Isaac Ginsburg

Role of lipoteichoic acid in infection and inflammation

Lipoteichoic acid (LTA) is a surface-associated adhesion amphiphile from Gram-positive bacteria and regulator of autolytic wall enzymes (muramidases). It is released from the bacterial cells mainly after bacteriolysis induced by lysozyme, cationic peptides from leucocytes, or beta-lactam antibiotics. It binds to target cells either non-specifically, to membrane phospholipids, or specifically, to CD14 and to Toll-like receptors. LTA bound to targets can interact with circulating antibodies and activate the complement cascade to induce a passive immune kill phenomenon. It also triggers the release from neutrophils and macrophages of reactive oxygen and nitrogen species, acid hydrolases, highly cationic proteinases, bactericidal cationic peptides, growth factors, and cytotoxic cytokines, which may act in synergy to amplify cell damage. Thus, LTA shares with endotoxin (lipopolysaccharide) many of its pathogenetic properties. In animal studies, LTA has induced arthritis, nephritis, uveitis, encephalomyelitis, meningeal inflammation, and periodontal lesions, and also triggered cascades resulting in septic shock and multiorgan failure. Binding of LTA to targets can be inhibited by antibodies, phospholipids, and specific antibodies to CD14 and Toll, and in vitro its release can be inhibited by non-bacteriolytic antibiotics and by polysulphates such as heparin, which probably interfere with the activation of autolysis. From all this evidence, LTA can be considered a virulence factor that has an important role in infections and in postinfectious sequelae caused by Gram-positive bacteria. The future development of effective antibacteriolitic drugs and multidrug strategies to attenuate LTA-induced secretion of proinflammatory agonists is of great importance to combat septic shock and multiorgan failure caused by Gram-positive bacteria.

The role of bacteriolysis in the pathophysiology of inflammation, infection and post‐infectious sequelae

The literature dealing with the biochemical basis of bacteriolysis and its role in inflammation, infection and in post‐infectious sequelae is reviewed and discussed. Bacteriolysis is an event that may occur when normal microbial multiplication is altered due to an uncontrolled activation of a series of autolytic cell‐wall breaking enzymes (muramidases). While a low‐level bacteriolysis sometimes occurs physiologically, due to “mistakes” in cell separation, a pronounced cell wall breakdown may occur following bacteriolysis induced either by beta‐lactam antibiotics or by a large variety of bacteriolysis‐inducing cationic peptides. These include spermine, spermidine, bactericidal peptides defensins, bacterial permeability increasing peptides from neutrophils, cationic proteins from eosinophils, lysozyme, myeloperoxidase, lactoferrin, the highly cationic proteinases elastase and cathepsins, PLA2, and certain synthetic polyamino acids. The cationic agents probably function by deregulating lipoteichoic acid (LTA) in Gram‐positive bacteria and phospholipids in Gram‐negative bacteria, the presumed regulators of the autolytic enzyme systems (muramidases). When bacteriolysis occurs in vivo, cell‐wall‐ and ‐membrane‐associated lipopolysaccharide (LPS (endotoxin)), lipoteichoic acid (LTA) and peptidoglycan (PPG), are released. These highly phlogistic agents can act on macrophages, either individually or in synergy, to induce the generation and release of reactive oxygen and nitrogen species, cytotoxic cytokines, hydrolases, proteinases, and also to activate the coagulation and complement cascades. All these agents and processes are involved in the pathophysiology of septic shock and multiple organ failure resulting from severe microbial infections. Bacteriolysis induced in in vitro models, either by polycations or by beta‐lactams, could be effectively inhibited by sulfated polysaccharides, by D‐amino acids as well as by certain anti‐bacteriolytic antibiotics. However, within phagocytic cells in inflammatory sites, bacteriolysis tends to be strongly inhibited presumably due to the inactivation by oxidants and proteinases of the bacterial muramidases. This might results in a long persistence of non‐biodegradable cell‐wall components causing granulomatous inflammation. However, persistence of microbial cell walls in vivo may also boost innate immunity against infections and against tumor‐cell proliferation. Therapeutic strategies to cope with the deleterious effects of bacteriolysis in vivo include combinations of autolysin inhibitors with combinations of certain anti‐inflammatory agents. These might inhibit the synergistic tissue‐ and‐ organ‐damaging “cross talks” which lead to septic shock and to additional post‐infectious sequelae.

Polyphenols enhance total oxidant-scavenging capacities of human blood by binding to red blood cells

The present study offers a new look at the role of erythrocytes and of erythrocytes–polyphenol complexes as potent ‘sinks’ for reactive oxygen species. We hereby show that human erythrocytes have the capacity not only to carry oxygen, but also to bind avidly to their surfaces a large variety of polyphenol antioxidants, which endows upon such complexes enhanced total oxidant-scavenging capacities (TOSC). This was proven by using confocal microscopy, 2,2-diphenyl-1-picrylhydrazyl radical, Folin-Ciocalteus reagent, cyclic voltammetry and chemiluminescence techniques. The results presented suggest that the true TOSC of blood is the sum of intracellular antioxidants of red blood cells and other blood cells (mainly due to catalase), the polyphenols bound to their surfaces and the antioxidant agents present in plasma. Since erythrocytes can avidly bind and rapidly remove circulating polyphenols, the rule of the thumb to quantify antioxidants in health and disease processes exclusively in plasma as customary in clinical settings, does not represent the true TOSC of whole blood. We also postulate that circulating erythrocytes and possibly also other blood cells might be constantly coated by polyphenols from supplemented nutrients, which act as antioxidant depots and can thus act as protectors against the harmful consequences of oxidative stress. Further studies are needed to determine the faith of polyphenols in the circulation and their sequestration in the spleen.

Mechanism of visible light phototoxicity on Porphyromonas gingivalis and Fusobacterium nucleatum.

Abstract Phototoxicity of visible light laser on the porphyrin-producing bacteria, Porphyromonas gingivalis, in the absence of photosensitizers and under aerobic conditions was shown in previous studies. Recently, we found that the noncoherent visible light sources at wavelengths of 400–500 nm, commonly used in restorative dentistry, induced a phototoxic effect on P. gingivalis, as well as on Fusobacterium nucleatum, and to a lesser extent on the Streptococci sp. To elucidate the mechanism of this phototoxic effect, P. gingivalis and F. nucleatum were exposed to light (1) under aerobic and anaerobic environments and (2) in the presence of scavengers of reactive oxygen species (ROS). Phototoxic effect was not observed when the bacteria were exposed to light under anaerobic conditions. Dimethyl thiourea, a hydroxyl radical scavenger, was effective in reducing phototoxicity (P ≤ 0.05). Other scavengers, such as catalase, superoxide dismutase and ascorbic acid, were less effective when applied separately. These results support the assumption that the phototoxic effect of blue light on the periopathogenic bacteria is oxygen dependent and that hydroxyl radicals play an important role in this process.

Cell wall degradation ofStaphylococcus aureus by lysozyme

In contrast to former findings lysozyme was able to attack the cell walls ofStaphylococcus aureus under acid conditions. However, experiments with14C-labelled cell walls and ribonuclease indicated that, under these conditions, lysozyme acted less as an muralytic enzyme but more as an activator of pre-existing autolytic wall enzymes. Electron microscopic studies showed that under these acid conditions the cell walls were degraded by a new mechanism (i.e. “attack from the inside”). This attack on the cell wall started asymmetrically within the region of the cross wall and induced the formation of periodically arranged lytic sites between the cytoplasmic membrane and the cell wall proper. Subsequently, a gap between the cell wall and the cytoplasmic membrane resulted and large cell wall segments became detached and suspended in the medium. The sequence of lytic events corresponded to processes known to take place during wall regeneration and wall formation. In the final stage of lysozyme action at pH 5 no cell debris but “stabilized protoplasts” were to be seen without detectable alterations of the primary shape of the cells. At the same time long extended ribbon-like structures appeared outside the bacteria. The origin as well as the chemical nature of this material is discussed. Furthermore, immunological implications are considered.

Inhibition of wall autolysis of staphylococci by sodium polyanethole sulfonate "liquoid".

SummaryLiquoid (polyanethole sulfonate) was neither capable of influencing the growth nor the viability of staphylococci. But liquoid induced a suppression of the activity of different autolytic wall systems of normally growing staphylococci, i.e., autolysins which participate in cross wall separation as well as autolysins which are responsible for cell wall turnover. Additionally, the lysostaphin-induced wall disintegration of staphylococci was inhibited by liquoid.However, no indication could be found for a direct inhibition of lytic wall enzymes by liquoid; rather an interaction of liquoid with the target structure for the autolytic wall enzymes, the cell wall itself, was postulated. On the basis of the experimental data with the teichoic acid- mutant S. aureus 52A5 the sites of wall teichoic acid were supposed to be an important target for the binding of liquoid to the staphylococcal cell wall.

Role of leukocyte factors and cationic polyelectrolytes in phagocytosis of group A streptococci and Candida albicans by neutrophils, macrophages, fibroblasts and epithelial cells: modulation by anionic polyelectrolytes in relation to pathogenesis of chronic inflammation.

A variety of cationic polyelectrolytes opsonized group A streptococci andCandida albicans to phagocytosis by human polymorphonuclear leukocytes and by mouse peritoneal macrophages. The most potent opsonins for streptococci were specific antibodies supplemented with complement, nuclear histone, polylysine, polyarginine, ribonuclease, leukocyte lysates, leukocyte cationic protein and, to a lesser extent, lysozyme and myeloperoxidase. Histone, RNAse, leukocyte extracts, and platelet extracts also functioned as opsonins for phagocytosis of streptococci in the peritoneal cavity, where phagocytic indices, higher than those obtained for the in vitro phagocytosis, were obtained. Fresh serum, polylysine, polyarginine, and nuclear histone acted as good opsonins forCandida, but none of the other factors tested were active. In order for the cationic proteins and leukocyte extracts to function as opsonins, they must be present on the particle surface. These agents were poor opsonins when applied on the macrophages. Nuclear histone, polylysine, polyarginine, and fresh human serum also functioned as good opsonins for the uptake ofCandida by mouse fibroblasts. On the other hand, none of the other substances which opsonized streptococci were effective withCandida. The phagocytic capabilities of fibroblast polykaryons were much higher than those of ordinary spindle-shaped mouse fibroblasts. Histone also functioned as a good opsonic agent for the uptake ofCandida by human fibroblasts, HeLa cells, epithelial cells, monkey kidney cells, and rat heart cells. On the other hand, neither leukocyte extracts nor ribonuclease LCP or MPO functioned as opsonins for these mammalian cells.Candida, taken up by fibroblasts, were present within tight phagosomes, but no fusion of lysosomes with the phagosome occurred. A small proportion of the internalized yeast cells underwent partial plasmolysis, but little damage to the rigid cell walls was observed within 24–48 h of internalization. Phagocytosis of streptococci andCandida by macrophages and the uptake ofCandida by fibroblasts were both strongly inhibited by liquoid (polyanethole sulfonic acid sodium salt). This anionic polyelectrolyte also markedly inhibited the release ofN-acetylglucosaminidase from macrophages without affecting cell viability (LDH release). Hyaluronic acid, DNA, and dextran sulfate markedly inhibited the uptake of histone-coated particles by macrophages. On the other hand, hyaluronic acid and DNA enhanced the uptake ofCandida by fibroblasts. The effect of these anionic polyelectrolytes on phagocytosis of serum-opsonized particles by macrophages was not consistent. While in some experiments it blocked phagocytosis, in others it either had no effect or even enhanced the uptake of the particles. Phagocytosis of microorganisms by “nonprofessional” phagocytes like fibroblasts and the paucity in these cells of hydrolases capable of breaking down microbial cell wall components may contribute to the persistence of non-biodegradable components of bacteria in tissues and to the perpetuation of chronic inflammatory sequellae. Cationic polyelectrolytes may also prove important as “helper” opsonins and as agents capable of enhancing the penetration into cells of both viable and nonviable particles, genetic material, and drugs.A variety of cationic polyelectrolytes opsonized group A streptococci andCandida albicans to phagocytosis by human polymorphonuclear leukocytes and by mouse peritoneal macrophages. The most potent opsonins for streptococci were specific antibodies supplemented with complement, nuclear histone, polylysine, polyarginine, ribonuclease, leukocyte lysates, leukocyte cationic protein and, to a lesser extent, lysozyme and myeloperoxidase. Histone, RNAse, leukocyte extracts, and platelet extracts also functioned as opsonins for phagocytosis of streptococci in the peritoneal cavity, where phagocytic indices, higher than those obtained for the in vitro phagocytosis, were obtained. Fresh serum, polylysine, polyarginine, and nuclear histone acted as good opsonins forCandida, but none of the other factors tested were active. In order for the cationic proteins and leukocyte extracts to function as opsonins, they must be present on the particle surface. These agents were poor opsonins when applied on the macrophages. Nuclear histone, polylysine, polyarginine, and fresh human serum also functioned as good opsonins for the uptake ofCandida by mouse fibroblasts. On the other hand, none of the other substances which opsonized streptococci were effective withCandida. The phagocytic capabilities of fibroblast polykaryons were much higher than those of ordinary spindle-shaped mouse fibroblasts. Histone also functioned as a good opsonic agent for the uptake ofCandida by human fibroblasts, HeLa cells, epithelial cells, monkey kidney cells, and rat heart cells. On the other hand, neither leukocyte extracts nor ribonuclease LCP or MPO functioned as opsonins for these mammalian cells.Candida, taken up by fibroblasts, were present within tight phagosomes, but no fusion of lysosomes with the phagosome occurred. A small proportion of the internalized yeast cells underwent partial plasmolysis, but little damage to the rigid cell walls was observed within 24–48 h of internalization. Phagocytosis of streptococci andCandida by macrophages and the uptake ofCandida by fibroblasts were both strongly inhibited by liquoid (polyanethole sulfonic acid sodium salt). This anionic polyelectrolyte also markedly inhibited the release ofN-acetylglucosaminidase from macrophages without affecting cell viability (LDH release). Hyaluronic acid, DNA, and dextran sulfate markedly inhibited the uptake of histone-coated particles by macrophages. On the other hand, hyaluronic acid and DNA enhanced the uptake ofCandida by fibroblasts. The effect of these anionic polyelectrolytes on phagocytosis of serum-opsonized particles by macrophages was not consistent. While in some experiments it blocked phagocytosis, in others it either had no effect or even enhanced the uptake of the particles. Phagocytosis of microorganisms by “nonprofessional” phagocytes like fibroblasts and the paucity in these cells of hydrolases capable of breaking down microbial cell wall components may contribute to the persistence of non-biodegradable components of bacteria in tissues and to the perpetuation of chronic inflammatory sequellae. Cationic polyelectrolytes may also prove important as “helper” opsonins and as agents capable of enhancing the penetration into cells of both viable and nonviable particles, genetic material, and drugs.

H2O2 renders cells accessible to lysis by exogenous phospholipase A2: a novel mechanism for cell damage in inflammatory processes

Phospholipase A2 (PLA2) and H2O2, secreted from activated inflammatory cells, play a central role in the tissue damage occurring in inflammatory processes. However, while exogenous PLA2 alone does not cause cell lysis, it readily does so when acting with H2O2. We have found that H2O2 degrades cell surface proteoglycans, thus rendering the membrane PL accessible to hydrolysis by exogenous PLA2. This novel mechanism introduces a role for cell surface proteoglycans in protection of cells from damage by pro‐inflammatory agents, and may assign a central role for the combined action of H2O2 and PLA2 in inflammatory and bacteriocidal processes.

Lysophosphatides enhance superoxide responses of stimulated human neutrophils

Human neutrophils which are pretreated with subtoxic concentrations of a variety of lysophosphatides (lysophosphatidytcholine, lysophosphatidylcholine oleoyl, lysophosphatidylcholine myrioyl, lysophosphatidylcholine stearoyl, lysophosphatidylcholine gamma-O-hexadecyl, lysophosphatidylinositol, and lysophosphatidylglycerol) act synergistically with neutrophil agonists phorbol myristate acetate, immune complexes, poly-L-histidine, phytohemagglutinin, andN-formyl methionyl-leucyl-phenyalanine to cause enhanced generation of superoxide (O2−). None of the lyso compounds by themselves caused generation of O2−. The lyso compounds strongly bound to the neutrophils and could not be washed away. All of the lyso compounds that collaborated with agonists to stimulate O2− generation were hemolytic for human red blood cells. On the other hand, lyso compounds that were nonhemolytic for red blood cells (lysophosphatidylcholine caproate, lysophosphatidylcholine decanoyl, lysophosphatidylethanolamine, lysophosphatidylserine) failed to collaborate with agonists to generate synergistic amounts of O2−. However, in the presence of cytochalasin B, both lysophosphatidyiethanolamine and lysophosphatidylserine also markedly enhanced O2− generation induced by immune complexes. O2− generation was also very markedly enhanced when substimulatory amounts of arachidonic acid or eicosapentanoic acid were added to PMNs in the presence of a variety of agonists. On the other hand, neither phospholipase C, streptolysin S (highly hemolytic), phospholipase A2, phosphatidylcholine, nor phosphatidylcholine dipalmitoyl (all nonhemolytic) had the capacity to synergize with any of the agonists tested to generate enhanced amounts of O2−. The data suggest that in addition to long-chain fatty acids, only those lyso compounds that possess fatty acids with more than 10 carbons and that are also highly hemolytic can cause enhanced generation of O2− in stimulated PMNs.

The effect of leukocyte hydrolases on bacteria. II. The synergistic action of lysozyme and extracts of PMN, macrophages, lymphocytes, and platelets in bacteriolysis.

Summary Extracts containing acid hydrolases and lysozyme derived from human and rabbit blood leukocytes, from rabbit peritoneal and lung macrophages and from peritoneal PMN are highly lytic for relabeled Staphylococcus albus, Streptococcus faecalis, and Streptococcus mutans. On the other hand, extracts of human and rabbit platelets and of rabbit lymphocytes, thymocytes, synovia and muscle are not lytic for these bacteria. A phenomenon is described in which lysozyme which is not lytic to these bacteria collaborates with nonlytic extracts of lymphocytes platelets, thymocytes, synovia and muscle in the lysis of Gram positive bacteria. Lysozyme also enhances the lysis of bacteria by extracts of PMN and macrophages. It is postulated that the bacteriolytic system present in PMN and macrophages comprises a group of preparatory nonlytic enzymes, also present in lymphocytes, thymocytes, platelets, and other tissues that prepare the peptidoglycan to cleavage by lysozyme. The preparatory enzyme systems of leukocytes and tissues have a pH optimum of 5.0 and are strongly inhibited by heparin and chondroitin sulfate. The relationship of the synergism between lysozyme and tissue enzymes in the degradation of microbial cells is discussed in relation to the pathogenesis of granulomatous inflammation.