by Gérard V. Sunnen,
M.D.
Abstract
Hepatitis C (HCV) is a global
disease with an expanding incidence and prevalence base. Of massive public
health importance, hepatitis C presents supremely challenging problems in view
of its adaptability and its pathogenic capacity. The unique strategies that HCV
utilizes to parasitize its host make it a formidable enemy and therapeutic
interventions need considerable honing to counter its progress. Ozone, because
of its special biological properties, has theoretical and practical attributes
to make it a potent HCV inactivator.
History of the virus A form of hepatitis became
recognized in the 1970's that resembled hepatitis B, serum hepatitis, and to a lesser
extent hepatitis A, infectious hepatitis. It had, however, novel features,
amongst them, a distinctive serological profile. In 1989, the genome of
hepatitis C (HCV) was deciphered.
It is possible, by means of extrapolation from the genetic evolution of a
virus, to approximate its age. Sequence genetic analysis points to the
diversification of different HCV genotypes 200 to 400 years ago. Ancestors to
these genotypes probably date back 100,000 or so years when viruses co-evolved
with modern humans. Further analysis of genetic viral trees and Old and
Today, in the context of human population growth, migration, and global travel,
the hepatitis C virus has expanded its territories, geographically, and
demographically. There is every indication that the evolution of this virus, in
all its forms, is currently manifesting an accelerated phase.
Virion architecture and molecular biology The HCV
particle is composed of a nucleocapsid containing its
genome, an RNA single strand composed of approximately 9600 nucleotides, and
its protein coating. The nucleocapsid is surrounded
by an envelope which allows attachment and penetration into host cells. The
genome encodes structural proteins designated as core (C), envelope 1 (E1),
envelope 2 (E2), and P7 (unknown function), providing for virion
architecture, and nonstructural proteins, mainly enzymes essential to the virion's life cycle, designated as NS2, NS3, NS4A, NS4B,
NS5A, and NS5B. Proteases release structural and nonstructural proteins. Helicases unwind viral nucleic acid. Polymerases replicate
RNA. Within this genome is located a hypervariable
region implying an area of intensive genetic fluidity and mutational potential.
HCV displays great genotypic flexibility which makes for sophisticated
evasiveness to host defenses.
The nucleocapsid is surrounded by an envelope, a
lipid bilayer associated with a union of
carbohydrates and proteins, glycoproteins. Up to 60%
of the lipid component of the envelope is phospholipid
and the remainder is mostly cholesterol. It possesses projections called peplomers which facilitate attachment to host cells. One
protein on peplomers of the HCV particle which is thought
to be instrumental in the attachment process is designated CD-81.
The sequence of nucleotides within the HCV genome shows significant variations.
Strains obtained from different parts of the world, for example, may differ
substantially in their structural and nonstructural protein compositions. This
has lead to a system of classification of the HCV family into 6 genotypes (1 to
6), and approximately 100 subtypes (designated a, b, c, ect.). Genotypes vary from each other by a factor of
30% over the entire genome. Subtypes vary by about 20%. Genotypes 1 to 3 have
global distribution, while genotype 4 and 5 are found mainly in
Within any one afflicted individual, HCV particles do not show a homogeneous
population. Instead, they function as a pool of genetically variant strains
known as quasispecies. This is due to the high
replication error inherent in the function of the polymerase enzymes. Herein lies one of the important armaments of HCV. Continuously
generated genetic diversity gives it great advantage in negotiating and
conquering immune defense and therapeutic strategies. Furthermore, the
antigenic differences between genotypes may have implications regarding the
proper evaluation and the therapeutic regimen of patients.
Viral life cycle A freely circulating virion enters a host cell by binding to a cell surface
receptor. In the case of HCV the host cell is a hepatocyte.
However, bone marrow, kidney cells, macrophages, lymphocytes, and granulocytes
may also be trespassed.
Once cell entry is achieved, the virion sheds its
envelope to commence its replication. It binds to cellular ribosomes
and released viral polymerase begins the RNA replication cycle. Newly formed nucleocapsids continue their assembly with the acquisition
of new envelopes by means of budding through membranes of the cell's endoplamic reticulum. Newly formed virions
may number in the range of 10 billion daily. The average life span of virions is in the order of a few hours.
Virions are then released into the general blood and
lymphatic circulation, ready to infect new cells, re-infect already diseased
cells, or a new host, mainly through bodily fluid transmission pathways. HCV
RNA, as measured by polymerase chain reaction (PCR) may show 10 million or more
virions per ml. As little as 0.0001 ml of blood may
be sufficient to impart infection. The evolution of hepatitis C is
characterized by phases of accentuated viremia
punctuated by periods of relative quiescence. The presence and timely detection
of these viremic waves may offer novel therapeutic
considerations.
Clinical and laboratory manifestations Hepatitis, from anyone of the several
viruses capable of inducing liver inflammation, produce a spectrum of clinical
and laboratory manifestations. Hepatitis C distinguishes itself by the low
incidence of acute phases and by the high incidence of progression to chronicity. Acute hepatitis C progresses from exposure, to
incubation, to pre-icteric, icteric,
and convalescent phases. With an incubation period of about 6 weeks, the first
and sometimes only symptoms include weakness, fatigue, indolence, headache,
nausea, poor appetite, and vague abdominal pain. The pre-icteric
period extends from the onset of symptoms to the appearance of jaundice,
ranging usually from
Chronic hepatitis C is characterized by the presence of HCV RNA and the
elevation of liver enzymes for 6 months or longer. Patients may be
asymptomatic, or at times suffer an acute exacerbation with a return of
symptoms. Approximately 75% of acutely ill patients continue into a chronic
phase evidenced by parameters of viral presence.
Hepatitis C can only be distinguished from other viral hepatic conditions by
serological and virological determinations. Liver
enzymes characteristically affected by HCV infection include serum alanine transfesferase (ALT), aspartate aminotransferase (AST),
gamma- glutamyl transpeptidase
(GGTP), and alkaline phosphatase; in addition, there
may be abnormalities in bilirubin, serum albumin, prothrombin time, and platelet density.
Cirrhosis, a diffuse disruption of liver tissue architecture with regenerative
nodules surrounded by fibrosis, is an important sequel to hepatitis C. Within
20 years post HCV infection 20 to 25% of patients will develop cirrhosis.
Hepatic decompensation ensues with ascites as the salient marker.
Hepatocellular carcinoma, another notable outcome of
HCV infection is present in approximately 5% of patients post infection. The
presence of cirrhosis is central to its genesis. Although the mechanisms by
which cirrhosis ushers carcinoma are unknown, it is likely that chronic
inflammation and the sustained pressure of cellular regeneration play important
roles.
Up to 10% of patients appear to have fully conquered the disease. HCV
antibodies are undetectable, as is HCV RNA. Liver enzymes are fully normalized,
but liver biopsy may show lingering areas of stagnant inflammation and spotty
necrosis. It is thus possible for host immunocompetence
to vanquish HCV infection and therapeutic strategies aim to assist the host
immune system to achieve this goal.
Immunological response to the virus HCV particles are detected early in the
infection, usually 1 to 2 weeks following exposure. Antibodies to HCV core,
nonstructural, and envelope elements appear about 6 weeks after exposure. A
broad range of cytokines are mobilized. Cellular immunity is activated with
broad recruitment of neutrophils, natural killer
(NK), macrophages, and CD4 and CD8 T helper cells.
Current and experimental treatment strategies As of
this date the main treatment strategies for hepatitis C include interferon and ribavirin. Interferons are
natural cellular products which activate macrophages, neutrophils
and natural killer cells. There is controversy as to interferon's biological
effects, be they mostly immunoregulatory or directly
antiviral. Ribavirin is a guanosine
analog that represses messenger RNA formation thus inhibiting the replication
of many DNA and RNA viruses. It is, however, mutagenic to mammalian cells. Ribavirin and interferon have significant medical and
psychiatric side effects.
Treatment response is defined as undetectable viral load 6 months following
therapy. Contemporary detection methods of quantitative HCV RNA determinations
are capable of detecting approximately 1000 viral copies per serum ml.
Resistance to antiviral therapies is a particularly vexing problem in anti HCV
treatment. Novel and experimental antiviral compounds include inhibitors of
protease, polymerase and helicase.
Vaccine development needs to take into account HCV's
antigenic rainbow and its high mutability. High mutation
rates in this condition implies a dauntingly diverse and variable array
of viral antigenic components. It is estimated, for example, that HCV mutates
significantly in its own host approximately a thousand times a year. This
implies that within any one afflicted individual there exists an awesomely
large array of viral quasispecies, which in turn
creates commensurate difficulties in the creation of effective vaccines.
Ozone: Physical and physiological properties Ozone (O3) is a naturally
occurring configuration of three oxygen atoms. With a molecular weight of 48,
the ozone molecule contains a large excess of energy. It has a bond angle of
127° and resonates among several forms. At room temperature, ozone has a half
life of about one hour, reverting to oxygen. A powerful oxidant, ozone has
unique biological properties which are being investigated for applications in
various medical fields. Basic research on ozone's biological dynamics have
centered upon its effects on blood cellular elements (erythrocytes, leucocytes,
and platelets), and to its serum components (proteins, lipoproteins, lipids,
carbohydrates, electrolytes). Administrating increaing
dosages of ozone to whole blood shows that beyond a certain threshold there is
a rise in the rate of hemolysis. This threshold,
depending upon various parameters, begins to be reached at 40 to 60 micrograms
per milliliter, and becomes significant when higher levels are attained.
Precise ozone dosing capacity is therefore essential in clinical practice and
research.
Leucocytes show good resistance to ozone because they have enzymes which
protect them from oxidative stress. These enzymes include superoxide
dismutase, glutathione, and catalase.
Research has shown that platelets also maintain their integrity after ozone
administration. In ozone therapy, the doses applied to blood are gauged to
avoid disruption of its cellular elements. Serum components remain viable
during ozone therapy. Lipid and protein peroxides, produced in small amounts by
ozonation, have demonstrable antiviral properties.
Interestingly, ozone tends to stimulate leucocyte
function and cytokine production. Ozone increases the oxygen saturation (p02)
in erythrocytes and enhances their pliability so that capillary circulation is
facilitated.
Ozone: Antiviral properties Recently, there has surged
renewed interest in the potential of ozone for viral inactivation. It has long
been established that ozone neutralizes bacteria, viruses, and fungi in aqueous
media. This has prompted the creation of water purification processing plants
in many major municipalities worldwide.
Ozone's antiviral properties may also be applied to the treatment of biological
fluids, albeit in technologically and physiologically appropriate ways. Indeed,
it is noted that ozone, administered in such dosages designed to respect the
integrity of blood's cellular and constituent elements, is capable of
inactivating a spectrum of viral families.
Some viruses are much more susceptible to ozone's action than others. It has
been found that lipid-enveloped viruses are the most sensitive. This group
includes, amongst others, HCV, Herpes 1 and 2, Cytomegalus,
HIV1 and 2.
The envelopes of viruses provide for intricate cell attachment, penetration,
and cell exit strategies. Peplomers, finely tuned to adjust to changing receptors on a variety
of host cells, constantly elaborate new glycoproteins
under the direction of E1 and E2 portions of the HCV genome. Envelopes are
fragile. They can be disrupted by ozone and its by-products.
In HCV, viral load appears to be a major factor in the invasiveness and
virulence of the disease process. Preliminary research has shown that reduction
of viral load in Hepatitis C by means of ozone therapy can significantly
normalize hepatic enzymes and improve measures of global patient health.
Volunteers administered ozone therapy according to the method outlined below
achieved a viral load reduction in the order of 5 log, or 99.9%, along with a
normalization of liver enzyme levels.
Ozone: Clinical methodology Ozone may be utilized for the therapy of a spectrum
of clinical conditions. Routes of administration are varied and include
external and internal (blood interfacing) methods. In the technique of ozone
major autohemotherapy for hepatitis C, an aliquot of
blood is withdrawn from a virally-afflicted patient, anticoagulated,
interfaced with an ozone/oxygen mixture, then
re-infused. This process is repeated serially until viral load reduction is
documented.
The aliquots of blood range from 50 ml. to 300 ml. Ozone dosages and treatment
frequency vary according to treatment protocols. The reason aliquots of blood
are treated and not, as one would propose, the entire blood volume,
is that in the latter case the total ozone dosage administered would exceed
toxic limits.
The average adult has 4 to 6 liters of blood, accounting for about 7% of body
weight. How can the viral load reduction observed via ozone therapy be
explained in the face of a technique that treats relatively small amount of
blood, albeit serially?
Ozone: Possible mechanisms of anti-viral action
The viral culling effects of
ozone in infected blood may recruit the following mechanisms:
Denaturation of virions
through direct contact with ozone. Ozone, via this mechanism, disrupts viral
envelope proteins, lipoproteins, lipids, and glycoproteins.
The presence of numerous double bonds in these unsaturated molecules makes them
vulnerable to the oxidizing effects of ozone which readily donates its oxygen
atom and accepts electrons in these redox reactions.
Double bonds are thus reconfigured, molecular architecture is disrupted and
widespread breakage of the envelope ensues. Deprived of an envelope, virions cannot sustain nor replicate themselves.
Ozone proper, and the peroxide compounds it creates, may directly alter
structures on the viral envelope which are necessary for attachment to host
cells. Peplomers, the viral glycoproteins
protuberances which connect to host cell receptors are likely sites of ozone
action. Alteration in peplomer integrity impairs
attachment to host cellular membranes foiling viral attachment and penetration.
Introduction of ozone into the serum portion of whole blood induces the
formation of lipid and protein peroxides. While these peroxides are not toxic
to the host in quantities produced by ozone therapy, they nevertheless possess
oxidizing properties of their own which persist in the bloodstream for several
hours. Peroxides created by ozone administration show long-term antiviral
effects which serve to further reduce viral load. This factor may explain in
part the reason for the fact that ozonated blood in
the amount processed in usual treatment protocols is able to reduce viral load
values in the total blood volume.
Immunological effects of ozone have been documented. Cytokines are proteins
manufactured by several different types of cells which regulate the functions
of other cells. Mostly released by leucocytes, they are important in mobilizing
the immune response. It has been found that ozone induces the release of
cytokines which in turn activate a spectrum of immune cells. This is likely to
constitute a significant avenue for the reduction of circulating virions.
Ozone action on viral particles in infected blood yield several possible
outcomes. One outcome is the modification of virions
so that they remain structurally grossly intact yet sufficiently dysfunctional
as to be nonpathogenic. This attenuation of viral particle functionality through
slight modifications of the viral envelope, and possibly the viral genome
itself, modifies pathogenicity and allows the host to
increase the sophistication of its immune response. The creation of
dysfunctional viruses by ozone offers unique therapeutic possibilities. In view
of the fact that so many mutational variants exist in any one afflicted
individual, the creation of an antigenic spectrum of crippled virions could provide for a unique host-specific
stimulation of the immune system, thus designing what may be called a
host-specific autovaccine.
Summary
Viruses are far from being
static entities. As quintessential intracellular parasites they have developed,
through millions of years of cohabitation with their hosts, astoundingly
sophisticated structures, survival, and propagation mechanisms. They have
adapted, modified their biological strategies, and evolved impressive genetic
diversity and mutational capacity to cope with the changing ecology of
planetary life.
HCV has an extremely high rate of mutation and within any one individual there
may exist millions of antigenic quasispecies. The
disease process is marked by periods of viral quiescence alternating with viremic waves whereby billions of virions
are poured into the blood and lymphatic reservoirs. Their astounding numbers
stress the immune system relentlessly and produce an inexorable compromise in
all parameters of its functioning.
Viral load reduction by means of ozone blood treatment alleviates immune system
fatigue. Ozone-mediated viral culling may be achieved by anyone of a number of
possible mechanisms. Direct virion denaturation, peplomer
alteration, lipid and protein peroxide formation, cytokine induction, host pan-humoral activation, and host-specific autovaccine
creation are suggested mechanisms. Due to the excess energy contained within
the ozone molecule, it is theoretically likely that ozone, unlike antiviral
options available today, will show effectiveness across the entire genotype and
subtype spectrum.
Ozone embodies unique physico-chemical and biological
properties which suggest an important role in the therapy of hepatitis C,
either as a monotherapy, or as an adjunct to standard
treatment regimens.
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