Publications

1985
Poly-L-arginine 'opsonizes' nuclei for phagocytosis by mouse fibroblasts
Hubner GE, Voigt W-H, Schlumberger HD, Ginsburg I. Poly-L-arginine 'opsonizes' nuclei for phagocytosis by mouse fibroblasts. IRCS Medical Science. 1985;13 (10) :934-935. poly-l-arginine_opsonizes.pdf
Superoxide generation by human blood leucocytes under the effect of cytolytic agents
Ginsburg I, Borinski R, Pabst M. Superoxide generation by human blood leucocytes under the effect of cytolytic agents. International journal of tissue reactions. 1985;7 (2) :143-147.Abstract
Human blood leucocytes generate large amounts of superoxide following stimulation by polyarginine, polyanetholesulphonate and mixtures of a variety of soluble agents. Generation of O2-. by the various "cocktails" of soluble ligands is markedly enhanced by cytochalasins A, B, C, D, E and F. The efficiency of cytochalasin A is, however, at least 50-fold greater than that of the other cytochalasins. Leucocytes that have been treated for a few minutes with the cytolytic agents saponin, digitonin and lysolecithin undergo lysis and lose their superoxide-producing capacities, when a variety of soluble ligands are employed to stimulate superoxide production. A partial reactivation of the superoxide-producing capacities of the leucocytes can be achieved by adding NADPH. However, as the concentration of the cytolytic agents increases, reactivation of the cytochrome C reduction is less inhibitable by SOD, suggesting that cell lysis releases reductases of cytochrome C not connected with the superoxide-producing system of the leucocytes. Both saponin and digitonin can totally replace polyarginine as ingredients of the "cocktail," suggesting that these agents may also function as "priming agents" for superoxide production which can, however, further be enhanced by the addition of mixtures of soluble agents. Thus, leucocytes which had been lysed by membrane-active agents can nevertheless produce superoxide if adequate amounts of NADPH are added.
Persistence of staphylococcal cell-wall components in inflammatory sites may be due to the modulation by sulphated polyelectrolytes of autolytic wall enzymes: a working hypothesis
Ginsburg I, Lahav M, Sadovnik M, Goultchin J, Wecke J, Giesbrecht P. Persistence of staphylococcal cell-wall components in inflammatory sites may be due to the modulation by sulphated polyelectrolytes of autolytic wall enzymes: a working hypothesis. International journal of tissue reactions. 1985;7 (4) :255-261.Abstract
The interaction of leucocytes with Staphylococcus aureus results in killing of the bacterial cells, but large portions of the bacterial cell walls persist apparently phagocytic cells for long periods. The mechanisms of biodegradation of staphylococci by leucocyte factors have shown that degradation of cell walls in vitro may be the result of the activation, by leucocyte kationic proteins, of the bacterial autolytic wall enzymes that are responsible for degrading the cell walls from within. This process is markedly inhibited by sulphated polysaccharides like dextran sulphate, by heparin, or by polyanetholesulfonate (liquoid). These anionic polyelectrolytes have also been shown to inhibit the lysis of staphylococci treated with bacteriolytic concentrations of penicillin G. Staphylococci injected intraarticularly into the knee joint of rats underwent massive plasmolysis, but structures compatible with cell walls (peptidoglycan) persisted within macrophages in the inflammatory sites, for long periods. It is postulated that the inability of leucocytes to degrade staphylococcal cell-wall components may be the result of the interference, by anionic polyelectrolytes likely to accumulate in the inflammatory sites, with the activation of the autolytic systems. Alternatively, anionic polyelectrolytes may coat the bacterial cells and interfere with the binding of the autolytic enzymes with their corresponding substrates.
NADPH and "cocktails" containing polyarginine reactivate superoxide generation in leukocytes lysed by membrane-damaging agents
Ginsburg I, Borinski R, Pabst M. NADPH and "cocktails" containing polyarginine reactivate superoxide generation in leukocytes lysed by membrane-damaging agents. Inflammation. 1985;9 (4) :341-363.Abstract
Human blood leukocytes generated large amounts of superoxide (O2-) following stimulation by certain "cocktails" of soluble agents consisting of poly-L-arginine (PARG), phytohemagglutinin, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine and polyanethole sulfanote (liquoid). A variety of cytochalasins, which markedly boosted O2- generation by the soluble cocktails, markedly depressed luminol-dependent chemiluminescence (LDCL) which had been induced either by opsonized streptococci or by soluble agents. Glutathione, which totally reversed the inhibition of LDCL induced by cytochalasin A, failed to reverse the inhibition of LDCL induced by cytochalasin B. Generation of O2- by all the soluble agents employed, except PMA, was strongly inhibited either by the omission of extracellular calcium and magnesium or by treatment with the calcium blocker TMB-8. Generation of O2- was enhanced following stimulation of leukocytes with soluble agents if the cells had been exposed to slightly hypotonic buffers. Leukocytes, which had been preincubated for short periods (5 min) with PARG, saponin, digitonin, or lysolecithin (LL) and which lost their viability, and their O2- and LDCL-generating capacities following stimulation by soluble agents containing cytochalasin B, nevertheless regained these activities by the addition of NADPH. It is suggested that the lytic agents induced the leakage out of NADPH rather than acting as inactivators of the oxidase in the leukocyte membranes. Prolonged incubation of leukocytes with lytic agents failed to allow restoration, by NADPH, of the generation of SOD-inhibitable O2- generation. Since PARG acted both as a cytolytic agent and as a inducer of O2- generation, we postulate that lytic agents might also act as "primers" of the nascent membrane oxidase which could, however, be further potentiated and activated by soluble agents acting in "multiple hits," PARG could be totally replaced either by LL or by digitonin in the generation of O2- provided that both PHA and cytochalasin B were present in the reaction mixtures. We suggest that the various ingredients of the soluble "cocktails" may help to assemble components of the NADPH oxidase. Such an assembly and regulations are prerequisite for stimulation of the NADPH oxidase and the generation of oxygen radicals in leukocytes.
Chemiluminescence and superoxide generation by leukocytes stimulated by polyelectrolyte-opsonized bacteria. Role of histones, polyarginine, polylysine, polyhistidine, cytochalasins, and inflammatory exudates as modulators of oxygen burst
Ginsburg I, Borinski R, Malamud D, Struckmeier F, Klimetzek V. Chemiluminescence and superoxide generation by leukocytes stimulated by polyelectrolyte-opsonized bacteria. Role of histones, polyarginine, polylysine, polyhistidine, cytochalasins, and inflammatory exudates as modulators of oxygen burst. Inflammation. 1985;9 (3) :245-271.Abstract
Human blood leukocytes generate intense luminol-dependent chemiluminescence (LDCL) following stimulation by streptococci and by Gram negative rods which had been preopsonized by cationic polyelectrolytes (histone, poly L-arginine-PARG, poly L-histidine-PHSTD). Streptococci but not Gram negative rods or hyaluronic acid-rich streptococci (group C) also induced intense LDCL following opsonization with the anionic polyelectrolytes-dextran sulfate or polyanethole sulfonate (liquoid) suggesting that the outer surfaces of different bacteria bound anionic polyelectrolytes to different extents. Both normal and immune serum, synovial fluids and pooled human saliva inhibited the LDCL responses induced by streptococci preopsonized with poly cations. On the other hand, bacteria which had been first preopsonized by the various body fluids and then subjected to a second opsonization by cationic ligands ("sandwiches"), induced a very intense LDCL response in leukocytes. Streptococci which had been preopsonized by PARG, histone or by PHSTD also triggered superoxide generation by blood leukocytes, which was markedly enhanced by a series of cytochalasins. PHSTD alone induced the formation of very large amounts of superoxide. Paradoxically, the same concentrations of cytochalasins B or C which markedly boosted the generation of superoxide following stimulation of leukocytes with soluble or particulate ligands, had a strong inhibitory effect on the generation of LDCL. On the other hand cycochalasins failed to inhibit LDCL which had been induced by phorbol myristate acetate (PMA). Peritoneal macrophages which had been harvested from C. parvum-stimulated mice, generated more LDCL and superoxide following stimulation by PARG than macrophages obtained from proteose peptone-stimulated mice. Macrophages which had been activated either by proteose peptone or by C. parvum and cultivated for 2 hours on teflon surfaces, generated much more LDCL than macrophages which had been cultivated for 24 hours on teflon surfaces. Both cationic and anionic polyelectrolytes mimic the effects of antibodies as activators of the oxygen burst in blood leukocytes and in macrophages. Such polyelectrolytes can serve as models to further study leukocyte-bacteria interactions in infectious and inflammatory sites.
Antibiotics and Polyelectrolytes Modulate Bacteriolysis and the Capacity of Bacteria to Trigger an Oxygen Burst in Neutrophils
Ginsburg I, Borinski R, Sadovnik M, Shauli S, Wecke J, Giesbrecht P, Lahav M. Antibiotics and Polyelectrolytes Modulate Bacteriolysis and the Capacity of Bacteria to Trigger an Oxygen Burst in Neutrophils. In: The Influence of Antibiotics on the Host-Parasite Relationship II. Vol. 2. Springer Berlin Heidelberg ; 1985. pp. 141-151.Abstract
The invasions of tissues by pathogenic microorganisms is followed by a sequence of events which culminate in phagocytosis and the intracellular killing of the ingested agents, by “professional” phagocytes [19]. It is also expected that the rich arsenal of hydrolases present in neutrophils and macrophages, including the muralytic enzyme lysozyme is adequate to degrade the complex architecture of the bacterial cells. Surprisingly, however, most pathogenic bacteria are extremely resistant to lysozyme action [14,21] and the fate of phagocytosed bacteria in vivo is not fully known [7,8,16,23]. The sequelae of the lack of bacterial degradation by leukocytes may be the “storage” of peptidoglycan-polysaccharide or peptidoglycan-lipopolysaccharide complexes within macrophages leading to the generation of granulomas and to the initiation of prolonged immune responses. This is pivotal to the initiation of immunopathological reactions [7, 8, 16, 23]. We have recently proposed [10, 11, 12, 13, 15, 29] that the biodegradation of certain microorganisms can be mediated through the activation, by cationic agents and phospholipases, of the bacterial own autolytic wall enzymes (suicidal phenomenon) which leads to the breakdown of the rigid cell walls. On the other hand, a variety of sulfonated anionic polyelectrolytes [11–13, 15] likely to be present in inflamed issues, may inhibit the biodegradation of the walls by the autolytic enzymes.
1984
T-2 toxin effect on bacterial infection and leukocyte functions
Yarom R, Sherman Y, More R, Ginsburg I, Borinski R, Yagen B. T-2 toxin effect on bacterial infection and leukocyte functions. Toxicology and Applied Pharmacology. 1984;75 (1) :60-68.Abstract
The effects of T-2 toxin on bacterial infection and leukocyte function and structure were examined in vivo and in vitro. Rats were innoculated with staphylococci after pretreatment with or without T-2 toxin. The T-2 pretreated rats failed to mount a cellular response to the bacteria. Blood and bone marrow cells were markedly suppressed by the T-2 toxin, the myeloid series being the most affected. In vitro studies with human leukocytes showed that small, nonkilling doses of T-2 toxin inhibited chemotaxis, chemiluminescence stimulated by bacteria, and phagocytosis of bacteria. It was concluded that this inhibition may contribute towards sepsis and rapid onset of death in T-2 toxin poisoning.
Poly-L-arginine and an N-formylated chemotactic peptide act synergistically with lectins and calcium ionophore to induce intense chemiluminescence and superoxide production in human blood leukocytes.
Ginsburg I, Borinski R, Lahav M, Y M, I E, P C, D M. Poly-L-arginine and an N-formylated chemotactic peptide act synergistically with lectins and calcium ionophore to induce intense chemiluminescence and superoxide production in human blood leukocytes. Inflammation. 1984;8 (1) :1-26.Abstract
Various cationic polyelectrolytes (poly-alpha-amino acids and histones), lectins, the chemotactic peptide, f-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were investigated regarding their capacity to induce luminol-dependent chemiluminescence (LDCL) and superoxide production by human blood leukocytes. Although when tested individually, poly-L-arginine (PARG), phytohemagglutinin (PHA), concanavalin A (Con A), or fMLP induced only a low to moderate LDCL response, very intense synergistic CL reactions were obtained by mixtures of PARG + PHA, PARG + Con A, PARG + PHA + fMLP, Ca2 + ionophore + PARG + PHA + fMLP, and PARG + PMA. The sequence of addition of the various agents to WBC in the presence of luminol absolutely determined the intensity of the LDCL signals obtained, the highest reactions being achieved when the WBC were preincubated for 2-3 min with A23187 followed by the sequential addition of fMLP, PARG, and PHA. These "multiple hits" induced CL reactions which were many times higher than those obtained by each factor alone. On the other hand, neither poly-L-lysine, poly-L-ornithine, poly-L-histidine, nor poly-L-asparagine, when employed at equimolar concentrations, cooperated efficiently with PHA and fMLP to trigger synergistic LDCL responses in leukocytes. Concomitantly with the induction of LDCL, certain ligand mixtures also triggered the production of superoxide. The LDCL which was induced by the "cocktail" of agents was markedly inhibited by sodium azide (93% inhibition), but to a lesser extent by catalase (10% inhibition) or by superoxide dismutase (20%-60% inhibition). On the other hand, scavengers of singlet oxygen and OH (sodium benzoate, histidine) did not affect the synergistic LDCL responses induced by these multiple ligands. Cytochalasin B also markedly inhibited the LDCL responses induced either by soluble stimuli or by streptococci preopsonized either with histone or with polyanethole sulfonate. The LDCL responses which were induced by mixtures of PARG and concanavalin A were also strongly inhibited by mannose, alpha-methyl mannoside, and poly-L-glutamic acid. The data suggest that the LDCL responses induced by the soluble ligands involved a myeloperoxidase-catalyzed reaction. The possible employment of "cocktails" of ligands to enhance the bactericidal effects of PMNs, macrophages, and natural killer cells on microbial cells and mammalian targets is discussed.
1983
The role of Ieucocyte and serum factors and of cationic polyelectrolytes in the lysis andbiodeg radation of Staphylococcus aureus: relation to the pathogenesis of staphylococcal infections
Ginsburg I, Sela MN, Neeman N, Lahav M. The role of Ieucocyte and serum factors and of cationic polyelectrolytes in the lysis andbiodeg radation of Staphylococcus aureus: relation to the pathogenesis of staphylococcal infections. STAPHYLOCOCCI AND STAPHYLOCOCCAL INFECTIONS. 1983 :325-355.Abstract
INTRODUCTION. Although much is known today about the mechanisms by which virulent Staphylococcus aureus induce tissue lesions and cause clinical manifestations in mammals, our knowledge of the role played by “professional” phagocytes in host and parasite interrelationships in staphylococcal infections, is not fully understood. The extensive literature on staphylococci and their role in human disease has been the subject of several excellent comprehensive reviews (Whipple, 1965; Cohen, 1972; Jeljaszewicz, 1976). It is accepted that the interception of staphylococci by granulocytes (PMNs) takes place soon after the penetration of the cocci into the tissues. This involves the release of chemotactic factors, by the bacteria themselves (Pusell et al., 1975) or the activation, by staphylococcal factors, of chemotactic agents from complement (Ginsburg and Quie, 1980). Subsequent phagocytosis is markedly enhanced by opsonins (Koenig, 1972; Ekstadt, 1974), and in most cases the engulfed bacteria may be killed intracellularly by a variety of bactericidal agents generated by activated PMNs (Klebanoff, 1972). It is also suggested that certain lysosomal enzymes, which are released into the phagolysosome, may digest the staphylococcal cells (Cohn, 1963a; De Voe et al., 1973; Ginsburg and Sela 1976; Ginsburg, 1979) (see Sections II.B and VI). Several reports have, however, shown that intracellular staphylococci may sometimes survive within PMNs, where they multiply and eventually kill the cell (Koenig, 1972; Cohen, 1972; Pearce et al., 1976). Surprisingly, very little is known about the fate and mechanism of biodegradation of staphylococcal cell constituents, once they have been sequestered within the phagolysosomes of leucocytes. The importance of this field of research stems from the findings that non-biodegradable cell wall components of a variety of microbial species may persist within macrophges for prolonged periods, to trigger chronic infiammatory sequelae (Dannenberg, 1968; Kanai and Kondo, 1974; Ginsburg et al., 1975a; 1975b; 1976a; Ginsburg and Sela, 1976; Adams, 1976; Page et al., 1978; Ginsburg, 1979). The inability of “professional” phagocytes to degrade intracellular bacteria may be due (1) to the presence, on the surface of certain microorganisms, of shielding capsular material (Dossett et al., 1969; Smith, 1977; Wilkinson, et al., 1979; Densen and Mandell, 1980), (2) to the lack of fusion between lysosomes and phagosomes (Goren et al., 1976; Densen and Mandell, 1980), (3) to the production of leucocidins (Gladstone and Van Heyningen, 1957; Woodin, 1960; Ginsburg, 1970; Van Heyningen, 1970; Bernheimer. 1970; Ginsburg, 1972), (4) to the lack of adequate lysosomal enzymes capable of cleaving bacterial peptidoglycans (Dannenberg, 1968; Ginsburg, 1972; Ginsburg and Sela, 1976; Page et al., 1978; Ginsburg, 1979), or (5) to the presence, in serum and in inflammatory exudates, of agents which interfere with the interaction of bactericidal and bacteriolytic age with engulfed bacteria. These fields were comprehensively reviewed EW’ Jeljaszewicz, 1976; Smith, 1977; Ginsburg, 1979; and by Densen and Mandell, 1980. During the last 8 years our laboratory has been studying the host- and-parasite interrelationships in streptococcal and staphylococcal infections using biochemical, electron microscopical and tissue culture techniques. In particular we studied the mechanisms by which leu- cocytes and their isolated lysosomal agents bring about the degrada- tion of staphylococcal cell wall components, and the role which may be played by such degradation products in the initiation of chronic inflammation. The present report is an updated overview of these studies.
CATIONIC POLYELECTROLYTES ACTIVATE AUTOLYTIC WALL ENZYMES IN STAPHYLOCOCCUSAUREUS: Modulation by anionic polyelectrolytes in relation to the survivalof bacterial constituents in tissues
Ginsburg I, Lahav M. CATIONIC POLYELECTROLYTES ACTIVATE AUTOLYTIC WALL ENZYMES IN STAPHYLOCOCCUSAUREUS: Modulation by anionic polyelectrolytes in relation to the survivalof bacterial constituents in tissues. The Target of Penicillin. 1983 :341-346.Abstract
Introduction. Although a wealth of knowledge exists today on the biochemical pathways of biosynthesis, turnover and autolysis of bacterial cell wall components in vitro (1, 2), surprisingly very little is actually known about the mechanisms of biodegradation of microbial constituents in_vivo. One should differentiate between bactericidal and bacteriolytic processes induced by leukocytes since killed, but non-degraded, microbial cells may persist within macrophages to trigger chronic inflammation (3, 4). The present communication further supports our contention (5, 6) that the degradation of microbial cell wall components by leukocytes may be due to activation, by leukocytic cationic proteins, of autolytic wall enzymes rather than to the direct cleavage of the cells by lysosomal hydrolases. The modulation of bacteriolysis by anionic polyelectrolytes will be described and discussed in relation to the pathogenesis of chronic inflammation and afequelae.
INDUCED AUTOLYTIC WALL PROCESSES IN HEAT-INACTIVATEDSTAPHYLOCOCCUS AUREUS
Lahav M, Ginsburg I. INDUCED AUTOLYTIC WALL PROCESSES IN HEAT-INACTIVATEDSTAPHYLOCOCCUS AUREUS. The Target of Penicillin. 1983 :335-340.Abstract
Introduction. In previous studies it could be shown that autolytic wall enzymes of bacteria can be activated by some cationic proteins (1). Recently we determination of firming our data lytic enzyme but obtained results from chemical and end group determination of the cleavage products from peptidoglycan confirming our data that even lysozyme acted not only as a muralytic enzyme but also as a cationic protein (2). In order to elucidate the mechanisms of the activation of autolytic wall processes by cationic proteins and of the direct respectively indirect muralytic actions of lysozyme we performed investigations on heated cells.
Lysis and biodegradation of microorganisms in infectious sites may involve cooperation between leukocyte, serum factors and bacterial wall autolysins: A working hypothesis
Ginsburg I, Lahav M. Lysis and biodegradation of microorganisms in infectious sites may involve cooperation between leukocyte, serum factors and bacterial wall autolysins: A working hypothesis. European Journal of Clinical Microbiology. 1983;2 (3) :186-191.Abstract
Although a voluminous literature exists today on the mechanisms by which "professional" leukocytes (granulocytes and maerophages) intercept with, engulf and eventually kill phagocytosed microorganisms ~ (1, 2), surprisingly very little is known about the mechanisms of degradation and elimination of bacteria from tissues. It is well established that phagocytic cells are endowed with numerous hydrolyric enzymes, including the key cell wall splitting enzyme-lysozyme, which can theoretically cleave, surface, eeU wall and cytoplasmic constituents of bacteria. Also, fresh mammalian sera are known to contain a complex mixture of heat-labile complement components (3) heat-stable lysozyme and platelet-derived cationic proteins (~-lysins) (4) which have been shown to kill and partially lyse certain microbial constituents. Surprisingly, however, the majority of virulent microorganisms are highly refractory to both leukocyte and serum lyric agents. Throughout this communication we shall use the general term bacteriolysis to denote the degradation of the cell walls, the outer membranes and the cytoplasmic constituents of bacteria. The term cell wall lysis will be used to describe the specific biochemical degradation of the bacterial peptidoglycan. This may also be accompanied by the rupture of the protoplast membrane and the release of cytoplasmic constituents. The inability of leukocyte and serum factors to induce bacteriolysis is linked to the presence, upon most bacterial surfaces, of'lipopolysaccharides, polysaccharides-teichoic complexes and certain lipids and waxes, which hinder the accessibility of the major cell wall splitting enzyme-lysozyme to the peptidoglycan (1). Once however this obstacle is overcome, the peptidoglycan ist degraded, and the protoplasts burst due to their high osmotic pressure, releasing degradation products of both cell wall and cytoplasmic constituents into the surrounding medium. The very extensive literature on these subjects has been recently summarized and reviewed (5-7). It has als0 been suggested that the process of killing and biochemical degradation of microbial constituents, either following phagocytosis or following treatment with fresh complement and lysozyme-sufficient serum are probably mediated by different mechanisms (6-8). While extensive loss of wall material and cytoplasmic entities is usually accompanied by a bactericidal reaction, the killing of bacteria either by the oxygen-dependent (9) or by the non-oxygen dependent bactericidal systems of leukocytes (5, 10) and serum (3), is not necessarily accompanied by a substantial bacteriolysis. The distinction between a bactericidal and a bacteriolyric process is important, in view of the observations that poorly degraded non-viable microbial constituents may persist for long periods both extracellularly and within phagocytic cells, to trigger and perpetuate chronic inflammatory sequellae (6, 7, 11-13). Furthermore, while degradation products of grampositive and acid-fast bacteria have been shown to be endowed with distinct pharmacological properties (14), to modulate the immune responses (15), to activate the complement cascade (16) and to be cytopathic for mammalian cells (14), the in vivo release of lipopolysaccharides of the outer membrane of gramnegative bacteria may result in severe coagulation defects (Shwartzman phenomenon) and in endotoxic shock (17). Poorly degraded cell wall components of bacteria have also been shown to be translocated within macrophages from one tissue site to another, thus contributing perhaps to the dissemination of granulomatosis (18-20). Although the nature of the biochemical pathways involved in bacterial biodegradation in tissues has not been fully elucidated, it has been recently suggested that a cooperation among leukocyte factors (21, 22), serum components (23), the bacterial own autolytic wall enzymes (21, 22) and certain antibiotics (24), may act in accord to induce a massive breakdown of cell wall and cytoplasmic constituents of bacteria. It is well established that the autolytic systems present in every bacterial cell, control cell division, the deposition of new cell wall material and the regeneration after treatment with certain antibiotics (25, 36). Autolytic enzymes have been isolated from many bacterial species and were found to possess muramidase, Nacetyl glucosaminidase, amidase and peptidase activities (25). It is also known that certain antibiotics, mainly of the penicillin and cephalosporin series, are capable of killing microorganisms, presumably by activating their autolytic wall enzymes (27). These intraceUular enzymes are thought to be controlled by endogenous lipid material (e.g.- phospholipids, lipoteichoic acid) (27). Thus, any agent present in leukocytes or in tissue fluids, which will disrupt the balance between autolytic enzymes and their naturally occurring inhibitors, may lead to the activation of autolysins, and concomitantly to the release of toxic bacterial agents. In view of the complex interrelationship which exist between bacteria and host factors in infectious and imflammatory sites, it was of interest to clarify some of the mechanisms and the factors involved in the biodegradation and persistence of microbial constituents in tissues. The following is an overview of our studies on this subject, employing staphylococci and streptococci as model systems, and using biochemical and electron microscopical techniques (28). The peptidoglycan of Staphylococcus aureus was labelled during the logarithmic phase of growth with 14C-N-acetylglucosamine. When such labelled cells were incubated for several hours at 37 ~ in acetate buffer pH 5.0, with small amounts of crude human leukocyte extracts or with more purified lysosomal extracts, a substantial amount of the radioactivity, associated with the cell walls was solubilized. Electron microscopical analysis of these reaction mixtures revealed the accumulation of cell wall fragments, and both amorphous and intact cytoplasmic constituents still retaining their typical morphologies (6, 7, 21, 22, 28, 29). Since the pH optimum for this reaction process was found to be on the acid side and compatible with the pH optima of many of the acid hydrolases known to be present in the leukocyte preparation (1) we postulated that the breakdown of the bacterial ceils was mediated by acid hydrolases. The findings, however, that heat treatment did not destroy the capacity of the leukocyte extracts to induce cell wall degradation, and that purified radiolabelled staphylococcal cell walls became completely refractory to the lytic effect of the extracts (21, 22), suggested that the wall degradation observed was probably not caused by the leukocyte hydrolases. Since leukocyte lysosomes are known to be rich in heat-stable argininerich bactericidal cationic proteins (LCP) (30) and in myeloperoxidase (MPO) (1) (also a cationic protein) it was reasonable to try and employ them, instead of the total leukocyte mixture, to lyse the staphylococci. Indeed we found that as little as 0.5-1/zg/ml of nuclear histone, poly-L-lysine, poly-L-arginine or MPO and 10-50/ag/ml of crystalline pancreatic ribonuclease or cytochrome C (all cationic in nature) were sufficient to induce massive loss of cell wall material from 108 log-phase staphylococci. Furthermore, small amounts of the membrane.damaging agents phospholipase A2, and polymyxins B and E were also capable of inducing cell wall lysis, as determined by the release of N-acetyl-glucosamine (8, 31-33). Since purified staphylococcal cell walls (devoid of cytoplasmic structures) were extremely refractory to any of the cationic polyelectrolytes or to the membrane-damaging agents, we postulated that perturbation of the staphylococcal membrane by these agents might have resulted in the activation of endogenous enzymes presumably associated with the autolytic systems (21, 22, 25, 26). As autolytic wall enzymes are known to be heat-labile (25, 26), it was of in,terest to try and reactivate the lytic process by the addition of freshly-harvested viable staphylococci (as donors of autolysins) to the heat killed radiolabeled staphylococci or to the purified labelled cell walls, in the presence of several of the cationic proteins or the membrane-damaging agents. Indeed, such mixtures resulted in a substantial loss of radiolabelled wall material. This process was completely blocked by anionic polyelectrolytes. We suggested, therefore, that the activators of autolysins interacted with the viable bacteria to release the autolytic enzymes, which in turn attacked and degraded the radiolabelled substances. Similar results were recently described with Bacillus subtilis (34). It has also been suggested that pneumococci, gonococci, meningococci, Streptococcus faecalis and perhaps listeriae may also likewise be degraded in vivo following the activation of their autolysins, and not through the direct action of lysosomal enzymes (28). Further experiments showed that Staphylococcus aureus, which had been cultivated in the presence of sub-inhibitory concentrations of penicillin G, became much more susceptible to wall lysis, following treatment with leukocyte extracts (24,35) suggesting a collaboration between/3-1actam antibiotics, leukocyte factors and bacterial autolysins in bacteriolysis (see 27). Other studies from our laboratory (23) have also shown that contrary to the accepted belief, both fresh and heat-treated human serum, when properly diluted, also lysed log-phase grampositive staphylococci and Streptococcus faecalis at pH 5.0. Since anionic polyelectrolytes also inhibited cell lysis induced by serum (23), and since heat-killed bacteria became resistant to lysis by serum, we postulated that, as in the case of leukocyte extracts, lysis was induced by a heat-stable factor, presumably ~-lysin of platelet origin (4) present in serum, which activated the autolytic systems of the bacteria. To further elucidate the mechanism of bacteriolysis, we have analyzed this process by electron microscopy. In collaborative studies with Prof. P. Giesbrecht and Dr. J. Wecke of the Robert Koch Institute in Berlin, we found (36) that a few hours after the addition of either crystalline lysozyme (500/ag) or pancreatic ribonuclease (50/ag/ml) to log-phase staphylococci, the first signs of cell damage could already be seen. These consisted of the formation of small, periodically-arranged lytic sites between the cell wall and the cytoplasmic membrane of the cross wall. This was followed by the formation of a distinct gap between the cell wall proper and the cytoplasmic membrane. The degradation of the peripheral cell wall continued to the opposite side of the cell, and extended gaps underneath the wall could be detected long before the cell wall itself was peeled off as large ribbons. The cross wall often appeared to be already disintegrated during the early phase of lysozyme or ribonuclease action. The release of the wall left apparently intact protoplasts, which still retained their original shape, and even the invagination of the cross walls were conserved. At this point over 70 % of the toal radioactivity associated with the cell wall was solubilized after three to four hours, and the radioactivity could not be sedimented at 100,000 • g suggesting the formation of solubilized peptidoglycan. Since lysozyme which had been heated to destroy its enzymatic activity still retained its ability to induce cell wall lysis, we postulated that lysozyme in this system did not act as an enzyme but as a cationic protein. Further studies from our laboratory (28) and in collaboration with the Robert Koch Institute (to be published in detail) have revealed that very similar ultrastructural changes in the staphylococci also took place following phagocytosis by mouse non-elicited macrophages in culture. On the other hand, although staphylococci, which had been injected into mouse or rat tissues, and which were phagocytosed by both PMNs and macrophages, underwent rapid loss of their cytoplasmic constituents, presumably by digestion with lysosomal hydrolases (37-39), no apparent damageto the cell walls was evident for many days, suggesting that the autolytic wall enzymes might have been inhibited. Since the degradation of the staphylococcal cell walls in vitro was completely inhibited by a variety of anionic polyelectrolytes like heparin, dextran sulfate, polyanethole sulfonate (a synthetic heparin), as well as by cationic polyelectrolytes like histones, poly-L-lysine, poly-L-ar~inine, etc., when used at 10-100 /ag/ml/10 ~ staphylococci (concentrations 10- 100 fold higher than those employed to activate the autolytic wall enzymes (see above), we also postulated that a delicate balance between activators and inhibitors may determine whether or not bacterial wall material may be degraded in tissue lesions in vivo (40,41). Finally, iecent studies (42) have also shown that high-molecular-weight degradation products of staphylococcal cell walls derived following treatment of the bacteria either in buffers (spontaneous wall lysis) or by small amounts of leukocyte extracts, were found to possess very strong chemot~ctic activities for PMNs in vitro, and to induce severe inflammatory lesions when injected intraarticularly to rats. Thus, it may be concluded that the fate of bacterial peptidoglycans in leukocytes in inflamed tissues may be dependent on the one hand on the availability of agents capable of activating autolytic wall enzymes in bacteria, and on the other hand on the presence in tissues of inhibitory substances (polyelectrolytes) which are capable of blocking bacteriolysis. It is, however, not aimed t9 rule out the possibility that other still unknown mechanisms may function in the complex milieu of inflammation, which may bring about the biodegradation of bacteria. The employment of certain antibiotics and other pharmacological agents, yet to be discovered, which will be capable of changing the balance between activators and inhibitors of autolytic enzymes, may contribute to a better understanding of the mechanisms involved in the survial and persistence of microbial agents in vivo. It is also obvious that the release of large quantities of microbial constituents following incomplete biodegradation may prove to be deleterious to tissues. Finally, our studies on staphylococci do not shed light on the mechanisms of biodegradation of other microbial species of medical imprtance, and is only a reflection of one possible mechanism, which may or may not be common to all microorganisms.
How are bacterial cells degraded by leukocytes in vivo? An enigma
Ginsburg I, Lahav M. How are bacterial cells degraded by leukocytes in vivo? An enigma. Clinical Immunology Newsletter. 1983;4 (11) :147-153.Abstract
This year marks the centennial anniversary of Elie Metchnikoff's discovery of the pivotal role played by "professional" phagocytes in body defenses against invading microorganisms. His cellular theory dealt with the phagocytic events and the postphagocytic killing, and also alluded to the digestion of the internalized bacteria by the "cystases," later shown to be associated with the lysosomal apparatus of leukocytes. Fo date, despite the fact that numerous studies have described in great detail the mechanisms by which serum and leukocytes kill microorganisms (1, 8, 13, 24), surprisingly little is actually known about the biochemical pathways of degradation and mechanisms of disposal of microbial constituents once they have been sequestered within phagolysosomes (3, 9, 10, 13, 24). It is usually taken for granted that the numerous hydrolytic enzymes, including the key bacteriolytic enzyme lysozyme (muramidase), present in lysosomes of "professional" phagocytic cells [granulocytes or polymorphonuclear neutrophils (PMNs), and macrophages] are capable, (at least theoretically) of stripping off bacterial coats, thus exposing the peptidoglycan to cleavage by touramidase. Yet, the majority of pathogenic microorganisms are highly refractory to lysozyme action (9, 10, 17). One should also bear in mind that, while a massive breakdown of microbial cell walls eventually may lead to a bactericidal reaction, the mere killing of a microorganisms, either by leukocyte or by serum factors, may not necessarily be followed by a bacteriolytic reaction. The importance of elucidating the mechanism of microbial biodegradation in tissues stems from the observation that in many infectious diseases there is "storage" of nonbiodegraded microbial cell wall components within macrophages for long periods, which may be responsible for the perpetuation and propagation of chronic inflammatory sequelae and tissue destruction (10, 18). We have recently postulated (11, 14, 15) that bacteriolysis, and the biochemical degradation that ensues after bacteria have been attacked by serum or by leukocytes, may involve close cooperation among heat-stable serum factors, cationic proteins and phospholipase A2 of leukocytes, and heat-labile endogenous bacterial autolyric wall enzymes. This cooperation is affected markedly by anionic polyelectrolytes, likely to accumulate in inflammatory exudates, which may shut down autolysis and, thus, contribute to unfavorable postinfectious sequelae (10, 19). The present communication is a summary of efforts from our laboratory to gain insight into the mechanisms of lysis of Staphylococcus attreus, chosen as a model, by lysosomal enzymes of human blood leukocytes (3, 8-11, 14, 15, 19).
1982
Effect of Antibiotics on the Lysis of Staphylococci and Streptococci by Leukocyte Factors, on the Production of Cellular and Extracellular Factors by Streptococci, and on the Solubilization of Cell-Sensitizing Agents from Gram-negative Rods
Ginsburg I, Lahav M, Bergner-Rabinowitz S, Ferne M. Effect of Antibiotics on the Lysis of Staphylococci and Streptococci by Leukocyte Factors, on the Production of Cellular and Extracellular Factors by Streptococci, and on the Solubilization of Cell-Sensitizing Agents from Gram-negative Rods. In: The Influence of Antibiotics on the Host-Parasite Relationship. ; 1982. pp. 219-227.Abstract
Although much is known today about the mode of action of antibiotics on microorganisms, relatively little has been done to evaluate the possible collaboration between antibiotics and the host defenses in the containment and elimination of pathogens from host tissues. Since certain antibiotics are known to interfere with the biosynthesis of bacterial cellular and extracellular components, it is conceivable that such modified bacterial cells may be more readily intercepted, killed, and eventually digested by professional phagocytes. On the other hand, certain antibiotics may have adverse effects on mammalian cells by interfering with their normal metabolism and subsequently with their antimicrobial functions. Although the role of bacteriolysis in host and parasite interrelationships has been recognized for over a decade, this field of research has surprisingly been almost totally neglected. The importance of understanding the mechanisms of biodegradation of microbial cells in vivo stems from the recognition that the inability of the enzymes of the host to degrade the rigid cell wall of microorganisms is a contributory factor to the formation of chronic granulomatous responses, and to the destruction of tissues [1, 6, 16, 17, 22, 30]. Thus, any antibiotics which will collaborate with leukocytes or with serum factors in the elimination of bacterial constitutents from infected tissues may greatly contribute to the well-being of the individual.
Effects of subminimal inhibitory concentrations of chloramphenicol, erythromycin and penicillin on group A streptococci
Michel J, Ferne M, Borinski R, Kornberg Z, Bergner-Rabinowitz S, Ginsburg I. Effects of subminimal inhibitory concentrations of chloramphenicol, erythromycin and penicillin on group A streptococci. European Journal of Clinical Microbiology. 1982;1 (6) :375-380.Abstract
Group A streptococci strains were grown in broth containing subminimal inhibitory concentrations of chloramphenicol, erythromycin and penicillin, and tested for possible changes in colonial morphology, activity and amount of cellular and extracellular components. The following components were tested: T protein, M protein, opacity factor, lipoteichoic acid, hyaluronic acid, streptolysin S, streptolysin O, DNase, hyaluronidase and NADase. Sub-MICs of these drugs produced variable changes in the bacteria. They increased the amount of hyaluronic acid and hyaluronidase, decreased the amount of M protein, and enhanced phagocytosis and the release of lipoteichoic acid. The results indicate that sub-MICs of chloramphenicol, erythromycin and penicillin may affect the pathogenicity and toxinogenicity of group A streptococci.
Isolation and characterization of rat skeletal muscle and cytoplasmic actin genes
Nudel U, Katcoff D, Zakut R, Shani M, Carmon Y, Finer M, Czosnek H, Ginsburg I, Yaffe D. Isolation and characterization of rat skeletal muscle and cytoplasmic actin genes. Proceedings of the National Academy of Sciences of the United States of America. 1982;79 (9) :2763-2767.Abstract
Southern blots of rat genomic DNA indicate the existence of at least 12 EcoRI DNA fragments containing actin gene sequences. By using specific probes and stringent conditions of hybridization, it was found that only one of these fragments contains sequences of the skeletal muscle alpha-actin gene. Recombinant bacteriophages originating from eight different actin genes were isolated from rat genomic DNA libraries. One of them, Act 15, contains the skeletal muscle actin gene. Another clone, Act I, contains a gene coding for a cytoplasmic actin, identified tentatively as the beta-actin gene. Both genes have a large intron very close to the 5' end of their transcribed region, followed by several small introns. DNA sequence analysis and comparison with the available data on actin genes in other organisms indicated an interesting relationship between the positions of introns and the evolutionary relatedness. Several intron sites are conserved from at least the echinoderms to the vertebrates; others appear to be present in some actin genes and not in others.
The role of cationic proteins and anionic polyelectrolytes in the control of bacterial infections. Cooperation between PMNS and macrophages
Ginsburg I, Lahav M. The role of cationic proteins and anionic polyelectrolytes in the control of bacterial infections. Cooperation between PMNS and macrophages. International Journal of Immunopharmacology. 1982;4 (4) :372.Abstract
It is postulated that lysosomal enzymes of leukocytes are capable of breaking down bacterial constituents and to cause bacteriolyasis. Studies from our laboratory have shown that radiolabled staphylococci (log-phase) arm readily lysed by leukocyte extracts at pH 5.0. On the other hand, old cells or heat-killed young cells are resistant degradation. The leukocytes extracts can be affectively replaced by cationic proteins as well as by membrane-damaging agents (phospholipase A2, polymaxins). Studies on the mechanisms of bacteriolysis have suggested that the cationic proteins act by activating the bacterial autolytic enzymes leading to bacteriolysis. This proces can be inhibited by a series of anionic polalalectrolytes likely to preent in inflammation, presumably by inactivating autolytic enzymes. The cooperation between PHNs and macrophages in bacteriolysis and control of bacterial growth by polalalectrolytes will be discussed in relation to the phatogenesis of the infection and inflammation.
Cationic Polyelectrolytes and Leukocyte Factors Function as Opsonins, Triggers of Chemiluminescence and Activators of Autolytic Enzymes in Bacteria: Modulation by Anionic Polyelectrolytes in Relation to Inflammation
Ginsburg I, Lahav M, Ferne M, Müller S. Cationic Polyelectrolytes and Leukocyte Factors Function as Opsonins, Triggers of Chemiluminescence and Activators of Autolytic Enzymes in Bacteria: Modulation by Anionic Polyelectrolytes in Relation to Inflammation. Advances in experimental medicine and biology. 1982;155 :151-160.Abstract
Both antibodies and complement components are essential for successful phagocytosis of many virulent microorganisms (1,2). Although the mechanisms by which opsonins promote particle uptake are not fully understood, it has been suggested that both electrostatic and hydrophobic forces act in concert with specific receptors for Fc and C3b to facilitate interiorization of particles (2,3). In the case of group A streptococci, opsonization by immunoglobulins abolishes the anti-phagocytic properties of the M-antigen (4,5). Since one mechanism by which opsonins may act is to decrease repulsion forces between negative charges present on the surface of the particle and phagocyte, cationic ligands may function as effective opsonins (6–11). In addition, cationic substances may participate in bacteriolysis. We recently suggested (11) that the breakdown of bacterial cells following phagocytosis is mediated indirectly by leukocyte cationic proteins and phospholipases which activate autolytic enzymes and not by lysosomal enzymes directly.
Phospholipids inhibit cytotoxic effects of Actinobacillus actinomycetemcomitans leukotoxin on human polymorphonuclear leukocytes
Ginsburg I, Tsai CC, SM W, NS T. Phospholipids inhibit cytotoxic effects of Actinobacillus actinomycetemcomitans leukotoxin on human polymorphonuclear leukocytes. Inflammation. 1982;6 (4) :365-370.Abstract
Isolated human peripheral blood neutrophils were exposed to sonic extracts of Actinobacillus actinomycetemcomitans. Such bacterial preparations contain a potent leukotoxin which rapidly kills the leukocytes as reflected by cellular uptake of trypan blue, extracellular release of lactate dehydrogenase, or discharge of 51Cr from pre-labeled cells. Exogenous phospholipids with a glycerol skeleton esterified by fatty acids or positively charged liposomes inhibited cytotoxic phenomena. The data suggest that cell damage may involve the interaction of leukotoxin with phospholipids in the neutrophil cell membrane and that exogenous lipids either compete for or sterically block binding of the leukotoxin to these moieties in the membrane.
Modulation of human lymphocyte transformation by bacterial products and leukocyte lysates
Sela MN, Ginsburg I, Dishon T, Duchan Z, Garfunkel AA. Modulation of human lymphocyte transformation by bacterial products and leukocyte lysates. Inflammation. 1982;6 (1) :31-38.Abstract
Blast transformation of human peripheral blood lymphocytes by PHA is shown to be modulated by lipoteichoic acid (LTA) of Streptococcus mutans, by a cell-sensitizing factor of Actinomyces viscosus, as well as by a frozen and thawed extract of human leukocytes (LE). While small amounts of LE (5-50 micrograms/10(6) cells) significantly enhanced PHA-induced transformation, higher amounts showed a lesser effect on the blastogenic response. Both LTA and the A. viscosus extract did not cause any lymphocyte blastogenic effect when used alone. On the other hand LTA had an inhibitory effect and the A. viscosus extract had an enhancing effect when lymphocytes were pretreated by these agents and then exposed to PHA.

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