 Are worry-free transfusions just a whiff of ozone away?

 Albert C. Baggs, BSc.

 From Medical Science News
 Can Med Assoc J 1993: 148(7),pp. 1155-1160
 April 1993


 Scientists in Canada and the United States are investigating the 
 use of ozone to destroy the human immunodeficiency virus (HIV), 
 the hepatitis and herpes viruses and other infectious agents in 
 the blood used for transfusion.  The studies were endorsed by 
 medical circles of the North Atlantic Treaty Organization (NATO) 
 because of a concern that viral pandemics have compromised the 
 ability of world blood banks to meet urgent and heavy military 
 demands.

 NATO's fears are justified.  The World Health Organization 
 recently estimated that more than 200 million people are long-
 term carriers of hepatitis B virus and that about 13 million 
 people are not infected with known HIV strains (1,2).  The 
 spread of HIV (and thus the threat to blood banks and military 
 organizations) is being impelled by socioeconomic factors and 
 the worldwide recession.  In Southeast Asia, for example, a 
 substantial increase in the number of reported cases of acquired 
 immunodeficiency syndrome (AIDS) has been directly attributed to 
 the sale of prepubescent Burmese girls to prostitution lords and 
 madams in Thailand.  The children, who are obliged to service up 
 to 15 customers daily (British Broadcasting Corporation World 
 Service News, November 1992), are sold by their parents to pay 
 for food and to finance drug addiction.  An epidemic of AIDS in 
 the Thai military population seems to be associated with 
 prostitution in the country.  As HIV spreads among heterosexual 
 men and women (3) a concomitant increase in the world-wide 
 rejection rate of donors and blood, which now reaches 20% to 
 30%, must be expected (1).

 Laboratories in Canada, the United States and other countries 
 have preliminary evidence that sterilization with ozone is 
 feasible.  In a brief to the NATO Blood Committee (1) the 
 surgeon general of the Canadian Armed Forces reported on 
 Canadian findings that within the sensitivity of the screening 
 methods used, a 3 minute ozonation of serum spiked with one 
 million HIV-1 particles per millilitre would achieve virtually 
 100% viral inactivation (loss of infectivity).  It was also 
 found that the procedure would destroy several other lipid-
 encapsulated viruses, including simian immunodeficiency virus 
 and various strains of interest to veterinarians (Table 1).

 The concept of gas sterilization originated in Germany in the 
 1050s: more than 10,000 units of human blood containing 
 hepatitis virus were rendered safe through treatment with a 
 mixture of ozone and oxygen and then used for transfusion.

 Canadian interest in the technique evolved partly from the early 
 German successes but mainly from in-vitro studies with ozone by 
 Captain Michael E. Shannon, a scientist in the Department of 
 National Defence, in collaboration with virologist Dr. Michael 
 O'Shaughnessy, at the Laboratory and Research Services Bureau, 
 Laboratory Centre for Disease Control, Ottawa.  Their 
 experiments led to a pilot study using ozone-treated blood in a 
 volunteer group of 24 patients with AIDS at the Ottawa General 
 Hospital (approved by the Health Protection Branch and the 
 hospital's ethics committee)(4).

 The Canadian experiments with HIV and other viruses used gas-
 exchange technology from Mueller Medical International Inc. 
 (Oakville, Ont.), with help from Medizone International Inc., 
 New York.  Generated from oxygen by high-voltage or ultraviolet 
 light, ozone can be delivered through a gas-exchange cartridge 
 or other system of media diffusion, at controlled 
 concentrations, to infected culture media, blood and Factor VIII 
 or other blood products.  If gas sterilization can be shown to 
 met Red Cross standards the technique could augment exiting 
 methods--micropore filtration, centrifugation and washing(5).

 Experimental results

 Two teams of US virologists have used comparable gas-diffusion 
 techniques to confirm the Canadian findings, with the following 
 results.

 * HIV-1 at concentrations of 10(13th) virions/l, was inactivated 
   in cell-culture media, plasma and purified Factor VIII 
   preparations through ozonation (1200 ppm of ozone for 2 hours) 
   by means of a hollow-fibre gas delivery system, with a minimal 
   loss (10%) of the biologic activity of clotting factors (6).

 * Preliminary assessment of erythrocyte function and life span 
   did not reveal any impairment (Michael E. Shannon, Department 
   of National Defence: personal communication, 1992).

 * The presence of cells in the ozonized media did not prevent 
   the inactivation of extracellular virions (7).

 * Ozonized biologic fluids, in which there is a presumed 
   abundance of superoxide, singlet oxygen, hydroxyl and peroxyl 
   radicals and other highly reactive species, had both 
   extracellular and intracellular virucidal properties (7).

 * Because of a defective glutathione peroxidase system, HIV-
   infected lymphocytes readily lysed after ozonation and 
   released presumably noninfective viral particles into the 
   extracellular medium (Michael E. Shannon: personal 
   communication, 1992) (8).

 * Other infected cells maintained in pre-ozonized media 
   exhibited a 40% reduction in the expression of HIV-1 core 
   antigen (p24 protein) (7).


 Mechanisms of Ozone action

 Ozone has long been used to destroy bacteria in municipal water 
 supplies.  Its destructiveness is partly attributed to the 
 oxidation of unsaturated bonds in the phospholipid and 
 lipoprotein architecture of bacteria, viruses and cells.  The 
 oxidation generates hydroperoxides, which are transformed to 
 peroxyl and hydroxyl radicals and to other reactive species, 
 including aldehydes.  Peroxyl radicals attack proteins, and 
 hydroxyl radicals induce disruptive structural changes in cell 
 membranes (9,10).  Virus-infected cells are considered less able 
 to withstand such oxidative influences than uninfected cells, 
 which have intact antioxidant mechanisms.  Thus, at relatively 
 low ozone concentrations required to sterilize whole blood, even 
 intracellular infective agents can likely be destroyed 
 selectively without uninfected tissue being substantially 
 altered.

 Several other specific mechanisms have been postulated for the 
 destruction of retroviruses, including HIV, by reactive oxygen 
 intermediates: inactivation of viral reverse transcriptase, 
 which would otherwise transcribe the viral RNA genome into host 
 DNA; oxidative inactivation of the essential cation cofactor of 
 the transcriptase; and interference with the ability of the HIV 
 envelope glycoprotein gp 120 to bind to the lymphocyte receptor 
 CD4 (7).  The lethal mutation of the genes (10,11) may 
 contribute to inactivation, especially of extracellular viruses, 
 whose genes are unprotected by the cellular endonucleases and 
 ligases that maintain DNA integrity.


 Primate studies

 As compelling as this research may be, the efficacy and safety 
 of blood sterilization with ozone remains to be proven.  To this 
 end, critical studies that make use of an isolate of a now fully 
 characterized simian immunodeficiency virus have been initiated.  
 These have involved extensive in-vitro research to clarify the 
 optimal dose of ozone required for viral destruction in 
 transfusion products.  Before extensive human trials can be 
 considered it will be necessary to perform dose-response and 
 toxicologic studies in animals to confirm the European evidence 
 that systemic ozone therapy is safe (Michael E. Shannon: 
 personal communication, 1992).  The experiments -- a 
 collaborative effort between scientists at the Department of 
 National Health and Welfare, the Department of National Defence, 
 the Canadian Animal Disease Research Institute and Cornell 
 University, Ithaca, NY -- will address the fundamental question 
 Will whole blood or specific blood fractions that have been 
 experimentally contaminated with a highly virulent strain of 
 simian immunodeficiency virus produce immunodeficiency disease 
 in primates if the blood is treated with ozone before 
 reinoculation?


 Discussion

 Military physicians ruefully accept that during war and 
 catastrophe-relief missions, especially in remote regions, 
 urgently needed blood and blood products must be obtained from 
 all available sources and that screening for infectious agents 
 will not always be possible.

 Although serologic tests for antibodies to HIV and viral 
 proteins and nucleic acid have increased the safety of blood 
 transfusions they do have limitations (12-14), particularly in 
 the detection of unknown immunodeficiency factors (15).  
 Clearly, there is a concern that transfusions and transplants 
 are not "HIV-proof (16,17).  In 1991 AIDS was diagnosed in 747 
 transfusion recipients and 347 patients with hemophilia in the 
 United States (18), there were an estimated 7200 such infections 
 in 1984, before screening for HIV became routine (19).  In 
 Canada, 279 of the 6560 patients with AIDS reported since 1979 
 were apparently infected through blood or blood products 
 (Federal Centre for AIDS, Department of National Health and 
 Welfare, Ottawa: personal communication, 1992).

 Given the concerns of the military, the medical profession and 
 the public there seems to be some urgency to the search for a 
 rapid, reliable, portable and cost-effective method of blood 
 decontamination that can destroy all known pathogens while 
 preserving the functions of erythrocytes, platelets, coagulation 
 factors and immune globulins.

 Various decontamination procedures and agents -- gamma 
 irradiation and the use of direct heat (60 degrees Celsius) and 
 steam, solvents and oxidizers (ether, alcohol, aldehyde and 
 surfactants), antibodies and enzymes -- have been shown to be 
 partially effective, but they have major disadvantages: 
 unsatisfactory sterilization, damage to erythrocytes (especially 
 from aldehydes) and the risk of mutagenic and potentially 
 carcinogenic contaminants being created.

 Published and unpublished laboratory evidence attesting to the 
 effectiveness of ozone against intracellular and extracellular 
 HIV in whole blood, plasma and Factor VIII products indicates 
 that ozonation, augmented by the filtration of leukocytes, may 
 meet at least some of the essential requirements for 
 decontamination.  However, the litmus tests will be studies of 
 infectivity risk in primates and the screening of HIV-spiked 
 blood, after ozonation, by means of the sensitive minute 
 quantities of viral nucleic acid to detectable levels (21-23); 
 the results of these investigations will be appraised with 
 interest by blood bank facilities in both Canada and the United 
 States.

 Caution is required in the treatment of human disease with ozone 
 because of the potential toxic effects of reactive oxygen 
 intermediates, which are known to mediate damage to tissue.  
 Indeed, several mechanisms of carcinogenesis associated with 
 oxygen radicals have been defined (10,11,24), even though ozone 
 has been found to inhibit cancer cells in vitro (25).

 Ozonized blood and topical preparations have been extensively 
 used for pantherapeutic purposes in Europe.  Indeed, in a review 
 of 300,000 patients (who had been given more than 5 million 
 ozone treatments) Jacob (26) concluded that when strict 
 protocols were followed the rate of adverse effects was 0.6% and 
 that there was no evidence of carcinogenicity.  Similarly, the 
 recent Canadian clinical trial of ozone in the treatment of AIDS 
 patients (4) (by means of phlebotomy, ozonation of the blood 
 sample and intramuscular reinoculation) showed no evidence of 
 serious side effects.

 Although vascular endothelial cells are susceptible to attack by 
 oxygen radicals several biochemical species provide some 
 protection.  Vitamin C, uric acid, N-acetyl-L-cysteine and 
 glutathione are known to scavenge oxygen radicals in the aqueous 
 region of cell membranes, but these water-soluble antioxidants 
 are ineffective against peroxyl radicals in the lipid region, 
 where vitamin E exhibits rather limited antioxidant efficiency 
 (27).  Additional defence against superoxide is provided by 
 dismutase enzymes and against hydrogen peroxide by catalases and 
 peroxidases.

 In elderly people, those with HIV infection and possibly those 
 with certain other viral infections these antioxidant and 
 enzymatic mechanism may be less efficient that in young, healthy 
 people, so that peroxidative damage to endothelial cells becomes 
 more probable.  Extracellular oxidant injury seems to induce 
 anticlotting mechanisms (28).  Furthermore, the treatment of 
 occlusive vascular disease with ozonized blood appears to 
 stimulate the enzymatic conversion of L-arginine to citrulline, 
 nitrite and nitrate by phagocytic cells.  Small amounts of 
 nitric oxide, a platelet disaggregator and vasodilator, are 
 produced in this pathway.  The production of prostacyclin (a 
 vasodilator) is also induced by ozone (29,30).

 Thus, the various actions of endogenous and ozone-generated 
 oxygen radicals have mixed clinical implications: the prevention 
 of thrombus formation and tissue infarction is clearly 
 desirable; however, damage to the vascular lumen, obstruction of 
 reparative hemostasis and extravasation are potential 
 complications of therapy when ozone concentrations exceed 50 
 ug/ml (which may be necessary to destroy retroviruses 
 effectively).

 The adverse effects of endogenous reactive oxygen intermediates 
 probably vary with age, diet and physiologic state (31).  
 Oxygen-radical damage has been associated with the aging process 
 and with age-related disease of the central nervous and 
 cardiovascular systems, including hypertension, damage to blood 
 vessels in the brain and cerebral ischemia (12,33).  Radicals 
 may also oxidize catecholamines and mediate the actions of 
 neurotoxins (34).  These pathologic processes may be associated 
 with dementia, demyclination and other degenerative neurologic 
 disorders, particularly those attributed to HIV (and possibly 
 other infectious agents such as papovavirus and cytomegalovirus 
 (35)), which evokes an immune-driven accumulation of oxidants.  
 If these adverse reactions complicate future clinical trials it 
 may be possible to counteract them with the use of covalently 
 modified or lipid-encapsulated antioxidants and protective 
 enzymes with prolonged circulatory half-lives (36,37).

 Oxygen radicals present something of a paradox in biology: the 
 accumulation of endogenous or xenogenous species is exceedingly 
 harmful to tissues; yet from a defensive perspective radicals 
 may be highly functional.  In its response to malignant cells 
 and infectious agents the repertoire of the immune system 
 usually includes the generation of reactive oxygen 
 intermediates.  Thus, macrophages reduce oxygen to superoxide as 
 they attempt to eliminated tumours (38), and various phagocytic 
 cells produce superoxide, hydrogen peroxide and hydroxyl when 
 killing bacteria (39) and in responding to irritant chemicals or 
 inorganic particles (40).  Similarly, the generations of 
 hydroxyl by natural killer cells seems to be critical to their 
 cytotoxic capability (41).  Oxygen radicals are also thought to 
 function in the early events leading to the activation and 
 proliferation of T lymphocytes (42).

 It appears that HIV infection intensifies these normal 
 immunologic oxidative mechanisms to a destructive scale.  A 
 state of chronic oxidative stress can be induced in vitro in 
 lymphocyte (T4) subpopulations by inflammatory cytokines 
 (interleukins 1 and 6 and tumour necrosis factor-x), which 
 stimulates mass production of reactive oxygen intermediates in 
 infected cells.  Glutathione peroxidase (a cellular antioxidant) 
 is rapidly depleted by these accumulating intermediates.  The 
 virus-laden cells thereby lose their ability to detoxify 
 amassing radicals and xenobiotics (8).  This oxidative stress 
 may also occur in macrophages and other infected cell lines 
 (e.g. in the skin, intestines, central nervous system and 
 spleen).

 This evidence is critical to the strategy behind the use of 
 ozone for decontaminating blood.  Since cells infected with HIV-
 1 are less able than healthy cells to cope with oxidative stress 
 they quickly lyse in the presence of ozone, and their viral 
 contents are exposed to oxidative disinfection.  Similarly, 
 extracellular retrovirus may be fully deactivated by direct 
 oxidative assault on the viral capsule and RNA.

 There are other complicating factors: respiratory syncytial 
 virus infection is partly mediated by the superoxide produced by 
 neutrophils (43); reactive oxygen intermediates cause extensive 
 damage to pulmonary tissue as well as bronchial 
 hyperresponsiveness (including bronchoconstriction (44)); and 
 evidence of oxygen radical involvement can often be found in 
 plasma as lipid peroxidation products (45).  Such evidence 
 supports the idea that superabundant immune-generated oxygen 
 radicals may be culprits or accomplices in many of the 
 cytopathologic features observed in AIDS, such as the fusion of 
 infected with uninfected cells to form multinucleated giants 
 that produce HIV particles in abundance (46), impaired T-
 lymphocyte proliferation, altered differentiation of T and B 
 cells and impaired activity of natural killer lines (8).

 Perhaps the firmest indictment of oxygen radicals and hydrogen 
 peroxide as mediators of HIV infection has come recently from 
 the Ludwig-Maximilians University, in Munich, where molecular 
 biologists have suggested that the expression and replication of 
 nine or more HIV-1 genes in human T lymphocytes results from the 
 oxidation of cytoplasmic initiators.  These "activated" proteins 
 bind to initiation sites on the viral genome (8,47,48).

 The possibility that oxygen radicals and peroxides generated by 
 ozone might contribute to oxidative stress and thereby 
 potentiate HIV transcription and translation in dormant (i.e., 
 provirus +) T cells is a clinical concern.  Inflammatory 
 cytokine responses were not evaluated in the Canadian clinical 
 studies, but the individual p24 viral antigen profiles of 
 patients were unaffected by low does of ozone.  This suggests 
 that the treatment did not increase (or decrease) HIV 
 transcription (Michael E. Shannon: personal communication, 
 1992)(4).

 A further concern contemplated by Canadian scientists is that 
 ultraviolet light, which is used to irradiate ozonized blood in 
 one technology now under evaluation, has been found to activate 
 both laboratory-hybridized and latent HIV genes in cultured 
 human cells (49).  However, an absorption spectrum for solar 
 radiation (50) indicates that at atmospheric (normal ozone 
 layer) concentrations oxygen-ozone may completely absorb 
 ultraviolet light at 253.7 nm (the energy fluence being 
 considered for blood decontamination).  The simultaneous 
 perfusion of blood with ozone may therefore prevent the 
 decondensation of DNA and the activation of integrated HIV genes 
 by ultraviolet energy (49).  Treating blood with a combination 
 of ultraviolet light and ozone does not appear to initiate 
 spontaneous cell division among lymphocytes and monocytes (Dr. 
 Anthony Bolton, consultant to Intermune Life Sciences Inc., 
 Etobicoke, Ontario: personal communication, 1992); this suggests 
 that the potently virucidal combination of ultraviolet energy 
 and ozone in blood decontamination devices is unlikely to switch 
 on viral genes.  (The HIV-activation issue has also been 
 enlivened by findings that other viruses [vaccinia virus and 
 herpesvirus-6](51,52) and tumour necrosis factor-x (53) can 
 stimulate HIV expression.)

 The systemic use of ozone in the treatment of AIDS could not 
 only reduce the virus load but also possibly revitalize the 
 immune system.  Although ozone therapy may seem rather novel, 
 the concept of oxygen radicals being used to destroy viruses in 
 biologic fluids and in cells is not entirely foreign to 
 pharmacology: it is believed, for example, that some 
 antimalarial drugs eliminate Plasmodium by inducing 
 intraerythrocytic oxidative stress (54).  With regard to the 
 effect of ozone on immune responses to HIV, several German and 
 Italian studies have suggested that ozone enhances the 
 production of interleukin-2 (the T-lymphocyte population growth 
 factor) and of at least one antiviral immune peptide, 
 interferon-y (55).  There is clinical evidence that a 
 (recombinant) sister peptide, interferon-x, can inhibit the 
 expression by infected cells of p24 viral core protein (56).  
 Interferon-stimulated macrophages may generate less superoxide 
 and hydrogen peroxide in response to HIV infection, and their 
 suppressive effects on T-cell proliferation and lymphokine 
 production may be restrained (57).


 Conclusion

 The potency of biologic oxidizing agents is well known and may 
 be easily and vividly demonstrated by the classic microbiologic 
 test in which a few drops of 3% hydrogen peroxide solution are 
 added to an agar culture of Staphylococcus aureus (catalase-
 positive).

 Findings at laboratories in North America and Europe have 
 demonstrated that ozone has remarkable potency against disease 
 factors in blood products.  However, the importance of animal 
 studies in evaluating the efficacy and safety of ozone in blood 
 decontamination and perhaps, eventually, in treating human 
 immunodeficiency is clear.  Blood approved for transfusion on 
 the basis that it has undergone ozone sterilization, alone or in 
 combination with another method, should ideally carry a risk of 
 infection at least as low as that of the blood that passes the 
 most rigorous screening methods now in use, and it should not 
 cause viral or other disease or immune complications.  Thus, to 
 determine toxicologic indices is a necessary step toward finding 
 the optimum therapeutic range.  Concentrations of ozone 
 exceeding 100 ug/ml, which are noxious to cells in vitro (55), 
 may be required to overcome the effects of circulating 
 antioxidants (27) and HIV-shielding blood proteins (6).  
 Preliminary findings suggest, however, that less than a fifth of 
 this concentration may be effective (Michael E. Shannon: 
 personal communication, 1992).


 Table 1: Pathogens considered to be susceptible to ozonation in 
          vitro.

 Lipid-encapsulated viruses
    Herpes viridae (simplex, varicella-zoster,
      cytomegalovirus, Epstein-Barr virus)
    Paramyxoviridae (mumps, measles)
    Orthomyxoviridae (Influenza)
    Rhabdoviridae (rabies)
    Retroviridae (e.g. human immunodeficiency virus,
      simian immunodeficiency virus, equine infectious
      anemia virus)
 Hepatitis viruses
 Polioviridae
 Echovirus
 Coxsackievirus
 Bacteria
    Coliform bacteria
    Staphylococcus aureus
    Aeromonas hydrophila
 Fungi
    Candida utilis



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