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Results for “Complement Pathway, Alternative”

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PubMed1976

Alternative complement pathway activity in sera from patients with sickle cell disease.

The low molecular weight cobra venom factor (CoVF) was used to activate the terminal sequence of the alternative complement pathway in thirty-one sera from patients with sickle cell disease (SCD). The SCD sera were compared with normal sera as a source of the alternative complement pathway factors C3 proactivator (C3PA) and C3PA convertase. These factors are required for formation of the enzymatically active CoVF-C3PA complex which is capable of cleaving C3 and thus initiating generation of the cytolytic C5b-9 complex. CoVF cofactor activity was significantly less than normal in SCD sera as measured in an indirect lysis assay, indicating reduced C3PA or C3PA convertase activity in these sera. Qualitative (immunoelectrophoresis) and quantitative (radial immunodiffusion) measurement of C3PA showed, however, that this protein is normal or elevated in SCD sera. Taken together, the reduced CoVF cofactor activity and normal or elevated C3PA in SCD sera suggests that sera from patients with sickle cell disease have reduced C3PA convertase activity.

Adolescent↗

PubMed1986

Mechanism of resistance to lysis by the alternative complement pathway in Trypanosoma cruzi trypomastigotes: effect of specific monoclonal antibody.

Trypanosoma cruzi G strain epimastigotes were lysed by normal human serum (NHS) through activation of the alternative complement pathway (ACP), whereas metacyclic trypomastigotes were resistant to lysis. Epimastigotes and metacyclics with equivalent amounts of C3b deposited on their surface bound factor B with similar affinities. In contrast, factor H bound with higher affinity to metacyclics than to epimastigotes. Both T. cruzi forms with bound C3b were extensively (60 to 80%) lysed after formation of surface C3-convertase and the addition of a C3-C9 complement source. In the presence of factors H and I, or incubation with NHS with EDTA, the percentage of lysis of metacyclics decreased faster than that of epimastigotes with increasing incubation times. These data suggest, as a possible mechanism of resistance to lysis in metacyclic trypomastigotes, the higher binding affinity of factor H to C3b and the inactivation of the latter by serum regulatory proteins. Metacyclics were lysed by NHS, through ACP, in the presence of human immune serum to T. cruzi or anti-T. cruzi monoclonal antibody, but not with the Fab fragment of the latter, which recognizes a 90,000 m.w. antigen from T. cruzi metacyclics. Protection of parasite-bound C3b from serum control proteins was observed when parasites were incubated, before C3 deposition, with the lytic monoclonal antibody but not with its Fab fragment or a nonrelated IgG control. When C3b was deposited on metacyclics before antibody binding, C3b inactivation occurred. In the lysis of metacyclics, through ACP activation, binding of antibody apparently creates new acceptor sites which prevent the activity of serum regulatory proteins.

Animals↗

PubMed1979

Activity of the alternative complement pathway after splenectomy: comparison to activity in sickle cell disease and hypogammaglobulinemia.

Patients with sickle cell disease and individuals who have undergone splenectomy share defects of certain host defense mechanisms and a predisposition to severe pyogenic bacterial infections. Since patients with sickle cell disease can have deficient activity of the alternative complement pathway, we have tested such activity in sera from splenectomized children and adults. A new kinetic hemolytic assay has been used, and we have compared results to those obtained with sera from patients with sickle cell disease or hypogammaglobulinemia. Sera from six of 58 splenectomized individuals (10%) had defective function of the alternative pathway, compared to 10 of 62 sera from patients with sickle cell disease (16%) and 10 of 18 sera from hypogammaglobulinemic patients (56%). Deficiency of antibody, a rate-influencing component of alternative pathway activity in this system, appears responsible for deficient activity in the hypogammaglobulinemic sera. The molecular basis for the deficiency found in sickle cell disease or after splenectomy is not clear. Defective function of the alternative complement pathway could contribute to the increased predisposition to bacterial infection that exists in these three patient groups.

Adolescent↗

PubMed1989

Polymerization of factor B of the alternative complement pathway via disulfide bond(s) in the presence of Cu2+ and stimulation by C3b, the major fragment of C3.

Factor B(B) of the alternative complement pathway has been found to dimerize via disulfide bond(s) in the presence of CuCl2. Poly B has no B activity. The Bb fragment was also dimerized, indicating that one free sulfhydryl group on the Bb portion might be involved in polymerization. The Ba fragment was not dimerized. C3b, the major fragment of C3, has the capacity to stimulate polymerization of B. Incubation of C3b, B and factor D in the presence of Mg2+ and Cu2+ resulted in the formation of poly B and diminished cleavage of B. These results suggest that polymerization of B due to Cu2+ might be partly responsible for the impairment of C3 convertase activity of the alternative pathway.

Complement C3↗

PubMed1983

The relative importance of the classical and alternative complement pathways in serum bactericidal activity against Escherichia coli.

The relative importance of the classical and alternative complement pathways in serum bactericidal activity against Escherichia coli strains of the common urinary O-serogroups has been assessed with strains that differ widely in their sensitivity to normal human serum. With most promptly serum-sensitive strains, rapid killing occurred, mediated by the classical pathway and, when this pathway was eliminated, delayed killing by alternative-pathway activity occurred. However, one strain of serogroup O1 was affected by the classical pathway only and a strain of serogroup O9 was killed rapidly by the alternative pathway. Strains with delayed sensitivity to normal human serum were largely, and in some cases exclusively, affected by the classical pathway. When added to heat-inactivated serum, some strains showed no significant growth whereas the viable numbers of other test strains increased more than 50-fold in 3 h. Whether this variation is due to differences in nutritional requirements or sensitivity to some non-complement-dependent bacteriostatic mechanism remains to be determined.

Complement System Proteins↗

PubMed1983

The alternative complement pathway control protein H binds to immune complexes and serves their detection.

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound 125I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of 125I-H; when fresh serum was chelated with 10 mM EDTA, 125I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), 125I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while 125I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.

Antigen-Antibody Complex↗

PubMedDate not supplied

Classical and alternative complement pathway activation in paracoccidioidomycosis.

The following study presents evidence of complement activation in paracoccidioidomycosis (PCM). Twenty-eight untreated patients were studied from endemic areas of Parana in southern Brazil. The activation of the classical pathway, evaluated by the C4d/C4 ratio, was significantly elevated in the patients compared to the control population (p < 0.005). Six patients were examined prospectively with the C4d/C4 assay during treatment and they showed a decrease in this ratio associated with clinical improvement. The activation of the alternative pathway was determined by rocket immunoelectrophoresis of fragment Ba. These levels were also significantly higher in the patient group in comparison to the controls (p < 0.0005). The prospective study also showed a significant variation in the Ba levels associated with clinical improvement (p < 0.01). Furthermore, the levels of C3, C4, CH50 and anti-Paracoccidioides brasiliensis IgG were determined in all patients. The anti-P. brasiliensis IgG levels showed a weak positive correlation with the C4d/C4 ratio (r[S] = 0.45; p < 0.03). The C3, C4 and CH50 levels did not show significant variations from the normal ranges. Our results suggest the involvement of both complement pathways, classical and alternative, in PCM and their association with disease activity.

Adolescent↗

PubMed1982

Guinea pig erythrocytes, after their contact with influenza virus, acquire the ability to activate the human alternative complement pathway through virus-induced desialation of the cells.

Guinea pig erythrocytes that had been exposed to influenza A virus activated the alternative complement pathway in whole human serum in the absence of natural antibodies. Because all virus particles were eluted from the treated cells, activation was not dependent on antiviral antibodies or on virus particles themselves. The relative capacity of treated erythrocytes to activate the alternative pathway was dependent on the amount of virus to which the cells had been exposed and was directly related to the amount of sialic acid removed from the erythrocyte membrane during incubation with either whole virus particles or purified viral sialidase. C3b bound to cells that had been treated with virus, and P-stabilized amplification convertase sites P,C3b,Bb formed on these cells, exhibited increased resistance to the action of the regulatory proteins beta-1H and C3b Ina compared with C3b and P,C3b,Bb on untreated, nonactivating cells. The acquired resistance of the cell-bound, P-stabilized amplification convertase to decay-dissociation by beta-1H was directly related to the activating capacity of the treated cells in whole serum (r = 0.95) and to the amount of sialic acid removed from the cells by the virus (r = 0.98). Desialation represents a specific alteration of the cell surface by which a nonimmune host, through activation of the alternative pathway, may deposit C3b on a target cell that had been exposed to influenza virus and may lyse virus virus-modified cells during orthomyxovirus infections.

Animals↗

PubMed1983

Activation of the alternate complement pathway by peptidoglycan of Actinomyces viscosus, a potentially pathogenic oral bacterium.

Peptidoglycans and cells walls from Actinomyces viscosus, Staphylococcus aureus, and group A streptococcus were compared for their relative abilities to activate the alternate complement pathway (ACP). On the dry-weight basis, the peptidoglycan from A. viscosus was 3.5 times more active than group A streptococcal peptidoglycan and 15.6 times more active than Staph. aureus peptidoglycan in activating the ACP. Consequently A. viscosus peptidoglycan is one of the most potent ACP-activators reported to date. For both A. viscosus and group A streptococcus, the peptidoglycan was a better ACP activator than cell walls from the same organism (125- and 52-fold, respectively) indicating that the peptidoglycan is probably the most important subcellular ACP-activator in these microorganisms. In contrast, cell walls from Staph, aureus were 9 times more active than peptidoglycan from Staph. aureus in activating the ACP, presumably because teichoic acids are the most important subcellular ACP activator in this microorganism.

Actinomyces↗

PubMed1989

Activation and binding of opsonic fragments of C3 on encapsulated Cryptococcus neoformans by using an alternative complement pathway reconstituted from six isolated proteins.

Encapsulated Cryptococcus neoformans yeast cells are potent activators of the complement system. We examined the interaction of the yeast cells with an alternative complement pathway reconstituted from isolated factor D, factor B, factor H, factor I, C3, and properdin. Incubation of encapsulated cryptococci with the reconstituted pathway led to activation and binding of C3 fragments to the yeast cells that was quantitatively and qualitatively identical to that observed with normal human serum. Incubation with either normal serum or a mixture of isolated proteins led to binding of 4 x 10(7) to 5 x 10(7) C3 molecules to the yeast cells. The kinetics for activation and binding of C3 were identical, with maximum binding observed after a 20-min incubation. Immunoglobulin G was not needed for optimal activation kinetics. C3 fragments eluted from the yeast cells by treatment with hydroxylamine and subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the presence primarily of iC3b on yeast cells incubated with either normal serum or the reconstituted pathway. Ultrastructural examination of the opsonized yeast cells showed that the cryptococcal capsule was the site for binding of C3 activated from normal serum or the reconstituted pathway, with a dense accumulation of C3 at the periphery of the capsule. Thus, incubation of encapsulated cryptococci in the reconstituted pathway led to deposition of opsonic complement fragments at a site that was appropriate for interaction with phagocyte receptors. Cryptococci opsonized with the reconstituted pathway showed a markedly enhanced interaction with cultured human monocytes compared with unopsonized yeast cells, indicating that the alternative pathway alone is opsonic for yeast cells. However, the results indicate that additional serum factors are needed for optimal opsonization of yeast cells because a 35% reduction in the number of cryptococci bound to macrophages was observed with cryptococci opsonized with the reconstituted pathway compared with that observed when yeast cells were opsonized with normal serum.

Complement Activation↗

PubMed1990

Host-specific evasion of the alternative complement pathway by schistosomes correlates with the presence of a phospholipase C-sensitive surface molecule resembling human decay accelerating factor.

Schistosoma mansoni parasites recovered from the blood stream were found to be nonactivators of the alternative complement pathway (ACP) when exposed to sera of homologous but not heterologous host species. Schistosomes could be converted into activators of the homologous ACP by treatment with phospholipase C. Antibodies to either human or guinea pig decay accelerating factor (DAF), a 70-kDa glycosylphosphatidylinositol anchored membrane glycoprotein which controls ACP activation on the mammalian cell plasma membrane, bound to the surface of immature schistosomes and immunoprecipitated a molecule of similar molecular mass from detergent extracts of surface iodinated parasites. Phospholipase C treatment drastically reduced the reactivity of the worms with the anti-DAF antibodies. These data suggest that schistosomes evade the ACP by inserting functional host DAF into their surfaces, possibly through adsorption of the molecule's lipophilic diacyglycerol membrane anchor moiety into the outer lipid bilayer of the parasite.

Adsorption↗

PubMed1983

Activation of the alternative complement pathway by natural antibody to glycolipids in guinea-pig serum.

Liposomes containing paragloboside (PG) on their membrane were readily lysed by C4-deficient guinea-pig serum (C4D-GPS) through activation of the alternative complement pathway (ACP). Therefore we examined the reactivity of several types of guinea-pig serum (GPS) on PG-liposomes and determined that all GPS except that from specific pathogen-free (SPF) Hartley guinea-pigs had lytic capacity in Mg-EGTA-GVB (gelatin veronal-buffered saline containing Mg++ and ethyleneglycol-bis(beta-aminoethyl ether)N,N'-tetraacetate). This lytic capacity of GPS corresponded with the amount of natural antibody to PG in those sera. Although GPS of SPF guinea-pigs (SPF-GPS) could not lyse PG-liposomes in Mg-EGTA-GVB, it could lyse the liposomes when heated C4D-GPS or Hartley GPS was added. Natural antibody to PG in the heated sera was regarded to have sensitized PG-liposomes to lysis by SPF-GPS via ACP activation. Since the antibody to PG-liposomes was removed by lacto-N-nor-hexaosylceramide which has the same chemical structure in the terminal oligosaccharide, the antibody to PG in GPS was suggested to have a specificity to the terminal structure of oligosaccharide shared by lacto-N-nor-hexaosylceramide. Furthermore, the IgM fraction, which had been prepared by gel filtration of heated C4D-GPS on a Sephadex G200 column, could also sensitize PG-liposomes to lytic reaction of SPF-GPS in Mg-EGTA-GVB. This sensitizing capacity of heated C4D-GPS was suppressed by absorption of the serum or its IgM fraction with anti-guinea-pig mu-chain antibody coupled to Sepharose. Therefore, it was concluded that the lysis of PG-liposomes by GPS in Mg-EGTA-GVB was a result of ACP activation mediated by natural antibodies to PG of the IgM type which are present in usual GPS. This conclusion indicated that natural antibodies of the IgM type might play a role with ACP in host defence, especially in C4-deficient guinea-pigs where the classical complement pathway is impaired.

Animals↗

PubMed1986

Production of nephritic factor of the alternative complement pathway by Epstein Barr virus-transformed B cell lines derived from a patient with membranoproliferative glomerulonephritis.

Nephritic factor of the alternative complement pathway (C3NeF) is an IgG autoantibody which binds to and stabilizes the C3 convertase (C3bBb) enzyme, and which has been detected mainly in sera from patients with membranoproliferative glomerulonephritis (MPGN) and partial lipodystrophy. To study the production of C3NeF, mononuclear cells isolated from the peripheral blood of patients with MPGN and C3NeF activity in their sera were infected with Epstein Barr virus (EBV) to establish active B lymphocyte cell lines. By using a modified C3NeF screening assay, we detected C3NeF activity in the supernatant of a B cell line derived from a patient with MPGN Type II, but in none of the supernatants of B cell lines derived from normal individuals. C3NeF-positive supernatants were investigated for their ability to conserve classical or alternative pathway C3 convertase activity by using EAC3bBb and EAC4b2a stabilization assays. C3NeF-positive supernatants stabilized the C3bBb convertase activity, but not the C4b2a convertase activity. Studies of the supernatants, using anti-human IgG affinity columns, showed that the C3NeF activity was in the IgG fraction; furthermore, C3NeF antibody agglutinated sheep erythrocytes coated with C3bBb, but not with C3b alone. On gel electrophoresis, both heavy and light chains of the C3NeF were comparable in size to that of normal human IgG molecules. We conclude that C3NeF, produced in vitro by EBV-transformed B cell lines derived from a patient with MPGN Type II, is functionally identical to the conventional C3NeF in serum. In vitro preparation of homogeneous NeF(s) should greatly facilitate the studies of the role of these autoantibodies in complement dysmetabolism.

B-Lymphocytes↗

PubMed1979

Heterotoxicity of human serum. IV. Role of the alternative complement pathway and natural antibody in the lethal toxicity of human serum for mice.

The toxic and often lethal reaction caused by intravascular injection of fresh human serum (FHS) was studied in mice. Decomplementation experiments indicated that the alternative complement pathway plays an essential role in the production of 'serum shock'. In vivo activation of human factor B and C3, following the administration of FHS into mice, was detected by immunoelectrophoresis. Absorption experiments suggested that natural antibodies were not required for toxicity, although they may amplify the lethal potential of normal sera by feedback mechanisms of C activation. Injections of FHS caused intravascular hemolysis of mouse erythrocytes and platelet aggregation in mice. The relationship of these observations to the mechanism(s) underlying lethality will be discussed in the following article.

Animals↗

PubMed1984

Activation of the alternative complement pathway by isolated human glomerular basement membrane.

The capacity of isolated human glomerular basement membrane (GBM) to initiate surface activation of the human alternative complement pathway was defined by the deposition of C3b under circumstances in which the classical complement pathway was inoperative. The deposition of C3b from normal or C2-deficient serum was time- and magnesium-dependent, implying a role for the alternative pathway. Normal human serum rendered deficient in D did not sustain C3b deposition until its reconstitution with D, indicating an absolute requirement for a protein unique to the alternative pathway and essential to the cleavage activation of the C3 amplification convertase of that pathway. The capacity of the excess control proteins H and I to prevent C3b deposition onto GBM incubated in C2-deficient serum provided further evidence for the direct activation of the alternative pathway in this system. The use of radiolabeled monoclonal antibody to localize the deposited C3b afforded specificity and quantitation of about 100 ng of C3b/mg of GBM. Immunohistochemical analysis with a monoclonal antibody to detect C3b demonstrated its deposition to be confined to the epithelial surface of the GBM.

Antibodies, Monoclonal↗

PubMed1978

Kinetic assessment of alternative complement pathway activity in a hemolytic system. II. Influence of antibody on alternative pathway activation.

By using a kinetic assay, we have examined the role of antibody in the lysis of rabbit erythrocytes (RaRBC) through the alternative complement (C) pathway. Sera from some hypogammaglobulinemic (Hgamma) humans and all agammaglobulinemic chickens tested had subnormal activity in the assay. Heated normal human or chicken sera, but not heated Hgamma sera, restored activity to deficient Hgamma serum and initiated hemolysis in the presence of rabbit serum as C source. Absorption of heated normal human serum with RaRBC, but not with sheep erythrocytes or zymosan, removed its ability to reconstitute deficient Hgamma serum. Normal hemolytic activity could be resotred to Hgamma serum with human IgM, IgG, or colostral IgA, with goat anti-RaRBC IgG, or with an eluate from serum-sensitized RaRBC, but not with myeloma IgA. Restoration of hemolytic activity to Hgamma serum could be achieved in a dose-dependent fashion with the F(ab')2 fragment of IgG. These results suggest that antibody exerts a significant rate-limiting effect on alternative pathway activity in the RaRBC lytic system. This raises the possibility that antibody may be required for efficient alternative pathway activity in vivo and that the pyogenic infections that occur in Hgamma individuals are due to inefficient activation and fixation of C3 through either the classical or alternative pathway.

Agammaglobulinemia↗

PubMed1986

Evasion of the alternative complement pathway by metacyclic trypomastigotes of Trypanosoma cruzi: dependence on the developmentally regulated synthesis of surface protein and N-linked carbohydrate.

Epimastigotes (EPI) of Trypanosoma cruzi are highly sensitive to lysis in fresh normal human serum by the alternative complement pathway (ACP). In contrast, metacyclic trypomastigotes (CMT) derived from EPI in stationary culture fail to activate the ACP and are thus resistant to serum-mediated lysis. To investigate the nature of the parasitic surface molecules which enable infective metacyclic trypomastigotes to evade the ACP, CMT were treated with a variety of different proteolytic and glycosidic enzymes, and their sensitivity to ACP-dependent lysis was tested. Pretreatment with pronase was found to cause a near complete reversal in the resistance of CMT to serum lysis, whereas trypsin or chymotrypsin induced smaller increases in complement sensitivity. Similarly, pretreatment with N-glycanase or neuraminidase also partially abrogated the resistance of CMT to ACP-dependent lysis. The effect of these enzymes on susceptibility to complement-mediated lysis was paralleled in increased C3 and C9 deposition on the organism. In addition, electrophoretic analysis of parasite-bound C3 indicated that the hemolytically inactive fragment, iC3b, was the major form of the molecule on CMT, while the hemolytically active fragment, C3b, predominated on pronase-treated CMT. Furthermore, when C3 was deposited on the parasite surface by means of purified ACP components, 80% of C3b on pronase-pretreated CMT but only 14% of the C3b on CMT bound the amplification protein factor B with high affinity, a prerequisite for efficient ACP activation. When cultured at 37 degrees C after pronase treatment, CMT gradually regained their resistance to ACP-mediated lysis. This process was blocked if puromycin, cycloheximide, or tunicamycin were included in the culture medium. The above findings suggest that evasion of the ACP by CMT is dependent on the developmentally regulated synthesis of protein as well as N-linked carbohydrate chains. A stage-specific 90,000 to 115,000 m.w. glycoprotein doublet present on the surface of CMT was shown to be uniquely sensitive to pronase digestion. Thus, this complex, which is also recognized by a CMT-specific monoclonal antibody, may be the glycoprotein component responsible for control of ACP activation

Animals↗

PubMed1987

Direct evidence that decreased serum opsonization of Streptococcus pneumoniae via the alternative complement pathway in sickle cell disease is related to antibody deficiency.

Two approaches were used to demonstrate that reduction in serum opsonization of Streptococcus pneumoniae via the alternative complement pathway in children with sickle cell disease is related to a deficiency of antibodies to pneumococcal capsular polysaccharide. First, opsonization of S. pneumoniae mediated by the alternative pathway in patients' sera was restored to normal by addition of the purified IgG or IgM fraction of goat antiserum to capsular polysaccharide of the homologous serotype. Secondly, IgG antibody titers to capsular polysaccharide in patients' sera correlated significantly with alternative pathway-mediated opsonization; the correlation between titers of IgM anticapsular antibodies and opsonization approached statistical significance. The sum of the IgG and IgM anticapsular antibody titers correlated most significantly with opsonization. Our results suggest that reduction in alternative pathway-mediated opsonization in sera from children with sickle cell disease is related to low levels of both IgG and IgM anticapsular antibodies.

Anemia, Sickle Cell↗