THE INFLUENCE OF THE WAR FACTOR ON AGE CHANGES IN THE SUBPOPULATION STRUCTURE OF LEUCOCYTES AS A COMPONENT OF INFLAMMAGING IN THE GROUPS OF MILITARY PERSONNEL AND CLEAN-UP WORKERS OF THE CHORNOBYL ACCIDENT. / ВПЛИВ ФАКТОРА ВІЙНИ НА ВІКОВІ ЗМІНИ СУБПОПУЛЯЦІЙНОЇ СТРУКТУРИ ЛЕЙКОЦИТІВ ЯК СКЛАДОВОЇ ІНФЛАМЕЙДЖИНГУ У ГРУПАХ ВІЙСЬКОВОСЛУЖБОВЦІВ ТА УЧАСНИКІВ ЛІКВІДАЦІЇ НАСЛІДКІВ АВАРІЇ НА ЧАЕС.

OBJECTIVE: to evaluate the subpopulation structure of peripheral blood leukocytes as a component of inflammaging in the servicemen of the Armed Forces of Ukraine and clean-up workers of the accident at the Chornobyl nuclear power plant. MATERIALS AND METHODS: Three hundred ninety-seven males aged 30-82 (54.99 ± 8.85) years examined. Among them: clean-up workers of the accident at the Chornobyl nuclear power plant, aged 54-64 (59.95 ± 1.88) years, who were examined before the beginning and during the Russian military invasion of Ukraine; 24 civilians of the control group, aged 50-77 (58.87 ± 7.18) years and 15 persons of the age control group, aged 42-49 (46.00 ± 2.26) years, who examined before the war has started; the comparison group-W - 27 civilians (age: 51-82 (65.44 ± 9.13)) and the age control group-W - 14 civilians (age: 31-49 (40.64 ± 5.59)) who examined after the war has started; 73 servicemen of the Armed Forces of Ukraine aged 30-49 (42.20 ± 5.35) years and 54 servicemen aged 50-59 (54.07 ± 2.86) years. The subpopulation distribution of peripheral blood leukocytes was analyzed using flow cytometry. RESULTS: A decrease in the relative number of CD45+ 14- granulocytes, CD3+ HLA-DR+ T-lymphocytes, and CD3-57+ NK against the background of an increase in the number of CD45+ 14+ monocytes, CD3-HLA-DR+ B-lymphocytes found in the majority of people in the examination groups, the study of which conducted after the beginning of the war, CD4+ T-lymphocytes and CD3+ 57+ NKT. The relative number of CD45+ 14- granulocytes increased against the background of a decrease in CD45+ 14- lymphocytes was noted in clean-up workers who were examined before the war started.About 30% of civilians over 50 years of age who were examined before the war started had a decrease in CD19+ B- lymphocytes, CD4+ and CD8+ T-lymphocytes, CD3-57+ NK, CD3+ 57+ NKT, and the CD4+ / CD8+ ratio. Conducted correlation analysis revealed a weak to moderate inverse correlation between the relative number of CD19+ B-lymphocytes and age in civilians, regardless of when they were examined, and the servicemen of the Armed Forces of Ukraine.Inverse correlations of HLA-DR+ B-lymphocytes, CD45+ 14+ monocytes, CD4+ T-lymphocytes, CD4+ / CD8+ ratio, and CD3-57+ NK and CD3+ 57+ NKT with age were noted in civilians who were examined after the war has started. A direct correlation between CD3+ 57+ NKT and age was found in the servicemen of the Armed Forces of Ukraine. CONCLUSIONS: The immunological profile of both clean-up workers in the Chornobyl nuclear power plant and the servicemen of the Armed Forces of Ukraine or civilians was similar compared to the group with reference indicators. Anintergroup study revealed changes in the subpopulation distribution of peripheral blood leukocytes, which may be associated with both aging and changes in living conditions during military operations. Specific changes in cellular immunity can either tip the balance towards inflammaging or indicate already existing immunopathologicalreactions.

IMMUNOPHENOTYPE OF LEUKEMIC CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS WITH NOTCH1 AND SF3B1 GENE MUTATIONS.

BACKGROUND: The typical chronic lymphocytic leukemia (CLL) immunophenotype is vital for diagnosis, but the expression of some antigens varies and has prognostic value. There are data that reduced CD20 expression is associated with NOTCH1 and SF3B1 gene mutations. AIM: To determine a high-risk group of CLL patients for prediction of unfavorable NOTCH1 and SF3B1 gene mutations based on immunophenotyping of leukemic cells. MATERIALS AND METHODS: Flow cytometric and molecular-genetic analysis (mutations of NOTCH1, SF3B1, and TP53 genes using the polymerase chain reaction followed by direct sequencing) was performed in a group of 86 previously untreated CLL patients. RESULTS: The immunophenotype of leukemic cells of all examined patients met the criteria of CLL diagnosis. NOTCH1 gene mutations were found in 21 patients (24.4%), and SF3B1 gene mutations - in 7 patients (8.1%). There were no TP53 gene mutations among the examined patients. A decreased number of CD20+CD5+ cells and a downward trend in the relative index of mean fluorescence intensity (iMFI) of CD20+ cells were found in patients with NOTCH1 and SF3B1 gene mutations. Based on the iMFI level (higher and/or lower than 3.0) and the number of CD20+CD5+ cells among all B-cells (higher and/or lower than 50%), we distinguished CLL cases with low and relatively high levels of CD20 antigen expression. Using ROC analysis and the parameter of low CD20 antigen expression, we could predict the presence of NOTCH1 and SF3B1 gene mutations in 73.3 ± 0.06% of patients (p = 0.001). The risk of NOTCH1 and SF3B1 gene mutations in cases with low CD20 antigen expression was 6.96 (95% CI = 2.53-19.18; p = 0.0001). The revealed regularities were statistically significant for patients in whom the diagnosis was established in all Binet - Rai stages except A0-AI. CONCLUSION: Our data confirmed a reduced CD20 expression in CLL patients with NOTCH1 and SF3B1 mutations. In addition, an approach was proposed to identify high-risk CLL patients for prediction of such mutations: previously untreated CLL patients at advanced Binet - Rai stages (BII, CIII, CIV) with a reduced number of double-positive CD20+CD5+ cells in peripheral blood and/or low iMFI of CD20+ cells.

STATUS OF THE CELLULAR IMMUNITY IN CLEAN-UP WORKERS OF THE CHORNOBYL ACCIDENT AND PATIENTS WITH CANCER OF THE MOUTH CAVITY, ORAL AND LARYNGEAL PART OF THE PHARYNX. / STAN KLITYNNOGO IMUNITETU V UChASNYKIV LIKVIDATsIÏ NASLIDKIV AVARIÏ NA ChAES TA KhVORYKh NA ZLOIaKISNI NOVOUTVORENNIa POROZhNYNY ROTA, ROTOVOÏ TA GORTANNOÏ ChASTYN GLOTKY.

OBJECTIVE: to establish cellular immunity in clean-up workers of the Chornobyl accident compared to patients with malignant neoplasms of the oral cavity, oral and laryngeal parts of the pharynx according to the subpopulation organization of peripheral blood leukocytes. MATERIALS AND METHODS: We examined 112 males, age (56,92 ± 6,17) years (M ± SD), including 26 male clean-up workers exposed at the radiation dose 10-500 mSv; 20 male clean-up workers exposed at the dose range 504-990mSv; 33 patients with malignant neoplasms of the oral cavity, oral and laryngeal parts of the pharynx and 33 non-exposed subjects of the control group. Immune cell subsets analysis was performed by the expression of differential and activation antigens of peripheral blood leukocytes using flow cytometry. RESULTS: In assessing each group's cellular immunity, there was a decrease in the number of CD19+ B-lymphocytes, CD3+ HLA-DR+ T- and CD3- HLA-DR+ B-lymphocytes, CD3- 16+ 56+ natural killer cells, combined with an increase in the number of CD8+ T-lymphocytes. An increase in the relative number of CD4+ CD8+ T-lymphocytes was determined in clean-up workers (D < 500 mSv) and cancer patients. An increase in the number of CD8+ T-lymphocytes and a decrease in the number of CD4+ T-lymphocytes were observed in clean-up workers (D > 500 mSv), as well as in cancer patients. In addition, a decrease of monocytes, CD3+ 16+ 56+ , and CD3+ TCRαß+ T-lymphocytes was determined in patients with oncological pathology. CONCLUSIONS: The obtained results show the unidirectionality of changes in cellular immunity in the participants of the liquidation of the consequences of the accident at the Chornobyl nuclear power plant and patients with the investigated oncological pathology, which indicates the formation of persistent violations of antitumor protection in the participants of the liquidation of the consequences of the accident as the basis of oncogenesis. Determining changes in the number of lymphocytes, monocytes, granulocytes, CD4+ T-lymphocytes, CD4+ CD8+ immature T-lymphocytes, the immunoregulatory ratio CD4+ / CD8+, CD3+TCRαß+ T-lymphocytes and CD3+ 16+ 56+ CTL can have an additional effect on the effectiveness antitumor protection and the probability of oncogenesis risk in immunocompromised individuals.

СHARACTERISTICS OF CYCLIN D1-MEDIATED REGULATION OF CELL CYCLE OF PERIPHERAL BLOOD LIMPHOCYTES OF CHORNOBYL CLEAN-UP WORKERS AND PERSONS WITH MALIGNANT NEOPLASMS OF THE ORAL CAVITY, OROPHARYNX AND LARYNGOPHARYNX. / KhARAKTERYSTYKA TsYKLIN D1-OPOSEREDKOVANOÏ REGULIaTsIÏ KLITYNNOGO TsYKLU LIMFOTsYTIV PERYFERYChNOÏ KROVI UChASNYKIV LIKVIDATsIÏ NASLIDKIV AVARIÏ NA ChAES TA OSIB, KhVORYKh NA ZLOIaKISNI NOVOUTVORENNIa POROZhNYNY ROTA, ROTOVOÏ TA GORTANNOÏ ChASTYN GLOTKY.

OBJECTIVE: to explore proliferative potential of peripheral blood lymphocytes of Chornobyl clean-up workers and persons with malignant neoplasms of the oral cavity, oropharynx and laryngopharynx by level of expression of cyclin D1 and quantitative parameters of cell cycle. MATERIALS AND METHODS: A total of 294 men aged (58.47 ± 7.32) were surveyed, 215 of them were Chornobyl clean"up workers (1986-1987), exposed at the dose range 10.43-3623.31 mSv; 49 persons of the control group and 30persons with malignant neoplasms of the oral cavity, oropharynx and laryngopharynx at III, IVА and IVВ stages ofthe disease. The analysis of parameters of cell cycle and proliferative activity of peripheral blood (PB) lymphocyteswas performed using the flow cytometry. The evaluation of distribution of cells by G0/G1, S, G2/M cell cycle phaseswas done in vivo and in in vitro. Proliferative potential was analyzed by level of expression of cytoplasmic protein ofcyclin D1. RESULTS: Proliferative potential of PB lymphocytes of Chornobyl clean"up workers and persons with malignant neo"plasms of the oral cavity, oropharynx and laryngopharynx was assessed. An increase in the level of spontaneousсyclin D1 expression and disturbance of сyclin D1-dependent regulation of cell cycle of PB lymphocytes after mito"gen activation were determined in the Chornobyl clean-up workers. An increase in pool of cells in the S" and G2/M"phases of cell cycle was detected, which characterizes high proliferative potential of PB lymphocytes. These changesare most pronounced in the subgroup of persons with a radiation dose of D > 500 mSv, and in persons with oncolo"gical pathology. CONCLUSIONS: A positive linear dependence has been established between the radiation dose and the number of cellsin the S"phase of cell cycle in the subgroup of Chornobyl clean"up workers with a radiation dose of D > 500 mSv. The detected changes of cyclin D1-dependent regulation of cell cycle and proliferative status of lymphocytes depend on the radiation dose, can be a manifestation of genome instability and be a cause for risks of oncogenesis, in a remote period after radiation exposure.

CHANGES OF CYCLIN D1-DEPENDENT REGULATION OF CELL CYCLE IN PERIPHERAL BLOOD LYMPHOCYTES OF CHORNOBYL CLEAN-UP WORKERS AT A REMOTE PERIOD AFTER RADIATION EXPOSURE. / PORUShENNIa TsYKLIN D1-ZALEZhNOÏ REGULIaTsIÏ KLITYNNOGO TsYKLU LIMFOTsYTIV PERYFERYChNOÏ KROVI UChASNYKIV LIKVIDATsIÏ NASLIDKIV AVARIÏ NA ChAES U VIDDALENOMU PERIODI PISLIa OPROMINENNIa.

OBJECTIVE: To study proliferative potential of peripheral blood lymphocytes of Chornobyl clean-up workers by levelof expression of cyclin D1 and quantitative parameters of cell cycle at a remote period after radiation exposure. MATERIALS AND METHODS: The research subject was the peripheral blood lymphocytes (PB) of Chornobyl clean-upworkers 30-33 years after radiation exposure. A total of 207 men were surveyed, 164 of them were clean-up workers exposed in the dose range 10.43-3623.31 mSv and 43 persons of the control group. Analysis of proliferationpotential (cell cycle initiation) and cyclin D1 expression in PB lymphocytes were performed in vitro by a micro methodof whole blood leukocytes culture with phytohemagglutinine-P (PHA). Sample preparation was performed by a standard immunofluorescent assay for intracellular proteins using the FITC labelled Mouse Anti-Human Cyclin D1Antibody Set. Cell distribution by cell cycle phases studied by propidium iodide DNA staining and analysis onFACSCalibur laser flow cytometer in histogram mode with separation of G0/G1-, S- and G2/M-regions and Sub-G0/G1-region (apoptotic cells). RESULTS AND CONCLUSIONS: An increase in the level of spontaneous сyclin D1 expression and disturbance of сyclinD1-dependent regulation of cell cycle of PB lymphocytes after mitogen activation were determined in a remote period after radiation exposure. An increase in the level of cyclin D1 expression was accompanied by increase in pool ofcells in the S- and G2/M-phases of cell cycle which characterizes the high proliferative potential of PB lymphocytes.Mitogen-induced delay of cell cycle of lymphocytes in G1/S check point and reduction of S-phase was revealed.These changes are a manifestation of genomic instability caused by the effect of radiation and depend on the radiation dose. The results confirm the hypothesis about the significance of levels of cyclin D1 expression, as a criterion for manifestations of genome instability and risks of oncogenesis in a remote period after irradiation.

EXPRESSION OF BIOLOGICAL MARKERS INDUCED BY IONIZING RADIATION AT THE LATE PERIOD AFTER EXPOSURE IN A WIDE RANGE OF DOSES. / EKSPRESIIa RADIATsIINO-INDUKOVANYKh BIOLOGIChNYKh MARKERIV U VIDDALENOMU PERIODI PISLIa OPROMINENNIa U ShYROKOMU INTERVALI DOZ.

OBJECTIVE: To study radiation induced biological markers of the late period after exposure. SUBJECTS AND METHODS: A study was performed in 235 Chornobyl accident male clean-up workers exposed in 1986-1987 (doses of external exposure: (M ± SD: 419.48 ± 654.60; range 0.10-3,500 mSv); age 58,34 ± 6,57 years. Controlgroup included 45 non-exposed subjects (mean age: 50.60 ± 5.37 (M ± SD). Gene expression was performed by RT-PCR on 7900HT Analyzer using TLDA for BCL2, CDKN2A, CLSTN2, GSTM1, IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2,TERT, TNF, TP53, CCND1 genes. Relative telomere length (RTL) was analysed by flow-FISH; immune cell subsets,γ-H2AÐ¥ and CyclinD1 expression by flow cytometry. RESULTS: A statistically significant and dose-dependent decrease in expression of the BCL2, SERPINB9, CDKN2A, andSTAT3 genes was demonstrated in parallel to a dose-dependent overexpression of MCF2L and upregulation of TP53 (upto 100 mSv). IL1B expression was the highest in exposed to doses from 0.1 to 100 mSv with a negative correlationbetween at IL1B expression and CD19+3-, CD3-HLA-DR+, CD4+8- cell counts and CD4+/CD8+ ratio. Hyperexpression ofTNF gene in doses above 100 mSv to 1,000 mSv was shown, and in higher doses a combination of TNF downregula-tion with increase in IFNG gene expression were demonstrated with correlations with numbers of CD3+16+56+ andCD25+ lymphocytes and inhibition of expression CLSTN2. An increased expression of γ-H2AÐ¥ and Cyclin D1 corre-lated to radiation dose, telomere shortening to age and concommittant pathology. CONCLUSIONS: Cellular immunity, gene expression, telomere length, intracellular protein parameters are shown to beamong perspective biological markers at a late period after radiation exposure.

BIOLOGICAL MARKERS OF EXTERNAL AND INTERNAL EXPOSURE IN SHELTER CONSTRUCTION WORKERS: A 13-YEAR EXPERIENCE.

OBJECTIVE: The aim of this study is to investigate the cellular, molecular, genetic and non-invasive functional biomarkers of occupational irradiation in workers exposed to a combination of external gamma-radiation and incorporation of transuranium elements. RESULTS: A study was performed in 688 radiation workers of Shelter object conversion into a safe system with mean shift dose of external exposure of 26.06 mSv (range: 0.1-113.35) and risk of internal exposure with transuranium elements. Several biological parameters could serve as biomarkers of exposure at radiation doses below 100 mSv and even in 20-50 mSv interval. The parallel changes were shown in decline of brain electric activity, telomere length, differences in CCND1, CDKN1A, CDKN2A, VEGFA, TP53, DDB2 genes expression. An increase in counts of dicentrics, pair fragments and TCR-variant lymphocytes at doses over professional limits shows the need of biological dosimetry. The most sensitive markers include TCR-CD4+, γ-H2AX+ and CyclinD1+ cell counts. Implementation of flow cytometry approach for these markers enables quick obtaining of quantitative data. Confounding factors included respiratory function and smoking. The study of the radiation workers with the history of chronic exposure in radiation area during 3-5 years demonstrates changes of compensatory origin, i.e. absence of telomere shortening, increased number of NK-cells in combination with lower expression of intracellular γ-H2AX. CONCLUSION: This study confirms the presence of radiation-induced changes in gene regulation of cell proliferation, telomere function and apoptosis in radiation workers exposed to external and internal exposure at doses above professional limits and increase of compensatory changes.