Part #1
Bocci V; Paulesu L
Studies on the biological effects of ozone
1. Induction of interferon gamma on human leucocytes.
Istituto di Fisiologia Generale, Universita di Siena, Italy.
Haematologica 1990 Nov-Dec;75(6):510-5
Unique Identifier: MEDLINE 91276379

Abstract: 
In this study we have investigated the effects of ozone on human
blood, as well as on resuspended buffy coats and Ficoll-purified
mononuclear cells. Samples were exposed at different ozone
concentrations (from 2.2 micrograms to 108 micrograms/ml) for 30 sec
and then incubated for different times at 37 degrees C in a 95%
air-5% CO2 humidified atmosphere. Supernatants were collected and
frozen at-20 degrees C until tested for interferon (IFN) activity. We
have determined that the ozone concentration is critical for
lymphokine induction. In fact, while low concentrations (2.2
micrograms/ml) are effective in lymphocytes, they do not induce IFN
in either whole or diluted (1:1) human blood, or resuspended buffy
coats. In such cases levels as high as 42 micrograms/ml are required.
On the other hand, a very high ozone concentration (108
micrograms/ml) is not effective and probably toxic. Maximal IFN
production occurs 72-96 h after ozone exposure, and the kinetics of
IFN release is similar to that after Staphylococcal Enterotoxin B
addition. Because ozonization of blood is a medical procedure
followed in several countries for treatment of viral diseases, this
study can open a new field of investigation that may yield useful
results both in biological and practical terms.

Part #2
Paulesu L; Luzzi E; Bocci V
Studies on the biological effects of ozone:
2. Induction of tumor necrosis factor (TNF-alpha) on human leucocytes.
Institute of General Physiology, University of Siena, Italy.
Lymphokine Cytokine Res 1991 Oct;10(5):409-12
Unique Identifier: MEDLINE 92118999

Abstract: 
The effect of ozone as a probable inducer of tumor necrosis factor
(TNF-alpha) has been investigated on human blood and on
Ficoll-purified blood mononuclear cells (PBMC). Samples were exposed
at different ozone concentrations ranging from 2.2 to 108
micrograms/ml and incubated at 37 degrees C in an 95% air-5% CO2
atmosphere. At predetermined times, all cell supernatants were tested
for TNF activity and some PBMC cultures were examined for DNA
synthesis. We have shown that ozone concentration is critical in
terms of TNF production and of cell mitogenesis and that, owing to
the presence of erythrocytes, higher ozone concentrations are
required to be effective in blood than in PBMC. Because ozonization
of blood is a procedure followed in several European countries for
the treatment of viral diseases and tumors, the release of factors
with antiviral and immunomodulatory activities by leukocytes may
explain the mechanism of action of ozone and of autohemotherapy.

Part #3
Bocci V; Luzzi E; Corradeschi F; Paulesu L; Di Stefano A
Studies on the biological effects of ozone:
3. An attempt to define conditions for optimal induction of cytokines.
Institute of General Physiology, Faculty of Pharmacy,
University of Siena, Italy.
Lymphokine Cytokine Res 1993 Apr;12(2):121-6
Unique Identifier: MEDLINE 93312993

Abstract: 
Ozonization of blood, normally carried out with citrated blood, may be
fine for the autohemotherapy of ischemic diseases but it may be at a
loss when employed in viral diseases or in immunodeficiencies. We
have shown that heparin, used as an anticoagulant, with the addition
of 5 mM CaCl2 favors production of cytokines by leukocytes with only
a modest increase in hemolysis. High plasmatic levels of glucose,
glutathione, and ascorbic acid decrease cytokine's yield because
these compounds act as antioxidants and quench the inducing activity
of ozone. Autohemotherapy with heparinized and Ca(2+)-supplemented
blood has not revealed any side effects in volunteers.

Part #4
Bocci V; Luzzi E; Corradeschi F; Paulesu L; et al
Studies on the biological effects of ozone:
4. Cytokine production and glutathione levels in human erythrocytes.
Institute of General Physiology, Faculty of Pharmacy,
University of Siena, Italy.
J Biol Regul Homeost Agents 1993 Oct-Dec;7(4):133-8
Unique Identifier: MEDLINE 94295419

Abstract: 
We have investigated the effect of various concentrations of ozone on
human blood aiming to correlate the production of cytokines with
depletion of reduced glutathione and hemolysis. As erythrocytes
constitute the bulk of blood cells and represent the main target of
ozone they have been taken as a useful marker of its oxidative
activity. It appears that a transient exposure (30 sec) of blood of
up to 78 micrograms ozone per ml of blood does not depress the
production of cytokines even though there is a slight increase of
hemolysis and a small decrease of intracellular reduced glutathione.
In contrast either a constant (up to 30 sec) exposure to an ozone
flux or a high ozone concentration (108 micrograms/ml) markedly
decreases reduced glutathione levels and depresses cytokine
production.

Part #5
Bocci V; Luzzi E; Corradeschi F; Paulesu L
Studies on the biological effects of ozone:
5. Evaluation of immunological parameters and tolerability
in normal volunteers receiving ambulatory autohaemotherapy.
Faculties of Pharmacy, Institute of General Physiology, Siena, Italy.
Biotherapy 1993-94;7(2):83-90
Unique Identifier: MEDLINE 95101410

Abstract: 
Autohaemotherapy, after a bland treatment ex vivo of blood with ozone,
is a fairly unknown medical procedure claimed to have therapeutic
value in viral diseases and neoplasms. Having already shown that
ozone acts as a mild inducer of cytokines, we have undertaken an
investigation in normal rabbits and in normal volunteers aiming to
evaluate eventual changes of some cytokine levels in plasma as well
as of immunological parameters such as the Mx protein, neopterin,
beta 2-microglobulin and of some acute-phase proteins after single or
repeated autohaemotherapy. We have also evaluated the potential
development of side-effects. This study is the first one to show
that autohaemotherapy can activate an immunological marker in normal
subjects without procuring any toxic effects.

Part #6
Bocci V; Luzzi E; Corradeschi F; Silvestri S
Studies on the biological effects of ozone:
6. Production of transforming growth factor 1
by human blood after ozone treatment.
Institute of General Physiology and Nutritional Sciences, Siena, Italy.
J Biol Regul Homeost Agents 1994 Oct-Dec;8(4):108-12
Unique Identifier: MEDLINE 95389925

Abstract: 
After exposing human whole blood from normal volunteers to ozone
concentrations ranging from 22 to 156 micrograms/ml, we have shown
that, upon incubation of up to 8 hours, there is a significant
release of transforming growth factor beta (TGF-beta 1). In
comparison to TGF-beta 1, TGF-beta 2 production is not influenced by
ozone concentrations. In line with our previous findings it appears
that blood, in the presence of heparin and 5mM Ca,2+ allows a
consistent production of tumor necrosis factor a (TNF alpha) and the
release of low and non-hazardous levels of free hemoglobin. These
data support the contention that autohemotherapy performed after
treating blood with ozone followed by reinfusion into the donor, may
represent a valuable therapeutic approach for achieving
immunoregulatory effects.

Part #7
Bocci V; Valacchi G; Corradeschi F; Aldinucci C; et al
Studies on the biological effects of ozone:
7. Generation of reactive oxygen species (ROS)
after exposure of human blood to ozone.
Institute of General Physiology, University of Siena, Italy.
J Biol Regul Homeost Agents 1998 Jul-Sep;12(3):67-75
Unique Identifier: MEDLINE 99011926

Abstract: 
The acceptance of any complementary medical approach is conditioned by
the results obtained after the same scientific scrutiny applied in
orthodox medicine. Otherwise any claim of efficacy remains in the
realm of fiction. In the case of ozone therapy, the mechanisms of
action have remained nebulous and in a series of publications we are
trying to present the biochemical, immunological and morphological
evidence in favour or against ozone therapy. We have now shown that
ozone (O3) dissolved in the water of either plasma or serum or
physiological saline generates reactive oxygen species (ROS), of
which hydrogen peroxide (H2O2) can be unequivocally demonstrated by
using specific methods for its detection. Lipids present in plasma
preferentially those present in lipoproteins, undergo peroxidation
that is somewhat O3-dose dependent and can be observed by the
measurement of thiobarbituric acid reactive substances (TBARS). While
the generation of H2O2 is crucial in activating both biochemical
(hexose monophosphate shunt) and immunological (via the transcription
factor NF-kB) mechanisms, the role of lipid oxidation products (LOP)
remains to be investigated. We have shown here that there is a small
but consistent induction of some cytokines (TNF-alpha, IFN-gamma and
IL-2) when human blood is directly exposed to O3 concentrations up to
100 micrograms/ml per g of blood. On the other hand, isolated blood
mononuclear cells (PBMC) in tissue culture medium are far more
sensitive to the oxidant action of O3 as shown by a progressive
reduction of the proliferation index with comparatively far lower O3,
concentrations. On the whole, these results support the concept that
much of the O3 toxicity is neutralized by the powerful antioxidant
system of blood. The minimal hemolysis supports this idea but as far
as platelets are concerned, we must mention that they tend to
aggregate in heparinized blood, even when it is exposed to an O3
concentration of 40 micrograms/ml. In spite of the lack of
side-effects after autohemotherapy, this drawback must be kept in
mind and avoided in clinical practice.

Part #8
Bocci V; Valacchi G; Corradeschi F; Fanetti G
Studies on the biological effects of ozone:
8. Effects on the total antioxidant status
and on interleukin-8 production.
Institute of General Physiology, University of Siena, Italy.
fisgen@unisi.it
Mediators Inflamm 1998;7(5):313-7
Unique Identifier: MEDLINE 99098434

Abstract: 
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.

Part #10
Valacchi G; Bocci V
Studies on the biological effects of ozone:
10. Release of factors from ozonated human platelets.
Institute of General Physiology, University of Siena, Italy.
Mediators Inflamm 1999;8(4-5):205-9
Unique Identifier: MEDLINE 20166840

Abstract: 
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).