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A Erdei

Publications and source records attributed to A Erdei.

At least 55 records · Page 3Linked to original sources
PubMed1988

Two populations of complement factor H differ in their ability to bind to cell surfaces.

Using hydrophobic affinity chromatography on phenyl-Sepharose, human complement factor H can be separated into two subpopulations, phi 1 and phi 2. Although phi 1 and phi 2 are known to differ in their aggregation properties under non-physiological low ionic strength conditions, no difference in aggregation state was detected under the conditions used for cell-binding experiments. We have investigated these two subpopulations further to determine whether functional differences exist between them. The subpopulation phi 2 was found to bind specifically and saturably to the surface of Raji cells. The binding of the other subpopulation, phi 1, was low, and essentially non-specific. A monoclonal anti-factor H antibody, BGH-1, was raised which recognizes preferentially the phi 2 subpopulation and inhibits the binding of factor H to cell surfaces.

Antibodies, Monoclonal↗
PubMed1988

Reversible biotinylation of C1q with a cleavable biotinyl derivative. Application in C1q receptor (C1qR) purification.

Reversible biotinylation of human C1q without impairment of its physiologic functions has allowed us to develop a simple and rapid purification method for C1q receptor (C1qR). The biotinylating reagent, NHS-SS-biotin (Mr 606.7) contains an extended connector or cross-linker arm which limits steric hindrance and is bridged by a cleavable disulfide bond to the biotin component. Biotinylation was achieved by mixing C1q (in PBS, pH 7.4) with NHS-SS-biotin (dissolved in dimethyl formamide) in a 50:1 v/v and 1:25 mol/mol ratio and allowing the reaction to continue at room temperature for 4 h. The mixture was then dialyzed against PBS pH 7.4 (2 X 1 liter) and analyzed by SDS-PAGE and hemolytic assay using C1q depleted serum. Under these conditions neither denaturation of the protein nor loss of hemolytic activity was evident. Such biotinylated C1q (Bio-C1q) was used to pull out the C1qR from detergent-solubilized (1% NP-40 in PBS, pH 7.4 plus inhibitors) 125I-surface labeled membrane solution that had been first centrifuged (1 h, 45,000 X g, 4 degrees C) and then sequentially precleared with immobilized protein A, protein A-IgG and gelatin. The mixture of Bio-C1q and membrane solution was then incubated (20 h, 4 degrees C), applied to immobilized avidin (equilibrated with PBS, pH 7.4, 0.1% NP-40) and after washing, the bound C1qR was eluted with equilibrating buffer containing 1 M NaCl, and the C1q by same buffer containing 100 mM DTT. The eluted C1qR contained a major Mr 70,000 molecule which upon reduction electrophoresed with an apparent Mr of 85,000-90,000 as assessed by SDS-PAGE analysis. In addition, a faint single chain band of 30-40 kDa was eluted with the major band and may represent a non-covalently associated part of the C1qR molecule.

Avidin↗
PubMed1988

Appearance of acceptor-bound C3b on HLA-DR positive macrophages and on stimulated U937 cells; inhibition of Fc gamma-receptors by the covalently fixed C3 fragments.

The appearance and the functional role of acceptor-bound C3b during differentiation of human monocytes into macrophages were studied. Acceptor-bound C3b could be detected by the immune adherence (IA) test parallel to the expression of antigenic determinants specific to mature cells--i.e. on days 4-5 of culture. Consequently, the capacity of these phagocytes to fix C3b covalently via C3b-acceptors (C3bAs) can be considered as one of the signs of their activation/differentiation. All the mature macrophages positive in the IA test were also found to express HLA-DR antigens on their membrane. Using solubilized extracts of stimulated, 35S-cysteine-labelled cells of the human monocytic cell line, U937, we demonstrate that C3 synthesized by these cells can bind to C3bAs of the same cells. Covalently fixed C3 fragments were found to inhibit Fc gamma-receptor-mediated ingestion of immune complexes and also antibody-dependent cellular cytotoxicity of monocyte-derived macrophages.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1988

The C1q receptor.

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Amino Acids↗
PubMed1988

Shedding of Fc receptor from mononuclear cells and their ability of binding IgG1 and IgG3 anti-Rh/D antibodies.

Fc receptors for IgG1 and IgG3 on peripheral blood lymphocytes and monocytes were studied before and after temperature shift from 4-37 degrees C. The investigations were performed in the EA test using human erythrocytes sensitized with anti-Rh/D/antibodies of IgG1 (EA IgG1) and IgG3 (EA IgG3) subclasses. It occurred that lymphocytes and monocytes were able to bind IgG1 and IgG3 antibodies before and after shedding, however, lower percentage of rosette was observed after temperature shift. This decrease was similar in the EAIgG1 and EAIgG3 tests. The supernatants obtained during shedding occurred to contain active Fc receptors since the inhibition of rosette formation was obtained after the incubation of sensitized erythrocytes with these supernatants. IgG1 as well as IgG3 myeloma proteins inhibited rosette formation in both EAIgG1 and EAIgG3 tests. Our data might suggest that IgG1 and IgG3 anti-D antibodies are able to bind to the same Fc receptor on lymphocytes as well as on monocytes.

Antigen-Antibody Reactions↗
PubMed1987

Complement factor H-binding protein of Raji cells and tonsil B lymphocytes.

Several reports have indicated that Factor H has specific effects on certain cell populations, suggesting that Factor H receptors may exist. Lambris & Ross [(1982) J. Exp. Med. 155, 1400-1411] purified a protein from Raji B-lymphoblastoid cell culture supernatants, using Factor H-Sepharose affinity chromatography. This species appeared to consist of two disulphide-linked components each of Mr 50,000, with an additional 50,000-Mr chain attached non-covalently. The existence of cell-surface Factor H-binding proteins has now been re-investigated with 125I surface-labelled Raji and tonsil B cells. Non-ionic-detergent extracts of the cells, in 0.1% Nonidet P40/10 mM-sodium phosphate buffer, pH 7.4, were incubated with Factor H-Sepharose in the presence of proteinase inhibitors. After the beads had been washed, bound components were eluted with 50 mM-NaCl. A single radioactive species was eluted from the resin, which migrates identically with Factor H (apparent Mr 170,000) in SDS/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions. Biosynthetic radiolabelling studies confirmed that this species was synthesized by Raji cells. Examination of culture supernatants from biosynthetically radiolabelled Raji cells showed again the presence of a single soluble species that bound to Factor H-Sepharose, but this species was of lower Mr (approx. 105,000) than the membrane-derived protein. The soluble form may be produced by proteolysis of the membrane form, or may be of separate origin. The similarity in size of the cell-surface protein to Factor H was initially confusing, but it is distinct from cell-surface Factor H on the basis of three criteria: (1) it is not recognized by anti-(Factor H) monoclonal antibodies MRC OX23 and MRC OX24, nor by polyclonal F(ab')2 anti-(Factor H); (2) it does not bind to Zn2+-chelate resin, whereas Factor H does; (3) cell-surface Factor H present on U937 cells does not bind to Factor H-Sepharose.

B-Lymphocytes↗
PubMed1987

Structure and specificity of complement receptors.

Fifteen to 16 cell surface proteins which interact with soluble components of the complement system have now been identified. Most of these--CR1, CR2, CR3, "CR4", DAF, HSV-1 c glycoprotein, Gp 45-70, p150,95, cell-surface Factor H, and a 90 kD protein--interact with C3 or C4 and their degradation products. Other receptors for C1q, Factor H, C5a, and the C5b-9 complex have been identified. Receptors for additional complement proteins such as Factor B or its fragments are likely to exist. Complement receptors have a wide tissue distribution and have major roles in controlling the turnover of the complement system, and regulating behaviour and growth of leukocytes.

Animals↗
PubMed1987

Involvement of complement in B-cell, T-cell and monocyte/macrophage activation.

In the early 70's it had been shown, that for the immune response against T-dependent antigens C3 was necessary, while T-independent antigens, although activating the alternative pathway of complement, triggered antibody formation also in C-deficient mice. During recent years functional and biochemical knowledge about complement binding structures on B-cells and monocytes/macrophages continuously increased and, also, on T-cells C3 binding entities have been detected. In the case of B-cells and, at least in special experimental conditions, in the case of T-cells C3 can exert a proliferative response as long as the cells are prestimulated (excited) by anti-Ig or IL-2, respectively. Monocytes can bind C3b- or iC3b-carrying particles, but only when progressed to macrophages can they phagocytose such particles. Thus the concept evolves that B-cells, T-cells and monocytes can acquire competence for a C3-driven response when excited properly. The involvement of molecules such as CR1, CR2, factor H, IL-2-receptor and others with a basic structure of repeating units of 61 amino acids in the triggering processes is a surprising finding and certainly suggests their functional importance. In the case of T-independent antigens the structures triggering the alternative pathway of complement are the structures triggering monocytes directly. Whether these two functions have a causal relationship has to be shown.

Animals↗
PubMed1987

Effector or target cell selection mediated by C3 bridges.

Potential effector cells (including stimulated lymphocytes and cultured monocytes) and potential target cells of NK and AK type cytotoxic reactions (including several lymphoblastoid cell lines) cleave the third complement component (C3). As a result of expression of C3bA sites such cells are able to bind covalently the activated C3b through its metastable binding site and thereby become "armed" by the C3b. This permits C3b-bridge formation between these cells and CR1-bearing cells. The "effector selection" (i.e. when C3b is bound covalently to potential target cells) or "target selection" (when C3b is covalently bound to C3bA sites on potential effector cells) mediated by C3b bridges results in enhanced killing capacity. Macrophages activate and bind C3b as well; but the covalent binding of C3b by these cells inhibits Fc receptor mediated ADCC type killing.

Animals↗
PubMed1986

Cell cycle control of activated, synchronized murine B lymphocytes--roles of macrophages and complement C3.

Three restriction points control the cell cycle of activated murine B lymphocytes in a synergistic way. The first is controlled by the occupancy of surface immunoglobulin either by antigen- or by immunoglobulin-specific antibodies. The second is controlled by the complement C3d receptor CR2 which can be occupied by cross-linked C3b or C3d to stimulate the entry into S phase, or by soluble C3d or a C3 alpha-chain peptide, binding to the CR2 receptor, which inhibit the entry into S phase. Macrophages produce so-called alpha factors which also control the B-cell cycle at the same point. Thus, it is suspected that macrophages produce components of the early pathway of complement activation which finally lead to cross-linking of CR2 receptors on B cells. The third restriction point is controlled by unknown receptors that recognize so-called beta factors produced by helper T lymphocytes.

Animals↗
PubMed1986

[Comparative studies of Fc receptors for IgG on resting and activated mouse T lymphocytes using different methods].

Fc-receptors for IgG (Fc gamma R) on resting (i.e. freshly prepared) and mitogen (Con A) or alloantigen-activated mouse spleen T cells were compared using binding of different markers such as 125J-labelled immune complexes, 125J-labelled anti Fc gamma R monoclonal antibody, FITC-labelled aggr. IgG and sheep erythrocytes covered with specific antibody (EA rosetting). C3b receptors were detected by rosetting with sheep erythrocytes covered with antibody and complement (EAC rosetting). The electrophoretic mobility of the cells without or after binding of aggr. IgG was also tested. A number of differences between resting and activated T cells were found: After activation of T cells by mitogen or alloantigen, a proportion of Fc gamma R-positive cells increased two to four times. Fc gamma R number per Fc gamma R-positive cell seemed to be higher on activated then on resting cells. Fc gamma R-positive resting cells did not shed their Fc gamma R upon incubation at 4 degrees C followed by incubation at 37 degrees C, but Fc gamma R-positive activated cells shed a remarkable proportion of their Fc gamma R on the same conditions. Binding of aggr. IgG caused a decrease of electrophoretic mobility of activated but not resting cells. Fc gamma R-positive resting cells were also C3b receptor-positive, whereas Fc gamma R-positive activated cells had no detectable C3b receptors.

Animals↗
PubMed1985

The action of human C3 in soluble or cross-linked form with resting and activated murine B lymphocytes.

The third component of complement C3 has been implied in the stimulation of B lymphocytes to proliferation and maturation for Ig secretion. We have reinvestigated the extent of this activation with either activated or resting murine splenic lymphocytes in serum-substituted cultures. Human C3 was used in either soluble or cross-linked form. Soluble as well as Sepharose-bound or glutaraldehyde-cross-linked C3, over a range of concentrations, was inactive with resting splenic lymphocytes of (C57BL/6J X DBA/2)F1, C3H/HeJ and C57BL/6J nu/nu mice. However, lipopolysaccharide-activated spleen cells, enriched for B cell blasts, were stimulated by immobilized and cross-linked C3, while they did not respond to soluble C3. The extent of restimulation was comparable to that induced by lipopolysaccharide and resulted in both increased proliferation and maturation to Ig-secreting cells. The stimulation of the blast cells appears to be C3 specific, since it can be inhibited by free C3.

Animals↗
PubMed1985

Functional cooperation of C3b-acceptors, Fc gamma-receptors and cell-surface proteases on macrophages.

Macrophages are FcR-positive cells, synthetize complement components and express proteolytic enzymes on their surface. In this paper a functional cooperation of C3b acceptor (C3bA) sites, which bind covalently nascent C3b molecules via their metastable binding site, IgG FcRs and cell surface proteases are described and the possible importance of this cooperation in regulation of immune response is discussed. It was found that isolated monocytes did not express C3bA in contrast to cultured macrophages which showed immune adherence positivity. Stimulation of macrophages resulted in enhanced expression of C3bA. C3 synthetized by macrophages was shown to be cleaved by cellular proteases which resulted in the binding of nascent C3b to C3bA. C3bA-nascent C3b interaction inhibited FcR-dependent effector functions, such as immune complex phagocytosis and antibody-dependent cellular cytotoxicity.

Animals↗
PubMed1985

A possible self-regulating mechanism mediated by C3b-acceptor-bound C3b generated by stimulated macrophages.

Macrophages have been shown to produce C3 and to bear Fc receptors (FcR), and besides the various C3 receptors, they possess C3b acceptors (C3bA) as well as surface proteases capable of cleaving C3. Using the immune adherence method, we demonstrated that the amount of covalently fixed (i.e., C3bA-bound) C3b is markedly increased upon cell stimulation by phorbol myristate acetate or aggregated IgG, even in the absence of C3. The enhancement of nascent C3b (C3bx) binding to C3bA on these cells could be reversed by inhibiting the process at different stages, using either cycloheximide, phenyl-methyl-sulphonyl-fluoride, salycil hydroxamic acid, or methylamine. On the basis of our present results and earlier results, we propose a self-regulatory mechanism by which activated, C3-producing macrophages cleave C3 by their surface proteases. C3bx generated in this way fixes covalently to C3bA of the producer cells, resulting in the inhibition of FcR on these cells.

Animals↗
PubMed1985

Antisera to gamma-aminobutyric acid. II. Immunocytochemical application to the central nervous system.

An antiserum to gamma-aminobutyric acid (GABA) was tested for the localization of GABAergic neurons in the central nervous system using the unlabeled antibody enzyme method under pre- and postembedding conditions. GABA immunostaining was compared with glutamate decarboxylase (GAD) immunoreactivity in the cerebellar cortex and in normal and colchicine-injected neocortex and hippocampus of cat. The types, distribution, and proportion of neurons and nerve terminals stained with either sera showed good agreement in all areas. Colchicine treatment had little effect on the density of GABA-immunoreactive cells but increased the number of GAD-positive cells to the level of GABA-positive neurons in normal tissue. GABA immunoreactivity was abolished by solid phase adsorption to GABA and it was attenuated by adsorption to beta-alanine or gamma-amino-beta-hydroxybutyric acid, but without selective loss of immunostaining. Reactivity was not affected by adsorption to glutamate, aspartate, taurine, glycine, cholecystokinin, or bovine serum albumin. The concentration (0.05-2.5%) of glutaraldehyde in the fixative was not critical. The antiserum allows the demonstration of immunoreactive GABA in neurons containing other neuroactive substances; cholecystokinin and GABA immunoreactivities have been shown in the same neurons of the hippocampus. In conclusion, antisera to GABA are good markers for the localization of GABAergic neuronal circuits.

Adsorption↗
PubMed1985

Antisera to gamma-aminobutyric acid. I. Production and characterization using a new model system.

Antisera to the amino acid gamma-aminobutyric acid (GABA) have been developed with the aim of immunohistochemical visualization of neurons that use it as a neurotransmitter. GABA bound to bovine serum albumin was the immunogen. The reactivities of the sera to GABA and a variety of structurally related compounds were tested by coupling these compounds to nitrocellulose paper activated with polylysine and glutaraldehyde and incubating the paper with the unlabeled antibody enzyme method, thus simulating immunohistochemistry of tissue sections. The antisera did not react with L-glutamate, L-aspartate, D-aspartate, glycine, taurine, L-glutamine, L-lysine, L-threonine, L-alanine, alpha-aminobutyrate, beta-aminobutyrate, putrescine, or delta-aminolevulinate. There was cross-reaction with gamma-amino-beta-hydroxybutyrate, 1-10%, and the homologues of GABA: beta-alanine, 1-10%, delta-aminovalerate, approximately 10%, and epsilon-amino-caproate, approximately 10%. The antisera reacted slightly with the dipeptide gamma-aminobutyrylleucine, but not carnosine or homocarnosine. Immunostaining of GABA was completely abolished by adsorption of the sera to GABA coupled to polyacrylamide beads by glutaraldehyde. The immunohistochemical model is simple, amino acids and peptides are bound in the same way as in aldehyde-fixed tissue and, in contrast to radioimmunoassay, it uses an immunohistochemical detection system. This method has enabled us to define the high specificity of anti-GABA sera and to use them in some novel ways. The model should prove useful in assessing the specificity of other antisera.

Adsorption↗