[Assessment of the Status of Effector and Regulatory Components of Immune System in Chelyabinsk Region Residents Exposed to Radiation Due to Residence in the Area Contaminated as a Result of the Radiation Accident at Mayak PA and in Their Offspring].

Immune status was studied in the framework of the current work and the results of the analysis of concentration of 26 characteristic parameters of innate and acquired immunity in 140 individuals from 56 trios (fathers, mothers and their Ist generation offspring that were included in 2 groups) are presented. Fathers and mothers of the children under study in the main groip Were exposed due to a long-term residence (from childhood to maturity) in the areas of Chelyabinsk region contaminated as a result of the-accident at Mayak PA (contamination included long-lived isotopes - 9°Sr and, to a smaller extent, ¹³7Cs and ²³9Pu) and then migrated into Ozyorsk prior to the conception of their children (75 individuals, 33 family trios). Comparison group (control) included parents and their offspring who are Ozyorsk residents never residing in the areas contaminated by radionuclides (65 individuals, 23 family trios). All the investigated individuals from the compared groups were of the corresponding age and gender and had never worked at nuclear facilities. Blood samples were obtained in the periods that excluded oncological, acute infectious and inflammatory diseases of any acute stages of chronic processes. Concentration of immune cells was measured by flow cytometer (Beckman Coulter, USA) using special monoclonal antibodies of the same manufacturer in a licensed medical center "Familia" (Chelyabinsk). The objective of the work is to assess the immune status in parents who migrated from contaminated areas prior to the conception of children and in their 1st generation offspring not exposed to radiation. Alterations of the immune status in the form of increase or, to a smaller extent, decrease of concentration of lymphocytes with effector and/or regulatory functions (B-1, T-helpers, NK, T-NK, late precursors of T-1 and T-1 of late activation) in blood of exposed parents and their offspring were detected in comparison with the results in the control group; that could possibly be related to the stimulation effect of low doses that support activation, proliferation and development of compensatory imbalance in the immune system and immunodeficiency in parents of the main group and in their offspring. In order to reveal the mechanisms of the detected alterations the interrelation between immune damage and incidence, of diseases among the cohorts involved in the current work will be studied further.

[Radiobiological Human Tissue repository: progress and perspectives for solving the problems of radiation safety and health protection of personnel and population].

Radiobiological Human Tissue repository was established in order to obtain and store biological material from Mayak PA workers occupationally exposed to ionizing (α- and/or γ-) radiation in a wide dose range, from the residents exposed to long term radiation due to radiation accidents and transfer of the samples to scientists for the purpose of studying the effects of radiation for people and their offspring. The accumulated biomaterial is the informational and research potential that form the basis for the work of the scientists in different spheres of biology and medicine. The repository comprises 5 sections: tumor and non-tumor tissues obtained in the course of autopsies, biopsies, surgeries, samples of blood and its components, of DNA, induced sputum, saliva, and other from people exposed or unexposed (control) to radiation. The biomaterial is stored in formalin, in paraffin blocks, slides, as well as in the freezers under low temperatures. All the information on the samples and the registrants (medical, dosimetry, demographic, and occupational data) was obtained and entered into the electronic database. A constantly updated website of the repository was developed in order to provide a possibility to get acquainted with the material and proceed with application for biosamples for scientists from Russia and abroad. Some data obtained in the course of scientific research works on the basis of the biomaterial from the Repository are briefly introduced in the review.

[Variability of DNA simple sequence repeats in peripheral blood of humans subjected to prolonged exposures of ionizing radiation].

Long-term post-radiation changes in the level of microsatellite DNA polymorphism in peripheral blood of the male "Mayak" employees (Ozyorsk, Russia), who had been exposed to prolonged gamma-irradiation during professional activities, were studied. DNA samples were obtained from the Radiobiology Repository of Human Tissue (Southern-Urals Biophysics Institute FMBA) and used as templates for arbitrarily primed PCR. Comparative analysis of the obtained samples of DNA fragments showed a significant increase in the number of high-molecular fragments and reduction in the number of amplified low molecular weight DNA fragments in comparison with the control. However, a direct correlation of the level of DNA polymorphism with the accumulated total dose of radiation was not found. The study of the polymorphism of microsatellite DNA repeats can be used for qualitative assessment of the levels of genetic variability.

X-ray fluorescence microscopy for investigation of archival tissues.

Several recent efforts in the radiation biology community worldwide have amassed records and archival tissues from animals exposed to different radionuclides and external beam irradiation. In most cases, these samples come from lifelong studies on large animal populations conducted in national laboratories and equivalent institutions throughout Europe, North America, and Japan. While many of these tissues were used for histopathological analyses, much more information may still be obtained from these samples. A new technique suitable for imaging of these tissues is x-ray fluorescence microscopy (XFM). Following development of third generation synchrotrons, XFM has emerged as an ideal technique for the study of metal content, speciation, and localization in cells, tissues, and organs. Here the authors review some of the recent XFM literature pertinent to tissue sample studies and present examples of XFM data obtained from tissue sections of beagle dog samples, which show that the quality of archival tissues allows XFM investigation.

[Assessment of bronchopulmonary immunity status for Mayak PA workers at the late period after prolonged exposure].

Samples of induced sputum from 187 individuals were used to estimate the bronchopulmonary immunity status after a prolonged radiation exposure (120 individuals who are the Mayak main plant workers were exposed to combined internal a-radiation due to incorporated plutonium (239Pu) and external y-radiation during their career). The control group included 67 Ozersk residents of a corresponding age and gender, without any occupational exposure, who were examined at the same period. The immune system is the most important component in homeostasis. In occupational workers, the status of the systemic and bronchopulmonary immunity after 30-55 years of career was estimated by the level of regulatory proteins in the samples of blood and the induced sputum supernatant stored at low temperatures in freezers of the Radiobiological human tissue Repository, SUBI (interleukins IL-1beta, IFN-gamma, IL-12+p70, IL-15, IL-17A and growth factors EGF, TGF-beta1, FGF and PDGF-AA), as well as by the content of effector and regulatory lymphocytes in blood. After the examination period, each registrant was provided with dosimetry data: 239Pu body burden was from 0.03 kBq to 11.89 kBq, the absorbed dose to the lung from incorporated radionuclide - from 0.01 Gy to 1.38 Gy, and accumulated doses of external y-radiation during the occupational career - from 0.02 Gy to 7.91 Gy. In the case of the prolonged combined radiation, the tensioned mode of immunity cell link functioning and the increase of activated lymphocyte forms were detected. Comparison of correlation coefficients between the content oflymphocytes, the level ofinterleukins and growth factors and the dose load allowed us to find relation of these ratios to the type of exposure, Pu body burden, the absorbed 239Pu dose to the lung and the accumulated external dose. The reduced concentration of the main growth factors in the group of Mayak workers is the evidence for the declined control function of proteins and immunodeficiency. The analysis of the regulatory proteins content in blood and sputum following occupational exposure allowed us to find specific features of the protein expression in support of the local and systemic immune homeostasis.

[Delayed and transgenerational molecular and genetic effects of prolonged influence of ionizing radiation in nuclear plant workers].

Genome variability and changes in immune homeostasis, induced in man in the course of long-term industrial contact with ionizing radiation (IR) sources were studied by using unique biomaterials stored in the Radiobiological Repository for Human Tissues at the Southern Urals Biophysics Institute, FMBA. The biomaterials, peripheral blood samples and blood DNA were obtained from the "Mayak" PA employers occupationally exposed to prolonged external gamma-radiation and/or internal alpha-radiation from incorporated 239Pu in a wide range of accumulated doses. A significant increase in the polymorphism of microsatellite-associated peripheral blood DNA repeats was revealed in a group of persons with accumulated doses of external gamma-radiation above 2.0 Gy, as well as in the descendants of parents with preconceptive doses of higher than 2.0 Gy. In persons whose parents had a preconceptive dose above 2.0 Gy, an increase in the gene p53 mutation rate was observed, and descendants of persons with dose of 3.0 Gy and higher showed mtDNA heteroplasmy, regardless of the sex of an exposed parent. Changes in the expression of membrane markers for the effector and regulatory T-lymphocytes depending on radiation type and dose load were determined. The growth factor level variations (TGF-beta1, EGF, HGF, FGF) in peripheral blood serum in persons exposed to radiation from gamma- or alpha-sources, allow us to consider them as biomarkers of radiation-induced disturbances in immune homeostasis. The concentration changes of TGF-beta1, apoptosis proteins (p53, TPA-cyk, sAPO-1/Fas), and the adhesion molecule sCD27 in the case of cardiovascular diseases in the serum of both irradiated and non-irradiated "Mayak" PA employers point to the information value of these immune response characteristics as specific biomarkers of cardiac disorders. It is proposed that the revealed changes in immune homeostasis and in the variability of somatic cell genome may provoke development of tumors and cardiovascular diseases in man in delayed periods after prolonged exposure to IR.

[Comparative study of hazards of energy plutonium and standard Pu-239 using non stochastic effects]. / Sravnitel'naia opasnost' énergeticheskogo plutoniia i standartnogo 239Pu po nestokhastichesksim éffektom.

In experiment with rats it was found that the "energy" Pu is more toxic than standard 239Pu when entered endotracheally. The comparison was made by the non-stochastic effects. The toxicity in respiratory system and blood system was 1.8 and 1.6 times higher when calculated per 1sGy of absorbed dose and 1.6 and 1.1 times higher when calculated per 1kBg/kg of the amount taken, respectively.

[State of cellular immunity effectors of the lung in inhalation exposure to low-transportable Pu-239]. / Sostoianie populiatsii éffektorov kletochnogo immuniteta legkikh pri ingaliatsionnom postuplenii nizkotransportabel'nogo 239Pu.

The reduction of populations of lung cell immunity effectors as well as changes in their cytotoxic and phagocytic activity was observed in rats after inhalation exposure to polymeric 239Pu. Dose-response curves described as a function of absorbed dose of alpha-irradiation in lung.

[Modifying effect of tributylphosphate (TBP) and hexachlorbutadien (HCLB) on non-stochastic effects of inhaled Pu-239]. / Modifitsiruiushchee vliianie tributilfosfata (TBF) i geksakhlorbutadiena (GKhBD) na nestokhasticheskie éffecty ot ingaliruemogo 239 Pu.

It has been established in experiments on rats that under combined inhalation exposure to 239Pu along with TBP and HCLB in amounts which may cause the changes in blood and immune systems the enhancing of different non-stochastic effects on the average by two times on a basis of a unit absorbed dose in comparison with isolated nuclide inhalation.

[An ultramicro method for determining pyrophosphatase using malachite green B]. / Ul'tramikrometod opredeleniia pirofosfatazy s pomoshch'iu malakhitovogo zelenogo B.

A modified sensitive micromethod is described for estimation of pyrophosphatase activity both in homogenate and suspensions of nuclear and mitochondrial fractions of rat liver tissue using a kit of reagents containing malachite green B. The analysis may be carried out in the preparation containing only 0.2 mg of biological material; the procedure is reproducible, rapid and allows to the requisite amount of reagents to be decreased. The activity of pyrophosphatase in homogenate, nuclear and mitochondrial fractions of rat liver tissue was higher at acid pH values as compared with that in the neutral medium; the highest enzymatic activity was detected in the nuclear fraction.

[The effect of granulocyte-macrophage colony-stimulating factor on hemopoiesis recovery and the survival of irradiated mice]. / Vliianie rekombinantnogo granulotsitarno-makrofagal'nogo koloniestimuliruiushchego faktora na vosstanovlenie krovetvoreniia i vyzhivaemost' obluchennykh myshei.

A human recombinant granulocytic-and-macrophagic colony-stimulating factor (rGM-CSF) administered repeatedly to irradiated (10 Gy) CBA mice increased CFUs and CFU-GM content, the number of bone marrow granulocytes and erythronormoblasts, and spleen and peripheral blood cellularity. The survival rate of exposed (9.7 Gy) mice repeatedly injected with rGM-CSF increased from 25% (control) to 90%.

[The cytotoxic activity of natural killer cells exposed to the joint and separate actions on the body of 239Pu, hexachlorobutadiene and tributyl phosphate]. / Tsitotoksicheskaia aktivnost' natural'nykh killerov pri sovmestom i razdel'nom deistvii na organizm 239Pu, geksakhlorbutadiena i tributilfosfata.

Wistar rats were subjected to a single exposure to 239Pu nitrate through inhalation with the subsequent procedure of imitation of inhalation or without it (the amount of 239Pu deposited in the lungs in 24 hr was 8.9 +/- 1.9 kBq/lung) and inhalation of hexachlorobutadiene and tributyl phosphate within one month in a combination with the radionuclide or without it. There was a 1.5-fold increase, above additive, in the harmful effect of 239Pu and chemical agents on the function of natural killers as observed 15--30 days after the combined exposure as compared to individual inhalation. On days 120 to 240 cell cytotoxic activity in rats of all groups was normalized to reach or to exceed the intact control.

[The immunity indices of rats after the long-term action of tritium oxide or gamma irradiation]. / Pokazateli immuniteta u krys posle dlitel'nogo deistviia okisi tritiia ili gamma-oblucheniia.

In experiments with Wistar rats, a study was made of the content of antibody-forming cells and cytotoxic activity of normal killers after long-term administration of tritium oxide (3HOH) (370 kBq.g-1 of body mass daily, cumulative dose, 8.1 Gy, and dose rate, 8.5 cGy/day), and after gamma irradiation with corresponding doses. The long-term radiation effect caused a decrease in the immunity indices: the impairment of the immune reactions was more pronounced after the effect of 3HOH than after gamma irradiation. Damages to the immune system of mice and rats after irradiation with similar doses were compared.

[The significance of immunity disorders in the development of tumorous effects during the long-term action of tritium in mice]. / Znachenie narushenii immuniteta v razvitii opukholevykh éffektov pri dlitel'nom deistvii tritiia u myshei.

In experiments with CBA mice it was shown that the long-term influence of tritium oxide administered with drinking water daily during 180 days (370 kBq.g-1 of body mass, cumulative dose 8.7 Gy, dose rate 4.5 cGy.day-1) causes the development of immunity deficiency 90 days after the onset of the administration that persists for up to 270 days. There is a 34% decrease in the average life of irradiated animals and an increase in the number of malignant neoplasms (postmortem examination was performed after natural death or killing on days 250, 350 and 450). There is a direct relationship between the occurrence of malignant neoplasms and the immunity deficiency.

[The early changes in humoral immunity under the prolonged action of tritium oxide with different dose rates]. / Rannie izmeneniia gumoral'nogo immuniteta pri dlitel'nom deistvii okisi tritiia s raznoi moshchnost'iu dozy.

Within the dose range from 0.2 to 1 Gy, early changes in the humoral immunity of mice exposed to tritium oxide at varying dose rates have been investigated. The study of the immunity impairment at different stages of immunopoiesis permits to reveal the points (a lymphocyte precursor department) that are mostly affected by radiation, to find the causes of the decrease in the antibody production, and to reveal the relationship between the damages observed the dose absorbed and dose rate of beta radiation.

[The antibody-producing cell count of rats exposed to polymeric 239Pu]. / Soderzhanie antiteloobrazuiushchikh kletok u krys pri deistvii polimernogo 239Pu.

In experiments with Wistar rats a study was made of the content of antibody-forming cells (AFC) in the spleen at remote times (3 to 12 months) after intravenous injection of 239Pu(IV) in doses of 166, 55, and 18 kBq/kg body mass. The doses absorbed in the central and peripheral immunity organs were defined. Pronounced spleen hypoplasia and profound inhibition of humoral immunity were displayed 1 year after the injection of a small amount of the radionuclide. AFC deficiency in animals was amounted to 11-32 per cent throughout the entire period of observation.

[Hematopoietic precursor cells in Wistar rats with acute myeloblastic leukemia]. / Krovetvornye kletki-predshestvenniki u krys porody Wistar s ostrym mieloblastnym leikozom.

The studies were performed on Wistar rats with acute myeloblastic leukemia induced by a prolonged administration of tritiated water (370 MBq/kg/day-1; an absorbed dose--25.3 Gy) and by repeated pyrogenal injections. A reduction of the pool of polypotent hemopoietic cells (CFUs) with a simultaneous increase of their proliferative activity and a decline of their ability to self-supporting were observed in the leukemia-bearing animals. Populations of CFUdc in DC and of CFUc were reduced in all cases; their colony-forming ability was also found to decrease. All animals with leukemia showed a lowered amount of CFU-BL in the bone marrow and of CFU-TL in the thymus, although the content of these cells in the peripheral blood remained unchanged.

[Effect of prolonged continuous external irradiation on humoral immunity indices of mice]. / Vliianie dlitel'nogo nepreryvnogo vneshnego oblucheniia na pokazateli gumoral'nogo immuniteta u myshei.

Changes in the content and function of cell populations and subpopulations involved in the humoral response of mice to the thymus-dependent antigen were investigated. The effect was followed during a prolonged continuous exposure to 137Cs gamma-emitter (total dose--5 Gy and daily dose--12 cGy for 22 hours) and after its termination. The data obtained give evidence for a decrease of the pool of polypotent lymphocyte precursors (CFUs), stable moderate hypoplasia of central and peripheral organs of the immune system, distinct inhibition of antibody production at the expense of reduced activity of precursors of lymphocytes, B-lymphocytes and T-helpers. In the remote postirradiation period residual radiation damage was seen in polypotent and committed precursors of lymphocytes and T-helpers which was responsible for the trend towards the decline of antibody production, hypoplasia in the spleen and lymph nodes being persistent.

[Cloning of murine B- and T-lymphocytes in vitro after the long-term action of tritium oxide]. / Klonirovanie B- i T-limfotsitov myshei in vitro posle dlitel'nogo deistviia okisi tritiia.

The cloning method was used to study the content of B- and T-lymphocyte committed precursors in central and peripheral organs of the immune system of mice at different times after long-term exposure to tritium oxide (a cumulative dose of approximately 9 Gy). It was shown that recovery of the colony-forming ability of the committed lymphocyte precursors was different in central and peripheral lymphoid organs; the dynamics and degree of restoration of the pools of B--(CFU--LB) and T--(CFU--LT) lymphocyte precursors were different.