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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↗
PubMed1995

Covalent linkage of C3 to properdin during complement activation.

Activation of the alternative complement pathway of serum produces complexes of properdin (P) and C3 as measured in a double antibody enzyme-linked immunosorbent assay. When purified from serum, these complexes decrease factor B hemolytic activity in serum and do not restore the alternative pathway hemolytic activity of serum deficient in P. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of activated serum containing biotinylated P, followed by blotting to nitrocellulose and development with streptavidin-alkaline phosphatase revealed a band at 53 kDa for monomeric P and an additional band at 160 kDa. P samples eluted from zymosan and purified from activated serum revealed a band at 116 kDa for C3 alpha and 74 kDa for C3 beta, and an additional band at 160 kDa when analyzed by SDS-PAGE, Western blotting and development with antibody to C3. The appearance of a 160 kDa band containing P and C3 indicates that these proteins are contained in a complex formed during activation of the alternative pathway. Activation of a purified reagent mixture containing factors B, D, and H, and 125I-labeled P or 125I-labeled C3, followed by SDS-PAGE and autoradiography confirmed the presence of a 160-kDa band which disappeared following hydroxylamine treatment of the sample. These data are consistent with a covalent linkage of C3 to P via the C3 alpha chain, producing the 160-kDa complex.

Complement C2↗
PubMed1982

Functional bacterial opsonic activity of human amniotic fluid.

There are some data to suggest that amniotic fluid protects the fetus from invasion by pathogenic bacteria. To examine methods by which amniotic fluid may offer such protection, quantitative antibody, complement activity, and functional opsonic capacity were measured. Immunoglobulins were measured by laser nephelometry and total hemolytic complement was determined by radial diffusion; results suggested activity adequate for bactericidal capacity. The chemiluminescence assay was used to quantitate the functional interaction between polymorphonuclear leukocytes and E. coli, group B streptococci (GBS), or zymosan particles preopsonized with amniotic fluid obtained at different stages of gestation. Results were compared to those for normal serum. Data were analyzed by evaluation of the initial slope, area under the curve, and peak chemiluminescence response. Opsonic activity of amniotic fluid for E. coli and GBS was demonstrated, with E. coli showing greater reactivity (maximum = 15,000 to 25,000 cpm) than GBS (10,000 to 20,000 cpm). Specific, as well as nonspecific, opsonic activity was demonstrated by absorption of amniotic fluid with killed bacteria. Concentration of amniotic fluid did not result in an increase in chemiluminescent activity, which demonstrates that optimal opsonic activity already exists. The classical and alternate pathways of complement were assessed for E. coli and GBS. Preterm amniotic fluid did not differ in response from that of amniotic fluid obtained from term pregnancies. This study demonstrates that amniotic fluid can provide the fetus with protection from bacterial pathogens and delineates mechanisms for such protection.

Amniotic Fluid↗
PubMed1988

An improved sensitive and simple microassay of mouse complement.

A simple and fast hemolytic microassay was developed for the determination of classical pathway complement activity in mouse serum. The assay is based on hemolysis of sheep red blood cells (SRBC) that are sensitized with polyclonal mouse antibodies. The degree of hemolysis was measured in the reaction supernatants by photometric reading in an ELISA plate scanner at 405 nm wavelength. It was found that some batches of unpurified mouse anti-SRBC antibodies gave insufficient hemolysis. Analysis of two antibody preparations indicated that this might be caused by anti-complementary factors in the ascites fluid, and by an excess of non-complement fixing IgG1 antibodies. For optimal and standardized results, removal of anticomplementary factors and enrichment for complement fixing IgG2 antibodies was required and was achieved using protein A purified anti-SRBC IgG. In our assay it is possible to determine CH50 titers in triplicate in 80 microliters samples of individual mouse sera with high sensitivity. Using this rapid one-step method large numbers of tests could be performed in 1 day.

Animals↗
PubMed1991

A microassay for the pore-forming activity of complement, perforin, and other cytolytic proteins based on confocal laser scanning microscopy.

A fluorescence microscopic assay for the activity of complement, perforin, and other cytolytic proteins which form transmembrane pores in cellular membranes is described. The assay was worked out and tested with red blood cell membranes (ghosts) and was then applied to intact hemoglobin-free cells. Resealed human erythrocyte ghosts were incubated with complement or perforin. A small polar fluorescent probe (fluorescein-labeled 1-kDa dextran, FD1) which permeates through complement and perforin pores but not through normal cell membranes was added to the samples. The capability of the confocal laser scanning microscope (CLSM) to generate thin optical sections was exploited to visualize and quantitate fluorescence inside single ghosts and thus determine the fraction of ghosts which had become permeable for FD1. The activity of complement or perforin was quantitated by plotting the fraction of permeable cells versus the concentration of the pore-forming protein. The results were in good agreement with those of a conventional hemolytic assay. The CLSM-based assay was then applied to intact hemoglobin-free cells for which only few alternative assays are available. Compared to conventional hemolytic assays for the activity of pore-forming proteins the assay described here can be applied to a large variety of natural and artificial membrane systems. The assay can be performed under nonlysing conditions. Furthermore, the assay is simple, relatively fast, and requires only extremely small amounts of cells and pore-forming proteins.

Animals↗
PubMed2004

Functional analysis in serum from atypical Hemolytic Uremic Syndrome patients reveals impaired protection of host cells associated with mutations in factor H.

A subgroup of patients with the most severe form of the Hemolytic Uremic Syndrome (HUS) presents mutations in the complement regulatory protein factor H. The functional analyses of the factor H mutant proteins purified from some of these patients have shown a specific defect in the capacity to control complement activation on cellular surfaces. Here, we show that these factor H-related complement regulatory defects can be detected in the patients' serum with a simple hemolytic assay. Data obtained from HUS patients and control individuals indicate that this assay is a useful tool for the molecular diagnosis of factor H-related HUS.

Animals↗
PubMed1999

Complement defects in aged mice compromise phagocytosis of Pseudomonas aeruginosa.

PURPOSE: The role of complement in phagocytosis and killing of P. aeruginosa was examined using serum from aged vs young donor mice. METHODS: Phagocytosis, complement hemolytic and microbicidal assays were used. RESULTS: Serum from young donor mice contained a heat-labile factor which significantly enhanced phagocytic activity of cells from young mice compared with similarly treated aged donor serum. Use of cobra venom factor (CVF) to destroy C3 and the terminal complement components in serum from young or aged donor mice also significantly decreased the phagocytic activity of young cells. EGTA treatment of young or aged donor serum, to activate the alternative pathway and selectively inhibit activation of the classical pathway, resulted in a significant decrease in phagocytosis by young cells in the presence of donor serum from either group. Alternative pathway mediated hemolysis also was measured and was significantly reduced in aged vs young donor serum. PMN microbicidal activity was tested using cells from young mice in the presence of aged vs young donor serum, but no significant differences were noted. CONCLUSION: These data provide evidence that defects in the alternative pathway of complement in the serum of aged animals lead to decreased phagocytic activity of cells from young mice, but not impaired bacterial killing.

Aging↗
PubMed1983

Activation of C1 by soluble IgG aggregates as detected by a novel one-step hemolytic assay that specifically measures the proenzyme form of C1s.

A new hemolytic assay is described that specifically measures the precursor form of the C1s subcomponent of the complement system. The assay employs a C1s-depleted reagent obtained by immunoadsorption of fresh human plasma on immobilized goat anti-human C1s antibodies. Linear Z plots are obtained with nanogram levels of precursor C1s, whereas C1s completely fails to induce hemolysis in the assay. Because low concentrations of C1s do not interfere with the activity of precursor C1s, the assay can be used for the stoichiometric measurement of C1 activation. The precursor C1s assay was applied to the study of C1 binding and activation by soluble aggregates of human IgG (AIgG). Incubation of purified human C1 with AIgG caused a temperature-independent consumption of whole C1 hemolytic activity, indicating binding of C1, but almost no consumption of the total (precursor + activated) C1s activity. On the other hand, activation of C1, measured as the time- and temperature-dependent consumption of precursor C1s, could greatly exceed the binding of C1. These findings can be explained by using recent findings concerning the association-dissociation equilibrium between C1q and the tetrameric complex of C1r and C1s.

Animals↗
PubMed1981

Induction of autoantibody-producing cells after the coculture of haptenated and normal human mononuclear leukocytes.

The coculture of normal human peripheral blood mononuclear leukocytes (PBL) and autologous mononuclear leukocytes coupled to the trinitrophenyl (TNP) hapten (TNP-PBL) was found to induce a polyclonal activation of antibody-producing cells. The polyclonal activation of antibody-producing cells was demonstrated by detecting the induction of cells producing antibody to sheep red blood cells using a complement-dependent, direct, hemolytic plaque-forming cell (PFC) assay. A ratio of four normal to one haptenated mononuclear leukocyte was found to be optimal for inducing the polyclonal activation of antibody-producing cell in these cultures. The plaque-forming cells assay in these experiments utilized monolayers of indicator red cells. Further evidence for the polyclonal induction of antibody-producing cells by TNP-PBL was provided by demonstrating PFC on monolayers of not only sheep red blood cells, but also autologous human red cells, bromelain-treated autologous red cells, TNP-coupled human and sheep red cells, and human autologous red cells coupled to human heat-aggregated IgG with chromic chloride. Thus cells secreting antibody to TNP, human red cells, and human IgG were induced. Anti-IgG and anti-human red cell-producing cells were first detected on Day 2 of culture and were still present on Day 9. Mononuclear leukocytes altered by chemical haptenation polyclonally stimulate normal mononuclear leukocytes to become antibody-producing cells. This polyclonal stimulation of antibody-producing cells includes cells producing antibodies to human IgG and human autologous red blood cells suggesting that autoantibody-producing cells are induced.

Animals↗
PubMed1977

Hereditary deficiency of the third component of complement in a child with fever, skin rash, and arthralgias: response to transfusion of whole blood.

A previously well 34-month-old male presenting with fever, skin rash, and arthralgias was found to lack C3 by immunochemical (undetectable) and hemolytic (1% normal) assays. No infectious agent could be demonstrated. Protein levels of Clq. C4, C5, properdin, and C3b-INA and hemolytic activities of complement components C1 to C9 except C3 were normal or elevated; total hemolytic complement activity was 13% of normal and was reconstituted by purified C3. Properdin factor B was 702 (normal 175 to 275) mug/ml, and was not cleaver upon addition of zymosan or cobra venom factor. The serum had normal immune adherence activity, but was deficient in ability to opsonize Candida albicans for uptake and Escherichia coli for killing by neurophils, generate neutrophil chemotactic factors and inhibit the growth of E. coli; these activities were restored by purified C3. A transfusion of 320 ml 1-hour-old normal whole blood on the fifty-second day resulted in transitory elevation of the C3 level to 25 mg/dl with a fall-off (approximately 2 1/2% per hour) to undetectable levels by 69 hours; it was followed by disappearance of the skin rash and arthralgias and return to normal of the previously elevated temperature and CRP levels. C3 levels in family members (seven of 24 half-normal), lack of anti-C3 activity, normal C3b-INA levels and a normal rate of catabolism of transfused C3 indicated that the deficiency was inherited with autosomal codominance and involved decreased synthesis of C3. Thus, this child is a unique individual with inherited C3 deficiency presenting with absence of repeated infections, whose symptoms of fever, skin rash, and arthralgia were abated by whole blood transfusion.

Blood Transfusion↗
PubMed1990

Recurrent meningitis in a patient with congenital deficiency of the C9 component of complement. First case of C9 deficiency in Europe.

We describe the first cases, to our knowledge, of C9 deficiency in Europe that were detected in a Swiss family, of which two members--one with a complete deficiency and the other with approximately half-normal C9 levels--experienced bacterial meningitis. The index patient, a 56-year-old white man with a history of purulent meningitis at the age of 23 years, presented with an acute meningococcal meningitis. No impairment of cellular immunity or immunoglobulin deficiency could be found. Complement assays showed a complete deficiency of the C9 component, while the other individual component levels were normal and the hemolytic activity (measured using the CH50 assay) was only slightly reduced. A family study revealed complete C9 deficiency in the patient's healthy brother and half-normal C9 concentrations in his sister, his son (who also had experienced an episode of bacterial meningitis), and his niece, consistent with an inherited C9 deficiency. This first case of recurrent meningitis in a white patient with complete C9 deficiency suggests that this complement defect may also be a risk factor for bacterial, especially neisserial, infections.

Complement C9↗
PubMed1997

Effects of synthetic oligonucleotides on human complement and coagulation.

Oligodeoxynucleotide phosphorothioates (PS-oligos) are being studied as novel therapeutic agents based on their ability to inhibit gene expression. Preclinical studies produced unanticipated complement and coagulation effects in monkeys receiving high-dose PS-oligo. In the present in vitro studies, PS-oligo inhibited normal human blood clotting as well as subsequent assays for prothrombin fragment PF(1+2) and hemolytic complement. PS-oligo treatment of normal donor plasma produced concentration-dependent prolongations of clotting times, with the activated partial thromboplastin time more sensitive than prothrombin time or thrombin clotting time. PS-oligo treatment of normal donor serum similarly reduced hemolytic complement activity in a concentration-dependent manner. Reduced hemolysis correlated with increased levels of complement fragment C4d. The anti-heparin drug protamine sulfate inhibited in vitro effects of PS-oligo in both complement and coagulation assays, suggesting that charged residues in internucleotide linkages of PS-oligo mediated the observed activities. Therefore, oligonucleotides with varying internucleotide linkages, nucleotide sequence, or secondary structure were compared. Both complement and coagulation effects appeared to be independent of nucleotide sequence but were strongly related to the nature of internucleotide linkages. Several of these modified oligonucleotides have been shown previously to retain potent antisense activity and thus may represent viable alternatives for antisense therapeutics.

Blood↗
PubMedDate not supplied

Selective C3 deficiency due to C3 nephritic factor in an apparently healthy girl.

Routine laboratory investigations performed on the serum of an 8-year-old girl examined because of a moderate degree of iron-deficiency anemia showed a markedly reduced C3 level. More detailed complement studies revealed a selective C3 deficiency, as indicated by the almost undetectable C3 concentration tested by both hemolytic and immunochemical assays and by the normal or slightly reduced levels of all the other complement components. The hemolytic activity of the serum was restored by the addition of partially purified C3 component. The isolated C3 deficiency could be attributed to the presence of a C3-cleaving activity in the serum of the propositus. This activity was identified as C3 nephritic factor (C3NeF) since it was heat-stable, was absorbed by Cowan I strain of Staphylococcus aureus and was-eluted in the IgG fraction after DEAE-chromatography of the serum. The levels of H and I factors of the alternative pathway in the serum of the propositus and of C3 in the serum samples of her parents and two siblings were found to be within the normal range. The previous clinical history of the girl and the follow-up for a period of approximately 5 years showed that she was apparently healthy and did not reveal clinical and/or laboratory evidence of glomerulonephritis, lipodystrophy or repeated bacterial infections usually associated with the presence of C3NeF in the serum.

Anemia, Hypochromic↗
PubMed1991

Bordetella pertussis adenylate cyclase toxin and hemolytic activities require a second gene, cyaC, for activation.

In these studies, the Bordetella pertussis adenylate cyclase toxin-hemolysin homology to the Escherichia coli hemolysin is extended with the finding of cyaC, a homolog to the E. coli hlyC gene, which is required for the production of a functional hemolysin molecule in E. coli. Mutations produced in the chromosome of B. pertussis upstream from the structural gene for the adenylate cyclase toxin revealed a region which was necessary for toxin and hemolytic activities of the molecule. These mutants produced the 216-kDa adenylate cyclase toxin as determined by Western blot (immunoblot) analysis. The adenylate cyclase enzymatic activities of these mutants were equivalent to that of wild type, but toxin activities were less than 1% of that of wild type, and the mutants were nonhemolytic on blood agar plates and in in vitro assays. The upstream region restored hemolytic activity when returned in trans to the mutant strains. This genetic complementation defined a gene which acts in trans to activate the adenylate cyclase toxin posttranslationally. Sequence analysis of the upstream region defined an open reading frame with homology to the E. coli hlyC gene. In contrast to E. coli, this open reading frame is oriented oppositely from the adenylate cyclase toxin structural gene.

Adenylyl Cyclases↗
PubMed1985

Unusual complement-mediated hemolytic kinetics at low ionic strength.

The dilution of human serum in relatively low ionic strength buffer (mu = 0.070) caused the spontaneous activation of C1 and a limited activation of C4 and C3 in the fluid phase. The unusual degree of activation of the complement system in the fluid phase suggested that the optimal functions of complement-regulatory systems such as C1 inhibitor might be reduced. As a function of the time of preincubation (PI) of diluted serum at 37 degrees C, under low ionic strength conditions, an unusual complement-mediated hemolytic kinetic pattern was observed upon adding sensitized erythrocytes (EA). For a 1:36 dilution of human serum, there was an initial progressive decrease in complement hemolytic activity (from 3 to 20 min PI, phase I), followed by an apparent functional reversal (increase) in hemolytic activity (20-50 min PI, phase II) and finally a gradual irreversible depletion of the hemolytic activity (after 50 min PI, phase III). This hemolytic pattern could only be adequately demonstrated using a kinetic assay which followed the course of lysis of EA in the presence of low dilutions of human serum as a complement source. Others might have missed this observation due to the use of end-point titration methods which required the use of relatively elevated serum dilutions at the time of EA addition. Mechanisms which governed the variations in hemolytic activity at low ionic strength were not clear. Speculatively, partial consumption of early complement components, generation of free C1q and generation of complement fragments might have accounted for the initial decrease in the hemolytic activity observed in phase I. The apparent functional reversal of hemolytic activity observed in phase II might have involved a critical depletion of C1 inhibitor which occurred secondary to C1 inhibitor binding to C1 (activated by low ionic strength effects) and to the C1 activated at the time of EA addition. Without sufficient regulation, a rapid unrestricted C1-mediated complement activation could have occurred, which resulted in a rapid deposition of complement on the EA. Finally, prolonged exposure of serum to low ionic strength effects appeared to induce a significant complement consumption, which caused a time-dependent irreversible depletion of complement hemolytic activity (phase III). Excess exogenous C1 inhibitor, when co-incubated with diluted serum at low ionic strength, reversed the time-dependent effects of low ionic strength and enhanced the subsequent specific complement-mediated hemolytic activity as compared to controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activation↗
PubMed2004

Multimeric tyrosine sulfate acts as an endothelial cell protectant and prevents complement activation in xenotransplantation models.

BACKGROUND: Activation of endothelial cells (EC) in xenotransplantation is mostly induced through binding of antibodies (Ab) and activation of the complement system. Activated EC lose their heparan sulfate proteoglycan (HSPG) layer and exhibit a procoagulant and pro-inflammatory cell surface. We have recently shown that the semi-synthetic proteoglycan analog dextran sulfate (DXS, MW 5000) blocks activation of the complement cascade and acts as an EC-protectant both in vitro and in vivo. However, DXS is a strong anticoagulant and systemic use of this substance in a clinical setting might therefore be compromised. It was the aim of this study to investigate a novel, fully synthetic EC-protectant with reduced inhibition of the coagulation system. METHOD: By screening with standard complement (CH50) and coagulation assays (activated partial thromboplastin time, aPTT), a conjugate of tyrosine sulfate to a polymer-backbone (sTyr-PAA) was identified as a candidate EC-protectant. The pathway-specificity of complement inhibition by sTyr-PAA was tested in hemolytic assays. To further characterize the substance, the effects of sTyr-PAA and DXS on complement deposition on pig cells were compared by flow cytometry and cytotoxicity assays. Using fluorescein-labeled sTyr-PAA (sTyr-PAA-Fluo), the binding of sTyr-PAA to cell surfaces was also investigated. RESULTS: Of all tested compounds, sTyr-PAA was the most effective substance in inhibiting all three pathways of complement activation. Its capacity to inhibit the coagulation cascade was significantly reduced as compared with DXS. sTyr-PAA also dose-dependently inhibited deposition of human complement on pig cells and this inhibition correlated with the binding of sTyr-PAA to the cells. Moreover, we were able to demonstrate that sTyr-PAA binds preferentially and dose-dependently to damaged EC. CONCLUSIONS: We could show that sTyr-PAA acts as an EC-protectant by binding to the cells and protecting them from complement-mediated damage. It has less effect on the coagulation system than DXS and may therefore have potential for in vivo application.

Adult↗
PubMed1995

Complement activation and histamine release following administration of roentgen contrast media.

Anaphylactoid reactions following administration of reontgen contrast media (CM) have occasionally been described. In this investigation, blood samples for nonallergic human volunteers were exposed to the CM iodixanol (Visipaque), iohexol (Omnipaque), ioxaglate (Hexabrix) and metrizoate (Isopaque 350). The degree of activation of the complement cascade and the amount of free histamine in the samples were estimated. By using a hemolytic assay, a dose-independent complement consumption was detected when salt-free dilutions of the CM were added to human serum. Very little complement consumption was detectable when the concentrations, indicating that in the CM solutions were adjusted toward normal plasma concentrations, indicating that the lack of salts in the CM formulations was responsible for causing the consumption of complement rather than the CM molecules themselves. By using ELISA assay for determination of the terminal complement complex (TCC), no increase in TCC level was detected following the addition of iodixanol to human serum. The results indicate that iodixanol does not activate the complement cascade when added to human serum, and that it is unlikely that anaphylactoid reactions observed in man after CM administration are caused by CM-induced anaphylatoxins. No histamine release was observed following the addition of ioxaglate, metrizoate, iohexol or iodixanol to blood from nonallergic individuals.

Anaphylaxis↗
PubMed1979

Deficiency of the fifth component of complement in human subjects. Clinical, genetic and immunologic studies in a large kindred.

The discovery of a large kindred with a heritable deficiency of the fifth component of complement (C5) has permitted the accumulation of new clinical, genetic and immunologic data concerning the role of C5 in human subjects. The proband, who has had nine episodes of disseminated gonococcal infection, has a hemolytic C5 level of approximately 0.5 per cent of normal. No C5 protein was detectable, but low levels of functional C5 activity could be found using a sensitive bactericidal assay. The proband's twin as well as another sister also had extremely low levels of hemolytic C5(approximately 0.5 per cent normal), but both these subjects have been healthy. Hemolytic complement and bacteriolytic activity could be restored by the addition of purified C5. No chemotactic activity for polymorphonuclear leukocytes could be generated in the C5-deficient serums upon activation of either the classic or alternative pathways, again demonstrating the importance of C5 in human subjects for the production of chemotactic factors. The chemotactic responsiveness of the patients' polymorphonuclear leukocytes and monocytes to preformed chemotactic factors was not depressed. Twenty-two of 32 other family members from three generations had depressed whole hemolytic complement levels. In 19 of 30 family members, levels of hemolytic C5 ranged from 13 to 64 per cent of normal. No linkage for C5 deficiency and the A or B loci of the major histocompatibility complex could be found. These data suggest an autosomal codominant mode of inheritance of C5 deficiency. Deficiency of C5 is compatible with good health, but it can be associated with repeated disseminated gonococcal infection.

Adult↗