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PubMed1978

A radial hemolysis method in agarose for the functional assay of properdin.

A simple one-step radial hemolytic assay for properdin has been devised. In this assay, the test material is introduced into a well in an agarose plate containing optimal concentrations of normal human serum immunochemically depleted of properdin (RP), EGTA, magnesium ions and unsensitized guinea pig erythrocytes. Following radial diffusion, the area of the hemolytic zones resulting from the activation of the alternative complement pathway and bystander lysis of guinea pig erythrocytes was directly proportional to the concentration of properdin in the test material. The assay is specific reproducible and sensitive and the correlation with the radioimmunoassay for properdin is very good. The assay has been used to measure properdin activity in animal sera.

Animals↗
PubMed2001

Hepatocyte nuclear factor 1alpha controls the expression of terminal complement genes.

The terminal components of the complement system contribute to host defense by forming the multiprotein membrane attack complex (MAC) which is responsible for cell lysis and several noncytotoxic effects. Most of the complement proteins are synthesized in the liver, but the mechanisms controlling their tissue-specific expression have not been elucidated. In this study we show that mice lacking the hepatic transcription factor hepatocyte nuclear factor 1alpha (HNF1alpha) fail to transcribe C5 and C8A complement genes. In addition, mRNAs encoding for several other terminal complement components or subunits are expressed at lower levels, including C8beta, C8gamma, and C9. We next used a reconstitution assay involving human sera with selective complement deficiencies to assess mouse complement activity. Sera from HNF1alpha-deficient mice showed negligible hemolytic activity of both C5 and C8alpha-gamma subunits. The activity of C8beta was severely affected despite only a 50% reduction in C8beta mRNA levels in the liver. This is reminiscent of C8alpha-gamma-deficient patients who accumulate extremely low levels of the C8beta subunit. Our results demonstrate that HNF1alpha plays a key role in the expression of C5 and C8A genes, two terminal complement component genes that are essential for the assembly of MAC as a result of complement activation.

Animals↗
PubMed1985

Antibodies to O-antigen of lipopolysaccharide are protective against neonatal infection with Escherichia coli K1.

Monoclonal IgM specific for the O18 antigen conferred passive protection to 1-week-old rats against bacteremia and killing after oral challenge with O18:K1 Escherichia coli. Specific protection of the pups was also achieved by immunizing the pregnant rats with purified O18 lipopolysaccharide. We suppose that most human newborns that are colonized by potentially invasive K1 E. coli are protected by the transplacental transfer of anti-lipopolysaccharide immunoglobulin G, and we suggest that treatment with such antibodies might in the future be considered a therapeutic option. Rat serum from 1-week-old animals had only about one-third of the complement hemolytic activity of adult rat serum. This low level of hemolytic activity correlated with a relatively poor bactericidal activity in antibody-dependent and antibody-independent bactericidal in vitro assays. Monoclonal anti-O18 immunoglobulin M, although protective in vivo and bactericidal when added to adult rat serum, only poorly inhibited the multiplication of O18:K1 cells in serum from 1-week-old rats. This suggests that other elements of host defense besides complement participate in antibody-mediated in vivo protection.

Agglutination Tests↗
PubMed1987

Studies on the hemolytic activity of the classical and alternative pathway of complement in various animal species.

The classical complement pathway (CP) activity (CH50) of sera of 13 animal species was compared by parallel assays using rabbit erythrocytes sensitized with immune antibody (RaE-Ab) and sensitized sheep erythrocytes (ShE-Ab). The alternative complement pathway (AP) activity (ACH50) in various species of animals was also measured using 13 species of erythrocytes and RaE-Ab as target cells. The results have demonstrated that virtually all species of animals studied possess CP and AP activity levels comparable with or higher than those in human sera. Concomitant measurement of the hemagglutinin titer to various species of erythrocytes carried out in the screening ACH50 assays on various species of sera with these erythrocytes disclosed a significant correlation between natural antibody level and AP activity (p less than 0.05).

Animals↗
PubMed1978

Genetic polymorphism of the second component of human complement (C2): presentation of a modified typing technique and data on C2 phenotype distribution, linkage genetics, and haplotype associations a Norwegian family material.

Proteins were separated by prolonged isoelectric focusing in polyacrylamide gels, whereupon C2 bands were detected by a specific hemolytic assay. This was performed by treating the gel with iodine to increase C2 activity, and then developing C2 bands with an agarose gel overlay containing sensitized sheep cells and diluted human serum as a complement source deficient in functional C2. The gene frequencies observed in a material of 122 unrelated adults were: C2(1): 0.97 and C2(2): 0.03. C2 linkage relations and C2 haplotype associations have been examined a family material. It is concluded that C2 is very closely linked to HLA loci.

Adult↗
PubMed1985

Disease-associated loss of erythrocyte complement receptors (CR1, C3b receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation.

Although surface membrane density of complement receptor type one (CR1) on erythrocytes (E) is probably an inherited trait among normal individuals, recent evidence from our laboratories suggests that the reduced number of CR1 per E observed in patients with systemic lupus erythematosus (SLE) results from acquired as well as genetic factors. In the present investigation, the number of CR1 per E was quantitated with 125I-monoclonal anti-CR1 and was found to vary inversely with disease activity in patients with SLE who were followed serially for as long as 14 mo. Although evidence for E surface-bound immune complexes or fixed C3b/iC3b was not obtained, periods of disease activity and low amounts of CR1 per E correlated with the presence of 100 to 800 molecules per E of fixed C3dg fragments (less than 100 C3dg per E in normal subjects). Reduced CR1 and excess fixed C3dg on E also were observed in patients with other disorders associated with complement activation, including chronic cold agglutinin disease, autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria (PNH), Sjögren's syndrome, and mycoplasma pneumonia. A significant negative correlation (r = -0.498) between CR1/E and fixed C3dg/E was demonstrable in 255 individual assays evaluated by regression analysis. CR1 decreased and fixed C3dg increased during active disease; the converse was obtained during remission. In patients with active SLE, both serum complement activity and E CR1 decreased, whereas fixed C3dg fragments increased. By piecewise linear regression analysis, the appearance of 100 to 400 C3dg molecules on patients' E corresponded to a 27 to 60%, reduction in the number of CR1 per E (p less than 0.0002), confirming that fixation of C3 to E was correlated with a loss of CR1. In patients with PNH, low values for CR1 were observed on moderately complement-sensitive PNH type II E in association with increased fixed C3 fragments; however, the markedly complement-sensitive PNH type III E had essentially normal amounts of CR1 and bore little fixed C3. The addition of soluble DNA/anti-DNA immune complexes to normal blood generated levels of fixed C3dg fragments on E comparable to those observed on E from patients with SLE. Kinetic experiments indicated that C3b was fixed to E during the process of immune complex binding and release from E CR1, and that this fixed C3b was subsequently degraded rapidly to fixed iC3b and more slowly to fixed C3dg without the loss of CR1 that occurs in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Anemia, Hemolytic↗
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↗
PubMed1982

Quantitative studies of the secretion of complement component C3 by resident, elicited and activated macrophages. Comparison with C2, C4 and lysosomal enzyme release.

To quantitate the secretion of complement component C3 by guinea pig peritoneal macrophages an enzyme-linked immunosorbent assay was developed. C3 secretion was studied in resident, elicited and activated macrophages and compared with release of hemolytically active C2 and C4, as well as the lysosomal enzyme beta-D-2-acetamido-2-deoxyglucosidase. Resident macrophages secreted about 6 ng C3/10(6) cells/h into culture supernatants over a period of 12 h. Corynebacterium parvum-activated cells were found to secrete 3 times that amount at nearly constant rates. There was a stepwise increase in secretion of functional C2 and C4 when comparing resident, elicited and activated macrophages; secretion was 2--4 times higher in activated than in resident cells.

Acetylglucosaminidase↗
PubMed1983

A simple alternative pathway for hemolytic assay of human complement component C3 using methylamine-treated plasma.

A quantitative, alternative pathway (AP) hemolytic assay for human complement component C3 has been developed. This AP-C3 assay is inexpensive, rapid, simple, reproducible and insensitive to C3 degradation products. The AP-C3 assay uses rabbit erythrocytes as complement activator and methylamine-treated plasma, depleted of C3 and C4, as complement source. Rabbit erythrocyte sensitivity varies little from batch to batch, and remains unaltered for at least one month in Alsever's solution. Methylamine plasma may be stored at -20 degrees C for 3 months. AP-C3 lysis of 5-25% of erythrocytes is complete in 20 min and does not change subsequently. The AP-C3 assay is optimally stable at 2 mM Mg2+, 5 mM EGTA and at 5 X 10(7) erythrocytes/ml, yet insensitive to at least 20% deviation in these concentrations. The AP-C3 assay is specific to functional C3 and well suited for determination in plasma samples and during C3 preparation. Adding excessive amounts of C3c or plasma components other than C3 does not change the hemolytic response. The level of native C3 in plasma from 14 donors relative to a reference plasma pool ranged from 0.78 to 1.23. The standard deviation of relative C3 determinations did not exceed 2%.

Animals↗
PubMed1980

Quantitative determination of circulating immune complexes by inhibition of the hemolytic activity of polyclonal IgM rheumatoid factor.

A practical and sensitive method for detection and quantification of soluble complement-fixing immune complexes in sera of patients with various disease states has been developed. The assay is based on inhibition of complement-dependent sheep red cell hemolysis mediated by polyclonal IgM rheumatoid factor. Aggregated human IgG was used as an in vitro model of C-fixing human immune complexes and was quantified by its ability to inhibit hemolysis of sensitized sheep red cells by isolated IgM rheumatoid factor. The limit of sensitivity of this assay was 1--3 micrograms/ml. Fixation of complement in competition with isolated IgM rheumatoid factor, resulting in inhibition of hemolysis of sensitized sheep red cells, was used for detection and quantification of immune complexes in human sera. IgM rheumatoid factor was incubated with sensitised sheep red cells followed by addition of test sera; guinea pig complement was added; and the amount of IgM rheumatoid factor mediated hemolysis was determined spectrophotometrically and referred to a standard curve of inhibition of hemolysis by increasing amounts of aggregated human gamma-globulin. Good discrimination between sero-positive rheumatoid arthritis and systemic lupus erythematosus patients compared with normal and hospitalized subjects was found.

Antigen-Antibody Complex↗
PubMedDate not supplied

Complement-fixing hidden rheumatoid factor in juvenile rheumatoid arthritis.

Fifteen to twenty percent of patients with juvenile rheumatoid arthritis (JRA) have positive latex fixation tests (LFT), whereas approximately 46% have previously been demonstrated to have hidden rheumatoid factors (RF), i.e., 19S IgM RF which can be detected by the LFT after acid separation of the IgM-containing fraction from serum. In this study, hidden RF were found in 59% of patients with seronegative JRA by use of a complement-dependent hemolytic assay. The median titer of JRA patients was 1:42, and in healthy and disease controls it was 1:7. The difference was significant at P less than 0.001. When data from patients with active disease were analyzed separately, the median titer for polyarticular JRA was 1:97 and for pauciarticular JRA, 1:91. The differences due to active disease were significant at P less than 0.001 and P less than 0.005, respectively. The results demonstrate that the hemolytic assay is more sensitive than the LFT in determining the presence of hidden RF, and activity of disease correlates well with high hemolytic RF titers.

Adolescent↗
PubMed1986

Activation of the alternative complement pathway by mumps infected cells: relationship to viral neuraminidase activity.

An inverse relationship exists between the sialic acid content of a particle and its ability to activate the alternative complement pathway. The present studies were performed to determine if the neuraminidase (NANase) activities of different mumps virus strains could influence the ability of mumps virus infected cells to activate the alternative pathway. CV-1 cells were infected with three different mumps virus strains (RW, O'Take, and Kilham) and after 24 hours, 10 percent guinea pig serum (GPS) treated with EGTA/MgCl2 or GPS lacking the 4th component of complement (C4DGPS) was added to the cell monolayers. After 30 minutes, the percentage C3 consumed was determined by a functional hemolytic assay. Cells infected with RW (high NANase) consumed significantly more C3 (23.2 per cent) than cells infected with Kilham (5.7 percent, low NANase). Cells infected with O'Take were intermediate in their ability to activate C3. The degree of C3 deposition on the surface of infected cells, detected by fluorescence microscopy, was also greater for cells infected with the RW than the Kilham strain of mumps virus. These studies suggest that the NANase activity of mumps virus can influence the ability of infected cells to activate the alternative pathway and thereby, the ability of complement to participate in host defense against mumps virus infection.

Animals↗
PubMed1984

Antibody-dependent alternative pathway killing of Haemophilus influenzae type b.

The bactericidal activities of human complement and human antibody directed against specific Haemophilus influenzae type b cell surface determinants were investigated. Strain Eagan, a laboratory isolate, and strain Kn, a clinical isolate, were used as the test organisms and gave qualitatively similar results. In the absence of antibody, both isolates were resistant to killing by 60% agammaglobulinemic serum (AGS) containing normal complement levels. The addition of affinity-purified immunoglobulin G anticapsular antibody was bactericidal with 15% AGS as the complement source. Bactericidal activity was also demonstrated with this antibody when the complement source was AGS-Mg-EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid], C2-deficient human serum (alternative complement pathway), or AGS in which factor D and properdin had been selectively inactivated (classical pathway). Immunoglobulin G fractions from a human serum pool or from serum from an adult who had recovered from H. influenzae type b (Kn) sepsis were absorbed to remove anticapsular antibody. The absorbed fractions containing noncapsular antibodies also activated complement-dependent bactericidal activity. But, in contrast to the results with anticapsular antibody, noncapsular antibodies did not elicit alternative pathway bactericidal activity. Incubation of cells of H. influenzae type b in C2-deficient serum or AGS-Mg-EGTA did not cause complement consumption (total hemolytic complement and C3). The addition of immunoglobulin G anticapsular antibody (but not noncapsular antibody) increased consumption of total complement and C3, paralleling the results of the bactericidal assays. These studies demonstrated an absolute requirement for anticapsular antibody in alternative pathway activation and killing of H. influenzae type b.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1976

Activation of therminal components of human complement by a trypsin-activated complex of human factor B and cobra venom factor.

Cleavage of C3 by CVF-B was demonstrated by hemolytic, immunoelectrophoretic and immune adherence reactions. No cleavage of C5 was detected by immunoelectrophoresis, but C5 hemolytic activity, assayed with EAC1423, decreased although less than C3 hemolytic activity. The co-existence of C3 with limiting amounts of C5 did not reduce the final degree of hemolysis of guinea pig erythrocytes (GPE) induced by late-acting components C6 through C9 and CVF-B. Thus, a CVF-B hemolytic system composed of GPE, C5 through C9 and CVF-B provided a method for titration of terminal components of human complement. CVF-B was able to generate hemolytically active sites of C567 on GPE by activation of C5, C6 and C7. The complex C567 in the fluid-phase decayed within 1 min but C567 on GPE was quite stable. Originally insensitive sheep erythrocytes became sensitive to the CVF-B hemolytic system if C3b sites were present, suggesting that cell-bound C3b played a role in orienting the positions of C567 to be fixed. CVF-B could be recovered quantitatively from the supernatant of the reaction mixture in which the hemolytically active intermediate GPEC-5678 had been formed through the interaction between C5 to C8 and CVF-B.

Animals↗
PubMedDate not supplied

Complement system in healthy term newborns: reference values in umbilical cord blood.

Activation of the complement system occurs in several diseases. For reliable identification of complement activation in neonates, we establish reference ranges of several components in cord blood of healthy term newborns. For this study, cord blood samples were taken from 125 healthy term newborns. Concentrations of C1r, C2, C5, C7, Properdin, and factors D, H, and I were determined by single radial immunodiffusion. C3a and C5a were measured by specific EIA and complement function was measured by hemolytic assays. The results were expressed as 5th percentile, median, and 95th percentile. The following respective concentrations were found: C1r: 27, 47, 65 mg/l; C2: 12.0, 18.0, 24.0 mg/l; C5: 64, 92, 127 mg/l; C7: 32, 60, 89 mg/l; Properdin: 5.6, 9.7, 14.2 mg/l; factor D: 3.6, 5.2, 7.3 mg/l; factor H: 178, 234, 296 mg/l; and factor I: 15, 24, 32 mg/l. The functional activity of the whole complement system was 24%, 43%, 97% and for the alternative pathway 39%, 58%, 76%. The concentration of the activated split products C3a was 4, 65, 255 microg/l and of C5a, 0.11, 0.26, 1.19 microg/l. These reference values may be important for the detection of deficiencies of native complement proteins or perinatal processes leading to an activation of the complement system.

Adult↗
PubMed1999

Active sites in complement components C5 and C3 identified by proximity to indels in the C3/4/5 protein family.

We recently suggested that sites of length polymorphisms in protein families (indels) might serve as useful guides for locating protein:protein interaction sites. This report describes additional site-specific mutagenesis and synthetic peptide inhibition studies aimed at testing this idea for the paralogous complement C3, C4, and C5 proteins. A series of C5 mutants was constructed by altering the C5 sequence at each of the 27 indels in this protein family. Mutants were expressed in COS cells and were assayed for hemolytic activity and protease sensitivity. Mutants at five indels showed relatively normal expression but substantially reduced sp. act., indicating that the mutations damaged sites important for C5 function. Twenty-three synthetic peptides with C5 sequences and 10 with C3 sequences were also tested for the ability to inhibit C hemolytic activity. Three of the C5 peptides and one of the C3 peptides showed 50% inhibition of both C hemolytic and bactericidal activities at a concentration of 100 microM. In several cases both the mutational and peptide methods implicated the same indel site. Overall, the results suggest that regions important for function of both C3 and C5 lie proximal to residues 150-200 and 1600-1620 in the precursor sequences. Additional sites potentially important for C5 function are near residue 500 in the beta-chain and at two or three sites between the N-terminus of the alpha'-chain and the C5d fragment. One of the latter sites, near residue 865, appears to be important for proteolytic activation of C5.

Amino Acid Sequence↗
PubMed1986

Homogeneous, liposome-based assay for total complement activity in serum.

This is a rapid, homogeneous, liposome-based assay for total complement activity in human serum. Liposome-encapsulated enzyme is unmasked by the action of complement on liposomes carrying surface-bound immune complexes. The amount of unmasked enzyme, proportional to the concentration of added complement, is quantified by measuring the absorbance of enzymically produced product at 410 nm. Complement activity in serum samples is extrapolated from a standard curve generated from dilutions of a guinea pig serum containing a known activity of complement. Interassay CVs were less than 7.0% and intra-assay CVs less than 2.8% for serum pools with complement activities spanning the normal range. Test results correlate as well with those of the hemolytic complement test (r = 0.80) as the latter correlates with itself (r = 0.82), and also correlate reasonably with measurements of complement components C3 (r = 0.62) and C4 (r = 0.74). Values for a normal population are reported. Advantages of this test include stability of reagents, speed, accuracy, simplicity, and avoidance of radioisotopes.

Alkaline Phosphatase↗