

    PubMed Central

    2013-01-01

    Background Maytenus royleanus is traditionally used in gastro-intestinal disorders. The aim of this study was to evaluate the methanol extract of leaves and its derived fractions for various antioxidant assays and for its potential against lipid peroxidation and hemolytic activity. Methods Various parameters including scavenging of free-radicals (DPPH, ABTS, hydroxyl and superoxide radical), hydrogen peroxide scavenging, Fe3+ to Fe2+ reducing capacity, total antioxidant capacity, anti-lipid peroxidation and anti-hemolytic activity were investigated. Methanol extract and its derived fractions were also subjected for chemical constituents. LC-MS was also performed on the methanol extract. Results Qualitative analysis of methanol extract exhibited the presence of alkaloids, anthraquinones, cardiac glycosides, coumarins, flavonoids, saponins, phlobatannins, tannins and terpenoids. LC-MS chromatogram indicated the composition of diverse compounds including flavonoids, phenolics and phytoestrogens. Methanol extract, its ethyl acetate and n-butanol fractions constituted the highest amount of total phenolic and flavonoid contents and showed a strong correlation coefficient with the IC50 values for the scavenging of DPPH, hydrogen peroxide radicals, superoxide radicals, anti-lipid peroxidation and anti-hemolytic efficacy. Moreover, n-butanol fraction showed the highest scavenging activity for ABTS radicals and for reduction of Fe3+ to Fe2+. Conclusions Present results suggested the therapeutic potential of Maytenus royleanus leaves, in particular, methanol extract, ethyl acetate and n-butanol fraction as therapeutic agent against free-radical associated damages. The protective potential of the extract and or fraction may be attributed due to the high concentration of phenolic, flavonoid, tannins and terpenoids. PMID:23800043

  13. 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. PMID:19356139

  14. A thermoactive secreted phospholipase A₂ purified from the venom glands of Scorpio maurus: relation between the kinetic properties and the hemolytic activity.

    PubMed

    Louati, Hanen; Krayem, Najeh; Fendri, Ahmed; Aissa, Imen; Sellami, Mohamed; Bezzine, Sofiane; Gargouri, Youssef

    2013-09-01

    A lipolytic activity was located in the scorpion venom glands (telsons), from which a phospholipase A₂ (Sm-PLVG) was purified. Like known phospholipases A₂ from scorpion venom, which are 14-18 kDa proteins, the purified Scorpio maurus-Phospholipase from Venom Glands (Sm-PLVG) has a molecular mass of 17 kDa containing long and short chains linked by disulfide bridge. It has a specific activity of 5500 U/mg measured at 47 °C and pH 8.5 using phosphatidylcholine as a substrate in presence of 8 mM NaTDC and 12 mM CaCl₂. The NH₂-terminal amino acid sequences of the purified Sm-PLVG showed similarities with those of long and short chains of some previously purified phospholipases from venom scorpions. Moreover, the Sm-PLVG exhibits hemolytic activity toward human, rabbit or rat erythrocytes. This hemolytic activity was related to its ability to interact with phospholipids' monolayer at high surface pressure. These properties are similar to those of phospholipases isolated from snake venoms. PMID:23831286

  15. Local and systemic activation of the whole complement cascade in human leukocytoclastic cutaneous vasculitis; C3d,g and terminal complement complex as sensitive markers.

    PubMed Central

    Dauchel, H; Joly, P; Delpech, A; Thomine, E; Sauger, F; Le Loet, X; Lauret, P; Tron, F; Fontaine, M; Ripoche, J

    1993-01-01

    We have studied complement activation both in plasma samples and in lesional skin from patients with leukocytoclastic cutaneous vasculitis (LCV). Enzyme immunoassay (EIA) quantification of the complement activation markers, C3d,g and the terminal complement complex (TCC) in plasma, showed that their levels were significantly increased in 66% and 55% of the patients, respectively (n = 29) compared with healthy controls, whereas the standard measurements of C3, factor B, C1q, C4 and C2 were generally within normal range. Elevations of C3d,g and TCC levels in plasma were significantly correlated. Importantly, a significant correlation was found between the severity of the vasculitis and both C3d,g and TCC plasma levels. Immunofluorescence studies of skin biopsy specimens demonstrated simultaneous presence of perivascular dermal deposits of C3d,g and TCC in lesional skin from 96% and 80% respectively of the patients (n = 25). There was a significant correlation between the intensity of the deposits of both markers. Clusterin, a TCC inhibitory protein, was always found at the same sites of perivascular TCC deposits. Immunofluorescence studies at the epidermal basement membrane zone (BMZ) revealed in each case deposits of C3d,g which were accompanied by TCC deposits in 52% of the biopsy specimens. These data demonstrate that there is a local and systemic activation of the whole complement cascade in human LCV. The presence of both C3d,g and clusterin-associated TCC perivascular deposits suggests an intervention of a regulatory mechanism of local complement activation in LCV. Finally, measurement of plasma C3d,g and TCC appears to be a sensitive indicator of systemic complement activation and disease severity in LCV. Images Fig. 1 Fig. 2 PMID:8485913

  16. Alginate microsphere compositions dictate different mechanisms of complement activation with consequences for cytokine release and leukocyte activation.

    PubMed

    Ørning, Pontus; Hoem, Kine Samset; Coron, Abba Elizabeth; Skjåk-Bræk, Gudmund; Mollnes, Tom Eirik; Brekke, Ole-Lars; Espevik, Terje; Rokstad, Anne Mari

    2016-05-10

    The inflammatory potential of 12 types of alginate-based microspheres was assessed in a human whole blood model. The inflammatory potential could be categorized from low to high based on the four main alginate microsphere types; alginate microbeads, liquefied core poly-l-ornithine (PLO)-containing microcapsules, liquefied core poly-l-lysine (PLL)-containing microcapsules, and solid core PLL-containing microcapsules. No complement or inflammatory cytokine activation was detected for the Ca/Ba alginate microbeads. Liquefied core PLO- and PLL-containing microcapsules induced significant fluid phase complement activation (TCC), but with low complement surface deposition (anti-C3c), and a low proinflammatory cytokine secretion, with exception of an elevated MCP-1(CCL2) secretion. The solid core PLL-containing microcapsules generated lower TCC but a marked complement surface deposition and significant induction of the proinflammatory cytokines interleukin (IL-1)β, TNF, IL-6, the chemokines IL-8 (CXCL8), and MIP-1α (CCL3) and MCP-1(CCL2). Inhibition with compstatin (C3 inhibitor) completely abolished complement surface deposition, leukocyte adhesion and the proinflammatory cytokines. The C5 inhibitions partly lead to a reduction of the proinflammatory cytokines. The leukocyte adhesion was abolished by inhibitory antibodies against CD18 and partly reduced by CD11b, but not by CD11c. Anti-CD18 significantly reduced the (IL-1)β, TNF, IL-6 and MIP-1α and anti-CD11b significantly reduced the IL-6 and VEGF secretion. MCP-1 was strongly activated by anti-CD18 and anti-CD11b. In conclusion the initial proinflammatory cytokine responses are driven by the microspheres potential to trigger complement C3 (C3b/iC3b) deposition, leukocyte activation and binding through complement receptor CR3 (CD11b/CD18). MCP-1 is one exception dependent on the fluid phase complement activation mediated through CR3. PMID:26993426

  17. 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

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

    PubMed

    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

  19. Complement factor H related proteins (CFHRs).

    PubMed

    Skerka, Christine; Chen, Qian; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T

    2013-12-15

    Factor H related proteins comprise a group of five plasma proteins: CFHR1, CFHR2, CFHR3, CFHR4 and CFHR5, and each member of this group binds to the central complement component C3b. Mutations, genetic deletions, duplications or rearrangements in the individual CFHR genes are associated with a number of diseases including atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathies (C3 glomerulonephritis (C3GN), dense deposit disease (DDD) and CFHR5 nephropathy), IgA nephropathy, age related macular degeneration (AMD) and systemic lupus erythematosus (SLE). Although complement regulatory functions were attributed to most of the members of the CFHR protein family, the precise role of each CFHR protein in complement activation and the exact contribution to disease pathology is still unclear. Recent publications show that CFHR proteins form homo- as well as heterodimers. Genetic abnormalities within the CFHR gene locus can result in hybrid proteins with affected dimerization or recognition domains which cause defective functions. Here we summarize the recent data about CFHR genes and proteins in order to better understand the role of CFHR proteins in complement activation and in complement associated diseases. PMID:23830046

  20. Relative contribution of contact and complement activation to inflammatory reactions in arthritic joints.

    PubMed Central

    Abbink, J J; Kamp, A M; Nuijens, J H; Erenberg, A J; Swaak, A J; Hack, C E

    1992-01-01

    Although both the complement and contact system are thought to contribute to the inflammatory reaction in arthritic joints, only activation of complement has so far been well established, whereas contact activation and its contribution to arthritis has not been systematically explored. Complement and contact activation were assessed in 71 patients with inflammatory arthropathies and 11 with osteoarthritis using sensitive assays for C3a, and C1-inhibitor (C1INH)-kallikrein and C1INH-factor XIIa complexes respectively. Increased plasma concentrations of kallikrein-and factor XIIa-C1INH complexes were found in two and seven of the 71 patients with inflammatory arthropathies, respectively, and in none of the patients with osteoarthritis. Increased synovial fluid concentrations of kallikrein and factor XIIa complexes occurred in 13 and 15 patients with inflammatory joint diseases respectively, and in two patients with osteoarthritis. Contact system parameters did not correlate with clinical symptoms, local activity, or neutrophil activation. In contrast, synovial fluid concentrations of C3a and C1INH-C1 complexes were increased in all patients and in 20 patients with inflammatory arthropathies respectively, and were higher in patients with a higher local activity score. Synovial fluid C3a correlated with parameters of neutrophil activation such as lactoferrin. Increased plasma concentrations of C3a and C1INH-C1 complexes occurred in 13 and 11 patients with inflammatory joint diseases, and in one and two patients with osteoarthritis respectively. Plasma concentrations of C3a correlated with the number of painful joints. Thus contact activation occurs only sporadically in patients with arthritis and contributes little if anything to the local inflammatory reaction and neutrophil activation. These latter events are significantly related to the extent of complement activation. PMID:1444625

  1. Therapeutic control of complement activation at the level of the central component C3.

    PubMed

    Ricklin, Daniel; Lambris, John D

    2016-06-01

    The increasing recognition of the complement system's association with diseases of the inflammatory spectrum and with biomaterial and transplant-related complications has generated growing interest in the therapeutic modulation of this innate immune cascade. As a central functional hub that largely drives the activation, amplification, and effector generation of the complement response, the plasma protein C3 has long been recognized as an attractive target. While pharmacological modulation of C3 activation may offer a powerful opportunity to interfere with or even prevent complement-driven pathologies, the development of C3 inhibitors has often been accompanied by concerns regarding the safety and feasibility of this approach. Although no C3-targeted inhibitors have thus far been approved for clinical use, several promising concepts and candidates have emerged in recent years. At the same time, experiences from preclinical development and clinical trials are slowly providing a more detailed picture of therapeutic complement inhibition at the level of C3. This review highlights the current therapeutic strategies to control C3 activation and discusses the possibilities and challenges on the road to bringing C3-targeted therapeutics to the clinic. PMID:26101137

  2. Levan-induced glomerulitis in rabbits: a possible role for direct complement activation in situ.

    PubMed Central

    Stark, H.; Alkalay, A.; Ben-Bassat, M.; Hazaz, B.; Joshua, H.

    1985-01-01

    Since high-molecular-weight levan is known to reduce capillary permeability to large molecules, an experiment was designed to investigate whether this agent may attenuate the glomerulonephritis associated with acute serum sickness in rabbits. The study, in fact, demonstrated an enhancing effect of levan, which caused increased glomerular proliferative changes and leucocyte infiltration and, possibly, increased IgG deposition in this experimental model. In addition, rabbits injected only with levan also demonstrated mild glomerulitis and C3 deposition. In one of four rabbits examined, this was accompanied by a marked fall in the serum level of total haemolysing complement. Levan was demonstrated to cause activation of complement when incubated with normal rabbit serum in vitro. We believe that these findings are best explained on the basis of complement activation in situ by levan in the glomeruli, probably via the alternative pathway, with the resulting inflammatory response. In the case of BSA-injected rabbits, this response is believed to be additive to that of the classical immune complex-mediated complement activation. Images Fig. 1 PMID:3986130

  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. 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

  5. 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

  6. [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. PMID:26575109

  7. Using an in vitro xenoantibody-mediated complement-dependent cytotoxicity model to evaluate the complement inhibitory activity of the peptidic C3 inhibitor Cp40

    PubMed Central

    Wang, Junxiang; Wang, Lu; Xiang, Ying; Ricklin, Daniel; Lambris, John D.; Chen, Gang

    2016-01-01

    Simple and reliable methods for evaluating the inhibitory effects of drug candidates on complement activation are essential for preclinical development. Here, using an immortalized porcine aortic endothelial cell line (iPEC) as target, we evaluated the feasibility and effectiveness of an in vitro xenoantibody-mediated complement-dependent cytotoxicity (CDC) model for evaluating the complement inhibitory activity of Cp40, a potent analog of the peptidic C3 inhibitor compstatin. The binding of human xenoantibodies to iPECs led to serum dilution-dependent cell death. Pretreatment of the human serum with Cp40 almost completely inhibited the deposition of C3 fragments and C5b-9 on the cells, resulting in a dose-dependent inhibition of CDC against the iPECs. Using the same method to compare the effects of Cp40 on complement activation in humans, rhesus and cynomolgus monkeys, we found that the inhibitory patterns were similar overall. Thus, the in vitro xenoantibody-mediated CDC assay may have considerable potential for future clinical use. PMID:26548839

  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. Human complement C3 deficiency: Th1 induction requires T cell-derived complement C3a and CD46 activation.

    PubMed

    Ghannam, Arije; Fauquert, Jean-Luc; Thomas, Caroline; Kemper, Claudia; Drouet, Christian

    2014-03-01

    Human T helper type 1 (Th1) responses are essential in defense. Although T cell receptor (TCR) and co-stimulator engagement are indispensable for T cell activation, stimulation of additional receptor pathways are also necessary for effector induction. For example, engagement of the complement regulator CD46 by its ligand C3b generated upon TCR activation is required for IFN-γ production as CD46-deficient patients lack Th1 responses. Utilizing T cells from two C3-deficient patients we demonstrate here that normal Th1 responses also depend on signals mediated by the anaphylatoxin C3a receptor (C3aR). Importantly, and like in CD46-deficient patients, whilst Th1 induction are impaired in C3-deficient patients in vitro, their Th2 responses are unaffected. Furthermore, C3-deficient CD4(+) T cells present with reduced expression of CD25 and CD122, further substantiating the growing notion that complement fragments regulate interleukin-2 receptor (IL-2R) assembly and that disturbance of complement-guided IL-2R assembly contributes to aberrant Th1 effector responses. Lastly, sustained intrinsic production of complement fragments may participate in the Th1 contraction phase as both C3a and CD46 engagement regulate IL-10 co-expression in Th1 cells. These data suggest that C3aR and CD46 activation via intrinsic generation of their respective ligands is an integral part of human Th1 (but not Th2) immunity. PMID:24321396

  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.