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Biomedical subjects

S Bhakdi

Publications and source records attributed to S Bhakdi.

At least 181 records · Page 10Linked to original sources
PubMed1987

Quantitation of monomeric and oligomeric forms of membrane-bound staphylococcal alpha-toxin by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody.

A murine monoclonal antibody generated against staphylococcal alpha-toxin was shown to react only with the monomeric (native), 3S form of the toxin. A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) constructed with this antibody permitted detection of 0.25 to 0.5 ng of native toxin per ml. Toxin oligomers formed either by heat aggregation in solution, on target erythrocyte membranes, or on phosphatidylcholine-cholesterol liposomes were unreactive in the ELISA when membranes were solubilized with the nondenaturing detergent Triton X-100. After dissociation of the oligomers by boiling in sodium dodecyl sulfate, however, the ELISA reactivity of the liberated 3S toxin was fully restored. Parallel determinations of membrane-bound toxin with sodium dodecyl sulfate and Triton X-100 solubilization thus permitted direct quantitation of total and monomeric toxin, respectively; the difference between these two values was represented by toxin oligomers. The detection limits for membrane-bound oligomeric and monomeric toxin on erythrocyte membranes are in the order of 100 molecules and 1 molecule per cell, respectively. Using this ELISA, we show that over 90% of alpha-toxin molecules bound to target membranes at 37 degrees C are in oligomeric form. Evidence is given that the monoclonal antibody neutralizes alpha-toxin by inhibiting its binding to both rabbit and human erythrocytes. This ELISA is the first assay that quantitatively discriminates between mono- and oligomeric forms of a pore-forming protein on target cell membranes.

Antibodies, Monoclonal↗
PubMed1987

Quantitative analysis of the binding and oligomerization of staphylococcal alpha-toxin in target erythrocyte membranes.

The binding of staphylococcal alpha-toxin to rabbit and human erythrocytes was quantitated over a wide range of toxin concentrations (3 x 10(-11) to 3 x 10(-6) M) with the use of an enzyme-linked immunosorbent assay that permitted simultaneous quantitation of monomeric and oligomeric toxin forms. Three basic observations were made. First, in no range of concentrations did the binding of alpha-toxin to rabbit erythrocytes display characteristics of a receptor-ligand interaction. Net binding to rabbit cells was nil at sublytic concentrations (10(-10) M or 3 ng/ml). The onset of binding occurred at around 10 ng/ml and remained fairly constant and ineffective (5 to 8% of toxin offered) over a wide concentration range (up to 10 micrograms/ml). Second, hemolysis of rabbit and human erythrocytes at 37 degrees C was always accompanied by the formation of toxin oligomers in the membrane. Third, overall toxin binding at 0 degree C followed a pattern similar to that at 37 degrees C. However, oligomer formation and cell lysis were retarded (but not totally inhibited) at 0 degree C. When rabbit erythrocytes were incubated with low levels of toxin at 0 degree C (0.5 microgram/ml) for 30 min, the toxin became bound exclusively in monomer form, and no lysis occurred. When cells thus treated were washed and suspended at 37 degrees C, lysis rapidly ensued, and native monomeric toxin was replaced by oligomeric toxin. The collective results directly support the oligomer pore concept of toxin action and also indicate that toxin oligomers form by lateral aggregation of bound monomers in the bilayer. They speak against the existence of specific binding sites for alpha-toxin on rabbit erythrocytes.

Animals↗
PubMed1987

Identification with monoclonal antibodies of hemolysin produced by clinical isolates of Escherichia coli.

Murine monoclonal antibodies were generated against the 107,000-dalton hemolysin encoded by the hemolytic determinant from Escherichia coli LE 2001, and colony blotting was used to assay for production of the hemolysin by 35 hemolytic strains of E. coli and other hemolytic members of the family Enterobacteriaceae of clinical origin. All hemolytic E. coli strains gave positive reactions with two monoclonal antibodies. In contrast, none of the hemolytic, non-E. coli isolates yielded positive colony blots. In addition, Western blotting showed that the hemolysins produced by all clinical E. coli isolates had a similar molecular weight of about 107,000. Discrete antigenic variation may occur in the molecule, since a third monoclonal antibody did not react with the hemolysin from a number of wild-type E. coli strains. Western blot analysis was used to assess the presence of immunoglobulin G (IgG), IgA, and IgM antibodies to E. coli hemolysin in human sera. All 20 of the tested sera from healthy adults contained antibodies to the toxin, with various constellations among the antibody classes. In contrast, sera from five of eight infants aged 8 to 36 months contained no antihemolysin antibodies. We conclude that the 107,000-dalton hemolysin of E. coli is a widespread immunogen that is produced by most or all hemolytic E. coli strains in the human host.

Animals↗
PubMed1987

Complement complex C5b-8 induces PGI2 formation in cultured endothelial cells.

The effects of the terminal complement sequence on prostacyclin (PGI2) generation in antibody-sensitized pulmonary arterial endothelial cells were examined. Whereas C5b-7 complement complexes induced no PGI2 formation, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI2 release in the absence of overt cell damage. Formation of the complete terminal complement complex C5b-9 enhanced PGI2 release but was accompanied by cytolysis. Extracellular Ca2+ was required for C5b-8-dependent PGI2 formation. Three different blockers of physiological calcium channels failed to suppress the observed stimulatory effect. In contrast, W7 [N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide] and trifluoperazine, inhibitors of calmodulin activity, all reduced the C5b-8-dependent PGI2 generation. None of the inhibitors used impaired Ca2+ flux into the cells. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six- to seven-fold enhanced passive influx of 45Ca2+ into the cells was noted. An enhanced passive influx was also observed for 51Cr O4(2-), [3H] aminobutyric acid, and [3H]sucrose, but not for [3H]inulin and [3H]dextran. These data together suggest that complement C5b-8 complexes may serve as Ca2+ bypass gates in endothelial cells, the ensuing influx of Ca2+ leading to subsequent activation of the arachidonic acid pathway.

Animals↗
PubMed1987

A reason for the cytolytic inefficiency of murine serum.

Murine serum exhibits very poor haemolytic and bactericidal activity. We report that this is due, at least in part, to the presence of a potent, naturally occurring plasma inhibitor of the terminal complement sequence. The inhibitor is a heat-stable euglobulin. It is highly effective in suppressing haemolysis following complement activation on target erythrocytes with heterologous serum. It also inhibits C3-independent reactive haemolysis of guinea-pig erythrocytes with human C5b-9. Current evidence indicates that the inhibitory factor acts at the C5b-7 stage by preventing binding of the terminal complement complex to cells undergoing complement attack. In this respect, the inhibitor differs from the previously recognized regulators of the terminal complement sequence including plasma S-protein. The inhibitor does not protect C5b-7-laden cells from the action of C8 and C9, and also does not suppress formation of haemolytically inactive SC5b-9 in the fluid phase. The action of murine inhibitory factor is not confined to the red cell, and its presence can totally abolish the bactericidal activity of human serum on a sensitive, rough E. coli K12 strain.

Animals↗
PubMed1987

Quantitative evaluation of the terminal C5b-9 complement complex by ELISA in human atherosclerotic arteries.

A quantitative ELISA using monoclonal and polyclonal antibodies against neoantigens of the terminal C5b-9 complement complex was used to evaluate the presence of terminal complexes in 68 human arterial samples with or without atherosclerotic involvement. Plasma levels of SC5b-9 were directly compared with the corresponding levels eluted from the femoral arteries in six patients undergoing surgical procedures. The plasma concentration of SC5b-9 in these donors was in the range of 30-90 arbitrary units (AU)/ml, equivalent to 100-300 ng/ml SC5b-9 or 45-130 AU/100 mg plasma protein. All the arterial samples contained detectable amounts of C5b-9. The aortic normal and fatty streaks intimae presented a minimum mean value of 65 +/- 12 AU/100 mg total protein, in the range of normal plasma SC5b-9 levels. The corresponding media contained significantly higher amounts of terminal complexes (115 +/- 30 AU/100 mg protein). Markedly increased levels of C5b-9 were eluted from aortic intimal thickenings (350 +/- 100 AU/100 mg protein) and the corresponding media (300 +/- 53 AU/100 mg protein). Similar concentrations were found in aortic fibrous plaques (340 +/- 80 AU/100 mg protein). The observed correlation between C5b-9 levels and atherosclerotic alterations in arterial walls is suggestive of chronic complement activation with involvement of the terminal complement sequence at these sites. These processes may contribute to progression of the arteriosclerotic lesions.

Adult↗
PubMed1986

Deposition of the terminal C5b-9 complement complex in infarcted areas of human myocardium.

Poly- and monoclonal antibodies to neoantigens of the human C5b-9 complement complex, as well as polyclonal antibodies to C5, C8, and C9, were used to detect and identify C5b-9 deposits in human myocardial tissue. Immunocytochemical studies were performed on fresh-frozen autopsy material derived from patients with myocardial infarctions; in addition, in 17 of these patients, paraffin sections of formalin-fixed tissue were investigated. Sixteen autopsies from patients with noncardiac diseases were analyzed as controls. Without exception, C5b-9 positivity was registered selectively and exclusively on and in myocardial cells located within the zones of infarction. The selectivity of staining was confirmed by control reactions for succinic dehydrogenase activity performed in adjacent, respective double-stained sections. Most intensive staining with anti-neoantigen antibodies was observed in the peripheral areas of the infarctions. Weak staining for C3d, rather strong staining for C5 and C9, and intermediate staining with anti-C8 antibodies were observed in the same localizations. Stainings for C4 and IgA were negative, whereas immunocytochemical reactions for IgG and IgM revealed an irregular and very weak staining. Only very weak staining was also observed with a monoclonal antibody to complement S-protein, indicating that the terminal complement components were deposited mainly in the form of membrane-damaging C5b-9 complexes. Immunocytochemical staining for C5b-9 was found to represent a most sensitive tool for detection of ischemic myocardial lesions, permitting easy detection even of single cell necroses. As a working hypothesis, we suggest that initial ischemia may cause loss of the ability of the heart muscle cells to regulate complement turnover at the membrane level. The resulting deposition of C5b-9 on the cell membranes may contribute to functional disturbance and irreversible damage of myocardial cells during the infarction process.

Adult↗
PubMed1986

Noncytolytic terminal complement complexes may serve as calcium gates to elicit leukotriene B4 generation in human polymorphonuclear leukocytes.

Complement effects on human polymorphonuclear leukocytes (PMN) have generally been ascribed to the anaphylatoxin C5a, which induces degranulation, superoxide anion generation, migration, and cell aggregation via interaction with membrane receptors. We here report that complement activation on the surface of antibody-sensitized human PMN provokes generation of the potent lipid mediator leukotriene B4 (LTB4) in strict dependence on complement component C8, but in the absence of detectable C9. The kinetics of LT generation are rapid, comparable with those observed after challenge with the calcium-ionophore A23187. LTB4 release is a distinct event that is dissociable from cytotoxicity as assessed by lactate dehydrogenase (LDH) release (dependent on C9) and from superoxide generation (independent of C8 and C9). It is dose dependent on extracellular calcium and is not observed in the absence of calcium. It is inhibited by substances interfering with calcium-calmodulin function (trifluoperazine and W7), but not by blockers of physiologic calcium channels (nimodipine, verapamil, and D 888). Addition of purified C8 to cells bearing C5b-7 induces a severalfold increase in their passive permeability to 45calcium. Sieving experiments with the use of marker molecules of different sizes collectively indicate the existence of small hydrophilic channels consisting exclusively or predominantly of C5b-8 complexes, which allow passive transmembrane flux of small molecules with Mr less than 200. Thus, noncytolytic terminal complement complexes may serve as a biological bypass gate for calcium in PMN membranes, triggering the arachidonic acid cascade with generation of LTB4 at doses well below the threshold required to invoke overt cell damage.

Calcium↗
PubMed1986

C5b-9 assembly: average binding of one C9 molecule to C5b-8 without poly-C9 formation generates a stable transmembrane pore.

Membrane attack by serum complement normally results in the formation of C5b-9 complexes that are heterogeneous with respect to their C9 content. We here report that an apparently homogeneous population of C5b-9 complexes can be generated through treatment of C5b-7-laden sheep erythrocytes with C8 and C9 for 60 min at 0 degree C. Experiments performed by using radioiodinated C8 and C9 components have indicated that binding of C8 to these target cells is essentially temperature independent. In contrast, when a surplus of C9 molecules is offered to C5b-8 cells, an approximately fourfold to 4.5-fold higher number of C9 molecules become cell bound at 37 degrees C as opposed to 0 degree C. C5b-9 complexes isolated from target membranes treated with C9 at 0 degree C contain no polymerized C9 and do not exhibit the ring structure characteristic of the classical complement lesion. Nevertheless, these complexes generate stable transmembrane channels and cause hemolysis at 37 degrees C. The pores have been sized to 1 to 3 nm effective diameter by osmotic protection experiments. SDS-PAGE of the isolated complexes indicates an average stoichiometry of only one molecule C9 bound per C5b-8 complex. The results show that oligomerization of C9 with formation of ring lesions is not a basic requirement for the generation of stable transmembrane complement pores in sheep erythrocytes. They indirectly support the contention that terminal complement components other than C9 contribute to the intramembrane domains of C5b-9 pores.

Animals↗
PubMed1986

Immunohistochemical study of the C5b-9 complex of complement in human kidneys.

The presence and localization of the C5b-9 neoantigens of the terminal complement sequence, of antigens expressed by cleavage fragments of C3, and of Factor H antigens have been studied by immunohistochemical techniques in morphologically normal adult human kidneys and in biopsy specimens from patients with a wide range of renal diseases with and without immune deposits. In morphologically normal kidneys, C5b-9 neoantigens were observed within all connective matrices (arteriolar media, glomerular basement membrane (GBM), mesangial matrix and tubular basement membrane). The C3d and C3g antigens of the C3dg, and C3bi cleavage fragments of C3 and Factor H antigens were found in similar locations. None of the matrices stained for immunoglobulins. Immunoelectron microscopy demonstrated that C3d, C3g, H antigens and the C5b-9 neoantigens were localized on membranous and vesicular structures embedded in the connective matrices. These structures represent cell membranes shed from adjacent cells as evidenced by their ultrastructural appearance and by the fact that those which were in close vicinity to pedicles within the GBM expressed the C3b receptor antigen, a specific marker for podocyte membranes. Formation of C5b-9 complexes in the shielded environment of connective matrices may explain their persistence over long periods of time in the absence of apparent immunopathological consequences. Biopsies from pathological kidneys were classified into three groups based on the pattern of glomerular staining with anti-C5b-9 antibodies. In the first group, a sparse mesangial labeling was seen, similar to that observed in normal kidneys. In the second group, abundant clusters of C5b-9 were seen in the same location as immune deposits. Activation of the complement system to completion could be documented in the absence of detectable C3 (C3c) antigen in glomeruli. Immunoelectron microscopy demonstrated that C5b-9 neoantigens were present on cell remnants in connective matrices in all specimens that were studied. Labeled cell remnants were present in large amounts in sclerotic matrices. C5b-9 neoantigens were constantly found on old and large immune deposits, and absent or occasionally present on recent and small immune deposits. In membranous nephropathy stage I, proteinuria appeared to be independent of the presence or absence of detectable C5b-9 neoantigens on immune deposits. Thus, the presence of C5b-9 neoantigens in pathological renal tissue does not have an univocal significance, and requires analysis of the localization of the antigens and appropriate controls in order to assess the potential role of C5b-9 in tissue damage.

Adult↗
PubMed1986

Production of listeriolysin by beta-hemolytic strains of Listeria monocytogenes.

Listeriolysin was isolated from target rabbit erythrocyte membranes after lysis of the cells with partially purified toxin derived from a culture supernatant of Listeria ivanovii. The membrane form of the toxin exhibited properties similar to those previously found for streptolysin O. Detergent-solubilized, delipidated listeriolysin was found to comprise a heterogeneous population of partially and fully circularized, amphiphilic oligomers whose embedment within the lipid bilayer generated large transmembrane pores. The molecular weight of the toxin monomer was estimated to be 55,000 to 60,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological cross-reactions between the toxin and streptolysin O were demonstrable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. An immunoblot assay for detecting listeriolysin in agar-incorporated, lysed erythrocyte membranes was developed, and 28 defined, clinical isolates of Listeria monocytogenes were examined for toxin production. These isolates caused beta-hemolysis on the agar plates and had previously been regarded as listeriolysin producers. However, we found that only two isolates produced genuine listeriolysin, since the sensitive immunoblot assay entirely failed to detect the toxin in all other cases. We excluded that this finding derived from proteolytic degradation of membrane-bound toxin. Thus, the great majority of human pathogenic Listeria strains appear to produce one or several hemolysins that are immunologically and, by inference, molecularly distinct from the streptolysin O-related listeriolysin. We propose that the streptolysin O-related toxin be designated alpha-listeriolysin and that the other hemolysin(s) be termed beta-listeriolysin.

Animals↗
PubMed1986

Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores.

Escherichia coli hemolysin is secreted as a water-soluble polypeptide of Mr 107,000. After binding to target erythrocytes, the membrane-bound toxin resembled an integral membrane protein in that it was refractory towards extraction with salt solutions of low ionic strength. Toxin-induced hemolysis could be totally inhibited by addition of 30 mM dextran 4 (mean Mr, 4,000; molecular diameter approximately 3 nm) to the extracellular medium. Uncharged molecules of smaller size (e.g., sucrose, with a molecular diameter of 0.9 nm, or raffinose, with a molecular diameter of 1.2 to 1.3 nm) did not afford such protection. Treatment of erythrocytes suspended in dextran-containing buffer with the toxin induced rapid efflux of cellular K+ and influx of 45Ca2+, as well as influx of [14C]mannitol and [3H]sucrose. [3H]inulin only slowly permeated into toxin-treated cells, and [3H]dextran uptake was virtually nil. Membranes lysed with high doses of E. coli hemolysin exhibited no recognizable ultrastructural lesions when examined by negative-staining electron microscopy. Sucrose density gradient centrifugation of deoxycholate-solubilized target membranes led to recovery of the toxin exclusively in monomer form. Incubation of toxin-treated cells with trypsin caused limited proteolysis with the generation of membrane-bound, toxin-derived polypeptides of Mr approximately 80,000 without destroying the functional pore. We suggest that E. coli hemolysin may damage cell membranes by partial insertion into the lipid bilayer and generation of a discrete, hydrophilic transmembrane pore with an effective diameter of approximately 3 nm. In contrast to the structured pores generated by cytolysins of gram-positive bacteria such as staphylococcal alpha-toxin and streptolysin O, pore formation by E. coli hemolysin may be caused by the insertion of toxin monomers into the target lipid bilayers.

Cell Membrane Permeability↗
PubMed1986

Use of a monoclonal antibody to determine the mode of transmembrane pore formation by streptolysin O.

Murine monoclonal antibodies were generated against streptolysin O. One out of 10 tested immunoglobulin clones exhibited strong neutralizing activity; in solution, the presence of approximately two to four antibody molecules per toxin monomer effected 50% neutralization of hemolytic toxin activity. An enzyme-linked immunosorbent assay performed with target cell membranes that were treated with streptolysin O in the presence and absence of neutralizing antibodies showed that the antibodies did not block primary binding of the toxin to the cells. When membranes were solubilized in deoxycholate detergent and centrifuged in sucrose density gradients, those lysed with streptolysin O contained detergent-resistant, high-molecular-weight oligomers identical to the pore lesions, whereas those given toxin and neutralizing antibody contained the toxin exclusively in low-molecular-weight, nonoligomerized form. The process of pore formation by streptolysin O must thus involve two distinct steps, i.e., the primary binding of toxin molecules to the membrane followed by oligomerization of bound toxin monomers by lateral aggregation in the lipid bilayer to form the transmembrane pores.

Animals↗
PubMed1986

Immunohistochemical analysis of C3 cleavage fragments, factor H, and the C5b-9 terminal complex of complement in de novo membranous glomerulonephritis occurring in patients with renal transplant.

Fifteen renal biopsies from 13 transplanted patients with de novo membranous nephropathy (DNMN) were investigated by immunofluorescence for the presence of C5b-9 neoantigens of the terminal sequence of complement and for antigens expressed by C3 cleavage fragments. DNMN lesions were classified as stage I, II or III upon light and electron microscopy examination. Seven biopsies were classified as stage I DNMN and 8 stage II-III. All patients were proteinuric. In six biopsies with stage I DNMN, staining for C5b-9 neoantigens was restricted to a fine granular labeling in mesangial areas which was analogous to that seen in normal kidneys in contrast with extensive parietal labeling for IgG, C3d and factor H antigens. In eight biopsies with stage II-III DNMN, the pattern of staining with anti-C5b-9 neoantigens antibodies was similar to that obtained with anti-IgG, anti-C3d and anti-factor H antibodies. These results suggest that in situ activation of the whole complement sequence throughout C5b-9 only occurs on large immune deposits (stage II-III DNMN).

Adolescent↗
PubMed1986

Does complement kill E. coli by producing transmural pores?

Three lines of evidence are presented to indicate that C5b-9 kills serum-sensitive E. coli K 12 cells by generating functional pores across the outer and inner bacterial membrane. First, viable cells carrying C5b-8 complexes are impermeable to o-nitrophenyl-beta-D-galactoside (ONPG), but lose viability and become permeable to this marker upon post-treatment with purified C9 in the absence of lysozyme. Cells killed with colicin E1 or gentamicin are also impermeable to ONPG but take up the marker if they are post-treated with lysozyme-free serum. Second, killing by C5b-9 is highly effective, deposition of only a small number of complexes being lethal. This has been demonstrated in experiments where viable cells carrying 2000-4000 C5b-7 complexes per CFU were permitted to multiply in broth culture, and the daughter generations subsequently treated with purified C8 and C9. Fifty percent killing was observed in the fifth to sixth generation, corresponding to a dilution of C5b-7 complexes to 50-100 molecules/CFU. In the presence of 2 mM EDTA, further dilution of C5b-7 down to 8-30 complexes/CFU still caused 50% killing of daughter cells. Third, treatment of C5b-7 cells with purified CC8 and C9 results in the release of intracellular K+, which commences immediately after addition of C8/C9. This was shown in experiments where C5b-7 cells were packed to high density in saline, post-treated with C8 + C9, and K+ directly measured in the cell supernatants. Based on these results, we propose that C5b-9 pores deposited in the outer bacterial membrane periodically fuse with the inner membrane, the transmural pores thus generated permitting rapid K+ efflux, with cell death ensuing through the collapse of membrane potential.

Bacteriolysis↗