Biomedical subjects

I Walev

Publications and source records attributed to I Walev.

32 records · Page 2Linked to original sources

PubMed1995

On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety.

Combined treatment with trypsin, cholesterol esterase, and neuraminidase transforms LDL, but not HDL or VLDL, to particles with properties akin to those of lipid extracted from atherosclerotic lesions. Single or double enzyme modifications, or treatment with phospholipase C, or simple vortexing are ineffective. Triple enzyme treatment disrupts the ordered and uniform structure of LDL particles, and gives rise to the formation of inhomogeneous lipid droplets 10-200 nm in diameter with a pronounced net negative charge, but lacking significant amounts of oxidized lipid. Enzymatically modified LDL (E-LDL), but not oxidatively modified LDL (ox-LDL), is endowed with potent complement-activating capacity. As previously found for lipid isolated from atherosclerotic lesions, complement activation occurs to completion via the alternative pathway and is independent of antibody. E-LDL is rapidly taken up by human macrophages to an extent exceeding the uptake of acetylated LDL (ac-LDL) or oxidatively modified LDL. After 16 h, cholesteryl oleate ester formation induced by E-LDL (50 micrograms/ml cholesterol) was in the range of 6-10 nmol/mg protein compared with 3-6 nmol/mg induced by an equivalent amount of acetylated LDL. At this concentration, E-LDL was essentially devoid of direct cytotoxic effects. Competition experiments indicated that uptake of E-LDL was mediated in part by ox-LDL receptor(s). Thus, approximately 90% of 125I-ox-LDL degradation was inhibited by a 2-fold excess of unlabeled E-LDL. Uptake of 125I-LDL was not inhibited by E-LDL. We hypothesize that extracellular enzymatic modification may represent an important step linking subendothelial deposition of LDL to the initiation of atherosclerosis.

Arteriosclerosis↗

PubMed1995

Potassium-inhibited processing of IL-1 beta in human monocytes.

Agents that deplete cells of K+ without grossly disrupting the plasma membrane were found to stimulate the cleavage of pro-interleukin (IL)-1 beta to mature IL-1 beta. Agents examined in this study included staphylococcal alpha-toxin and gramicidin, both of which selectively permeabilize plasma membranes for monovalent ions, the ionophores nigericin and valinomycin, and the Na+/K+ ATPase inhibitor ouabain. K+ depletion by brief hypotonic shock also triggered processing of pro-IL-1 beta. The central role of K+ depletion for inducing IL-1 beta maturation was demonstrated in cells permeabilized with alpha-toxin: processing of pro-IL-1 beta was totally blocked when cells were suspended in medium that contained high K+, but could be induced by replacing extracellular K+ with Na+, choline+ or sucrose. To test whether K+ flux might also be important in physiological situations, monocytes were stimulated with lipopolysaccharide (LPS) for 1-2 h to trigger pro-IL-1 beta synthesis, and transferred to K(+)-rich medium. This maneuver totally suppressed IL-1 beta maturation. Even after 16 h, however, removal of K+ from the medium resulted in rapid processing and export of IL-1 beta. Ongoing export of mature IL-1 beta from cells stimulated with LPS for 2-6 h could also be arrested by transfer to K(+)-rich medium. Moreover, a combination of two K+ channel blockers inhibited processing of IL-1 beta in LPS-stimulated monocytes. We hypothesize that K+ movement and local K+ concentrations directly or indirectly influence the action of interleukin-1 beta-converting enzyme (ICE) and, possibly, of related intracellular proteases.

Antibody Specificity↗

PubMed1995

Characterization of Vibrio cholerae El Tor cytolysin as an oligomerizing pore-forming toxin.

V. cholerae El Tor cytolysin is a secreted, water-soluble protein of M(r) 60,000 that may be relevant to the pathogenesis of acute diarrhea. In this communication, we demonstrate that the toxin binds to and oligomerizes in target membranes to form SDS-stable aggregates of M(r) 200,000-250,000 that generate small transmembrane pores. Pores formed in erythrocytes were approximately 0.7 nm in size, as demonstrated by osmotic protection experiments. Binding was shown to occur in a temperature-independent manner preceding the temperature-dependent oligomerization step. Pores were also shown to be formed in L929 and HEp-2 cells, human fibroblasts and keratinocytes, albeit with highly varying efficacy. At neutral pH and in the presence of serum, human fibroblasts were able to repair a limited number of lesions. The collective data identify V. cholerae El Tor cytolysin as an oligomerizing toxin that damages cells by creating small transmembrane pores.

Adenosine Triphosphate↗

PubMed1995

Enhancement by TNF-alpha of reactivation and replication of latent herpes simplex virus from trigeminal ganglia of mice.

The influence of tumor-necrosis-factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukine-1 (IL-1) and IL-3 on the in vitro reactivation frequency and replication rate of trigeminal ganglia of mice latently infected with herpes simplex virus (HSV) strain KOS was studied. It could be demonstrated that TNF-alpha and possibility GM-CSF, but not IL-1 and IL-3, enhanced the reactivation frequency and replication of HSV. Interferon alpha/beta (IFN alpha/beta) prevented reactivation and replication.

Animals↗

PubMed1995

Correlation of virus replication, cytokine (TNF-alpha and IL-1) producing cells, neuronal necrosis and inflammation after intranasal infection of mice with herpes simplex virus strains of different virulence.

The number of TNF-alpha and IL-1 beta producing cells was investigated during the acute replication phase of herpes simplex virus (HSV) in trigeminal ganglia after intranasal infection with strains of different virulence. The highly virulent strain WAL replicated strongly and induced many cytokine producing cells early in the ganglia. The low virulent strain HFEM replicated less, only few cytokine producing cells were detected late. The thymidine-kinase negative (TK-) virus 1301 did not replicate but produced some lymphocytic inflammation. The higher the virulence of strains of HSV-1 or -2 was, the stronger was the extent of histopathological lesions; moreover, a dissociation in time between replication and cellular reaction (granulocytic and lymphocytic) could be observed after infection with strains HFEM and TK- virus 1301. CD4 and CD8 positive cells could be detected mainly at the rim of necrotic areas, TNF-alpha and IL-1 beta producing cells, however, were scattered throughout the ganglia.

Administration, Intranasal↗

PubMed1995

Binding, oligomerization, and pore formation by streptolysin O in erythrocytes and fibroblast membranes: detection of nonlytic polymers.

Streptolysin O (SLO) is a representative of the family of cholesterol-binding cytolysins that form large pores in target cell membranes. Aggregation of the toxin to polymeric structures is required for pore formation. However, it is not known whether, vice versa, polymers may under certain circumstances remain nonfunctional, and whether this might be the cause underlying the relative resistance of certain cells towards toxin action. In the present study, we applied radioiodinated, functionally active SLO to human, rabbit, and mouse erythrocytes and to human fibroblasts and keratinocytes. Binding and polymerization were quantified and correlated with membrane damage. At low toxin concentrations, human and rabbit but not mouse erythrocytes were lysed, but binding and polymerization of SLO were essentially identical in all cases. Nonlytic polymers were also detected on human fibroblasts and keratinocytes treated with subcytotoxic concentrations of SLO, and quantitative estimates indicated that nonpermeabilized cells could carry hundreds of polymers on their surface. When applied at low concentrations to fibroblasts, much of the toxin remained in monomer form and was subsequently shed from the cells. This was shown by monitoring the fate of radioiodinated toxin and also by using a sensitive cell enzyme-linked immunosorbent assay that permitted immunological detection of surface-exposed SLO. Thus, relative resistance of cells towards the permeabilizing action of SLO may be due to their ability to tolerate formation of a limited number of SLO polymers and to shedding of nonoligomerized toxin from their surface.

Animals↗

PubMed1994

Recovery of human fibroblasts from attack by the pore-forming alpha-toxin of Staphylococcus aureus.

When applied at low concentrations (< 10 micrograms/ml), staphylococcal alpha-toxin generates a small channel in keratinocyte and lymphocyte membranes that permits selective transmembrane flux of monovalent ions. Here we show that a moderate concentration (1-50 micrograms/ml) of alpha-toxin similarly produces a small pore in membranes of human fibroblasts. This process leads to rapid leakage of K+ and to a drop in cellular ATP to 10-20% of normal levels in 2 h. In the presence of medium supplemented with serum and at pH 7.4, the cells are able to recover from toxin attack, so that normal levels of K+ and ATP are reached after 6-8 h at 37 degrees C. The repair process is dependent on the presence of serum in the medium and is very sensitive towards pH. Decreases of pH in the medium to < or = 7.0 as well as increases to > or = 7.8 causes the repair mechanism to fail. The fate of cell-bound toxin molecules was investigated by using a radiolabelled tracer and by immunological detection of toxin exposed at the cell surface. The results indicated that 50-70% of the toxin was shed from cell membranes. However, there was no clear correlation between shedding and recovery, and shedding was also observed in cells that died at pH 7.8. Shedding was not decisive for repair, since cells that had recovered from toxin attack continued to carry 30-40% of initially bound toxin on their cell surface. Blockade of Na+/K(+)-ATPases with ouabain evoked similar kinetics of K(+)-depletion in control cells, compared with cells that had just recuperated from toxin attack and that still carried 30-40% alpha-toxin on their surface. We therefore tentatively concluded that repair of alpha-toxin lesions was due to closure of small pores, rather than from compensation of membrane leaks by up-regulation of Na+/K(+)-ATPase activity. We speculate that repair of small membrane lesions may extend to other agents that produce channels of similar nature in nucleated cells. Larger pores created by E. coli hemolysin or streptolysin O, both of which form larger functional transmembrane lesions, could not be repaired by fibroblasts.

Adenosine Triphosphate↗

PubMed1994

Cyclosporin A resistance of herpes simplex virus-induced "fusion from within" as a phenotypical marker of mutations in the Syn 3 locus of the glycoprotein B gene.

We here report research in which nine strains of Herpes simplex virus (HSV) with fusing activity were investigated in order to establish precise phenotypical markers of mutations in the carboxy terminus of glycoprotein B (gB). The gene region encoding the carboxy terminus of gB was isolated, then cloned, and finally sequenced. Our investigation showed that seven strains have different mutations in the syn 3 locus. We observed no base difference in the gB gene region encoding the carboxy terminus of gB of two other strains. Strains with a mutation in the carboxy terminus of gB induced fusion from within (FFWI) in the presence of Cyclosporin A (CyA) at a concentration up to 150 microM. There are two clusters of mutations correlated with the syn 3 locus and selected in the presence of CyA: One group comprised of amino acid substitutions at position 816, the other of changes at positions 853, 854, and 857. In contrast, the fusion induced by strains with mutations in other syn loci is CyA sensitive. CyA inhibits the FFWI at concentrations of 20-60 microM. The results demonstrate the CyA resistance of HSV-induced FFWI should serve as a phenotypical marker of mutations in the carboxy terminus of gB. Moreover, our investigations revealed that fusion from without (FFWO) does not always serve as a phenotypical marker of mutations in the syn 3 locus. On the one hand, all FFWO-positive strains possess a syn 3 locus mutation, whilst, on the other hand, five strains with mutations in the carboxy terminus of gB are FFWO negative.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation.

Peripheral-blood human T lymphocytes were treated with Staphylococcus aureus alpha-toxin. Membrane permeabilization was assessed by measuring efflux of K+ and Rb+ and influx of Na+, Ca2+, and propidium iodide. Cellular ATP and [3H]thymidine incorporation following lectin stimulation were measured as parameters for cell viability. Internucleosomal cleavage characteristic of programmed cell death was assessed by agarose gel electrophoresis and by quantifying low-molecular-weight, [3H]thymidine-labeled DNA fragments. Nanomolar concentrations of alpha-toxin evoked protracted, irreversible ATP depletion in both activated and resting T lymphocytes. Toxin-damaged cells also lost their ability to incorporate [3H]thymidine upon subsequent stimulation with phytohemagglutinin. These cells carried toxin hexamers, and their plasma membranes became permeable for monovalent ions but not for Ca2+ and propidium iodide. The permeabilization event was followed by internucleosomal DNA degradation characteristic of programmed cell death. Membranes of cells treated with high toxin doses (> 300 nM) became permeable to both Ca2+ and propidium iodide. In this case, ATP depletion occurred within minutes and no DNA degradation was observed. When cells were suspended in Na(+)-free buffer, alpha-toxin applied at low doses still bound and formed hexamers. However, these cells displayed neither DNA degradation nor loss of viability. The data indicate that formation of very small but not of large alpha-toxin pores may trigger programmed cell death in lymphocytes and that uncontrolled flux of Na+ ions may be an important event precipitating the suicide cascade.

Adenosine Triphosphate↗

PubMed1993

A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes.

Depending on the size of the pores one wishes to produce in plasma membranes, the choice will probably fall on one of the three toxins discussed above. S. aureus alpha-toxin should be tried first when pores of 1-1.5 nm diameter are required. This is generally the case when Ca2+ and nucleotide dependence of a given process is being studied. If alpha-toxin does not work, this is probably due to the fact that the toxin either does not produce pores, or that the pores are too small. In this case, high concentrations of alpha-toxin should be tried. If this still does not work, we recommend the use of HlyA. When very large pores are to be created, e.g. for introduction of antibodies into the cells, SLO or another member of this toxin family are the agents of choice. SLO preparations need to be checked for presence of protease contaminants. Tetanolysin currently offers advantages since it is protease-free, and the size of the pores can probably be controlled by varying the toxin dose. Methods for assessing the size of pores created by such agents have been published in the recent literature, and the appropriate papers can be consulted whenever the need arises.

Bacterial Proteins↗

PubMed1993

Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions.

Incubation of human keratinocytes with nanomolar concentrations of Staphylococcus aureus alpha-toxin leads to irreversible depletion of cellular ATP. The toxin forms hexamers in the target cell membranes, and rapid transmembrane flux of K+, Na+, and 86Rb+ is observed. Unexpectedly, pores formed in keratinocytes through application of low but lethal doses of alpha-toxin appeared to be considerably smaller than those formed in erythrocyte membranes. They permitted neither rapid influx of Ca2+ or propidium iodide, nor efflux of carboxyfluorescein. Larger pores allowing flux of all three markers did form when the toxin was applied at high concentrations. Flux of monovalent ions and reduction in cellular ATP levels evoked by low toxin doses correlated temporally with a fall in oxygen consumption, which was interpreted to reflect breakdown of mitochondrial respiration. The lethal event could not be thwarted by manipulating the extracellular K+ or Ca2+ concentrations. Realization that alpha-toxin may form very small pores in nucleated cells is important for future research on cellular toxin effects and membrane repair processes.

Bacterial Toxins↗

PubMed1992

Relationship between HLA I surface expression and different cytopathic effects produced after herpes simplex virus infection in vitro.

In the present study, we investigated the effects of herpes simplex virus (HSV) infection on the expression of HLA class I antigens and beta 2-microglobulin in human fibroblasts. The mRNA abundance for HLA class I was shown to be strongly reduced after infection with HSV strains either producing cell rounding or fusion from within (FFWI), however, HLA class I expression on the surface of cells is strongly reduced only after appearance of FFWI. Using a ts mutant (ts 78R) or CyA in combination with a fusion from without (FFWO) inducing strain of HSV, this loss of HLA class I antigens is assumed to be correlated to the rearrangement of the cell membrane during the fusion process itself as a late event of cytopathogenicity.

Blotting, Northern↗

PubMed1991

Characterization of fusion from without induced by herpes simplex virus.

The process of fusion from without (FFWO) induced by herpes simplex virus (HSV) was analyzed by using various inhibitors and compared to fusion from within (FFWI). The fate of certain elements of the cytoskeleton after FFWO was also investigated. Our experiments demonstrate FFWO as a very suitable system for study of early virus-cell interactions. Zn++ ions proved inhibitory for penetration whilst pretreatment of cells with Ca++ ions before infection enhanced FFWO activity. Dissociation of penetration from the fusion process itself was possible by use of Zn++ ions, low pH-treatment and antiserum on the one hand and N-ethylmaleimide and cytochalasin D on the other. Penetration itself needs only 6 min or less to proceed. FFWO is independent of inhibitors of glycosylation (tunicamycin) and intracellular vesicular traffic (monensin), protein-synthesis (cycloheximide) and energy-delivery (2.4 dinitrophenol and Na-azide). Analyzed strains of HSV-1 and -2 producing FFWI could be subgrouped into three categories: Strain ANG with high, strain HFEM and Lux with low and strains IES, Len, MP, US with no FFWO activity. The results of these experiments indicate that the property of FFWO is not purely a consequence of the number of PFU but depends on certain inherent properties of the virus particles. Addition of heparin as well as treatment of cells with heparitinase effectively prevented FFWO, indicating identical virus receptors for entrance of virus into cells and FFWO. During our studies several calf sera were found to inhibit FFWO-activity. Inhibition of FFWO by a glycoconjugate (ferritin coupled with oleic acid) indicates specific stereochemical hindrance of FFWO by this compound. Shortly after FFWO the actin filaments rearrange to form long fibres and surface fibronectin is being lost from the cell membrane.

Animals↗

PubMed1991

Differentiation of herpes simplex virus-induced fusion from without and fusion from within by cyclosporin A and compound 48/80.

Treating strains of herpes simplex virus (HSV) in culture with either cyclosporin A or compound 48/80, allowed the strains to be divided into two groups. Group 1 contains the strains ANG and HFEM of HSV-1 and Lux syn (HSV-2) producing fusion from within (FFWI) and fusion from without (FFWO). Cyclosporin A fails to inhibit both types of fusion at concentrations up to 100 microM. Strains ANG and HFEM belong to the syn 3 marker locus group identified for HSV-1. Group 2 contains all other fusion-producing strains of HSV tested so far. Cyclosporin A inhibits FFWI at concentrations as low as 10 to 20 microM. These strains belong to the syn locus marker groups 1, 2, 4 and 5. From the fact that mutations in glycoprotein B belong to the syn 3 marker group we conclude that glycoprotein B is of major importance for FFWO. Compound 48/80 also differentiates between these two groups of viruses. O-Acetyl cyclosporin A is unable to inhibit FFWI induced by group 2 viruses; in contrast, cyclosporin H and the Ca2+ ionophore A23187 exert inhibition effects similar to those exerted by cyclosporin A. We conclude from the effects of these compounds that binding properties of the OH group of cyclosporin A and an increase of Ca2+ ions may be preconditions for the observed effects. Binding of cyclosporin A to cyclophilin does not appear to be responsible for these effects.

Animals↗