Mycoplasma penetrans invades HeLa cells and survives within them for prolonged periods of time. The intracellular distribution of M. penetrans within HeLa cells was studied utilizing the acidotropic dye LysoTracker (green), which permeates cell membranes and upon protonation remains trapped in acidic compartments. The excitation and emission spectra of the green LysoTracker are suitable for colocalization studies with rabbit anti- M. penetrans antibodies and red Cy5 goat anti-rabbit IgG. The images collected by confocal laser scanning microscopy revealed that in the infected HeLa cells almost all Cy5 fluorescent foci (red) were located within the LysoTrack-labelled intracellular compartments, apparently endosomes. Viable mycoplasmas were detected within endosomes for prolonged periods of time, apparently due to a potent antioxidant activity detected in M. penetrans.

The growth factors basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF-I) have been implicated in the pathophysiology of atherosclerosis and restenosis. The Tibetan herbal preparation PADMA-28 (a mixture of 22 plants which is used as an anti-atherosclerosis agent) was tested for its ability to inhibit the mitogenic activity of bFGF and IGF-I, growth factors involved in restenosis, atherosclerosis and tumour progression. DNA synthesis and proliferation of vascular smooth muscle cells, in response to serum bFGF, thrombin, or combinations thereof, were abrogated in the presence of microgram amounts of both the aqueous and organic, partially purified, extracts of PADMA-28. These fractions also inhibited IGF-I-mediated proliferation, migration and invasion of tumour cells responsive to IGF-I. The inhibition by PADMA 28 was reversible upon removal of the PADMA extracts, indicating that the effects were not related to cell toxicity. These and other properties (i.e., anti-oxidant activity) of PADMA-28 may be responsible for its beneficial effect as an anti-atherosclerotic agent, suggesting that this herbal preparation may have potential applications in the prevention of intimal hyperplasia and arterial stenosis secondary to coronary angioplasty and bypass surgery, as well as in the prevention and treatment of other vascular diseases and tumour growth and metastasis.

It is known that many agents influence the capacity of cells to produce reactive oxygen species. However, assaying these agents, both those that stimulate and those that inhibit reactive oxygen production, can be complicated and time consuming. Here, a method is described in which two different cocktails are employed to stimulate luminol-dependent chemiluminescence (LDCL). These cocktails are comprised of luminol, with either sodium selenite [IV] (SEL) or tellurite [IV] (TEL) (where IV and VI refer to the 4+ or 6+ oxidation state of selenium or tellurium salts, respectively), morpholinosidonimine (SIN-1), serum albumin and Co(2+), called the SIN-1a (with selenite) and SIN1b (with tellurite) cocktails, respectively; or luminol with glucose oxidase (GO), sodium selenite [IV] and Co(2+), called the GO cocktail. The cocktails functioned best in Hank's balanced salt solution (HBSS) containing 1% glucose at pH 7.4, incubated at approximately 22 degrees C. Within 30-60 s there was a burst of luminescence, which lasted for 7-10 min. In 100% ethanol, the SIN-1 cocktails also generated LDCL to 70% of that produced in HBSS. Neither selenite [VI], seleno-cystine, seleno-methionine, nor the selenium-containing drug, ebselen, could replace SEL. Moreover, the effects of the NO-donor, SIN-1, could not be replicated by the oxyradical generators, xanthine-xanthine oxidase or hypochlorous acid. Only low levels of luminescence were generated by combinations of the peroxyl radical generator, 2,2'-azobis-2-amidinopropane dihydrochloride (AAPH) with either SEL or TEL. It is suggested that light emission induced by the SIN1 cocktail results from the oxidation of SEL [IV] to the [VI] state, possibly due to the generation of mixtures of superoxide, peroxide, peroxynitrite and also of unidentified oxidant species, catalyzed by CoCo(2+). However, the involvement of hydroxyl radicals in LDCL could not be confirmed by use of either dimethyl thiourea or by electron spin resonance (ESR). LDCL induced by the two cocktails is strongly reduced by phosphates, EDTA, deferoxamine, CuCo(2+), MnCo(2+), as well as by the "classical" antioxidants superoxide dismutase (SOD), ascorbate, vitamin E, uric acid or thiols. It is suggested that these chemiluminescence cocktail systems can be used to determine the total anti-oxidant capacities of biological fluids and commercially available anti-oxidants.

Using two luminescence-inducing cocktails, two distinct patterns of inhibition of light by different anti-oxidants have been identified, comprising Group A, in which a complete inhibition of light emission which is then followed by re-emergence of light, forming apparent S-shaped curves or similar shapes. This light pattern is induced by the "classical" anti-oxidants, ascorbate, vitamin E, uric acid, thiols, deferoxamine, as well as by anti-oxidant agents present in plasma, saliva, urine and in extracts derived from black coffee, and Group B, in which a gradually emerging "mound"-shaped pattern of light was seen with extracts from the Tibetan plant mixture PADMA-28, elderberry (Sambucol), grape seeds, green and black teas, apple, parsimony, red wines, edible oils and SOD. While the results with the Group A agents point to the presence of probably a single, major, anti-oxidants relatively sensitive to oxidation, Group B agents probably include a mixture of anti-oxidants which are more resistant to oxidation. It was also shown that agents from Group B could protect agents from Group A against consumption by the oxidants generated by the cocktails. It is proposed that these simple to use cocktails which probably generate a multiplicity of oxidants mimicking those generated by activated phagocytes, can rapidly assess the total anti-oxidant capacities (TAOC) in body fluids derived from patients suffering of excessive oxidative stress. Also, this technique may be useful in determining the content of dietary anti-oxidants recommended as supplements to enhance the resistance against excessive oxidation of lipids.

Although there is a general consensus that highly cationic peptides kill bacteria primarily by injuring their membranes, an additional hypothesis is proposed suggesting that a large variety of cationic peptides might also render bacteria non viable by activating their autolytic wall enzymes - muramidases (a "Trojan Horse" phenomenon), resulting in bacteriolysis. This group of cationic peptides includes: lysozyme, lactoferrin, neutrophil-derived permeability increasing peptides, defensins, elastase, cathepsin G, and secretory phopholipase A2. In this respect, cationic peptides mimic the bactericidal/bacteriolytic effects exerted by of beta-lactam antibiotics. Bacteriolysis results in a massive release of the pro-inflammatory cell-wall components, endotoxin (LPS), lipoteichoic acid (LTA) and peptidoglycan (PPG), which if not effectively controlled, can trigger the coagulation and complement cascades, the release from phagocytes of inflammatory cytokines, reactive oxygen and nitrogen species, and proteinases. Synergism (a "cross-talk") among such agonists released following bacteriolysis, is probably the main cause for septic shock and multiple organ failure. It is proposed that a use of bacteriolysis-inducing antibiotics should be avoided in bacteremic patients and particularly in those patients already suspected of developing shock symptoms as these might further enhance bacteriolysis and the release of LPS, LTA and PPG. Furthermore, in additonal to the supportive regimen exercised in intensive care settings, a use of non bacteriolysis-inducing antibiotics when combined with highly sulfated compounds (e.g. heparin, and other clinically certified polysufates) should be considered instead, as these might prevent the activation of the microbial own autolytic systems induced either by highly cationic peptides released by activated phagocytes or by the highly bacteriolytic beta-lactams. Polysulfates might also depress the deleterious effects of the complement cascade and the use of combinations among anti-oxidants ( N-acetyl cysteine), proteinase inhibitors and phospholipids might prove effective to depress the synergistic cytotoxic effects induced by inflammatory agonists. Also, a use of gamma globulin enriched either in anti-LPS or in anti-LTA activities might serve to prevent the binding of these toxins to receptors upon macrophage which upon activation generate inflammatory cytokines. Thus, a use of "cocktails" of anti-inflammatory agents might replace the unsuccessful use of single antagonists proven in scores of clinical trials of sepsis to by ineffective in prolonging the lives of patients. It is enigmatic why the concept, and the publications which support a role for cationic peptides also as potent inducers of bacteriolysis, an arch evil and a deleterious phenomenon which undoubtedly plays a pivotal role in the pathophysiology of post-infectious sequelae, has been consistently disregarded.

BACKGROUND: Previous studies have shown that PADMA-28, a multicomponent, traditional Tibetan herbal plant preparation possesses a variety of beneficial effects on several experimental models of inflammatory and immune processes, including autoimmune diabetes and autoimmune encephalomyelitis. In humans, PADMA-28 attenuated the symptoms associated with intermittent claudications in atherosclerotic patients. OBJECTIVE: To assess the effect of PADMA 28 on the immune system, e.g. cytokine (interleukins) production. DESIGN: Cytokine production by human blood monocytes (derived from 12 healthy donors) stimulated in vitro, either by endotoxin (LPS) from Salmonella typhi or by lipoteichoic acid (LTA) from group A Streptococci was modulated by PADMA-28. RESULTS: The present study showed that an aqueous extract of PADMA-28 strongly decreased the production of the inflammatory cytokines IL-1beta, IL-6, IL-8 and TNF-alpha, and more moderately, also decreased the anti-inflammatory cytokine IL-10 induced by LPS. However, the LTA - induced IL-10 production was [not significantly] increased by the low dose PADMA-28, while not effected at all by the higher dose of PADMA-28. CONCLUSIONS: The data from these finding suggest a possible clinical efficacy of PADMA-28 either in autoimmune and in inflammatory conditions or in post-inflammatory sequelae, as previously shown in in vivo and human studies, probably by decreasing inflammatory cytokines.