Complement Pathway In Human Serum Research Articles

A study of the xenoantigenicity of adult pig islets cells

The pig pancreas is considered to be the most suitable source of islets for xenotransplantation into patients with type I diabetes. The purpose of this study was to assess the antigenicity of pig islets, including the Galalpha1-3Galbeta1-4GlcNAc-R (the alpha-Gal) and Hanganutziu-Deicher (H-D) antigens, and the pathway involved in human complement activation. The expression of alpha-Gal on islets from adult pigs was investigated by immunohistochemical staining and flowcytometric analysis. The alpha1,3 galactosyltransferase (alpha1,3GT) activity of islets was determined by high-performance liquid chromatography. Antigenicity to human natural antibodies, including the H-D antigen of pig islets was next examined by treatment of pig islets with tunicamycin, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and/or neuraminidase. In addition, complement-mediated islets lysis was examined using factor D-deficient and C1-deficient sera. Adult pig islets expressed negligible amounts of alpha-Gal epitope, and alpha1,3GT activity was also undetectable. However, human natural antibodies, immunoglobulin G and M, and the anti H-D antibody react to the adult islet. Treatment of pig islets with tunicamycin, but not PDMP, led to a drastic reduction in antigenicity to human serum, indicating the importance of N-linked sugars on the islets. Neuraminidase treatment indicated the presence of, not only the H-D antigen, but also other sialic acid antigens that reacted with the human natural antibody. The complement deposition of C4, C3 and factor B on islets was demonstrated. The alternative pathway-mediated pig islet killing accounted for approximately 30% of that by the total complement pathway. The origin of antigenicity of pig islets is mainly N-linked sugars including sialic acid antigens, but not the alpha-Gal, and pig islets can be injured by both the classical and the alternative complement pathway in human serum.

Collectin in a non-mammalian species: isolation and characterization of mannan-binding protein (MBP) from chicken serum.

A chicken serum lectin was isolated by affinity chromatography on TSK-75 beads derivatized with the monosaccharide N-acetyl-D-mannosamine (ManNAc). Serum was applied to the column in a Ca(2+)-containing buffer and proteins were eluted with EDTA. After recalcification, the eluate was passed through a new ManNAc-derivatized column. Bound proteins were eluted with 50 mM ManNAc. Anti-carbohydrate antibodies present in the eluate were removed by passage through a rabbit anti-chicken immunoglobulin derivatized column, and the lectin was further purified by ion-exchange chromatography and gel-permeation chromatography. The purified chicken lectin shows an overall structure similar to mammalian mannan-binding protein (MBP). SDS-PAGE revealed two polypeptides of M(r) 33 and 34 kDa (reduced) with identical sequence for the first 30 NH2-terminal residues. The NH2-terminal sequence shows 43% identity with the human MBP. Like mammalian MBP, the polypeptides of the chicken lectin are degraded by treatment with collagenase. Residues 26-30 (G-L-P(OH)-G-D) are likely to represent the beginning of the collagenous region. Mobilities on SDS-PAGE of the COOH-terminal collagenase-resistant fragment under reduced and non-reduced conditions indicate the presence of intrachain disulphide bonds, as are also found in mammalian MBP. Gel chromatography showed an intact mol. wt of 750 kDa. Binding of the chicken MBP to mannan was inhibited by monosaccharides in the following order of potency: ManNAc > L-fucose > mannose > N-acetylglucosamine. Other monosaccharides inhibited poorly or not at all. Chicken MBP, bound to mannan, activated the classical complement pathway in human serum. Electron micrographs show structures and dimensions resembling human MBP. Overall, the results show that the purified lectin is the chicken homologue to mammalian MBP and indicate the presence of a MBP-like clearance system outside mammals.

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°C resulted in loss of activation when the adsorbed serum was interacted with fresh Sephadex beads. Acid eluted proteins from adsorbed 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.

Activation of the classical complement pathway by BioRex-70

The cation exchange resin BioRex-70 was able to activate the classical complement pathway in human serum at 37°C over the resin concentration range 0–5% (v/v). Using zymosan-treated human serum, it was found that the activation proceeded as far as complement protein C3.

The use of monoclonal antibodies as probes of the three-dimensional structure of human complement factor D.

The monoclonal antibodies (MAb) were studied for their binding specificities and their effects on the hemolytic and proteolytic activities of human D. One Mab, FD10-1, was obtained from mice immunized with native D; the other, JA4-2, was similarly obtained from mice immunized with BSA coupled to a synthetic nonapeptide (Arg-Ile-Leu-Gly-Gly-Arg-Glu-Ala-Tyr) containing the seven NH2-terminal amino acids of D. By using a PEG-precipitation assay system, lactoperoxidase-iodinated D was precipitated by FD10-1 cell culture supernatant as well as by the purified MAb. Conversely, with the use of the same assay system, neither the JA4-2 cell culture supernatant nor the purified MAb precipitated any significant amount of 125I-D. In contrast, by using a solid-phase binding assay, radiolabeled JA4-2 as well as FD10-1 bound to D-coated wells. In addition, the binding of purified JA4-2 to D-coated wells could be completely inhibited by the nonapeptide, whereas the binding of purified FD10-1 at equivalent concentrations was unaffected. These binding studies correlated with the results of functional assays. FD10-1 was found to inhibit the hemolytic activity of the alternative complement pathway in human serum as well as the cleavage of radiolabeled B by D in the presence of cobra venom factor, whereas JA4-2 had no significant inhibitory effect in either of these assays. These data suggest that the NH2-terminus of native D, like that of other active serine proteases, is buried inside the molecule where it is inaccessible to the bulky JA4-2 MAb.

Neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement.

All normal human sera examined neutralized WS/33 H1N1 influenza virus efficiently by one of two antibody-dependent mechanisms. A minority of the sera contained moderate levels of IgG antibody directed against the viral hemagglutinin that had the ability to directly neutralize the virus. The majority of sera tested contained very low levels of IgG anti-hemagglutinin antibody, which was detectable with a specific ELISA but not by conventional HAI assays. Such IgG antibody was unable to directly neutralize the virus. Studies with agammaglobulinemic serum and with sera depleted of and reconstituted with complement components established essential roles for IgG and the components of the classical complement pathway through C3 for neutralization. The components of the alternative and membrane attack pathways were not needed for neutralization. As anticipated from the requirement for IgG and exclusive mediation of neutralization by the classical pathway, the virus-IgG immune complex activated purified C1. Binding of C3 and C4 to the virus was demonstrated, as was classical pathway-mediated triggering of the alternative pathway, with recruitment of properdin. In addition, the H1N1 influenza virus also directly activated the alternative complement pathway in human serum, leading to C3 and properdin deposition on the viral envelope. Such direct alternative pathway activation also required immunoglobulin. However, the alternative pathway alone was unable to neutralize the virus. Thus, most normal sera examined contain low levels of IgG anti-hemagglutinin antibody, which activate the classical pathway of the complement system and neutralize WS/33 influenza virus by deposition of C3 and C4 on the viral envelope.

Mechanism of activation of the classical complement pathway in human serum by a lectin from Euonymus europeus

Mechanism of activation of the classical complement pathway in human serum by a lectin from Euonymus europeus

Decreased complement activity after bilateral renal ischemia in rats.

The bilateral occlusion of the renal arteries for 2h resulted in a marked decrease of serum complement activity after recirculation. The release of a proteinic factor with anticomplementary activity detectable in vitro in normal kidney supernate is considered a probable cause of the phenomenon. This factor is able to activate the alternative complement pathway in human serum.

Malignant cells isolated from Burkitt's lymphoma but not other forms of leukemia activate the alternative complement pathway in human serum.

Cells isolated directly from primary biopsies of Burkitt's lymphoma were found to activate the alternative complement pathway in hypogammaglobulinemic human serum. In contrast, cells isolated from patients with either acute lymphoblastic leukemia, chronic lymphocytic leukemia or chronic myeloid leukemia did not activate. No defects in the ability of Burkitt's lymphoma sera to support alternative pathway activation were detected.

Evaluation of a hemolytic assay of the alternative complement pathway in human serum.

Alternative pathway activity of human serum was titrated by use of unsensitized rabbit erythrocytes (RE). Under the conditions of the assay, the von Krogh equation could be used to relate the proportion of RE lysed to the level of alternative pathway activity. The use of a 50% hemolytic endpoint provided maximum sensitivity in the assay. The 50% hemolytic endpoint could be calculated from a single measurement in the region of 20% to 80% lysis or RE. Factor B was required for lysis of RE in the test, but neither C2 nor C8 was limiting under the conditions of the assay. Alternative pathway activities of three sera with abnormal IgG levels were in the normal range, but normal serum absorbed with RE at 0 C before testing had diminished lytic activity with the test. Lysis of RE in acute-phase sera of 16 patients who had bacteremic pneumococcal pneumonia was significantly below normal (P < 0.01). Results with Re lysis in these patients correlated well with levels of Factor B that were measured immunochemically and with consumption of whole complement by zymosan.

Activation of the Classical Complement Pathway in Human Serum by a Small Oligosaccharide

Abstract Two small oligosaccharides were hydrolyzed from a native polysaccharide by polysaccharidases indigenous to the venom of the tropical ant Pseudomyrmex sp. after incubation for 5 hr at 37°C. They were purified by passage through an ultrafiltration membrane (m.w. cut-off, 500 daltons), cation exchange chromatography at pH 3 to separate the oligosaccharides from peptides, and gel filtration chromatography. The highly purified oligosaccharides contained no amino acids and no polar or nonpolar lipids. The larger oligosaccharide had an estimated m.w. of 840 daltons, and the smaller 430 daltons. The larger species was a potent activator of C1 in normal human serum at 37°C, resulting in the consumption of its two natural substrates C4 and C2. It had no effect on the C1 INH, and its action was destroyed by treatment with sodium periodate. Analysis of both oligosaccharides by gas chromatography revealed five sugars: glucose, mannose, galactose, N-acetyl-glucosamine, and N-acetyl-galactosamine. Chemical analyses revealed the presence of reducing sugars in the larger species, but neither contained hexuronic acids. The latter were found in the native polysaccharide. The smaller oligosaccharide may be a monomer of the larger species. After electrophoresis on paper at pH 1.9, the 840-dalton oligosaccharide separated into two species: the first, which remained at the origin, contained reducing carbon atoms. The second migrated toward the cathode and contained no reducing sugars. Both species caused the consumption of C2 in human serum. The oligosaccharide(s) with a m.w. of 840 daltons is the smallest carbohydrate described that causes activation of the classical complement pathway.

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.

Kinetic analysis of chemotactic factor generation in human serum via activation of the classical and alternate complement pathways

The kinetics of complement-related chemotactic factor generation in human serum were evaluated. Addition of immune complexes to serum resulted in formation of chemotactic activity within 5 min of incubation while activation with S. typhosa endotoxin of serum from normal subjects resulted in a prolonged (15–20 min) latent period prior to generation of chemotactic activity. In contrast to S. typhosa, endotoxin incubation of serum with E. coli endotoxin resulted in rapid formation of chemotactic activity with a kinetic pattern similar to that noted with immune complexes. In these sera antibody to E. coli but not S. typhosa endotoxin was demonstrable; analysis of sera from immunized individuals with demonstrable S. typhosa antibody showed a pattern of chemotactic factor formation similar to that of immune complexes and E. coli endotoxin. Activation of human serum deficient in C2 (<0.05% normal C2) with either immune complexes or E. coli endotoxin resulted in a prolonged latent period before formation of chemotactic activity although total chemotactic activity at 60 min was normal; addition of purified C2 restored normal kinetics. The addition of Mg 2+-EGTA to normal serum activated with immune complexes or E. coli endotoxin changed the pattern from rapid to delayed formation of chemotactic activity. EDTA completely inhibited generation of chemotactic factor with immune complexes or endotoxin. Activation with immune complexes of serum heated for 20 min at 50°C resulted in the rapid formation of chemotactic activity, while activation with S. typhosa endotoxin failed to generate chemotactic activity. These results indicate that in human serum generation of chemotactic factors proceeds rapidly when mediated by antibody and components of the classical pathway, but only after a latent period when mediated by the alternate pathway. Further studies demonstrated that activation of either complement pathway in human serum results in the generation of a chemotactic factor, which is heat stable (30 min at 56°C), has an estimated molecular weight of 15,500 daltons, is inhibited by goat antibody to human C5 but not C3 and, therefore, represents the C5 cleavage product C5a. Thus, the classical and alternate complement pathways in human serum can be easily distinguished by kinetic analysis. Since normal levels of chemotactic factor may be present after 60 min of incubation, despite complete block of one complement pathway, kinetic analysis may be required to detect clinically important abnormalities.