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PubMed1991

Simple quantitative haemolytic microassay for determination of complement alternative pathway activation (AP50).

We describe here a simple, reliable and quantitative method to measure the alternative pathway (AP) dependent mode of complement activation for the lysis of rabbit erythrocytes. In the test the reciprocal of the plasma volume needed to destroy 50% of available rabbit erythrocytes is defined as the functional measure of this activity (AP50). The test was found to be highly reproducible both within and between assays with a coefficient of variation which was less than 5%. Sensitivity was also shown to be satisfactory and all of the plasma samples from the healthy blood donors which were tested could be assayed with precision. The specificity of the AP50 assay for AP complement activation was verified by the fact that a C4 affinity-depleted plasma gave an AP50 value within the normal range (52.8 U/ml) while a similar aliquot of the same plasma, affinity-depleted of factor B, gave an undetectable AP50 value (less than 10 U/ml). Furthermore, a sample was unable to lyse the target cells when heated to 50 degrees C or 56 degrees C, treatments which are known to destroy factor B and total haemolytic complement, respectively. To ensure inhibition of the classical pathway of activation. EGTA and MgCl2 were added during the assay. An advantage of this assay is that it is possible, using microplates, multipipettes and a spectrophotometer coupled to a computer, to semi-automate the procedure.

Animals↗

PubMed1992

Determination of optimal in vitro conditions for caprine alternative complement pathway assay.

Optimal in vitro testing conditions for caprine alternative complement pathway assay were determined. Effects of the following variables were tested: heterologous erythrocytes; pH, ionic strength and Mg2+ ion concentration of the complement diluent; incubation time and temperature. Rabbit erythrocytes were the optimal target cells. The optimal buffer conditions were: pH 8.0, ionic strength 0.06 mmol NaCl and 5mmol Mg2+ ion. Optimal incubation time and temperature were 75 min and 30 degrees C, respectively.

Animals↗

PubMed1989

Influence of surface modulations by enzymes and monoclonal antibodies on alternative complement pathway activation by Yersinia enterocolitica.

Effector mechanisms resulting from alternative complement pathway (ACP) activation cannot act efficiently against Yersinia enterocolitica serotype O3, as indicated by poor C3 to C9 consumption and by survival in EGTA (ethyleneglycoldiaminetetraacetic acid) Mg-serum. These results were not influenced by the lack or presence of plasmid-encoded outer membrane proteins or lipopolysaccharides (LPS) with different amounts of side chains or by treatment of the bacteria with pronase or neuraminidase. Surface modulation of Y. enterocolitica with polyclonal immunoglobulin G or the immunoglobulin G fragments F(ab')2 and Fab always converted Y. enterocolitica to a high ACP activator, with strong C3 to C9 consumption and surface deposition of activated C3. Killing of Y. enterocolitica as a result of antibody-mediated ACP activation was observed only with bacteria grown at 22 degrees C but not with bacteria from 37 degrees C cultures. The expression of complement resistance in Y. enterocolitica grown at 37 degrees C was not influenced by the presence or absence of plasmids. Using different monoclonal antibodies (MAb), we found that MAb with LPS specificity mediated ACP activation, whereas MAb specific for different plasmid-encoded outer membrane proteins were ineffective, despite surface binding. These results suggest a major inhibitory role of LPS on ACP activation which was neutralized by LPS-specific antibodies.

Animals↗

PubMed1987

Quantitation of C3 nephritic factor of alternative complement pathway by an enzyme-linked immunosorbent assay.

We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of C3 nephritic factor of the alternative pathway of complement (NeFA). Incubation of the NeFA-positive serum (patient KS serum) with normal human serum (NHS) in Mg-EGTA resulted in the formation of C3-B-IgG complex. No complex was formed in EDTA. At first this was detected as three types of complexes: C3-IgG, B-IgG and B-C3, by the combination of antibodies. The reaction mixture in Mg-EGTA was filtered through an ACA 22 column, from which the complexes were eluted in the same part as the first protein peak. When IgG purified from KS serum was incubated with NHS in Mg-EGTA, B-C3 complex increased in proportion to the dose of IgG. These results indicated that only one kind of complex consisting of IgG, C3 and B (IgG-C3-B) was generated by the addition of NeFA to NHS. Serum NeFA could be quantified as the titer of B-C3 complex formed after its incubation with NHS in Mg-EGTA. Using the ELISA method, NeFA was positive in five out of six patients with membranoproliferative glomerulonephritis (MPGN) type II and in only one of 17 with MPGN type I. Titers obtained by the new method were in good accordance with those by C3 conversion and C3bBb stabilization assays for NeFA, and the new method was more exact and simple than the conventional methods.

Antigen-Antibody Complex↗

PubMed1978

Regulation by membrane sialic acid of beta1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway.

Sheep erythrocytes in their native state did not activate the alternative complement pathway, as measured by lysis in dilutions of normal human serum containing [ethylenebis(oxyethylenenitrilo)] tetraacetic acid but acquired this capacity after membrane sialic acid residues had been removed (by sialidase) or modified (by NaIO(4)). Activation of the alternative pathway by sheep erythrocytes required removal or modification of at least 40% of the membrane sialic acid to reach threshold, and it increased proportionately when larger amounts of sialic acid had been affected. Studies with isolated proteins of the alternative pathway demonstrated that the altered erythrocyte membranes resembled natural activators in protecting bound C3b from inactivation by C3b inactivator and beta1H and protecting bound amplification C3 convertase (C3b,Bb) from decay-dissociation by beta1H. A 1% decrease in intact sialic acid was associated with a 1% decrease in beta1H activity in decay-dissociation of membrane bound C3b,Bb. Because removal of the C8 and C9 carbon atoms from the polyhydroxylated side chain of sialic acid by oxidation with NaIO(4) was functionally equivalent to removal of the entire sialic acid moiety, secondary effects of the latter reaction, such as diminution of the negative charge of the membrane or exposure of penultimate galactose residues, were not considered to be responsible for the altered activity of beta1H. These studies suggest that facilitation, by membrane sialic acid residues, of the interaction between bound C3b and beta1H is essential to prevent the particle from effectively activating the alternative pathway.

Animals↗

PubMed1976

Nonspecific complement activation by streptococcal structures. II. Properdin-independent initiation of the alternate pathway.

Complement consumption by isolated membranes and walls from Group A streptococci and various other gram-positive microbes has been tested. These microbial structures were found to activate the alternate complement pathway. However, unlike endotoxin, inulin, or other plant polysaccharides, activation of complement by our material was found to bypass properdin. The activating factor(s) also differs from cobra venom in its/their requirement for factor D. Preliminary experiments suggest this factor isolated from membranes to be a protein and to have a mol wt greater than 40-60,000 daltons. Our studies have led us to speculate that the phylogenetic role of the alternate complement pathway may be the primordial nonspecific defense system which has retained certain fundamental aspects up to the present time.

Antigens, Bacterial↗

PubMed1979

The activation of the alternative complement pathway by fetal calf serum and mouse thymocytes.

Mouse thymocytes activated the alternative complement pathway of mouse serum in the presence of heated fetal calf serum. The activation required C3 from the fetal calf serum but was independent of antibody either in the murine or bovine serum. No other murine cells tested, including erythrocytes, peripheral blood lymphocytes, lymph node cells, spleen cells, and various cultured cell lines, activated the alternative complement pathway as effectively as thymocytes. In addition, sera from species other than cows could not substitute for fetal calf serum. The C3 deposited on thymocytes was in the form of both C3b (immune adherence positive) and C3bi (conglutinable). We propose that the basis of activation in this system is the specific protection of bovine C3b on mouse thymocyte surface.

Absorption↗

PubMed1976

Serum factors activating the alternative complement pathway in autoimmune disease: description of two different factors from patients with systemic lupus erythematosus.

Serum factors which activated the alternative pathway of complement were detected in 10 of 26 patients with systemic lupus erythematosus (SLE), three of 18 patients with mixed connective tissue disease, one patient with scleroderma, and one with Sjögren's syndrome. This activation was detected by conversion of factor B and C3 into split products and by lysis of glutathione-sensitized human erythrocytes under conditions where classical pathway activation was blocked. The serum factors capable of activating the alternative pathway could be seperated into 7S and 19S-type molecules by sucrose density gradient ultracentrifugation. In two patients with SLE, serum factors were isolated by ion-exchange chromatography, preparative electrophoresis, and molecular sieve chromatography. The serum 7S factor was immunochemically identical to the C3 nephritic factor (C3NeF) of hypocomplementemic chronic glomerulonephritis. Antisera to other complement components failed to react with this purified material. The 7S factor was able to activate the alternative pathway in C2 deficient serum, and in normal human serum under conditions where classical pathway was blocked, but was unable to do so in sera depleted of factor B, factor D, and C3. The activity could be removed by antiserum to C3NeF. In contrast, the serum 19S activator of the alternative pathway was not reactive with antiserum to C3NeF. It had a fast gamma mobility on electrophoresis and the activity could be removed by absorption with anti-IgG and anti-IgM. It was suggested that the 19S factors could consist, in part, of immune complexes.

Absorption↗

PubMed1978

Activation of the alternate complement pathway by peptidoglycan from streptococcal cell wall.

Activation of the alternate complement pathway in human serum by several bacterial components was compared. Peptidoglycan from group A streptococcal cell walls was the most active material, on a weight basis, followed by cell walls, protoplast membranes, and whole cells. The group-specific carbohydrate was inactive. Treatment of peptidoglycan with low concentrations of lysozyme or short periods of sonic treatment enhanced complement activation. High concentrations of lysozyme or extended sonic treatment of peptidoglycan destroyed or greatly reduced the capacity to activate complement. Lysozyme treatment of group A streptococcal cell walls or lipopolysaccharide had no measurable effect. Activation of the alternate complement pathway by group D streptococcal cell walls was destroyed by lysozyme. Activity of peptidoglycan was not inhibited by N-acetyl glucosamine, N-acetyl muramic acid, or D-alanine-D-alanine. Conversion of C3 and factored B by peptidoglycan was shown to occur by immunoelectrophoresis and crossed immunoelectrophoresis.

Animals↗

PubMedDate not supplied

Heparin free hemodialysis does not cause fibrin consumptive coagulopathy and maintains alternate pathway complement activation.

Heparin-free hemodialysis allows demonstration of the intradialytic coagulation status not previously possible during standard heparin anticoagulant hemodialysis protocols in patients with end-stage renal disease. No evidence for consumptive coagulopathy was noted in the absence of heparin during hemodialysis with cuprophane hollow fiber dialyzers. Fibrinogen levels, euglobulin lysis time, and platelet counts remained stable throughout 3 hours of heparin-free hemodialysis. Serial thrombin times and platelet aggregation improved during heparin-free hemodialysis. Hemodialyzer volumes and the ultrafiltration coefficient decreased only minimally. The alternate pathway of complement activation associated with hemodialysis blood-membrane interaction is not prevented by heparin-free hemodialysis.

Adult↗

PubMed1979

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

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

Animals↗

PubMed1985

Properties of glycans that activate the human alternative complement pathway and interact with the human monocyte beta-glucan receptor.

The glycans used in an earlier study to define the ligand specificity of the human monocyte phagocytic receptor for unopsonized particulate activators were assessed for their capacities to activate the proteins of the human alternative complement pathway. Normal human serum was preincubated with glycans under conditions of chelation to prevent activation of the classical complement pathway, and the activation-depletion of the alternative complement pathway was determined by the subsequent capacity of the serum to lyse rabbit erythrocytes (Er). When serum was preincubated at a 1/2 dilution in 8 mM EGTA/2 mM Mg with increasing numbers of yeast glucan or zymosan particles, and was evaluated at final serum dilutions of 1/8, its capacity to lyse Er was found to be reduced by 50% with 1.9 X 10(6)/ml yeast glucan particles and 1.4 X 10(6)/ml zymosan particles. At 2 mg/ml of serum diluted 1/2 in 8 mM EGTA/2 mM Mg, nonturbid preparations of mannan, laminarin, or pyrogen-free inulin and turbid suspensions of cellulose, Sephadex, agarose, or purified inulin failed to activate the alternative complement pathway. In contrast, activation-depletion of the alternative pathway was induced by turbid preparations of crude inulin, nigeran, pachyman, barley beta-glucan, and pustulan, which at 700 micrograms/ml, 500 micrograms/ml, 350 micrograms/ml, 60 micrograms/ml, and 27 micrograms/ml, respectively, effected 50% reductions in the subsequent lysis of Er. After centrifugation of 2 mg/ml suspensions of barley beta-glucan at 1100 X G for 5 min and at 15,000 X G for 15 min, the supernatants contained 90 to 92% and 65% of the barley beta-glucan, respectively, as determined by the anthrone method. On a weight basis, the 1100 X G supernatant exhibited the same capacity to activate the alternative pathway as the corresponding original suspension, whereas the 15,000 X G supernatants had less than 3% of the original anti-complementary activity. Preincubation of adherent human monocytes with increasing concentrations of barley beta-glucan suspensions, 100,000 X G supernatants containing 64% of the original beta-glucan, and laminarin all decreased subsequent ingestion of 1.25 X 10(6) zymosan particles in a dose-related fashion. The numbers of monocytes from three different donors phagocytosing zymosan were reduced by 50% after pretreatment with 30 to 65 micrograms/ml, 25 to 48 micrograms/ml, and 12 to 15 micrograms/ml of barley beta-glucan suspensions, 100,000 X G supernatants of barley beta-glucan, and laminarin, respectively, even though the latter two preparations were fully soluble and had no capacity to activate the alternative pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activation↗

PubMed1976

Alternative complement pathway: activity levels in allogeneic pregnancy.

Classical and alternative complement pathway activities have been evaluated in sera of women in progressive stages of gestation and in pregnant mice belonging to outbred or inbred matings, as compared to suitable controls. While classical C pathway was found to be unmodified, the alternative one attained in pregnancy significantly higher activity levels. Results are discussed in the light of mother-conceptus relationships.

Animals↗

PubMed1978

Autosomal locus regulates inverse relationship between sialic acid content and capacity of mouse erythrocytes to activate human alternative complement pathway.

The observation that mouse erythrocytes (E(m)) from 21 inbred strains had variable capabilities to activate the human alternative complement pathway permitted the demonstration that membrane sialic acid content was inversely related to activating capacity and was regulated by codominant alleles of a single autosomal locus. Linear regression analysis also demonstrated a significant inverse correlation between the sialic acid content of E(m) from four inbred strains and the concentration of beta1H required for decay-dissociation of the properdin-stabilized amplification convertase on the E(m). E(m) from F(1) hybrids derived from strains with high and low alternative pathway activating capacities and from their backcrosses exhibited the alternative pathway activating capacities expected if the activity were regulated by alleles of a single autosomal locus. That this same locus predominantly regulated the sialic acid content of E(m) was established by the significant inverse correlation between the sialic acid content and the alternative pathway activating capacity of E(m) from mice of the F(1) and backcross generations. Although the fluid phase interaction of C3, B, and [unk]D continuously generates C3b in a reaction augmented by properdin, it is the covalent attachment of C3b to bystander surfaces deficient in sialic acid that activates the alternative complement pathway at that site because of impaired binding of beta1H to C3b on such surfaces. Thus, discrimination between activating and nonactivating surfaces occurs after C3b deposition, and sialic acid deficiency represents the molecular basis for our earlier finding that activating particles circumvent the regulatory actions of the control proteins of the alternative pathway.

Animals↗

PubMed1978

Activation of the alternative complement pathway by human B cell lymphoma lines is associated with Epstein-Barr virus transformation of the cells.

Activation of the alternative complement pathway by human B cell lymphoma lines is correlated with the presence of Epstein Barr virus (EBV) in the cell genome. EBV-negative B cell lymphoma lines produce little activation of the alternative pathway as measured either by C3 deposition on the cell surface or C3 conversion and consumption of alternative pathway activity in the supernatant serum. By contrast, EBV-positive sublines derived by in vitro EBV conversion of EBV-negative parental lines produce considerable activation of the alternative pathway. This membrane-associated complement-activating mechanism reflects an EBV-induced membrane change in these cells and may provide a mechanism whereby EBV-transformed cells are controlled in vivo.

B-Lymphocytes↗

PubMed1978

Activation of the alternative complement pathway by a streptococcal lipoteichoic acid.

A glycerol lipoteichoic acid antigen from Streptococcus pyogenes 1-RP41 was found by rabbit erythrocyte hemolytic assay to activate the alternative complement pathway in human sera. Over a narrow concentration range of the teichoic acid, complement consumption was dose dependent, whereas at higher concentrations of the acid complement consumption could not be detected.

Antigens, Bacterial↗

PubMed1988

Specific antibodies enhance Sephadex-induced activation of the alternative complement pathway in human serum.

Sephadex beads, which resemble cellulose in their basic chemical structure, were used to study the molecular mechanisms by which cellulosic dialysis membranes activate the alternative complement pathway in normal human serum. Sera from different individuals were found to vary in the extent of activation which occurred following incubation with a fixed amount (surface area) of polymer. Preadsorption of serum with an excess of Sephadex at 2 degrees C resulted in loss of activation when the absorbed serum was interacted with fresh Sephadex beads. Acid eluted proteins from absorbed Sephadex restored the capacity of preadsorbed serum to activate complement in the presence of fresh Sephadex. Adsorption of the immunoglobulin G (IgG) fraction and of F(ab')2 fragments from IgG prepared from the plasma of a normal individual with Sephadex, resulted in the specific binding of some IgG and F(ab'2) molecules to the particles. IgG and F(ab')2 coated beads activated complement in Sephadex-adsorbed serum. Thus, specific anti-dextran IgG antibodies trigger activation of the alternative complement pathway by Sephadex in human serum. The effect is independent of the Fc region of IgG. These results suggest that specific antibodies could be important in determining complement activation in vivo in patients undergoing haemodialysis with cellulosic membranes.

Adsorption↗

PubMed1987

Alternative complement pathway activation fragment Ba binds to C3b. Evidence that formation of the factor B-C3b complex involves two discrete points of contact.

Alternative complement pathway C3 convertase formation involves the cleavage of C3b-associated factor B into fragments Ba and Bb. Whereas Bb, in complex with C3b, has proteolytic specificity toward native C3, the function of the Ba moiety in the formation and/or decay of alternative complement pathway C3 convertase is uncertain. Therefore, we have examined the effect of purified Ba fragment on both fluid-phase and surface-bound enzymatic activity and showed that whereas Ba could inhibit the rate of C3 convertase formation, the rate of intrinsic decay remained unaffected. A specific, metal ion-independent interaction between Ba and C3b was subsequently demonstrated by use of the cross-linking reagent dithiobis(succinimidyl propionate). When cell-associated 125I-B was activated by D, the dissociation of Bb fragment displayed simple first-order kinetics with a half-time of 2.4 min, this value being in reasonable agreement with the hemolytically determined decay rate of 1.8 min. In contrast, most of the Ba fragment undergoes rapid dissociation, but there is also evidence to suggest the establishment of a new equilibrium due to the ability of Ba to rebind to C3b. Cumulatively, these data are consistent with a model in which the attachment of intact B to C3b is mediated by two points of contact, one being in the Ba domain and the other in the Bb domain. Due to avidity effects, each of these interactions could be of relatively low intrinsic affinity, and the characteristic unidirectionality of alternative complement pathway C3 convertase decay may simply result from the low intrinsic association of "univalent" Bb for the C3b subunit.

Chemical Phenomena↗