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PubMed1995

The development of PCR based assays for the detection and differentiation of simian immunodeficiency virus in vivo.

Polymerase chain reaction based assays, which amplify a region of the gag gene, have been developed for the direct detection of simian immunodeficiency virus (SIV) DNA sequences in the blood of experimentally infected cynomolgus macaques. In macaques infected with a characterised virus pool (11/88 pool SIVmac 32H), an assay employing a single round of amplification was found to be highly sensitive and specific. However, in animals infected with the SIV molecular clones J5 and C8 (Rud et al., J. Gen. Virol. 75, 529-543), it was necessary to use two rounds of amplification and nested primer pairs in order to achieve sensitivity > 90%. In order to differentiate macaques infected with either of the two genetically distinct SIV clones, J5 or C8, a third PCR based assay has been developed, which amplifies a 492 bp region of the nef gene. Sequence differences between the nef genes of the two molecular clones enabled the PCR product amplified from each virus to be distinguished by restriction analysis. These sensitive and specific assays complement virological detection of SIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines.

Animals↗
PubMed1989

The release of C5a in complement-activated serum does not require C6.

The influence of terminal complement components on the generation and release of the complement C5a fragment was investigated by comparing the levels of C5a in complement-activated serum with the levels of C5a produced in serum depleted of complement C6. In order to investigate the release of C5a, a modified C5a assay was developed that utilizes an anti-C5b monoclonal antibody to remove C5, C5b, and C5b-C5a complexes from samples prior to C5a assay. The modified assay was developed because the standard methodology, which includes an acid-precipitation step designed to dissociate C5a and C5b, cannot distinguish free C5a from the C5a that is bound to C5b. Therefore, the standard methodology is not capable of monitoring the influence of terminal components on C5a/C5b dissociation. Levels of C5a were measured in complement-activated whole human serum, in serum depleted of C6, and in serum containing inhibitory levels of anti-C6 Fab using both the modified C5a assay and the standard methodology. Sera were complement-activated with either zymosan to activate the alternative complement pathway or with antibody-coated sheep erythrocytes to activate the classical pathway. The levels of free C5a in C6-depleted sera after activation were equivalent to the C5a levels in activated whole serum, indicating that C6 is not required for the release of C5a from C5b. In addition, the quantity of C5a detected in zymosan-activated sera using the standard acid-precipitation methodology was greater than C5a levels when assayed using the modified immunoadsorption technique, confirming that acid-treatment enhances the C5a dissociation and promotes C5a recovery. Since the other terminal components, C7, C8, and C9, bind to C5b only after C5b only after C6 is bound, these results indicate that none of the terminal components are required for the release of C5a. Although the terminal components could influence the rate of C5a release, the quantity of C5a released in serum was entirely independent of terminal components.

Animals↗
PubMed1981

How complement kills E. coli. I. Location of the lethal lesion.

We have studied the action of human complement (C) on E. coli membranes. We find, as have others, that C disrupts the outer membrane (OM), allowing the release of periplasmic proteins. In addition, we have found 1) that in the complete absence of lysozyme, C damages the inner membrane (IM), 2) IM damage is different from OM damage in that only small molecules traverse a damaged IM whereas macromolecules traverse damaged OM, 3) IM damage and OM damage occur with identical kinetics and dose response, suggesting that IM and OM damage are closely coupled events, and 4) upon the addition of purified C8 and C9 to the washed cellular intermediate, E. coli C 1-7, both IM and OM are damaged coordinately. These results, taken together, suggest that C damages E. coli membranes by acting at a site contiguous with both membranes. We speculate that C may simultaneously gain access to both membranes by acting at the junctions between IM and OM.

Alkaline Phosphatase↗
PubMed1995

A rapid method for the isolation of analogues of human CD59 by preparative SDS-PAGE: application to pig CD59.

A method for the rapid isolation of functionally active analogues of human CD59 from erythrocytes (E) is described. The method, here applied to pig E, is based on the fractionation of a butanol extract of E ghosts by preparative SDS-PAGE followed by gel filtration on Superose 12. Purification was monitored using a functional complement inhibition assay. SDS-PAGE analysis of the product of this procedure indicated a single protein band with apparent M(r) of 20 kDa under reducing and non-reducing conditions. The preparation could be incorporated into guinea pig E to inhibit both CVF-reactive lysis and lysis through C8 and C9 using preformed C5b-7 sites, demonstrating that it contained a CD59-like activity. PIPLC treatment of the isolated protein abolished the inhibition. In contrast to SDS-PAGE analysis, amino-terminal sequence analysis of the preparation showed that it comprised two components. One was identified from databank searches as a fragment of pig glycophorin. These two components could not be separated by either standard or affinity chromatographic techniques. The second component was novel and had high sequence homology with human CD59, identifying it as the pig analogue. Further functional studies showed that the pig analogue of human CD59 was effective in the protection of guinea pig E against lysis by serum from a variety of species, including human.

Amino Acid Sequence↗
PubMed1999

Immunoglobulin G and complement immunoreactivity in the cerebral cortex of patients with Rasmussen's encephalitis.

OBJECTIVE: To provide evidence that complement (C')-dependent processes may be involved in Rasmussen's encephalitis (RE). BACKGROUND: RE is a rare, progressive, childhood epilepsy syndrome associated with inflammation and neuronal cell loss in a single cerebral hemisphere. Recent work suggests an autoimmune immunoglobulin (Ig) G-mediated process is important in disease pathogenesis. METHODS: Brain samples from RE and complex partial epilepsy control patients were analyzed immunohistochemically. Sections were stained for IgG and the C' factors C4, C8, and the membrane attack complex (MAC). RESULTS: Brain samples from three of five patients with active, progressive RE but neither of two chronic RE nor five control epilepsy patients demonstrated immunoreactivity for IgG, C4, C8, and MAC on discrete patches of cerebrocortical neurons. Intensely activated glial fibrillary acid protein-positive astrocytes were found in areas overlapping these patches. CONCLUSION: Focally distributed IgG- and C'-positive neurons were found to colocalize with activated astrocytes, suggesting focal IgG-dependent classical C' cascade pathway activation with attendant tissue damage in this subset of RE patients. Intraparenchymal C' activation triggered by pathogenic antibodies may contribute to the development of focal inflammation, neuronal cell loss, and pharmacoresistant seizures in some patients with this disease. This process may be an important component in the initial, active phase of RE.

Cerebral Cortex↗
PubMed1992

IgA nephropathy in patients with congenital C9 deficiency.

The clinical, histologic, and immunopathological findings of three young Japanese males with congenital C9 deficiency and primary IgA nephropathy are reported. The C9 deficiency was discovered either through mass complement screening, or when low hemolytic activity for CH50 and normal C3 levels were detected in plasma. Hematuria and proteinuria were detected at the age of 8 or 9 years as a result of annual urinary screening tests for school children. Renal biopsy showed focal and segmental mesangial proliferation with small epithelial crescents in one patient, and mild, diffuse mesangial proliferation in two. IgA and C3 were deposited predominantly in the mesangial area, and staining for C9 was negative in these patients. Electron microscopy revealed electron dense deposits predominantly in mesangial and paramesangial zones. Immunohistochemical staining in renal biopsy tissues from two patients showed mesangial staining for C5, C8, and S-protein, but staining for C5b-9 neoantigen was completely negative. These results show that the formation of C5b-9 complex is not essential for the induction of human IgA nephropathy, and also for the proliferation of mesangial and even parietal epithelial cells.

Child↗
PubMed2001

Complement-mediated lipopolysaccharide release and outer membrane damage in Escherichia coli J5: requirement for C9.

Lipopolysaccharides (LPS) are major antigenic components of the outer membrane of Gram-negative bacteria and can stimulate activation of the complement system. Such activation leads to formation of the complement membrane attack complex (MAC) on the cell walls, LPS release and, in serum-sensitive strains, to cell death. In this study, Escherichia coli J5 strains, which incorporate exogenous galactose exclusively into LPS, were used to generate target strains with different LPS chemotypes, and the LPS of the strains was labelled with tritium (3H-LPS). The ability of normal human serum (NHS) and human complement-deficient sera to release LPS was subsequently monitored. NHS-induced release of 64-95.7% of 3H-LPS within 30 min; overall, no significant difference was observed between release of LPS from E. coli J5 strains with different LPS chemotypes. In functional assays, maximum LPS release had occurred by 30 min and before maximum bacterial killing. Electron microscopy revealed NHS-induced outer-membrane disruption in the form of blebs at 15 min; at this time-point the inner membrane remained intact. Background LPS release and no bactericidal activity were detected in heat-inactivated serum or human sera deficient in C6, C7 or C8. The C9-deficient (C9D) serum had low bactericidal activity and failed to induce LPS release; however, addition of purified human C9 reconstituted its ability to release LPS. This study demonstrated the need for functional C9 molecules for LPS-releasing activities in serum-sensitive E. coli J5 strains.

Blood Bactericidal Activity↗
PubMed1992

Bactericidal activity of C9-deficient human serum.

Escherichia coli B/SM, strain 1-1, was killed dose dependently by human hereditary C9-deficient serum (C9DHS), which was shown to contain no C9 Ag by an ELISA method. On the other hand, human hereditary C7-deficient serum did not kill the bacteria under similar conditions. The bactericidal activity of C9DHS was inhibited by rabbit anti-C5 antibody but not by murine anti-C9 mAb. The anti-C9 antibody decreased the bactericidal activity of normal human serum (NHS) to the level of that with C9DHS. Sheep anti-human lysozyme antibody did not affect the bactericidal activity of C9DHS or NHS even when added at more than twice the concentration required to block the serum lysozyme activity on Micrococcus luteus. After treatment with C9DHS and washing, surviving Escherichia coli were killed by C9, but not by lysozyme, transferrin, or both. Other strains of E. coli (K12 W3110, C600, and NIHJ) and Salmonella typhimurium (strain NCTC 74), all maintained in the laboratory, were also killed by C9DHS. However, pathogenic strains recently isolated from patients with traveler's diarrhea and some strains of S. typhimurium were resistant to both C9DHS and NHS, at least at the serum concentration tested. A concentration of 0.1 M Tris did not increase the susceptibility of serum-resistant strains of bacteria to C9DHS, but made one strain of S. typhimurium tested susceptible to NHS, but not to C9DHS. These results clearly showed that C9DHS kills bacteria that are sensitive to NHS through activation of C up to the step of C8 in the same way that C9-deficient C serum lyzed sensitized erythrocytes.

Blood Bactericidal Activity↗
PubMed1997

The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity.

The membrane attack complex of complement (C) in sublytic concentrations stimulates endothelial cells (EC) to express adhesion molecules and to release biologically active products. We have examined the ability of a cytolytically inactive form of this complex, which is incapable of inserting into the cell membrane, to upregulate the expression of adhesion molecules and of tissue factor (TF) procoagulant activity. The inactive terminal C complex (iTCC) was prepared by mixing C5b6, C7, C8, and C9 and was purified by fast protein liquid chromatography on a Superose 12 column. Binding of this complex to EC was found to be dose dependent and was inhibited by anti-C9 antibodies, as assessed both by ELISA using an mAb anti-C9 neoantigen and by measuring cell-bound 125I-labeled iTCC. Exposure of EC to iTCC resulted in a dose- and time-dependent expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 accompanied by increased levels of the corresponding mRNA, but not in the rapid expression of P-selectin. Inactive TCC also induced increased TF activity evaluated by a chromogenic assay that measures the formation of factor Xa. These effects were inhibited by anti-C9 antibodies. The data support the conclusion that iTCC may induce proinflammatory and procoagulant activities on EC.

Cell Adhesion Molecules↗
PubMed1998

Molecular analysis of polyreactive monoclonal antibodies from rheumatic carditis: human anti-N-acetylglucosamine/anti-myosin antibody V region genes.

Anti-myosin Abs are associated with inflammatory heart diseases such as rheumatic carditis and myocarditis. In this study, human cross-reactive anti-streptococcal/anti-myosin mAbs 1.C8, 1.H9, 5.G3, and 3.B6, produced from peripheral blood lymphocytes of patients with rheumatic carditis, and mAb 10.2.5, produced from a tonsil, were characterized, and the nucleotide sequences of their V(H) and V(H)L genes were analyzed. Human mAbs 1.C8, 1.H9, 10.2.5, and 3.B6 reacted with human cardiac myosin while mAb 5.G3 did not. The mAbs were strongly reactive with N-acetyl-beta-D-glucosamine, the dominant epitope of the group A streptococcal carbohydrate. mAb 1.H9 was moderately cytotoxic to rat heart cells in vitro in the presence of complement. The anti-myosin mAbs from rheumatic carditis were found to react with specific peptides from the light meromyosin region of the human cardiac myosin molecule. Anti-streptococcal/anti-myosin mAbs from normal individuals reacted with distinctly different light meromyosin peptides. The mAbs were encoded by V(H)3 gene segments V3-8, V3-23, and V3-30 and by the V(H)4 gene segment V4-59. The variable region genes encoding the anti-streptococcal/anti-myosin repertoire were heterogeneous and exhibited little evidence of Ag-driven somatic mutation.

Acetylglucosamine↗
PubMed1995

Complement expression in human brain. Biosynthesis of terminal pathway components and regulators in human glial cells and cell lines.

C biosynthesis at extrahepatic sites remote from plasma C may be important in the protection of tissues against inflammation and infection but may also contribute to tissue injury. This latter possibility is particularly relevant in the central nervous system (CNS), where several cell types are susceptible to damage by C. We have previously shown that human astrocyte-derived tumor cell lines synthesize and secrete all of the components of the activation pathways of C. In this study, we demonstrate that these cells also produce the components (C6, C7, C8, and C9) and regulators (S-protein and clusterin) of the lytic terminal C pathway. The terminal components produced are hemolytically active, and secretion is markedly up-regulated by the inflammatory cytokine IFN-gamma. Primary human fetal astrocytes also expressed C6, C7, S-protein, and clusterin. The human monocyte/macrophage cell line, used here as a model for microglia, also produced all terminal components and regulators when appropriately stimulated. These studies raise the prospect of the intrathecal synthesis of a complete, functional C system and its regulators in the inflamed CNS. Intrathecal C synthesis may be important in the resolution of infection and inflammation but, given the C susceptibility of some CNS cell types, may also exacerbate damage in demyelination and neurodegeneration.

Antibodies, Monoclonal↗
PubMed1978

Leukocyte complement: neoantigens of the membrane attack complex on the surface of human leukocytes prepared from defibrinated blood.

The neoantigenic determinants (neoAg) which have been identified in the human C5b-9 membranolytic C complex were detected here by the direct fluorescent antibody technique on the surface of 27 +/- 11% of viable peripheral blood leukocytes (PBL). The cells were prepared from defibrinated blood by sedimentation on Ficoll-Hypaque. Specificity of the antisera was established by quantitative inhibition of the fluorescent staining reaction, and of agglutination of EAC1-7, by highly purified C5b-9 complex. No inhibition was observed with fresh normal human serum. The majority of the PBL with surface neoAg was found in the B lymphocyte subpopulation that failed to form rosettes with sheep erythrocytes. NeoAg on B lymphocytes was removed to differing degrees by trypsin, papain, or pepsin treatment, and by maintaining the cells at 4 degree C for 20 hr in serum-free medium. The individual components, C5, C6, C7, C8, and C9, were also detected on the surface of PBL. With differential fluorescent stains, C5 and neoAg as well as C8 and neoAg could be detected on the same cells. The results indicate that viable B lymphocytes prepared from defibrinated blood, have the components of the membrane attack complex of C on their surface. The concomitant occurrence of the neoAg indicates that these proteins are present at least in part in the form of the assembled terminal complex.

Antigens↗
PubMed1991

Complement and antibody mediate enhancement of HIV infection by increasing virus binding and provirus formation.

Previous studies have shown that infection of complement receptor (CR2)-bearing cells with HIV pretreated with antibody (Ab) plus complement (C) resulted in increased virus expression. The current study was designed to determine whether C-mediated 'enhancement' of HIV-1 production was the result of increased virus infection of cells as assessed by provirus formation and virus binding. Virus was incubated with anti-HIV Ab and/or C and added to CR2-positive MT-2 cells. Increased virus expression by MT-2 cells correlated with increased numbers of HIV-immunofluorescent-positive cells at 24 and 48 h and higher levels of provirus detected 8-28 h after infection. MT-2 cells also bound threefold more Ab-plus-C-treated virus than untreated virus. Serial dilutions of C showed that high levels of C with Ab did not enhance but rather suppressed virus expression. Studies were also performed which showed that terminal C components C5 and C8 were not necessary for the enhancing effect. The increased binding of C-coated HIV to CR-positive cells has important implications for the fate of virus in vivo.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1990

The complement SC5b-9 complex mediates cell adhesion through a vitronectin receptor.

Adhesion of cells to the terminal complement complex of C5b through C9 containing the serum S-protein (SC5b-9) was investigated using a microtiter plate attachment assay with L8 myoblast indicator cells. The skeletal muscle-derived L8 myoblasts bound and spread on substratum coated with SC5b-9, and with the vitronectin/S-protein component of SC5b-9. The myoblasts did not adhere to substratum coated with collagen, laminin, or fibronectin. The cell attachment was blocked by antibody to vitronectin/S-protein, whereas antibody to the other components C5, C6, C7, C8, or C9 had minimal effect. The cells were not bound to free vitronectin because attachment activity was removed by adsorption with an anti-C6 antibody column. The L8 cell attachment was dependent on divalent cations, was blocked by synthetic peptides containing the amino acid sequence Arg-Gly-Asp, and was inhibited by antivitronectin receptor antibody. These results indicate that cells adhere to the SC5b-9 complex through interaction of the vitronectin component with an integrin vitronectin receptor. Cell attachment to terminal C complexes could be used for leukocyte adherence and migration during inflammation, and also for attachment of tissue cells during regeneration after disease or traumatic injury.

Animals↗
PubMed1993

Generation of diacylglycerol and ceramide during homologous complement activation.

Formation of sublytic terminal complement complexes (TCC) on nucleated cells produces transient increase in [Ca2+]i and activates protein kinase C. The present study is to evaluate whether TCC can generate endogenous signal messengers other than Ca2+ that regulate cell activities by measuring mass-levels of sn-1,2-diacylglycerol (DAG) and ceramide. As targets, lymphoblastoid human B cell lines JY25 and its mutant JY5 were used. JY5, cells deficient in glycosylphosphatidylinositol-anchored proteins with higher lytic susceptibility to human complement, are four times more efficient in forming C5b-9. When cells sensitized with limited anti-class II IgG were exposed to human serum to generate sublytic TCC, a sustained increase in DAG and ceramide was observed with a maximum 3.6-fold DAG increase over basal level in JY25 and 2.8-fold in JY5, and 6.3-fold ceramide increase in JY25 and 2.8-fold in JY5. The effect of TCC was evaluated with C7-deficient human serum (C7D) +/- C7 and also with C5b6, C7, C8, and C9 proteins. The DAG and ceramide increase by C7D + C7 over C7D control were 1.6- and 1.8-fold, respectively, in JY25, and 2.3-, and two-fold in JY5. TCC activation also induced an increased hydrolysis of sphyingomyelin and phosphatidylcholine. In addition, DAG increase by TCC was primarily achieved by C5b-7 and preincubation of cells with pertussis toxininhibited DAG increase, suggesting an involvement of a pertussis toxin-sensitive GTP-binding protein. As important signal transduction molecules, DAG and ceramide generated in response to TCC assembly, could participate in cell activation during inflammation and repair.

Animals↗
PubMed1994

Analysis of C3 deposition and degradation on Neisseria meningitidis and Neisseria gonorrhoeae.

The deposition and degradation of human complement component C3 on the cell surfaces of Neisseria meningitidis and Neisseria gonorrhoeae were studied. Bacteria were incubated in human serum, and ester-linked C3 fragments were analyzed by hydroxylamine release and immunoblot detection. Similar patterns of C3 degradation were found for both serum-resistant and serum-sensitive meningococcal strains of serogroups A, B, C, Y, and W135, as well as for serum-sensitive gonococcal strains and their sialylated serum-resistant variants. The predominant fragments in all cases were the 40-kDa alpha' 2 chain of iC3b and the 75-kDa beta chain common to both C3b and iC3b. The 67-kDa alpha' 1 chain of iC3b was also detected. The 105-kDa alpha' chain of intact C3b represented a minor proportion of deposited C3. Capsule-specific immunoglobulin G or immunoglobulin A1 did not alter the observed degradation patterns, nor did incubation of meningococci in properdin-deficient serum. The degradation of C3 in C5-, C6-, or C8-deficient serum was the same as that in normal serum, although the deposition of C3 was severely limited, based as indicated by the intensity of the fragments. With the use of an enzyme-linked immunosorbent assay that measured total iC3b and C3, I found that both iC3b deposition and C3 deposition varied among meningococcal and gonococcal strains and that the amounts of iC3b and C3 were independent of the relative quantities of cell surface sialic acid and of serum sensitivity for meningococci but not for gonococci. I conclude that complement activation on neisserial cell surface results in the formation of an identical repertoire of predominantly iC3b fragments of ester-linked C3b molecules regardless of the presence of sialic acid in either the capsule or the lipooligosaccharide or of the sensitivity of the organism to complement-mediated lysis but that the quantities of both ester- and amide-linked iC3b molecules deposited exhibit strain variability.

Complement Activation↗
PubMed1979

An anticomplementary agent, K-76 monocarboxylic acid: its site and mechanism of inhibition of the complement activation cascade.

A monocarboxylic acid derivative (K-76 COOH) of K-76, purified from the culture filtrate of Stachybotrys complement I nov. sp. K-76, inhibits complement (C) activity. Its inhibitory action is mainly on C5 step. It strongly inhibits the generation of EAC1,4b,2a,3b,5b from C5 and EAC1,4b,2a,3b, and accelerates the decay of EAC1,4b,2a,3b,5b. It also causes some inhibition of the reactions of the reactions of C2,C3,C6,C7 and C9 with their respective preceding intermediate cells. It has no effect on the generation of EAC1,4b from C4 and EAC1, or of EAC-8 from C8 and EAC-7, and apparently increases the generation of EAC1,4b from C1 and EAC4b probably by inhibiting transfer or turnover of C1. It does not affect the rate of decay of EAC1,4b,2a or the T max of generation of EAC1,4b,2a, and it inhibits immune adherence only at high concentration. K-76 COOH also strongly inhibits hemolysis through the alternative pathway of C activation by cobra venom factor, but it does not seem to inhibit the early steps of the alternative pathway, because it has little affect on the consumption of C3 or the conversion of beta 1C to beta 1A on treatment of C serum with zymosan. K-76 COOH probably combines with C5 molecules, forming the inactive complexes, or it causes the structural alteration of C5.

Animals↗
PubMed1991

Urinary excretion of terminal complement complexes in glomerular disease.

To evaluate renal terminal complement activation in patients with glomerular diseases, we measured terminal complement complexes (TCCs) in plasma and urine with sandwich enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against a C9 neoepitope expressed on TCC and a polyclonal antihuman C7 antibody. TCCs were detectable in plasma but not in urine in most of normal controls. In plasma, TCC levels were elevated in 4 of 22 patients with lupus nephritis and in 6 of 12 with membranoproliferative glomerulonephritis. However all patients with IgA nephritis, focal glomerulosclerosis, idiopathic membranous nephritis and idiopathic minimal change nephrotic syndrome (MC) showed normal values. In urine, TCCs were detectable in almost all patients with heavy proteinuria (greater than or equal to 100 mg/ml) except MC. The TCCs present in urine were partially purified by gel filtration using Sepharose 6B and were found to contain C5, C6, C7, C8, C9 and S protein by ELISA. Although the molecular weight of TCC is similar to that of IgM, the fractional excretion rate of TCC was about 100 times higher than that of IgM. These results suggest that TCCs detectable in urine contain SC5b-9 complexes and are mostly of renal origin.

Adolescent↗