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Limited tryptic cleavage of complement factor H abrogates recognition of sialic acid-containing surfaces by the alternative pathway of complement.

The potency of complement factor H (H) in accelerating the decay of the alternative pathway C3 convertase, C3b,Bb (decay-accelerating activity), was used as a measure of the affinity of native versus trypsin-treated H for the complement protein C3b bound to surfaces. When about 99% of H was cleaved at the primary tryptic cleavage site 34 kDa from the N-terminus, its decay-accelerating activity on C3b,Bb on sheep erythrocytes fell about 60-fold, whereas the trypsin-treated H was only 3-4 times less potent than native H in dissociating C3b,Bb on Sepharose 4B. The residual decay-accelerating activity, remaining after the primary cleavage, was not affected by secondary cleavage at a site 120 kDa from the N-terminus, as shown with H preparations cleaved to different degrees. Because cell surface sialic acid is known to be responsible for the high affinity of H for C3b bound on sheep erythrocytes, the results strongly suggest that the integrity of the primary tryptic cleavage site of H is essential for the recognition of sialic acid-containing surfaces by the C3b-H complex.

Animals

Complement activation in acne vulgaris: consumption of complement by comedones.

Comedones, the contents of acne lesions, were shown to consume scomplement hemolytic activity in normal serum. This consumption was stimulated by the addition of serum from patients with inflammatory acne. Absorption of acne serum with Propionibacterium acnes cells removed all stimulating activity. Immunoelectrophoretic analysis of serum incubated with comedones revealed the conversion of C3 and factor B in normal serum. The addition of acne serum resulted in cleavage of C4. In serum treated with ethylene glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, only C3 and factor B were converted. This indicates that comedones may activate complement by either the classical or the alternative pathway. It is suggested that P. acnes cells in comedonal material are responsible for the complement activation.

Acne Vulgaris

Studies on rat complement. II. Complement level in experimental tumor in rats.

In the course of methylcholanthrene induced carcinogenesis in rats, CIA50, C4 and C3 increased as compared with control and correlation of tumor size with increase in CIA50, C4 and C3 was observed. In the course of dimethylaminoazobenzen carcinogenesis of rats, complement level and C3 level decreased but in splenectomized rats fed by dimethylaminoazobenzen showed elevated level of complement system. After Corynebacterium infection, CIA50 and C3 of rats increased. This phenomenon is considered to be one of the essential factors to induce high resistance against tumor inoculation.

Animals

Complement activation and bioincompatibility. The terminal complement complex for evaluation and surface modification with heparin for improvement of biomaterials.

The degree of biocompatibility of biomaterials can be evaluated using various assay systems detecting activation of the blood cascade systems, leukocytes or platelets. Activation of complement is one mechanism associated with adverse effects observed when bioincompatible materials are used. We present data showing that the terminal complement complex, an indicator of terminal pathway activation, is suitable for evaluation of biocompatibility of biomaterials such as cardiopulmonary bypass devices. Furthermore, our results suggest that bioincompatibility is improved when artificial surfaces are modified with end point attached functionally active heparin.

Complement Activation

Opsonin-dependent and independent surface phagocytosis of S. aureus proceeds independently of complement and complement receptors.

We examined the mechanism of surface phagocytosis of Staphylococcus aureus by human polymorphonuclear leucocytes (PMN). Surface phagocytosis of unopsonized bacteria occurred, but was significantly enhanced by the presence of serum. The serum requirement was low, and a maximal effect occurred with serum concentrations of 0.25-0.5%. The opsonic effect of serum was not removed by heat inactivation of complement but was adsorbed, at low serum concentrations, by protein A, indicating that opsonin-dependent surface phagocytosis requires IgG but not C3. The requirement of opsonin-dependent surface phagocytosis for IgG was demonstrated further with purified IgG preparations as the sole opsonin. Activation of PMN by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA) increased opsonin-independent surface phagocytosis by 47% and 66%, respectively, but had no effect on opsonin-dependent surface phagocytosis. Blockade of the PMN iC3b receptor (CR3), which has lectin-like properties, by a panel of monoclonal antibodies against the alpha- and beta-chains of CR3 did not inhibit the surface phagocytosis of opsonized or unopsonized S. aureus, and one antibody (NIMP-R10) enhanced opsonin-independent surface phagocytosis. These results indicate that the mechanism of surface phagocytosis is quite different to that observed in suspension assays. Opsonin-independent surface phagocytosis occurs and is enhanced by PMN activation, opsonin-dependent surface phagocytosis is dependent on IgG and not complement, and neither opsonin-independent nor -dependent surface phagocytosis proceeds through CR3.

Antibodies, Monoclonal

[Complement activation in patients with chronic polyarthritis, measured by the level of complement fraction C3a in plasma].

Using a new C3a-radioimmunoassay, we could show a significantly increased complement activity in patients with active rheumatoid arthritis. The C3a-plasma level in rheumatoid patients was 529 ng/ml, whereas in patients with psoriatic arthropathy it was 164 ng/ml, in patients with noninflammatory muscular pain syndromes 170 ng/ml and in healthy blood donors we found a mean level of 221 ng/ml. The reproducibility was high, therefore it seems that the possibility to calculate the risks of complement-activating immunological disorders can be increased by using the C3a-radioimmunoassay. On the other hand the C3a-increase in rheumatoid patients may be an expression of the immunological activity of the disease.

Arthritis

Complement system in human colostrum: presence of nine complement components and factors of alternative pathway in human colostrum.

Evidence has been obtained for the presence in human colostrum of all nine components of complement (C), C1 through C9, and factors of the alternative pathway. Samples of colostrums collected from five women at 1-4 days after normal parturition were assayed for the haemolytic activities of individual components. As compared with normal human sera, the activities of each component ranged from 0.03 to 7% of those in sera. The activities of C4, C7 and C9 were relatively high, while that of C1 was extremely low. In most of the cases, the activities of individual components gradually increased following delivery, when expressed as the activity per unit weight (g) of protein in the colostrum. When the colostrums were treated with cobra venom factor, most of the colostrums showed 10-20% reduction in the C3 activity. This finding indicates the presence of factors such as B and D which are involved in the activation of C through the alternative pathway. The role as a defense factor of the C system in human colostrum and milk is discussed in connection with the ability of secretory IgA to react with C.

Colostrum

CELISA (complement-enzyme linked immuno sorbent assay) a new method for the estimation of complement fixing antibodies; its use for Chagas' disease.

A description is given of a new method for the estimation of complement fixing antibodies. This is a modification of the Enzyme Linked Immuno Sorbent Assay (ELISA) to a Complement-Enzyme Linked Immuno Sorbent Assay (CILISA) using an anti-C3 conjugate. The method is independent of the haemolytic system. To test the sensitivity of CELISA, comparative studies were carried out with the Haemolytic Complement Fixation Test (CFT), indirect haemagglutination test and ELISA with sera from patients with Chagas' Disease. The results with CELISA showed significant correlations with CFT titers whereas no correlations were observed with the other serological tests.

Antibodies

Regulation of human and murine complement: comparison of 5' structural and functional elements regulating human and murine complement factor B gene expression.

The serine protease complement factor B (Bf), an acute phase plasma protein, is a component of the alternative pathway of complement activation. Previous studies revealed that several cytokines including IFN-gamma and IL-1 are involved in mediating acute phase Bf expression. To determine the molecular details of Bf expression we isolated, sequenced and characterized the 5' flanking regions of the human and murine Bf genes. In both species the Bf transcriptional start site in liver was located less than 400 bp 3' to the polyadenylation site of the upstream C2 gene. This upstream intergenic region contained greater than 65% nucleotide homology between species. Within this region, an IRS and three heat shock consensus elements were found in the murine sequence in an identical position to that of the human. To examine the functional details of Bf expression, a series of mouse and human Bf promoter-chloramphenicol acetyltransferase (CAT) chimeric gene constructs were transfected into mouse L or human HepG2 cells. Analysis of expression of these fusion gene constructs revealed that 1) cis-acting DNA sequences identified, at least in part, in the 3' untranslated region of the C2 gene (within the 400 bp upstream of the Bf cap site) mediate responsiveness to IL-1 and IFN-gamma, 2) the responsiveness to each mediator appears to be conferred by separate upstream regions similar in position and homologous in man and mouse, and 3) the IL-1 responsive region in both species appears to have the characteristics of an enhancer element. The results of this analysis suggest a selective pressure to conserve the intergenic sequence between C2 and Bf genes and that further studies of these sequences will be useful in elucidating mechanisms controlling the acute phase response.

Animals

2-Chloroadenosine inhibits complement-induced reactive oxygen metabolite production and recovery of human polymorphonuclear leucocytes attacked by complement.

Luminol-enhanced chemiluminescence demonstrated that 2-chloroadenosine inhibited complement-induced reactive oxygen metabolite production in human polymorphonuclear leucocytes. This inhibition was reversed by 8-phenyltheophylline. Binding of a [125I]-labelled monoclonal antibody against C9 to human PMN demonstrated removal of membrane attack complexes from the cell membranes, and in addition indicated inhibition of this process by 2-chloroadenosine. This compound also increased the percentage of complement-induced cell lysis of polymorphonuclear leucocytes.

2-Chloroadenosine

A quantitative method for assessing the third complement factor (C3) attached to the surface of opsonized Pseudomonas aeruginosa: interrelationship between C3 fixation, phagocytosis and complement consumption.

A direct enzyme-linked immunoassay is described for the determination of C3 that becomes attached to the surface of bacteria upon incubation in serum. The assay uses horseradish peroxidase-conjugated rabbit antiserum specific for human C3. In this study various Pseudomonas aeruginosa strains, Escherichia coli and Staphylococcus aureus were opsonized under different conditions and the amount of C3 fixed to the bacterial surface was measured directly. The extent of C3 fixation was compared with the percentage of phagocytosis by human polymorphonuclear leukocytes and with the complement consumption in the fluid phase. It was demonstrated that C3 fixation closely correlated with the percentage of phagocytosis but not with complement consumption. The method can be performed with relatively simple equipment and could be used in routine laboratories in order to determine the extent of opsonization of Pseudomonas aeruginosa and other bacteria and to detect opsonic defects in the sera of patients.

Complement Activation

Complement, the immune-complex lattice, and the pathophysiology of complement-deficiency syndromes.

The role of the components of the complement classical pathway in maintaining antigen-antibody complexes in solution has implications for the understanding of the pathophysiology of complement-deficiency syndromes. It is proposed that this mechanism may normally keep immune complexes soluble for a sufficient time for their safe elimination by the mononuclear phagocyte system. When an early component of the classical pathway is deficient or depleted antigen-antibody complexes would be more likely to precipitate at or near their site of formation and lead to immunologically mediated inflammation. This hypothesis is supported by the predisposition to immune-complex diseases of patients with genetically determined deficiencies of components of the classical pathway.

Antigen-Antibody Complex

Complement-dependent cellular cytotoxicity: lymphoblastoid lines that activate complement component 3 (C3) and express C3 receptors have increased sensitivity to lymphocyte-mediated lysis in the presence of fresh human serum.

Lymphocyte-mediated lysis of cells of the Raji, Daudi, Jijoye, and Bjab lines was elevated when fresh human serum was added to the assay. A higher proportion of effector-target conjugates was observed in the presence of human serum. In similar experiments lysis of 1301, Rael, and P3HR-1 cells was unaltered. All cell lines activated the alternative pathway of complement but they varied in the expression of receptors for complement component 3 (C3) and in the ability to fix the C3 cleavage products on their membrane. The enhancement of lysis in the presence of human serum occurred only with those cells that bound C3. This characteristic was correlated to the expression of C3 receptors. Analysis of the nature of the deposited C3 was performed with Raji cells. Raji cells exposed to human serum bound C3b as indicated by the immunoadherence test. The C3b was further processed to C3bi, because the immunoadherence declined with time and conjugate formation increased with Daudi cells, which carry the C3 receptors CR2 and CR3. This suggests that in the lytic assay lymphocytes with C3bi receptors are recruited in the presence of human serum. We assume that the bridge of C3 molecules between targets and effectors increases the avidity of their interaction.

Burkitt Lymphoma

C3bi receptor (complement receptor type 3) recognizes a region of complement protein C3 containing the sequence Arg-Gly-Asp.

Human phagocytes express a receptor termed complement receptor type 3 (CR3) that recognizes the complement protein fragment C3bi. We show here that CR3 recognizes a region of C3 that contains the sequence Arg-Gly-Asp (RGD). CR3 is down-modulated upon spreading of macrophages on surfaces coated with a synthetic 21-residue peptide from C3 (residues 1383-1403). This peptide was also attached to erythrocytes by coupling myristic acid to its amino terminus and allowing the myristoylated peptide to bind to erythrocytes through hydrophobic interactions. Erythrocytes coated with this RGD-containing segment of C3 were bound by macrophages, and binding could be blocked by specific monoclonal antibodies against CR3. Since CR3 recognizes a peptide sequence that contains the RGD triplet, it appears to be a member of a larger family of adhesion-promoting receptors that recognize RGD-containing proteins. However, since CR3 does not recognize a hexapeptide containing RGD, we presume that residues beyond the RGD triplet contribute to binding. We have compared the RGD-containing region of fibronectin and vitronectin, proteins known to be recognized by means of their RGD-containing regions, with those in human and murine C3. A striking homology is observed over an approximately equal to 50 amino acid sequence present in all four proteins. We suggest that this extended region of homology contains a structure recognized by adhesion-promoting receptors.

Amino Acid Sequence

The fixation of complement and the activated first component (C1) of complement by complexes formed between antibody and divalent hapten.

Hapten-antibody complexes prepared at equivalence with the bivalent hapten bis-DNP-octamethylene-diamine and purified rabbit anti-DNP antibody were fractionated by Sepharose gel-filtration and the fractions examined by electron microscopy. Individual fractions were tested for whole-complement fixation and C1 fixation. Dimer forms did not show this type of biological activity, while fractions containing tetramers and larger polymers exhibited both C and C1 fixation, which could be inhibited by prior exposure of the complexes to the univalent hapten epsilon-DNP-caproic acid. The dose-response result indicated that the C-fixation observed was not due to interpolymeric cooperative effects. It was concluded that in the generation of biological activity by soluble antigen-antibody complexes made with complement-fixing antibody, quaternary structural changes following specific combination with antigen may be as important as any tertiary structural alterations that occur in the individual immunoglobulin molecule.

Animals

Macrophage type 3 complement receptors mediate serum-independent binding of Leishmania donovani. Detection of macrophage-derived complement on the parasite surface by immunoelectron microscopy.

In this study, direct visual evidence for local opsonization of L. donovani by macrophage (M phi)-derived complement components was obtained using immunoelectron microscopy. C3 deposition was detected on the surface of both promastigotes and amastigotes after 20 min serum-free incubation with murine resident peritoneal M phi (RPM), followed by fixation and incubation first with specific antibody directed against C3 and then with gold-labelled protein A. Gold deposition was not observed around either form of the parasite if the anti-C3 antibody was omitted. For promastigotes, the degree of C3 deposition under serum-free conditions was comparable with that observed in the presence of an exogenous (serum) source of C3, but did not result in the same severe damage to the parasite as did the latter. Addition of sodium salicyl hydroxamate, which prevents covalent binding of C3 to activator surfaces, abrogated promastigote binding. Hence, although the anti-C3 antibody did not distinguish between native C3 and its breakdown product iC3b, these data support our earlier conclusion that promastigote binding to the CR3 of murine RPM is complement dependent. For amastigotes, gold deposition and binding to murine RPM were not eliminated by sodium salicyl hydroxamate. The presence of normal mouse serum resulted in increased gold deposition, but did not mediate either enhanced binding to M phi or damage to the amastigote. These data suggest that a proportion of C3 binding to the amastigote surface may be via noncovalent linkages, and that the C3 bound may not be in the correct form to mediate binding to CR3.

Animals

Identification of an alternative polyadenylation site in the human C3b/C4b receptor (complement receptor type 1) transcriptional unit and prediction of a secreted form of complement receptor type 1.

The human C3b/C4b receptor or complement receptor type one (CR1) is an approximately 200-kD single chain membrane glycoprotein of human peripheral blood cells that mediates the binding, processing, and transport of C3b-bearing immune complexes and regulates the activity of the complement cascade. Analysis of partial cDNA clones has shown that the COOH terminus is composed predominantly of three tandemly repeated regions of 450 amino acids each (15). In this report, we present a cDNA sequence that encodes the NH2 terminus of CR1. It appears to have been derived from an alternatively processed transcript, caused by polyadenylation occurring at a site within an intron in the CR1 transcriptional unit. The resulting truncated messenger carries an open reading frame that would produce a short, secreted CR1 form. We present genomic sequences and Northern blots which support this hypothesis and we propose that the NH2-terminal end of CR1 is a likely location for active sites. In addition, we report evidence for a CR1-like sequence in the human genome and we present a model for the organization of CR1.

Amino Acid Sequence

Studies on complement-fixation reaction in equine infectious anemia. II. Identification of complement-fixing inhibitors.

The substances responsible for inhibiting complement-fixation (CF) reaction of the late-stage serum of an equine infectious anemia (EIA)-infected horse were investigated. It was found that the IgG and IgG(T) classes in the late-stage serum were responsible for the CF inhibition. IgA could not be detected in partially purified IgG(T) by an immunodiffusion test using rabbit anti-human IgA serum. Other serum components could not be demonstrated in purified IgG by immunoelectrophoresis using rabbit anti-horse serum. The IgG class simultaneously showed CF and CF-inhibiting (CFI) activities, whereas the IgG(T) class showed only CFI activity. The IgG(T) class could exert CFI activity only when it had been reacted with the EIA antigen before addition of the reference CF serum and complement. In contrast, the IgG class converted the CF-active reference serum into a non-CF-reactive one irrespective of whether it was simultaneously reacted with the EIA antigen and the reference CF serum, whether it was added to the reaction mixture of the EIA antigen and the reference CF serum, or whether it was sensitized with the EIA antigen before addition of the reference CF serum. Inhibitory activities of the IgG and IgG(T) classes seemed to be different from each other in their reaction pattern as far as tested under our experimental conditions. Their CFI activities seemed to be specific for EIA, being negative in CFI activity in reaction with other antigens.

Animals