PADMA 28 is a multi-component herbal mixture formulated according to an ancient Tibetan recipe. PADMA 28 is known to stimulate collagen production and reduced levels of collagen-degrading matrix metalloproteinases (MMPs). The goal of the present study was to determine whether topical treatment of rat skin with PADMA 28 would improve skin structure/function, and whether subsequently induced abrasion wounds would heal more rapidly in skin that had been pretreated with PADMA 28. Hairless rats were exposed to a potent topical corticosteroid (Temovate) in combination with either DMSO alone or with PADMA 28 given topically. At the end of the treatment period, superficial wounds were created in the skin, and time to wound closure was assessed. Collagen production and matrix-degrading MMPs were assessed. Abrasion wounds in skin that had been pretreated with PADMA 28 healed more rapidly than did wounds in Temovate plus DMSO-treated skin. Under conditions in which improved wound healing was observed, there was an increased collagen production and decreased MMP expression, but no significant epidermal hyperplasia and no evidence of skin irritation. The ability to stimulate collagen production and inhibit collagen-degrading enzymes in skin and facilitate more rapid wound closure without irritation should provide a rationale for development of the herbal preparation as a "skin-repair" agent.

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-Ciocalteu’s 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.

Hepatic fibrosis is the end-stage consequence of chronic liver disease, affecting many people worldwide. Unlike the anti-fibrotic effect of natural killer (NK) cells, CD8 and NK T subsets are considered as profibrogenic subsets. Padma Hepaten is a multi-compound herbal preparation derived from Tibetan medicine and has proven efficacy in some clinical trials and tests at the cellular level. In this study, we evaluate the immune efficacy of Padma Hepaten administered intraperitoneally (i.p.) and/or orally in a mice model of hepatic fibrosis. Hepatic fibrosis was induced by 6 weeks of biweekly i.p. carbon tetrachloride (CCl4) injections in male C57Bl6 mice. There were four groups, including naive mice, non-treated fibrotic mice and fibrotic mice treated by Padma Hepaten at weeks 5–6 of fibrosis induction either orally or by i.p. injections. Padma Hepaten was prepared at 10 mg/ml in saline and 250 µl (2·5 mg) were administered four times per week. After week 6, animals were killed. To isolate a Padma Hepaten-associated effect on lymphocytes, splenocytes were harvested from either naive or Padma Hepaten-treated non-fibrotic donors. Isolated splenocytes were therefore reconstituted into two groups of irradiated recipients. Recipients were then administered the same CCl4 regimen. Hepatic fibrosis was determined by sirius red staining of liver sections and by assessment of alpha smooth muscle actin expression compared with β-actin (both by mRNA as well as the protein liver extract western blotting). Hepatic fibrosis and alanine aminotransferase serum levels were decreased significantly in both Padma Hepaten-treated groups compared with the non-treated fibrotic group. Padma Hepaten treatment was associated with attenuation of lymphocyte subsets in both treated groups. Using a chemiluminescence technique to assess total anti-oxidant capacities (TAC), it was found that both the plasmas and livers of mice treated by CCl4 had significantly higher TAC compared with controls. However, the levels of TAC in animals treated either by CCl4 alone or CCl4 with Padma Hepaten were similar. Adoptive transfer of Padma Hepaten-treated lymphocytes was associated with fibrosis amelioration compared with recipients with naive lymphocytes. CCl4 generates higher levels of anti-oxidant capacities, probably as a response to oxidative stress. Padma Hepaten administration attenuated hepatic fibrogenesis significantly, accompanied by attenuation of lymphocyte but not anti-oxidant capacities.

Several microbial species, including probiotic lactic acid bacteria, have the ability to irreversibly bind a large variety of polyphenols (flavonoids) and anthocyanidins found in many colored fruits and vegetables and to enhance their total oxidant-scavenging capacities (TOSC). The binding of flavonoids to microbial surfaces was further increased by the cationic polyelectrolytes ligands poly-L-histidine, chlorhexidine and Copaxone. This phenomenon was confirmed visually, by the FRAP, DPPH, cyclic voltammetry, Folin-Ciocalteu as well as by luminol-dependent chemiluminescence techniques employed to assay TOSC. The possibility is considered that clinically, microbial cells in the oral cavity and in the gastro intestinal tract, complexed with antioxidant polyphenols from nutrients and with cationic ligands, might increase the protection of mammalian cells against damage induced by excessive generation of reactive oxygen species during infections and inflammation.

A novel assay was developed to measure the capacity of polyphenols to chelate cobalt(II) by using the reduction of the tetrazolium salts, NBT (nitroblue tetrazolium chloride), MTT (methylthiazolyldiphenyl-tetrazolium bromide), and XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) to formazan products. The reduction of the salts is initiated by a cocktail comprised of cobalt(II), H(2)O(2), and selenium(IV), which generates hydroxyl radical, peroxide, and superoxide ions. However, because cobalt(II) could not be replaced either by Fe(II), Mn(II), or Cu(II), the classical Fenton transitional metals, it indicates that cobalt is the key player in the tetrazolium salt reduction. Micromolar concentrations of a large variety of antioxidant polyphenols and minute amounts of fruit beverages rich in polyphenols can readily chelate cobalt, resulting in the inhibition of the reduction of tetrazolium salt to formazan, in a dose-dependent manner. However, this method is unsuitable to measure low molecular weight antioxidants such as ascorbate, uric acid, and vitamin E since these have no chelating properties for cobalt(II). The newly described tetrazolium reduction method is as sensitive as the ferric ion reducing antioxidant power, 2,2-diphenyl-2-picrylhydrazyl hydrate, and the luminol-dependent chemiluminescence antioxidant assays. The practical advantages of using the newly described method to quantify polyphenol levels from various sources are briefly discussed.

It has been suggested that the very low incidence of atherosclerosis in glycogen storage disease type Ia (GSD Ia) subjects might be attributed to elevated levels of uric acid, one of the potent low molecular- weight antioxidants found in plasma. The present communication describes a use of two analytical methods-cyclic voltammetry and ferric reducing ability of plasma-and also two chemiluminescence methods to evaluate the total oxidant-scavenging capacities (TOSC) of plasma from GSD Ia patients. Our results verified the elevation of TOSC in GSD Ia patients and we propose the inclusion of luminescence and cyclic voltammetry assays as reliable methods for estimating TOSC in a variety of clinical disorders. Our findings with the cyclic voltammetry method add support to the assumption that the elevated uric acid levels might be the main contributor to plasma antioxidant capacity and possible protection against atherosclerosis.

The present view focuses on the possibility that cationic antimicrobial peptides (CAMPs) might, in addition to their killing effects due to permeabilization of microbial membranes, also function similarly to beta-lactam antibiotics to activate nascent autolytic wall enzymes, leading to bacteriolysis. Since the massive release of microbial cell wall components is a major cause of postinfectious sequelae, the in vivo process of bacteriolysis must be controlled. Due to the emergence of antibiotic resistance in pathogenic bacteria, CAMPs might be useful as an alternative to antibiotics. However, they should be used with caution, since they might also function as a ’double-edged sword’ by injuring both the bacteria and host.

ZusammenfassungInflammaging bezeichnet einen chronischen Entzündungszustand, der durch altersbedingte Veränderungen des Immunsystems entsteht. Dieser wird als Grundlage vieler chronisch-entzündlicher Alterskrankheiten wie z.B. Arteriosklerose, Diabetes mellitus Typ 2, Morbus Alzheimer und Krebserkrankungen vermutet. Die Entstehung des vorherrschenden proinflammatorischen Milieus und der Verlauf des Entzündungsprozesses wird durch oxidativen Stress beschleunigt und in manchen Fällen verstärkt. Bei Entzündungsprozessen schütten aktivierte Immunzellen ein Arsenal an bioaktiven Stoffen aus, welche synergistisch zusammenwirken und neben positiven Wirkungen auch zu Zell- und Gewebeschäden führen. Im Fall einer manifesten Infektion sind ähnliche Prozesse beschrieben worden. Komplexe Phytotherapeutika (z.B. Padma 28) sind als pleiotrop wirkende Gemische geeignet, diesen krankmachenden Prozess an unterschiedlichen Wirkorten zu unterbinden. So sind etwa neben dem klinischen Effekt von Padma 28 bei Arteriosklerose auch verschiedene antiatherogene Wirkungsmechanismen des Präparats gut dokumentiert. Die Resultate stützen die Hypothese eines «multi- target»-Behandlungsansatzes von chronisch-entzündlichen Erkrankungen mit pflanzlichen Vielstoffgemischen, die hier einen wertvollen Betrag für neue Präventions- und Therapieverfahren liefern können. Sie sind somit geeignet, den Formenkreis von Inflammaging günstig zu beeinflussen, die Entstehung von Folgeerkrankheiten zu verlangsamen bzw. in manchen Fällen zu verhindern.

MDI 301 is a picolinic acid-substituted ester of 9-cis retinoic acid. It has been shown in the past that MDI 301 increases epidermal thickness, decreases matrix metalloproteinase (MMP) activity, and increases procollagen synthesis in organ-cultured human skin. Unlike all-trans retinoic acid (RA), MDI 301 does not induce expression of proinflammatory cytokines or induce expression of leukocyte adhesion molecules in human skin. In the present study we examined topical MDI 301 treatment for ability to improve the structure and function of skin in three models of skin damage in rodents and for ability to improve abrasion wound healing in these models. MDI 301 was applied daily to the skin of rats treated with the potent corticosteroid, clobetasol propionate, to the skin of diabetic rats (8 weeks posttreatment with streptozotocin) and to the skin of aged (14-16-month-old) rats. In all three models, subsequently induced abrasion wounds healed more rapidly in the retinoid-treated animals than in vehicle-treated controls. Immediately after complete wound closure, tissue from the wound site (as well as from a control site) was put into organ culture and maintained for 3 days. At the end of the incubation period, culture fluids were assessed for soluble type I collagen and for MMPs-2 and -9. In all three models, the level of type I collagen was increased and MMP levels were decreased by MDI 301. In all three models, skin irritation during the retinoid-treatment phase was virtually nonexistent.

Low molecular weight antioxidants (LMWA) supplements are a popular and routine approach to assist the cell and the whole organism to cope with increasing oxidative stress. Numerous experiments have been conducted in which exogenous antioxidants were supplemented to cells, animals and humans to prevent and delay pathological disorders associated with reactive oxygen species. Recently, many meta-analysis publications have demonstrated the failure of this approach and in some cases even showed an increase in the severity of the disease and all-cause mortality. The reasons for the lack of success are not fully understood and the concept of antioxidant therapy is questionable. We suggest a new explanation concerning the way antioxidants function in the living cells that can elucidate some of the conflicting data published. The aim of this study was to examine the hypothesis that the overall antioxidant capacities of cells in culture remains constant and since the cells tightly regulate this antioxidant network, supplementation with exogenous antioxidants cannot enhance the total antioxidant capacity of the cells. This assumption was examined in HaCaT, Hep3B, PC3 and Caco-2 cells using several types of antioxidant supplements. It has been shown that while the levels of the specific administrated antioxidant increased significantly intracellularly, the overall antioxidant capacity of the cells as evaluated by various methods did not increase, and in some cases, even decreased. These results support the hypothesis and demonstrate that the total antioxidant capacity of these cells in culture is kept under tight regulation and cannot be enhanced by exogenous LMWA.

The pathogenicity of skin disorders involves a complexity of physiological, immunological, environmental, and genetic phenomena. This review focuses on cross-talks between two main agents, the oxidants and cytokines network, which have recently been found to play important roles in the pathophysiology of a large variety of skin disorders, including carcinogenesis, UVB irradiation damages, inflammatory processes, and a series of diseases such as, psoriasis, pyoderma gangrenosum, atopic dermatitis, irritant contact dermatitis, and bacterial skin infections. In particular the review discusses the question how an interplay between oxidants and cytokines might be beneficial in wound-healing and in therapeutic strategies in clinical settings. These involve topical applications and oral administration of antioxidant and inflammatory-cytokines-neutralizing antibodies. Monitoring cytokine expression in skin disorders (inflammatory versus anti-inflammatory, or Th1 versus Th2 types of cytokines) will definitely help to evaluate the severity of injury, its type, and its role in therapy. Furthermore, it is expected that future studies should explore the possible roles of the synergistic interactions between antioxidants and cytokines and their impact on the Th1/Th2 cytokine networks balances.

Mycoplasma fermentans is an extracellular microorganism capable of adhering to the surface of host cells. It has been recently shown that plasminogen binding to M. fermentans in the presence of the urokinase-type plasminogen activator promotes the invasion of host cells by this organism. In this report, we show that viable mycoplasmas persist within the infected HeLa cells for prolonged periods of time despite the expectation that within host cells the organism may be exposed to oxidative stress. Using cyclic voltammetry and luminol-enhanced chemiluminescence assays, we detected a potent reducing antioxidant activity in M. fermentans. The reducing antioxidant activity was heat stable, not affected by proteolysis and was almost totally lost upon dialysis suggesting that the activity is due to a nonproteinaceus low molecular weight antioxidant. This antioxidant was partially purified by Bio-Gel column chromatography followed by high-pressure liquid chromatographic analysis. We suggest that the high reducing antioxidant capacity in M. fermentans is a principal defense mechanism playing a major role in the battle of the organism against oxidative stress within the host cells.

PADMA 28, a multi-component herbal mixture formulated according to an ancient Tibetan recipe, was assessed for effects on human dermal fibroblasts and epidermal keratinocytes in monolayer culture, and for effects on human skin in organ culture. PADMA 28 stimulated survival of fibroblasts in monolayer culture. In fibroblast monolayer culture and human skin organ culture, levels of matrix metalloproteinase-1 (MMP-1; interstitial collagenase) were reduced and type I procollagen production was increased. When keratinocytes were examined, there was no evidence of growth stimulation over a wide range of PADMA 28 concentrations. At high concentration, PADMA 28 inhibited keratinocyte proliferation. When organ cultures of human skin were treated with PADMA 28, there was no evidence of hyperplastic growth in the epidermis. Topical treatment of rhino mice with PADMA 28 failed to induce epidermal hyperplasia and was completely non-irritating. The ability to stimulate collagen production and inhibit the major collagen-degrading enzyme in skin without inducing a hyperplastic response in the epidermis may provide a basis for development of the herbal preparation as a "skin-repair" agent.

Chlorhexidine (CHX) and Hydrogen peroxide (HP) are potent antibacterial agents that are used in controlling dental plaque. However, both agents bear undesired side-effects. We have tested the hypothesis that an antibacterial synergistic effect can occur between the two agents against Streptococcus sobrinus, Streptococcus faecalis and Staphylococcus aureus. We have found that at several combinations of HP and CHX an antibacterial synergistic effect does occur, while at other combinations a on-significant synergism was noticed. No antagonism between the two agents was found in our experimental system. It can be postulated that the mechanism of this synergistic effect is via alteration of the bacterial cell-surface by CHX thereby allowing for an increased amount of HP to penetrate and to react with the intercellular organelles of the bacteria. These results suggest that CHX and HP can be of use in controlling the dental plaque in the oral cavity.

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.

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 objective of the present communication is to describe the role played by combinations between diethydithiocarbamate (DDC) and divalent metals in hemolysis of human RBC. RBC which had been treated with DDC (10-50 microM) were moderately hemolyzed (about 50%) upon the addition of subtoxic amounts of Cu2+ (50 microM). However, a much stronger and a faster hemolysis occurred either if mixtures of RBC-DDC were immediately treated either by Co2+ (50 microM) or by a premixture of Cu2+ and Co2+ (Cu:Co) (50 microM). While Fe2+ and Ni2+, at 50 microM, initiated 30-50% hemolysis when combined with DDC (50 microM), on a molar basis, Cd2+ was at least 50 fold more efficient than any of the other metals in the initiation of hemolysis by DDC. On the other hand, neither Mn2+ nor Zn2+, had any hemolysis-initiating effects. Co2+ was the only metal which totally blocked hemolysis if added to DDC prior to the addition of the other metals. Hemolysis by mixtures of DDC + (Cu:Co) was strongly inhibited by anaerobiosis (flushing with nitrogen gas), by the reducing agents glutathione, N-acetyl cysteine, mercaptosuccinate, ascorbate, TEMPO, and alpha-tocopherol, by the PLA2 inhibitorbromophenacylbromide (BrPACBr), by tetracycline as well as by phosphatidyl choline, cholesterol and by trypan blue. However, TEMPO, BrPACBr and PC were the only agents which inhibited hemolysis induced by DDC: Cd2+ complexes. On the other hand, none of the classical scavengers of reactive oxygen species (ROS) employed e.g dimethylthiourea, catalase, histidine, mannitol, sodium benzoate, nor the metal chelators desferal and phenanthroline, had any appreciable inhibitory effects on hemolysis induced by DDC + (Cu:Co). DDC oxidized by H2O2 lost its capacity to act in concert either with Cu2+ or with Cd2+ to hemolyze RBC. While either heating RBC to temperatures greater than 37 degrees C or exposure of the cells to glucose-oxidase-generated peroxide diminished their susceptibility to hemolysis, exposure to the peroxyl radical from AAPH, enhanced hemolysis by DDC + (Cu:Co). The cyclovoltammetry patterns of DDC were drastically changed either by Cu2+, Co2+ or by Cd2+ suggesting a strong interaction of the metals with DDC. Also, while the absorbance spectrum of DDC at 280 nm was decreased by 50% either by Co2+, Cd2+ or by H2O2, a 90% reduction in absorbance occurred if DDC + H2O2 mixtures were treated either by Cu2+ or by Co2+, but not by Cd2+. Taken together, it is suggested that DDC-metal chelates can induce hemolysis by affecting the stability and the integrity of the RBC membrane, and possibly also of the cytoskeleton and the role played by reducing agents as inhibitors might be related to their ability to deplete oxygen which is also supported by the inhibitory effects of anaeobiosis.

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 mode of action of antitumour anthraquinone derivatives (i.e. mitoxantrone) is not clearly established yet. It includes, among others, intercalation and binding to DNA, bioreduction and aerobic redox cycling. A series of anthraquinone derivatives, with potentially bioreducible groups sited in the side chain, have been synthesized and biologically evaluated. Their redox and cytotoxic activities were screened. Derivatives which bear a 2-(dimethylamino)ethylamino substituent, known to confer high DNA affinity, demonstrated cytotoxicity but not redox activity (beside the anthraquinone reduction). Conversely, derivatives which showed redox activity were not cytotoxic toward the P388 cell line. The results suggest that bioreduction is not the main mode of action in the cytotoxicity of anthraquinones.