Institute of General Physiology,
University of Siena,
53100 Siena,
Italy

There are several reasons explaining why autohaemotherapy after
ozone treatment has remained in a scientific limbo: firstly, the
biological basis have remained nebulous and have been barely
studied [1,2], partly because of lack of financial support by the
pharmaceutical industry, secondly the therapeutic schemes have
been elaborated on personal bias rather than on the evaluation of
objective parameters [3,4], thirdly clinical experience, although
vast, had remained limited to private practice with the
consequence that results have been reported on a anecdotal fashion
and never published in peer-reviewed journals [1,3-8]; even worse,
a few quacks have completely discredited ozonetherapy by claiming
wonderful results and exploiting patients. Fourthly, ozone has
often been used unproperly without knowing exactly its
physicochemical properties [9] and fifthly, the awareness that
ozone is one of tile worst pollutant and can generate formation of
oxidizing compounds [10-14] has comprehensibly originated a strong
prejudice for its use. Thus, it is not surprising that the use of
ozone for medical purposes is only allowed in Germany, Austria,
Switzerland, Italy, Russia, Cuba and in a few States of USA.
England and France, although widely using ozone for the treatment
of drinking water, refrain from using it in Medicine. This
involutive state of affairs has now undergone a critical
reappraisal by considering that:

a)   of the various routes of ozone administration, the exposure
of a
     volume of blood to a precise ozone dose offers and meaningful
and
     reproducible delivery system.
b)   Ozone is now considered a citokine inducer with a definite
     benefit/risk ratio. As a consequence, autohaemotherapy may
favour
     immunorestoration in secondary cellular immune deficiencies.
c)   Blood cellular components display such a marked functional
     heterogeneity that under ozone action quite different
biological
     responses have to be expected. This does not exclude however,
     that therapeutic activity in each disease has a
multifactorial
     origin.

Routes of ozone administration in different pathological
conditions.

The solubility of ozone in water, is about 50% higher than oxygen
and it follows Henry's law [1,15]. However when ozone dissolves in
the plasma, Henry's law is no longer valid because ozone
decomposes in about one second and generates a cascade of unstable
and highly reactive oxygen intermediates (ROI) among which are
aldehydes, ozonides, hydrogen peroxide and lipid hydroperoxides
that almost instantaneously react with membranous and cytoplasmic
components [1,14-17]. On the other hand, cells and body fluids
contain a wealth of antioxidant compounds such as vitamins, low
molecular weight compounds and proteins [18] as well as a number
of enzymes able to either scavenge ROI or to rapidly regenerate
reducing compounds. Both parenteral (intravenous, intra-arterial,
intramuscular, subcutaneous and intra-articular) and local (nasal,
tubal, oral, vaginal, vesical, colorectal and cutaneous) routes
have been used [1,3] for the treatment of a number of pathological
states with ozone (Table 1 [19-26]). One is surprised to note
that, with the exception of the intravenous route, only minor
side-effects have been observed probably because the potential
toxicity of ozone is largely quenched by the body antioxidant
reservoir and because the initial irritating (burning feeling)
action of ozone in tissues is within few minutes followed by
analgesia.

Use of the intravenous administration route is extremely dangerous
because even if the gaseous mixture of oxygen-ozone is
administered very slowly with a pump, it frequently procures lung
embolization and serious side effects, particularly when daily
dosing is up to 120 ml. Moreover, to the best of my knowledge,
this procedure does not yield clinical benefit and no data have
been published.

Jacobs [27] has analysed the type of accidents and side-effects
after ozonetherapy in Germany in a total of 5,579,238 applications
performed in 384,775 patients up to 1982. Although the percentage
of accident due to ozone was 0.0007 %, it nonetheless included
four fatalities most likely due to lung embolization while the
remaining were minor accidents due to technical mistakes. During
the last decade the administration of ozone via the intravenous
route has been prohibited in Europe but regrettably someone still
uses it in USA.

Intraarterial administration of 02-03, useful for treating chronic
limb ischemia, is far less risky probably because only a small
volume of gas is injected and because there is rapid gas
solubilization and absorption at the capillary level [3,4,6,8].

Administration of up to 150 ml of 02-03, via intramuscular and
subcutaneous routes is painful for a few minutes, and although
therapeutic efficacy is claimed to be good, convincing clinical
data are lacking [3]. My prejudice against these routes,
particularly when used for immunomodulatory purposes, is that
ozone dosing is totally empirical because no stoichiometric
relationship can be defined between the ozone dose and the unknown
volume of blood exposed in an internal cavity or tissue.

Of the local treatments, the cutaneous one (for torpid ulcers or
necrotic lesions due to vascular limb disorders), is free of side
effects and effective particularly when combined to
autohaemotherapy [3,6,8,15].

Colorectal insufflation over a period of one minute of up to 800
ml of 02-03 (ozone concentration 20 ug/ml) is reported to be free
of side-effects and effective in AIDS patients with intractable
diarrhoea and in hepatitis patients [28-30]. This route ought to
be borne in mind because the procedure of execution is simple, it
can be done by the patient at home and it is a practical
alternative when there is not a venous access. However, so far
neither endpoints are available for establishing an optimal dosage
for this route, nor do we know how ozone insufflated into the
colon lumen works. While there will occur an improved oxygenation
of portal and peripheral blood [29] and possibly of intestinal
lymph, we can only speculate on the activation of the colon-
associated lymphoid system. It remains also unknown whether the
bactericidal activity of ozone enhances absorption of bacterial
compounds, similar to muramyl/ peptides, with immunoenhancing
properties.

In conclusion, all of these routes have the disadvantage that
ozone dosing has to be defined on a trial and error basis which is
difficult and time-consuming to assess. Thus, at present time,
ozonated autohaemotherapy, first described by Wehrli and Steinbart
[31], represents the best option because it implies an almost
stoichiometric relationship between a known dose of ozone versus a
volume of blood measured by weight (about 105 g correspond to 100
ml) and because we can correlate several biochemical and
immunological parameters with ozone dosing. These are crucial
advantages that associated with the simplicity of the procedure,
portray ozonated autohaemotherapy as a very important therapeutic
possibility. After the discovery [32] that endothelial and other
cells synthesize NO, that is an inorganic free radical gas and use
it as a mediator and immune modulator, we ought to examine without
any bias the biological activity of ozone.

Induction of cytokines from blood mononuclear cells (BMC) during
ozonation.

Autohaemotherapy represents an almost ideal model because there is
a reasonable stoichiometric relationship between ozone and blood.
However, even in this case, among blood samples there are
unavoidable uncertainties such as a variable amount of antioxidant
compounds [18,33], of intracellular reducing enzymes [33,34], as
well as quantitative and qualitative differences in cell
components. As we have found [35,36] already great variability
among normal donors, we expect an even greater variability among
patients and for this reason, rather than suggesting an optimal
ozone concentration (around 70 ug/ml ozone per ml of blood at
normal barometric pressure), we propose that effective ozone
concentrations range between 50 and 80 ug/ml without any evident
toxicity when erythrocyte counts range between 3.5-5.0 x
10(6)/mm(3). Within these concentrations there is no formation of
metahaemoglobin, either haemolysis or intraerythrocylic reduced
glutathione levels remain below 3.0 or 10%, respectively [35,36],
cell viability is normal and no morphologic cell damage is evident
by electron microscopic analysis (Bocci et al., manuscript in
preparation).

The new result which has confirmed the hypothesis that ozone can
act as a cytokine's inducer [37] is that blood, after being
exposed for a few minutes to concentrations of ozone ranging from
10 up to 90 ug/ml (per ml of blood) at normal barometric pressure,
upon the usual incubation in air/CO, (95/5%) for up to 72 hours,
progressively releases small amounts of cytokines such as
interferon (IFN) B and y, tumor necrosis factor (TNFx),
interleukins (IL) 1B, 2, 4, 6, 8 and 10, granulocyte-macrophage,
colony-stimulating factor (GM-CSF) and activated transforming
growth factor (TGF) B1 [35,36,38-40]. IFNx has been barely
detectable and other cytokines will be measured as soon as we have
suitable reagents. At least for IFNs B/y and TNFx there is
correspondence between biological and immunoenzymatic activities.
In spite of great variability among normal blood samples, that is
a normal fact due to the existence of either low or high
responders [41], there is an ozone dose/cytokine response effect
that tends to level off when ozone concentration reaches the 70
ug/ml mark. At the 105 ug/ml level, that is the maximum ozone
concentration delivered by the generator, we have often noted a
depressed cytokine production suggesting a possible cytotoxic
effect [40]. Cytokine production is markedly depressed when blood
is collected in the usual Ca++ chelating solution (citrate-
phosphate-dextrose) and incubated in the absence of extracellular
Ca.++  This finding led us to test the physiological
anticoagulant, i.e., heparin (25 U/ml of blood) additioned with
CaCl2, to a final concentration of 5mM [35]. Thus, a five-fold
surplus of extracellular Ca++ (physiological Ca++ level is about 1
mM) displays a superinducing effect [2] but it must be noted that
further Ca++ addition up to 50 mM, although modestly increasing
cytokine's production, yields a prohibitive haemolysis [35].
However, heparin occasionally presents the problem of excessive
anticoagulation as it has recently occurred in two hepatitis
patients leading us to re-evaluate whether CPD treated blood, once
heparinized and recalcified at physiological levels, is
sufficiently activated.  Data to be reported soon have shown that,
after Ca++ addition, the production of cytokine is partially
restored whereas blood sample incubated without Ca++ are
consistently inhibited.  These results have practical importance
because they indicate that either CPD- or heparin-treated blood
after ozonation and reinfusion will release similar amounts of
cytokines in vivo.  As a rule now, for patients under
anticoagulant therapy, or aspirin, or prone to the haemorrhagic
syndrome, or thrombocytopenia or hepatic dysfunction, we collect
blood in CPD only thus avoiding any risk of dyscoagulation.

How ozone acts at the cellular level remains uncertain and for the
time being, it is a matter of speculation.  Probably ozone may
oxidize unspecifically some carbohydrates, probably galactose
[42,43], present on the cell membrane lectins leading to a
coupling with transducer proteins and to an enhanced Ca++ influx.
Moreover ROI can passively diffuse through the cell membrane and
activate the gene-regulatory Nuclear Factor-Kappa B (NF-Kappa B)
that appears to play many roles in the immune cells and certainly
causes gene activation for several cytokines [44,45].  At the
present our working hypothesis is that ozone acts unspecifically:
the bulk of generated ROI is consumed by antioxidant substances in
plasma and by the huge amounts of phospholipids present in
erythrocytes because their membranous surface is as large as 70
m(2) per 100 ml of blood.  As there is about 1 BMC every 3000
erythrocytes, there should occur a transient overproduction of
ROI, for them to reach the trans-activating factor in the
cytoplams and release the inhibitory subunit I Kappa B.  Schreck
et al [44] have indicated a threshold of about 30 uM of hydrogen
peroxide to be effective in their system, implying that if ROI are
not homogenously distributed, BMC will not be activated.  We do
not envisage and actually we do not notice cell damage because
intracellular catalase, superoxide dismutase as well as other
reducing enzymes and chainbraking compounds [14,17,18,33], are
capable of quickly neutralizing or scavenging residual ROI
terminating the ozone action.  In conclusion it would seem that
ozone dosing is critical in the sense that if it is too low it is
probably ineffective while if it is excessive, it may induce
oxidative stress and eventually cell apoptosis [46].

Another interesting characteristic of this approach is that blood,
after being thouroughly exposed to O2/O3 ex vivo for 5 minutes can
be reinfused in the donor without practically any trace of ozone.
Only the pO2 level, from a baseline value of 33-40 mmHg reaches in
a few minutes a plateau level of about 400 presumably returning to
the original value after gas exchange in the capillaries. Lipid
hydroperoxide levels in plasma increase about 3-fold immediately
after ozonation and return to baseline values within 3-4 hours
when blood is incubated in vitro. However it is most important to
note that in vivo, even 5 min after reinfusion, peroxides in the
plasma remain at baseline value.

Both CPD- and heparin-treated blood are occasionally mixed to
minimize erythrocyte sedimentation and reinfused fairly rapidly
without any vascular or respiratory distress. In order to
stabilize the plasma levels of antioxidants and to make sure that
patients receive a normal vitaminic support we have always
described a daily multivitamin (including vitamin C and E)
supplement.

Mechanisms of action of ozonated autohaemotherapy.

Until recently it was thought that in chronic viral diseases, the
virucidal properties of ozone were of paramount importance
[1,3,7,21,47-49] for the therapeutic effect, neglecting the fact
that the viral reservoirs are the internal organs (liver for
hepatitis, lymph nodes and spleen for HIV infection, neuronal
ganglia for herpes viruses etc) and that less viral particles are
free in the plasma, implying a minimal direct effect during
exposure of a small aliquot of plasma to ozone [50]. Moreover it
was reported that after ozonation, leukocytes improved their
phagocytic activity and that immunoglobulin levels could increase
without underlying the causes of these biological effects [51].
The breakthrough has come with the demonstration that ozone acts
as an inducer of cytokine's production [35,36,38-40]. Since then
the approach of ozonized autohaemotherapy has gained a rational
basis and one can understand why synthesis of antibodies can be
stimulated by IL-6. enhanced phagocytic functions and leukocytosis
can be due to IL-8 and GM-CSF and how both direct and indirect
antiviral activities can be stimulated by IFNB, y and TNFx. In
viral diseases, however, it cannot be excluded that small amounts
of free virus inactivated in the plasma during ozonization may act
either as an endogenous immunogen or/and an activator of cell-
mediated immunity. It is worth while mentioning that
autohaemotherapy has been surprisingly used also for the treatment
of autoimmune diseases such as rheumatoid arthritis [52] and it
would be desirable to investigate whether particular ozone
concentrations may enhance the release of inhibitory cytokines
leading either to the suppression of autoreactive cytotoxic T cell
clones or/and to the blocking of inflammatory cytokines by the
release of either soluble receptors or/and cytokine antagonists.
It is possible that the release of IL-lO [53] and TGFB1[54,55] can
serve the useful purpose of quenching an excessive immune
stimulation, thus leading to an orderly reprogramming of immune
responses.

Distribution and fate of ozunatcd blood cells after reinfusion.

With the exception of the most aged erythrocytes that, being more
susceptible to ozone,,re likely to be taken up by the reticulo-
endothelial system [56],it is reasonable to speculate that most
erythrocytes will continue to circulate in the vascular system. On
the other hand, activated BMC may home in various lymphoid and
non-lymphoid organs and we plan to verify this possibility with
Indium-III labelled cells as soon as possible. If this happens, as
we have shown during in vitro incubation [35,36], BMC will release
around the pericellular environment various cytokines which can
bind to the appropriate receptors on neighbouring stationary or in
transit cells. In comparison to classical mitogens, which can
easily activate 10-20% of the isolated BMC causing the synthesis
of large amounts of cytokines, depending upon the fairly critical
ozone dosing, a smaller percentage of BMC appears activated so
that the minute amount of released cytokines is consumed in
cellular microenvironments and does not emerge in the general
circulation via the lymphatic system. This tentative explanation
is supported by two pieces of evidence: firstly, we have never
detected any significant change in cytokine's levels in the plasma
of healthy volunteers at 1, 3, 6, 9 and 24 hours after reinfusion
of ozonated blood [57]. In contrast, after injection of as little
as 4 ng/Kg body weight of endotoxin in patients, there is a
massive release of pyrogenic cytokines with IL-1, IL-6 and TNFx
peaking a few hours after endotoxin administration [58]. The
second point is that although autohaemotherapy does not allow the
release of endogenous cytokines in the circulation, which is an
important advantage. it has a real biological effect because 48-72
hours after reinfusion we have measured [57] in BMC an increase of
the Mx protein that is one of the best indicators of IFN release
[59]. The progressive enlargement of the area under curve of Mx
protein versus time throughout autohaemotherapeutic treatments
suggests that in vivo there occurs a progressive amplification of
the priming and IFN production [57]. Thus, ozonated
autohaemotherapy may be assimilated to a slightly enhanced
physiological response [41,60] due to a gaseous inducer which acts
rapidly and disappears. Further support to this interpretation is
that typical side effects such as chills, fever, fatigue and
nausea never occur after autohaemotherapy and actually the
patients often report a sense of well-being and euphoria that may
be due to improved oxygenation or/and release of hormonal factors
not yet identified. As far as the number of treatments to be
carried out is concerned, one treatment only can be hardly
effective as the number of ozonated BMC present in 300 mi of blood
is probably less than 0.1% of the total BMC mass [61]. If this
calculation is correct, only the prolonged repetition of
autohaemotherapy (two treatments weekly for several months) can
allow a progressively amplified activation of the immune system
via numerous pathways such as activation of either major
histocompatibility complex-restricted cytolysis, or unspecific
killing and removal of viral-infected and metastatic cells. Table
I reports a list of chronic viral diseases or pathological
situations accompanied by either immune deficiency or immune
dysregulation, where ozonated autohaernotherapy has and could play
a beneficial role.

Autohaemotherapy may prove to be very useful as an adjuvant
treatment particularly if either chemotherapy or/and radiotherapy
have been effective in reducing the tumor mass. Several
immunotherapeutic approaches such as: a) therapy with monospecific
or bispecific antibodies [62]; b) adoptive immunotherapy with more
or less genetically engineered cells [63]; c) exogenous
administration of either an array of immunostimulants [64] or/and
d) of recombinant cytokines such as IFNs, IL-1, 2, 3, 6 and 12,
TNFx and leukopoietins are being actively pursued. Approaches a to
c are in a more or less advanced experimental phase while therapy
with cytokines has dominated the scene in the last decade.
Unfortunately pharmacological administration of cytokines, that
physiologically are not present in the circulation, causes a
substantial increase of their plasma levels which correlate well
with fairly severe toxicity [65]. Clinical results in solid
neoplasms have been far below expectations and the cost/benefit
ratio is very high [66].

The variety of biological effects depends upon the heterogeneity
of blood cells.

While immunomodulatory effects are due to BMC activation, the
beneficial effect of autohaemotherapy in chronic limb ischemia, in
cardiac, ophthalmologic and cerebral vasculopathies [3,4,6,8,22]
in infections and in burns [1,3,4,6,8,15,19,20] can be explained
by improved transport of oxygen due either to increased oxygen
availability, or oxygen delivery to hypoxic tissues due to an
increase of 2-3 diphosphoglycerate in erythrocytes. The rapid
healing of torpid ulcers in ischemic disorders is becoming
particularly interesting since the demonstration that transforming
growth factors (TGF) B1 accelerates the healing process [67-69]
and that levels of activated TGF B1 increase substantially after
ozonation of blood, probably due to partial degranulation of
platelets [40]. There is no doubt that improved oxygenation
enhances cell metabolism and proliferation and that the local
application of ozone inhibits bacterial infections but, until now,
the crucial role of proteins such as TGF B1, vascular endothelial
growth factor (VEGF) for enhancing neovascularization [70] and
healing has not been taken into account. Thus it appears that
clinical benefits possibly derive from the activation of
erythrocyte function, the release of either TGF B1, other growth
factors and by improved leukocytic functions in terms of removal
of necrotic tissue and bactericidal activities.

Final remarks

It has been pointed out that ozonated autohaemotherapy performed
with an optimized procedure represents a powerful therapeutic
approach. Its main advantages are the lack of toxicity, often a
feeling of well-being and the equilibrated, although slow,
stimulation of cytokine production accompanied by improved
oxygenation and metabolism. Both in the treatment of neoplasia,
particularly after chemiotherapy, and of chronic viral diseases
the frequent report of well-being after treatment is relevant
because the quality of life of these patients is generally poor.
We are planning to investigate the reason of euphoria and we
believe that it may be due to an immune-neuroendocrine response
elicited by the ozonated blood. On clinical ground there is also
the need to carry out extensive and well-controlled clinical
trials in several diseases including HIV infection [50]. The
treatment is simple to execute, safe, far less expensive that
comparable procedures and could be carried out easily also in
Third-World Countries where it could be applied also to several
parasitic diseases.

Aknowledgements

I am very grateful to Miss Patrizia Marrocchesi for her skill and
patience in preparing the manuscript. This work has been partially
supported by MURST (40%) and by the National Research Council
(CNR), Rome, Targeted Project "Applicazioni cliniche della ricerca
oncologica" (ACRO).

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