Both aqueous and methanolic fractions derived from the Tibetan preparation PADMA-28 (a mixture of 22 plants) used as an anti-atherosclerotic agent, and which is non-cytolytic to a variety of mammalian cells, were found to strongly inhibit (1) the killing of epithelial cells in culture induced by 'cocktails' comprising oxidants, membrane perforating agents and proteinases; (2) the generation of luminol-dependent chemiluminescence in human neutrophils stimulated by opsonized bacteria; (3) the peroxidation of intralipid (a preparation rich in phopholipids) induced in the presence of copper; and (4) the activity of neutrophil elastase. It is proposed that PADMA-28 might prove beneficial for the prevention of cell damage induced by synergism among pro-inflammatory agonists which is central in the initiation of tissue destruction in inflammatory and infectious conditions.

The paper discusses the principal evidence that supports the concept that cell and tissue injury in infectious and post-infectious and inflammatory sequelae might involve a deleterious synergistic interaction among microbial- and host-derived pro-inflammatory agonists. Experimental models had proposed that a rapid cell and tissue injury might be induced by combinations among subtoxic amounts of three major groups of agonists generated both by microorganisms and by the host's own defense systems. These include: (1) oxidants: Superoxide, H(2)O(2), OH', oxidants generated by xanthine-xanthine-oxidase, ROO; HOC1, NO, OONO'-, (2) the membrane-injuring and perforating agents, microbial hemolysins, phospholipases A(2) and C, lysophosphatides, bactericidal cationic proteins, fatty acids, bile salts and the attack complex of complement a, certain xenobics and (3) the highly cationic proteinases, elastase and cathepsin G, as well as collagenase, plasmin, trypsin and a variety of microbial proteinases. Cell killing by combinations among the various agonists also results in the release of membrane-associated arachidonate and metabolites. Cell damage might be further enhanced by certain cytokines either acting directly on targets or through their capacity to prime phagocytes to generate excessive amounts of oxidants. The microbial cell wall components, lipoteichoic acid (LTA), lipopolysaccharides (LPS) and peptidoglycan (PPG), released following bacteriolysis, induced either by cationic proteins from neutrophils and eosinophils or by beta lactam antibiotics, are potent activators of macrophages which can release oxidants, cytolytic cytokines and NO. The microbial cell wall components can also activate the cascades of coagulation, complement and fibrinolysis. All these cascades might further synergize with microbial toxins and metabolites and with phagocyte-derived agonsits to amplify tissue damage and to induce septic shock, multiple organ failure, 'flesh-eating' syndromes, etc. The long persistence of non-biodegradable bacterial cell wall components within activated macrophages in granulomatous inflammation might be the result of the inactivation by oxidants and proteinases of bacterial autolytic wall enzymes (muramidases). The unsuccessful attempts in recent clinical trials to prevent septic shock by the administration of single antagonists is disconcerting. It does suggest however that, since tissue damage in post-infectious syndromes is most probably the end result of synergistic interactions among a multiplicity of agents, only agents which might depress bacteriolysis in vivo and 'cocktails' of appropriate antagonists, but not single antagonists, if administered at the early phases of infection especially to patients at high risk, might help to control the development of post-infectious syndromes. However, the use of adequate predictive markers for sepsis and other post-infectious complications is highly desirable. Although it is conceivable that anti-inflammatory strategies might also be counter-productive as they might act as 'double-edge swords', intensive investigations to devise combination therapies are warranted. The present review also lists the major anti-inflammatory agents and strategies and combinations among them which have been proposed in the last few years for clinical treatments of sepsis and other post-infectious complications.

The possible role played by streptolysin S (SLS) of group A streptococci in the pathophysiology of streptococcal infections and in post-streptococcal sequelae is discussed. The following properties of SLS justify its definition as a distinct virulence factor: 1) its presence on the streptococcus surface in a cell-bound form, 2) its continuous and prolonged synthesis by resting streptococci, 3) its non-immunogenicity, 4) its extractability by serum proteins (albumin, alpha lipoprotein), 5) its ability to become transferred directly to target cells while being protected from inhibitory agents in the milieu of inflammation, 6) its ability to bore holes in the membrane phospholipids in a large variety of mammalian cells, 7) its ability to synergize with oxidants, proteolytic enzymes, and with additional host-derived proinflammatory agonists, and 8) its absence in streptococcal mutants associated with a lower pathogenicity for animals. Because tissue damage in streptococcal and post-streptococcal sequelae might be the end result of a distinct synergism between streptococcal and host-derived proinflammatory agonists it is proposed that only cocktails of anti-inflammatory agents including distinct inhibitors of SLS (phospholipids), gamma globulin, inhibitors of reactive oxygen species, proteinases, cationic proteins cytokines etc., will be effective in inhibiting the multiple synergistic interactions which lead to fasciitis, myositis and the flesh-eating syndromes, and often develop into sepsis, septic shock and multiple organ failure. The creation of mutants deficient in SLS and in proteases will help shed light on the specific role played by SLS in the virulence of group A hemolytic streptococci.

Comment on Proteolytic enzymes and airway diseases. [Eur Respir J. 1998] Neutrophil serine proteinases and defensins in chronic obstructive pulmonary disease: effects on pulmonary epithelium. [Eur Respir J. 1998] To the Editor: I have recently read with much interest two excellent reports in the European Respiratory Journal which discussed the role of neutrophil proteinases and defensins in chronic obstructive pulmonary disease [1] and in airway diseases [2]. Reading through these articles, it was surprising not to find any considerations of a major aspect related to the elucidation of the possible mechanisms of tissue damage in the lungs during inflammation. I refer to extensive studies from several laboratories which had proposed that tissue damage in inflammatory and infectious processes may primarily be the result of a synergistic "cross talk" among a multiplicity of pro-inflammatory agents (a multi-component system) [3, 4]. A series of publications [5±14] have shown that a severe and rapid membrane injury (necrosis) could be initiated in mammalian cells by a synergism among subtoxic concen- trations of three major groups of agonists. These included a) oxidants (H2O2, peroxyl radical, oxidants generated by xanthine-xanthine-oxidase, NO, HOCl, OONO-), b) mem- brane -perforating agents (microbial haemolysins/phospho- lipases A2 and C, lysophosphatides, free fatty acids, cationic proteins, histone [9] and defensins [5], and c) highly cationic proteolytic enzymes, (elastase, cathepsin G) [3, 4, 12]. These synergistic cytotoxic effects can be further amplified by certain cytokines. Furthermore, combinations of oxidants and elastase have also been shown to synergize to cause severe lung damage in animal models [6±10]. It has also been proposed that a deleterious synergism among microbial and host-derived pro-inflammatory agonists may frequently contribute to tissue injury in many infectious and post- infection complications [3, 4]. A notable example is, sepsis and the "flesh-eating" syndrome caused by highly toxigenic and invasive bacteria. Other studies had also shown that subtoxic amounts of the membrane-active xenobiotics, ethanol, methanol, n-butanol and the pesticide linden [13], could also synergize with subtoxic concentrations of peroxide, proteinases and cationic agents to amplify the damage to endothelial cells in culture. The results with the xenobiotics are of especial interest and concern to pulmonologists as these volatile agents may be inhaled and might then synergize with oxidants, proteinases and cationic proteins released either by accumulating neu- trophils or by activated lung macrophages to cause damage to both epithelial and endothelial cells. It has also been documented that ˜-lactam antibiotics and a large variety of cationic agents including, elastase, cath- epsin G, defensins, lysozyme, myeloperoxidase, spermine, spermidine, histones, polymyxin B and chlorhexidine are all capable of activating the autolytic wall enzymes (murami- dases) in bacteria leading to bacteriolysis [14]. Bacteriolysis at least in Gram-positive bacteria induced either by ˜- lactams or by cationic agents can, however, be strongly inhibited by sulphated polyanions presumably by inactivat- ing the autolytic wall enzymes responsible for breaking down the rigid cell wall. It is accepted that the massive release widely of bacterial wall components (lipopolysac- charide, lipoteichoic acid (LTA), peptidoglycan), in vivo, can activate macrophages to release cytotoxic cytokines, NO and also to activate the complement and coagulation cascades leading to sepsis, systemic inflammatory response syndrome (SIRS), multiple organ disfunction syndrome (MODS) and multiple organ failure (MOF) [15]. Today there are controversial opinions and hot debates regarding the approaches to treat sepsis, adult respiratory distress syndrome (ARDS) and additional post-infectious and inflammatory sequelae [15]. Unfortunately, the exclu- sive use of single antagonists to treat these syndromes has yielded poor results. Such failures may principally be due to, a) the lack of adequate and rapid tests to predict the onset of such complications so that treatment of patients usually starts too late, and b) a lack of sufficient awareness that fighting the deleterious effects caused by synergistic cytotoxic mechan- isms necessitates the use not of single antagonists but of cocktails comprised of a multiplicity of anti-inflammatory agents. Hopefully, a wider recognition of synergism concept of cellular injury [3, 4, 11±13] might offer a new and more realistic approach to this complex and still unsolved clinical problem. I. Ginsburg Dept of Oral Biology, Hebrew University - Hadassah Faculty of Dental Medicine, Jerusalem, Israel. Fax: 972 26758583.

The purpose of this review-hypothesis is to discuss the literature which had proposed the concept that the mechanisms by which infectious and inflammatory processes induce cell and tissue injury, in vivo, might paradoxically involve a deleterious synergistic ‘cross-talk’, among microbial- and host-derived pro-inflammatory agonists. This argument is based on studies of the mechanisms of tissue damage caused by catalase-negative group A hemolytic streptococci and also on a large body of evidence describing synergistic interactions among a multiplicity of agonists leading to cell and tissue damage in inflammatory and infectious processes. A very rapid cell damage (necrosis), accompanied by the release of large amounts of arachidonic acid and metabolites, could be induced when subtoxic amounts of oxidants (superoxide, oxidants generated by xanthine-xanthine oxidase, HOCl, NO), synergized with subtoxic amounts of a large series of membrane-perforating agents (streptococcal and other bacterial-derived hemolysins, phospholipases A2 and C, lysophosphatides, cationic proteins, fatty acids, xenobiotics, the attack complex of complement and certain cytokines). Subtoxic amounts of proteinases (elastase, cathepsin G, plasmin, trypsin) very dramatically further enhanced cell damage induced by combinations between oxidants and the membrane perforators. Thus, irrespective of the source of agonists, whether derived from microorganisms or from the hosts, a triad comprised of an oxidant, a membrane perforator, and a proteinase constitutes a potent cytolytic cocktail the activity of which may be further enhanced by certain cytokines. The role played by non-biodegradable microbial cell wall components (lipopolysaccharide, lipoteichoic acid, peptidoglycan) released following polycation- and antibiotic-induced bacteriolysis in the activation of macrophages to release oxidants, cytolytic cytokines and NO is also discussed in relation to the pathophysiology of granulomatous inflammation and sepsis. The recent failures to prevent septic shock by the administration of only single antagonists is disconcerting. It suggests, however, that since tissue damage in post-infectious syndromes is caused by synergistic interactions among a multiplicity of agents, only cocktails of appropriate antagonists, if administered at the early phase of infection and to patients at high risk, might prevent the development of post-infectious syndromes.

BACKGROUND. Changes in the sympathetic nervous system may be a cause of postoperative cardiovascular complications. The authors hypothesized that changes in both beta-adrenergic receptor (betaAR) function (as assessed in lymphocytes) and in sympathetic activity (assessed by plasma catecholamines and by heart rate variability [HRV] measurements obtained from Holter recordings) occur after operation. METHODS: The HRV parameters were measured in 28 patients having thoracotomy (n = 14) or laparotomy (n = 14) before and for as long as 6 days after operation. Transthoracic echocardiography was performed before and on postoperative day 2. Lymphocytes were also isolated from blood obtained before anesthesia and again on postoperative days 1, 2, 3, and 5 (or 6). They were used to examine betaAR number (Bmax) and cyclic adenosine monophosphate (cAMP) production after stimulation with isoproterenol and prostaglandin E1. In addition, plasma epinephrine, norepinephrine, and cortisol concentrations were determined at similar intervals. RESULTS: After abdominal and thoracic surgery, most time and all frequency indices of HRV decreased significantly, as did Bmax and basal and isoproterenol-stimulated cAMP production. The decrements in HRV correlated with those of Bmax and isoproterenol-stimulated cAMP throughout the first postoperative week and inversely correlated with the increase in heart rate. Plasma catecholamine concentrations did not change significantly from baseline values, but plasma cortisol levels did increase after operation in both groups. Left ventricular ejection fraction was normal in both groups and unaffected by surgery. CONCLUSIONS: Persistent downregulation and desensitization of the lymphocyte betaAR/adenylyl cyclase system correlated with decrements in time and frequency domain indices of HRV throughout the first week after major abdominal or thoracic surgery. These physiologic alterations suggest the continued presence of adaptive autonomic regulatory mechanisms and may explain why the at-risk period after major surgery appears to be about 1 week or more.

Comment on Tissue injury in neutrophilic inflammation. [Inflamm Res. 1997]

An in vitro model was employed to study the potential role of streptococcal extra-cellular products, rich in streptolysin O, in cellular injury as related to streptococcal infections and post-streptococcal sequelae. Extra-cellular products (EXPA) rich in streptolysin O were isolated from type 4, group A hemolytic streptococci grown in a chemostat, in a synthetic medium. EXPA induced moderate cytopathogenic changes in monkey kidney epithelial cells and in rat heart cells pre-labeled with 3H-arachidonate. However very strong toxic effects were induced when EXP was combined with oxidants (glucose oxides generated H2O2, AAPH-induced peroxyl radical (ROO.), NO generated by sodium nitroprusside) and proteinases (plasmin, trypsin). Cell killing was distinctly synergistic in nature. Cell damage induced by the multi-component cocktails was strongly inhibited either by micromolar amounts of gamma globulin, and Evan's blue which neutralized SLO activity, by tetracycline, trasylol (aprotinin), epsilon amino caproic acid and by soybean trypsin inhibitor, all proteinase inhibitors as well as by a non-penetrating PLA2 inhibitor A. The results suggest that fasciitis, myositis and sepsis resulting from infections with hemolytic streptococci might be caused by a coordinated 'cross-talk' among microbial, leukocyte and additional host-derived pro-inflammatory agents. Since attempts to prolong lives of septic patients by the exclusive administration of single antagonists invariably failed, it is proposed that the administration of 'cocktails' of putative inhibitors against major pro-inflammatory agonizes generated in inflammation and infection might protect against the deleterious effects caused by the biochemical and pharmacological cascades which are known to be activated in sepsis.

I read with much interest the review article ‘‘Mechanisms of neutrophil-induced parenchymal cell injury’’(H. Jaeschke and C.W. Smith, J. Leukoc. Biol. 61, 647–653). As I read through the text it became apparent that very basic and relevant concepts as well as publications regarding the possible mecha- nisms by which phagocytes kill targets had not been included in the review. The authors rightfully write, ‘‘The question regarding the molecular mechanism of neutrophil-induced target cell injury is controversial.’’ Yet despite the common knowledge and understanding that the mechanisms of cell damage most probably involve an interaction among a multiplicity of agonists (a multicomponent system), the section, ‘‘Mechanisms of neutro- phil-induced parenchymal cell injury’’ had adopted an ex- tremely reductionist and oversimplified approach to the prob- lem. It considered (see Fig. 1) what seems to be the exclusive role of oxidants and proteinases as potential cell injuring agents, as if these are the sole noxious agents generated by activated phagocytes. Although I fully respect the prerogatives and choices by the authors to refer exclusively to hepatocytes and parenchymal cells and to select citations from the literature pertaining to these tissues in order to support their thesis, it is still intriguing why not a single word was mentioned about the obvious possibility that oxidants, proteinases, and additional agonists might perhaps act mainly in concert (synergize) to injure any cell type? To the best of our knowledge and experience in this field of research (see list of recommended literature), even a normal cell line, as well as some of the tumor cells tested in vitro by us and by others, which could not readily be killed by physiologi- cal amounts of oxidants (H2O2 ROO, HCIO, NO) alone, were nevertheless rapidly killed in a synergistic manner if the oxidants were combined with any of a long list of membrane- perforating agents. These included phospholipase A2, phospho- lipase C, lysophosphatides, fatty acids, microbial hemolysins, cationic peptides and proteins, bile salts, complement compo- nents, and xenobiotics such as ethanol, methanol, and lindane. The inclusion of proteinases (trypsin, plasmin, elastase chymotrypsin), together with oxidants and the membrane perforators, further significantly enhanced cellular damage. It is also of great interest and is perhaps paradoxical that microbial agents might also synergize with phagocyte-derived agonists, but in an adverse fashion, to injure host tissues. It is also important to consider that all these proinflammatory agonists might be simultaneously present in infectious and inflammatory sites. Our studies also suggested that the induction of a sublethal membrane injury abolished, to a large extent, the potent antioxidant defenses of the cells— a finding of great significance. The readers of the Journal of Leukocyte Biology might be interested in a series of publications dealing with the ‘‘syner- gism’’ concept of cellular injury as related to infectious and inflammatory conditions, which have been published since 1986 (see list of recommended literature). An invited overview by Ginsburg and Kohen [8] undertook to discuss, in great detail, those papers that described the role of synergism in cellular injury. Unfortunately and enigmatically, publications that have described the ‘‘synergism concept’’ of cellular injury published since 1986 are hardly ever cited. If the synergism concept of cellular injury is logical and conforms with the current knowledge in the field, publications describing this phenom- enon should be quoted. If on the other hand these ideas are extreme, bizarre, and scientifically unacceptable, such papers should be discussed and challenged properly and even ridi- culed. However, it is totally unacceptable that such publica- tions be simply ignored. Approaching the third millennium, the readers of scientific journals deserve not an oversimplified approach to complicated scientific issues, but more realistic, integrated, and updated appraisals of the literature even if these might not always fully conform with the investigator’s own concepts or with the prevailing paradoxes, dogmas, cliches, and myths. It is also very surprising, and of great concern, why the referees of the papers did not bring any of these publications and concepts to the attention of the authors. After all, the main task of the referees and the editorial board is to criticize the validity and novelty of investigations brought to their attention and to strongly instruct negligent authors to give proper credit to relevant papers and concepts in their field of research. It is regrettable that this has not happened. Unfortunately, this is how, for the sake of brevity and a reductionist approach to the solution of complex biological phenomena, very basic and pioneering investigations and ‘‘novel’’ concepts may be simply ignored and buried for good. It is obvious that the ones who might suffer most from such an approach to the compilation of reviews and papers are the investigators, the readers, and perhaps most importantly, the credibility of journals at large. I shall greatly appreciate receiving comments and sugges- tions about these matters.

The mechanisms of cellular damage caused by infectious and inflammatory processes are complex and are still not fully understood. There is, however, a consensus that reactive oxygen species (ROS) generated by phagocytes migrating to injured tissues might be the main agents responsible for cellular damage in inflammatory processes. However, because both activated phagocytes and catalase-negative, peroxide-producing, toxigenic bacteria (Streptococci, Clostridiae) secrete a near-identical array of proinflammatory agonists, including reactive oxygen species (ROS), and because these microbial species might kill their targets by a synergism among several of their secreted enzymes (a multicomponent system), we postulated that activated phagocytes might also function in the same way. Using radiolabeled targets, in culture, we demonstrated that subtoxic amounts of a variety of oxidants (H2O2, radicals produced by xanthine-xanthine-oxidase, peroxyl radical, NO) acted synergistically with subtoxic amounts of a large series of membrane-perforating agents (microbial hemolysins, phospholipases, fatty acids, cationic proteins, proteinases, bile salts, the attack complex of complement, the xenobiotics, lindane, ethanol, methanol) to kill cells in culture and to release large amounts of arachidonic acid and metabolites. Membrane perforators might act primarily to overcome the potent antioxidant systems present in all mammalian cells and scavengers of ROS and inhibitors of the additional agonists might act to abolish the synergism among ROS and the membrane-damaging agents. It is also proposed that protection against tissue damage in vivo should also include 'cocktails' of appropriate antagonists. It is enigmatic that those publications which do describe both in-vitro and in-vivo models proposing that a synergism among a multiplicity of agonists might truly represent the mechanisms by which tissues are injured, in vivo, are hardly ever quoted in the current literature.

Some non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the production or actions of oxygen radicals generated by polymorphonuclear leucocytes (PMNs); this mechanism may contribute towards their anti-inflammatory activity. In the present study, the effects of a new enolcarboxamide NSAID, meloxicam, on oxyradical production by human PMNs exposed to various stimuli in vitro were compared with those of other standard NSAIDs. The various stimuli employed were intended to mimic the likely synergies which occur with cytokines and bacterial production (e.g. f-met-leu-phe (fMLP) peptide) in inflamed tissues and to give an insight into the site and mechanism of action of meloxicam and related drugs on the cellular processes involved in oxyradical generation. The results show that meloxicam is a potent inhibitor of oxyradical production at drug concentrations comparable with those encountered during therapy. Its mechanism of action appears similar to that of other enolcarboxamides and, while relatively complex, involves effects which are stimulus dependent and myeloperoxidase sensitive. They probably do not involve inhibition of fMLP-Gi protein receptor activation but may involve tumour necrosis factor-⇌ post-receptor activation. Enolcarboxamides have variable effects on phorbol myristate acetate-protein kinase C3-mediated oxyradical production.

The effect of diethyldithiocarbamate (DDC) and sodium nitroprusside (SNP) on the killing of endothelial cells and on the release of arachidonate by mixtures of oxidants and membrane-damaging agents was studied in a tissue culture model employing bovine aortic endothelial cells labeled either with 51Chromium or 3arachidonic acid. While exposure to low, subtoxic concentrations of oxidants (reagent H2O2, glucose-oxidase generated peroxide, xanthine xanthine oxidase, AAPH-generated peroxyl radical, menadione-generated oxidants) did not result either in cell death or in the loss of membrane-associated arachidonic acid, the addition of subtoxic amounts of a variety of membrane-damaging agents (streptolysin S, PLA2, histone, taurocholate, wheatgerm agglutinin) resulted in a synergistic cell death. However, no significant amounts of arachidonate were released unless proteinases were also present. The addition to these reaction mixtures of subtoxic amounts of DDC (an SOD inhibitor and a copper chelator) not only very markedly enhanced cell death but also resulted in the release of large amounts of arachidonate (in the complete absence of added proteinases). Furthermore, the inclusion in DDC-containing reaction mixtures of subtoxic amounts of SNP, a generator of NO, further enhanced, in a synergistic manner, both cell killing and the release of arachidonate. Cell killing and the release of arachidonate induced by the DDC and SNP-containing mixtures of agonists were strongly inhibited by catalase, glutathione, N-acetyl cysteine, vitamin A, and by a nonpenetrating PLA2 inhibitor as well as by tetracyclines. A partial inhibition of cell killing was also obtained by 1,10-phenanthroline and by antimycin. It is suggested that DDC might amplify cell damage by forming intracellular, loosely-bound complexes with copper and probably also by depleting antioxidant thiols. It is also suggested that "cocktails" containing oxidants, membrane-damaging agents, DDC, and SNP might be beneficial for killing of tumor cells in vivo and for the assessment of the toxicity of xenobiotics in vitro.

Background/airns: Carbobenzoxy-phenylalanyl-methionine (CBZ-Phe-Met), a known inhibitor of the chemotactic peptide N- formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro, has not been evaluated as a topical anti-inflammatory agent in vivo. In order to measure the effect of CBZ-phe-met, one needs a repeat- able, quantitative, easily obtainable standard measurement of the edema formation. In this study, a caliper designed for measuring soft materials was used to evaluate the edema, in- duced by croton oil on rabbit ears, as well as the effects of CBZ- phe-met. Methods: The model used in this study was croton-induced in- flammation on rabbit ears. A caliper for measuring soft materials ( European standard DIN 863 part 3, manufactured by TESA Ltd., Renens, Switzerland) was used to evaluate the edema, which is part of the inflammatory effect . The action of CBZ-phe- met and two other anti-inflammatory agents; hydrocortisone and Na-ibuprofen, were compared. Results: CBZ-phe-met 1-5% was found to reduce the edema on rabbit ears induced by croton oil by 15 to 93%. 5% CBZ-phe- met was found to be as effective as 5% Na-ibuprofen and 0.1% hydrocortisone. Conclusion: The caliper for soft materials was found to be suit- able for measuring the edema induced by croton-oil, as well as the reducing edema due to anti-inflammatory treatment. It was also found that CBZ-phe-met is a potent topical anti-inflamma- tory agent in the croton-oil-induced inflammatory model. This may indicate a new approach in the treatment of inflammation. Key words: Soft material caliper - inflammation - N-formyl-me- thionyl-leucyl-phenylalanine (fMLP) - carbobenzoxy-phenylala- nyl-methionyl (CBZ-phe-met).

The invention relates to a compound of formula (I) in which X1 and X2 each independently represents an oxygen or nitrogen atom; p, m and n are each independently an integer of at least 2; R, R1 and R2 each independently represents a hydrogen atom; a halogen atom; an optionally substituted straight-chained or branched alkyl, alkenyl or alkynyl radical; a group R3O in which R3 is hydrogen atom, an optionally substituted straight-chained or branched alkyl, alkenyl or alkynyl radical; optionally substituted acyl or optionally substituted aryl or heteroaryl; a group R4O(O)C in which R4 is a hydrogen atom or an optionally substituted straight-chained or branched alkyl, alkenyl or alkynyl radical; a group -SR5 in which R5 is a hydrogen atom or an optionally substituted straight-chained or branched alkyl, alkenyl or alkynyl radical; a group -NR6R7 in which R6 and R7 each independently represents a hydrogen atom, an optionally substituted straight-chained or branched alkyl, alkenyl or alkynyl radical; optionally substituted acyl; or an optionally substituted phosphate ester group. The invention also relates to processes for the preparation of compounds of the formula and to pharmaceutical compositions containing the same.

An attempt was made to incorporate alpha-tocopherol in negatively and positively-charged submicron emulsions, with the aim of providing an effective topical preparation against skin oxidative damage. In cell culture toxicity experiments using human fibroblast it was shown that the positively-charged alpha-tocopherol emulsion did not exhibit any toxic effect despite the low dilution and respective high concentration used. Negatively and positively-charged submicron emulsions of alpha-tocopherol and their respective blank emulsions were topically applied to rats that were subjected to UVA irradiation under different experimental conditions. No difference was observed between the negatively and positively-charged alpha-tocopherol submicron emulsions regarding the rate of oxidation and peroxyl radical scavenging ability of skin homogenates and both were able to protect rat skin against oxidative stress. However, in a non-invasive evaluation of the lipid hydroperoxidation process in rat skin following exposure to UVA irradiation, the positively-charged alpha-tocopherol submicron emulsion elicited a significantly better protective effect than the corresponding negatively-charged emulsion. These results suggest that the positively-charged emulsion exhibits a more prolonged residence time in the uppermost layers of the skin than the negatively-charged emulsion.

This work describes the synthesis and characterization of a new family of antioxidants. The molecules have the same active group, but different oil-to-water and octanol-to-water partition coefficients due to different substituents. Three new molecules were synthesized based on the chemical structure of the primary amide attached to a thiophosphate group forming an amidothionophosphate. The amidothionophosphate molecules were exposed to the oxidative stress of hydrogen peroxide and sodium hypochlorite, and the chemical changes following the exposure were monitored by 31P NMR. The reaction constants with the reactive oxygen species hydroxyl radical and superoxide radical were also calculated and found to be 1.5 x 10(9) M-1s-1 and 8.1 x 10(2) M-1s-1, respectively. To elucidate the ability of amidothionophosphates to act as antioxidants in protecting lipids and proteins, we examined damage prevention in bovine serum albumin, egg phosphatidylcholine liposomes, and lipid emulsions following oxidative stress. Amidothionophosphate showed unique protection properties in these models. In contrast to other antioxidant molecules (ascorbic acid, cysteine, and alpha-tocopherol) the new group did not have any pro-oxidative effects as measured by oxygen consumption from buffer solutions containing amidothionophosphates and cupric sulfate as a source of redox-active metal ions. Amidothionophosphates reduced significantly and in a dose-dependent manner the oxidative burst in human neutrophils as measured by luminol-dependent chemiluminescence, and they also markedly depressed the killing of human fibroblasts by mixtures of glucose oxidase and streptolysin S. The toxicity of these molecules was tested by IP injection of doses up to 1000 mg/kg to white Sabra mice. No mortality was observed 30 d after administration of up to 500 mg/kg.

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.

A novel approach to the assessment of the toxicity of the chlorinated pesticide hexachlorocyclohexane (lindane) and the organic solvents methanol and w-butanol, employing endothelial cells in culture, is presented. This highly reproducible system involves the simultaneous treatment of [51Cr]. and [3H]arachidonic acid-labeled rat pulmonary endothelial cells with xenobiotics combined with glucose oxidase-generated H2O2, phospholipase c, streptolysin S, diethyldithiocarbamate (DDC), sodium nitroprusside (NP), histone, and trypsin. Such treatment leads to synergistic cell killing and the release of arachidonic acid (Ginsburg and Kohen, 1995b). Thus, subtoxic amounts of xenobiotics that failed to kill the cells became highly cytolytic when combined with the various mixtures of agonists. Cytotoxicity and the release of membrane lipids are strongly inhibited by catalase, by Mn2+, and by soybean trypsin inhibitor. The "synergism" concept of cellular toxicity is relevant, in particular, in infectious and inflammatory sites where phagocyte- and tissue-derived proinflammatory agonists are generated in large amounts as a result of cellular damage induced either by pathogenic microorganisms, by activated phagocytes, or by xenobiotics. This simple and inexpensive in vitro model of cellular cytotoxicity might supplement and even replace the more costly animal experimentations involved in the assessment of the toxicity and safety of newly designed drugs.

Cell-impermeable inhibitors of phospholipase A2 were prepared by linking inhibiting molecules to macromolecular carriers which prevent the inhibitor's internalization. These preparations inhibit the release of oxygen reactive species from neutrophils and cell death induced by inflammatory agents, as well as bleomycin-induced lung injury.

BACKGROUND: Oxygen-derived radicals are implicated in the pathogenesis of tissue damage and ulcerogenesis. This study aimed to examine the effect of manganese, glycine, and carotene, oxygen radical scavengers, on ethanol-induced gastric lesions in the rat and on ethanol cytotoxicity in epithelial cell culture. METHODS: MnCl2 + glycine (12.5-50 mg/rat) were injected subcutaneously up to 6 h before oral administration of 1 ml of 96% ethanol, and 0.5 ml carrot juice or beta-carotene was given orally 30 min before the ethanol. Mucosal injury was evaluated 1 h later by gross and microscopic scoring. The effect of Mn2+ and carrot juice was also tested in monolayers of radiolabeled epithelial cells exposed to H2O2 + ethanol injury as expressed by the extent of the isotope leakage. RESULTS: Mn2+ and glycine pretreatment dose-dependently reduced ethanol-induced gastric lesion formation. Protection was maximal when treatment was applied 4 h before the insult. Gross damage was also markedly prevented by pretreatment with carotenes and dimethylthiourea (DMTU, 75 mg/kg intraperitoneally) but not by allopurinol. Mixtures of subtoxic concentrations of ethanol and H2O2 were highly lethal for epithelial cell monolayers. In this model, cell death was markedly attenuated by catalase, DMTU, Mn2+, and carrot juice. CONCLUSIONS: Ethanol-induced gastric mucosal damage may involve generation of oxygen-derived radicals, independent of the xanthine oxidase system. By acting as oxygen radical scavengers, Mn2+, glycine, and carotenes, like catalase and DMTU, provide significant gastroprotection.