CORonavirus-19 mild to moderate pneumonia Management with blood Ozonization in patients with Respiratory failure (CORMOR) multicentric prospective randomized clinical trial.

BACKGROUND: Following positive experience on the use of blood ozonation in SARS-CoV-2, the CORMOR randomized trial was designed to evaluate the adjuvant role of oxygen/ozone therapy in mild to moderate SARS-CoV-2 pneumonia. METHODS: The trial (ClinicalTrial.gov NCT04388514) was conducted in four different Italian centers (April-October 2020). Patients were treated according to best available standard of care (SoC) therapy, with or without O3-autohemotherapy (O3-AHT). RESULTS: A total of 92 patients were enrolled: SoC + O3-AHT (48 patients) were compared to the SoC treatment (44 patients). The two groups differed in steroids therapy administration (72.7% in SoC arm vs. 50.0% in O3-AHT arm; p = 0.044). Steroid therapy was routinely started when it was subsequently deemed as effective for the treatment of COVID-19 disease. No significant differences in mortality rates, length of hospital stay, mechanical ventilation requirement and ICU admission were observed. Clinical improvement in patients with pneumonia was assessed according to a specifically designed score (decrease in SIMEU class, improvement in radiology imaging, improvement in PaO2/FiO2, reduction in LDH and requirement of oxygen therapy ≤ 5 days). Score assessment was performed on day-3 (T3) and day-7 (TEnd) of O3-AHT treatment. A significant increase in the score was reported at TEnd, in the O3-AHT treatment arm (0 [0-1] in the SoC arm vs. 2 [1-3] the O3-AHT arm; p = 0.018). No adverse events related O3-AHT treatment was observed. CONCLUSION: In mild-to-moderate pneumonia due to SARS-CoV-2, adjuvant oxygen/ozone therapy did not show any effect on mortality, or mechanical intubation but show a clinical improvement a day 7 from randomization in a composite clinical endpoint. Larger Randomized prospective studies alone or in combination with steroids are needed to confirm our results.

Effects of ambient ozone exposure on circulating extracellular vehicle microRNA levels in coronary artery disease patients.

Exposure to ambient air pollutants such as ozone (O3) and particulate matter (PM) is associated with increased cardiovascular morbidity and rate of mortality, but the underlying biological mechanisms have yet to be described. Emerging evidence shows that extracellular vehicle (EV) microRNAs (miRNAs) may facilitate cell-to-cell and organ-to-organ communications and play a role in the air pollution-induced cardiovascular effects. This study aims to explore the association between air pollutant exposure and miRNA changes related to cardiovascular diseases. Using a panel study design, 14 participants with coronary artery diseases were enrolled in this study. Each participant had up to 10 clinical visits and their plasma samples were collected and measured for expression of miRNA-21 (miR-21), miR-126, miR-146, miR-150, and miR-155. Mixed effects models adjusted for temperature, humidity, and season were used to examine the association between miRNA levels and exposure to 8-hr O3 or 24-hr PM2.5 up to 4 days prior. Results demonstrated that miR-150 expression was positively associated with O3 exposure at 1-4 days lag and 5day moving average while miR-155 expression tracked with O3 exposure at lag 0. No significant association was found between miRNA expression and ambient PM2.5 at any time point. ß-blocker and diabetic medication usage significantly modified the correlation between O3 exposure and miR-150 expression where the link was more prominent among non-users. In conclusion, evidence indicated an association between exposure to ambient O3 and circulating levels of EV miR-150 and miR-155 was observed. These findings pointed to a future research direction involving miRNA-mediated mechanisms of O3-induced cardiovascular effects.

Ozone Exposure Increases Circulating Stress Hormones and Lipid Metabolites in Humans.

RATIONALE: Air pollution has been associated with increased prevalence of type 2 diabetes; however, the mechanisms remain unknown. We have shown that acute ozone exposure in rats induces release of stress hormones, hyperglycemia, leptinemia, and glucose intolerance that are associated with global changes in peripheral glucose, lipid, and amino acid metabolism. OBJECTIVES: To examine ozone-induced metabolic derangement in humans using serum metabolomic assessment, establish human-to-rodent coherence, and identify novel nonprotein biomarkers. METHODS: Serum samples were obtained from a crossover clinical study that included two clinic visits (n = 24 each) where each subject was blindly exposed in the morning to either filtered air or 0.3 parts per million ozone for 2 hours during 15-minute on-off exercise. Serum samples collected within 1 hour after exposure were assessed for changes in metabolites using a metabolomic approach. MEASUREMENTS AND MAIN RESULTS: Metabolomic analysis revealed that ozone exposure markedly increased serum cortisol and corticosterone together with increases in monoacylglycerol, glycerol, and medium- and long-chain free fatty acids, reflective of lipid mobilization and catabolism. Additionally, ozone exposure increased serum lysolipids, potentially originating from membrane lipid breakdown. Ozone exposure also increased circulating mitochondrial ß-oxidation-derived metabolites, such as acylcarnitines, together with increases in the ketone body 3-hydroxybutyrate. These changes suggested saturation of ß-oxidation by ozone in exercising humans. CONCLUSIONS: As in rodents, acute ozone exposure increased stress hormones and globally altered peripheral lipid metabolism in humans, likely through activation of a neurohormonally mediated stress response pathway. The metabolomic assessment revealed new biomarkers and allowed for establishment of rodent-to-human coherence. Clinical trial registered with www.clinicaltrials.gov (NCT 01492517).

Ozone: A Multifaceted Molecule with Unexpected Therapeutic Activity.

A comprehensive outline for understanding and recommending the therapeutic use of ozone in combination with established therapy in diseases characterized by a chronic oxidative stress is currently available. The view of the absolute ozone toxicity is incorrect, because it has been based either on lung or on studies performed in artificial environments that do not correspond to the real antioxidant capacity of body compartments. In fact, ozone exerts either a potent toxic activity or it can stimulate biological responses of vital importance, analogously to gases with prospective therapeutic value such as NO, CO, H2S, H2, as well as O2 itself. Such a crucial difference has increasingly become evident during the last decade. The purpose of this review is to explain the aspects still poorly understood, highlighting the divergent activity of ozone on the various biological districts. It will be clarified that such a dual effect does not depend only upon the final gas concentration, but also on the particular biological system where ozone acts. The real significance of ozone as adjuvant therapeutic treatment concerns severe chronic pathologies among which are cardiovascular diseases, chronic obstructive pulmonary diseases, multiple sclerosis, and the dry form of age-related macular degeneration. It is time for a full insertion of ozone therapy within pharmaceutical sciences, responding to all the requirements of quality, efficacy and safety, rather than as either an alternative or an esoteric approach.

Validity of Oxygen-Ozone Therapy as Integrated Medication Form in Chronic Inflammatory Diseases.

The state-of-the-art of oxygen-ozone therapy is now clarified and all the mechanisms of action of medical ozone are within classical biochemistry and molecular biology. The outcomes of standard treatments in peripheral arterial occlusive disease (PAOD) and dry-form of age-related macular degeneration (AMD) have been compared with the documented therapeutic results achieved with ozonated autohemotherapy (O-AHT). On the other hand, the clinical data of O-AHT on stroke remain indicative. As the cost of O-AHT is almost irrelevant, its application in all public hospitals, especially those of poor Countries, would allow two advantages: the first is for the patient, who will improve her/his conditions, and the second is for Health Authorities burdened with increasing costs. The aim of this paper is to report to clinical scientists that O-AHT is a scientific-based therapeutic approach without side effects. The integration of O-AHT with effective approved drugs is likely to yield the best clinical results in several chronic inflammatory diseases.

An integrated medical treatment for type-2 diabetes.

This paper tries to emphasize two relevant concepts: the first is that type 2 diabetes is a chronic diseases characterized by both a dysmetabolism and a chronic oxidative stress. A variety of orthodox drugs are somewhat able to correct the metabolic alterations, but do not deal with the chronic inflammation. Consequently, as the validity of precisely treating blood with therapeutic ozone concentrations in restoring a redox homeostasis has been now demonstrated, the integration of ozone therapy appears essential for a rational treatment of type 2 diabetes. Such a combination may be able to reduce the diabetic epidemic.

Combination of LC-MS- and GC-MS-based metabolomics to study the effect of ozonated autohemotherapy on human blood.

Ozonated autohemotherapy (O3-AHT) is a medical approach during which blood obtained from the patient is ozonated and injected back into the body. Despite an increasing number of evidence that O3-AHT is safe, this type of therapy remains controversial. To extend knowledge about the changes in blood evoked by O3-AHT, LC-MS- and GC-MS-based metabolic fingerprinting was used to compare plasma samples obtained from blood before and after the treatment with potentially therapeutic concentrations of ozone. The procedure was performed in PVC bags utilized for blood storage to study also possible interactions between ozone and plastic. By use of GC-MS, an increase in lactic acid and pyruvic acid was observed, which indicated an increased rate of glycolysis. With LC-MS, changes in plasma antioxidants were observed. Moreover, concentrations of lipid oxidation products (LOP) and lysophospholipids were increased after ozone treatment. This is the first report of increased LOPs metabolites after ozonation of blood. Seven metabolites detected by LC-QTOF-MS only in ozonated samples could be considered as novel biomarkers of oxidative stress. Several plasticizers have been detected by both techniques in blood stored in PVC bags. PVC is known to be an ozone resistant material, but ozonation of blood in PVC bags stimulates leaching of plasticizers into the blood.

How a calculated oxidative stress can yield multiple therapeutic effects.

It is proposed to discuss how ozonetherapy acts on patients affected by vascular and degenerative diseases. Ozone is a strong oxidant but, if used in small dosages on human blood ex vivo, acts as an acceptable stressor. By instantly reacting with PUFA bound to albumin, ozone is entirely consumed but generates two messengers acting in an early and in a late phase: the former is due to hydrogen peroxide, which triggers biochemical pathways on blood cells and the latter is due to alkenals which are infused into the donor patient. After undergoing a partial catabolism, alkenals enter into a great number of body's cells, where they react with Nrf2-Keap1 protein: the transfer of activated Nrf2 into the nucleus and its binding to antioxidant response element (ARE) is the crucial event able to upregulate the synthesis of antioxidant proteins, phase II enzymes and HO-1. With the progress of ozonetherapy, these protective enzymes are able to reverse the oxidative stress induced by chronic inflammation. Consequently, the repetition of graduated stresses induces a multiform adaptive response able to block the progress of the disease and to improve the quality of life.

Randomised, double-blinded, placebo-controlled, clinical trial of ozone therapy as treatment of sudden sensorineural hearing loss.

OBJECTIVE: To investigate the safety and efficacy of ozone therapy in adult patients with sudden sensorineural hearing loss. DESIGN: Prospective, randomised, double-blinded, placebo-controlled, parallel group, clinical trial. SUBJECTS: Forty-five adult patients presented with sudden sensorineural hearing loss, and were randomly allocated to receive either placebo (15 patients) or ozone therapy (auto-haemotherapy; 30 patients). For the latter treatment, 100 ml of the patient's blood was treated immediately with a 1:1 volume, gaseous mixture of oxygen and ozone (from an ozone generator) and re-injected into the patient by intravenous infusion. Treatments were administered twice weekly for 10 sessions. The following data were recorded: pre- and post-treatment mean hearing gains; air and bone pure tone averages; speech reception thresholds; speech discrimination scores; and subjective recovery rates. RESULTS: Significant recovery was observed in 23 patients (77 per cent) receiving ozone treatment, compared with six (40 per cent) patients receiving placebo (p < 0.05). Mean hearing gains, pure tone averages, speech reception thresholds and subjective recovery rates were significantly better in ozone-treated patients compared with placebo-treated patients (p < 0.05). CONCLUSION: Ozone therapy is a significant modality for treatment of sudden sensorineural hearing loss; no complications were observed.

Is there an association between lifetime cumulative exposure and acute pulmonary responses to ozone?

OBJECTIVE: To investigate the potential effects of lifetime cumulative ozone (O3) exposure on acute pulmonary responses to O3. METHODS: Fifteen healthy subjects from a larger cohort of young adults were exposed to 200 ppb O3 for 4 hours followed by bronchoscopy and bronchoalveolar lavage 18 hours later. Lung function, symptom questionnaires, and blood samples were obtained before and after each exposure. Subjects' lifetime cumulative O3 exposures were estimated from residential histories and air-quality monitoring data. RESULTS: Acute exposure to O3 caused decrements in forced expiratory volume in 1 second (FEV1), maximal mid-expiratory flow rate (FEF25-75), and forced expiratory flow rate at 75% of forced vital capacity (FEF75), and an increase in plasma clara cell protein (CC16) level. Changes in CC16 and lower respiratory symptoms, but not in lung function, were positively correlated with lifetime cumulative O3 exposure. CONCLUSION: Higher lifetime cumulative O3 exposure was associated with airway injury and respiratory symptom responses, but not with airway inflammatory or lung function responses, to acute O3 exposure.

Oxygenation-ozonation of blood during extracorporeal circulation: in vitro efficiency of a new gas exchange device.

We have investigated the performance of a new gas exchange device (GED), named L001, specifically devised for the ozonation of human blood during extracorporeal circulation. This procedure, defined with the acronym "EBOO," means "extracorporeal blood oxygenation-ozonation." The innovative GED is made of microporous, ozone-resistant, polipropylene hollow fibers with an external diameter of 200 microm, a thickness of 50 microm, and a membrane surface area of 0.22 m(2). The material is coated with phosphorylcholine on the external side in contact with the circulating blood, while a gas mixture, necessarily composed of medical oxygen and ozone (about 99 and 1%, respectively), flows inside the fibers in opposite direction. The new GED has been tested by using a buffered saline solution containing KI and by varying several parameters, and it has shown to be very versatile and efficient. Its main characteristics are minimal foreign surface contact, high gas transfer, and negligible priming volume. This device appears to be a practical, nontoxic, and rather inexpensive tool for performing ozonation of blood for already defined human diseases.

Fistula function and dialysis adequacy during ozonotherapy in chronically hemodialyzed patients.

There are a variety of complications related to chronic hemodialysis treatment, including thrombosis in hemodialysis access leading to blood recirculation, and in turn to the deterioration in hemodialysis effectiveness. Given that ozone decreases blood viscosity, increases erythrocyte deformability, and inhibits coagulation, the periodic blood ozonation may be of benefit in the attenuation of these disturbances. To gain insight into this issue,we originally evaluated the impact of ozonated autohemotherapy on recirculation in arteriovenous fistula, hemodialysis adequacy, and the frequency of dialyzer reuse. Twelve chronically hemodialyzed patients with peripheral arterial disease were enrolled in the prospective, placebo-controlled study. Nine sessions of autohemotherapy with the exposure of blood to oxygen, as a control, and nine sessions of autohemotherapy where the blood is exposed to ozone in a concentration of 50 microg/mL are administered in a single-blind manner. Access recirculation is measured by means of spectral technology(Crit Line Monitor, HemaMetrics, Kaysville, UT, U.S.A.), and hemodialysis adequacy is calculated using the Daugirdas formula and expressed as the Kt/V index. The Kt/V index and the frequency of dialyzer reuse do not change after ozonated autohemotherapy. Recirculation decreases after ozonotherapy in the majority of patients.on average by 35.3%, but the change does not reach the level of statistical significance (P = 0.064). We demonstrate that ozonated autohemotherapy does not influence dialysis adequacy and the frequency of dialyzer reuse. The improvement of fistula function, expressed as a decrease in recirculation, is not significant, although seen in the majority of patients.

Effect of O(2)/O(3) atmosphere on the rate of osmotic hemolysis of bovine erythrocytes in the presence of some antioxidants.

Purified red blood cells, exposed to an ozone atmosphere, show an increased rate of hemolysis on sudden osmotic stress. To determine this effect of ozone in the presence of natural antioxidants, bovine red blood cells, used as models, were suspended in blood plasma, or in physiological saline with one of the following antioxidants: albumin, glutathione, uric acid, glucose and a vitamin E analog (trolox). After exposure of the suspensions to oxygen and oxygen/ozone atmospheres the rates of osmotic hemolysis were measured, using a stop-flow technique, and compared with rates measured in air-exposed controls. Blood plasma, containing all natural antioxidants, caused a decreased rate of osmotic hemolysis of cells exposed to oxygen and also decreased the effect of ozone. Trolox cancelled the oxygen effects only. Albumin, glutathione and uric acid tended to protect the cells from the hemolytic effects of ozone. The antihemolytic effect of glucose, seen only in some samples, may depend on uncontrolled factors. The alteration of the rates correlates with an increased fluidity of red cell membranes exposed to ozone.

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.

A comparison of biomarkers of ozone exposure in human plasma, nasal lavage, and sputum.

We examined ozone-induced upper and lower airway inflammatory responses and the concentrations of hydroxylated salicylate metabolites using nasal lavage fluid and induced sputum, in order to identify noninvasive and sensitive biomarkers for ozone exposure and effects. A time course for plasma concentration of 2, 3-dihydroxybenzoic acid (2,3-DHBA, a salicylate metabolite and an indicator for hydroxyl radical) in response to 0.12 ppm ozone was also studied. Healthy, young, nonsmoking volunteers were given acetylsalicylic acid (ASA, 975 mg) or placebo orally. Subjects were exposed to ozone (0.12 or 0.4 ppm) or filtered air in an environmental chamber for 2 h, while performing intermittent exercise. Blood was collected hourly over a 4-h period. After exposure, nasal lavage fluid was collected, and sputum was induced using hypertonic saline. Results show that in sputum the percentage of neutrophils was significantly higher after the subjects were exposed to 0.4 ppm ozone (p<.05) than after they were exposed to filtered air or 0.12 ppm ozone. The absolute number and the percentage of macrophages were significantly lower at 0.4 ppm ozone than for filtered air control or 0.12 ppm ozone. The percentage of lymphocytes in sputum was also significantly lower at 0.4 ppm ozone than for filtered air control or 0.12 ppm ozone. The sputum cellular responses to ozone were not significantly altered by ASA treatment. In nasal lavage, cell counts and differentials did not change significantly after exposure to ozone in comparison to filtered air control. The cellular data indicate an acute inflammation developed during ozone exposure in the lower respiratory tract. The concentrations of total protein and interleukin-8 and the activity of N-acetyl-beta-D-glucosaminidase (a lysosomal enzyme) in nasal lavage and sputum did not change significantly following exposure to ozone in comparison to filtered air control. Plasma 2,3-DHBA concentration increased significantly following exposure to 0.12 ppm ozone in an exposure-dependent temporal pattern. Salicylate metabolites in nasal lavage fluid and sputum did not increase significantly following exposure to ozone. There was a marked variation of 2,3-DHBA concentrations in airway fluids. Data suggest that plasma 2,3-DHBA is a sensitive marker indicating acute ozone exposure, even at an ozone concentration that causes minimal observable airway effects in healthy subjects.

Studies on the biological effects of ozone: 8. Effects on the total antioxidant status and on interleukin-8 production.

Ozone (O3) is a controversial gas because, owing to its potent oxidant properties, it exerts damaging effects on the respiratory tract and yet it has been used for four decades as a therapy. While the disinfectant activity of O3 is understandable, it is less clear how other biological effects can be elicited in human blood with practically no toxicity. On the other hand plasma and cells are endowed with a powerful antioxidant system so that a fairly wide range of O3 concentrations between 40 and 80 microg/ml per gram of blood (approximately 0.83-1.66 mM) are effective but not deleterious. After blood ozonation total antioxidant status (TAS) and plasma protein thiol groups (PTG) decrease by 20% and 25%, respectively, while thiobarbituric acid reactive substances (TBARS) increases up to five-fold. The increase of haemolysis is negligible suggesting that the erythrocyte membrane is spared at the expense of other sacrificial substrates. While there is a clear relationship between the ozone dose and IL-8 levels, we have noticed that high TAS and PTG values inhibit the cytokine production. This is in line with the current idea that hydrogen peroxide, as a byproduct of O3 decomposition, acts as a messenger for the cytokine induction.

Oxidative damage to extracellular fluids by ozone and possible protective effects of thiols.

Environmental levels of ozone (O3) frequently exceed air quality standards in many urban areas, and much research has been devoted to pathophysiological effects of O3 inhalation. Inhaled O3 will interact primarily with respiratory tract lining fluids (RTLF) and with constituents therein. It is believed that interaction of O3 with constituents in RTLF occurs by reactive absorption. We investigated interactions of O3 with human blood plasma, used as a model extracellular fluid representing RTLF, and studied oxidation of plasma antioxidants, proteins, and lipids. Plasma was exposed to various concentrations of O3 in humidified air, supplied at a continuous flow, in a system that mimics exposure of RTLF to inhaled O3 in vivo. Interaction of O3 with plasma appeared to be caused by reactive absorption of O3 by plasma. It was found that O3 reacts primarily with the aqueous antioxidants ascorbate and urate. Reactive absorption of O3 by plasma ascorbate and urate was found to be more efficient at low (2 ppm) O3 levels than at high (16 ppm) levels. We were also able to detect oxidative damage to plasma proteins and lipids after prolonged exposure to O3. Second, we investigated whether GSH or dihydrolipoic acid (DHLA) could prevent oxidative damage to plasma proteins and lipids by O3, under our exposure conditions. In contrast to plasma, RTLF contain relatively high amounts of GSH, which may contribute to antioxidant protection to respiratory tract epithelial cells. DHLA is an endogenous dithiol and has potent antioxidant properties. Addition of either GSH or DHLA to plasma (at concentrations up to 1 mM) prior to O3 exposure did not inhibit oxidation of plasma proteins and lipids during exposure to O3, nor did it attenuate depletion rates of ascorbate or urate. Our results indicate that added thiols cause increased reactive absorption of O3, rather than preventing reaction of O3 with other plasma constituents. Thiol supplementation could afford protection against O3-induced injury in vivo by increasing reactive absorption of O3 in the upper respiratory tract, thereby protecting the epithelia of lower airways and gas-exchanging portions of the lungs from exposure to toxic levels of O3.(ABSTRACT TRUNCATED AT 400 WORDS)

Red blood cell antioxidants in human volunteers exposed to ozone.

Indices of red blood cell (RBC) antioxidant capacity can undergo changes upon exposure to oxidants, either acutely or chronically. To investigate whether these changes might provide a biochemical marker for acute environmental ozone exposure, we assessed RBC glutathione (GSH) and catalase (CAT) responses in seven normal volunteers exposed to 0.16 ppm ozone for 7.5 hours compared to the same measurements following sham exposure to clean air. For each subject, an interim period of two weeks separated the two exposure studies. Investigators performing the RBC assays were unaware of the environmental conditions. No changes in either GSH or CAT were observed for any study condition when compared to pre-study values. Our conclusion is that RBC antioxidants do not accurately reflect in vivo exposure to ozone at concentrations readily attainable during periods of heavy urban pollution. Our data dispute the value of these indices as markers of acute environmental photochemical oxidant exposure.