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S Bhakdi

Publications and source records attributed to S Bhakdi.

At least 19 recordsLinked to original sources
PubMed1992

Sphingolipid transport from the trans-Golgi network to the apical surface in permeabilized MDCK cells.

We have measured the transport of de novo synthesized fluorescent analogs of sphingomyelin and glucosylceramide from the trans-Golgi network (TGN) to the apical membrane in basolaterally permeabilized Madin-Darby canine kidney (MDCK) cells. Sphingolipid transport was temperature, ATP and cytosol dependent. Introduction of bovine serum albumin (BSA), which binds fluorescent sphingolipid monomer, into the permeabilized cells, did not affect lipid transport to the apical membrane. Both fluorescent sphingomyelin and glucosylceramide analogs were localized to the lumenal bilayer leaflet of isolated TGN-derived vesicles. These results strongly suggest that both sphingolipids are transported from the TGN to the apical membrane via vesicular traffic.

Animals↗
PubMed1992

Flow cytometric assay for estimating fungicidal activity of amphotericin B in human serum.

We describe a simple and rapid bioassay for estimating fungicidal activity of Amphotericin B in human serum using flow cytometry. The method exploits the fact that Candida albicans damaged by Amphotericin B show a decrease in size and take up propidium iodide to exhibit a red fluorescence after deoxycholate treatment. These phenomena display characteristic dose dependencies, and their assessment permits serum fungicidal activity to be broadly grouped into three categories: (1) subfungicidal; (2) fungicidal; and (3) strongly fungicidal. In normal human serum, these three categories correspond to Amphotericin B concentrations of 0 less than or equal to 0.5 micrograms/ml, 0.75-1.5 micrograms/ml, and greater than 2 micrograms/ml, respectively. Pilot analysis of serum samples obtained from four patients undergoing Amphotericin B therapy confirmed the feasibility of using the flow cytometric assay for estimating drug fungicidal activity ex vivo. The method is very simple, generates results within 5 h, and could prove useful for monitoring therapy with this effective but toxic drug.

Amphotericin B↗
PubMed1992

CD59 (homologous restriction factor 20), a plasma membrane protein that protects against complement C5b-9 attack, in human atherosclerotic lesions.

Blood cells express a cell membrane protein, termed homologous restriction factor 20 (HRF20) and identical to CD59, that can inhibit complement C5b-9 insertion into their membranes. In this report, we investigated by immunohistochemistry whether CD59 was present on cells in human atherosclerotic lesions since membranous C5b-9(m) has been found in lesions. Using a monoclonal anti-CD59 antibody, a cellular CD59 staining pattern was apparent in nearly all lesion specimens. CD59 stain co-localised with macrophage (CD14), T lymphocyte (CD7), endothelial cell (anti-factor VIII related antigen) and smooth muscle cell cytoskeletal-specific antigens (anti-alpha actin and muscle myosin). Endothelial cells always exhibited a more intense stain than the other cell types. CD59 antigen was not localised to any one area of the lesions. Usually CD59-positive cells occurred in clusters rather than as randomly spaced individual cells. CD59 did not stain all cells of the lesion and in particular did not appear to stain all smooth muscle cells. Areas of CD59-negative cells were sometimes observed to exhibit a cellular C5b-9 staining pattern. C5b-9 deposits were also observed in CD59-positive regions. Normal saphenous vein stained strongly for CD59 at the endothelial lining and weakly in the media. Capillaries in atherosclerotic intima always stained strongly for CD59. We conclude that HRF20 is constitutively expressed on endothelium and is under regulatory control in smooth muscle cells. Cellular C5b-9 attack in atherosclerotic lesions is therefore most likely to occur on smooth muscle cells.

Actins↗
PubMed1992

Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes.

We describe a novel flow cytometric method for quantifying opsonophagocytosis and killing of Staphylococcus aureus in cell-rich plasma obtained after dextran sedimentation of erythrocytes. To analyze opsonophagocytosis, phagocytes were labeled with a phycoerythrin-conjugated monoclonal antibody and were incubated with viable staphylococci containing carboxyfluorescein as a vital fluorescent dye. Phagocytosing cells assumed a dual, orange-green fluorescence. The relative numbers of bacteria associating with phagocytes could be determined by quantifying the decrease of free green fluorescent particles. A parallel incubation of fluorescent bacteria with unlabeled cell-rich plasma was performed to assess phagocytic killing. Blood cells were lysed with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate. This detergent spared viable bacteria, and residual green fluorescent particles were counted. The decrease in the number of these particles relative to the controls yielded the degree of killing. At bacteria-to-phagocyte ratios of 1:1 and 10:1, approximately 36 and 75% of the phagocytes participated in opsonophagocytosis, respectively. Over 90% of the staphylococci were phagocyte associated after 30 to 60 min. Killing rates were on the order of 66% +/- 12% and 80% +/- 7% after 1 and 2 h of incubation, respectively. These numbers, which were confirmed by colony countings, were significantly lower than those reported in the majority of past reports.

Antibodies, Monoclonal↗
PubMed1992

Induction of severe vascular leakage by low doses of Escherichia coli hemolysin in perfused rabbit lungs.

S. aureus alpha-toxin and E. coli hemolysin (Hly) represent two prototypes of pore-forming cytolysins. Both are established virulence factors and have been implicated in the development of septic lung failure. Low doses of these agents cause thromboxane-mediated vasoconstriction and edema formation in isolated perfused rabbit lungs. In a preceding investigation, we observed that alpha-toxin causes overt endothelial cell damage in these lungs, as demonstrable by electron microscopy (Seeger W, Birkemeyer RG, Ermert L, Suttorp N, Bhakdi S, Duncker HR: Lab Invest 63:341, 1990). Here, we present results of a parallel study conducted with E. coli hemolysin. Thromboxane-dependent pulmonary hypertension was suppressed by the addition of acetylsalicylic acid to the perfusion fluid in all cases. Administration of 0.2 hemolytic units (HU; i.e., 20 ng/ml protein) resulted in progressive weight gain after a lag period of 10 to 15 minutes, and 30 minutes after toxin application the gravimetrically determined capillary filtration coefficients (Kfc) were increased greater than 10-fold. Perfusion was terminated when the total lung weight gain surpassed 20 gm. 0.12 HU/ml E. coli hemolysin caused 2- to 3-fold increased capillary filtration coefficients values within 110 minutes, concomitant with intermediate quantities of edema formation (9.7 +/- 2.7 gm). Potassium liberation in the absence of lactate dehydrogenase release occurred in all toxin treated lungs. Electron microscopic examination after perfusion fixation revealed interstitial edema formation in areas remote from the blood-gas exchange barrier. Increased numbers of endothelial plasmalemmal vesicles were visualized at the very onset of edema formation in lungs exposed to 0.2 HU/ml, and after a 110-minute exposure to 0.12 HU/ml of the toxin, but not in lungs exhibiting severe edema (greater than 20 gm weight gain). In contrast to our previous results with alpha-toxin, endothelial cells displayed normal electron density here and were not detached from the fused basal lamina. Hence, although both pore formers provoke severe vascular leakage in our experimental model, the underlying pathways probably divert fundamentally from each other.

Animals↗
PubMed1991

Staphylococcus aureus alpha-toxin. Dual mechanism of binding to target cells.

Staphylococcal alpha-toxin was radiolabeled to high specific radioactivity (1,500-3,000 Ci/mmol) under retention of its hemolytic activity. Binding studies with susceptible rabbit erythrocytes and highly resistant human erythrocytes revealed that binding of alpha-toxin to target cells can occur via two different mechanisms. Binding of alpha-toxin to rabbit erythrocytes initially involves specific binding sites and occurs at low concentrations, with half-maximal binding at 1-2 nM. In contrast, toxin binding to human erythrocytes is absorptive and nonspecific, in this case, significant binding as well as hemolysis occur only at alpha-toxin concentrations exceeding 1 microM. Autoradiographic analyses of membrane-associated alpha-toxin from either cell species proved that hemolysis was inevitably associated with the formation of toxin hexamers. Our data indicate that the high susceptibility of certain target cells toward alpha-toxin is caused by the presence of specific binding sites. However, membrane damage of both susceptible and nonsusceptible target cells occurs via a common mechanism involving toxin oligomerization and pore formation.

Animals↗
PubMed1991

Subhemolytic doses of Escherichia coli hemolysin evoke large quantities of lipoxygenase products in human neutrophils.

Polymorphonuclear leukocytes (PMN) have been identified as preferred target cells for Escherichia coli hemolysin in human blood (Bhakdi, S., Greulich, S., Muhly, M., Eberspächer, B., Becker, H., Thiele, A., and Hugo, F. (1989) J. Exp. Med. 169, 737-754). Leukotriene and 5-hydroxyeicosatetraenoic acid generation was investigated in human PMN challenged with E. coli hemolysin in the absence or presence of free arachidonic acid or eicosapentaenoic acid (EPA). In the absence of exogenous free fatty acids, E. coli hemolysin (0.01-10 hemolytic units/ml) induced moderate generation of leukotriene B4 (LTB4) and its omega-oxidation products. The presence of free arachidonic acid (10 microM) during E. coli hemolysin (0.1 hemolytic unit/ml) challenge evoked the generation of large quantities of these products (greater than 100 pmol/1.5 x 10(7) PMN). In parallel, large amounts of 5-hydroxyeicosatetraenoic acid and nonenzymatic LTA4 hydrolysis products appeared. Product release peaked or plateaued 5-10 min after E. coli hemolysin challenge. The presence of exogenous EPA upon E. coli hemolysin challenge resulted in the exclusive generation of LTB5 and metabolites, LTA5 decay products and 5-hydroxyeicosapentaenoic acid. Dose and time dependences corresponded to those with arachidonic acid provision, and the total of EPA-derived products surpassed that of arachidonic acid metabolites in corresponding experiments approximately 2-fold. Increasing the time between free fatty acid provision and E. coli hemolysin challenge resulted in a rapid decline in the generation of arachidonic acid or EPA metabolites. Thus, subhemolytic doses of E. coli hemolysin evoke marked PMN eicosanoid generation that is dependent on exogenous free fatty acid supply, with total amounts approximating those found in calcium ionophore-stimulated neutrophils.

Bacterial Proteins↗
PubMed1991

Complement pore genesis observed in erythrocyte membranes by fluorescence microscopic single-channel recording.

The formation and opening of single complement pores could be directly observed in erythrocyte ghosts by confocal laser-scanning microscopy employing the recently introduced method of fluorescence microscopic single-channel recording. Resealed sheep erythrocyte ghosts were incubated with human complement. By limiting the concentration of C8, the eighth component of complement, the fraction of cells rendered permeable for the small polar fluorescent probe Lucifer Yellow was varied between 0.50 and 0.90. Under each condition the flux rate, k, of Lucifer Yellow was determined for a substantial number of ghosts. By analysing the sample population distribution of k the flux rate k1 of ghosts with a single pore was found to be (4.8 +/- 1.1) x 10(-3) s-1 consistent with a pore radius of about 3.5 nm (35 A). The genesis of single complement pores was studied by continuous influx measurements while triggering pore formation by a temperature shift. Pore genesis was found to be a very slow process, proceeding on a time scale of several minutes. During pore genesis the influx curves had a sigmoid shape, which excluded the possibility that the pore was preformed on the membrane surface and subsequently inserted. However, the influx curves could be well simulated by a model which assumed that pores grow stepwise by sequential incorporation of C9 monomers. The model predicts conditions under which the incorporation of single monomers can be directly revealed.

Animals↗
PubMed1991

Functional size of complement and perforin pores compared by confocal laser scanning microscopy and fluorescence microphotolysis.

Confocal laser scanning microscopy and fluorescence microphotolysis (also referred to as fluorescence photobleaching recovery) were employed to study the transport of hydrophilic fluorescent tracers through complement and perforin pores. By optimizing the confocal effect it was possible to determine the exclusion limit of the pores in situ, i.e. without separation of cells and tracer solution. Single-cell flux measurements by fluorescence microphotolysis yielded information on the sample population distribution of flux rates. By these means a direct comparison of complement and perforin pores was made in sheep erythrocyte membranes. In accordance with previous studies employing a variety of different techniques complement pores were found to have a functional radius of approx. 50 A when generated at high complement concentrations. The flux rate distribution indicated that pore size heterogeneity was rather small under these conditions. Perforin pores, generated in sheep erythrocyte membranes at high perforin concentrations, were found to have a functional size very similar to complement pores. Furthermore, the functional size of the perforin pore seemed to be relatively independent of the dynamic properties of the target membrane since in two cell membranes which are very different in this regard, the human erythrocyte membrane and the plasma membrane of erythroleukemic cells, the functional radius of the perforin pore was also close to 50 A. A perforin-specific antibody reduced the functional radius of perforin pores to 45 A.

Animals↗
PubMed1991

Analysis of complement C3 activation products in human atherosclerotic lesions.

Cleavage of the complement C3 protein is essential for complement activation. Saline extracts of human atherosclerotic lesions were examined by various techniques for the presence of C3 cleavage fragments. Crossed intermediate gel immunoelectrophoresis revealed that native C3 was the predominate C3 protein in extracts and that the C3dg fragment was also detected. SDS-PAGE/Western blot analyses of lesion extracts employing monoclonal antibodies directed at C3c and C3dg fragment determinants demonstrated molecular weight bands corresponding to the known molecular weights of all the physiologic C3 cleavage fragments, except C3b which is known to have a short half-life. After C3, the two most common fragments observed were C3c and C3dg. No bands other than those corresponding to known C3 cleavage fragments were observed and control antibody stains were always negative. In some blots bands with a greater molecular mass than C3 were evident, indicating that some of the C3 in lesions may be covalently bound to an activator. We have previously identified a large (100-500 nm) nonapoprotein containing lipid particle (LCA) as a major complement activating structure in human atherosclerotic lesions. Fractionation of lesion extracts by molecular sieve chromatography and sucrose density gradient centrifugation failed to reveal a concordance between LCA and C3 antigens. The results indicate that complement activation, i.e. C3 convertase formation, takes place in human atherosclerotic lesions and that activated C3 is degraded according to normal complement regulatory mechanisms.

Arteries↗
PubMed1991

Stimulation of monokine production by lipoteichoic acids.

Lipoteichoic acids (LTAs) isolated from bacterial species, including Staphylococcus aureus, Streptococcus pyogenes A, Enterococcus faecalis, Streptococcus pneumoniae, and Listeria monocytogenes, were tested for their ability to stimulate the production of interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha in cultured human monocytes. LTAs from S. aureus and S. pneumoniae failed to induce monokine production when applied in the concentration range of 0.05 to 5.0 micrograms/ml. However, LTAs from several enterococcal species (0.5 to 5 micrograms/ml) induced the release of all three monokines at levels similar to those observed after lipopolysaccharide stimulation. The kinetics of IL-1 beta and tumor necrosis factor alpha release elicited by LTAs closely resembled those observed following lipopolysaccharide application. Cytokine production occurred in the presence of both fetal calf serum and autologous human serum. Hence, it was not dependent on complement activation and could not be suppressed by naturally occurring human antibodies. Deacylation caused the total loss of monocyte stimulatory capacity. Deacylated LTAs were unable to prevent monocyte activation by intact LTAs, so primary binding of these molecules probably does not involve a simple interaction of a membrane receptor with the hydrophilic portion of the molecule. The results identify some species of LTAs as inducers of monokine production in human monocytes.

Acylation↗
PubMed1991

Superoxide generation by human neutrophils induced by low doses of Escherichia coli hemolysin.

Escherichia coli hemolysin (Hly) was isolated from bacterial culture supernatants by polyethylene glycol precipitation and centrifugation in glycerol density gradients. The toxin preparations contained less than 1 mol of lipopolysaccharide per 10 mol of protein, and they had no fatty acids. The capacity of purified hemolysin to stimulate superoxide anion production in polymorphonuclear leukocytes was monitored kinetically in a lumimeter by using the lucigenin assay and was correlated with the kinetics of transmembrane pore formation. When applied to leukocytes suspended in protein-free buffer, very low concentrations (0.02 to 0.1 HU/ml) of the toxin strongly stimulated the production of superoxide anions; shortly thereafter, irreversible membrane permeabilization occurred. When the toxin was applied at concentrations exceeding 0.2 to 0.3 HU/ml, membrane permeabilization was so rapid that the cells were unable to mount a respiratory burst. When applied in the narrow range of 0.05 to 0.1 HU/ml, E. coli hemolysin rivaled phorbol myristate acetate in its capacity to stimulate production of superoxide anions. Additionally, hemolysin applied at doses that elicited no pore formation (0.01 to 0.02 HU/ml) primed leukocytes for an augmented response to subsequent challenge by the phorbol ester. These data demonstrate that very low doses of E. coli hemolysin can evoke cellular reactions that appear independent of and precede transmembrane pore formation and cell death.

Bacterial Proteins↗
PubMed1991

Quantitative analysis of opsonophagocytosis and of killing of Candida albicans by human peripheral blood leukocytes by using flow cytometry.

We describe a simple, rapid, automated procedure for measuring opsonophagocytosis and killing of Candida albicans by human peripheral blood leukocytes. Yeast cells are labelled by allowing uptake and cleavage of membrane-permeable bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester to its membrane-impermeable fluorescent derivative bis-carboxyethyl-carboxyfluorescein. The yeast cells are added to cell-rich plasma obtained after dextran sedimentation of erythrocytes. Opsonophagocytosis and killing are quantified by using automated fluorescent cell analysis, and the following parameters can be obtained: (i) relative percentage of phagocytes that participate in opsonophagocytosis, (ii) relative percentage of yeast cells that become associated with phagocytes, and (iii) percentage of killing of C. albicans. The first two parameters are obtained through the additional use of a phycoerythrin-conjugated monoclonal antibody that selectively labels monocytes and polymorphonuclear granulocytes in peripheral blood. Killing is assessed by solubilizing blood cells with deoxycholate to liberate yeast cells from the phagocytes. Viable yeast cells retain carboxyfluorescein, but nonviable cells lose the fluorescent marker; thus, the reduction in number of fluorescent particles directly reflects phagocytic killing. Results obtained by the present method correlated excellently with parallel enumerations by colony counting. Test results with seven healthy individuals revealed a marked dissociation between the process of opsonophagocytosis, which was essentially complete after 20 min at 37 degrees C, and killing rates, which were 48% +/- 11% and 63% +/- 9% (standard deviation) after 1 and 2 h, respectively, when yeast cell-to-phagocyte ratios were in the range of 0.5:1 to 2:1. The described assay is unrivaled in simplicity, rapidity, and reproducibility and generates results for a large number of samples within hours.

Candida albicans↗
PubMed1991

Alpha-toxin of Staphylococcus aureus.

Alpha-toxin, the major cytotoxic agent elaborated by Staphylococcus aureus, was the first bacterial exotoxin to be identified as a pore former. The protein is secreted as a single-chain, water-soluble molecule of Mr 33,000. At low concentrations (less than 100 nM), the toxin binds to as yet unidentified, high-affinity acceptor sites that have been detected on a variety of cells including rabbit erythrocytes, human platelets, monocytes and endothelial cells. At high concentrations, the toxin additionally binds via nonspecific absorption to lipid bilayers; it can thus damage both cells lacking significant numbers of the acceptor and protein-free artificial lipid bilayers. Membrane damage occurs in both cases after membrane-bound toxin molecules collide via lateral diffusion to form ring-structured hexamers. The latter insert spontaneously into the lipid bilayer to form discrete transmembrane pores of effective diameter 1 to 2 nm. A hypothetical model is advanced in which the pore is lined by amphiphilic beta-sheets, one surface of which interacts with lipids whereas the other repels apolar membrane constitutents to force open an aqueous passage. The detrimental effects of alpha-toxin are due not only to the death of susceptible targets, but also to the presence of secondary cellular reactions that can be triggered via Ca2+ influx through the pores. Well-studied phenomena include the stimulation of arachidonic acid metabolism, triggering of granule exocytosis, and contractile dysfunction. Such processes cause profound long-range disturbances such as development of pulmonary edema and promotion of blood coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Lung vascular injury after administration of viable hemolysin-forming Escherichia coli in isolated rabbit lungs.

Escherichia coli hemolysin, a transmembrane pore-forming exotoxin, is considered an important virulence factor. In the present study, the possible significance of hemolysin production was investigated in a model of septic lung failure through infusion of viable bacteria in isolated rabbit lungs; 10(4) to 10(7) E. coli/ml perfusate caused a dose- and time-dependent appearance of hemolysin, accompanied by release of potassium, thromboxane A2, and PGI2 into the perfusate. Concomitantly, marked pulmonary hypertension developed. Inhibitor studies suggested that the pressor response was predominantly mediated by pulmonary thromboxane generation. Administration of hemolysin-forming E. coli additionally caused a protracted, dose-dependent increase in the lung capillary filtration coefficient, followed by severe edema formation. The permeability increase was independent of lung prostanoid generation. An E. coli strain that releases an inactive form of hemolysin completely failed to provoke the described biophysical and biochemical responses. Preapplication of 2 x 10(8) human granulocytes was without effect in the present experimental model. We conclude that the hemolysin produced by low numbers of E. coli organisms can provoke thromboxane-mediated pulmonary hypertension and severe vascular leakage. E. coli hemolysin and, possibly, other related cytolysins may thus contribute directly to the pathogenesis of acute respiratory failure under conditions of sepsis or pneumonia.

6-Ketoprostaglandin F1 alpha↗
PubMed1991

Escherichia coli hemolysin is a potent inductor of phosphoinositide hydrolysis and related metabolic responses in human neutrophils.

Escherichia coli hemolysin (Hly) is a proteinaceous pore-forming exotoxin that probably represents a significant virulence factor in E. coli infections. We investigated its influence on human polymorphonuclear neutrophils (PMN), previously identified as highly susceptible targets. Hly provoked rapid secretion of elastase and myeloperoxidase, generation of superoxide, and synthesis of platelet-activating factor (PAF) and lyso-PAF. Concomitantly, marked phosphatidylinositol (PtdIns) hydrolysis with sequential appearance of the inositol-phosphates, inositol-phosphates, inositol triphosphate, diphosphate, and monophosphate, respectively, and formation of diacylglycerol, occurred. The metabolic responses displayed distinct bell-shaped dose dependencies, with maximum events noted at low toxin concentrations of 0.1-0.5 hemolytic units per milliliter. PtdIns hydrolysis and metabolic responses elicited by Hly exceeded those evoked by optimal concentrations of formylmethionyl-leucyl phenylalanine, PAF, leukotriene B4, A23187, or staphylococcal alpha-toxin. The toxin-induced effects were sensitive toward modulators of PMN stimulus transmission pathways (pertussis toxin, the kinase C inhibitor H7, and phorbol myristate acetate "priming"). We conclude that the marked capacity of low doses of Hly to elicit degranulation, respiratory burst, and lipid mediator generation in human PMN probably envolves signal transduction via PtdIns hydrolysis.

Bacterial Proteins↗
PubMed1990

Inflammatory lipid mediator generation elicited by viable hemolysin-forming Escherichia coli in lung vasculature.

Escherichia coli hemolysin, a transmembrane pore-forming exotoxin, is considered an important virulence factor for E. coli-related extraintestinal infections and sepsis. The possible significance of hemolysin liberation for induction of inflammatory lipid mediators was investigated in isolated rabbit lungs infused with viable bacteria (concentration range, 10(4)-10(7)/ml). Hemolysin-secreting E. coli (E. coli-Hly+), but not an E. coli strain that releases an inactive form of the exotoxin, induced marked lung leukotriene (LT) generation with predominance of cysteinyl LTs. Eicosanoid synthesis was not inhibited in the presence of plasma with toxin-neutralizing capacity. Pre-application of 2 x 10(8) human granulocytes, which sequestered in the lung microvasculature, caused a severalfold increase in leukotriene generation in response to E. coli-Hly+ challenge both in the absence and presence of plasma. Data are presented indicating neutrophil-endothelial cell cooperation in arachidonic acid lipoxygenase metabolism as an underlying mechanism. We conclude that liberation of hemolysin from viable E. coli induces marked lipid mediator generation in lung vasculature, which is potentiated in the presence of neutrophil sequestration and may contribute to microcirculatory disturbances during the course of severe infections.

Animals↗