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PubMed1992

Regulation of complement activity by vaccinia virus complement-control protein.

A major protein secreted by vaccinia virus-infected cells has structural similarity to the super-family of complement-control proteins. This vaccinia complement-control protein (VCP) was studied to determine how it regulates complement activation. VCP was bound by C4b and C3b and served as a cofactor with factor I in cleaving these two molecules. VCP inhibited the formation and accelerated the decay of the classical C3 convertase. It also accelerated decay of the alternative pathway convertase, although higher concentrations were apparently needed. In vitro, therefore, VCP interfered with the classical and alternative complement pathways at several steps. In vivo, this interference may increase the virulence of vaccinia virus by enabling it to escape attack by the host's complement system.

Complement Activation↗
PubMed1982

Alternate pathway of complement and bactericidal response of the channel catfish to Salmonella paratyphi.

Fresh channel catfish (Ictalurus punctatus) serum from unimmunized catfish exhibited 100% bactericidal activity against Salmonella paratyphi. Components responsible for bactericidal activity could be absorbed from the fresh catfish serum with S. paratyphi. The bactericidal system of the fresh catfish serum showed a need for magnesium rather than for calcium after EDTA treatment. The addition of salicylaldoxime or ammonium hydroxide to catfish serum indicated the alternate rather than the classical pathway of complement activation to be important in bactericidal activity against S. paratyphi. Bactericidal activity of catfish serum was labile when incubated at 47 degrees C for 30 min., stable for at least 4 mo. at -80 degrees C and could be absorbed with S. paratyphi at 25 degrees C. Very minimal bactericidal activity was present in the descending portion of the first 13.7S peak with most activity being found in the descending portion of the second 7.1S peak and throughout the entire 3.4S peak after Sephadex G-200 catfish serum fractionation.

Ammonium Hydroxide↗
PubMed1978

In vitro activation of the alternative pathway of complement by settled grain dust.

Settled grain dust was collected from several active grain elevators in the Superior-Duluth areas of the United States. Particle size distribution (47% less than 5 micrometer) and endotoxin contamination (429 ng/gm) of the dust were similar to those reported for the airborne parent dust. Human complement uas activated in vitro in a dose-response manner which could be quantified. This hemolytic consumption was via the alternative pathway as defined by ethylenediaminetertraacetic acid/ethylene-glycol-bis-(beta-amino-ethyl ether) N,N'-tetraacetic acid (EDTA/EGTA) differential serum chelation, factor B conversion, and complement reductions in serum from guinea pigs deficient in C4. It is proposed that continuous low-dose exposure to aerosolized, biologically active rafter dust could contribute to the respiratory insult of grain workers.

Complement Activation↗
PubMed2001

Isolation and characterization of a human alternative complement pathway-inhibiting protein from larval hemolymph of the silkworm, Bombyx mori.

An alternative complement pathway-inhibiting protein (ACPIP), which inhibits the activation of the alternative complement pathway (ACP) of the human serum, was isolated from larval hemolymph of the silkworm, Bombyx mori, by using ammonium sulfate fractionation and column chromatographies to homogeneity. About 400microg of ACPIP was routinely obtained from 20ml hemolymph. The purified ACPIP preparation consisted of two distinct polypeptides (34 and 32kDa) on SDS-PAGE. The amino acid compositions of the two polypeptides were nearly identical; 21% of the amino acid residues were acidic. The amino terminal amino acid sequences up to 20 residues in these two polypeptides were also identical. Addition of the ACPIP to human serum resulted in a dose-dependent inhibition of the hemolysis of intact rabbit erythrocytes via the ACP, whereas in no inhibition of hemolysis of sensitized-sheep erythrocytes (EA) via the classical pathway.

Amino Acid Sequence↗
PubMed1986

The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing.

We have examined the killing of E. coli and kinetics of lipopolysaccharide (LPS) release after the exposure of the bacteria to normal human serum (NHS) and sera deficient in complement components, or with inactivated complement components. LPS of the galactose epimerase-deficient strain E. coli J5 were specifically radiolabeled by growing the bacteria in a medium containing [3H]galactose. Exposure of the washed bacteria to NHS resulted in a significant reduction (greater than 99%) in viability within 15 min and the concomitant release of radiolabeled LPS. However, maximal release of LPS was consistently 30% of the total radiolabel incorporated into the LPS molecules. The amount of tritium-labeled LPS released was shown to be directly proportional to the concentration of bacteria exposed to NHS, suggesting that release of LPS was not limited by the availability of some critical serum component(s). The consumption of complement in NHS by incubation with E. coli was demonstrated by decreased alternative and classical pathway-specific hemolytic activity. The use of Factor D-depleted and VEM-treated human sera demonstrated that, with these bacteria, both the alternative and classical pathways of complement contribute to bacterial killing and release of LPS. It is noteworthy that, in VEM-treated and Factor D-depleted sera, the rate of killing and the kinetics of LPS release were somewhat slower as compared to control serum. Bacterial killing in C7-depleted and C9-deficient human sera was minimal. Neither killing nor LPS release occurred in heat-inactivated (56 degrees C, 30 min) human serum. The amount of [3H]LPS released by C9-deficient serum was qualitatively similar to the amount released by the action of NHS. Tritium-labeled LPS was not released in C7-depleted serum. These data indicate that bacterial killing can be dissociated from LPS release, and suggest that, whereas LPS release may be necessary for the bactericidal effects of serum complement, it is probably not sufficient to effect killing. Furthermore, a significant fraction of LPS can be removed from the outer membrane of the bacteria without an apparent affect on viability.

Adult↗
PubMed1979

Activation of human complement by liposomes: a model for membrane activation of the alternative pathway.

Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.

Agammaglobulinemia↗
PubMed1990

Activation of the alternative pathway of complement by twelve different rabbit-mouse chimeric transfectoma IgA isotypes.

Previous experiments have resulted in the identification and cloning of 13 nonallelic genes encoding the constant region of rabbit IgA H chains. The genes, C alpha 1 to C alpha 13, were each cloned into an expression vector containing the VDJ gene of a dansyl (DNS)-binding murine hybridoma and the constructs were then transfected into SP2/0 cells that were producing murine kappa-L chains from the DNS-binding hybridoma. Of the 13 resulting transfectomas, 12 were shown, by ELISA, to secrete DNS-binding chimeric rabbit-mouse IgA molecules. These transfectoma antibodies, representing 12 different isotypes, are of high affinity and provide a unique source of Ag-specific IgA for comparison of the functions of the multiple IgA isotypes. One such function for antibodies is activation of C by either the classical or alternative pathway. We have used the DNS-binding IgA transfectoma antibodies in C assays based on binding of rabbit C3 to IgA-Ag complexes in an ELISA. The results demonstrated that all 12 IgA isotypes are capable of activating C by the alternative pathway but that none can activate C by the classical pathway. Control experiments demonstrated that activation was hapten dependent and was not caused by endotoxin contamination. These data demonstrate that Ag-specific IgA molecules, unmodified by heat or chemical aggregation, activate C by the alternative pathway but not by the classical pathway.

Animals↗
PubMedDate not supplied

Lambda prophage decreases Escherichia coli sensitivity to human serum bactericidal effect.

Escherichia coli C600 and C600(lambda) strains were tested for their susceptibility to the bactericidal action of 4% normal human serum. C600 survival was reduced to 30%, 23% and 16% after 60, 150 and 180 min of exposure to serum, respectively, whereas the percentage of survival of C600(lambda) was 199, 109 and 65% at the same times. The estimated exposition times for 50% killing showed an eight-fold difference, they were 23 and 202 min for C600 and C600(lambda), respectively. None of the two strains tested was killed when incubated with serum whose alternative complement pathway was inactivated by heating at 50 degrees C for 20 min, showing that this pathway, and not the classical one, was responsible of the bactericidal action, a conclusion further supported by the finding that both strains were differentially killed by the alternative complement pathway, C600 showing a 14X, 10X and 4X greater susceptibility than C600(lambda) at 60, 120 and 180 min of exposure to serum whose classical pathway was selectively inhibited by chelation with 10 mM EGTA plus 2 mM MgCl2. We feel that lambda phage may lower the serum sensitivity of its lysogen by altering the bacterial external surface, perhaps by the inclusion of some protein encoded by an accessory gene of the lambda genome, and thus interfering with either the formation, deposition or activity of the membrane attack complex.

Bacteriophage lambda↗
PubMed1988

Structure-function relationships in the inhibitory effect of heparin on complement activation: independency of the anti-coagulant and anti-complementary sites on the heparin molecule.

Fluid phase heparin inhibits formation of the classical and alternative pathway C3 convertase of complement in assays performed either with purified complement proteins or in whole serum. Experiments using oligosaccharides of homogeneous mol. wt obtained by mild nitrous hydrolysis of heparin, demonstrated that the inhibitory activity of heparin increased exponentially with mol. wt for fragments containing between 4 and 14 saccharidic units and that fragments of mol. wt above 4700 (greater than 14 saccharidic units) had a similar anti-complementary activity to that of native heparin. Fragments of homogeneous mol. wt (octasaccharides) separated by ion exchange chromatography on the basis of negative charges, exhibited increasing inhibitory activity with increasing sulfate content. Over-sulfation of fragments of defined mol. wt resulted in a constant enhancement of the relative capacity of each fragment species to inhibit formation of the classical and alternative pathway C3 convertases. A synthetic pentasaccharide representing the minimal critical sequence responsible for the binding of heparin to anti-thrombin III exhibited a similar inhibitory capacity on formation of the C3 convertases as another synthetic pentasaccharide that was devoid of anti-Xa activity. These studies contribute to define a minimal structure of the heparin molecule with C3b- and C4b-binding capacity and definitively establish the independency of the anti-coagulant and anti-complementary sites on the heparin molecule.

Blood Coagulation↗
PubMed1985

Phagocytosis and killing of Gardnerella vaginalis by human neutrophils.

Gardnerella vaginalis was ingested and killed by neutrophils in the presence of normal human serum. Heat inactivation of the serum inhibited these processes. The opsonisation of some but not all G vaginalis strains was enhanced by immune rabbit serum. Immune serum did not, however, enhance intracellular killing. Blockade of the classical pathway of complement activation had no effect on the opsonic activity of human serum. These results suggest that the opsonisation, phagocytosis, and killing of G vaginalis by human serum and neutrophils is primarily mediated by complement activated by the alternative pathway. Serum without neutrophils has little effect on the viability of G vaginalis.

Animals↗
PubMed1990

Generation of the membrane attack complex during haemodialysis: impact of classical and alternative pathway components.

1. The generation of the plasma SC5b-9 complex (the soluble form of the membrane attack complex) during haemodialysis was studied in 29 patients together with markers of the classical (C4d fragment) and alternative (Bb fragment) pathways and the common iC3b fragment. 2. In the patients dialysed with Hemophan and Cuprophan membranes, a rapid increase in the plasma levels of SC5b-9 complex, Bb fragment and iC3b fragment occurred within 15 min of the initiation of the haemodialysis procedure. The SC5b-9 complex and the Bb fragment concentrations remained at a plateau until 120 min thereafter. The iC3b fragment level showed a continual decline. 3. The C4d fragment concentrations remained unchanged, indicating that generation of the membrane attack complex is most likely due only to activation of the alternative pathway. 4. The generation of the membrane attack complex occurred throughout the full haemodialysis period. In patients treated with Polysulfon membranes no statistically significant variation in these components was noted. 5. The study shows that the membrane attack complex may be generated during the full period of the haemodialysis session and is a stable index of biocompatibility. 6. Moreover, it seems be involved directly in haemodialysis-associated phenomena.

Complement Activation↗
PubMed1994

Reconstitution of the alternative pathway of complement by plasma infusions given to a patient with an SLE-like syndrome associated with a hereditary C3 dysfunction.

OBJECTIVE: To reconstitute a dysfunctional form of complement factor C3 in a patient with a systemic lupus erythematosus (SLE)-like syndrome. METHODS: The propositus was treated with plasma infusions during five sessions over a period of eight months. RESULTS: The alternative pathway was reconstituted to normal levels for approximately two to three days after each infusion. C3 fragments were incorporated into previously detected deposits of IgG and IgM at the dermal-epidermal junction and the immune complex levels gradually decreased during the whole treatment period. CONCLUSION: The reconstitution appears to result in the solubilisation of tissue immune complexes and a subsequent transportation to the fixed macrophage system.

Antigen-Antibody Complex↗
PubMed1987

Contribution of the complement system to antibody-mediated binding of Trypanosoma gambiense to macrophages.

The role of complement in the process of binding of trypanosomes to macrophages in the presence of specific antibody was studied. The aggregation of trypanosomes observed at the optimal antigen-antibody ratio or in the presence of excess antigen inhibited the binding. Complement caused clumped trypanosomes to dissociate, and the free trypanosomes, which were presumed to be coated with antibody that had fixed complement, readily attached to surfaces of phagocytes. Thus, complement was shown to contribute at the site of the antigen-antibody reaction to the creation of an environment suitable for the binding. It seems likely that the trypanosomes dissociated by complement adhered to C3 receptors of the macrophage. However, in the absence of complement and in regions of antibody excess, free trypanosomes also attached to phagocytes. Thus phagocytes may also have receptors for the Fc portion of aggregated antibody. Complement activated by the alternate pathway also enhanced attachment of trypanosomes to phagocytes, but the effect was not as rapid as it was when complement was activated by classical means.

Animals↗
PubMed1991

An assay for complement factor B in species at different levels of evolution.

The alternative complement pathway is regarded as the evolutionary ancestral complement system. It is therefore of interest to analyze and characterize factor B of the alternative complement pathway in animals at different levels of evolution. We describe a functional assay which is based on the interaction of factor B from various animal species with Cobra Venom Factor, the snake C3b-analog. The signal system in the assay is C3-conversion in factor B-depleted chicken serum. The results show that a functional C3-convertase can be created with factor B from mammals, aves, and amphibians.

Animals↗
PubMed1987

Alternative pathway of complement activation by stimulated T lymphocytes. II. Elevation of cytotoxic potential against complement receptor-carrying cell lines.

Exposure of lectin-stimulated (concanavalin A, phytohemagglutinin and pokeweed mitogen) blood lymphocytes to human serum or to purified C3 increased their cytotoxic capacity towards complement receptor positive targets such as Raji and Daudi cells. The lysis of complement receptor-negative lymphoblastoid cell lines was not influenced. The lytic capacity of lymphocytes exposed to 12-O-tetradecanoylphorbol 13-acetate was not elevated by human serum. Lectin-stimulated lymphocytes were previously shown to activate and bind C3. The results using lymphocytes activated in different ways and targets with or without complement receptor expression suggest that the C3b deposited on lymphocytes binds to the complement receptor on the targets. This contact elevates the avidity between the two cells as indicated also by the increased frequency of the lymphocyte-target conjugates. On the basis of immune adherence the C3 fragment bound on the lymphocytes was identified as C3b. The increase of the conjugate formation and cytotoxicity was abrogated when the target cells, Raji, were pre-exposed to purified C3d which occupy the CR2 receptor. The majority of lymphocytes responsible for the cytotoxicity were CD8+.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1993

Activation of the complement system by Francisella tularensis lipopolysaccharide.

We have shown by combining lipopolysaccharide (LPS) extracted and purified from Francisella tularensis live vaccine strain (LVS) with normal complement and back titrating with sensitised sheep red blood cells that the LPS activates complement. Deionising the LPS and converting it into the single salt forms of pyridine, ethanolamine and triethylamine altered the ability to activate complement according to the apparent molecular weight due to aggregation. Francisella tularensis LPS activated complement deficient in a component of the alternative pathway (factor B) but failed to activate complement deficient in a component of the classical pathway (C1q). In addition normal complement suspended in ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) which inactivates the classic pathway was not activated by LPS, and we concluded that the LPS activates complement predominantly via the classical pathway. LPS bound to specific monoclonal antibodies activated complement more than LPS alone. An anti-core monoclonal antibody was approximately tenfold more potent when bound to LPS then an anti-O side chain monoclonal antibody in activating complement.

Antibodies, Bacterial↗
PubMed1997

Detection and quantification of the control proteins of the alternative pathway of complement in 3T3-L1 adipocytes.

The complement peptide C3a desarg is identical to acylation-stimulating protein (ASP), a human plasma protein that potently stimulates adipocyte triacylglycerol synthesis and glucose transport. Both human and murine adipocytes express mRNA and/or protein for the complement components C3 and factors B and D (adipsin) required to generate ASP. However, the regulatory mechanisms controlling this process are unknown. We have established a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique to demonstrate the presence in mouse 3T3-L1 adipocytes of mRNA for all components of the alternative pathway, including the control proteins factors I and H, CR1 and properdin. On differentiation, mRNA for C3 (fivefold) and factor D (> 50-fold) increased, whereas stimulation with tumour necrosis factor (TNF)-alpha and interleukin (IL) 1 beta led to eightfold increases in factor B mRNA. Metabolic labelling followed by immunoprecipitation showed that factor B protein is normally present in small quantities, and is greatly increased by cytokine stimulation. The larger quantities of C3 and H proteins present were little affected, whereas levels of C3a increased on cytokine stimulation. These results suggest that the rate-limiting step in the cytokine-induced production of ASP in adipocytes is factor B synthesis.

3T3 Cells↗