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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↗
PubMed1981

'Nephritic factor' may be an autoantibody to C3b or C4b.

We attempted to clarify the character of 'Nephritic Factor' in the alternative (NeFA) and classical pathways (NeFC) of the complement, using an agglutinating and a hemolytic activity assay. The results suggested a possibility that NeFA and NeFC were different in their ability but might appear at the same time in some cases, and that NeFA was anti-C3b autoantibody and NeFC anti-C4b autoantibody.

Autoantibodies↗
PubMed2001

In situ complement activation by polyethylene wear debris.

A frequent long-term complication of total joint arthroplasty is aseptic loosening, the end result of wear debris accumulation, synovitis, and osteolysis about the implant-bone or cement-bone interface. Complement, an effector system in plasma, synovial fluid, and tissue, has powerful chemotactic, inflammatory, and osteoclast-activating potentials. This study explored the complement-activating ability of polyethylene, a material used in joint implants. In vitro hemolytic assays using sheep red blood cells (E(sh)), human serum, and particulate polyethylene suggested alternative pathway complement activation, as well as polyethylene adsorption of activated complement components. These results were confirmed by enzyme-linked immunosorbent assay (ELISA) quantification of activated complement factors Bb and C3b. In situ double antibody immunoperoxidase staining for factors Bb, C3a, iC3b, and SC5-9 in synovial tissue from revision hip specimens showed localized alternative pathway activation and component adsorption. These results introduce a likely role for complement activation in particle-mediated recruitment, proliferation, and activation of macrophages during early events in osteolysis and implant loosening.

Biocompatible Materials↗
PubMed1979

The presence of complement in human cervical mucus and its possible relevance to infertility in women with complement-dependent sperm-immobilizing antibodies.

Full-complement component lytic activity was measured in human midcycle cervical mucus, using a sensitive 51Cr release hemolytic assay. The level measured was 11.5% of the activity of complement in an equal volume of undiluted human serum. The relevance of this level of complement to complement-dependent sperm-immobilizing antibody activity was studied. After 1 hour's incubation with mucus levels of complement, immobilization of about 50% of spermatozoa occurred and after 3 hours' incubation, immobilization of about 70% of spermatozoa occurred.

Antibodies↗
PubMedDate not supplied

PEG-modified protamine with improved pharmacological/pharmaceutical properties as a potential protamine substitute: synthesis and in vitro evaluation.

Cardiopulmonary bypass (CPB) procedures are frequently associated with massive inflammatory responses, resulting in a high rate of morbidity and mortality in routine cardiac operations. One recognized attribute of these deleterious responses is the synergic effect of heparin and protamine, which elicit the activation of the complement system in vivo. To circumvent such toxic effects following protamine reversal of heparin anticoagulation in the CPB procedures, we proposed that poly(ethylene glycol) (PEG)-modified protamine could retain the heparin-neutralization ability and yet diminish the induced complement activation by the formed heparin-protamine complexes (HPC), thereby providing highly improved pharmacological properties. PEGylation of protamine was carried out by utilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. Size exclusion chromatography (SEC), reverse-phase high performance liquid chromatography (RP-HPLC), and matrix-assisted laser desorption mass spectrometry (MALDI-MS) were used to assess the conjugation stiochiometry, the purity of the conjugates, and the site of PEG modification, respectively. The heparin-neutralizing activity was determined by using heparin affinity chromatography and various biological assays including the plasma-activated partial thromboplastin time (aPTT), anti-Xa, and anti-IIa methods. The potency in inducing complement activation was examined in vitro using the CH50 hemolytic assay. The PEG-modified protamine was successfully synthesized with a PEG/protamine stiochiometry of 1:1. Only one conjugation site for PEG that was located at the N-terminal end of protamine was obtained. In the biological evaluations, the PEG-modified protamine displayed a full retention of the heparin-neutralizing ability of protamine and a significantly reduced activity in complement activation following its complexation with heparin. Results from studies of the particle size and zeta potential indicated that the PEG-modified protamine formed substantially smaller aggregates with heparin, rendering them less effective in triggering the size-dependent complement responses. As with protamine, PEG-modified protamine exhibited an enhanced aqueous solubility, therefore attaining significantly improved pharmaceutical properties. These preliminary results suggested that the PEG-modified protamine conjugate might serve as a potential protamine substitute with improved therapeutic and pharmaceutical properties in heparin reversal.

Blood Coagulation↗
PubMed1981

The role of C9 in complement-mediated killing of Neisseria.

During the routine examination of a healthy 31-yr-old woman, we found an incomplete deficiency of the 9th component of complement (C9). By hemolytic assay her serum C9 activity was 10 to 15% of normal. Limited family studies suggested that she inherited the deficiency as an autosomal codominant trait. She had no history of unusual or severe infections. When tested for bactericidal activity against serum-sensitive Neisseria gonorrhoeae and N. meningitidis, her serum reacted comparably to normal serum. Normal serum depleted immunochemically of C9 and sera from congenitally C9-deficient patients were also bactericidal against serum-sensitive Neisseria but required 120 min to kill the same numbers of gonococci that intact serum killed within 30 min. In the electron microscope, N. gonorrhoeae incubated with C9-depleted serum were fragmented but lacked the typical C lesions. Therefore, serum lacking C9 can kill serum-sensitive Neisseria, unlike sera deficient in the other terminal C components.

Adult↗
PubMed1993

Properdin, a positive regulator of complement activation, is expressed in human T cell lines and peripheral blood T cells.

Properdin plays an important regulatory role in the activation of the complement system. Here we report the biosynthesis of properdin in four different human T cell lines and in T cells purified from peripheral blood. Cell sorting experiments, in conjunction with Northern blotting, showed that both CD4- and CD8-bearing populations of T cells have the potential to synthesize properdin. The functional activity of properdin secreted from these T cell lines was determined in a hemolytic assay. In view of the numerous ways in which complement activation may influence cellular immune response, the present results indicate, for the first time, a possible interaction between the complement and the T cell system.

Adult↗
PubMed1981

Activation of mouse complement by monoclonal mouse antibodies.

The ability of monoclonal mouse IgM, IgD and IgG anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) antibodies to activate mouse complement was studied using a hemolytic assay. Efficient hemolysis was obtained with IgG2a, IgG2b and IgG3 antibodies but no lysis was observed using a monoclonal IgD. Of several IgG1 antibodies tested, three gave no detectable hemolysis, although weak but significant hemolysis was obtained with two other IgG1. IgM was found to be powerfully hemolytic in that it was effective at lower concentrations than were IgG. However, using complement from mouse strains carrying the H-2k haplotype it was found that, under conditions of complement limitation and saturating antibody, a fixed amount of complement could lyse about 3 to 4 times as many IgG-coated sheep red cells as IgM-coated red cells. This discrimination between IgM and IgG as regards the efficiency of complement utilization is controlled by a gene in the S region of H-2 and is not apparent with complement from mice carrying the b, d or s allele at that locus.

Animals↗
PubMed1990

Isolation of the fifth component of the bovine complement system.

Bovine C5 has been isolated from fresh bovine serum by a five-step procedure: polyethylene glycol precipitation, sequential ion-exchange chromatography on DEAE-Sephacel and CM-Sephadex, hydroxylapatite chromatography, and affinity chromatography. The purified C5 was a protein of apparent molecular weight 202,000 +/- 9,000 composed of two chains: an alpha-chain of molecular weight 127,000 +/- 5,000 and a beta-chain of molecular weight 74,000 +/- 2,000. The alpha-chain was cleaved by Sepharose-CVF.Bb (a cobra venom factor (CVF)-induced C3/C5 alternative pathway convertase) in the absence of any C3 or C3b. The monocarboxylic acid form of K-76, a sesquiterpene compound isolated from the culture filtrates of Stachybotris complementi, inhibited the alternative pathway of bovine serum, and the inhibited hemolytic activity was restored, in a dose dependent manner, by bovine C5. This provided the basis for a C5 functional assay throughout the purification procedure. The purified C5 showed species specificity and was functionally distinct from bovine C3.

Animals↗
PubMed1981

Detection of shedding of human blood monocyte Fc receptor during in vitro culture.

Human monocytes, isolated from peripheral blood and placed in culture, produce supernatants that have Fc receptor activity. This activity is detectable using a complement-dependent hemolytic assay. Inhibition of hemolysis is detected with undiluted supernatants, is temperature-dependent and is not demonstrable with culture supernatants from Fc-receptor-negative endothelial cells. To more completely determine their monocyte supernatants contained shed Fc receptors, inhibition of direct Fc rosettes by the same supernatants was demonstrated. The supernatants are most active for Fc receptor activity after monocytes are cultured for 7-9 days. The activity of Fc receptor material varied among donors. Monocyte culture supernatants may be utilized as sources of monocyte Fc receptor material for future evaluation.

Adult↗
PubMedDate not supplied

Early recipient-donor switch of the complement type after liver xenotransplantation.

Liver transplantation is an immunological peculiarity with respect to the resistance of the graft to humoral rejection. We undertook a kinetic analysis of molecules involved in humoral rejection for a period of one week following xenografting in the hamster to rat model system. A complement-dependent lymphocytotoxicity test (CDC) was used to detect anti-donor antibodies in the recipient rats. Complement was studied by two methods. Function of the classical complement pathway was evaluated with a hemolytic assay, and C3 was measured by radial immunodiffusion. Conversion of the major plasma proteins from recipient to donor profile was studied by zone electrophoresis on agarose. CDC showed antibody titers rose during the first week post-transplantation, and they were of complement-activating isotypes. Zone electrophoresis showed almost complete replacement of rat C3 by hamster C3 within 72 hours. Hemolytic assay of complement on day 6 post-transplant showed serum of the xenograft recipients could lyse erythrocytes sensitized with rat antibody with 80% of efficiency of normal rat serum. Our data show the effector molecules for humoral rejection, rat antibodies with anti-hamster specificity and a functional complement cascade, were present within the first week following transplantation. Rapid conversion of serum complement to hamster proteins maintains compatibility with the species-specific membrane inhibitors of complement activation expressed by the xenografted hepatocytes, and could limit complement-mediated damage.

Animals↗
PubMed1978

Inherited deficiency of the seventh component of complement associated with nephritis. Propensity to formation of C56 and related C7-consuming activity.

A 46-yr-old female with chronic pyelonephritis was found to lack complement (C) activity by the use of hemolytic screen assays in agarose gels. These assays also revealed a propensity of patient serum to form an activated complex of the fifth and sixth components of C, C56. Each of the C component hemolytic activities was present in normal or elevated amounts with the exception of C7, which was undetectable; addition of purified C7 led to the restoration of hemolytic activity. C-dependent phagocytosis, immune adherence, and neutrophil chemotaxis were normal. Family studies demonstrated that the defect was transmitted as an autosomal codominant apparently not linked with alleles at the HLA-A or HLA-B loci. Persisting C56 was readily formed in this as compared to normal serum upon incubation with multiple C activators including zymosan, inulin, immune complexes, heat-aggregated human gamma globulin, endotoxin, and agarose. A heat-stable (56 degrees C, 30 min) activity which consumed C7 with time-and temperature-dependent kinetics was detected in plasma and serum, and seemed to be similar to a "C7 inactivator" previously described in another C7-deficient individual. However, this activity was found to have properties identical to those of C56 during low ionic strength precipitation and chromatography on Sephadex G-200, to be specifically removed upon passage through an anti-C5 immunoadsorbent column, and to be associated with a small amount of C56, suggesting that it represents an expression of small amounts of C56 rather than a new C-inhibitory activity. Thus, an individual with chronic nephritis lacking C7 is reported; the utility of a hemolytic screen assay in agarose plates for the detection of such patients is emphasized; persisting C56 is shown readily to be formed in this serum; and the presence of C7-consuming activity which is associated with and in all likelihood attributable to C56 is shown.

Complement C5↗
PubMed1985

Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria.

Decay-accelerating factor (DAF) is a 70,000 Mr protein that has been isolated from the membrane of red cells. The function of DAF is to inhibit the assembly of amplifying enzymes of the complement cascade on the cell surface, thereby protecting them from damage by autologous complement. We raised monoclonal antibodies to DAF and used them to study its distribution in cells from the peripheral blood of normal individuals and of patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by the unusual susceptibility of red cells to the hemolytic activity of complement. The results of immunoradiometric assays and of fluorescence-activated cell sorter analysis showed that DAF was present not only on red cells but was widely distributed on the surface membrane of platelets, neutrophils, monocytes, and B and T lymphocytes. By Western blotting, we observed small but consistent differences in the Mr of DAF from the membranes of various cell types. Quantitative studies showed that phagocytes and B lymphocytes, which presumably enter more frequently in contact with immune complexes and other potential activators of complement, had the highest DAF levels. As previously reported by others, the red cells from PNH patients were DAF deficient. When the patients' red cells were incubated in acidified serum (Ham test), only the DAF-deficient cells were lysed. In addition, we detected defects in DAF expression on platelets and all types of leukocytes. The observed patterns of DAF deficiency in these patients were consistent with the concept that the PNH cells were of monoclonal origin. In one patient, abnormal and normal cells were found only in the erythroid, myeloid, and megakaryocytic lineages. In two other patients, the lymphocytes were also DAF deficient, suggesting that a mutation occurred in a totipotent stem cell. It appears, therefore, that the lesion leading to PNH can occur at various stages in the differentiation of hematopoietic cells.

Antibodies, Monoclonal↗
PubMed1979

Classical and alternate complement pathway activities in paired dairy cow--newborn calf sera.

Hemolytic assays were used to compare alternate and classical C pathway activities in sera obtained from clinically normal newborn dairy calves and their mothers at the time of delivery. Mean alternate and classical CH50 concentrations in sera from newborn calves were both significantly lower than in their dams (P less than 0.001). The titer of alternate C pathway activity, expressed as CH50 units/ml, in sera from 17 calves was 12.9 +/- 5.5, whereas for the cows it was 25.8 +/- 6.2. The ratio of cow: calf serum alternate CH50 titers averaged 2.25 +/- 0.80 and ranged from 0.88 to 4.14. Classical CH50 titers were 78.0 +/- 42.7 units/ml in calf sera and 246.0 +/- 44.5 in cow sera. The ratio of cow: calf serum classical CH50 titers averaged 3.71 +/- 1.49 and ranged from 1.19 to 6.87. The wide range of values, noted for both the alternate and classical C pathways, within maternal and neonatal groups was assumed to reflect the biologic variability of complement levels in bovine serum. The possible relationships between deficient levels of alternate and classical CH50 activity in newborn calves and their susceptibility to infections is discussed.

Animals↗
PubMed1998

Smokeless tobacco extracts activate complement in vitro: a potential pathogenic mechanism for initiating inflammation of the oral mucosa.

The use of smokeless tobacco has been linked to an increased incidence of inflammation of the buccal and gingival mucosa. However, the mechanisms by which smokeless tobacco initiates inflammation are not well understood. The complement cascade is a ubiquitous source of proinflammatory molecules and can be activated rapidly by a wide variety of agents. Therefore, the effect of smokeless tobacco on complement was investigated as a potential pathogenic mechanism for triggering inflammation of the oral mucosa. Aqueous extracts of loose leaf chewing tobacco (1S1), dry snuff (1S2), and moist snuff (1S3), added to normal human serum, depleted complement hemolytic activity in a dose-dependent manner. Experiments utilizing sera deficient in one specific complement component indicated that the smokeless tobacco-induced depletion of hemolytic activity was due largely to consumption of C3. Furthermore, assays designed to test the activity of the alternative pathway of complement clearly showed that all three extracts depleted the hemolytic activity of this pathway. Finally, all three smokeless tobacco extracts activated the alternative pathway since significantly elevated levels of the cleavage fragments iC3b and Bb were detected in extract-treated serum. High quantities of the classical pathway cleavage fragment C4d also were detected in serum treated with moist snuff (1S3). The results clearly demonstrate that smokeless tobacco extracts activate the alternative pathway and also suggest some measure of classical pathway activation. Activation of complement by smokeless tobacco may be a mechanism for initiating inflammation of the oral mucosa.

Complement Activation↗
PubMedDate not supplied

[Significance of immune complexes in children with severe hemophilia A in substitution treatment with factor VIII concentrates].

Sera and EDTA-Plasma of patients with severe Haemophilia A were analysed for immune complexes and the hemolytic activity of complement in relation to Factor VIII replacement, in order to confirm or possibly exclude a relationship to allergic reactions. Immune complexes were isolated by PEG precipitation and quantitated. In addition a solid phase ELISA assay was used to detect complement-binding complexes. Total hemolytic complement activity of the classical and the alternate pathway was measured in addition to the C3 splitproduct C3d. The results obtained from 12 patients with severe Haemophilia A showed slightly increased immune complex titers, no changes of the immune complex levels during Factor VIII replacement and no alteration of the complement system following the infusions. One patient developed an allergic reaction without evidence of complement activation.

Adolescent↗
PubMed1978

Complement activation in acne vulgaris: in vitro studies with Propionibacterium acnes and Propionibacterium granulosum.

To better define the role of bacteria in inflammatory acne vulgaris, we have investigated the ability of four strains of Propionibacterium acnes and three strains of Propionibacterium granulosum to activate complement. Complement activation was assayed by incubating normal human serum with varying concentrations of each strain and measuring residual total hemolytic complement activity. When serum was tested unaltered, P. acnes strains were approximately threefold more potent than an equal weight of P. granulosum in consuming complement, which could reflect classical and/or alternative pathway activation. All strains also consumed complement in serum chelated with ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, which selectively assays alternative pathway activation. Incubation of unaltered serum with both P. acnes and P. granulosum resulted in immunoelectrophoretic conversion of C4, C3, and factor B of the alternative pathway. Incubation of chelated serum resulted in conversion of C3 and factor B. These data taken together suggest that both species can activate complement through either pathway. Serum incubated with P. acnes was chemotactic for polymorphonuclear leukocytes, and this chemotactic activity was largely C5 dependent as shown by antibody inhibition. It is suggested that complement activation may occur in vivo in acne, and the inflammatory response may be contributed to by the generation of C5-dependent chemotactic factors.

Acne Vulgaris↗
PubMed2002

Transfer of prostasomal CD59 to CD59-deficient red blood cells results in protection against complement-mediated hemolysis.

PROBLEM: Prostasomes isolated from human seminal plasma have complement regulatory properties because of their content of CD59, a glycosylphosphatidylinositol (GPI)-anchored protein. We investigated a functional role of prostasomes by the possibility of transferring CD59 from prostasomes to rabbit erythrocytes (RE) and human erythrocytes obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH), both types of cells lacking CD59. METHOD OF STUDY: We used the assay of hemolytic activity of the alternative pathway of the complement system to compare the liability of the erythrocytes to hemolysis by the complement system with and without pre-incubation with prostasomes. CD59 gained by the RE and PNH erythrocytes was established by flow cytometry. The effect of phosphatidylinositol phospholipase C (PIPLC) on the GPI anchor of prostasomal CD59 and the effect of heat treatment on the prostasomes were also studied. Anti-CD59 antibodies were used to block the protective effect of prostasomes on erythrocytes. RESULTS: Both RE and PNH erythrocytes showed diminished complement-mediated hemolysis after incubation with prostasomes. This was because of the transfer of CD59 from prostasomes to the red blood cells during pre-incubation as evidenced by the hemolytic assay and flow-cytometry. The efficacy of the prostasomes was affected by heat treatment and was totally lost at 100 degrees C. Phosphatidylinositol phospholipase C broke the GPI anchor and released CD59 from prostasomes and the RE surface (after pre-incubation with prostasomes) but not from the human PNH erythrocytes. CONCLUSIONS: A transfer mechanism of CD59 takes place during pre-incubation from prostasomes to erythrocytes lacking CD59 which supports the idea that transfer of prostasomal CD59 can protect cells from lysis elicited by C5b-9. This might be a mechanism by which autologous and allogeneic cells are protected against complement attack in the genital tracts.

Animals↗