COVID-19: POTENTIAL FOR EXTRACORPOREAL BLOOD IRRADIATION WITH UV LIGHT Текст научной статьи по специальности «Фундаментальная медицина»

COVID-19: POTENTIAL FOR EXTRACORPOREAL BLOOD IRRADIATION WITH UV LIGHT

Mikhail J. Artamonov1; Thomas J. Lewis2; David Brownstein3; Anastasia Hatkevich4; Evgeniy L. Sokov5; Inessa A. Minenko6

1MJA Research and Development, East Stroudsburg, PA; GoMD, San Diego, CA

2

Health Revival Partners, Talbott, TN; GoMD, San Diego, CA Center for Holistic Medicine, West Bloomfield Township,

4Northwest Association of Physicians and Medical Organizations Using Medical Gases 5Association of Pain Medicine Specialists, President, Moscow, Russia 6First Sechenov Moscow State Medical University, Moscow, Russia

Abstract

SARS-CoV-2, the cause of COVID-19, is the newest member of the Coronaviridae family of viruses and is the third coronavirus crossing animal species barriers to infect human populations. Multiple studies have reported that multidrug-resistant pathogenic microorganisms are highly sensitive to UVC inactivation.

Here we report the history of Ultraviolet, its activity against infectious agents, and case studies which state that the use of UVC irradiation for the prevention and treatment of localized infections is still in the very early stages of development. Most of the studies are confined to in vitro and ex vivo levels, while in vivo animal studies and clinical studies are much rarer. However, the case study on 195 psoriasis patients treated with UVB did not provide evidence for increased skin cancer risk with up to 9 years of follow-up. An analysis of 3867 patients receiving UVB over an 18-year period, with a median number of 29 treatments and 352 patients receiving 100 or more treatments with more than 6 months of follow-up for each patient, showed no increase in skin cancers of any kind.

One advantage of using UVC over antibiotics is that UVC can eradicate microorganisms in a much faster manner, while antibiotics usually take several days to take effect, especially in burns and chronic wounds that frequently have impaired blood perfusion. UVC may also be much more cost effective than the commonly used antibiotics. Therefore, microorganisms are found to be more sensitive to UVC than mammalian cells. As a result, with appropriate doses, pathogenic microorganisms may be selectively inactivated by UVC with minimum nonspecific damage to mammalian cells.

Keywords: Ultraviolet (UV) Therapy; Extracorporeal Blood Treatment; Adaptive Immune System Augmentation; Vaccine Adjuvant; SARS-CoV-2; COVID-19 Treatment Support

Covid-19 is the newest member of the Coronaviridae family of viruses and is the third coronavirus crossing animal species barriers to infect human populations. The

previous two members of the family are the severe acute respiratory syndrome coronavirus (SARS-CoV), emerging in 2002, and the Middle East respiratory syndrome coronavirus (MERS-CoV), in 2012. All can cause severe, even fatal, sudden acute respiratory syndrome or severe cardiopulmonary distress. The SARS-CoV-2 may also be creating a systemic hypoxic environment by inhibiting the functioning of Heme.

Covid-19 attests to the high mutational capacities of Coronavirus family members. By extending their reservoir range to include other animal species (such as pangolins), by delaying the onset of symptoms while maintaining infectivity, and by further mastering human-to-human transmission and expanding vectoring routes to include droplet, oral-fecal, and body fluids modes (1), SARS viruses greatly expand their capacity to create epidemics and pandemics.

As with many viruses, coronaviruses have complex host invasion, replication, and transmission cycles. A crucial replication phase, known as the viremic phase, involves the explosive reproduction of viral particles and virions, exiting from infected and dying host cells, disgorging billions of viral progenies, daily, into many types of bodily fluids. Indeed, in these massive seedlings, all organs are suddenly virally overwhelmed and existentially challenged, leading to a cytokine storm and sepsis.

The present thesis proposes a low-cost and potentially highly effective method for reducing coronavirus blood stream onslaughts, Extracorporeal Blood Oxygenation-Ozonation (EHO). Bona fide COVID-19 pharmaceuticals are not proven, and the human immune response is too often incapable of abating since normal T-cell activation is known to be sluggish due to the body's naivete relative to novel SARS antigens. This results in delayed response times and often severe or fatal disease. Reduction of viral load, through ozone treatment, is posited to offer a plausible emergency viral abatement strategy. This approach may also be applied in prevention for asymptomatic but virus positive individuals, amelioration of disease severity, or curing current disease in non-emergency settings.

Coronavirus, genome and classification

Coronaviruses (CoV) are a member of a diverse group of RNA viruses comprised of a large genome size varying between 26 to 32 kb. They are named for the crown-like (or corona in Latin) spikes of the virus protruding to the periphery with a diameter of 60-160nm under electron microscopy. The largest group of CoVs belong to the Nidovirales order, which contains Coronaviridae, Arteriviridae, and Roniviridae families known to induce common colds and diarrheal illnesses in humans. (2) The viral genome is linear and monopartite with a positive sense ssRNA genome. The linear RNA genome of CoVs is capped and polyadenylated. The 5' end of the CoV genome encodes replicase gene which contains two large open reading frames (ORFs), ORF1a and ORF1b. They encompass around two-third or ~20 kb of the genome. Replicase gene translates two large polyproteins, pp1a and pp1ab. The polyprotein pp1ab is expressed as a result of the translational frameshift between ORF1a and ORF1ab. The replicase polyproteins are further cleaved into 16 proteins which include proteins related to enzymatic activities, protease activities, polymerases and helicases which fuses with a zinc finger complex at the N-terminus and a Zn-ribbon-containing papain-like proteinase. (3)

The recent 2019-nCoV or SARS-CoV-2 is a member of genus Betacoronavirus in the subgenus Sarbecovirus. The SARS-CoV and MERS-CoVs are quite distant at genomic levels to current 2019-nCoV but are part of the Betacoronavirus genus. The SARS-CoV-2 shows lower similarity (50-51.8%) with MERS-CoV and similarity is near to 79% with SARS-CoV (3).

Transmission, Incubation, Clinical Manifestation, and Risk Stratification

A study conducted by experts from the US Centers for Disease Control and Prevention (CDC), NIH, UCLA, and Princeton University investigated how long the virus remained infectious on different surfaces. The investigators found that SARS-CoV-2 is detectable in aerosols for up to 3 hours, on copper up to 4 hours, up to 24 hours on cardboard, and up to 3 days on plastic and stainless steel. "The results provide key information about the stability of SARS-CoV-2, which causes COVID-19 disease, and suggests that people may acquire the virus through the air and after touching contaminated objects," according to an NIH press release.

The SARS-CoV-1 and 2 exhibit similar stability ex vivo, however the contagious period appears to significantly precede symptoms when SARS-CoV-2 as compared to SARS-CoV-1. (4) estimated that the mean incubation period of infection with SARSCoV-2 was 5.2 days (95% CI, 4.1-7.0), with the majority of the cases showing symptoms within 12.5 days of exposure, justify 14 days of quarantine. However, recent reports suggest that more than 14 days incubation period was observed in exposed persons recommending a double quarantine period of 28 days. Recent epidemiological analysis indicates that the exposed person may act as a source of infection to others during the incubation period. According to the WHO the estimated reproductive number (R0) to be 2.2 (95% CI, 1.4-3.9); however, have determined an R0 between 3.6 and 4.0, and between 2.24 to 3.58 and 7.4 days (95% CI, 4.2-14), respectively (5).

Major initial clinical symptoms of COVID-19 include fever, most of which are high fevers that occur within several days and are not alleviated by routine anti-infective drugs, additionally coughing, headache and muscle pain or fatigue. (6) Other clinical symptoms observed at low frequency include elevated troponin levels, diarrhea, myalgia and myocarditis. (6) It should be emphasized that asymptomatic patients are also positive for 2019-nCoV (Chang et al 2020), so the presence of asymptomatic carriers requires due attention, and potentially treatment to reduce transmission. Nearly 20% of the patients appeared comorbidities with regard to the dysfunction of other organs, primarily renal impairment and patients with underlying cardiovascular diseases often demonstrated comorbid heart failure. (7) They gradually develop initial symptoms in the cardiovascular system, digestive system, and nervous system, which increased the difficulty of diagnosis (6).

The COVID-19 may present with no, mild, moderate, or severe illness. Among the severe clinical manifestations, there are severe pneumonia, ARDS, sepsis, hypoxia, and septic shock. The clinical course of the disease seems to predict a favorable trend in the majority of patients. In a percentage still to be defined of cases, after about a week there is a sudden worsening of clinical conditions with rapidly worsening respiratory failure and MOD/MOF. As a reference, the criteria of the severity of respiratory insufficiency and the diagnostic criteria of sepsis and septic shock can be used (8).

Serious cases can quickly progress to acute respiratory distress syndrome, septic shock, irreformable metabolic acidosis, coagulopathy and multiple organ failure. Nearly 80% of the patients have normal or decreased white blood cell counts, and 72.3% have lymphocytopenia. (6) Lung involvement was present in all cases (4), with most chest computed tomography (CT) scans showing lesions in multiple lung lobes, some of which are dense. Ground-glass opacity co-existed with consolidation shadows or cord-like shadows are observed. Since respiratory supports are administered to most of the patients, oxygen saturation can be maintained at above 90% as indicated by pulse oximetry monitoring. (6) It is reported that severe and critically ill patients have moderate to low fever, even without obvious fever. Mild patients show just low fever, mild fatigue, and no pneumonia. Judging from the current cases, most patients have a good prognosis but poor for the elderly and those with chronic underlying disease. Symptoms in children are relatively mild.

SARS-CoV-2 may work to create tissue damage by multiple mechanisms. Virus surface open reading frame glycoproteins may bind to the porphyrin of heme. It is also reported that these proteins may attack the heme on the 1-beta chain of hemoglobin to dissociate the iron to form the porphyrin. Biochemical examination of 99 patients with novel coronavirus pneumonia also showed abnormal hemoglobin-related biochemical indexes. (2) This report demonstrates that the hemoglobin and neutrophil counts of most patients have decreased, and the index values of serum ferritin, erythrocyte sedimentation rate, C-reactive protein, albumin, and lactate dehydrogenase of many patients increase significantly. This data implies that the patient's hemoglobin is decreasing, and the heme is increasing, as the body accumulates too many oxidizing iron ions, which will cause inflammation in the body and increase C-reactive protein, albumin, and other markers of inflammation.

Quantitatively measuring physiological health is an important component in determining aspects of immunity involved in controlling viral replication. It is also crucial in identifying and staging sufferers when resources are consumed as in periods of pandemics. Initial serology indicates changes to the following markers, that are current used to risk stratify hospitalized patients at Mass General Hospital: CBC with diff (trend total lymphocyte count), Complete metabolic panel, CPK (creatine kinase), (9), D-dimer, Ferritin, ns-CRP, ESR, LDH, Troponin, and baseline ECG. All patients in any must undergo a through medical workup when practical, for risk stratification, triage prioritization, and future screening for disease vulnerability (10).

ULTRAVIOLET: PHYSICAL AND PHYSIOLOGICAL PROPERTIES

Ultraviolet (UV) radiation is part of the electromagnetic spectrum with a wavelength range (100-400 nm) shorter than that of visible light (400-700 nm), but longer than x-rays (<100 nm). UV radiation is divided into four distinct spectral areas including vacuum UV (100-200 nm), UVC (200-280 nm), UVB (280-315 nm) and UVA (315-400 nm). Only part of UVB and UVA can reach on earth, because wavelengths shorter than 280 nm are filtered out by the atmosphere especially by the "ozone layer".

History

In 1801 Johann Wilhelm Ritter, a Polish physicist working at the University of Jena in Germany discovered a form of light beyond the violet end of the spectrum that

he called "Chemical Rays" and which later became "Ultraviolet" light [1]. In 1845, Bonnet [2] first reported that sunlight could be used to treat tuberculosis arthritis (a bacterial infection of the joints).

In the second half of the nineteenth century, the therapeutic application of sunlight known as heliotherapy gradually became popular. In 1855, Rikli from Switzerland opened a thermal station in Veldes in Slovenia for the provision of helio-therapy [3]. In 1877, Downes and Blunt discovered by chance that sunlight could kill bacteria [4]. They noted that sugar water placed on a window- sill turned cloudy in the shade but remained clear while in the sun. Upon microscopic examination of the two solutions, they realized that bacteria were growing in the shaded solution but not in the one exposed to sunlight.

In 1904, the Danish physician Niels Finsen was awarded the Nobel Prize in Physiology or Medicine for his work on UV treatment of various skin conditions. He had a success rate of 98% in thousands of cases, mostly the form of cutaneous tuberculosis known as lupus vulgaris [5]. Walter H Ude reported a series of 100 cases of erysipelas (a cutaneous infection caused by Streptococcus pyogenes) in the 1920s, with high cure rates using irradiation of the skin with UV light [6].

Emmett K Knott in Seattle, WA reasoned that the beneficial effects of UV irradiation to the skin obtained by Ude, might (at least partly) be explained by the irradiation of blood circulating in the superficial capillaries of the skin. With his collaborator Edblom, an irradiation chamber was constructed to allow direct exposure of the blood to UV. The irradiation chamber was circular and contained a labyrinthine set of channels that connected the inlet and outlet ports. All these channels were covered with a quartz window that formed the top of the chamber. The irradiation chamber was so designed as to provide maximum turbulence of the blood flowing through (see Fig. 25.2). This was done in order to: (a) prevent the formation of a thin film of blood on the chamber window that would absorb and filter out much of the UV light; (b) insure that all the blood passing through the chamber was equally exposed to UV [7].

Knott and co-workers then carried out a series of experiments using UV irradiation of blood extracted from dogs that had been intravenously infected with Staphylococcus aureus bacteria and hemolytic Streptococcus species, and then the treated blood was reinfused into the dogs. They found that it was unnecessary to deliver a sufficient exposure of UV light to the blood to directory kill all the bacteria in the circulation. It was also found unnecessary to expose the total blood volume in the dogs. The optimum amount of blood to be irradiated was determined to be only 5 -7% of the estimated blood volume or approximately 3.5 mL per kg of body weight. Exceeding these limits led to loss of the benefits of the therapy. All the dogs that were treated with the optimized dose of UV to the blood, recovered from an overwhelming infection (while many dogs in the control group died). None of the dogs that were treated and survived, showed any long-term ill effects after 4 months of observation [7].

Hancock and Knott [8] had similar success in another patient suffering from advanced hemolytic streptococcal septicemia. These workers noted that in the majority of cases, a marked cyanosis (blue tinge to the skin caused by a lack of oxygenated blood flow) was present at the time of initiation of UBI. It was noted that during (or

immediately following) the treatment a rapid relief of the cyanosis occurred, with improvement in respiration accompanied by a noticeable flushing of the skin, with a distinct loss of pallor.

These observations led to application of UBI in patients suffering from pneumonia. In a series of 75 cases in which the diagnoses of pneumonia were confirmed by X-rays, all patients responded well to UBI showing a rapid decrease in temperature, disappearance of cyanosis (often within 3-5 min), cessation of delirium if present, a marked reduction in pulse rate and a rapid resolution of pulmonary consolidation. A shortening of the time of hospitalizations and accelerated convalescence was regularly observed.

In cases of viral pneumonia, in 1943 they reported a complete disappearance of symptoms in 24 to 76 hours following a single treatment; a disappearance of coughing in 3 to 7 days; and lung clearing in 24 to 96 hours (as evident in subsequent X-rays).

Henry A Barrett at the Willard Parker Hospital in New York City in 1940 reported on 110 cases including a number of different infections. Twenty-nine different conditions were described as being responsive, including the following: infectious arthritis, septic abortion, osteoarthritis, tuberculosis glands, chronic blepharitis, mastoiditis, uveitis, furunculosis, chronic paranasal sinusitis, acne vulgaris, and secondary anemia [23, 24].

EV Rebbeck at the Shadyside Hospital in Pittsburgh, PA, reported the use of UBI in Escherichia coli septicemia, post-abortion sepsis, puerperal sepsis, peritonitis, and typhoid fever [25-29] and Robert C Olney at the Providence Hospital, Lincoln, NE, treated biliary disease, pelvic cellulitis and viral hepatitis [30-32].

Mechanisms of Action of UBI

UBI affects various functions of red blood cells and various different leukocytes as has been proven in various in vitro studies. A common model is stimulator cells in mixed leukocyte cultures; another is helper cells in mitogen- stimulated cultures. UV also reversed cytokine production and blocked cytokine release. UV can also disturb cell membrane mobilization (Fig. 25.3)

Effects on Red Blood Cells

Anaerobic conditions strongly inhibited the process by which long wave UV light induces the loss of K+ ions from red blood cells. Kabat proved that UV-irradiation could affect the osmotic properties of red blood cells, the submicroscopic structure and the metabolism of adenine nucleotides. Irradiation times (60, 120, 180, 240 and 300 minutes) were used; during the irradiation, ATP decreased while the amounts of ADP, AXP, adenine compounds all increased. UV also increased hypotonic Na + and K+ ion exchange and the hematocrit value increased [33].

When Rh-positive blood was irradiated with UV light there was a significant increase in immunosorption activity. Vasil'eva et al. [34] studied varying UV irradiation conditions on both red blood cells and leucocyte-thrombocyte suspensions. Immunosorption activity increased immediately after irradiation in whole blood and red blood cells; however the immunosorption capacity in leucocyte-thrombocyte suspensions was lost after 2 days.

A two-phase polymer system containing poly-dextran was used to show that the cell surface of circulating erythrocytes was reduced after UV irradiation. This

contributed to the prolongation of survival of transfused erythrocytes and was suggested to explain the more effective therapeutic activity of autotransfused blood [35]. Snopov et al. suggested that some structural alterations in the erythrocytes, particularly in the glycocalyx were related to the improved effect of autotrans-fused blood after UV-irradiation [36]. Ichiki et al. showed that the cellular volume and the membrane potential of erythrocytes could be changed by UV irradiation. However an excessive dose of UV could decrease the production of H2O2 [37].

Effects on Neutrophils

Lower doses of UV (<0.1 J/cm ) increased the production of peroxides (H2O2) by polymorphonuclear leukocytes (which is the largest amongst all the different blood cells). The ability of UBI to increase the production of reactive oxygen species (ROS) by neutrophils could be inhibited by addition of arachidonic acid or lysophosphatidylcholine (LPC), as well as the anti-oxidant, a-tocopherol [38]. In chronic inflammatory diseases, the concentration of large IC--IgG, IgM, and small IC-IgM showed an inverse linear correlation with increased UBI dose delivered to autotransfused blood [39].

Artiukhov suggested that the generation of nitric oxide (NO) by photomodified neutrophils was due to the activation of the iNOS enzyme. De novo NO synthesis was increased by UV-irradiation, which also affected TNF-alpha production. Irradiation with lower dose (75.5 J/ m2) allowed the maintenance of the physiological homeostasis. While higher dose (755 and 2265 J/m2) delivered to neutrophils led to potential damage, by increasing the concentration of NO metabolites. When UV-irradiated cells were incubated with the transcriptional inhibitor of protein synthesis, cycloheximide the activation of iNOS and NO synthesis was prevented. High doses of UV-irradiation (755 J/m2) on neutrophils, showed a positive correlation between NO and TNF-alpha concentrations [40].

Zorkina carried out a 30-day rabbit experiment, and suggested that the chronic stress produced with a combination of hypodynamia and UBI, affected neutrophils and eliminated coagulation. UBI contributed to improvement in the body's abilities to resist long-term hypodynamia and ameliorated chronic stress. UBI enhanced the adaptive process through activated neutrophils, prevented disseminated intravascular coagulation, and changed the atherogenic metabolic profile [41].

Effects on Lymphocytes (T-Cells and B-Cells)

UBI generally decreases lymphocyte viability. UVC irradiation is the most effective among the three UV spectral regions. UVB and UVC irradiation can abolish the proliferative and stimulatory ability as well as the accessory/ antigen-presenting ability of lymphocytes in vitro. The cell-surface properties, calcium mobilization, cytokine production and release, and other sub cellular processes could all be changed by UV irradiation [42]. Areltt et al. used the "Comet "assay to detect DNA-strand breakage (single cell gel electrophoresis) as an indicator of excision repair to prove that circulating human T-lymphocytes were exquisitely hyper-sensitive to the DNA-damaging and lethal effects of UV-B radiation, raising the possibility that UV-B may make a contribution to immunosuppression via a direct effect on extracapilliary T-lymphocytes [43].

Teunissen et al. suggested that UVB radiation neither selectively affects either Th1 or Th2 nor CD4 or CD8 T-cell subsets. Compared with different dose of UVB irradiation, although the phototoxic effect was not immediately apparent, a low dose of UVB (LD50: 0.5-1 mJ/cm ) irradiation was sufficient to kill most T cells after 48-72 h [44]. There was a dose dependent reduction in all measured cytokines (IL-2, IL-4, IL-5, IFN-y , TNF-a) in the same way 72 h after irradiation. This fall in production was indicated by a remarkable correlation between loss of viability and reduction of cytokine production that may be caused directly by cell death. However, CD4+ or CD8+ T cell subsets, expression of CD4 and CD8 as well as the CD4/CD8 ratio compared with the non-irradiated control, was not altered by UVB, suggesting that none of the T-cell subsets was selectively affected.

Schieven et al. observed that UV-induced tyrosine phosphorylation in B cells after surface immunoglobulin cross-linking. This observation was very similar to the

9+

production of Ca signals in T cells. It means that UV irradiation of lymphocytes

9+ 9+

could induce both tyrosine phosphorylation and Ca signals. Ca channels in lymphocyte membranes are sensitive to UV irradiation; UV radiation causes DNA damage through the activation of cellular signal-transduction processes. UV radiation (depending on dose and wavelength) not only induces tyrosine phosphorylation in

9+

lymphocytes but also Ca signals in Jurkat T cells. Furthermore, the pattern of surface immunoglobulin cross-linking was similar to the UV-induced tyrosine phosphorylation in B cells. The UBI effect on lymphocyte function may play an important role in

9+

tyrosine phosphorylation and Ca signals, which can escape from normal receptor control. They showed that both CD4+ and CD8+ T cells (normal human lymphocytes) gave strong reactions during UV-irradiation [45].

In a similar study, Spielberg et al. found that UV-induced lymphocyte inhibition

9+

showed a similar course in disruption of Ca homeostasis by comparing UV with gamma irradiation, which have different effects on lymphocyte membranes [46].

9+

Furthermore, the presence of Ca channels in lymphocyte membranes that are sensitive to UV irradiation was shown by indo-1 staining and cytofluorometry.

9+

Intracellular calcium [Ca ]i kinetics was measured in UVC or UVB-exposed human peripheral blood leukocytes (PBL) and Jurkat cells were in parallel with functional

9+

assays.

The UV-induced i[Ca2+] rise was predominantly due to an influx of extracellular calcium, and it was more pronounced in T-cells than in non-T cells. It was

9+

observed that [Ca ]i increased within 2-3 h of irradiation; these increases were UV-dose dependent and reached maxima of 240% and 180% above the baseline level (130

9+

nM) for UVB and UVC. UV induced a bigger [Ca ]i rise in T-cells than in non-T cells, due to the influx of extracellular calcium. UV-induced calcium shifts, and UV irradiation on the plasma membrane decreased the sensitivity to respond to phytohemagglutinin (PHA) in mixed leukocyte cultures.

A series of studies confirmed that UVR irradiated lymphocytes were not able to induce allogeneic cells in the mixed lymphocyte culture (MLC) as first reported by Lindahl-kiessling. [47-49]. Clusters formed by specialized accessory cells after mitogenic or allogenic stimulation, with dendritic cells (DC) are necessary for lymphocyte activation to occur. Aprile found that UV irradiation of DC before culture

completely abrogated accessory activity was capable to block both cluster formation and no lymphocyte proliferation occurred [50].

Kovacs et al. [51] found that induction of DNA repair mechanisms was dependent on the dose of UVC light between 2 and 16 J/cm . It was evaluated in irradiated and non-irradiated lymphocytes in 51 healthy blood donors. UVC irradiation (253.7 nm) at

9 -5

doses of 2, 4, 8 and 16 J/m by measuring [ H] thymidine incorporation in the presence of 2 mM hydroxyurea added 30 min before irradiation to inhibit DNA-replication synthesis. No significant age-related difference was found in donors between 17 and 74 years.

UV-induced differentiation in human lymphocytes, and accelerated the intensity of DNA repair in these cells [52]. Exposure to UV irradiation was more effective than methyl methane sulfonate (MMS) in increasing unscheduled DNA synthesis, especially when MMS was added prior to the UV-irradiation, at 2 h or 26 h before UVC, because MMS affects DNA repair by alkylating the DNA polymerase [53]. Photo-modification of HLA-D/ DR antigens could be a trigger mechanism for activation of immunocompetent cells by UV-irradiation. Lymphocytes were isolated from non-irradiated blood, irradiated blood and a mixture of the two in different ratios (1:10,1:40,1:160) [54].

UBI before transfusion can inhabit immune recognition and prevent bone marrow graft rejection in vivo. After 9.2 Gy of total body irradiation (TBI) and 2.8 ± 2.1 x

o

10 /Kg donor marrow cells were infused, whole blood was exposed for 30 minutes to UV light at a dose of 1.35 J/cm and then injected into the recipient dogs. The control group, which was transfused with sham-exposed blood, rejected the bone marrow grafts, while no rejection was found in the group, which received UV-exposed blood before the transplanted marrow. UV irradiation on blood inhibited lymphocyte activation by eliminating a critical DC-dependent signal [55].

Oluwole et al. suggested that transfusion of UV-irradiated blood into recipients prior to heart transplantation could be carried out, in order to inhibit immune response, and reduce lymphocyte- mediated rejection [56]. Three sets of different rat strains (ACI, Lewis, W/F) were used for heart transplantation in his research. In the series where ACI rats received a Lewis heart, 1 mL transfusion of donor-type blood with or without UV-irradiation was transfused at 1, 2, and 3 weeks prior to the transplantation. A mixed lymphocyte reaction showed that ACI lymphocytes were weaker responders to Lewis lymphocytes, and the same as the other two series of different type heart transplantations. UV irradiation of donor rhesus-positive blood can be used to increase the therapeutic effect of blood exchange transfusion in children with rhesus-conflict hemolytic disease [57].

Effects on Monocytes, Macrophages and Dendritic Cells

All these types of blood cells including monocytes, macrophages and dendritic arise from the myelocytic lineage of hematogenous stem cells, and act as phagocytes and antigen presenting cells. The phagocytic capacity of UV-B irradiated mononuclear cells derived from human peripheral blood could be enhanced by all four types of deoxyribonucleoside supplementation [58].

Stimulation of phagocytic activity (PhA) appears to be one of the earliest mechanisms in immuno-correction by UV-irradiation of blood therapy. In Samoilova's

research, non-irradiated blood, mixed with 1:10 irradiation blood, were tested for PhA of monocytes and granulocytes. Increase of 1.4-1.7 times of PhA compare with non-supplemented blood, because monocytes and granulocytes could be increase by adding UV-irradiated blood into healthy adults. The enhancement of PhA depends on its initial level and may occur simultaneously with structural changes of the cell surface components [59].

UV-irradiation increased the phagocytic activity of human monocytes and granulocytes, and the "integrated phagocytic index" increased in proportion to the irradiation dose, while a lower initial level would increase more than a higher initial rate after UV-irradiation [60].

Simon et al. [61] concluded that UVB could convert Langerhans cells (LC) or splenic adherent cells (SAC) from an immunogenic phenotype into a tolerogenic phenotype, as far as antigen presenting cells were concerned (LC or SAC). In his research, a single dose of irradiation (200 J/cm ) was delivered to LC and SAC. The loss of responsiveness was found when UV-LC or UV-SAC were incubated with Th1 cells that had been pre-incubated with keyhole limpet hemocyanin (KLH). Furthermore, such loss of responsiveness was not related to the release of soluble suppressor factors, but was Ag-specific, MHC-restricted, and long-lasting. The hypothesis to explain these results was that delivery of a costimulatory signal(s) had been interfered with by UVB irradiation, because unresponsiveness by UVB-LC or UVB-SAC could not be induced by non-irradiated allogeneic SAC.

Effects on Platelets

H2O2 production in platelets is low at very low UV dose, but it increased suddenly as the dose increased above 0.4 J/cm . Pamphilon reported that platelet concentrates (PC) could become non-immunogenic after UVR and after being stored for 5 days in DuPont Stericell containers. Lactate levels, p-thromboglobulin and platelet factor were higher after UV, while glucose levels decreased with an irradiation dose of 3000 J/m at a mean wavelength of 310 nm applied in DuPont Stericell bags [62]. Ultraviolet B (UVB) irradiation of platelet concentrate (PC) accelerated downregulation of CD14 and nonspecifically increased the loss of monocytes by inhibiting the upregulation of ICAM-1 and HLA-DR [63]. However, UV irradiation of platelet concentrates produced a reduction of immunological response in a cell suspension [64-66].

Redox Status

Artyukhov et al. [71] discovered that dose- dependent UV-irradiation could activate the myeloperoxidase (MPO) and the NADPH- oxidase systems in donor blood. Two doses of UV-light were used (75.5 and 151.0 J/m ) and the higher dose activated more free radicals and H2O2 than the lower dose, another two groups were divided by the type of relationship between MPO activity and UV light dose (from 75.5 to 1510 J/m), low enzyme activity (group 1) increased under the effect of UV exposure at doses of 75.5 and 151.0 J/m , while in group 2 this parameter (MPO activity) decreased. MPO activity showed the same results in dose dependent UV-irradiation, however, increasing the dose to 1510 J/m could not increase the activity of MPO. In the next experiments, lipid peroxidation (LPO) was evaluated after UV exposure of the blood. Two groups of donors were distinguished by the relationship between blood content of LPO products and UV exposure dose. UV irradiation at low doses (75.5-

151.0 J/m ) decreased initially high LPO values and increased initially low LPO levels. In phagocytes, NADPH-oxidase plays one of the most important roles as a photoacceptor for UV light. NADPH oxidase causes increased superoxide (O2^—) production after UV-irradiation of blood by activation of the enzyme complex. UV irradiation also decreases intracellular pH caused by activation of the NADPH-oxidase complex.

UBI can also protect against free radical damage by elevating the activity of various antioxidants after spinal cord injury in rabbits, 186 rabbits were randomly divided into 4 groups, (control, blood transfusion, injured and UV treatment). UV irradiation (wavelength 253.7 nm,

5.68 mW/m2)

was used in the treatment group at 4872 h after surgery for spinal cord injury. Free radical signals (FR), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were measured. In the treatment group, superoxide dismutase and glutathione peroxidase were much increased and showed significant differences compared with the other groups, while FR and MDA decreased significantly compared to other groups. Because UV irradiation of blood decreased the MDA and FR content in spinal cord tissue; they also suggested that these two factors contributed to higher SOD activity and increased GSH-PX [72]

Summary on Mechanisms

UBI has always caused much confusion, both in the general public and also in some medical professionals, because germicidal UV light (UVC) is used to sterilize water, disinfect surfaces, and as an aid to infection control in operating rooms, and food processing and packaging plants. Many people therefore assume that UBI must act by killing pathogens (bacteria, viruses or other microorganisms) circulating in the bloodstream. However there is no evidence that this is actually the case. Therefore the mechanisms of action must lie in some other action of UV on the various components of blood. Although the entire body of evidence on the mechanisms of action of UBI is very complex, as can be seen from the foregoing material, we can attempt to draw some general conclusions. Firstly UBI is clearly an example of the well-known phenomenon called "hormesis" or "biphasic dose response'. This phenomenon has been well reviewed by Edward Calabrese from U Mass Amherst [73, 74]. The basic concept states that any toxic chemical substance or drug, or any physical insult (such as ionizing radiation, hyperthermia, or oxidative stress) can be beneficial, protective or even therapeutic, provided the dose is low enough. If the dose is increased, the beneficial or protective effects disappear, and if the dose is even further increased, then the detrimental effects of the treatment become very evident. This is clearly shown by Knott's original experiments on dogs that led to the establishment of only 5-7% of total blood volume as the optimal amount of blood to be irradiated.

UBI appears to have three broadly different classes of effects on different blood components. In the case of neutrophils, monocytes, macrophages, and dendritic cells, UBI can activate phagocytosis, increase the secretion of NO and reactive nitrogen species, and convert the DC phenotype from an immunogenic one into a tolerogenic one, thus perhaps lessening the effects of a "cytokine storm" as is often found in sepsis. In the case of lymphocytes, the effects of UBI are to inhibit (or in fact kill) various classes of lymphocytes. This is not perhaps very surprising, considering the well-

established cell-death pathways and apoptotic signaling found in lymphocytes. However it is not impossible, that the killing of circulating lymphocytes could reduce systemic inflammation, which would again be beneficial in cases of sepsis. It is also clear that UBI can oxidize blood lipids and lipoproteins, and therefore increase oxidative stress. However it is also possible that a brief burst of oxidative stress, may be beneficial, whereas continued chronic levels of oxidative stress have been generally considered as detrimental. Many antioxidant defenses are up-regulated by brief exposure to oxidative stress, and this has been postulated to be one of the fundamental mechanisms responsible for may aspects of hormesis. The oxidative nature of UBI has encouraged us to draw parallels with ozone therapy and other forms of 'oxygen therapy".

UVC irradiation for infections

The mechanism of UVC inactivation of microorganisms is to cause cellular damage by inducing changes in the chemical structure of DNA chains [5]. The consequence is the production of cyclobutane pyrimidine dimers (CPDs) causing distortion of the DNA molecule, which might cause malfunctions in cell replication and lead to cell death.

In vitro/ex vivo studies

An ex vivo study was carried out by Taylor et al. to investigate the use of UVC irradiation (254 nm) for the prophylaxis of surgical site infections [8]. The authors modeled a 'clean' surgical wound lightly contaminated with airborne bacteria by using agar, ovine muscle and ovine adipose tissue, respectively. It was found that airborne bacteria were inhibited more rapidly and more completely on agar than on muscle. A coating of blood over the microorganisms on muscle substantially reduced the effectiveness of UVC. At an irradiance of 1.2 mW/cm calculated at the lamp aperture, 1 min UVC irradiation time reduced bacterial colony forming units (CFUs) by 99.1% on agar, 97.1% on muscle (p = 0.046) and 53.5% on muscle coated with blood (p < 0.001). The combination of pulsed jet lavage and UVC was tested with the intention to remove the blood coated over the bacteria prior to UVC irradiation. The bacterial CFUs were reduced by 97.7% with the combination of pulsed jet lavage and UVC.

Conner-Kerr et al. examined the effectiveness of UVC irradiation at 254 nm in inactivating antibiotic-resistant strains of Staphylococcus aureus and Enterococcus

o

faecalis in vitro [7]. Bacterial suspensions at 10 CFU/ml were prepared and plated on agar medium and then exposed to UVC irradiation. The calculated irradiance at the device aperture was 15.54 mW/cm and distance between UVC lamp and agar medium was 25.4 mm. For the methicillin-resistant strain of S. aureus (MRSA), inactivation rates were 99.9% at 5 s and 100% at 90 s. For vancomycin-resistant E. faecalis (VRE), inactivation rates were 99.9% at 5 s and 100% at 45 s. These findings suggest that UVC at 254 nm is bactericidal for antibiotic-resistant strains of S. aureus and E. faecalis at times as short as 5 s.

In a similar study, Rao et al. reported a complete (100%) eradication of the microorganisms on agar at the UVC doses ranging from a minimum of 5 s irradiation (methicillin-resistant, coagulase-negative Staphylococcus and Streptococcus pyogenes) to a maximum of 15 s irradiation (methicillin-susceptible S.

aureus and Enterococci species) [9]. The irradiance used was 5 mW/cm calculated at the lamp aperture and lamp-agar distance was 10 cm.

By using a prototype solid-state UVC light-emitting diode device at 265 nm, Dean et al. evaluated the efficacy of UVC for treating corneal bacterial infections in vitro [10]. Agar plate lawns of S. aureus, Escherichia coli, Pseudomonas aeruginosa and S. pyogenes were exposed to UVC irradiation at an irradiance of 1.93 mW/cm (calculated at the target surface) for varying length of time. The study demonstrated that a 1-s exposure to UVC (1.93 mJ/cm ) was sufficient to induce 100% inhibition of growth for all the bacterial species tested. In this study, human corneal epithelial cells cultured on glass cover slips were also exposed to corresponding doses of UVC from the same device.

An idea of using UVC irradiation for disinfection of catheter biofilms was reported by Bak et al. [11]. In this study, the investigators determined the dose requirement for UVC disinfection of catheter biofilms. Contaminated urinary catheters from patients (n = 67) were used as test samples. The microorganisms identified from the catheter biofilms included E. coli (n = 32), coagulase-negative Staphylococcus (n = 22), E. faecalis (n = 13), Streptococcus (n = 13), P. aeruginosa (n = 12), Coryneforms (n = 7) and so on. Mean killing rates of the bacteria in catheter biofilms were 89.6 (11.8

9 9 9

mJ/cm ), 98 (47 mJ/cm ) and 99% (1400 mJ/cm ). The UVC exposures were calculated at the target surface.

Mohr et al. described the use of UVC for pathogen reduction of platelet concentrates [12]. The application of strong agitation of loosely fixed platelet concentrate bags resolved one of the problems related to the use of UVC for pathogen inactivation: namely, the quenching of the irradiation in protein-containing and turbid solutions or cell suspensions. Agitation allowed the penetration of UVC irradiation for inactivation of six bacterial species, including Gram-positive Bacillus cereus, S. aureus and Staphylococcus epidermidis, and Gram-negative E. coli, Klebsiella pneumoniae, P. aeruginosa. All bacteria species tested were reduced by more than 4 log10 at 400 mJ/cm (calculated at the surface of quartz plate where platelet concentrate samples were placed). The study also proved that platelet damage by UVC irradiation was limited under the conditions used. The in vitro functions and the other variables measured were only moderately influenced, and the storage stability of the of platelet concentrates was not impaired. Glucose consumption was slightly enhanced and lactate accumulation was slightly increased in comparison to the untreated control samples. By contrast, irradiation of platelet concentrates with UVC irradiation leads to more enhanced platelet metabolism, as evidenced by lactate accumulation and a stronger decrease in pH during storage.

Sullivan and Conner-Kerr compared the inactivation efficacies of UVC on pathogenic bacteria and fungi, in both single suspensions and mixed suspensions in vitro [13]. The calculated irradiance at the device aperture was 15.54 mW/cm , with the distance between the UVC lamp and suspension surface 25.4 mm. Upon exposure to UVC, a 99.9% inactivation rate was obtained at 3-5 s for the bacteria (P. aeruginosa and Mycobacterium abscessus) tested. By contrast, 15-30 s of UVC treatment was required to obtain 99.9% inactivation of the fungi (Candida albicans, Aspergillus fumigatus) tested.

Dai et al. tested the ability of UVC to inactivate dermatophyte suspensions in vitro and to sterilize an ex vivo model of nail infection [14]. Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum canis suspensions were irradiated with UVC (254 nm) at a dose of 120 mJ/cm (calculated at the suspension surface) and surviving fungal cells quantified. T. rubrum infecting porcine hoof slices and human toenail clippings was irradiated with UVC at the doses of 36-864 J/cm . In vitro studies showed that 3-5 log10 of cell inactivation in dermatophyte suspensions were produced with 120 mJ/cm UVC irradiation. Depending on factors such as the thickness and infectious burden of the ex vivo cultures, the radiant exposure of UVC needed for complete sterilization was usually in the order of tens to hundreds of J/cm .

Animal studies

There has been, rather surprisingly, only one reported animal study on the use of UVC irradiation to treating infections. Dai et al. investigated the use of UVC irradiation (254 nm) for treatment of C. albicans infection in mouse third-degree burns [15]. The C. albicans strain was stably transformed with a version of the Gaussia princeps luciferase gene that allowed real-time bioluminescence imaging of the progression of C. albicans infection. UVC treatment with a single exposure carried out on day 0 (30 min postinfection) gave an average 2.16-log10 (99.2%) loss of fungal luminescence when 2.92 J/cm UVC had been delivered, while UVC at 24 h postinfection gave a 1.94-log10 (95.8%) reduction of fungal luminescence after 6.48 J/cm (Figure 2). The UVC exposures were calculated at the surfaces of mouse burns. Statistical analysis demonstrated that UVC treatment carried out on both day 0 and day 1 significantly reduced the fungal burden of infected burns by 99.2% (p = 0.003) and 97.7% (p = 0.004), respectively. UVC was found to be superior to a topical antifungal drug, nystatin cream (p = 0.028).

Clinical studies

The first clinical study was reported by Taylor et al. who used UVC irradiation (254 nm) for the disinfection of surgical wounds during total joint arthroplasty procedures [16]. UVC irradiation commenced 10 min after the operation, allowing the wound to be exposed to a conventional open-air environment. Two different UVC irradiances, 0.1 and 0.3 mW/cm (calculated at the lamp aperture), were used. Bacteria in wounds were measured by imprinting with 47-mm diameter 5-^m mixed cellulose acetate and nitrate membrane filters. After 10 min of UVC irradiation, the average bacterial CFU in wounds was reduced by 87% with 0.1 mW/cm (n = 18; p < 0.001) and 92% with 0.3 mW/cm (n = 13; p < 0.001), compared with that with conventional environment (n = 13).

Shimomura et al. examined the efficacies of UVC irradiation (254 nm) on the prevention of catheter exit-site infections [17]. First, bacterial cultures of swabbed fluid from the catheter exit site were obtained from 68 continuous ambulatory peritoneal dialysis outpatients six times during the 24-month observation period. Second, the bactericidal effects of UVC irradiation on the catheter exit-site were examined. The authors found that:

• In spite of disinfection of the catheter exit site by the strict application of povidone-iodine once or twice a day, 23-45% of the cases were found to be microorganism positive;

• In the nasal cavity, S. aureus was detected in 20-25% of patients. There was a high incidence of exit-site infection among the S. aureus nasal carriers;

• UVC irradiation was performed (twice per day, 30-60 s each time) in 18 cases that constantly revealed bacteria on culture at the catheter exit site. Ten cases (55%) became culture negative, three cases showed a microbial decrease and five cases remained unchanged. These results suggest that UVC can eliminate bacteria and can be of prophylactic use for exit-site infections.

Thai et al. investigated the use of UVC for the treatment of cutaneous ulcer infections [18]. In this study, three patients with chronic ulcers infected with MRSA were treated with UVC at 254 nm. UVC irradiation was applied to each wound for 180 s, with the irradiance of 15.54 mW/cm (calculated at the UVC device aperture) and wound-lamp distance of 25.4 mm. In all three patients, UVC treatment reduced the relative amount of bacteria in wounds and facilitated wound healing. Two patients had complete wound closure following 1 week of UVC treatment. In a later study performed by the same group, 22 patients with chronic ulcers exhibiting at least two signs of infection and critically colonized with bacteria received a single 180-s treatment of UVC. Semi-quantitative swabs taken immediately before and after UVC treatment were used to assess changes in the bacterial bioburden present within the wound bed [19]. A statistically significant (p < 0.0001) reduction in the relative amount of bacteria following a single treatment of UVC was observed. The greatest reduction in semi-quantitative swab scores following UVC treatment were observed for wounds colonized with P. aeruginosa and wounds colonized with only one species of bacteria. Significant (p < 0.05) reductions in the relative amount of bacteria were also observed in 12 ulcers in which MRSA was present.

A study using UVC to treat toenail onychomycosis was reported by Boker et al. [20]. Thirty patients with mild-to-moderate onychomycosis involving no more than 35% of the great toenail were equally randomized to receive 4 weekly UVC treatments with either a low-pressure mercury lamp delivering a total UVC dose of 22 J/cm at the surfaces of the treated toenails or via a xenon pulsed-light device delivering a total UVC dose of 2-4 J/cm2

at the surface of the treated toenails. The investigator's global assessment (IGA) scale was used to assess treatment efficacy. In total, 60% of patients treated with the xenon pulsed-light device showed an improvement of at least 1 point on their week-16 IGA scale compared with baseline (p < 0.01). An image depicting the result of a patient in the xenon-pulsed light group is presented in Figure 3. Of patients treated with the low-pressure mercury lamp, 26% had at least a 1-point improvement in their week-16 IGA score (p < 0.01). The treatments with both devices were well tolerated. Minor and uncommon side effects included temporary mild erythema of the irradiated toe.

Summary

In summary, in vitro studies have reported that multidrug-resistant pathogenic microorganisms are highly sensitive to UVC inactivation. Generally, bacterial cells are found to be more sensitive to UVC than fungal cells.

The UVC doses required to inactivate a therapeutically sufficient fraction of microorganisms in vivo (e.g., 180 s irradiation time in study of Thai et al. [19]) may be orders of magnitude higher than those for in vitro (e.g., 5 s irradiation time in the study of Conner-Kerr et al. [7]). This is because the energy of UVC irradiation attenuates exponentially when penetrating into tissue.

One advantage of using UVC over antibiotics is that UVC can eradicate microorganisms in a much faster manner (2-3-log10 eradication of microorganism population in vivo could be achieved in less than 1 h), while antibiotics usually take several days to take effect, especially in burns and chronic wounds that frequently have impaired blood perfusion. UVC may also be much more cost effective than the commonly used antibiotics.

In summary, microorganisms are found to be more sensitive to UVC than mammalian cells. As a result, with appropriate doses, pathogenic microorganisms may be selectively inactivated by UVC with minimum nonspecific damage to mammalian cells. This is crucial in the application of UVC irradiation for localized infections. Studies did find that UVC at the effective antimicrobial doses can cause DNA damage to mammalian cells to some extent. However, at that same time, it has been found that the UVC-induced DNA damages can be rapidly repaired by the DNA repairing enzymes of the host. To further minimize the UVC-induced DNA damage, one can combine the use of protective agents (e.g., green tea) [39] and DNA repair agents (e.g., DNA repair liposomes) [40] with UVC irradiation. Green tea could be applied to the UVC irradiated area during the UVC treatment and liposomes could be applied after the UVC treatment. In addition, the intact skin surrounding the area to be treated could be screened from UVC irradiation [15,19].

Extracorporeal Photochemotherapy (ECP)

Extracorporeal photochemotherapy (ECP) involves the addition of a photosensitizing drug 8-methoxypsoralen (8-MOP) into blood that is then treated with UVA light (320-360 nm). ECP was originally derived from the use of PUVA (psoralen and UVA) to treat psoriasis and other skin diseases. In the case of dermatology the psoralen was administered either orally (pills) or as a bath therapy. Often the whole body was exposed to light in a "PUVA box" containing UVA emitting fluorescent tube lights. ECP has been widely used as immunotherapy for cutaneous T cell lymphoma (CTCL) since it received US Food and Drug Administration (FDA) approval in 1988. As an apheresis-based immunomodulatory therapy which involves UVA irradiation of autologous peripheral blood mononuclear cells (PBMCs) exposed to the 8-MOP, there are a numbers of features of ECP that distinguish it from other immunologic therapy, which are beneficial in immune-stimulation against cancer and in the transplant setting as an immune-modulator; for induction of antigen presenting cells (APCS), to extracorporeal sequester and modify processed leukocytes, and so on. [76] It has used for treatment of other autoimmune-mediated disorders and organ allograft rejection, and is especially beneficial for cutaneous T-cell lymphoma (CTCL) and graft-versus host disease (GVHD). Both these indications require killing of lymphocytes

ECP Therapy Treatment

The standard schedule of ECP treatment involves 2 successive days at 4 week intervals Tens of thousands of patients afflicted with CTCL, organ transplant rejection,

GVHD, Crohn's disease and type 1 diabetes [77-82], have received benefits from treatment with ECP since the first report of the systemic efficacy of by Edelson [83]. In his studies, he carried out treatment of skin manifestations in patients with cutaneous T-cell lymphoma (CTCL) and achieved a response rate of greater than 70% compared with other forms of treatment. Wollnia tested ECP in fourteen patients (all male) aged 38-72 years with CTCL of the mycosis fungoides type, stage IIa/IIb, and achieved a total response rate of 56% [84].

Mechanism of ECP

It is known that both UVC and UVB can damage DNA strands, as well as UVA activated 8-MOP. However the types of DNA lesions produced are very different for these two different kinds of UV-mediated DNA damage (Fig. 25.4). UVC and UVB both produce defined UV photo-products which are mainly the cyclobutane pyrimidine dimers (particularly TT dimers [85]) and pyrimidine-pyrimidone (6-4) photoproducts [86]. On the other hand, PUVA or ECPBM as it is known today cross-links the pyrimidine bases of DNA in complementary sister strands (inter- strand cross-links). These two different mechanisms of action are shown in Fig. 25.2. DNA damage by whatever means it is caused is likely to cause apoptosis of the extracorporally targeted lymphocytes [87]. ECP can treat erythrodermic CTCL by killing malignant CD8 T-cells but also by stimulating an immune response against thee malignant cells [88]. Two major effects of ECP have been well-confirmed: one is its immunostimulatory effects against neoplastic cells in CTCL; the other is its immunosuppressive effects against T-cell-mediated disorders such as GVHD and rejection in organ transplantation

[89]

Modern Devices to Carry Out UBI

Although it is often said that UBI is "the cure that time forgot" [90, 91], it has not actually been completely forgotten. There are several companies, organizations and devices existing at the present time, which are being used or proposed (on a rather small scale) to carry out UBI, or as it often called "Photoluminescence Therapy (PT)" Several websites provide information on UBI and PT. Perhaps one of the most comprehensive is (http://www.mnwelldir.org/docs/uv light/uv light3.htm) that provides a listing of practitioners located in USA that offer UBI to patients. UBI medical (http://ubimedical.com/about-us.html) also has a lot of information available. The web-site entitled "Infections cured" (http://infectionscured.com) is also worth checking out. Physicians UBI Awareness Center (http://drsubi.com) even has a video posted online comparing different kinds of UBI machines.

Conclusion

UV irradiation of blood was hailed as a miracle therapy for treating serious infections in the 1940s and 1950s. In an ironic quirk of fate, this historical time period coincided with the widespread introduction of penicillin antibiotics, which were rapidly found to be an even bigger medical miracle therapy. Moreover another major success of UBI, which was becoming increasingly used to treat polio, was also eclipsed by the introduction of the Salk polio vaccine in 1955 [91]. UBI had originally been an American discovery, but then was transitioned to being more studied in Russia and other eastern countries, which had long concentrated on physical therapies for many diseases, which were more usually treated with drugs in the West.

However in the last decade the problem of multi-antibiotic resistant bacteria has grown relentlessly. Multidrug-resistant (MDR) and pandrug resistant (PDR) bacterial strains and their related infections are emerging threats to public health throughout the world [92]. These are associated with approximately two-fold higher mortality rates and considerably prolonged hospital admissions [93]. The infections caused by antibiotic resistant strains are often exceptionally hard to treat due to the limited range of therapeutic options [94]. Recently in Feb 2015, the Review on Antimicrobial Resistance stated "Drug- resistant infections could kill an extra 10 million people across the world every year by 2050 if they are not tackled. By this date they could also cost the world around $100 trillion in lost output: more than the size of the current world economy, and roughly equivalent to the world losing the output of the UK economy every year, for 35 years" [95].

Sepsis is an uncontrolled response to infection involving massive cytokine release, widespread inflammation, which leads to blood clots and leaky vessels. Multi-organ failure can follow. Every year, severe sepsis strikes more than a million Americans. It is estimated that between 28-50% percent of these people die. Patients with sepsis are usually treated in hospital intensive care units with broad-spectrum antibiotics, oxygen and intravenous fluids to maintain normal blood oxygen levels and blood pressure. Despite decades of research, no drugs that specifically target the aggressive immune response that characterizes sepsis have been developed.

We would like to propose that UBI be reconsidered and re-investigated as a treatment for systemic infections caused by multi-drug resistant Gram-positive and Gram-negative bacteria in patients who are running out of (or who have already run out) of options. Patients at risk of death from sepsis could also be considered as candidates for UBI. Further research is required into the mechanisms of action of UBI. The present confusion about exactly what is happening during and after the treatment is playing a large role in the controversy about whether UBI could ever be a mainstream medical therapy, or must remain side-lined in the "alternative and complementary" category where it has been allowed to be forgotten for the last 50 years.

Key issues

• Multidrug-resistant microorganisms are highly susceptible to ultraviolet C (UVC) inactivation.

With appropriate doses, UVC can selectively inactivate microorganisms while preserving viability of mammalian cells and, moreover, is reported to promote wound healing.

Animal studies have shown that UVC is less damaging to human tissue than UVB, which is an accepted option for a large number of cutaneous disorders in humans with an excellent safety profile.

• Under excessive repeated UVC irradiation, resistance of microorganisms to UVC inactivation may develop.

UVC should be investigated as an alternative approach for prophylaxis and treatment of localized infectious diseases, especially those caused by multidrug-resistant pathogens.

UVC should be used in a manner such that side effects are minimized and development of resistance of microorganisms to UVC avoided.

Infection produces inflammation, edema, and a significant lowering of oxygen tension and diffusion in the affected tissues, which is critical to immune cell functions. Benefits of higher oxygen tension can be seen in the 126 Int J. Biosocial Med Research Vol. 14(2) 115-32, 1996 accepted use of hyperbaric oxygen therapy for osteomyelitis, where healthy circulation is already slow. Deductive reasoning would suggest that any rise in oxygen tension would help the body's immune defenses. Such can be seen in anecdotal reports of hyperbaric oxygen therapy alone resolving necrotizing fascitis. German research (Table 5) documents a rise in oxygen consumption and oxidation within the body stimulation of mitochondrial oxidation results in greater ATP production. In effect, UV blood irradiation therapy may be providing an inactivation of bacteria, a more resistant terrain, improved circulation, alkalinization, etc. While perhaps not as dramatic a treatment as hyperbaric oxygen therapy, it may provide a similar and longer-lasting effect through the secondary emanations of the absorbed ultraviolet rays. Such emissions, which last for many weeks, may account for the observed cumulative effectiveness of the therapy. UV photons, absorbed by hemoglobin, are gradually released over time, continuing the stimulation to the body's physiology. For eons, nature has utilized the sun's ultraviolet energy as a cleansing agent for the earth. The lack of resistance of bacteria to ultraviolet treatment is not surprising, since if bacteria could develop resistance, they have had approximately 3 billion years to do so. Only two discrepancies in accounts of this therapy could be found between the older American and modern German literature. Venous oxygen tension was reported by Miley to be increased, even up to one month after treatment. Frick, on the other hand, reported a rise in Pa02, and a fall in PV02, suggesting greater oxygen delivery and absorption in the tissues. A rise in 2,3 DGP can account for the latter. Miley recommended the treatment for fevers of unknown origin,[34] yet Seng's article suggested that as a contraindication. Perhaps the German author feels the infections should be clearly diagnosed first, while Miley was so impressed with his results and the safety of the treatment, he thought it was proper to treat any presumed infection with the technique. For years, there have been anecdotes and reports of another oxidative therapy (ozone) helping a variety of chronic conditions including, but not limited to, rheumatoid diseases, arterial and circulatory disorders, osteoporosis pain, viruses, and immune deficiencies. Some recent findings shed light on how this particular oxidative therapy might help such a wide variety of conditions. Bocci has determined that exposure of blood to ozone at concentrations used by practitioners for years induces cytokines and interferons.[35,36] In fact, he went on to call ozone "an almost ideal cytokine inducer." He concluded that such immune system modulation could explain the benefits of ozone reported for decades on a very wide variety of conditions. Mattman has detailed hundreds of reports linking cell wall deficient bacteria to a wide span of disease states.[37] Autoimmune disease may not be autoimmune at all, but rather an immune attack a hidden infection with native tissue being damaged by a prolonged or dysfunctional immune response to these "stealth pathogens." The broad spectrum of biologic effects of these nonspecific oxidative therapies may explain the broad range of benefits. It is quite possible that all of the oxidative therapies may operate through similar mechanisms postulated by Bocci for

ozone (namely the generation of reactive oxygen species, which in turn induce some very exceptional biochemical events).

Ultraviolet has clearly been shown to be a superior anti-infective. It is possible that the secondary emanations previously described could inactivate pathogens deep in tissues. However, of possible greater import is its effect on the other various physiologic factors affecting the terrain. The improvement in oxygen delivery and consumption, rise in circulation, blood elements, stimulation of mitochondrial oxidation and shift towards alkalinity, while all nonspecific in themselves, may help hasten the cellular response in very many disease states.

Modern medicine has focused on drugs to suppress symptoms or inhibit certain physiology (NSAID drugs as prostaglandin inhibitors, hypertensive drugs as enzymatic blockers) to treat disease. As a result, we have seen the frightening rise of resistant organism and the side-effects of chemical pharmacology. Perhaps medicine should consider the concept of nonspecific modalities that encourage the body's healing response and immune system. What could be a safer or more effective agent against infection than the bacteriocidal capabilities of our own phagocytes and a properly functioning immune system? At least 20 American physicians are currently utilizing photooxidation and have advised me of dramatic cures of intractable infections, including osteomyelitis. Communications from these physicians are verifying my findings in the use of this modality with chronic fatigue. A German videotape related that several hundred physicians are currently employing the technique in Germany with hundreds of thousands of treatments having been performed through the years and never any reported incidents of toxicity (other than a mild Herxheimer reaction).

Ultraviolet irradiation of blood has been approved by the FDA for the treatment of cutaneous T-cell lymphoma. Thus, the method is legal within the context of FDA's definition of legality. It is also legal, from the standpoint of long (over 50 years) and continuous use by physicians in the United States as a commercially viable product before the present FDA was even in existence. "[39]

The technique is taught at workshops and seminars sponsored by the International Association of Oxidative Medicine (telephone: 405634-1310). The American Board of Oxidative Medicine (a member of the American Board of Specialties of Alternative Medicine) certifies doctors in the various techniques of oxidative medicine, including UBIT. 128 Int J. Biosocial Med Research Vol. 14(2) 115-32, 1996 Conclusion This simple, inexpensive, and nonspecific technique was clearly shown years ago to be a totally safe and extremely effective method of treating and curing infections; promoting oxygenation; vasodilation; improving asthma; enhancing body physiology, circulation, and treating a variety of specific diseases. Its use in hospitals and offices could significantly reduce mortality, morbidity, and human suffering. Much more research needs to be done in determining all of the potential uses of ultraviolet blood irradiation therapy and also its correlation with other oxidative therapies.

COVID-19, MERS, SARS, and UV: The Future of Research

Covid-19, MERS, and SARS are produced by novel coronaviruses that have succeeded in breaching the immunological defenses of our contemporary human populations. They appear to have developed an uncomfortable balance between viral propagation and lethality.

A universal strategy in mastering infections, whether bacterial or viral, is the body's culling of pathogenic organisms to the point where they no longer represent an invasive and replicative threat. This may be achieved by responsive systems of host immune counter-offense, with molecular memory capable of neutralizing future viral attacks.

Covid-19, SARS, and MERS are acute, rapidly progressing, pan-inflammatory infections that, predicated upon the coronavirus quasi-species involved, may present distressful morbidity and mortality outcomes. A salient clinical configuration in these infections stems from their acute involvement of the respiratory system and their rapid disruption of blood gas balance. When pO2 and pCO2 are sufficiently compromised, chemoreceptors in the medulla begin to fail and respiration stops.

Because of their galloping symptomatology, Covid-19, MERS, and SARS ideally would benefit from proactive emergency viral culling. With estimated 10 billion viral particles disgorged daily in the general circulation, viremic reproductive juggernauts commonly seen in lipid-enveloped viral cycles need modulation.

Can systemically administered oxygen/ozone mixtures assist in this process? In severe Covid-19 cases, the disease progression may be stunningly rapid. Present countermeasures are non-existent. Indeed, Covid-19 may need more intensive intervention and emergency abatement of viral aggressiveness.

CONCLUSION

• The use of UVC irradiation for the prevention and treatment of localized infections is still in the very early stages of development. Most of the studies are confined to in vitro and ex vivo levels, while in vivo animal studies and clinical studies are much rarer.

• Several in vitro studies have reported that multidrug-resistant pathogenic microorganisms are highly sensitive to UVC inactivation. Bacterial cells are found to be more sensitive to UVC than fungal cells. Microorganisms are found to be more sensitive to UVC than mammalian cells.

• One advantage of using UVC over antibiotics is that UVC can eradicate microorganisms in a much faster manner. UVC may also be much more cost effective than the commonly used antibiotics.

• UVC is generally much more effective and also safer for prophylaxis of wound infections than for treatment of already infected wounds. This is because when the infections get established, pathogenic microorganisms penetrate deep into the tissue and biofilms can often be formed. Higher doses of irradiation are needed to treat established infections (e.g., as reported by Dai et al. [15]) because irradiation is quickly attenuated when penetrating into tissue. Similarly, higher irradiation doses are needed when inactivating microorganisms within biofilm than are needed for their plank-tonic counterparts. It is likely that the use of higher irradiation doses is accompanied by higher side effects by compromising the inactivation selectivity and increasing the UVC-induced tissue damage.

• The effects of UVC irradiation on wound healing have also been investigated, and variable results have been reported. While pathogenic microorganisms impede the healing of infected wounds, one can expect that the eradication of microorganisms by UVC would enhance wound healing in infected wounds.

• Studies found that UVC at the effective antimicrobial doses can cause DNA damage to mammalian cells to some extent. However, it has also been found that the UVC-induced DNA damages can be rapidly repaired by the DNA repairing enzymes.

• In contrast to the large amount of studies regarding the chronic effects of UVB on human skin and tissue, there has been no similar report on the chronic effects of UVC. However, it has been suggested in an animal study that UVC is less carcinogenic than UVB [38] because of its more superficial penetration depth. The authors of this paper state that, "Abnormal differentiation of a layer of cells that is committed to being sloughed off anyway (UVC) is not harmful, whereas mutation of the basal cells (UVA or UVB) may result in skin cancer." [38]. On the other hand, it has been reported that UVB treatment is an effective options for a large number of cutaneous disorders in humans with excellent safety profiles. A retrospective study of 195 psoriasis patients treated with UVB did not provide evidence for increased skin cancer risk with up to 9 years of follow-up [33]. An analysis of 3867 patients receiving UVB over an 18-year period, with a median number of 29 treatments and 352 patients receiving 100 or more treatments with more than 6 months of follow-up for each patient, showed no increase in skin cancers of any kind [34].

• It has been found that resistance of microorganisms to UVC may develop after excessive repetition (e.g., 80 cycles) of UVC irradiation [41]. Therefore, similar to traditional antibiotics, excessive or long- term use of UVC should also be avoided.

• It is worth noting that the use of UVC for sun-sensitive patients (or lupus erythematosus patients) should be cautious. It is well known that solar irradiation, mainly the part of UVA and UVB, cause photosensitivity in lupus erythematosus patients [42], which is an abnormal reaction of human skin to solar irradiation. It was found that similar effects can also be induced by UVC [43].

• It is also worth noting that the penetration of UVC irradiation in human skin and tissue is limited, and as a result, topical irradiation of UVC may not be sufficient to reach deeply located infections and subsequently inactivate pathogenic microorganisms. However, with the advancement of modern optical fiber technologies [44,45], this limitation could be overcome by delivering the UVC irradiation interstitially to the infected sites. In addition, optical clearing techniques [46,47], which have attracted extensive attention recently, have provided another potential technique for improving the UVC penetration in human skin and tissue.

• In conclusion, we believe there are situations where the risk- benefit ratio is favorable for the use of UVC for treating localized infections, particularly when the microorganisms responsible are antibiotic resistant. While permissible UVC dose exposure limits for human tissue do not exist, it is expected that there would be an acceptable maximum number of repetition times for UVC irradiation and possibly a total lifetime cumulative exposure. For wound infections, we expect that only limited numbers of repeated doses of UVC irradiation would be required, while the UV-induced carcinogenic mutation is a long-term effect of prolonged use of UVC.

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