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Biomedical subjects

S Bhakdi

Publications and source records attributed to S Bhakdi.

At least 199 records · Page 11Linked to original sources
PubMed1985

Minimal requirements for exocytosis. A study using PC 12 cells permeabilized with staphylococcal alpha-toxin.

The membrane-permeabilizing effects of streptolysin O, staphylococcal alpha-toxin, and digitonin on cultured rat pheochromocytoma cells were studied. All three agents perturbed the plasma membrane, causing release of intracellular 86Rb+ and uptake of trypan blue. In addition, streptolysin O and digitonin also damaged the membranes of secretory vesicles, including a parallel release of dopamine. In contrast, the effects of alpha-toxin appeared to be strictly confined to the plasma membrane, and no dopamine release was observed with this agent. The exocytotic machinery, however, remained intact and could be triggered by subsequent introduction of micromolar concentrations of Ca2+ into the medium. Dopamine release was entirely Ca2+ specific and occurred independent of the presence or absence of other cations or anions including K+ glutamate, K+ acetate, or Na+ chloride. Ca2+-induced exocytosis did not require the presence of Mg2+-ATP in the medium. The process was insensitive to pH alterations in the range pH 6.6-7.2, and appeared optimal at an osmolarity of 300 mosm/kg. Toxin permeabilization seems to be an excellent method for studying the minimal requirements for exocytosis.

Adrenal Gland Neoplasms↗
PubMed1985

Alpha-toxin permeabilized rat pheochromocytoma cells: a new approach to investigate stimulus-secretion coupling.

The channel forming alpha-toxin of Staphylococcus aureus (about 50 micrograms/ml) markedly reduces the Ca2+ requirement for dopamine release by the rat pheochromocytoma cell line (PC 12). Maximal secretion by intact cells requires approximately 1 mM Ca2+, whereas release by alpha-toxin-permeabilized cells can already be triggered with microM concentrations of Ca2+. The latter process reaches a plateau at about 1 microM free Ca2+ and increases again with 10-20 microM free Ca2+. The sensitivity to low concentrations of Ca2+ indicates that the toxin, as a selective cell membrane permeabilizing agent, can be used as a powerful instrument to study stimulus-secretion coupling.

Adrenal Gland Neoplasms↗
PubMed1985

Electroimmunoassay-immunoblotting (EIA-IB) for the utilization of monoclonal antibodies in quantitative immunoelectrophoresis: the method and its applications.

Immunoprecipitates formed in conventional electroimmunoassays were solubilized by brief immersion of the agarose gels in sodium dodecylsulfate. The proteins were then transferred to nitrocellulose sheets by electroblotting. The blotted proteins were readily amenable to analyses by non-precipitating monoclonal antibodies and the immunoblots were developed with second antibody/biotin-streptavidin-peroxidase staining. The electroimmunoassay-immunoblot (EIA-IB) method is of value in (1) specificity assays of monoclonal antibodies in crossed immunoelectrophoresis; (2) analysis of specific molecular interactions between proteins; (3) rapid screening and simple identification of monoclonal antibodies by line immunoelectrophoresis-immunoblotting.

Animals↗
PubMed1985

Interaction of complement S-protein with thrombin-antithrombin complexes: a role for the S-protein in haemostasis.

Complement S-protein is known to function as an inhibitor of the terminal complement sequence (Bhakdi, S. and Tranum-Jensen, J., Biochim. Biophys. Acta 737, 343, 1983). We here report that the S-protein may also play a functional role in haemostasis. Electro-immunoassays performed with the use of poly- and monoclonal antibodies to the protein revealed that it binds to thrombin-anti-thrombin III (T X AT-III) to form stable S X T X AT-III complexes. These complexes form naturally during blood coagulation. They have been identified in human serum and have also been generated in vitro with the respective purified proteins. Formation of the complexes is paralleled by a net protection of thrombin towards the inactivating effects of AT-III, demonstrable in functional assays using a synthetic polypeptide or fibrinogen as substrates for the protease. S-protein may thus exert a promoting effect on blood coagulation and could emerge as a novel component of the blood coagulation system in the future.

Animals↗
PubMed1985

Monoclonal antibodies to human plasma protein X alias complement S-protein.

Protein X alias complement S-protein was isolated by dissociation from purified XC5b-9 (fluid-phase terminal C5b-9) complexes with 250 mM deoxycholate and subsequent sucrose density gradient centrifugation and Sephacryl gel chromatography. Polyclonal rabbit and monoclonal mouse antibodies were used to preliminarily characterize the protein in human serum and plasma. In plasma, Protein X yielded a symmetrical immunoprecipitate of alpha 2-mobility in a crossed immunoelectrophoresis assay. However, a second immunoprecipitate of alpha 1-mobility was observed when serum was analysed; this precipitate represented Protein X in complex with antithrombin-III. The co-precipitation of Protein X with serum antithrombin-III was exploited for establishing a simple screening test for unequivocal identification of monoclonal anti-Protein X antibodies. SDS-PAGE immunoblotting with monoclonal antibodies showed that Protein X exhibits pronounced microheterogeneity, migrating as a diffuse moiety of approx. Mr 80-90 000. Additionally, a small amount of polymeric aggregates appear to be present in plasma. Reduction of disulfide bonds led to liberation of a polypeptide of approx. 15 K as discerned by two-dimensional SDS-PAGE immunoblotting. Protein X is not cleaved to lower molecular weight entities during the process of blood coagulation or during formation of fluid-phase terminal complement complexes. The plasma concentrations in healthy adults were in the range of 500-700 micrograms/ml. The availability of methods for isolating Protein X and raising monoclonal antibodies will facilitate further studies on the dual role of this protein in the terminal complement and coagulation cascades.

Animals↗
PubMed1985

Monoclonal antibodies against neoantigens of the terminal C5b-9 complex of human complement.

Assembly of the terminal C5b-C9 complement components into the cytolytic C5b-9 complex is accompanied by exposure of characteristic neoantigens on the macromolecule. We report the production and characterization of mouse monoclonal antibodies to C9-dependent neoantigens of human C5b-9. Binding-inhibition assays with EDTA-human plasma and micro-ELISA assays with purified C9 showed that the antibodies did not react with native complement components and thus confirmed the specificity of the antibodies for the neoantigens. The monoclonal antibodies did, however, cross-react with cytolytically inactive, fluid-phase C5b-9 complexes. Thus, expression of the neoantigenic determinants was not dependent on the formation of high molecular weight C9 polymers with the complex, since these are absent in fluid-phase C5b-9. Radioiodinated antibodies could be utilized in immunoradiometric assays for the detection and quantitation of C5b-9 on cell membranes. Cross-reactivities of the antibodies with C9-dependent neoantigens of several other animal species were examined and antibody clones cross-reacting with rabbit (clones 3B1, 3D8, and 2F3), sheep (clones 3D8 and 2F3) and guinea-pig (clone 3D8) neoantigens were identified. Three of four tested clones (3D8, 2F3, 1A12) precipitated C5b-9 complexes in double-diffusion assays, probably due to their interaction with multiple and repeating C9-epitopes on the terminal complexes. The monoclonal antibodies will be of value for definitive identification and quantitation of C5b-9 on cell membranes and in tissues, and for establishing immunoassays for detection and quantitation of terminal fluid-phase C5b-9 complexes in plasma.

Animals↗
PubMed1985

Positive camp-phenomenon elicited by coagulase-negative staphylococci.

Fifty out of sixty-five isolates of coagulase-negative staphylococci displayed a positive CAMP-reaction due to synergism of an extracellular toxin with S. aureus beta-toxin on sheep erythrocytes. Of the CAMP-positive isolates, 40 were identified as S. epidermidis, 8 as S. hemolyticus, and 2 each as S. hominis and S. saprophyticus. CAMP-positivity did not appear to be an indication of pathogenicity, and the incidence of CAMP-positive strains derived from wound infections did not differ from that found in clinically insignificant isolates. In five cases of mixed wound infection with S. aureus and a CAMP-positive S. epidermidis, no synergism between the toxins of the respective, paired isolates could be demonstrated since the S. aureus strains in these cases did not produce beta-toxin. The results show that most coagulase-negative staphylococci produce one or more toxins, possibly delta-toxin, detectable by the CAMP-reaction. The biological significance of toxin production remains to be elucidated.

Bacterial Toxins↗
PubMed1985

Possible involvement of terminal complement complex in active Heymann nephritis.

We investigated whether the appearance of various complement components in renal deposits of immune complexes correlated with the development of proteinuria in rats with active Heymann nephritis. Sequential kidney biopsy specimens and serum samples were obtained from Lewis rats immunized with Fx1A in complete Freund's adjuvant. Circulating antibodies against purified auto-antigen renal tubular epithelial glycoprotein, as measured by ELISA, were found in the circulation together with a diffuse granular deposition of IgG1, IgG2a, and IgG2b in the glomeruli within 2 weeks after immunization. Biopsy specimens taken 4 weeks after immunization showed diffuse deposition of C4 and C3, which indicated that activation of complement by the classical pathway had occurred. The detection of the C5b-9 complex of complement in glomerular deposits coincided with the development of abnormal proteinuria indicating that the glomerular damage in this autoimmune disease may be caused by complement-mediated lesions in the glomerular capillary walls.

Animals↗
PubMed1985

A two-stage immunoradiometric assay with 125I-staphylococcal protein A for the detection of antibodies and complement on human blood cells.

A sensitive two-stage immunoradiometric assay (IRMA) using unlabeled specific antiglobulin sera followed by binding of radiolabeled staphylococcal protein A has been developed for detection of human IgG, IgM, IgA and complement C3 on human blood cells. In an experimental model system, purified human immunoglobulins (Ig) were coupled onto red blood cells, platelets, and leukocytes. The Ig-coated red blood cells were analyzed in parallel by three methods: the radioimmune antiglobulin test with radiolabeled anti-Ig; the radiolabeled staphyloccal protein A test, and the IRMA. Of the three methods evaluated, the latter was found to be sensitive and the easiest to perform. The applicability of the IRMA was established by investigating a group of 14 selected patients with autoimmune warm type hemolytic anemia. We observed that the IRMA detected cell-bound antibodies (IgG and/or IgM) in several cases where conventional assays yielded negative results.

Antibodies↗
PubMed1985

Decomplementation antigen, a possible determinant of staphylococcal pathogenicity.

We report the existence of an extracellular staphylococcal product, designated staphylococcal decomplementation antigen (DA), that causes rapid consumption of early-reacting complement components up to and including C5 in human serum. Complement activation occurs as a consequence of immune complex formation between DA and specific human immunoglobulin G antibodies and proceeds primarily via the classical pathway. The terminal components C7, C8, and C9 are not consumed during the process. Levels of DA production do not correlate with the expression of classical pathogenic factors, such as coagulase, clumping factor, protein A, or alpha-toxin. DA is a nondialyzable macromolecule eluting in a molecular-weight region of 70,000 to 120,000 on Sephacryl S-300 and displaying an apparent sedimentation coefficient of 3 to 4 S on sucrose density gradients. The molecule is remarkably stable and resists destruction upon boiling for 30 min or by treatment with pronase, lysostaphin, DNase, or RNase. We anticipate that DA protects staphylococci from complement attack through induction of abortive, complement-consuming reactions in the fluid phase.

Antigens, Bacterial↗
PubMed1985

Isolation and partial characterization of staphylococcal decomplementation antigen.

A substance with potent decomplementation activity was isolated from staphylococcal culture supernatants by polyethylene glycol precipitation, DEAE-ion-exchange and Sephacryl chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified substance exhibited all the characteristics of the decomplementation antigen (DA) previously detected in unfractionated culture supernatants. It contained glucosamine and phosphorus and was provisionally identified as extracellular, water-soluble teichoic acid of Staphylococcus aureus. DA was entirely resistant towards the action of proteases, DNase, RNase, or lysostaphin and withstood boiling for 30 min. Its electrophoretic mobility in agarose gels at pH 8.7 was approximately double that of human serum albumin. The molecule eluted in a molecular-weight region of 70,000 to 120,000 on Sephacryl S-300 and sedimented as a symmetrical 3 to 4 S moiety in sucrose density gradients. It migrated near the dye front on 12.5% sodium dodecyl sulfate-polyacrylamide gels and remained undenatured after boiling in sodium dodecyl sulfate. DA formed a symmetrical immunoprecipitate upon crossed immunoelectrophoresis against pooled human immunoglobulin G. It was identified as the major extracellular antigen present in unfractionated S. aureus culture supernatants that is precipitable by naturally occurring human immunoglobulin G antibodies. Immune complexes forming between DA and human immunoglobulin G exhibited an extraordinary capacity to activate the classical complement pathway. Micro- or nanogram amounts of purified antigen added to antibody-containing human serum effected rapid and complete consumption of C3, C4, and C5. The biochemical and biological properties of DA single out this molecule for an important role in suppressing the opsonizing activity of host complement through induction of abortive complement consumption in the fluid phase.

Antigens, Bacterial↗
PubMed1985

Mechanism of membrane damage by streptolysin-O.

Streptolysin-O (SLO) is a thiol-activated, membrane-damaging protein toxin of Mr 69,000 that is produced by most strains of beta-hemolytic group A streptococci. Native, primarily water-soluble toxin molecules bind to cholesterol-containing target membranes to assemble into supramolecular curved rod structures (25 to 100 nm long by ca. 7.5 nm wide), forming rings and arcs that penetrate into the apolar domain of the bilayer. Electron microscopic analyses of toxin polymers in their native and reconstituted membrane-bound form indicate that the convex surface of the rod structures is a hydrophobic, lipid-binding domain, whereas the concave surfaces appear to be hydrophilic. The embedment of the rings and arcs generates large transmembrane slits or pores of up to 30-nm diameter that can be directly visualized by negative staining and freeze-fracture electron microscopy. SLO oligomers were isolated in extensively delipidated form in detergent solution, and cholesterol was found not to detectably contribute to the observed rod structures. The rods are stable structures that resist prolonged exposure to trypsin and chymotrypsin. They can be reincorporated into cholesterol-free phosphatidylcholine liposomes to generate lesions identical to those observed on erythrocytes lysed by native SLO. Thus, although cholesterol plays a key role in the initial binding of SLO to the membrane, it does not directly participate in the formation of the membrane-penetrating toxin channels. Membrane damage by SLO is basically analogous to that mediated by previously studied channel formers, namely, the C5b-9 complement complex and staphylococcal alpha-toxin.

Animals↗
PubMed1985

Complement activation and attack on autologous cell membranes induced by streptolysin-O.

Streptolysin-O damages mammalian membranes through generation of large transmembrane channels formed by membrane-inserted polymers of the toxin (S. Bhakdi et al., Infect. Immun. 47:52-60, 1985). We here report that the native toxin binds naturally occurring human serum immunoglobulin G antibodies to form immune complexes with potent complement-activating capacity. Nanomolar concentrations of toxin added to antibody-containing serum cause rapid consumption of C4 and C5 hemolytic activity and 30 to 90% C3 conversion within 10 to 60 min at 37 degrees C. After binding to target membranes, streptolysin-O polymers serve as foci for antibody-dependent complement activation, which proceeds to completion with the formation of terminal C5b-9 complexes on the autologous cells. The binding and insertion of a primarily water-soluble bacterial product into a host cell membrane has thus been shown to generate a stable and hyperactive focus for activation of and self-attack by the complement system. We suggest that this process perpetuates local tissue damage, deviates host complement action away from the invading bacteria, and may possibly play a role in the pathogenesis of poststreptococcal disease.

Antigen-Antibody Complex↗
PubMed1985

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting as a serological tool in the diagnosis of syphilitic infections.

The utility of sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting as a serological tool in the diagnosis of human syphilitic infections was examined. In model experiments, rabbits were immunized with Treponema pallidum or T phagedenis, and the antisera were tested for cross-reactivities with both sets of antigens. A major T. pallidum antigen with a molecular weight of ca. 17,000 appeared to be the most reliable specific antigenic marker as assessed by the immunoblotting technique with peroxidase-labeled second antibodies. Antibodies to this antigen were never detected in hyperimmune rabbit anti-T. phagedenis sera or in the sera of nonsyphilitic humans. In contrast, reactive antibodies were found in all syphilitic human sera and also in liquor samples that were positive in the passive hemagglutination test. Differentiation between immunoglobulin M and immunoglobulin G antibodies was directly possible by applying the respective specific second antibodies. Immunoblotting tests were performed with sera exhibiting low passive hemagglutination test titers and equivocal fluorescent treponemal antibody and rapid plasma reagin card reactions. In more than 60% of these cases, immunoblot positivity with respect to the 17,000-molecular-weight antigen was found. The same results were obtained with partially purified 17,000-molecular-weight antigen. The immunoblot technique should be useful as an additional diagnostic tool for differentiating between true and false-positive serological reactions.

Animals↗
PubMed1985

Staphylococcal alpha-toxin-induced PGI2 production in endothelial cells: role of calcium.

Studies in erythrocytes indicate that staphylococcal alpha-toxin generates discrete transmembrane channels with an effective diameter of 2-3 nm. In cultured, confluent, pig pulmonary arterial endothelial cells we studied the triggering of the arachidonic acid cascade and its dependence on calcium influx, possibly through toxin-created pores. In endothelial cells alpha-toxin time dependently (5-30 min) and dose dependently (0.1-8 micrograms/ml) stimulated the release of radiolabeled arachidonic acid and prostacyclin (PGI2) production in similar amounts as the calcium ionophore A23187 (10 microM). Preincubation of alpha-toxin with neutralizing antibodies abolished the effect. The toxin response was strictly dose dependent on extracellular calcium but not on magnesium. The toxin effect was accompanied by an up to 10-fold increased passive permeability of pulmonary arterial endothelial cells for 45Ca. Interference with calcium-calmodulin function (trifluoperazine, W7) dose dependently reduced production of PGI2, but blockers of physiological calcium channels (verapamil, nimodipine, nisoldipine, and diltiazem) did not. In contrast to the effect of the ionophore A23187, the toxin effect was accompanied by a release of potassium, but in neither system was there a release of lactate dehydrogenase. In addition, alpha-toxin but not ionophore-exposed endothelial cells showed an increased passive influx of small radiolabeled markers (45Ca and [3H]sucrose) but not of large markers [( 3H]inulin and [3H]dextran). These data are consistent with the concept that alpha-toxin triggers the arachidonic acid cascade in pulmonary arterial endothelial cells by calcium influx and suggest that this calcium influx may proceed through toxin-created transmembrane channels.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Membrane damage by channel-forming proteins: staphylococcal alpha-toxin, streptolysin-O and the C5b-9 complement complex.

One mechanism through which cells can be damaged involves insertion of alien proteins into the membrane bilayer and the formation of hydrophilic transmembrane pores. Three examples for this process are discussed, namely membrane damage by staphylococcal alpha-toxin, streptolysin-O, and by the terminal C5b-9 complement complex. Common to all is the principle of a transition of the proteins from a water-soluble state to an amphiphilic state, occurring through the appearance or exposure of apolar surfaces during oligomerization of the protein molecules into supramolecular aggregates. The resulting complexes or protein oligomers insert spontaneously into the target lipid bilayer and assume properties akin to those of integral membrane proteins. The protein channels can be isolated from membranes after their solubilization by mild detergents and characterized on a bio-immunochemical and ultrastructural level.

Animals↗
PubMed1984

A simple method for quantitative measurement of C3d in human plasma.

We describe a simple and reliable method for quantitating C3d in human plasma. The method rests on the finding that native C3 and its activation/inactivation product C3c bind to Concanavalin A, whereas C3d does not. Rocket affinoimmunoelectrophoresis with Con-A Sepharose incorporated into an intermediate gel permits quantitation of free C3d in 2-20 microliters aliquots of EDTA-plasma without any manipulation prior to sample application. Using this method, we found that the level of circulating C3d in plasma of 30 healthy donors was usually well below 3% of maximally convertable C3d.

Complement C3↗