1

Advanced medical countermeasures for radiological accidents and nuclear disasters: prevention, prophylaxis, treatment and pre- and post-exposure management.
Dmitri Popov1 , Vecheslav Maliev23. 1- Advanced Medical Technologies & Systems, Richmond Hill, Ontario, L4E4W8 Canada [Email: dlpopov@rogers.com] 2- Vladicaucasian Scientific Center of Russian Academy of Sciences, Biotechnology Dept., 93 pr. Kosta Hetagyrova, Vladicaucas 362008 North OssetiaAlania, Russia. [Email: niobiot@mail.ru] Key words: Cerebrovascular Acute Radiation Syndrome (Cv ARS), Cardiovascular Acute Radiation Syndrome (Cr ARS), Gastrointestinal Acute Radiation Syndrome (GI ARS), Hematopoietic Acute Radiation Syndrome( Hp ARS), Radiation Neurotoxins (RNT), Neurotransmitters, Radiation Countermeasures, Antiradiation Vaccine (ArV), Antiradiation Blocking Antibodies, Antiradiation Antidote-Jg G. Psychoneuroimmunology, Neurotoxicity. Introduction: Countermeasures against nuclear terrorism to prevent or limit the number of irradiated human population or radiation intoxications include early identification of the nuclear terrorism event and all persons which exposed by radiation, decontamination program and procedures, radiation control, and medical countermeasures which include medical diagnosis, differential diagnosis of Acute Radiation Syndromes by Immune Enzyme Assay , pre-exposure vaccination with Human Antiradiation Vaccine, post-exposure specific treatment - de-intoxication with Radiation Antidote IgG (blocking Antiradiation Antibodies). Our Advanced Medical Technology elaborated as a part of effective countermeasure include Plan of Action. [ 31, 32, 34, 35, 36, 37, 38 ]. Countermeasures against nuclear terrorism to prevent or limit the number of high level of lethality and severe forms of radiation illness or intoxications include (a) early identification of the nuclear terrorism event and persons exposed, (b) appropriate decontamination, (c) radiation control, and (d) medical countermeasures and medical management of ARS. [5, 6, 13, 62, 63, 64 ]. Medical countermeasures, which include medical interventions such as active immune-prophylaxis with Human Antiradiation Vaccine , passive immune-prophylaxis with Antiradiation Antitoxins immuneglobulins IgG , and chemoprophylaxis - post-exposure antioxidants prophylaxis and antibiotic prophylaxis. Medical countermeasures with Antiradiation Vaccine could be initiated either before an exposure (if individuals are identified as being at high risk for exposure) or after a confirmed exposure event. [ 31, 32 ]. Pathology aspects - Modern radiobiology: contention of concepts for advanced technology and development of effective countermeasure: specific immune-prophylaxis, prevention and treatment of biological consequences after nuclear terrorism event. Radiation, Radiation Toxicity, Radiation Toxins. Radiation Toxicity and Inflammation. Acute Radiation Disease (ARD) or Acute Radiation Syndromes (ARS) are defined as the collective toxic clinical states observed from the acute pathological processes in various doses of irradiated mammals. [ 1, 2, 5, 13]. Central Nervous System, Cardiovascular System, Hematopoietic system, gastrointestinal mucosa, all cell with high mitotic and metabolic rate are highly sensitive to radiation exposure. [ 43, 63, 65, 76 ]. Radiation toxicity include generation of Reactive Oxygen Species, activation and hyper-activation of the

2 group of cytotoxic molecules ( Radiation Toxins- enzymes with high proteolytic activity), interaction of Inflammatory cells and Resident Tissue Cells, Leukocyte recruitment, trans-immigration, adhesion in Acute Systemic Inflammation, Endothelial Cell Barrier disruption. [ 1, 2, 24, 31, 32, 34, 35, 36, 37, 38, 62, 63, 64]. Radiation toxins isolated from the lymph or blood or cells of irradiated animals are classified as hematotoxic, neurotoxic, and enteric non-bacterial(GI) Radiation Toxins, and they play an important role in development of Hematopoietic, Cerebrovascular, Cardiovascular and Gastro- Intestinal Acute Radiation Syndromes. [ 31, 32, 37, 38]. Proteinases - proteolytic enzymes playing important role in the development of acute radiation toxicity. Proteolytic enzymes stored in cytoplasmic granules as inactive form and after irradiation migrate into intra-cellular space. Proteinases with different catalytic activity classified into four groups: serine proteinases; metallo-proteinases; cysteine proteinases; aspartatic proteinases. [ 84, 89, 90 ]. Four Groups and Seven most important distinct Toxins derived from post-irradiated animals have been designated as Specific Radiation Toxins (SRD): SRD-1 (Neuro-toxic radiation toxin generated by Cerebrovascular ARS), SRD-3 (Vascular-toxic radiation toxin generated by Cardiovascular ARS), SRD-3 (Enteric non-bacterial radiation toxins generated by the Gastrointestinal form of ARS), and SRD-4 (Hematotoxic radiation toxins generated by hematological, bone marrow form of ARS ). SRD-4 is further subdivided into four groups depending on the severity of the ARS induced: SRD-4/1, mild ARS; SRD-4/2, moderate ARS; SRD-4/3, severe ARS and SRD-4/4, extremely severe ARS. We conclude that the SRD-1 and the SRD-2 radiation toxins produce toxicity for central and peripheral nervous system. [ 31, 32, 62, 63]. Radiation Toxins possess high toxic properties. Radiation Neurotoxin isolated from lymphatic system of irradiated animals or from cells compartments and injected to healthy animals in toxic doses 0.03 mg/kg, 0.5 mg/kg, 10.0 mg/kg, 15.0 mg/kg have had initiated development of acute failure of blood circulation and breathing ventilation. Death of laboratory animals had occurred within 5 min-3 days after injection of toxic doses of Radiation Neuro-Toxins and depended on a concentration and a type of active substance of Radiation Toxins. [ 62, 63]. Role of Radiation Neurotoxins, Radiation Cytotoxins, Radiation Hematotoxins in triggering, developing of radiation Central Nervous System injury, Cardio-Vascular System disorder, Gastro-Intestinal System events, Hematopoietic System disease and white/red blood cells lysis were reviewed in literature. [ 1, 6, 20, 22, 26, 31, 32, 34, 35, 36, 37, 38, 42, 43, 48, 51, 56, 62, 63, 83, 84, 92]. Inflammatory processes in the vascular system and an increase in permeability of the endothelial cell barrier after severe damages of endothelial cells and function of endothelial cell. [42,62,63]. Radiation Neurotoxins - rapidly acting enzymatic blood toxic lethal agent, which concentrated in irradiated cells, migrated into tissues and circulated in interstitial fluid, lymph, blood with interactions with cell membranes, receptors and cell compartments. [62,63]. Radiation Toxins include three major types of toxins: 1. Proteins with high enzymatic activity. 2. Vasoactive Polypeptides. 3. The group of Lipids with toxic properties. [ 62, 63]. Radiation: Apoptosis and/or Necrosis? We postulate that Apoptosis and Necrosis are the two major types of programmed cell death after irradiation. Apoptosis and Necrosis initiated by programmed control mechanisms. Radiation induced cell death by triggering apoptosis pathways was described in many articles and supported by many scientists. [1, 11, 14, 17, 20, 43, 57, 58 ]. However, some scientists and some institutions described deadly processes developing in irradiated eukaryotic cells as only apoptosis and some scientists describing a variety of complex mechanisms and mentioned that radiation-induced cell death could developing under different mechanisms

3 of pathogenesis which include and apoptosis and\or necrosis. [1, 11, 17, 20, 43, 57, 58 ]. We postulate that necrosis or apoptosis could be developed for the same time and after same defined doses of radiation for different cells lines with different radio-sensitivity. The Cell Death Nomenclature Committee recommends the use of the appropriate diagnosis: apoptotic necrosis. [28]. Necrosis possesses the characteristics of accidental or externally-induced cell death from the influence of different environmental factors and triggering development of inflammation and elaborating of toxins, release of pro-inflammatory active and toxic cellular contents into the intracellular and extracellular fluids and concentrated in the lymphatic systems and blood circulation. [ 21, 24, 28, 56, 63]. Some scientists presenting results and conclusions that Caspases are important molecular mechanisms and central components of the necrosis processes. [ 17, 21, 57]. At the present time necrosis is considered as an alternative form of programmed cell death whose activation induces important biological consequences including immune reactions and induced inflammation. [ 17, 24, 27, 33]. Table 1. Radiation Pathology- Apoptosis and Necrosis: Comparison of Morphological and Biochemical features. [ 1, 4, 11, 14, 17, 20, 21, 22, 26, 28, 43, 57, 58, 76] Radiation Apoptosis ( cells suicide) Inflammation never present. Radiation Necrosis (cells homicide) Inflammation always present. Systemic inflammatory response syndrome (SIRS), toxic multiple organ injury (TMOI), toxic multiple organ dysfunction syndromes (TMODS), and finally, toxic multiple organ failure (TMOF). Toxic substances always present. Radiation Toxins specific group of proteins with high enzymatic activity, activated after irradiation. Radiation Toxins the group of lipids with toxic properties. Radiation Toxins the group of vasoactive peptides. Karyolysis, Pyknosis, Karyorhesis. Nuclear swelling, Chromatin granular, Chromatin flocculation, Types of radiation induced damage of DNA: Breaks of the strand, alteration to bases, destruction of sugar, cross-links interactions and formation of dimmers. Necrosis programmed cell death. Caspase activation always present pathway for necrosis. Caspase depended initiation of developing of necrosis possible. Caspase-8 initiation possible with apoptosis and necrosis Genetic control can initiate necrosis. Environmentally induce. The Blebbing phenomenon, increased membranes permeability. Cell membrane affected by external and internal factors.

Toxic substances never present.

Morphologically cells shrinks, become denser, condensation occur, original name shrinkage necrosis. Mitochondrias structure and functions are not affected as primary process. Apoptosis programmed cell death. Caspase activation always present.

Genetic control initiate apoptosis. Could be environmentally induce. The Budding phenomenon. Cell membrane not affected or affected as secondary process.

Acute Radiation Syndromes (ARS) are defined as the collective toxic clinical states observed from the acute pathological processes in various doses of irradiated mammals; to include: systemic inflammatory response syndrome (SIRS), toxic multiple organ injury (TMOI), toxic multiple organ dysfunction syndromes (TMODS), and finally, toxic multiple organ failure (TMOF). Toxicity and inflammation are special features of radiation which induced necrosis. [ 2, 20, 27, 43 ]. However, toxicity of radiation toxins and inflammation could induce additional apoptotic and/or necrotic processes and involve the huge group of innocent bystander cells. [ 3, 27, 58, 63 ] Blocking antibodies to radiation toxins demonstrate toxin neutralization. Interesting, immune neutralization of radiation toxins reduce clinical manifestation of radiation toxicity include behavior defects or neurological defects developed after irradiation. [ 33, 37, 63 ]. Radiation Syndromes: Cerebrovascular Acute Radiation Syndrome (Cv ARS) is an extremely severe, specific, dynamic injury of Central Nervous System (CNS) and involved interactions with Peripheral Nervous System (PNS). [ 2, 63 ] Cv ARS can be induced by neutron, heavy ions, or gamma radiation. [ 35, 63, 65, 66, 67, 68, 70, 71, 72, 73, 77, 78, 79 ] The Radiation alone and Radiation Neurotoxins even without irradiation induce increased permeability of blood vessels, disruption of the blood-brain barrier, blood-cerebrospinal fluid (CSF) barrier and developing severe disorder of blood macro- and microcirculation. [ 56, 83] Principles of Radiation Psychoneuro-immunology and Psychoneuro-allergology can be applied for determination of pathological processes developed after irradiation. [ 62, 63 ]. Selective administration of Radiation Neurotoxins to radiation naive mammals induce similar effects as after irradiation. The mammals after defined doses of irradiation or administration of radiation neurotoxins demonstrated a specific clinical features of Cerebrovascular ARS with development significant changes in motor function, behavior or develop neurological defects which include such acute reactions as dystonia, ataxia, ceizures, respiratory and cardiac arrest. [ 32, 63, 92 ]. Countermeasure development: Differential Diagnosis of Acute Radiation Syndromes by Immune Enzyme Assay. Differential diagnosis of Acute Radiation Syndromes by the method of Immune Enzyme Assay ( ELISA) is a very efficient diagnostic tool of biological dozimetry and evaluation of acute radiation disease. [6, 46, 59, 60, 64 ]. We use as biological markers the group of essential Radiotoxins enzymes - high molecular weight proteins and glycoproteins with specific antigenic properties. We postulate that the SRD-1 and the SRD2 radiation toxins produce toxicity for central and peripheral nervous system, cardiovascular system. Determination of high levels of SRD-1, SRD-2, SRD-3 and SRD-4 in the peripheral blood allowed to recognize early periods of Cerebrovascular, Cardiovascular, Gastrointestinal and Hematopoietic forms of ARS. [ 63 , 64 ] The important goal of an early assessment with Enzyme Immune Assay is the accurate description of the Acute Radiation Syndromes at initial phases. Early and precise differential diagnosis allow doctors to provide an effective medical management of ARS. Polyclonal rabbit antibodies against Radiation Toxins (RT), the main product of cell destruction after irradiation, were obtained and applied for immuneenzyme assay (ELISA). The developed ELISA can be applied for group specific determination of Radiation Toxins - proteins with high enzymatic, proteolytic activity and their toxic metabolites. [ 37, 64 ]. The method of detection Radiation Toxins and differential diagnosis of Acute Radiation Syndromes new Detection Kit offers extreme useful specificity and sensitivity. Acute toxic effects of these Radiation Toxins include acute and chronic damage to the cerebro-vascular

5 system, cardio-vascular system, respiratory tract, gastro-intestinal tract, hematopoietic system, immune system, lymphatic system. The additional reasons given above necessitate the use of effective techniques for detecting Radiation Toxins and related compounds in the dangerous environment that can handle large sample loads for big amount of patients or population with a rapid turnover time. Table 2. Experiments and technology of ELISA for differential diagnosis of ARS. SRD-1 Irradiation of mammal experimental and agricultural animals. Preparation of Toxoid Forms of Radiation Toxins SRD-1 Immunization of radiation nave rabbits Separation of antisera contained antibodies to SRD-1 proteins. Antisera Testing to SRD-1 by the ELISA Technique ELISA Testing for SRD-1 Detection in the blood of irradiated mammals of the SRD-1 proteins confirmed development of Acute CerebroVascular Radiation Syndrome. SRD- 2 Irradiation of mammal experimental and agricultural animals. Preparation of Toxoid Forms of Radiation Toxins SRD-2 Immunization of radiation nave rabbits Separation of antisera contained antibodies to SRD-2 proteins. Antisera Testing to SRD-2 by the ELISA Technique ELISA Testing for SRD-2 Detection in the blood of irradiated mammals of the SRD-2 proteins confirmed development of Acute CardioVascular Syndrome. SRD-3 Irradiation of mammal experimental and agricultural animals. Preparation of Toxoid Forms of Radiation Toxins SRD-3 Immunization of radiation nave rabbits Separation of antisera contained antibodies to SRD-3 proteins. Antisera Testing to SRD-3 by the ELISA Technique ELISA Testing for SRD-3 Detection in the blood of irradiated mammals of the SRD-3 proteins confirmed development of Acute GastroIntestinal Syndrome. SRD-4 Irradiation of mammal experimental and agricultural animals. Preparation of Toxoid Forms of Radiation Toxins SRD-4 Immunization of radiation nave rabbits Separation of antisera contained antibodies to SRD-4 proteins. Antisera Testing to SRD -4 by the ELISA Technique ELISA Testing for SRD-4 Detection in the blood of irradiated mammals of the SRD-4 proteins confirmed development of Acute Hematopoietic Syndrome.

The use of Radiation Toxins, a proteins with high enzymatic activity in the immunoassay of Acute Radiation Toxins has been proposed previously [ 34, 35, 37 ]. Although all types of Radiation toxins can be detected in same probes of irradiated species the concentration of Radiation Toxins could very different. The presence of SRD 1 always inform clinicians about development severe form of Acute Cerebro-Vascular Radiation Syndrome. The concentration of toxic molecules such as SRD-3, SRD-3, SRD-4 demonstrate different levels of radiation injury and for predicting the outcome of a radiation illness. Countermeasure development: Pre-exposure prevention, prophylaxis and treatment of Acute Radiation Syndromes with Experimental Human Antiradiation Vaccine: Description The Human Antiradiation Vaccine produced by Advanced Medical Technology and Systems Inc. in cooperation with Vladicaucazian Centre of Biotechnology, RAS RF only for experiments, is a sterile, stable, freeze-dried suspension of Antiradiation Toxins prepared from cells of irradiated mammal species. The Antiradiation Toxins is harvested from irradiated mammals cells, concentrated by

6 ultrafiltration and chemically or radiologically inactivated . One dose of Antiradiation Vaccine contains less than 100 mg proteins. This vaccine must only be used intramuscularly and as a single dose. The experimental vaccine can contain preservative or stabilizer. It should be used immediately after reconstitution, and if not administered promptly, discard contents. The potency of one dose (1.0 mL) Human Antiradiation Vaccine is under discovery. Pre-exposure immunization High titer antibody responses of the Experimental Antiradiation Vaccine made in Radiation Toxins isolated from irradiated cells have been demonstrated in trials conducted in Russia under independent control. Seroconversion was often obtained with only one dose but after two doses one month apart, 100% of the recipients developed specific antiradiation antibody. In the Russia, Experimental Antiradiation Vaccine resulted in geometric mean titers (GMT) of 13.9 IU/mL at Day 55 and 6.4 IU/mL at Day 110 when three doses were given intramuscularly during the course of one month. Indications and usage 1. Rationale of prophylaxis and pre-treatment. Physicians must evaluate each possible radiation exposure. Pre-exposure immunization may be offered to persons in high-risk groups, such as cancer patients, workers at the nuclear power stations, nuclear laboratory workers, and persons spending time at vessels with nuclear power engines where radiation is a constant potential threat. Persons whose professional roles such as pilots and personal of civic and military air jets bring them into contact with high altitude and higher doses of radiation potentially should also be considered for pre-exposure prophylaxis. Civic population and military personal could be considered for prophylaxis as a part of Active Plan countermeasures against nuclear terrorism. Radiation protection with Human Antiradiation Vaccine extremely useful for cancer patients which undergo to radiation therapy, for patients which undergo to medical diagnostic procedures like Computer Tomography Scan or Magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), or magnetic resonance tomography (MRT). Vaccination is recommended for children living which undergo radiation therapy or clinical diagnostic procedures where exposure to radiation is a constant threat. Worldwide statistics indicate children are more at risk to influence of radiation than adults. [ 5, 7, 8, 9, 12, 49, 50 ]. Pre-exposure specific antiradiation prophylaxis is given for several reasons. First, it may provide protection to persons with unexpected exposure to radiation or chronic low dose of radiation. Secondly, it may protect persons whose post-exposure therapy might be expected to be delayed. Especially after nuclear disaster, nuclear accident or nuclear terrorists attack. [ 13, 15, 16, 18]. Finally, although it does not eliminate the need for additional therapy after a radiation exposure, it make therapy more effective and decreasing the number of doses of specific Antiradiation Antidote IgG needed. This is of particular importance for persons at high risk of being exposed in countries where the available risk of nuclear terrorism or nuclear accidents. Pre-exposure immunization: Consists of the three doses of EHAV, 1.0 mL, intramuscularly (deltoid area), one each on Days 1; 7 and 28. Administration of routine booster doses of vaccine depends on exposure risk category as noted in Table 3. Pre-exposure immunization of immune-suppressed persons are possible, but should be controlled by ELISA. [ 31, 32, 47, 64]

Table 3. Criteria for pre-exposure immunization.

7 Risk category Continuous Nature of risk Irradiation present continuously Typical population Pilots, astronauts, workers of nuclear industry. Potentially could be exposed: personal of nuclear power stations, personal of vessels with nuclear power engine. Cancer patients. Pre-exposure regimen Primary pre-exposure immunization course. Serology annually. Booster immunization when antibody titer falls below acceptable level Primary pre-exposure immunization course. Booster immunization or serology every 3 years. Or before radiological treatment and after. Primary pre-exposure immunization course. No routine booster immunization or serology No pre-exposure immunization

Frequent

Irradiation episodic

Infrequent

Exposure nearly always episodic with source recognized

Patients who undergo to diagnostic procedures: CT scan, MRI, NMRI, MRT

Rare

General population, military personal in time of peace.

Pre-exposure booster immunization consists of one dose of Human Antiradiation Vaccine, 1.0 mL/dose, IM (deltoid area). Acceptable antibody level is 1: 10 titer. Countermeasure development: Pre-exposure and post-exposure Immunotherapy of Acute Radiation Syndromes with Human Antiradiation Antidote-Ig G: Indications and usage. 1. Rationale of treatment Physicians must evaluate each possible radiation exposure. 2. Differential diagnosis of Acute Radiation Syndromes by the method of Immune Enzyme Assay ( ELISA) is a very efficient diagnostic tool of biological dozimetry and evaluation of acute radiation disease. Immunoglobulins is an important part of Acquired Immunity and participate in such important immunological processes as recognition, regulation and elimination of foreign antigens. At the present time, Intravenous Immunoglobulins are used for an efficient therapy for immune deficiency syndromes, thrombocytopenias, inflammatory reactions, modulation of autoimmunity and a wide range of hematologic disorders. [31, 32, 34, 36, 37, 39 ]. Traditionally, the treatment of Acute Radiation Syndromes (ARS) includes supportive therapy, cytokine therapy, blood component transfusions and stem cell transplantation. [19, 49, 50, 51 ]. However, results of treatment of ARS remain limited and in cases of severe radiation injury insufficient. [ 19, 49 ]. Studies of therapy effects of Anti-radiation Immunoglobulin G in vivo have established that specific antibodies to Radiation Toxins of SRD group can be important, effective part of medical management of

8 ARS and can play a significant role in neutralization of radiation induced toxicity. Multiple-organ failure at Acute Radiation Syndromes is a major cause of mortality after high doses of gamma irradiation. [ 27, 49, 65, 67, 69, 76 ] Hyper-immunization of non-irradiated animals by non-toxic doses of Radiation Toxins was provided. The immunoglobulin fraction of pooled hyper-immune anti-radiation plasma was separated. Specific treatment Postexposure antiradiation immunization should always include administration of both antibody (preferably polyvalent Antiradiation Antidote IgG ) and vaccine, with one exception. The combination of globulin and vaccine is recommended for radiation exposures regardless of the interval between radiation exposure and specific treatment. The ASAP treatment is begun after radiation exposure, the more effective treatment and higher survival rate. However, there have been instances in which the decision to begin treatment was made as late as five or ten days or longer after the exposure due to delay in recognition or management problems that an exposure had occurred. If antiradiation treatment is indicated, both Antiradiation ImmuneGlobulin (AIG) and Human Antiradiation Vaccine (HAV) should be given as soon as possible regardless of the interval from exposure. Local reactions to vaccines are common and do not contraindicate continuing treatment. [31, 32,34, 38 ]. Contraindications. For radiation postexposure treatment, there are no known specific contraindications to the use of Human Antiradiation Vaccine. In cases of pre-exposure immunization, there are no known specific contraindications other than situations such as developing simple febrile illness. Drug interactions Corticosteroids, other immunosuppressive agents, and immunosuppressive illnesses can interfere with the development of active immunity and predispose the patient to developing Acute Radiation Syndromes. Immunosuppressive agents should not be administered during post-exposure therapy, unless essential for the treatment of other conditions. When Antiradiation post-exposure prophylaxis is administered to persons receiving steroids or other immunosuppressive therapy, it is especially important that serum be tested for antibodies to radiation toxins to ensure that an adequate response has developed. Human Antiradiation Vaccine and Antiradiation Antidote IgG could be given to patients together with antioxidants and antibiotics. Human Antiradiation Vaccine and Antiradiation Antidote IgG could be/should be given before and after Radiation Therapy for already immunologically suppressed cancer patients. Chemotherapy for cancer patients are not contraindication for pre-exposure vaccination with HAV and post-exposure therapy with AA IgG. Table 4. Experiments and technology of elaboration of multivalent Antiradiation Antidote IgG . Acute Cerebro Vascular Radiation Syndrome. Isolation Radiation Toxins from cells or central lymph of irradiated animals Cv RS Preparation of Toxoid Forms of Radiation Toxins Acute Cardiovascular Radiation Syndrome. Isolation Radiation Toxins from cells or central lymph of irradiated animals Cr RS Preparation of Toxoid Forms of Radiation Toxins Acute Gastrointestinal Radiation Syndrome. Isolation Radiation Toxins from cells or central lymph of irradiated animals GI RS Preparation of Toxoid Forms of Radiation Toxins Acute Hematopoietic Radiation Syndrome. Isolation Radiation Toxins from cells or central lymph of irradiated animals HP RS Preparation of Toxoid Forms of Radiation Toxins Control

Existing as nonactive form only in cells of radiation nave mammals. -

9 SRD-1 Immunization of radiation nave animals ( rabbits, horses) Separation of antisera contained antibodies to SRD-1 proteins ELISA Testing for SRD-1 Separation of IgG to SDR-1from antisera Antiradiation Antidote IgG with specific blocking antibodies to Radiation Toxins of SDR-1 group SRD-2 Immunization of radiation nave animals ( rabbits, horses) Separation of antisera contained antibodies to SRD-2 proteins ELISA Testing for SRD-2 Separation of IgG to SDR-2 from antisera Antiradiation Antidote IgG with specific blocking antibodies to Radiation Toxins of SDR-2 group SRD-3 Immunization of radiation nave animals ( rabbits, horses) Separation of antisera contained antibodies to SRD-3 proteins ELISA Testing for SRD-3 Separation of IgG to SDR-3 from antisera Antiradiation Antidote IgG with specific blocking antibodies to Radiation Toxins of SDR-3 group SRD-4 Immunization of radiation nave animals ( rabbits, horses) Separation of antisera contained antibodies to SRD-4 proteins ELISA Testing for SRD-4 Separation of IgG to SDR-4 from antisera Antiradiation Antidote IgG with specific blocking antibodies to Radiation Toxins of SDR-4 group

Natural IgG preparations.

Antiradiation Antidote IgG - Immunoglobulines to Radiation Toxins were used for a treatment of Acute Radiation Syndromes and the efficacy of this bio-pharmaceutical agent was initially evaluated. Therapeutic application of Specific Anti-Radiation Immunoglobulin had significantly diminished mortality rate at Acute Radiation Syndromes and was much more effective compare with natural immunoglobulins preparations and irradiated forms of natural immunoglobulins. [31, 32, 36]. If an immunotherapy treatment approach to treatment of acute radiation syndromes (ARS) were to be developed; consideration could be given to neutralization of radiation toxins (Specific Radiation Determinants- SRD) by specific antiradiation antibodies. [ 31, 32, 36] To accomplish this objective, irradiated animals were injected with a preparation of Anti-radiation Antidote Immunoglobulin Ig G obtained from hyper-immune donors. We tested several specific hyper-immune IgG preparations against these radiation toxins and observed that their toxic properties were neutralized by specific antibodies of antiradiation IgG. Material and Methods: Rabbits were inoculated with SRD radiation toxins to induce hyper-immune serum. The hyper-immune serum was pooled from several animals, purified, and concentrated. Enzyme-linked immune-sorbent assays of the hyper-immune serum revealed high titers of IgG with specific binding to Radiation Toxins. The Antiradiation Antidote IgG preparation was injected to laboratory animals one hour before and three hours after irradiation or only 24 hours after irradiation, and was evaluated for its ability to protect inoculated animals against the development of acute radiation syndromes. [ 31, 32 ]. Results: Animals that were inoculated with specific antiradiation antibodies Antiradiation Antidote IgG before receiving lethal irradiation at LD 100/30 exhibited 60-75% survival rate at 30 days, whereas all control animals expired by 30 days following exposure. These inoculated animals also exhibited markedly reduced clinical symptoms of ARS, even those that did not survive irradiation. [ 31, 36 ] Discussion:

10 The results of our experiments demonstrate that rabbit hyper-immune serum directed against SRD toxins afford significant, albeit incomplete, protection against high doses of radiation. In comparison, the mortality rate of irradiated control animals was 100% in the same time period. The mortality rates of hyper-immune serum-treated animals varied in different groups of animals and different forms of ARS. However, significant radioprotection was observed in each group treated with IgGs activated against specific radiation toxins. The survival rate was 80 % in groups of irradiated animals with Hematopoietic ARS, with Gastro Intestinal ARS. The survival rate was 55 -75 % in group with severe forms of Cerebrovascular ARS or Cardiovascular ARS. Countermeasure development: Antiradiation Vaccine Prophylaxis, prevention, treatment of biological sequelae after neutron irradiation. Introduction: The efficacy of an anti-radiation vaccine for the prophylaxis, prevention and therapy of acute radiation pathology was studied in a neutron exposure facility and compared to traditional radioprotectors. [ 6, 7, 8, 9, 18, 47,, 31, 32 39, 44, 44, 65, 76] The biological effects of fast neutrons include damage of the central nervous system and cardiovascular system with development of the cerebro-vascular form of acute radiation syndrome. [48, 65, 66, 67, 68] This is a result of the formation of neurotoxins in the central nervous system of irradiated animals after high doses of fast neutron exposure. Neutron irradiation generated cerebrovascular and cardiovascular radiation toxins (neurotoxins) which induce the development of clinical symptoms, syndromes and underlying patho-physiological processes. [56, 62, 63] Current methods of radiation protection and acute medical management are not effective against moderate and high doses of neutron irradiation. A novel vaccine against radiation-induced systemic toxins may be a more effective radioprotectant against high doses of neutron and gamma-radiation. The antigens for the vaccine are derived from neuro- and cyto-toxins isolated from the tissues of gammairradiated animals. Methods : 1) Prepare antiradiation vaccine: Standard Experimental Antiradiation Vaccine. 2) Neutron exposure at the Scientific Research Institute of Nuclear Physics, Dubna, Russia, employing the research reactor BBP-M, generating a mixed neutron beam contained 95 % fast neutron irradiation and 5% gamma-irradiation. Neutron energy: 1.98 2.30 Me- V energy; Dose/Dose rate 10.7 Gy, at 0.22 Gy/min. Table 5. Experimental Design and radioprotection efficacy after neutron irradiation. Groups: mammal in experiments Rabbits Group A Control: 5 rabbits Group B -Placebo: 5 rabbits Radioprotectants Doses of radioprotectants Time of administration: before or after irradiation - radiation naive Lethality rate/ survival period after irradiation. 100% survival 100% lethality after irradiation. Survival period two hours after irradiation 100% lethality after irradiation.

Without any intervention Standard placebo

Standard dose

Group C: 5 rabbits

Radioprotectant Cystamine

50 mg/kg

15 minutes before irradiation

11 Survival period 10 hours after 100% lethality after irradiation. Survival period 24 30 hours after irradiation. 100% lethality after irradiation. Survival period up to 10 days after irradiation.

Group D: 5 rabbits

Radioprotectant Mexamine

10 mg/kg

15 minutes before irradiation

Group E: 5 rabbits

Experimental Antiradiation Vaccine

1 ml first preexposure vaccination. 1 ml second preexposure vaccination

28 days before irradiation; 14 days before irradiation

Results: Group A: Control group with radiation nave animals which didnt underwent Neutron irradiation. Group B: 100% mortality within two hours after neutron irradiation with clinical symptoms of Acute Cerebrovascular Syndrome. Survival period after Neutron Irradiation - 2 hours following irradiation. Group C: 100% mortality within 8-10 hours following irradiation. Group D: 100% mortality within24 30 hours after irradiation. In groups B - D the development of Acute Radiation Cerebrovascular Syndrome produced rapid death. Group E - 100% mortality by 240 hours following neutron irradiation with animals exhibiting a combination or individual forms of Acute Cerebrovascular, Cardiovascular, and Gastrointestinal clinical syndromes. Discussion: Antiradiation Vaccine significantly prolonged the survival time of mammals after being exposed a high dose LD 100/30 with neutron radiation, from two hours in controls to 6-10 days. We postulate that the radiation toxins of SRD group isolated from the tissue or lymph of gamma-irradiated animals are similar in structure to radiation toxins generated and circulated in the blood and lymph of neutron-irradiated animals. Preliminary studies indicate that the toxico- kinetics and toxico-dynamics of radiation toxins generated following neutron-irradiation are similar to those following similar doses of gamma-irradiation. Antiradiation vaccine: Technology and development of prophylaxis, prevention and treatment of biological consequences from Heavy Ion irradiation. An anti-radiation vaccine could be an important part of a countermeasures regimen for effective radioprotection, immune-prophylaxis and immunotherapy of the acute radiation syndromes after heavy ion irradiation. Reliable protection of non-neoplastic regions of patients with different forms of cancer which undergo to heavy ion-therapy ( e.g. Hadron-therapy) can significantly extend the efficiency of the therapeutic course. [70, 71, 75, 77, 79 ]. The protection of cosmonauts, astronauts from the heavy ion radiation component of space radiation with specific immune-prophylaxis by the anti-radiation vaccine may be an important part of medical management for long term space missions. [31, 70, 71, 72, 73, 75, 77, 78 ]. Methods and experiments: The Antiradiation Vaccine preparation standard mixture of toxoid form of Radiation Toxins-SRD-group. Heavy ion exposure was accomplished at Department of Scientific Research Institute of Nuclear Physics, Dubna, Russia. The heavy ions irradiation was generated in heavy ion (Fe56) accelerator -UTI. Heavy Ion linear transfer energy -2000-2600 KeV mkm, 600 MeV U. Absorbed Dose - 2820 Rad.

12 Experimental Design: Rabbits from all groups were irradiated by heavy ion accelerator except group of experimental laboratory animals for control.

Table 6. Experimental Design and radioprotection efficacy after heavy ion irradiation. Groups: mammal in experiments Rabbits Group A control - 5 rabbits Group B placebo -5 rabbits. radioprotectants Doses of Time of radioprotectants. administration: before or after irradiation Without any protectants and iirradiation Standart Standart doses 15 minutes placebo of placebo before irradiation Lethality rate/ survival period after irradiation. Not lethality Specific clinical features -

100 % in first 15 - 24 hours

Group C 5 Cystamine rabbits radioprotectant C Group D 5 Gammafos rabbits: (Amifostine) radioprotectant

50 mg/kg

15 minutes before irradiation

400 mg/ kg

15 minutes before irradiation

Group E - 5 rabbits :

Experimental Antiradiation Vaccine.

1 ml first preexposure vaccination. 1 ml second pre-exposure vaccination

28 days before irradiation; 14 days before irradiation

100 % mortality in first 24 35 hour after irradiation 100 % mortality first 24 35 hour after irradiation 100 % mortality. Survival period up to 10 days.

Symptoms of Cerebrovascular, Cardiovascular extremely severe forms of ARS with extensive burns of skin Clinical picture same as in group B

Clinical picture same as in group B and C. Clinical picture include severe form of ARS.

In groups B-D, development of the acute radiation cerebrovascular and cardiovascular syndromes as well as extensive burns of skin caused rapid death. Group E -100% mortality in 280-290 hours (12 days) following heavy ion irradiation while animals were exhibiting a combination or individual forms of the

13 acute cerebrovascular, cardiovascular, and gastrointestinal forms and focal skin burns. Discussion: The Antiradiation Vaccine (ARV) and specific immune-prophylaxis are an effective method of neutralization of Radiation Toxins. Vaccination with the ARV significantly extended the survival time after irradiation with heavy ions from two hours up to 300 hours. Clinical signs, clinical features, symptoms were somewhat attenuated. Degree of clinical forms of the Acute Radiation Syndromes were diminished in their severity. Groups B-D demonstrated an extremely severe degree (Degree 4) of Cerebrovascular and Cardiovascular forms of the Acute Radiation Syndromes and lethality 100% was registered in a short time after irradiation. Radiation induced burns in this groups (with Cutaneous sub-syndrome of ARS Degree 4) that were deep with extensive and total dysfunction and possible muscle involvement developed. Animals from group E -Radioprotectant - Antiradiation Vaccine had demonstrated later development of the severe Degree 3 or even Degree 2-3 forms of Cerebrovascular and Cardiovascular forms of the ARS and a survival time of irradiated animals was significantly prolonged. Cutaneous subsyndrome developed in Degree 3 or Degree 2-3. Our results have demonstrated the potential radioprotection efficacy of specific immune-prophylaxis with the Antiradiation Vaccine against heavy ion irradiation. Countermeasure development: Specific Immune-prophylaxis and Immunotherapy of Combined Acute Radiation Syndromes. Combined Acute Radiation Syndromes (CARS) are extremely severe injuries. Combination of Radiation and Thermal factors induce development of the acute patho-physiological processes in mammals with severe form of injury, initiated by different types of physical factors. Both types of this types of injury share major patho-physiological processes like toxic systemic inflammatory response syndrome (TSIRS), toxic multiple organ injury (TMOI), toxic multiple organ dysfunction syndromes (TMOD), toxic multiple organ failure (TMOF). [2, 3, 23, 24, 25, 40, 41, 43, 44, 45, 62, 63]. Table 7. Classification of Radiation Toxins and Thermal Toxins. Radiation Toxins [ ]. Cerebrovascular Radiation Toxins - Nature of Toxins: Proteins with high enzymatic activity. Burn Toxins [ ]. Burn Toxins identified as lipid-protein complex in the cell-wall membrane which is present in nontoxic form in normal skin but is polimerazed by heat to a trimeric form with specific toxicity. Burn Toxins and precursor can be isolated from the serum of burned human patients. The toxins can be used for active immunization and production of antiburn serum. Antitoxic IgG effective treatment

Cardiovascular Radiation Toxins Nature of Toxins: Proteins with high enzymatic activity Gastrointestinal Radiation Toxins - Nature of Toxins: Proteins with high enzymatic activity Hematopoietic Radiation Toxins - Nature of Toxins: Proteins with high enzymatic activity

High doses of Radiation induce formation of Specific Radiation Toxins (SRT) which include four major group of Toxins: Cerebrovascular Radiation Toxins (Cv RT), Cardiovascular Radiation Toxins (Cr RT), Gastrointestinal Radiation Toxins (Gi RT), and Hematopoietic Radiation Toxins (Hp RT). CvRT, Cr RT, Gi RT groups of toxins are defined as Neurotoxins and Hp RT group is defined as Hematotoxins. [ 62, 63]. Thermal injury induce formation of Specific Burn Toxins which are highly toxic and induce systemic reactions. Circulated Specific Burn Toxins protein lipid complex pathogenic effect of SBT most probably due to generalized damage to the cell wall membranes and promote septicemia. [40, 41, 44].

14 Radiation injury induce formation of Specific Radiation Toxins which are highly toxic and also induce systemic reactions. Circulated Specific Radiation Toxins could be presented by several groups of biological active molecules such as proteins with high enzymatic activity, polypeptides and protein lipid complex. [62, 63, 56 ]. Specific Radiation Toxins (SRT) present in inactive form in almost all cells and after activation can induce specific processes and reactions programmed cell death or apoptosis or necrosis - terminology apoptotic necrosis should be considered. [ 62, 63]. Administration of Specific Burn Toxins (IV or IM) to healthy mammals induces development of lymphocytosis, leukocytosis, trombocytosis, and activation of blood coagulation cascade. Administration of Specific Radiation Toxins (IV or IM) to radiation naive animals induces leukopeina, thrombopenia, lymphopenia as a result of clonogenic programmed cell death. Blood coagulation cascade suppression is registered. [ 62, 63]. Materials and Methods: Rabbits, white rats, mice were used for different stages of our experiments. Animals were quarantined at laboratory conditions for three weeks prior to experimentation. Isolation of the SRT was provided from the central lymphatic duct of irradiated cows. Immunization of horses and rabbits to obtain Antiradiation Antibodies (Specific Antiradiation Antidote SAR) was provided. Animals in our experiments: rabbits, mice, white rats were irradiated in the VSRI (Kazan), Academy of Veterinary Medicine (Moscow), Scientific Research Institute of Radiobiology (Gomel), Scientific Research Nuclear Center (Dubna). Equipment for gamma-irradiation: " Pyma", "Panorama" -Co gamma radiation source. Irradiation was performed by different doses corresponding to induction of severe forms of the Acute Radiation Syndromes (ARS). Mice and rats were receiving the combined radiation and thermal injury. Model of the thermal injury: Burns -10% of total body surface. Third grade of burns was used as a model. Thermal Injury was given after irradiation. Preparations of Antiradiation Vaccine - contained a toxoid form of Radiation Toxins were used for immune-prophylaxis. Preparations of Antiradiation Antidote IgG contained antibodies to Radiation Toxins were used for immune-therapy. Tabl. 8. Experimental Design and radioprotection efficacy after combined Acute Radiation Syndromes. Group A Control Group B Control Group C Control Group D. Experiment Animals with ARS Animals with the thermal injury Animals with combined injury radiation + thermal injury Animals with ARS Treatment without treatment Treatment without treatment Treatment without treatment Pre-exposure ( 24 days before irradiation) vaccination with Experimental Antiradiation Vaccine Vaccination with Experimental Antiradiation vaccine - 24 days before thermal injury. Vaccination with Experimental Antiradiation Vaccine - 24 days before combined injury. Specific Immune-therapy with Antiradiation Antidote IgG

Group E. Experiment

Animals with thermal injury.

Group F. Experiment

Animals with combined injury: radiation + thermal injury. Animals with combined injury: radiation + thermal injury

Group G

15 Group H Group I Control Animals with Acute Radiation Syndromes Animals with thermal injury Animals without interventions Specific Immune-therapy with Antiradiation Antidote IgG Specific immune-therapy with Antiradiation Antidote IgG Without any treatment

The Lethality Doses (LD) 100/30 of radiation caused 100 % mortality rate in next 30 days after irradiation with development of Acute Cerebrovascular and/or Cardiovascular forms of the ARS in all groups. The thermal injury induced the third degree burns with area of dry necrosis in Group B. Mortality rate in this group with thermal injury without treatment was 100 % within next 30 days. Lethality rate at Combined Radiation and Thermal injury without any treatment in group C was 100 % within next 30 days mostly after seven days after intervention. Immune-prophylaxis by the specific AV was most effective for animals with the ARS and survival rate was up to 70 %. Immune-therapy by the specific AA IgG demonstrated less effectiveness and survival rate was about 65% in different groups of irradiated animals with lethal doses. For animals with the thermal injury only, immune-therapy by the Antiradiation Vaccine and immuneprophylaxis by Antiradiation Antitoxin IgG were in-effective and the survival rate had not exceeded 15 %. Results of specific immune-therapy and immune-prophylaxis provided at combined radiation thermal injury (CRTI) had demonstrated 35% of survival rate. Conclusion: Effects of Different Biological Response to specific immune-prophylaxis with Antiradiation Vaccine and specific immune-therapy with Antiradiation Antitoxin IgG had demonstrated effective radioprotection for irradiated animals with different forms of the ARS. The recovery phases demonstrated a shorter period of reconvalescence. Effects of the specific immune-prophylaxis by the AV and immunetherapy by AA IgG provided for animals with thermal and combined injury were less effective although probably useful. Immune-prophylaxis and Immune-therapy by the Specific Immune-modifiers used at combined and Thermal injury demonstrated a prolonged life time after immune-prophylaxis. Demarcation zone of burns and necrotic tissues rejection were more expressed after immune-therapy. Specific Immune-prophylaxis with the Thermal Injury Toxins and Specific Immune-therapy with the specific anti-thermal antibodies (serum of IgG preparation) can significantly improve results of therapy of thermal and combined injury. [ 15, 40, 41, 62, 63 ]. Conclusions and recommendations. Countries with advanced nuclear power industry coordinates important strategic research projects that support Countermeasure development. [13, 30, 31, 32, 39, ] In Europe the Commission of the European Communities accepted and supported a Concerted Action called Medical Treatment Protocols for Radiation Accident Victims as a Basis for a Computerised Guidance System, in short METREPOL. Main purposes of this interdisciplinary project was to develop a new approach in the medical management of radiation accidents with respect to diagnostic procedures and therapeutic options based on the recognition and evaluation of health impairments after acute radiation exposure [ 93, 94]. The U.S. government has taken significant steps toward developing and acquiring vaccines, drugs, and other medical countermeasures (MCMs) to protect and treat the population after a biological attack. [ 6, 7, 8, 9, 13, 19, 30, 34, 37]. As a part of Global System Countermeasures three advanced medical countermeasures were successfully developed. [13, 93, 94] Medical Countermeasure number One is a Differential Diagnosis of Acute Radiation Syndromes by Enzyme Linked Immune Assay (ELISA) as effective diagnostic procedure. [64].

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