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PubMedDate not supplied

[The complement system and circulating immune complexes in burn patients].

The results of the study of humoral immunity in 80 burn patients are presented. A typical feature of all burn patients was a decrease in the total activity of the alternative path of the activation of complement and its factors B and D, beginning from the first day after the trauma. The character of changes in the functional activity of components C1-C5 of the classical path depended on the area of damages with their activation if burn area was less than 20% of the skin surface and deactivation if the burn area exceeded 20%.

Antibody Formation↗
PubMed1974

A serum factor in chronic hypocomplementemic hephritis distinct from immunoglobulins and activating the alternate pathway of complement.

Nephritic factor (C3NeF) has been isolated from plasma of patients with hypocomplementemic chronic glomerulonephritis (HCG) by ion exchange and molecular sieve chromatography. This material was further treated with solidified anti-Ig antiserum. The purified material failed to react with antiserum to human IgG, IgG3, Fab, Fc, and kappa and lambda chains, but retained full C3NeF activity. The nonidentity of C3NeF with IgG was further demonstrated by Ouchterlony analysis using anti-IgG and anti-C3NeF. Isolated C3NeF was found to be a protein with a sedimentation coefficient of 7S and a mol wt of 150,000 daltons, which on microzone electrophoresis and gel electrophoresis at pH 8.6 behaved as a gamma-globulin. C3NeF is not a C1q precipitin and does not activate the classical complement pathway. Unlike cobra venom factor, it failed to enter into a complex with C3 proactivator (C3PA) when incubated with normal human serum (NHS) and then subjected to sucrose density gradient ultracentrifugation. The action of isolated C3NeF on C3 requires C3PA, C3PA convertase (C3PAse), and properdin (P). Similarly, C3PA conversion by C3NeF requires P, C3PAse, and C3. Total hemolytic activity was lost by incubation of 64 microg of C3NeF/1 ml NHS at 37 degrees C for 30 min. Both C3a and C5a anaphylatoxin could be generated by C3NeF in serum previously depleted of anaphylatoxin inactivator. Anti-C3NeF was found to detect an antigen in all NHS tested. Treatment of NHS with solidified anti-C3NeF caused impairment of the alternate complement pathway. It failed to sustain lysis of glutathione-treated human erythrocytes initiated by inulin. It is conceivable that the normal serum constituent which is removed by anti-C3NeF constitutes the inactive precursor of C3NeF, and a heretofore unrecognized component of the alternate pathway.

Adsorption↗
PubMedDate not supplied

[Assessment of total complement activity of blood plasma (serum) using reader for immunoenzyme analysis].

A method for instrumental assessment of the total complementary activity of human blood plasma (serum) by the classical and alternative activation is proposed. The test is carried out by the micromethod in round-bottom 96-well plates for immunological tests. The results are read by automated analyzer for enzyme immunoassay recording at 405 nm wavelength.

Complement Activation↗
PubMedDate not supplied

A C1q-binding 40 kDa porin from Aeromonas salmonicida: cloning, sequencing, role in serum susceptibility and fish immunoprotection.

A 40 kDa C1q-binding outer membrane protein from Aeromonas salmonicida was identified by direct-binding assay with biotinylated C1q, a subcomponent of the complement classical pathway component C1. The 40 kDa porin structural gene from the A450 A. salmonicida typical strain (A+:O+) was cloned in Escherichia coli and sequenced. The amino acid sequence of the 40 kDa A. salmonicida porin, its ability to bind C1q in an antibody independent process, and its immunological cross-reaction with the A. hydrophila AH-3 porin II, allow us to determine the role of this protein in serum susceptibility. Furthermore, we obtained defined A. salmonicida 40 kDa porin insertion mutants in serum sensitive or resistant strains, and we complemented these mutants with a plasmid harboring only the 40 kDa porin gene from A. salmonicida A450 in order to define its role as an important surface molecule involved in serum susceptibility and C1q binding. Similar complementation was obtained using the A. hydrophila AH-3 porin II gene. The 40 kDa porin gene and/or protein was present in all the A. salmonicida typical or atypical strains tested. Furthermore, the A. hydrophila AH-3 porin II seems to be an important molecule for fish immunoprotection against either A. salmonicida or A. hydrophila strains.

Aeromonas hydrophila↗
PubMed2005

Synergy between two active sites of human complement receptor type 1 (CD35) in complement regulation: implications for the structure of the classical pathway C3 convertase and generation of more potent inhibitors.

The extracellular domain of the complement receptor type 1 (CR1; CD35) consists entirely of 30 complement control protein repeats (CCPs). CR1 has two distinct functional sites, site 1 (CCPs 1-3) and two copies of site 2 (CCPs 8-10 and CCPs 15-17). In this report we further define the structural requirements for decay-accelerating activity (DAA) for the classical pathway (CP) C3 and C5 convertases and, using these results, generate more potent decay accelerators. Previously, we demonstrated that both sites 1 and 2, tandemly arranged, are required for efficient DAA for C5 convertases. We show that site 1 dissociates the CP C5 convertase, whereas the role of site 2 is to bind the C3b subunit. The intervening CCPs between two functional sites are required for optimal DAA, suggesting that a spatial orientation of the two sites is important. DAA for the CP C3 convertase is increased synergistically if two copies of site 1, particularly those carrying DAA-increasing mutations, are contained within one protein. DAA in such constructs may exceed that of long homologous repeat A (CCPs 1-7) by up to 58-fold. To explain this synergy, we propose a dimeric structure for the CP C3 convertase on cell surfaces. We also extended our previous studies of the amino acid requirements for DAA of site 1 and found that the CCP 1/CCP 2 junction is critical and that Phe82 may contact the C3 convertases. These observations increase our understanding of the mechanism of DAA. In addition, a more potent decay-accelerating form of CR1 was generated.

Binding Sites↗
PubMed1990

Antibody enhances killing of Tritrichomonas foetus by the alternative bovine complement pathway.

The role of bovine antibody and complement in host defense against Tritrichomonas foetus was measured by using an assay of trichomonad viability based on protozoal uptake of tritiated adenine. Moderate killing was measured in the absence of antibody only with high concentrations of complement-preserved hypogammaglobulinemic bovine serum. However, very low concentrations of hyperimmune serum promoted significant enhancement (P less than 0.05) of killing by complement. Heat inactivation of complement (56 degrees C for 30 min) eliminated antibody-dependent and -independent killing. Similarly, depletion of bovine factor B in serum by heat treatment (50 degrees C for 45 min) abolished antibody-dependent and -independent killing. However, selective inactivation of the classical complement pathway with magnesium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not affect antibody-dependent or -independent killing by complement. These findings demonstrate antibody enhancement of complement-mediated killing of T. foetus by the alternative pathway of bovine complement.

Adenine↗
PubMed1978

Activation of complement by stroma from normal and paroxysmal nocturnal haemoglobinuria red cells.

Stroma from normal, AET-treated and PNH red cells and their KC1-extracts (partially purified on Sephadex G-200) are able to trigger the activation of the alternative complement pathway. This fact has been demonstrated by: 1 - the lysis of PNH cells incubated in serum treated with stroma from normal or PHN-RBC or with their extracts; the addition of Mg2+ or Ca2+ or of their chelators (EDTA, EGTA) to the extract-treated serum enhances or abolishes the lysis 2 - the reduction of complement acitvity in fresh serum incubated for 60' with PNH-extract 3 - the appearance of C3 breakdown products in serum incubated with PNH-extract, demonstrated by crossed immunoelectrophoresis. In contrast, the same stroma (or extract) inhibits the sucrose lysis test, in which the lysis takes place through the classical complement pathway. No differences on the complement activation were observed between PNH and normal RBC stroma and between their chromatographic extracts. These findings may suggest the possible role of diurnal variation of Mg2+ and Ca2+ concentration in precipitating haemolytic attacks and the possibility that small amount of circulating red cell stroma might maintain the haemolysis on PNH RBC.

Acetylcholinesterase↗
PubMed1991

Antibody-dependent alternate pathway of complement activation in opsonophagocytosis of Porphyromonas gingivalis.

It has been suggested that the ability of Porphyromonas gingivalis to proteolyse complement, as well as its production of a capsule, contributes to resistance to phagocytosis by polymorphonuclear leukocytes. In this report, the opsonic role of serum complement and its activation pathways were investigated, using individual sera heat treated or depleted of factors B, C2, and C1q and the divalent cations Mg2+ and Ca2+. A fluorochrome microassay was used to quantitate phagocytosis of P. gingivalis A7436 by human polymorphonuclear leukocytes. Heat treatment of rabbit antiserum to P. gingivalis (RaPg) (56 degrees C, 30 min) resulted in a reduction in phagocytosis from 100% to 55% +/- 5%, while heat treatment of chronic adult periodontal disease serum abrogated phagocytosis. The heat-labile activity of RaPg was fully restored with MgEGTA-chelated rabbit serum but not EDTA- or EGTA-chelated rabbit serum. The addition of serum depleted of factor B but not C2 or C1q restored most of the heat-labile activity; however, the factor B-depleted serum was suspect, due to low-level opsonization of zymosan (inhibitable by EDTA but not MgEGTA). Adding C1q at 80 micrograms/ml to serum depleted of C1q restored much but not all of the activity lost through heat treatment or through depletion of C1q. A large part of opsonic activity with C2- and C1q-depleted sera was enhanced by the addition of 4 x 10(-3) M Mg2+. The data indicate that although opsonophagocytosis of P. gingivalis A7436 is dependent on the classical complement pathway, a significant contribution is made by an antibody-dependent alternate pathway.

Animals↗
PubMed2001

Activation of complement and contact system in Alzheimer's disease.

beta-Amyloid protein (betaA) has been implicated in the pathogenesis of Alzheimer's disease (AD) because of its neurotoxicity and ability to trigger a local inflammatory response. Although assembly of betaA in particular aggregates seems to be crucial event in AD pathogenesis, soluble, non-fibrillar betaA may also be involved. Non-fibrillar betaA1-42, and truncated peptide 1-28, induced dose-dependent activation of C4 sparing C3. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar betaA can still activate C4 in plasma genetically deficient in C1q. A C1q independent mechanism of complement classical pathway activation could be via the activation of contact/kinin system. The possible involvement of contact system in AD is suggested by the finding that this system is massively activated in CSF of AD patients. The mechanism of activation of contact system could be the result of an anionic interaction of residues within the region 1-11 of betaA1-42 with factor XII, and of kallikrein generation. Concomitant incubation of a small cationic peptide (lysine4) with betaA abrogated its ability to trigger the cleavage of high molecular weight kininogen. In vivo, prevention of contact system activation beside the reduction of kallikrein generation, can also decrease the activation of complement system and the release of interleukin-6, both factors being considered to play an important role in the inflammatory reactions in AD brain.

Alzheimer Disease↗
PubMed1980

Complement, opsonins, and the immune response to bacterial infection in burned patients.

Studies were performed to evaluate complement, opsonins, and the immune response to bacterial infection in burned patients. Concentrations and functional acitivities of components of the classical and alternative complement pathways were measured in the sera of four septic, two bacteremic, and four nonseptic burned patients. In addition, heat-labile and heat-stable opsonic activity and agglutinin titers directed against the infecting bacterial strains were measured in the sera of the four septic patients and in an additional group of 11 septic burned patients with abnormal complement profiles. Functional activity of the alternative complement pathway and the concentration of properdin were shown to be persistently decreased during eight weeks postburn in the septic, bacteremic, and nonseptic burned patients; reduced classical pathway activity was demonstrated during the initial postburn period only in the septic patients. Two of the 15 septic patients had decreased heat-labile serum opsonic activity for their infecting bacterial strains, which occurred only during the initial postburn period. Heat-stable opsonins and agglutinin titers in the patients' sera directed against the infecting bacterial strains were equivalent to those in normal human sera, except for the agglutinin titers to Streptococcus faecalis which were increased in the patients' sera in comparison to the normal sera. These results indicate that the multiple complement abnormalities which occur in septic burned patients do not predispose these patients to bacterial infection by decreasing serum opsonic activity. Moreover, heat-stable immune IgG antibodies are not produced during septicemia which facilitate opsonization of the infecting bacterial strains in the absence of an intact complement system.

Adolescent↗
PubMed1984

The role of different K antigens of Escherichia coli in phagocytosis by polymorphonuclear leukocytes.

The importance of K antigens from six strains of Escherichia coli for the interaction with polymorphonuclear leukocytes (PMNL) was studied. The major factor influencing this interaction was the ability of strains to activate complement by the classical route during opsonisation, this process being reduced for most K-positive strains. Interference of K antigens with the functioning of common pili as adhesions of eukaryotic cells was not observed nor a toxic effect of K antigens on PMNL.

Antibodies, Bacterial↗
PubMed1997

The oxidative burst triggered by Salmonella typhimurium in differentiated U937 cells requires complement and a complete bacterial lipopolysaccharide.

The bacterial and serum factors involved in the oxidative response triggered by Salmonella typhimurium in differentiated U937 cells were investigated. Complement activation was shown to be required, using sera deficient in complement factors. An original dot-blot technique was developed to study the activation of complement by either bacteria or purified lipopolysaccharide (LPS). Both O-specific and lipid A segments of LPS were found to play a role in the triggering of the oxidative response. Lipid A was responsible for bacterial C3-derived opsonization by inducing an antibody-independent activation of complement classical pathway, whereas O-specific polysaccharide chains (O-Ag) were involved in cellular activation. Inhibition experiments using anti-cell surface marker monoclonal antibodies showed the involvement of the alpha chain of CR3 (CD11b) in the oxidative response developed by differentiated U937 cells in response to S. typhimurium infection. Whether both iC3b and O-Ag interact with different domains of CR3 or whether the binding of O-Ag occurs via a not yet identified receptor remains to be determined.

Cell Differentiation↗
PubMed1999

[The interconnection between the severity of meningococcal infection and the endotoxicity levels and patient's blood complement].

Seventy-eight patients with severe systemic meningococcal disease admitted to the Intensive Care Unit of the Second Moscow Hospital for Infectious Diseases were divided into four groups by complications of their disease: patients with refractory septic shock (RSS)--group 1; patients with early septic shock (ESS)--group 2; patients without shock but with severe mental disorders--group 3; patients without any of these complications--group 4. The LPS concentration in plasma was assessed by chromogenic method. Initial LPS levels in plasma of group 3 or group 4 patients (170 ng/l, median value and 360 ng/l, respectively) were greater than those of healthy donors (LPS < 15 ng/l). LPS concentration was significantly greater in group 2 (920 ng/l) or group 1 (12,400 ng/l). LPS levels declined exponentially in all the patients. The half-life was calculated to be 1.4 (+) -0.3 h. In group 2 and 1, respectively, the classical pathway complement activity in patients' serum was 50 and 10% of normal control values. To estimate significant prognostic factors for fatality in our patients, specificity and factor fatality difference of various clinical and laboratory factors were calculated. The cut-off LPS value for development of ESS was 600 ng/l and that for development of RSS and death was 8000 ng/l. For the prediction of fatality using the former cut-off value of LPS, sensitivity was 84% and specificity 100%. Using plasma complement activity (cut-off--15% of normal value) for prediction, sensitivity was 75% and specificity was 100%. Other factors (platelet and WBC count, blood pH, BP, etc.) had lower predictive power. Thus to date plasma endotoxin level and complement activity are the best prognostic factors in meningococcal disease.

Adolescent↗
PubMed1988

Assembly of the membrane attack complex promotes decay of the alternative pathway C3 convertase on Neisseria gonorrhoeae.

C3, C4, factor B, properdin, and C2 binding to serum-sensitive and serum-resistant gonococci was quantitated in C8-deficient and normal human serum by using fluorescein-conjugated antibodies and 3H-labeled components. Organism and serum-specific differences were noted, the most striking of which involved factor B and properdin binding to the serum-sensitive strains in the different sera. C3 binding to these organisms was quantitatively and kinetically equivalent in C8-deficient and normal human serum. In contrast, factor B and properdin binding reached a plateau after 5 min in C8-deficient serum but peaked and fell to control values in normal human serum. Identical results were obtained with normal human serum immunochemically depleted of C8. Between 7 and 15% of the bound C3 participated in formation of the alternative pathway convertase C3bBb/P. Reconstitution of the C cascade by adding purified C8 to C8-deficient serum led to the loss of factor B previously bound to the organisms. Factor B loss occurred coincident with bacterial killing and membrane disruption as observed by electron microscopy. Prevention of membrane disruption by depleting normal human serum of lysozyme had no effect on killing and failed to prevent factor B loss. Stabilization of the C3bBb complex with Ni2+ prevented factor B loss as well as gross membrane disruption but not bacterial killing. C2 (the classical pathway analog of factor B) binding to gonococci was equivalent in C8-deficient and normal human serum peaking within 2.5 min and falling to control values in both sera thereafter. We conclude that the assembly of the membrane attack complex promotes decay of C3bBb/P with release of factor B and properdin but not C3 from the organism surface. Membrane disruption does not appear to be required for this effect. This activity may represent a mechanism to limit continued C consumption.

Blood Bactericidal Activity↗
PubMed1999

Complement C1s activation in degenerating articular cartilage of rheumatoid arthritis patients: immunohistochemical studies with an active form specific antibody.

OBJECTIVE: The first complement component C1s was reported to have novel functions to degrade matrix components, besides its activities in the classic complement pathway. This study explores participation of C1s in articular cartilage degradation in rheumatoid arthritis (RA). METHODS: Normal articular cartilage (n = 6) and cartilage obtained from joints with RA (n = 15) and osteoarthritis (OA, n = 10) were immunostained using anti-C1s monoclonal antibodies PG11, which recognises both active and inactive C1s, and M241, which is specifically reactive to activated C1s. The effects of inflammatory cytokines on C1s production by human articular chondrocytes were also examined by sandwich ELISA. RESULTS: In normal articular cartilage, C1s was negative in staining with both PG11 and M241. In contrast, degenerating cartilage of RA was stained with PG11 (14 of 15 cases), and in most of the cases (13 of 15 cases) C1s was activated as revealed by M241 staining. In OA, C1s staining was restricted in severely degrading part of cartilage (5 of 10 cases), and even in that part C1s was not activated. In addition, C1s production by chondrocytes in vitro was increased by an inflammatory cytokine, tumour necrosis factor alpha. CONCLUSION: These results suggest that C1s activated in degenerative cartilage matrix of RA but not in that of OA. C1s is thought to participate in the pathogenesis of RA through its collagenolytic activity in addition to the role in the classic cascade.

Adolescent↗
PubMed2005

Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood.

BACKGROUND: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. METHODS: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. RESULTS: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. CONCLUSIONS: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.

Animals↗
PubMed2004

Anti-C1q autoantibodies amplify pathogenic complement activation in systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) often develop glomerulonephritis (i.e., inflammation in the glomeruli of the kidney), commonly referred to as lupus nephritis. Patients with lupus nephritis typically have autoantibodies to the complement classical pathway protein C1q. Whether these anti-C1q antibodies play any role in the development of lupus nephritis has been unclear. In this issue of the JCI, a new study demonstrates that anti-C1q antibodies can amplify glomerular injury but only when they are bound within the glomerulus to C1q that has been already brought to that site by other types of glomerular-reactive autoantibodies. These studies are the first, to our knowledge, to provide a causal link between anti-C1q antibodies and target organ damage in SLE.

Animals↗
PubMed2000

A comparative study of mammalian and reptilian alternative pathway of complement-mediated killing of the Lyme disease spirochete (Borrelia burgdorferi).

The potential bactericidal activity of the alternative complement pathway of mammalian and reptilian sera to Borrelia burgdorferi sensu stricto (s.s.) was evaluated in vitro. Complement-mediated killing was observed when cultured spirochetes were inoculated into sera from the western fence lizard (Sceloporus occidentalis) and from the southern alligator lizard (Elgaria multicarinata), but not when they were inoculated into serum from either the deer mouse (Peromyscus maniculatus) or from humans. Spirochetes were still alive after 4 hr in lizard serum that had been preheated at 56 C for 30 min to inactivate complement. Furthermore, when lizard serum was chelated with 10 mM ethylenediaminetetraacetic acid to block all complement activation, borreliacidal activity was arrested. When lizard serum was chelated with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus 4 mM MgCl2 to block only classical complement pathway activation, >85% of spirochetes were immobilized within 1 hr. Differences in B. burgdorferi s.s. mortality were not observed when chelators with or without MgCl2 were added to serum from either deer mice or humans. Proteins comprising the alternative complement pathway are responsible for the borreliacidal activity observed in the blood of S. occidentalis and E. multicarinata.

Animals↗