SEARCH · PubMed Health

Results for “Complement C8”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources

PubMed1981

Screening for deficiencies in the classical and alternative pathways of complement by hemolysis in gel.

Two assays based on hemolysis in gel were assessed for screening complement (C) component deficiencies. In one assay sensitized sheep erythrocyte (EA) were incorporated in agarose gel containing Ca2+ and Mg2+, in the other guinea pig erythrocytes (GpE) were used in the presence of Mg2+and EGTA. With few exceptions, fresh samples from healthy individuals produced homogeneous areas of complete hemolysis in both assays. Clearly aberrant patterns were observed in approximately 4% of healthy blood donors. Sera from patients having complete deficiencies of Clq, C2 or C4 produced clear lysis of GpE only, whereas in sera lacking C3 or C8 lysis was grossly impaired in both assays. Properdin deficient serum produced very slight lysis of GpE but normal lysis of EA. Reconstitution of these C-deficient sera gave normal lysis. Together, the two assays supplement immunochemical C3 and C4 determinations for screening out C disorders.

Animals↗

PubMed1978

Leukocyte complement: assembly of the membrane attack complex of complement by human peripheral blood leukocytes in the presence and absence of serum.

The specific neoantigenic determinants (neoAg) that are indicative of the assembled C5b-9 C complex are generated on the surface of peripheral blood leukocytes (PBL) during collection and processing of blood. Formation of neoAg on PBL could be prevented by collecting blood directly into 20 mM EDTA and, could be induced in vitro by adding autologous serum to isolated PBL that lacked neoAg. When neoAg was induced by the addition of serum containing 125I-labeled C8, the C8 was incorporated into a 23S complex which could be eluted from PBL. A mechanism for neoAg formation on PBL independent of exogenous serum factors was detected when PBL were placed into culture in serum-free medium. Results with metabolic inhibitors and 14C-leucine suggest that PBL can synthesize C5 and assemble the C5b-9 complex. The possible relevance of these findings to the understanding of mechanisms of cell-mediated cytotoxicity is discussed.

Antigens↗

PubMed1995

Determination of the active site of CD59 with synthetic peptides.

CD59 inhibits the formation of membrane attack complex (MAC) of human complement by binding to C8 and C9 in the nascent membrane attack complex and inhibiting C9 binding to C8 in C5b-8 and C9 polymerization. Considering five disulfide bridges of CD59, we divided the molecule into two portions and synthesized the two peptides. One represented an amino-terminal half, P1-41, consisting of residues 1-41, while another represented a carboxyl-terminal half, P42-77, consisting of residues 42-77. P1-41 inhibited the MAC formation much more strongly than P42-77, indicating that the amino-terminal half contained the active site. We further synthesized P4-18 that consisted of residues 4-18 and P19-41 that consisted of residues 19-41. The activity of P4-18 was less than that of P19-41. Surprisingly, P19-41 showed higher activity than P1-41 and was comparable to urine CD59. Residues 19-41 were further divided into two portions: P20-25 which consisted of residues 20-25 and P27-38 which consisted of residues 27-38. Although their activities were significantly less than the activity of P19-41, P27-38 showed higher activity than P20-25. Residues 27-38 were further divided into three portions: P27-32 which consisted of residues 27-32, P30-34 which consisted of residues 30-34 and P33-38 which consisted of residues 33-38. When these peptides were assayed for the activities, all of them showed significant activities, even though they needed 10-fold more concentrations than P19-41. These data suggest that the portion made up of residues 27-38 is the active site constituting the binding site to C8 and C9.

Amino Acid Sequence↗

PubMed1979

Complement lysis of human erythrocytes. Differeing susceptibility of two types of paroxysmal nocturnal hemoglobinuria cells to C5b-9.

Although enhanced sensitivity of erythrocytes to complement-mediated lysis is a hallmark of paroxysmal nocturnal hemoglobinuria (PNH), subpopulations of erythrocytes in such patients vary significantly in this respect. One PNH erythrocyte subpopulation (termed type III) comprises exquisitely sensitive cells, whereas type II PNH erythrocytes are intermediate in complement sensitivity between PNH type III and normal human erythrocytes. Differences in the action of the terminal complement components that would account for the differing lytic behavior of types II and III PNH erythrocytes have been proposed but not directly demonstrated. The present studies, making use of carefully selected cases with pure populations of type II or type III erythrocytes, confirm a prior observation that antibody-coated PNH erythrocytes of both types II and III display comparably supranormal C3 binding in whole human serum. However, when lysis was induced by the isolated C5b-9 membrane attack mechanism, bypassing the requirement for C3 binding, only type III PNH cells exhibited greater than normal lysis. This finding suggests that type III PNH erythrocytes have an additional membrane abnormality not present in type II cells. Thus, the differing lytic behavior of these two cell types in whole serum may reflect the additive effects on type III cells of both exaggerated C3 binding and enhanced sensitivity to C5b-9, whereas the more moderate lysis of type II PNH cells may be determined mainly or entirely by the earlier-acting mechanism producing augmented C3 binding. The failure of guinea pig C8 and C9, as opposed to human C8 and C9, to reveal the true lytic sensitivity of PNH-III E in our earlier study is illustrated, and its implications briefly discussed.

Animals↗

PubMed1987

Inhibition of the lytic activity of perforin (cytolysin) and of late complement components by proteoglycans.

The complement components (C6, C7, C8 and C9) implicated in the lysis of target cells and the pore-forming, lytic protein from cytotoxic T-lymphocytes and NK-cells, perforin, contain an amino acid sequence which is highly homologous to a repeat unit identified in the LDL-receptor (Tschopp et al., 1986, Nature, 322, 831-834). The domain of the LDL-receptor, which is thought to interact with a positively charged segment of its ligands apoprotein B and E, is rich in cysteine residues and contains a cluster of negative charges. We show that the negatively charged molecules suramin and glycosaminoglycans, the positively charged peptides protamine and polylysine, all of which are known to abolish binding of LDL to its receptor (Goldstein et al., 1985, A. Rev. cell. Biol., 1, 1-39) inhibit the lytic activities of C6, C7, C8, C9 and perforin. Moreover, these negatively charged molecules are potent inhibitors of cytolytic T-lymphocyte-mediated lysis of target cells, suggesting a functionally crucial role for perforin in cell-mediated cytolysis. We propose that the negatively charged, cysteine-rich domain of these complement proteins and perforin interacts with an as yet unidentified positively charged segment of its ligand in a manner analogous to the LDL-LDL receptor interaction. Homologous cysteine-rich domains in functionally unrelated proteins may therefore be functionally conserved as ideal rigid interaction domains with the conserved cysteine residues as framework. Specificity of the domain for its ligand would be conferred by the non-conserved amino acid residues.

Cell Line↗

PubMed1994

Adherence of monocytes and polymorphonuclear cells to infective larvae of Strongyloides stercoralis after complement activation.

The activation of the complement system by living Strongyloides stercoralis filariform larvae and their antigenic preparation was demonstrated in vitro through both classical and alternative pathways. This activation does not require the presence of specific antibodies but promotes the adhesion of peripheral blood monocytes (MNC) and polymorphonuclear cells (PMNC) to the larval surface. Larvae, totally coated by PMNC, showed a visible loss of motility after 2 hr incubation. Ethylenediamine tetraacetic acid, in contrast to ethylene glycol-bis beta-aminoethylether N,N,N,N-tetraacetic acid, abolished the adherence activity, suggesting the involvement of the alternative pathway in this process. Deposition of complement components C1q, C3, C4, C8, and properdin on the larval surface was demonstrated by immunofluorescence assays. In addition, complement activation by the larvae was demonstrated through C3 conversion and C4 cleavage assays, both depending on the number of larvae. On the other hand, complement activation by S. stercoralis antigen was determined by factor B and C4 cleavage, as well as C3 conversion assays. Our results suggest that the complement system as a first line of defense, in association with the effector cells, plays an important role in the nonspecific immune response of the host to S. stercoralis infection, especially considering the constant parasite recycling through the host tissues.

Animals↗

PubMed1986

Does complement kill E. coli by producing transmural pores?

Three lines of evidence are presented to indicate that C5b-9 kills serum-sensitive E. coli K 12 cells by generating functional pores across the outer and inner bacterial membrane. First, viable cells carrying C5b-8 complexes are impermeable to o-nitrophenyl-beta-D-galactoside (ONPG), but lose viability and become permeable to this marker upon post-treatment with purified C9 in the absence of lysozyme. Cells killed with colicin E1 or gentamicin are also impermeable to ONPG but take up the marker if they are post-treated with lysozyme-free serum. Second, killing by C5b-9 is highly effective, deposition of only a small number of complexes being lethal. This has been demonstrated in experiments where viable cells carrying 2000-4000 C5b-7 complexes per CFU were permitted to multiply in broth culture, and the daughter generations subsequently treated with purified C8 and C9. Fifty percent killing was observed in the fifth to sixth generation, corresponding to a dilution of C5b-7 complexes to 50-100 molecules/CFU. In the presence of 2 mM EDTA, further dilution of C5b-7 down to 8-30 complexes/CFU still caused 50% killing of daughter cells. Third, treatment of C5b-7 cells with purified CC8 and C9 results in the release of intracellular K+, which commences immediately after addition of C8/C9. This was shown in experiments where C5b-7 cells were packed to high density in saline, post-treated with C8 + C9, and K+ directly measured in the cell supernatants. Based on these results, we propose that C5b-9 pores deposited in the outer bacterial membrane periodically fuse with the inner membrane, the transmural pores thus generated permitting rapid K+ efflux, with cell death ensuing through the collapse of membrane potential.

Bacteriolysis↗

PubMed1994

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

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

Acinetobacter Infections↗

PubMed1996

CD59 homologue regulates complement-dependent cytolysis of rat Schwann cells.

Antibody (Ab) sensitized sciatic nerve Schwann cells (SchC) of 2-day-old rats (SchC/2d) were significantly more susceptible to cytolysis by both heterologous, guinea pig (GP), and homologous rat serum complement (40 +/- 3.8% and 21.2 +/- 3.1%, respectively) than SchC of 6-day-old rats (SchC/6d) (7.9 +/- 5.9% and 2.6 +/- 3.1%, respectively). To determine if resistance to complement (C)-mediated cytolysis correlated with expression of membrane proteins which regulate C activation, we used Western blot and FACS analysis. Binding of specific polyclonal Ab demonstrated similar concentrations of Crry, a regulator of C3 convertase formation, on plasma membranes of SchC 2d and 6d. During C activation, both C3b deposition and iC3b formation were greater on SchC/6d than on SchC/2d and the C3b deposition did not correlate with enhanced cytolysis. In contrast, 2.1-fold more rat CD59, a regulator of C8 and C9 incorporation into C5b-9, detected with Western blot on SchC/6d compared with SchC/2d was confirmed by FACS. Further, both rat and GP C8/C9 lysed SchC/2d expressing human C5b-7 (20.1 +/- 3.7 and 21.6 +/- 4.7%, respectively), while only GP C8/C9 caused cytolysis of 10.7 +/- 4.3% SchC/6d expressing hu C5b-7 and rat C8/C9 did not (0.5 +/- 0.5%). Preincubation of SchC/6d with an F(ab)2 fragment of an mAb to rCD59 with blocking capacity, increased cytolysis mediated by rat serum C more than 6-fold to 16.7 +/- 3.0% but only 1.7-fold (maximum cytolysis 37.4 +/- 11.2%) in SchC/2d. Our data suggest that expression of rat CD59 on SchC increased almost two-fold between postnatal days 2 and 6, and this increased expression on more terminally differentiated SchC is a significant factor in regulating terminal complement complex formation and limiting cytolysis of rat SchC by homologous serum complement.

Animals↗

PubMed1982

Molecular organization of C9 within the membrane attack complex of complement. Induction of circular C9 polymerization by the C5b-8 assembly.

Evidence has been presented suggesting that during assembly of the membrane attack complex (MAC) of complement, the C5b-8 complex induces polymerization of C9. The C9 polymer was detected by sodium dodecyl sulfate (SDS) gel electrophoresis of MAC isolated from complement-lysed erythrocytes. It resembled the previously described polymerized C9 (poly C9) produced from isolated monomeric C9 by prolonged incubation at 37 degrees C in that it was resistant to dissociation by SDS and reducing agents and had an apparent molecular weight of approximately 1.1 million. The presence of poly C9 in the MAC was further supported by the expression of identical neoantigens by the MAC and poly C9 and by the high C9 content of the MAC relative to its other constituents. Isolated C8 in solution was found to have a single C9-binding site. In mixture, the two proteins formed a reversible equimolar complex that had a sedimentation coefficient of 10.5S. In contrast, a single, cell-bound C5b-8 complex was found to bind up to 12-15 C9 molecules and clusters of C5b- 8 bound 6-8 C9 molecules per C8 molecule. In either case, typical ultrastructural membrane lesions were observed, suggesting that the membrane lesion is identical with the tubular poly C9 consisting of 12-16 C9 molecules, and that the MAC can have either the composition (C5b-8)polyC9 or (CSb-8)(2)polyC9. When C9 input was restricted so that the molar C9/C8 ratio was less than or equal to 3, C9-induced aggregates of C5b-8 were observed but virtually no circular membrane lesions were found. We suggest, therefore, that C9, at low dosage, causes cross-linking of multiple C5b-8 complexes within the target membrane and that, at high dosage, C9 is polymerized by C5b-8 to form a transmembrane channel within the MAC assembly. It is primarily the C9 polymer that evokes the ultrastructural image of the MAC or of membrane lesions caused by complement.

Animals↗

PubMed1996

Discordant renal histopathologic findings and complement profiles in membranoproliferative glomerulonephritis type III.

Patients with membranoproliferative glomerulonephritis (MPGN) type III and a low serum C3 concentration tend to have evidence for a nephritic factor of the terminal complement pathway (Nft). Complement profiles were studied in three patients with MPGN type III and low serum C3 concentrations. Serum C3 concentrations were 52, 21, and 14 mg/dL (normal range, 83 to 177 mg/dL). Serum Clq, C2, C4, properdin, and C5 concentrations were normal in all patients, whereas two had a slight decrease of C7 or C8. This pattern of complement activation resembles that seen with MPGN type II in which a nephritic factor activates the amplification loop (NFa). We conclude that in patients with MPGN type III the previously reported profile/presence of Nft is not always found, at least in the chronic stage of the disease, despite a low C3 value.

Biopsy↗

PubMed2003

Formation of complement membrane attack complex in mammalian cerebral cortex evokes seizures and neurodegeneration.

The complement system consists of >30 proteins that interact in a carefully regulated manner to destroy invading bacteria and prevent the deposition of immune complexes in normal tissue. This complex system can be activated by diverse mechanisms proceeding through distinct pathways, yet all converge on a final common pathway in which five proteins assemble into a multimolecular complex, the membrane attack complex (MAC). The MAC inserts into cell membranes to form a functional pore, resulting in ion flux and ultimately osmotic lysis. Immunohistochemical evidence of the MAC decorating neurons in cortical gray matter has been identified in multiple CNS diseases, yet the deleterious consequences, if any, of MAC deposition in the cortex of mammalian brain in vivo are unknown. Here we demonstrate that the sequential infusion of individual proteins of the membrane attack pathway (C5b6, C7, C8, and C9) into the hippocampus of awake, freely moving rats induced both behavioral and electrographic seizures as well as cytotoxicity. The onset of seizures occurred during or shortly after the infusion of C8/C9. Neither seizures nor cytotoxicity resulted from the simultaneous infusion of all five proteins premixed in vitro. The requirement for the sequential infusion of all five proteins together with the temporal relationship of seizure onset to infusions of C8/C9 implies that the MAC was formed in vivo and triggered both seizures and cytotoxicity. Deposition of the complement MAC in cortical gray matter may contribute to epileptic seizures and cell death in diverse diseases of the human brain.

Animals↗

PubMed1996

C5b-8 step lysis of swine endothelial cells by human complement and functional feature of transfected CD59.

The authors established several swine endothelial cell (SEC) lines expressing human CD59 by transfection of cDNA, and assessed the function of the transfectant molecules in comparison with those of membrane cofactor protein (MCP) and decay-accelerating factor (DAF) in an in vitro hyperacute rejection model of swine to human discordant xenograft. At the usual expression rate, DAF and MCP protected SEC from human complement mediated cell lysis, but CD59 did not block human complement attack on SEC. However, CD59 protects SEC from cell lysis when sufficiently expressed as in human umbilical vein (HUVEC). The authors examined why CD59 needed so many molecules to protect human complement-mediated SEC lysis and found that SEC underwent lysis by human C5b-8. The degree of C5b-8 step lysis of SEC was approximately 70% of the total activation (C5b-9). Additionally, CD59 protected human complement activities less efficiently at the C5b-8 step than at the C9-step. Therefore, to overcome human complement mediated SEC lysis, C8 activity must be inhibited by dense expression of CD59.

Animals↗

PubMed2001

Complement components, but not complement inhibitors, are upregulated in atherosclerotic plaques.

Complement activation occurs in atherosclerotic plaques. The capacity of arterial tissue to inhibit this activation through generation of the complement regulators C1 inhibitor, decay accelerating factor, membrane cofactor protein (CD46), C4 binding protein (C4BP), and protectin (CD59) was evaluated in pairs of aortic atherosclerotic plaques and nearby normal artery from 11 human postmortem specimens. All 22 samples produced mRNAs for each of these proteins. The ratios of plaque versus normal artery pairs was not significantly different from unity for any of these inhibitors. However, in plaques, the mRNAs for C1r and C1s, the substrates for the C1 inhibitor, were increased 2.35- and 4.96-fold, respectively, compared with normal artery; mRNA for C4, the target for C4BP, was elevated l.34-fold; and mRNAs for C7 and C8, the targets for CD59, were elevated 2.61- and 3.25-fold, respectively. By Western blotting and immunohistochemistry, fraction Bb of factor B, a marker of alternative pathway activation, was barely detectable in plaque and normal arterial tissue. These data indicate that it is primarily the classical, not the alternative pathway, that is activated in plaques and that key inhibitors are not upregulated to defend against this activation.

Antigens, CD↗

PubMed1991

Complement-mediated killing of the Lyme disease spirochete Borrelia burgdorferi. Role of antibody in formation of an effective membrane attack complex.

Lyme disease is a multisystemic illness caused by the spirochete Borrelia burgdorferi. In the absence of specific antibody, the spirochete is resistant to the bactericidal activity of C, despite the capacity of B. burgdorferi to activate both C pathways. We examined the mechanism of serum resistance by measuring the deposition of C3 and terminal C components on B. burgdorferi in the presence and absence of immune IgG. In normal human serum antibody-sensitized borreliae bound similar amounts of C3, and similar or increased amounts of C8 and C9, in comparison to unsensitized bacteria. However, at comparable levels of C3, C8, or C9 uptake, only sensitized bacteria were killed. The requirement of antibody for killing could not be explained by differences in the rate of C deposition or by differences in the C9 to C8 ratio in the membrane attack complex (MAC). We found that bacteria incubated in C5-depleted human serum, but not in C6-depleted serum, were killed when this treatment was followed by antibody and the missing C components. Bacteria were also killed by reactive lysis (C5b-9) provided that antibody was present. Therefore, the effect of bactericidal IgG occurred at the stage of C5b binding to the bacterial surface. Elution studies of bound C9 indicated that the MAC was stably bound to the outer membrane of B. burgdorferi, whether or not the bacteria were treated with antibody. However, treatment with 0.1% trypsin released 48% of 125I-C9 from the surface of unsensitized borreliae and 24% from IgG-sensitized cells, demonstrating that the presence of the antibody changed the accessibility to trypsin of C9 in the MAC. These results indicate that the effect of antibody in the killing process is not to enhance the rate or extent of initial or terminal component binding, but rather to alter the bacterial outer membrane to allow effective MAC formation.

Antibodies, Bacterial↗

PubMed1979

Deficiency of the ninth component of complement in man.

The studies of serum from a case with C9 (the ninth component of complement) deficiency are described. A 29-year-old woman in good health was found to have low serum complement levels (CH50). C9 of her serum was undetectable by the hemolytic assay and by the immunochemical analysis but all other components were normal. It was demonstrated that low CH50 of her serum was due to the hemolysis of the sensitized sheep erythrocytes (EA) by the complement components from C1 to C8.

Adult↗

PubMed1998

Shark complement: an assessment.

The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA clone encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish (Triakis scyllia) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C8n and C9n, and is activated by immune complexes in the presence of Ca++ and Mg++ ions. The ACP is antibody independent, requiring Mg++ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C3 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q-like rather than like mannan-binding protein. N-terminal amino acid sequences of the alpha and beta chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4 gamma chain, shows little similarity to human C4 gamma chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and alpha 2-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.

Amino Acid Sequence↗

PubMed1993

Soluble complex of complement increases hydraulic conductivity in single microvessels of rat lung.

We determined the effect of sera enriched with the soluble complex of complement (SC5b-9), on hydraulic conductivity (Lp) of single pulmonary venules (diameter 20-30 microns). Sera free of anticoagulants and blood cells were prepared from rat and human blood. Lp were determined by our split drop technique in isolated, blood-perfused lungs prepared from anesthetized rats (2% halothane; Sprague Dawley, 500 g; n = 73). Zymosan-activated (ZAS) and control sera were used for Lp determinations. In ZAS prepared from human serum, SC5b-9 concentration was > 300 micrograms/ml (control: < 1 microgram/ml) as determined by ELISA. At baseline, Lp averaged 3.4 +/- .4 x 10(-7) ml/(cm2.s.cm H2O), but it increased by 217 +/- 32% with undiluted ZAS (P < 0.05). The Lp increase correlated significantly with different ZAS dilutions for rat serum and with SC5b-9 concentration for human serum. Lp did not increase significantly with ZAS prepared from heat-treated sera, C6- and C8-deficient sera; or with ZAS in which SC5b-9 had been depleted by immunoprecipitation. The ZAS-induced increase of Lp was blocked completely by venular preinfusion with the arginine-glycine-aspartic acid (RGD) tripeptide (1 mg/ml, 10 min). We report for the first time that: (a) SC5b-9 increases lung endothelial Lp; and (b) the increase of Lp is attributable to an integrin-dependent mechanism.

Animals↗