Complement-dependent Reaction Research Articles

Enzymatically-derived oligo-carrageenans interact with α-Gal antibodies and Galectin-3

Carrageenans are linear sulfated galactans synthesized in the Gigartinales, Rhodophyceae species with a varied range of biological properties that are of value to the pharmaceutical and cosmetic sectors. It is unknown how the fine structure of carrageenans dictates their capacity to affect molecular and cellular responses important to wound healing, or the ability to mitigate oxidative, hemostatic and inflammatory processes. Here we use specific endo-carrageenases, from the marine bacterium Zobellia galactanivorans, to produce enzymatically defined neo-series oligosaccharides from carrageenans with 3,6-anhydro-D-galactose on the non-reducing end. Further enzymatic modification of the oligosaccharides was done by treating with the 3,6-anhydro-D-galactosidases from the same bacterium which hydrolyze non-reducing end 3,6-anhydro-D-galactose moieties from neo-carrageenan oligosaccharides. Using the enzymatically produced oligosaccharides, we demonstrate binding to natural human serum antibodies and a monoclonal anti-αGal Ab (m86). The significant interactions with the Galα(1,3)Gal reactive antibodies produced by humans makes them potential potent inducers of complement-dependent reactions and attractive for therapeutic applications. We also demonstrate modulation of the galectin selectivity for the Gal-3 Carbohydrate Recognition Domain (CRD) relative to Gal-1 which has implications to targeting specific biological pathways regulated by the galectins.

B cells masquerade as dendritic cells

In a recent paper, Schriek et al. show that, in a complement-dependent reaction, marginal zone B cells (MZBs) steal MHC class II (MHCII) from dendritic cells (DCs). Activation of T cells by stolen MHCII complexes potentially allows MZBs to augment or regulate T cell activation in ways that are distinct from DCs.

Specific antibodies induce apoptosis in Trypanosoma cruzi epimastigotes

The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported. Mouse immune sera depleted complement-induced damage in epimastigotes characterized by morphological changes and death. The purpose of this work was to study the mechanism of death in epimastigotes exposed to decomplemented mouse immune serum. Epimastigotes were maintained in RPMI medium. Immune sera were prepared in mice by immunization with whole crude epimastigote extracts. Viable epimastigotes were incubated with decomplemented normal or immune sera at 37 degrees C. By electron microscopy, agglutinated parasites showed characteristic patterns of membrane fusion between two or more parasites; this fusion also produced interdigitation of the subpellicular microtubules. Apoptosis was determined by flow cytometry using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and annexin V assays. Nuclear features were examined by 4'-,6-diamidino-2'-phenylindole diHCI cytochemistry that demonstrated apoptotic nuclear condensation. Caspase activity was also measured. TUNEL results showed that parasites incubated with decomplemented immune sera took up 26% of specific fluorescence as compared to 1.3% in parasites incubated with decomplemented normal sera. The Annexin-V-Fluos staining kit revealed that epimastigotes incubated with decomplemented immune sera exposed phosphatidylserine on the external leaflet of the plasma membrane. The incubation of parasites with immune sera showed caspase 3 activity. We conclude that specific antibodies are able to induce agglutination and apoptosis in epimastigotes, although the pathway is not elucidated.

Ultrastructural damage of Trypanosoma cruzi epimastigotes exposed to decomplemented immune sera.

The susceptibility of Trypanosoma cruzi epimastigotes to lysis by normal or immune sera in a complement-dependent reaction has been reported, but the effects induced directly by immune serum depleted of complement remain unstudied. The aim of this work was to study the ultrastructural alterations induced in T. cruzi epimastigotes by immune mouse or rabbit sera with or without complement. A local isolate of T. cruzi (Queretaro) was used in all experiments. Immune sera were raised in both mouse and rabbit by immunization with T. cruzi epimastigote antigens. Light microscopy showed intense agglutination of epimastigotes when incubated with decomplemented mouse or rabbit immune sera. A distinctive ultrastructural feature of this agglutination pattern was the fusion of plasma membranes and a pattern of intercrossing between subpellicular microtubules. Agglutination was associated with fragmentation of nuclear membranes and swelling of cytoplasm, Golgi cisternae, endoplasmic reticulum, mitochondria and kinetoplast membranes. Agglutinated parasites also incorporated trypan blue stain. Results of [3H]-thymidine incorporation confirmed that epimastigotes exposed to specific antibodies in the absence of complement were incapable of proliferating. Ultrastructural changes observed in epimastigote micrographs incubated with decomplemented immune mouse sera were statistically significant (P<0.001) when compared with results obtained from images after incubation with decomplemented normal mouse sera.

Bactericidal effect of a whey protein concentrate with anti-Helicobacter pylori activity.

To investigate the effect of whey protein concentrate (WPC) enriched in anti-Helicobacter pylori antibodies on growth of the organism in vitro. A WPC rich in H. pylori-specific antibodies was produced by immunizing lactating cows against H. pylori and processing pooled bulk milk samples into whey powder. The antibodies bound several proteins within the bacterial homogenate and were active at pH 5. In a complement-dependent reaction, the immune WPC was highly bactericidal against four H. pylori strains tested in vitro. WPC produced with milk from H. pylori-immunized cows contains antibodies that are active at the pH of the stomach, and bactericidal against H. pylori in vitro, via the classical complement pathway. This study has demonstrated the potential for use of WPC in the prevention/treatment of H. pylori infections.

A beneficial role of bile pigments as an endogenous tissue protector: Anti-complement effects of biliverdin and conjugated bilirubin

Bile pigments possess and anti-complement property and could be involved in tissue protection. In this study, we examined the physiological actions of bile pigments, which had been generally regarded as waste catabolites. Biliverdin inhibited complement cascade reaction in vitro, especially at the C1 step in the classical pathway at low micromolar concentrations. Further, Forssman anaphylaxis in guinea pigs, being closely associated with complement reactions, was inhibited by oral or intravenous administration of biliverdin. Conjugated bilirubin also showed an inhibitory effect on complement-dependent reactions in vitro. From These observation, we propose a hypothesis that the pigments serves as endogenous tissue protectors by multiple lines of mechanisms including antioxidant and annti-complement actions.

Introduction: Genetic Deficiencies of Complement Proteins—Consequences and Molecular Basis

The complement system represents one of the major host defense mechanisms and contributes to the regulation of the physiological and pathological immune response. Insight into the organization of this system of soluble and membrane-associated proteins was obtained from numerous immunochemical studies. After the identification of a given protein and purification, its functional characterization in various, often sophisticated in vitro systems was required for the understanding of complement-dependent reactions. These studies revealed the importance of complement proteins or their fragments as both regulatory and effector elements within the immune system: complement has obviously a critical role in defense mechanisms like opsonization or cell lysis; it represents a major mediator of the inflammatory reaction; complement fragments or complexes regulate the functional status of lymphoid and non-lymphoid cell populations and affect thereby directly the course of the immune response; finally, the proper handling of immune complexes is complement-dependent . The corroboration of the in vitro findings by suitable in vivo systems attracted much attention. One experimental approach used depletion in vivo by induction of systemic complement activation, e.g. with cobra venom factor. Complement depletion-albeit transiently-is rapidly achieved, paralleled however by massive generation of biologically highly active complement fragments. These mediators potentially affect the homeostasis of the immune system. A direct correlation between the absence of a complement protein and the impairment of a given biological response or a pathological phenomenon is therefore difficult. For this reason, selective genetic deficiencies of individual complement proteins as experiments of nature provided a valuable model. The impairments observed in affected patients or animals provided crucial evidence for the biological role of a given component and represent a kind of substantiation for the laboratory findings. The relative importance of the protein within the physiological immune response becomes obvious in a deficient individual without the need for prior pretreatment or experimental manipulation. In addition, the contribution of the principal elements of the complement cascade is clearly revealed, e.g. classical vs. alternative pathway or terminal components.

IMMUNOPHARMACOLOGICAL STUDIES OF THE AQUEOUS EXTRACT OF CINNAMOMUM CASSIA (CCAq) I. ANTI-ALLERGIC ACTION

Effect of the aqueous extract of Cinnamomum Cassia (CCAq) on experimental allergic reaction was investigated. Ig E mediated reactions, homologous passive cutaneous anaphylaxis (PCA), degranulation of mast cells, and the release of histamine from sensitized lung tissues classified as the type I reaction by Coombs and Gell were not affected by CCAq. Complement dependent reactions including reversed cutaneous anaphylaxis (RCA), Forssman cutaneous vasculitis (FCV), and nephrotoxic serum (NTS) nephritis classified as type II and the Arthus reaction classified as type III were clearly inhibited by CCAq. However, CCAq did not affect the nephritis caused by the F(ab’)2 portion of the nephrotoxic IgG antibody. CCAq in a high concentration inhibited the immunological hemolysis, chemotactic migration of neutrophils in response to complement activated serum, and the generation of chemotactic factors. The type IV reaction, contact dermatitis, was not affected by CCAq. The production of hemolytic plaque forming cells was slightly inhibited by CCAq. These results suggest that CCAq has an anticomplement action and inhibits the complement dependent allergic reaction.

Immunopharmacological studies of the aqueous extract of Cinnamomum cassia (CCAq). I. Anti-allergic action.

Effect of the aqueous extract of Cinnamomum Cassia (CCAq) on experimental allergic reaction was investigated. IgE mediated reactions, homologous passive cutaneous anaphylaxis (PCA), degranulation of mast cells, and the release of histamine from sensitized lung tissues classified as the type I reaction by Coombs and Gell were not affected by CCAq. Complement dependent reactions including reversed cutaneous anaphylaxis (RCA), Forssman cutaneous vasculitis (FCV), and nephrotoxic serum (NTS) nephritis classified as type II and the Arthus reaction classified as type III were clearly inhibited by CCAq. However, CCAq did not affect the nephritis caused by the F(ab')2 portion of the nephrotoxic IgG antibody. CCAq in a high concentration inhibited the immunological hemolysis, chemotactic migration of neutrophils in response to complement activated serum, and the generation of chemotactic factors. The type IV reaction, contact dermatitis, was not affected by CCAq. The production of hemolytic plaque forming cells was slightly inhibited by CCAq. These results suggest that CCAq has an anticomplement action and inhibits the complement dependent allergic reaction.

Analysis of outer membrane components of Escherichia coli ML308 225 and of a serum-resistant mutant.

Escherichia coli ML308 225 in the early exponential phase of batch cultures was killed by human serum in a complement-dependent reaction. Under the same conditions, a mutant derived from this strain by multiple subcultures in human serum was resistant to killing by serum. The components of the outer membrane of both the parent and mutant strains were analyzed. The protein and phospholipid analyses were identical, but the mutant strain produced twice as much lipopolysaccharide as the parent strain. These results suggest that the extent of bacterial surface coverage by lipopolysaccharide determines the degree of sensitivity to serum.

Complement-dependence of platelet serotonin release test in polytransfused patients.

The research of platelet isoantibodies in patients with Cooley's anaemia was performed by simultaneous determination of the platelet-complement fixation test, platelet factor 3 availability assay and 14C-serotonin release test. In 93% of the examined patients we obtained positive results with the 5HT-release test, which appeared to be a complement-dependent reaction in most of the sera-containing isoantibodies, different from sera of patients affected by autoimmune thrombocytopenia.

Some approaches to mapping the redistribution of parent-cell H-2 antigens on the membrane of cell hybrids

Cell hybrids were created by Sendai virus-induced fusion of A 9 (H- 2 k) cells and peritoneal exudate cells (presumably macrophages, H- 2 d), and the distribution of parent-derived H- 2 specificities was followed using two indirect methods. Competition within pairs of non-crossreacting H- 2 specificities for the attachment of specific antibodies was measured by the blocking index. In cell hybrids, no blocking was detectable within the pairs H- 2·4–H- 2·11 and H- 2·4–H- 2·5 where the members of each pair were derived from different parents. When, however, both specificities of a pair (H- 2·5, H- 2·11) were from the same parent, their blocking relationship was similar as in the particular parent. In different experiments and with different types of cells, the presence of blocking between two H- 2 specificities was found to be confined to the “double cis” configuration of their genetic determinants. No blocking was found in “single trans”, “single cis”, and “double trans” configurations. The difference in blocking could not be simply traced back to a different cell surface density of the antigens under test. Trans specificities that do not compete (H- 2·4, H- 2·11) were shown to cooperate in a complement-dependent reaction with a specific antibody. The competition clearly indicates an intermixing of parent derived H- 2 antigens on the cell hybrid membrane. The possible bearing of the data on the understanding of the control of the specificity of the cell-surface architecture is discussed.

Role of Fumaropimaric Acid in Guinea Pig Complement Dependent and Non-Complement Dependent Biologic Reactions

Summary Fumaropimaric acid, one of a newly discovered group of potent, complement inhibitors has been shown to block the hemolytic activity of undiluted guinea pig serum when added in vitro, and after intravenous injection into guinea pigs. It also dissociates the activated trimolecular C′(5,6,7)a complex, preventing its chemotactic activity. When injected intravenously in doses of 600 mg/kg or less, fumaropimaric acid suppresses the complement dependent, systemic Forssman, cutaneous Forssman and reversed passive Arthus reactions. In the last reaction it was shown that complete inhibition could occur despite no interference with the formation of tissue-bound antigen-antibody complexes or the fixation of C′3 in vessel walls. Although the lysis of sensitized erythrocytes incubated with serum from guinea pigs injected with the drug was inhibited, this did not prevent the complement reaction from proceeding to and possibly through the EAC′la (4,2)a step. Thus, the ability of C′3 to be bound in sites of the Arthus reaction inhibited by fumaropimaric acid might indicate a block in the in vivo reaction beyond the C′3 step. It is not clear, however, whether the suppression of any of these complement dependent reactions is due to the effect of fumaropimaric acid on the complement system or on some other function(s) necessary for the expression of these responses. The PCA reaction in guinea pigs was markedly but incompletely inhibited by fumaropimaric acid. No difference was noted in the effects of the drug on the homocytotropic reaction caused by non-complement-fixing guinea pig antibodies or the heterocytotropic reaction caused by complement-fixing rabbit antibodies. In view of the evidence that guinea pig PCA given by these antibodies is not complement dependent, it is unlikely that fumaropimaric acid exerts its partial suppressive effect on the PCA by interferring with complement. Evidence is cited that makes it unlikely that the demonstrated direct antihistaminic effect of fumaropimaric acid is responsible for the suppression of the PCA.

Bactericidal method for the measurement in normal serum of antibody to gramnegative bacteria.

Landy, Maurice (National Cancer Institute, Bethesda, Md.), J. Gabriel Michael, and James L. Whitby. Bactericidal method for the measurement in normal serum of antibody to gram-negative bacteria. J. Bacteriol. 83:631-640. 1962.-The bactericidal effect of normal serum on gram-negative bacteria was investigated with the objective of determining whether this antibody-complement reaction could be used for the measurement of antibody. The major variables affecting the outcome of this complement-dependent reaction were explored systematically to establish the test conditions in which specific antibody was the limiting factor. Among these were the use of an excess of absorbed human serum as a complement source, inoculum, reaction conditions, and precise enumeration of surviving bacteria. It was demonstrated that the test conditions could be controlled so as to provide a sensitive and objective assay for antibody to representative strains of various gram-negative genera.

The effect of nasal secretions upon human serum.

The discovery of the resistance-lowering properties of hog gastric mucin (Nungester et al, 1932) has been followed by a number of studies on the properties of mucins lowering host resistance. Although the great majority of these studies have dealt with the production of infection in experimental animals, a number of workers have reported the alteration of the antibacterial properties of the blood following treatment with hog gastric mucin (Nungester et al, 1936; Keefer and Spink, 1938; Oerskov, 1940; Fothergill et al, 1937; Engley, 1954a and b). Although the mechanisms of the antibacterial effects of blood have not been fully described, the importance of serum complement has repeatedly been emphasized particularly as affecting the gram-negative organisms. An important antibacterial phenomenon which involves complement has recently been described by Pillemer (1954), who has reported the isolation of an antibacterial serum protein, properdin, which acts upon susceptible organisms in a complement-dependent reaction. The anticomplementary activity of hog gastric mucin has been reported by Lambert and Richley (1952). Also, the characterization of one of the active