How an ill-conceived methodological approach can condemn the medical use of ozone therapy.
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Blood↗
Biomedical subjects
V Bocci
Publications and source records attributed to V Bocci.
Can the combination of localized "proliferative therapy" with "minor ozonated autohemotherapy" restore the natural healing process?
Regenerative injection therapy (RIT), also known as proliferative therapy, has been used for over 30 years in the USA in patients with spinal and peripheral joint and ligamentous pathologies. It involves the injection of mildly irritating medications onto ligaments and tendons, most commonly at origins and insertions. These injections cause a mild inflammatory response which "turns on" the normal healing process and results in the regeneration of these structures. At the same time they strengthen and become less sensitive to pain through a combination of neurolysis of small nerve fibers (C-fibers) and increased stability of the underlying structures. Oxygen/ozone therapy is a well established complementary therapy practiced in many European countries. The ozone dissolves in body fluids and immediately reacts with biomolecules generating messengers responsible for biological and therapeutic activities. This results in an anti inflammatory response, which also results in a similar trophic reaction to that of RIT. It is logical to expect that combining these two modalities would result in enhanced healing and therefore improved clinical outcomes. Oxygen/ozone therapy, accomplished by autohemotherapy (AHT), is performed by either administering ozonated blood intravenously (Major AHT) or via intramuscular route (Minor AHT). These procedures result in stimulation of the immune and healing systems. Our concept is that the local injection of this activated blood injected directly to the ligamentous areas that are also being treated with RIT will act as a direct stimulation to the healing process. In addition, combining this with intravenous major AHT should stimulate the immune system to augment and support this process. RIT and oxygen/ozone therapy have been extensively studied separately. We propose a study of lumbosacral ligamentous pain to explore this therapeutic combination. We hope that this paper will stimulate general interest in this area of medicine and result in investigation of the "interface" between these treatment modalities.
Blood Transfusion, Autologous↗
Effects of ozone on isolated peripheral blood mononuclear cells.
We have investigated the release of cytokines from isolated peripheral human blood mononuclear cells (PBMC) exposed to various ozone concentrations for 10 min and the release of both proinflammatory and immunosuppressive cytokine after 24, 48 and 76 h incubation. Ozonation was performed by exposing for 10 min equal cell numbers and volumes of cell suspension to equal volumes of a gas mixture (1:1 ratio) composed of oxygen-ozone with precise ozone concentrations ranging from 1.0 up to 80 microg/ml (0.02 up to 1.68 mM). Markers of oxidative stress showed a significant relationship between ozone doses and both lipid peroxidation and protein thiol groups content. With the exception of the lowest ozone concentration, the cytokine production of PBMC was depressed particularly at concentrations from 40 mug/ml upwards. There was no significant effect on IL-6 production between exposed or unexposed cells, up to 72 h of incubation. IL-4 production was markedly affected by ozone exposure, showing a marked decrease even at the lowest ozone concentration (2.5 microg/ml) already after 24 h incubation. On the other hand, production of IFN-gamma and TNF-alpha was slightly stimulated by the lowest ozone dose either at all times or only after 72 h incubation, respectively. Analysis of the proliferation index (PI) is consistent with these results showing that, while the lowest concentration stimulates it, progressively increasing O(3) concentrations inhibit the PI. These data show that there is a significant relationship between cytokine production and ozone concentrations and that PBMC are very sensitive to oxidation particularly in presence of serum with low antioxidant capacity.
Antioxidants↗
The dual action of ozone on the skin.
The aim of this brief review is to summarize the recent literature on the effect of ozone (O3) on cutaneous tissues. Recently it has been reported that a chronic contact with O3 can be deleterious for the skin. Our group and others have shown a progressive depletion of antioxidant content in the stratum corneum and this can then lead to a cascade of effects resulting in an active cellular response in the deeper layers of the skin. Using an in vivo model we have shown an increase of proliferative, adaptive and proinflammatory cutaneous tissue responses. On the other hand the well known activity of O3 as a potent disinfectant and oxygen (O2) donor has been also studied for therapeutic use. Two approaches have been described. The first consists of a quasi-total body exposure in a thermostatically controlled cabin. This treatment has proved to be useful in patients with chronic limb ischaemia. The second approach is based on the topical application of ozonated olive oil in several kinds of skin infection (from soreness to diabetic ulcers, burns, traumatic and surgical wounds, abscesses and skin reactions after radiotherapy). We and other authors have observed a striking cleansing effect with improved oxygenation and enhanced healing of these conditions. It is now clear that, on the skin, O3, like other drugs, poisons and radiation, can display either a damaging effect from a long exposure or a beneficial effect after a brief exposure to O2 and O3 or to the application of ozonated oil to chronic wounds.
Humans↗
Extracorporeal blood oxygenation and ozonation (EBOO): a controlled trial in patients with peripheral artery disease.
BACKGROUND: Since 1990 our group has been using extracorporeal circulation to ozonate blood by an original method, known as extracorporeal blood oxygenation and ozonation (EBOO), with the aim of amplifying the results observed with ozone autohemotherapy. OBJECTIVE: To verify the hypothesis that EBOO improves the skin lesions typical of peripheral artery disease (PAD) patients. METHODS: Twenty-eight patients with PAD were randomized to receive EBOO or intravenous prostacyclin in a controlled clinical trial. The primary efficacy parameters were regression of skin lesions and pain,and improvement in quality of life and vascularisation. RESULTS: Patients treated with EBOO showed highly significant regression of skin lesions with respect to patients treated with prostacyclin. Other parameters that were significantly different in the two groups of patients were pain,pruritus, heavy legs and well-being. No significant differences in vascularisation of the lower limbs before and after treatment were found in either group. No side effects or complications were recorded during the 210 EBOO treatments. CONCLUSION: EBOO was much more effective than prostacyclin for treating skin lesions in PAD patients and also had a positive effect on patient general condition without any apparent change in arterial circulation. This suggests other mechanisms of action of EBOO.
Aged↗
Effect of 4-hydroxynonenal on antioxidant capacity and apoptosis induction in Jurkat T cells.
4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation and may have either physiological or pathological significance regulating cell proliferation. We studied some biochemical effects of HNE, at various concentrations (0.1-100 microM), on Jurkat T cells incubated thereafter for 24, 48 and 72 h. HNE at low concentrations significantly enhanced the proliferation index, whereas at higher concentrations progressively blocked cell proliferation. Caspase 3 activity increased significantly at HNE concentrations between 1 and 10 microM and decreased at higher concentrations. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) increased progressively with HNE concentrations, particularly GSH-Px. Glucose-6-phosphate dehydrogenase (G6PDH) showed a different pattern, increasing at low HNE (1-5 microM) concentrations and rapidly declined thereafter. These results show that HNE may induce growth inhibition of Jurkat T cells and regulate the activity of typical antioxidant enzymes. Furthermore, the protective effect of doubling the foetal calf serum still points out the risk that cultured cells undergo oxidative stress during incubation.
Aldehydes↗
The ozone tolerance: I) Enhancement of antioxidant enzymes is ozone dose-dependent in Jurkat cells.
We have begun to examine the biological and toxic effects of ozone on Jurkat T cells incubated thereafter for 24, 48 and 72 h. Tissue culture medium was strengthened by adding 20% fetal calf serum with an albumin content of about 6 mg/ml. Ozonization was performed by exposing for 10 min a volume of cell suspension (4 x 10(5)/ml) to an equal volume of a gas mixture composed of oxygen-ozone with precise ozone concentrations ranging from 1.5 up to 72 microg/ml (31.5-1512 microM). The proliferation index declined progressively and was ozone dose-dependent. The response of enzymatic activities varied depending upon the enzyme and ozone concentrations: glucose-6-phosphate dehydrogenase begins to increase at an ozone dose of 6 microg/ml (126 microM), reached a peak at 12 microg/ml (252 microM) and rapidly declined thereafter. On the other hand activities of superoxide dismutase, glutathione peroxidase and glutathione reductase increased progressively from the ozone concentration of 12 microg/ml. Thus, as we have observed in blood, the biological response is linked to the ozone dose that must reach a threshold to be effective.
Antioxidants↗
Effects of dietary yoghurt on immunological and clinical parameters of rhinopathic patients.
OBJECTIVES: To examine the immunological and clinical influence of 4 months' feeding with either yoghurt or partially skimmed milk or nothing, on 20 volunteers. SUBJECTS: Thirteen subjects had a demonstrated allergic rhinopathy and seven were healthy subjects and participated as controls. RESEARCH DESIGN: Either a group of seven or a group of six rhinopathic patients were fed either 450 g yoghurt or 450 g partially skimmed milk, respectively, for 4 months between March and October 1999. All subjects maintained their usual diet throughout the study. Peripheral blood mononuclear cells (PBMC) were isolated before and after the experimental period and cultured for periods of 40 and 64 h. Proliferation index assay and release of IFNgamma and IL-4 without and with PHA stimulation were assessed. Allergic rhinopathy was evaluated before and after the 4 months period by performing the nasal functionality tests (Active Anterior Rhinomanometry, Acoustic Rhinometry), the prick test, the nasal specific provocation test (NPT), the dosage of specific IgE blood levels, the evaluation of the symptomatological score and the nasal mucociliary transport test. RESULTS: No significant change of the proliferation index was noted among the three groups. Cultured PBMC of the group fed with yoghurt released more IFNgamma and less IL-4. Cytokine plasma levels were at and remained at basal levels. Prick test, specific serum IgEs and NPT remained immodified. Muco-ciliary transport time (MCTt) and symptomatological score showed a definitive improvement after yoghurt feeding. CONCLUSION: Yoghurt feeding appears to improve or prevent allergic recurrences in rhinopatic patients.
Adult↗
Necrotizing fasciitis successfully treated with extracorporeal blood oxygenation and ozonization (EBOO).
A case of necrotizing fasciitis in a dialysis patient is described. Since traditional therapies were unsuccessful, extracorporeal blood oxygenation and ozonation (EBOO) was tried. This technique is no longer in the experimental stage and is used routinely in our hospital. Patient condition improved radically after EBOO.
Aged↗
Ozonation of blood during extracorporeal circulation. II. Comparative analysis of several oxygenator-ozonators and selection of one type.
Exposure of human blood ex vivo to oxygen-ozone (O2-O3) using either dialysis exchangers or normal oxygenators gives rise to a number of problems, one of which linked to platelet activation, leads to rapid occlusion and no gas exchange. Semipermeable membranes were found unsuitable because, except for one, they were gas-transfer inefficient, allowed ultrafiltration and were more or less vulnerable to O3. Over the last three years we have examined several types of hydrophobic O3-resistant hollow fiber capillaries but, if the polypropylene surface is not properly coated, there is platelet aggregation and blood coagulation. These problems while far less relevant with O2 alone, become prohibitive in the presence of ozone. Recently new oxygenators have been prepared with special materials to make them more biocompatible and it has become possible to oxygenate and ozonate up to 5L of blood in about an hour, thus making the treatment of critical patients feasible.
Blood Gas Analysis↗
Studies on the biological effects of ozone: 11. Release of factors from human endothelial cells.
BACKGROUND: Empirical observations have shown that ozonated autohemotherapy markedly improves the symptoms of chronic limb ischemia (muscular pain at rest, intermittent claudication, etc) in atherosclerotic patients, but mechanisms of action remain unclear. AIMS: Human endothelial cells (HUVECs) are known to release nitrogen monoxide (NO) and we investigated the biological effects of human ozonated serum on HUVECs in culture. METHODS: We assessed the relevance of peroxidation, the release of NO as nitrite and of three classical cytokines. RESULTS: The treatment of HUVECs with ozonated serum yields a dose dependent increase of thiobarbituric acid reactive substances (TBARS) and of hydrogen peroxide (H2O2) and a decrease of protein thiol groups (PTG). Concomitantly, in comparison to either the control or the oxygenated sample, there is a significant and steady increase of nitric oxide (NO) production; this is markedly enhanced by the addition of L-arginine (20 microM) and inhibited in the presence of the NO inhibitor, L-NAME (20 mM). The main mediator of ozone action is H2O2 as it has been shown either after its direct measurement or by the addition of 20, 40 and 100 microM. Moreover, during 24 hours incubation we have investigated the production of endothelin 1 (ET-1), E-selectin and Interleukin 8 (IL-8) and it appears that ozonation enhances IL-8, inhibits E-selectin and hardly modifies ET-1 production. CONCLUSIONS: It appears that reinfusion of ozonated blood, by enhancing release of NO, may induce vasodilation in ischemic areas and reduce hypoxia.
Cells, Cultured↗
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 oxygenation 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.
Aged↗
Studies on the biological effects of ozone: 9. Effects of ozone on human platelets.
During the course of ozonated autohaemotherapy (O3-AHT) using heparin as an anticoagulant, it was occasionally observed that a few clots were retained in the filter during blood reinfusion. This observation prompted an investigation on the effect of ozone (O3) on human platelets. We have now shown, both by biochemical and morphological criteria, that heparin in the presence of O3 can promote platelet aggregation. In contrast, after Ca(2+) chelation with citrate, platelet aggregation is much reduced. The potential role of the transient formation of hydrogen peroxide (H2O2) in the presence of Ca2+ with the possible expression of adhesion molecules is briefly discussed.
Journal Article↗
Studies on the biological effects of ozone: 10. Release of factors from ozonated human platelets.
In a previous work we have shown that heparin, in the presence of ozone (O3), promotes a dose-dependent platelet aggregation, while after Ca2+ chelation with citrate, platelet aggregation is almost negligible. These results led us to think that aggregation may enhance the release of platelet components. We have here shown that indeed significantly higher amount of platelet-derived growth factor (PDGF), transforming growth factor beta1 (TGF-beta1) and interleukin-8 (IL-8) are released in a dose-dependent manner after ozonation of heparinised platelet-rich plasma samples. These findings may explain the enhanced healing of torpid ulcers in patients with chronic limb ischemia treated with O3 autohaemoteraphy (O3-AHT).
Adenosine Diphosphate↗
Prevention of renal injury after induction of ozone tolerance in rats submitted to warm ischaemia.
On the basis that ozone (O3) can upregulate cellular antioxidant enzymes, a morphological, biochemical and functional renal study was performed in rats undergoing a prolonged treatment with O3 before renal ischaemia. Rats were divided into four groups: (1) control, a medial abdominal incision was performed to expose the kidneys; (2) ischaemia, in animals undergoing a bilateral renal ischaemia (30 min), with subsequent reperfusion (3 h); (3) O3 + ischaemia, as group 2, but with previous treatment with O3 (0.5 mg/kg per day given in 2.5 ml O2) via rectal administration for 15 treatments; (4) O2 + ischaemia, as group 3, but using oxygen (O2) alone. Biochemical parameters as fructosamine level, phospholipase A, and superoxide dismutases (SOD) activities, as well as renal plasma flow (RPF) and glomerular filtration rate (GFR), were measured by means of plasma clearance of p-amino-hippurate and inulin, respectively. In comparison with groups 1 and 3, the RPF and GFR were significantly decreased in groups 2 and 4. Interestingly, renal homogenates of the latter groups yielded significantly higher values of phospholipase A activity and fructosamine level in comparison with either the control (1) and the O3 (3) treated groups. Moreover renal SOD activity showed a significant increase in group 3 without significant differences among groups 1, 2 and 4. Morphological alterations of the kidney were present in 100%, 88% and 30% of the animals in groups 2, 4 and 3, respectively. It is proposed that the O3 protective effect can be ascribed to the substantial possibility of upregulating the antioxidant defence system capable of counteracting the damaging effect of ischaemia. These findings suggest that, whenever possible, ozone preconditioning may represent a prophylactic approach for minimizing renal damage before transplantation.
Animals↗
The oropharyngeal delivery of interferons: where are we and where do we need to go?
The controversial concept of oral delivery of interferon (IFN) is reviewed in relation to possible mechanisms of action. Although there is a rational basis and some positive clinical results for believing that it may work, problems remain to understand which is the most effective IFN preparation and why. Owing to a minimal cost and lack of toxicity, the oral as well as other unconventional routes of IFN delivery deserve to be evaluated because they may become useful.
Administration, Oral↗
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 microg/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 countercurrent with respect to blood and the time necessary for perfusion in vivo. The biochemical parameters showed that an O3 concentration as low as 10 microg/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 microg/ml) but the exchanger became less effective (low pO2 values) due to progressive clogging with cells.
Animals↗