РАДИАЦИОННАЯ БИОЛОГИЯ. РАДИОЭКОЛОГИЯ, 2007, том 47, № 3, с. 286-291
PROCEEDINGS OF THE 4TH INTERNATIONAL WORKSHOP ON SPACE RADIATION RESEARCH = AND 17TH ANNUAL NASA SPACE RADIATION HEALTH =
INVESTIGATORS' WORKSHOP (Moscow-St.-Petersburg, June 5-9, 2006)
УДК 599:539.1.047
MECHANISMS OF ACTION FOR AN ANTI-RADIATION VACCINE IN REDUCING THE BIOLOGICAL IMPACT OF HIGH DOSE AND DOSE-RATE, LOW-LINEAR ENERGY TRANSFER
RADIATION EXPOSURE
© 2007 V. Maliev1, D. Popov2*, R. C. Casey3, J. A. Jones4
1 Vladicaucasian Scientific Center, Russian Academy of Sciences, Biotechnology Departament, Russia 2 Advanced Medical Technologies Systems, Toronto, Canada 3 Universities Space Research Association, Houston, TX 4 NASA-Johnson Space Center, Houston, TX
The development of an anti-radiation vaccine could be very useful in reducing acute radiation syndromes. Existing principles for the treatment of acute radiation syndromes are based on the amelioration of progressive pathophysiological changes, using the concept of replacement therapy. Active immunization by small quantities of the essential radiation-induced systemic toxins of what we call the Specific Radiation Determinant (SRD) before irradiation increased duration of life among animals that were irradiated by lethal or sub-lethal doses of y-radiation. The SRD toxins possess antigenic properties that are specific to different forms of acute radiation sickness. Intramuscular injection of larger quantities of the SRD toxins induce signs and symptoms in irradiated naive animals similar to those observed in acute radiation syndromes, including death. Providing passive immunization, at variable periods of time following radiation, with preparations of immune-globulins directed at the SRD molecules, can confer some protection in the development of clinical sequelae in irradiated animals. Improved survival rates and times were observed in animals that received lower, sublethal doses of the same SRDs prior to irradiation. Therefore, active immunization can be induced by SRD molecules as a prophylaxis. The protective effects of the immunization begin to manifest 15-35 days after an injection of a biologically active SDR preparation. The SRD molecules are a group of radiation toxins with antigenic properties that correlate specifically with different forms of radiation disease. The SRD molecules are composed of gly-coproteins and lipoproteins that accumulate in the lymphatic system of mammals in the first hours after irradiation, and preliminary analysis suggests that they may originate from cellular membrane components. The molecular weight of the SRD group ranges from 200-250 kDa. The SRD molecules were isolated from the lymphatic systems of laboratory animals that were irradiated with doses known to induce the development of cerebral (SRD-1), non-specific toxic effects (SRD-2), gastrointestinal (SRD-3) and hematological (bone marrow) (SRD-4) syndromes. Our results suggest that an anti-radiation vaccine can be developed for prophylactic use against radiation damage induced by acute exposure to significant doses of low Linear Energy Transfer (LET) radiation for humans, including nuclear power workers, commercial and military pilots, cosmonauts/astronauts, nuclear-powered engine vessel operators and possibly even the civilian population in the case of a nuclear terrorism event.
APC: antigen presenting cells; ARS: acute radiation syndrome; SRD: specific radiation determinant; SAV: specific anti-radiation vaccine; SRD-1: cerebral acute radiation syndrome; SRD-2: Specific acute radiation toxic effects; SRD-3: gastrointestinal acute radiation syndrome; SRD-4/1: mild typical ARS; SRD-4/2: moderate typical ARS; SRD-4/3: severe typical ARS; SRD-4/4: extremely severe typical bone marrow-ARS.
The goal of this research was the investigation of a group of radiotoxic molecules isolated from the lymph of irradiated animals. A group of radiotoxins, each with different biological activities were produced and collected from the lymph of animals with different doses
* Corresponding Adress: 82 Cozens Dr., Richmond Hill, Ontario, L4E4w8, Canada; ph.: 905-223-3879; e-mail: dlpopov@fci-broadband.com.
of low LET radiation. The isolation of these radiotoxic molecules, henceforth, called Specific Radiation Determinants (SRD), allowed researchers to reproduce animal models of the different radiation syndromes, without irradiation. Several SRD radiotoxins have been identified as the products of radiation-induced biochemical cross-linkage of cellular components. Perhaps more importantly, these radiotoxins play important roles in both the development and potential pro-
phylaxis of Acute Radiation Syndrome (ARS). The aim of this research was to isolate antigens that specifically drive the development of ARS and determine their efficacy as prophylactic measures against radiation injury [11, 17, 18]. To do this, we developed an ELISA for the serological diagnosis of the different syndromes of ARS, in addition to developing the SRDs as a vaccine to provide prophylaxis for the development of radiation-induced pathology.
MATERIAL AND METHODS
Our study includes the following experimental animals listed in Table 1. The experimental group consisted of animals with normal blood profiles and body temperatures, whose homeostatic constants of central lymph did not exceed the limits of normal variability. Mild, moderate, severe, and extremely severe acute radiation sickness of the hematological form, as well as the gastrointestinal, toxic and cerebral acute radiation syndromes, were induced in the experimental groups of animals. The animals were exposed to y-rays, based on body weight of the animals in doses up to 10 Gy, and were irradiated in RUM-17, Puma, and Panorama devices. The exposure dose rate ranged from 3-29 Ampere/kg. On the day preceding radiation exposure, and also 15, 30, and 45 days post-exposure, a lympho-venous anastomosis was created surgically.
Gel filtration and high-performance liquid chroma-tography were used to extract the immunochemical specific radiation determinant (SRD) from the central lymph of animals with cerebral (SRD-1), toxic (SRD-2), gastrointestinal syndromes (SRD-3) and hematological (bone marrow) (mild-SRD-4/1, moderate SRD-4/2; severe SRD-4/3; and extremely severe SRD-4/4) forms of ARS [11, 19].
The vaccine was produced from lyophilized SRD-1 or SRD-4/4 (isolated from the lymph of animals irradiated at doses inducing cerebral and extremely severe ARS), which were dissolved in an isotonic solution of NaCl. The dose of administered was based on computation of the amount of SRD per unit volume of central lymph and absorbed dose of radiation.
RESULTS
The radiomimetic properties of SRD preparations were evaluated on their ability to induce either positive or negative radiobiological effects in animals after they were administered parenterally. Symptoms of ARS that were induced with SRD-1 and SRD-4/4 in radiationnaive animals are comparable with similar symptoms induced by irradiation. Table 2 describes the composition of SRD-3 and SRD-4/4 i solated from the central lymph of animals with the gastrointestinal and extremely severe forms of ARS. Although both compounds have similar compositions, there are also distinct differences. SRD-4/4 c ontains more protein and less lipid and mineral than SRD-3, while both contain
Table 1. Experimental animals
Species Age Weight N
Black motley cattle 2.5-3.0 years 3QQ-35Q kg 134
Ukrainian pigs 6-12 months 35-9Q kg 142
Prekos sheep 3-12 months 1S-23 kg 156
Mixed breed dogs 2-4 years 6.Q-6.5 kg 162
Chinchilla rabbits 11-12 months 3.5-3.7 kg 1SQ
Latvian draft horses 3-8 years 35Q-55Q kg 32
Balb mice 2-3 months 2Q-22 g 2.636
Wistar rats 3-4 months 1SQ-22Qg 4.QQ2
Table 2. Macromolecular composition of SRD-3 and SRD-4/4
Component Percentage
SRD-3 (gastrointes- SRD-4/4 (extremely
tinal ARS) severe ARS)
Protein 5Q.1 ± Q.Q9 56.2 ± 0.12
Lipid 3S.2 ± Q.Q4 30.1 ± 0.09
Carbohydrate 1Q.2 ± Q.Q3 10.1 ± 0.07
Mineral 1.3 ± Q.Q4 3.4 ± 0.17
similar percentages of carbohydrate (Table 2). This suggests a relative homogeneity of the SRD preparations, and according to computations, the molecular mass of the isolated determinants were on the order of 200-250 kDa. Because the SRDs were discovered in the lymph tissues, we hypothesize that they are disseminated throughout the body via lymphatic system transport.
Table 2 shows macromolecular composition of SRD-3 and SRD-4/4.The high immunogenicity and specificity of the isolated SRD preparations allowed us to use them as antigens in experimental anti-radiation vaccines and to develop an enzyme-linked immune assay to evaluate the severity of radiation injuries in the exposed animals. The ELISA assay allowed the detection of "radiation" antigens (SRD 1-4) in the blood serum, urine, and organs of animals during the first 2 months after radiation exposure (Table 3) and the "radiation-induced" antibodies to the SRDs over the subsequent 2 years (observation period). The chemical composition of isolated determinants of SRD-3 and SRD-4/4 appeared to be mainly conjugated proteins with prosthetic groups containing lipid and carbohydrate components. This type of conjugated protein is typically found in plasma membranes and the intracel-lular biomembranes of the nucleus, mitochondria, and microsomes. In addition, conjugated proteins are also present in a free state in physiological transport media. Membrane lipids are vulnerable to oxidative damage [1-8]. The destructive action of radiation on biological macromolecules of the membrane can be direct as well
Table 3. The time required to develop maximum level of SRD depends on type/severity of acute radiation sickness
Type and severity of ARS
Time (hours) required for peak SRD levels after radiation
Rabbits
Dogs
lymph blood lymph blood lymph blood
Typical mild (SRD-4/1) 3-8 6-10 5-8 20-40 3-12 6-15
Moderate (SRD-4/2) 3-10 6-10 3-10 15-50 3-14 8-15
Sever
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