Hepatitis C and Ozone Therapy
(Ed. Note: This is a technical article. If you are not a scientist you can
skip to the parts on ozone and the summary at the end to get the gist. The
point is, ozone is effective against the Hep C
virus)
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 New
World primates take the primordial forms of these viruses to primate
speciation periods some 35 million years ago.
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 Africa, and 6 is distributed in Asia. Importantly,
genotype and subtype differences have shown varying susceptibility to
antiviral therapy.
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 2 to 12 days. The icteric phase
corresponds to the declaration of jaundice and darkened urine. The
convalescent phase is marked by the gradual disappearance of symptoms.
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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