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Biomedical subjects

S Bhakdi

Publications and source records attributed to S Bhakdi.

At least 145 records · Page 8Linked to original sources
PubMed1990

Effects of Escherichia coli hemolysin on endothelial cell function.

Escherichia coli hemolysin is considered an important virulence factor in extraintestinal E. coli infections. The present study demonstrates that cultured pulmonary artery endothelial cells are susceptible to attack by low concentrations of E. coli hemolysin (greater than or equal to 0.05 hemolytic units/ml; greater than or equal to 5 ng/ml). Sublytic amounts of hemolysin increased the permeability of endothelial cell monolayers in a time- and dose-dependent manner. The hydraulic conductivity increased approximately 30-fold and the reflection coefficient for large molecules dropped from 0.71 to less than 0.05, indicating a toxin-induced loss of endothelial barrier function. The alterations of endothelial monolayer permeability were accompanied by cell retraction and interendothelial gap formation. In addition, E. coli hemolysin stimulated prostacyclin synthesis in endothelial cells. This effect was strictly dependent on the presence of extracellular Ca2+ but not of Mg2+. An enhanced passive influx of 45Ca2+ and 3H-sucrose but not of tritiated inulin and dextran was noted in toxin-treated cells, indicating that small transmembrane pores comparable to those detected in rabbit erythrocytes had been generated in endothelial cell membranes. These pores may act as nonphysiologic Ca2+ gates, thereby initiating different Ca2+-dependent cellular processes. We conclude that endothelial cells are highly susceptible to E. coli hemolysin and that two major endothelial cell functions are altered by very low concentrations of hemolysin.

Animals↗
PubMed1990

Leukotriene and hydroxyeicosatetraenoic acid generation elicited by low doses of Escherichia coli hemolysin in rabbit lungs.

Low doses of Escherichia coli hemolysin cause thromboxane-mediated hypertension and vascular leakage in blood-free perfused rabbit lungs (W. Seeger, H. Walter, N. Suttorp, M. Muhly, and S. Bhakdi, J. Clin. Invest. 84:220-227, 1989). The recirculating buffer medium and bronchoalveolar lavage fluid from lungs exposed to hemolysin (2.5 hemolytic units per ml) in the presence of cyclooxygenase inhibitor were analyzed for leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) by reverse-phase and straight-phase high-pressure liquid chromatographic techniques combined with UV spectrum analysis and post-high-pressure liquid chromatography radioimmunoassay. A rapid release of large amounts of cysteinyl-LTs and leukotriene B4 (LTB4) into the intravascular space was noted (total sum, approximately 4 to 5 micrograms). Similar quantities have hitherto been elicited only by high concentrations of the artificial calcium ionophore A 23187. Moreover, a marked liberation of 5-HETE and 12-hydroxyheptadecatrienoic acid into the buffer medium occurred, whereas LTB4 represented the predominant compound in the lavage fluid. The hemolysin-induced burst of LT and HETE generation preceded the onset of vascular leakage. The outstanding capacity of E. coli hemolysin to produce the liberation of potent lipid mediators is probably relevant to the pathways of vascular injury and amplification of inflammatory events during severe infection with hemolytic E. coli strains.

Animals↗
PubMed1990

Effects of Escherichia coli hemolysin on human monocytes. Cytocidal action and stimulation of interleukin 1 release.

This study reports on the potent cytocidal and interleukin-1 releasing properties of Escherichia coli hemolysin (ECH) on human monocytes. Nanomolar concentrations of purified ECH (250-2,000 ng/ml) caused rapid and irreversible depletion of cellular ATP to levels below 20% of controls within 60 min. Subcytocidal doses (10-200 ng/ml) of ECH induced rapid release within 60-120 min of large amounts of interleukin 1 beta (IL-1 beta) from cultured monocytes. IL-1 beta release occurred in the presence of actinomycin D and cycloheximide, and was thus probably due to processing and export of intracellular IL-1 beta precursor. Incubation of toxin-producing E. coli at ratios of only 0.3-3 colony-forming units per monocyte evoked approximately 50% depletion of total cellular ATP within 90 min. Toxin producers also stimulated synthesis and release of large amounts of interleukin 1, but not of tumor necrosis factor within the same time span. In contrast, non-toxin producers caused neither cell death nor rapid interleukin 1 release. Stimulation of rapid interleukin 1 release coupled with potent cytocidal effects on cells of monocytic origin may represent pathogenetically significant events incurred by bacterial strains that produce ECH and related cytolysins.

Adenosine Triphosphate↗
PubMed1990

Pathogenesis of dengue: an alternative hypothesis.

This paper presents a novel but entirely hypothetical concept on the pathogenesis of the shock syndrome (DSS) associated with dengue hemorrhagic fever (DHF). Antibody-dependent enhancement (ADE) is widely thought to be central to the development of these clinical entities. Current views on the mechanisms underlying ADE centre on two major lines of thought: 1) Non-neutralizing antibodies to dengue virus (DV) can enhance viral uptake and replication in target cells (monocytes). 2) DHF/DSS are the consequences of enhanced viral replication, paired with immunopathological processes that are evoked by monocyte dysfunction and detrimental reactions caused by activated T-lymphocytes. The present hypothesis proposes, by contrast, a central role for the following processes: 1) Secondary infection of an individual who has sub or non-neutralizing antibody titers against DV leads to a booster antibody response and a steep rise in antibody levels. 2) Antibodies against DV bind to and direct a selective attack of the complement system onto cells expressing viral antigens on their surface. DHF/DSS are the direct and indirect consequences of complement activation on these cells. The advanced hypothesis, which departs from the mainstream of "immune enhancement" concepts, can easily be tested by experimentation.

Antigens, Viral↗
PubMed1990

[Functions and relevance of the terminal complement sequence].

The terminal complement sequence is initiated upon cleavage of C5 with liberation of C5a anaphylatoxin, and involves the assembly of macromolecular C5b-9 complexes either on cell surfaces or in plasma. Cell-bound C5b-9 complexes generate transmembrane pores that can cause cell death, or they can elicit secondary cellular reactions triggered, for example, by passive flux of calcium ions into the cells. In vivo functions of the fluid-phase SC5b-9 complex have not yet been defined, but the identity of S-protein with vitronectin (serum spreading factor) provokes the anticipation that significant biological functions of this complex do exist. The terminal complement sequence may fulfill protective functions when it is triggered on alien cells that are marked for destruction. Dysregulation in the complement sequence may, however, result in detrimental attack by C5b-9 on autologous cells. Examples include not only autoimmune disease states, but also the activation of complement on dead or dying cells, and bystander attack on blood cells during cardiopulmonary bypass. Methods for detecting and quantifying C5b-9 are outlined, and the potential usefulness of such assays in clinical research is discussed.

Animals↗
PubMed1990

Staphylococcal alpha-toxin-induced vascular leakage in isolated perfused rabbit lungs.

alpha-Toxin, the major, pore-forming exotoxin of Staphylococcus aureus, caused acute hypertension when perfused through blood-free rabbit lungs (21). This reaction is mediated by pulmonary thromboxane generation, for which toxin-induced calcium flux into target cells with subsequent stimulation of arachidonic acid metabolism is predominantly responsible. In the present study, we investigated the effects of alpha-toxin on the integrity of the lung microvasculature. Thromboxane generation was inhibited in all experiments to suppress the development of pulmonary hypertension. Application of low alpha-toxin concentrations (5 to 40 ng/ml) induced protracted, severe vascular leakage in a dose-dependent manner. After a lag period of 40 to 120 minutes, gravimetrically determined capillary filtration coefficients progressively increased to greater than 10-fold values, and this was followed by pronounced weight gain of the isolated organs. These physiologic alterations were paralleled by dose- and time-dependent structural changes documented by electron microscopic examination of perfusion-fixed lungs. Increasing electron density of microvascular endothelial cell nuclei and subsequently of their cytoplasma was noted, followed by detachment of these cells from the mutual endoepithelial basal lamina. Edema was localized in the blood-gas exchange area, in contrast to hydrostatically induced lung fluid accumulation. These results identify pulmonary microvascular endothelium cells as highly susceptible targets for attack by alpha-toxin. Given a similar sensitivity of human endothelial cells, alpha-toxin might directly contribute to the pathogenesis of acute respiratory failure under conditions of severe infection with Staphylococcus aureus.

Animals↗
PubMed1989

Biotinylation: a simple method for labelling complement component C8 with preservation of functional activity.

Biotinylation of human C8 with the water-soluble biotin derivative biotinylamidohexanoic acid, N-hydroxysulfosuccinimide ester is an excellent method for labelling this terminal complement component with preservation of its functional activity. The biotinylated product can be detected both in native form and also following its incorporation into the terminal complement complexes. Detection assays include Western blotting, crossed immunoblotting, ELISA, and immunocytochemistry. Biotinylation is an attractive alternative method for labelling C8 and may be used for detecting and quantifying C8 and C5b-9 complexes in their soluble and membrane-bound forms.

Biotin↗
PubMed1989

Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes.

The contribution of Escherichia coli hemolysin (ECH) to bacterial virulence has been considered mainly in context with its hemolytic properties. We here report that this prevalent bacterial cytolysin is the most potent leukocidin known to date. Very low concentrations (approximately 1 ng/ml) of ECH evoke membrane permeability defects in PMN (2-10 x 10(6) cells/ml) leading to an efflux of cellular ATP and influx of propidium iodide. The attacked cells do not appear to repair the membrane lesions. Human serum albumin, high density and low density lipoprotein, and IgG together protect erythrocytes and platelets against attack by even high doses (5-25 micrograms/ml) of ECH. In contrast, PMN are still permeabilized by ECH at low doses (50-250 ng/ml) in the presence of these plasma inactivators. Thus, PMN become preferred targets for attack by ECH in human blood and protein-rich body fluids. Kinetic studies demonstrate that membrane permeabilization is a rapid process, ATP-release commencing within seconds after application of toxin to leukocytes. It is estimated that membrane permeabilization ensues upon binding of approximately 300 molecules ECH/PMN. This process is paralleled by granule exocytosis, and by loss of phagocytic killing capacity of the cells. The recognition that ECH directly counteracts a major immune defence mechanism of the human organism through its attack on granulocytes under physiological conditions sheds new light on its possible role and potential importance as a virulence factor of E. coli.

Adenosine Triphosphate↗
PubMed1989

Introduction of macromolecules into bovine adrenal medullary chromaffin cells and rat pheochromocytoma cells (PC12) by permeabilization with streptolysin O: inhibitory effect of tetanus toxin on catecholamine secretion.

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.

Adrenal Medulla↗
PubMed1989

Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo.

Alpha-toxin, the major cytolysin of Staphylococcus aureus, preferentially attacks human platelets and cultured monocytes, thereby promoting coagulation and the release of interleukin-1 and tumor necrosis factor. Titers of naturally occurring antibodies in human blood are not high enough to substantially inhibit these pathological reactions. In the present study, F(ab')2 fragment preparations from hyperimmune globulin obtained from immunized volunteers were tested for their capacity to inhibit the cytotoxic action of alpha-toxin in vitro and in vivo. These antibody preparations exhibited neutralizing anti-alpha-toxin titers of 80 to 120 IU/ml, whereas titers in commercial immunoglobulin preparations were 1 to 4 IU/ml. In vitro, the presence of 2 to 4 mg of hyperimmune globulin per ml protected human platelets against the action of 1 to 2 micrograms of alpha-toxin per ml. Similarly, these antibodies fully protected human monocytes against the ATP-depleting and cytokine-liberating effects of 0.1 to 1 microgram of alpha-toxin per ml. Intravenous application of 0.5 mg (85 to 120 micrograms/kg of body weight) of alpha-toxin in cynomolgus monkeys elicited acute pathophysiological reactions which were heralded by a selective drop in blood platelet counts. Toxin doses of 1 to 2 mg (170 to 425 micrograms/kg) had a rapid lethal effect, the animals presenting with signs of cardiovascular collapse and pulmonary edema. Prior intravenous application of 4 ml of hyperimmune globulins per kg inhibited the systemic toxic and lethal effects of 1 mg (200 micrograms/kg) of alpha-toxin. In contrast, normal human immunoglobulins exhibited no substantial protective efficacy in vitro and only marginal effects in vivo. It is concluded that high-titered anti-alpha-toxin antibodies effectively protect against the cytotoxic actions of alpha-toxin.

Animals↗
PubMed1989

Release of interleukin-1 beta associated with potent cytocidal action of staphylococcal alpha-toxin on human monocytes.

The pathogenetic relevance of Staphylococcus aureus alpha-toxin in humans has been debated because human cells have been thought to display a natural resistance toward the cytotoxic action of this cytolysin. Following our previous demonstration that human platelets represent sensitive targets for toxin attack, we have now identified monocytes as a second, highly vulnerable human cell species that succumb to attack by low doses (20 ng/ml) of alpha-toxin. The cytotoxic action of alpha-toxin is reflected in a rapid depletion of cellular ATP that is essentially complete within 30 min. The presence of human plasma proteins affords some protection of monocytes against the action of the toxin. In 10% autologous serum, ATP depletion commences at 80 to 300 ng of toxin per ml. Subcytolytic doses stimulate the release of tumor necrosis factor alpha, a process that is slightly accentuated in the presence of 50% serum. Cytocidal toxin doses unfailingly cause the release of large amounts of interleukin-1 beta from cultured cells, with levels of this monokine generally exceeding 10 ng/ml in the cell supernatants 60 min after application of toxin. Initial evidence suggests that this is due to processing of intracellular interleukin-1 rather than to de novo synthesis of the cytokine. All noted effects are abrogated in the presence of a neutralizing monoclonal antibody against alpha-toxin. Through its capacity to provoke cytokine release from monocytes and its attack on platelets, alpha-toxin may initiate cellular events that are relevant to the pathogenesis of staphylococcal infection.

Adenosine Triphosphate↗
PubMed1989

Quantitative study of the binding and hemolytic efficiency of Escherichia coli hemolysin.

Mono- and polyclonal antibodies were used to construct a sandwich enzyme-linked immunosorbent assay that permitted quantitation of Escherichia coli hemolysin in soluble and membrane-bound forms. Toxin concentrations of 4 to 14 micrograms/ml were measured in culture supernatants of E. coli LE 2001 at times of peak hemolytic activity. Quantitative studies on the binding of E. coli hemolysin to rabbit erythrocytes were conducted at 0 and 37 degrees C. At 37 degrees C, 85 to 95% of bindable toxin was cell bound after 60 min, and no saturability of binding was observed in the studied range of concentrations, which resulted in deposition of approximately 100 to 50,000 toxin molecules per cell. Binding was slower and less effective at 0 degrees C; however, hemolysis did occur at low temperature. The number of cell-bound toxin molecules required to generate a hemolytic lesion within 60 min was estimated to be approximately 100 molecules per cell at 37 degrees C and 800 to 1,000 molecules per cell at 0 degrees C. Upon prolonged incubation (5 to 20 h, 37 degrees C), the number of molecules evoking a functional lesion decreased to approximately 5 to 20 per cell. These results are compatible with the concept that E. coli hemolysin first adsorbs to the cell surface, with membrane insertion and pore formation following in a second step that may be temporally dissociated from that of binding. The data support the pore concept of toxin action by showing that attachment of a low and finite number of toxin molecules to an erythrocyte will ultimately generate a cytolytic lesion.

Animals↗
PubMed1989

Local complement activation, thromboxane-mediated vasoconstriction, and vascular leakage in isolated lungs. Role of the terminal complement sequence.

In situ complement activation was induced in isolated, ventilated, and blood-free perfused rabbit lungs. Six to 11% human serum (vol/vol) was repeatedly admixed to the recirculating Krebs Henseleit buffer during 10-min periods, interrupted by rinsing phases with serum-free medium. This caused dose-dependent, reversible, and reproducible pulmonary artery pressor responses with pressure peaks or plateaus of 8 to 22 mm Hg. Pressor responses were paralleled by a marked release of thromboxane (TxA2) and prostaglandin I2 into the medium. Heat-inactivated serum, purified fluid-phase SC5b-9, and serum that had been activated with inulin in the absence or presence of a carboxypeptidase inhibitor all failed to elicit pressor responses. Both pressor response and prostanoid release were strictly dependent on complement factor C8, and evidence for C8 binding was obtained with the use of 125I-C8. Inhibitor studies collectively indicated that TxA2 was the predominant vasoconstrictive agent. Although repetitive short-term application of human serum induced no major lung weight gain, prolonged exposure to serum (40 to 80 min) without intermittent rinsing phases caused a delayed increase in the capillary filtration coefficient to maximally 3- to 4-fold values, with marked lung edema formation. We conclude that in situ activation of complement in the rabbit lung vasculature causes immediate intrapulmonary prostanoid generation and thromboxane-mediated vasoconstriction, independent of circulating leukocytes, but dependent on the formation of terminal membrane-complement complexes. Continuous complement activation results in a delayed increase in lung vascular permeability.

Animals↗
PubMed1989

Thromboxane-mediated hypertension and vascular leakage evoked by low doses of Escherichia coli hemolysin in rabbit lungs.

Escherichia coli hemolysin has been implicated as a pathogenicity factor in extraintestinal E. coli infections including sepsis. In the present study the effects of intravascular administration of hemolysin were investigated in isolated blood-free perfused rabbit lungs. Low concentrations of the toxin in the perfusate (0.05-5 hemolytic units/ml, corresponding to approximately 5-500 ng/ml), caused a dose- and time-dependent release of potassium, thromboxane A2, and prostaglandin I2, but not of lactate dehydrogenase, into the recirculating medium, as well as a dose-dependent liberation of the prostanoids into the bronchoalveolar space. These events were paralleled by a dose-dependent pulmonary hypertension, and studies with different inhibitors collectively indicated that the vasoconstrictor response was mediated predominantly by pulmonary thromboxane generation. In addition, E. coli hemolysin elicited a protracted, dose-dependent increase in the lung capillary filtration coefficient, which was independent of the prostanoid-mediated pressor response and resulted in severe pulmonary edema formation. We conclude that E. coli hemolysin can elicit thromboxane-mediated pulmonary hypertension combined with severe vascular leakage in isolated lungs in the absence of circulating inflammatory cells and humoral mediator systems, mimicking the key events in the development of acute respiratory failure in states of septicemia.

Animals↗
PubMed1989

Immunohistochemical study of complement S protein (Vitronectin) in normal and diseased human kidneys: relationship to neoantigens of the C5b-9 terminal complex.

The localization of S protein (Vitronectin) antigen was studied by indirect immunofluorescence and immunoelectron microscopy in normal adult human kidneys and in biopsy specimens from patients with a wide range of renal diseases, and compared with that of neoantigens of the C5b-9 terminal complement complex. S protein antigen was diffusely present in arteriolar perimyocytic matrices, the glomerular basement membrane and mesangial matrix, and tubular basement membranes in the cortex of normal and diseased kidneys without superimposable staining for C5b-9 neoantigens. Cell remnants embedded in normal and sclerotic extracellular matrices expressed S protein antigen and also stained for C5b-9 neoantigens. Several lines of evidence suggested that S protein present in connective matrices most likely represents S protein or C5b-9 complexes trapped from the circulation. Glomerular immune deposits and arteriolar hyalin deposits which contained C5b-9 neoantigens also contained S protein antigen in the same location. In a few specimens from patients with membranous nephritis stage I and IgA nephropathy, immune deposits contained neither detectable C5b-9 neoantigens nor S protein. The observed strong co-staining of immune deposits for S-protein and C5b-9 caution against the generalization that C5b-9 within glomerular immune deposits represent membrane-bound cytolytic complement complexes.

Adult↗
PubMed1989

Terminal complement complex and endothelial cells.

The effects of the terminal complement sequence on two endothelial cell functions (prostacyclin (PGI2) generation and permeability of an endothelial cell monolayer) were examined in antibody-sensitized pulmonary artery endothelial cells. Whereas C5b-7 complement complexes induced on PGI2 formation and no enhanced endothelial permeability, addition of purified complement component C8 resulted in a time- and dose-dependent burst of PGI2 release and in a substantially increased permeability of an endothelial cell monolayer 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. One minute after addition of C8 to endothelial cells carrying C5b-7 complexes, a six to sevenfold enhanced passive influx of 45Ca2+ into the cells was noted. An enhanced passive influx was also observed for 51CrO4(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 and the actin-myosin system.

Arachidonic Acid↗
PubMed1989

Prelesional complement activation in experimental atherosclerosis. Terminal C5b-9 complement deposition coincides with cholesterol accumulation in the aortic intima of hypercholesterolemic rabbits.

Cholesterol accumulation in the tunica intima of large and medium sized arteries is a characteristic of atherosclerotic lesion development. Cholesterol activates the complement system in vitro generating C5b-9 complexes, and C5b-9 antigens have been observed in atherosclerotic lesions. We therefore investigated whether complement activation correlates temporally with accumulation of intimal cholesterol in vivo. Frozen sections of aortic tissue from rabbits fed a 0.3% cholesterol diet for 0, 2, 6, and 10 weeks were stained with antibodies directed against neoantigens of the C5b-9 complex. Lipids were detected with oil red O, filipin, and antibodies against apoprotein B. C5b-9 complexes were observed to colocalize with extracellular lipid in the subendothelial space beginning at 2 weeks on the diet and extending past 10 weeks. Quantitation of aorta extracted C5b-9 complexes by an enzyme-linked immunosorbent assay revealed a progressive increase in aortic C5b-9 content as a function of time on the cholesterol diet. An approximately 30-fold increase in aortic C5b-9 occurred between 0 and 10 weeks on the diet, whereas plasma C5b-9 remained at normal levels throughout this time. Aortic cholesterol and triglyceride levels approximately doubled between 0 and 10 weeks on the diet and plasma cholesterol increased nearly 50-fold. Intimal C5b-9 deposition preceded monocyte infiltration and foam cell development. The data indicates that early cholesterol accumulation in the aortic intima results in complement activation. Since complement activation products are chemotactic for monocytes and promote their adhesion to the endothelium, complement may provide a mechanism by which monocytes are attracted to arterial regions of lipid accumulation.

Animals↗