Carlo Aldinucci

Ozone In Medicine

Abstract Ozone therapy has been used as a complementary medical approach for half a century but it has encountered skepticism by orthodox medicine because, particularly in the past, it has been used by practitioners and others without a rational basis and appropriate controls. With the advent of modern medical ozone generators incorporating a photometer, it has become possible to obtain precise ozone concentrations and to evaluate some mechanisms of action and possible toxicity. In contrast with the respiratory tract, human blood exposed to appropriate ozone concentrations is able to tame its strong oxidant properties and neither acute, nor chronic side effects have ensued in millions of patients treated with ozonated autohaemotherapy (O3-AHT). This review summarizes our studies aimed at clarifying biological effects, defining any possible damage, the therapeutic window and suitable doses able to express a therapeutic activity. A very interesting and promising aspect is the induction of the so-called heat stress proteins (HSP) leading to adaptation to a chronic oxidative stress. The use of ozone in human therapy has been reviewed but so far very few controlled clinical studies have been reported. Mostly on the basis of anecdotal results, ozone therapy appears useful in infectious diseases, immune depression, vascular disorders, degenerative diseases and orthopedics.

Role of intracellular Ca2+ and calmodulin/MAP kinase kinase/extracellular signal-regulated protein kinase signalling pathway in the mitogenic and antimitogenic effect of nitric oxide in glia- and neurone-derived cell lines.

To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen‐activated protein kinases [extracellular signal‐regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)‐1‐[2‐aminiethyl]‐N‐[2‐ammonioethyl]amino]diazen‐1‐ium‐1,2diolate (DETA/NO) concentrations (10 µm) determined a gradual, moderate elevation in [Ca2+]i (46.8 ± 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 ± 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 ± 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down‐regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50–300 µm) of DETA/NO negatively regulated cell proliferation via a Ca2+‐independent mechanism.

The effect of pulsed electromagnetic fields on the physiologic behaviour of a human astrocytoma cell line

We evaluated the effects of 50 Hz pulsed electromagnetic fields (EMFs) with a peak magnetic field of 3 mT on human astrocytoma cells. Our results clearly demonstrate that, after the cells were exposed to EMFs for 24 h, the basal [Ca(2+)](i) levels increased significantly from 124+/-51 nM to 200+/-79 nM. Pretreatment of the cells with 1.2 microM substance P increased the [Ca(2+)](i) to 555+/-278 nM, while EMF exposure caused a significant drop in [Ca(2+)](i) to 327+/-146 nM. The overall effect of EMFs probably depends on the prevailing Ca(2+) conditions of the cells. After exposure, the proliferative responses of both normal and substance P-pretreated cells increased slightly from 1.03 to 1.07 and 1.04 to 1.06, respectively. U-373 MG cells spontaneously released about 10 pg/ml of interleukin-6 which was significantly increased after the addition of substance P. Moreover, immediately after EMF exposure and 24 h thereafter, the interleukin-6 levels were more elevated (about 40%) than in controls. On the whole, our data suggest that, by changing the properties of cell membranes, EMFs can influence Ca(2+) transport processes and hence Ca(2+) homeostasis. The increased levels of interleukin-6 after 24 h of EMF exposure may confirm the complex connection between Ca(2+) levels, substance P and the cytokine network.

Potentiation of intracellular Ca2+ mobilization by hypoxia-induced NO generation in rat brain striatal slices and human astrocytoma U-373 MG cells and its involvement in tissue damage

The relationship between nitric oxide (NO) and intracellular Ca2+ in hypoxic–ischemic brain damage is not known in detail. Here we used rat striatal slices perfused under low‐oxygen and Ca2+‐free conditions and cultured human astrocytoma cells incubated under similar conditions as models to study the dynamics of intracellular NO and Ca2+ in hypoxia‐induced tissue damage. Exposure of rat striatal slices for 70 min to low oxygen tension elicited a delayed and sustained increase in the release of 45Ca2+. This was potentiated by the NO donors sodium nitroprusside (SNP) and spermine–NO and inhibited by N‐ω‐nitro‐L‐arginine methyl ester (L‐NAME) or by the NO scavenger 2‐phenyl‐4,4,5,5 tetramethylimidazoline‐1‐oxyl‐3‐oxide (PTIO). A membrane‐permeant form of heparin in combination with either ruthenium red (RR) or ryanodine (RY) also inhibited 45Ca2+ release. In human astrocytoma U‐373 MG cells, hypoxia increased intracellular Ca2+ concentration ([Ca2+]i) by 67.2 ± 13.1% compared to normoxic controls and this effect was inhibited by L‐NAME, PTIO or heparin plus RR. In striatal tissue, hypoxia increased NO production and LDH release and both effects were antagonized by L‐NAME. Although heparin plus RR or RY antagonized hypoxia‐induced increase in LDH release they failed to counteract increased NO production. These data therefore indicate that NO contributes to hypoxic damage through increased intracellular Ca2+ mobilization from endoplasmic reticulum and suggest that the NO–Ca2+ signalling might be a potential therapeutic target in hypoxia‐induced neuronal degeneration.

A novel CB2 agonist, COR167, potently protects rat brain cortical slices against OGD and reperfusion injury

Cannabinoid CB2 receptor activation has been shown to have many pharmacological but not psychotropic effects. The aim of this study was to investigate the potential protection of brain tissues afforded by the novel substituted 4-quinolone-3-carboxylic acid derivative COR167, a selective CB2 agonist, toward ischemia and reperfusion-induced injury, as well as the mechanism of this potential effect. Rat brain cortical slices subjected to oxygen and glucose deprivation (OGD) followed by re-oxygenation were used. Cell damage was quantified by measuring at the end of the reperfusion phase the release into the artificial cerebrospinal fluid (ACSF) of lactate dehydrogenase (LDH), glutamate, IL-6 and TNF-α and by evaluating in tissue the lipid-peroxides (thiobarbituric acid-reactive substances, TBARS), the free, reduced glutathione content (GSH) and the water gain (TWG), taken as an index of cell swelling. COR167 (10nM or 100 nM), added to ACSF during the entire reperfusion phase, markedly reduced LDH and glutamate release, as well as TWG. Lower (0.1-1 nM) or higher concentrations (1,000 nM) were ineffective, suggesting thereby an hormetic behavior. COR167 at 10nM concentration markedly reverted in tissues TBARS increase and GSH decrease, while reducing IL-6 and TNF-α release into ACSF. COR167 effects on glutamate and LDH release were abrogated by the selective CB2 inverse-agonists COR170 (1 nM) and AM630 (1μM) but not by the CB1 antagonist AM251 (1 μM). COR170 as well as AM630 per se were able to revert TWG. The CB2 receptor agonist COR167 potently protected rat brain cortical slices against OGD and reperfusion injury, partly through CB2 receptors activation.

Extracorporeal blood oxygenation and ozonation (EBOO) in man. preliminary report.

Autohemotherapy with ozone has been used for four decades with encouraging results but, owing to the lack of clinical studies, it has never been adopted by orthodox medicine. Confident of the valid principles of ozone therapy, we have endeavoured to increase its therapeutic efficacy. Over a ten-year period we have developed an apparatus that makes it possible to treat large quantities of blood with ozone in extracorporeal circulation (extracorporeal blood oxigenation and ozonation EBOO). One of us volunteered to test the system and after six treatments noted the disappearance of two lipomas. This prompted us to treat a patient with Madelung disease and several patients with atherosclerotic vasculopathy. Besides showing therapeutic effects, the preliminary results indicate that EBOO is clinically valid, without side-effects and worthy of testing in various diseases.

Short cycles of both static and pulsed electromagnetic fields have no effect on the induction of cytokines by peripheral blood mononuclear cells

We evaluated the effect of short cycles of static and pulsed electromagnetic field exposure on the eventual activation of peripheral blood mononuclear cells. The cells were subjected to three 15-min cycles of EMF, each exposure being followed by 105 min without a field, for a total of 6 hr. The results clearly demonstrate that the proliferative responses of both normal cells and cells stimulated with 1 microg/ml phytohemagglutinin were not distinguishable from control cells not exposed to EMF. Moreover, although the production of interleukin-2, interferon gamma and tumor necrosis factor alpha increased during the first 48 hr of incubation, the values remained unchanged with respect to controls. This indicates that brief exposure to an electromagnetic field has no significant effect on peripheral blood mononuclear cells. The comparison between biological activity and the cytokine antigen present in our samples indicated that the recovery of antigen corresponded to an equal recovery of biological activity, suggesting the absence of either qualitative differences in these proteins or the impairment of transcriptional and translational processes.

Rottlerin exhibits antiangiogenic effects in vitro.

Rottlerin, a natural product purified from Mallotus philippinensis, has a number of target molecules and biological effects. We recently found that Rottlerin caused growth arrest in MCF‐7 breast cancer cells and human immortalized keratinocytes, through inhibition of NFκB and downregulation of cyclin D‐1. To evaluate whether this effect could be generalized to primary cells, human microvascular endothelial cells were treated with Rottlerin. In this study, we demonstrated that Rottlerin prevents basal and TNFα‐stimulated NFκB nuclear migration and DNA binding also in human microvascular endothelial cell, where NFκB inhibition was accompanied by the downregulation of NFκB target gene products, such as cyclin D‐1 and endothelin‐1, which are essential molecules for endothelial cell proliferation and survival. Rottlerin, indeed, inhibited human microvascular endothelial cells proliferation and tube formation on Matrigel. Rottlerin also increases cytoplasmic free calcium and nitric oxide levels and downregulates endothelin converting enzyme‐1 expression, thus contributing to the drop in endothelin‐1 and growth arrest. These results suggest that Rottlerin may prove useful in the development of therapeutic agents against angiogenesis.

Ozonation of blood during extracorporeal circulation. I. Rationale, methodology and preliminary studies.

We investigated whether exposure of blood ex-vivo to oxygen-ozone (O2-O3) through a gas exchanger is feasible and practical. We first evaluated the classical dialysis-type technique but we soon realized that semipermeable membranes are unsuitable because they are hydrophilic and vulnerable to O3. We therefore adopted a system with hydrophobic O3-resistant hollow fibers enclosed in a polycarbonate housing with a membrane area of about 0.5 m2. First we tested the system with normal saline, determining the production of hydrogen peroxide (H2O2) at O3 concentrations from 5 to 40 μg/ml. We then evaluated critical parameters by circulating swine blood in vitro; this revealed that heparin is not an ideal anticoagulant for this system. Finally, we performed several experiments in sheep and defined optimal anticoagulant dose (sodium citrate, ACD), priming solution, volume of blood flow per min, volume and concentration of O2-O3 mixture flowing counter-current with respect to blood and the time necessary for perfusion in vivo. The biochemical parameters showed that an O3 concentration as low as 10 μg/ml is effective; this means that gas exchange and O3 reactivity are rapid and capable of inducing biological effects. The sheep showed no adverse effects even after 50 min of extracorporeal circulation at higher O3 concentrations (20 to 40 μg/ml) but the exchanger became less effective (low pO2 values) due to progressive clogging with cells.

Short-Term Hypoxia Enhances the Migratory Capability of Dendritic Cell Through HIF-1α and PI3K/Akt Pathway

Hypoxia represents an inadequate oxygen supply to tissues, which can modulate cell functions, primarily through the hypoxia‐inducible transcription factor HIF‐1α. Dendritic cells (DC) are professional antigen‐presenting cells and their migration maybe affected by hypoxia, since the local microenvironment in lymphoid organs, as well as in inflamed and tumor tissues, is characterized by low oxygen tensions. In this study we observed an enhanced migratory capability of human monocyte‐derived DC, using in vitro migration assays performed under hypoxic conditions. Such enhancement was independent on either the chemoattractants involved or the maturation level of DC. However, HIF‐1α appeared to be crucial for the migration only of immature DC and not for mature DC under hypoxia, as indicated by HIF‐1α siRNA approaches. Furthermore, we observed that while Akt phosphorylation was enhanced in both immature and mature DC exposed to hypoxia, other signaling pathways, such as p38 and p42/p44 MAPK, were differently affected during hypoxic treatment. More interestingly, aspecific and specific inhibition of PI3K/Akt indicated that such pathway was relevant for the migration of both immature and matured DC under hypoxia, even when DC were transfected with HIF‐1α siRNA. Our results indicate that, besides HIF‐1α, several other pathways, including PI3K/Akt, may be involved in the response to hypoxia of immature and, more specifically, of mature DC to sustain their trafficking and functions within hypoxic microenvironments. J. Cell. Physiol. 229: 2067–2076, 2014.