[Influence of chronic irradiation on the distribution of blood lymphocyte subpopulations among professionals of the atomic industry].

The aim of the study was to investigate the effects of chronic exposure to ionizing radiation on the cellular immunity of employees of the nuclear industry. Peripheral blood samples were studied in 195 employees of Physics and Power Engineering Institute (PPEI, Obninsk), who professionallycontacted with sources ofionizing radiation and were under individual dosimetric control. The median cumulative dose was 61.2 mSv, the average duration of work at the enterprise -27 ± 5 years. The control group consisted of 57 healthy individuals of a similar age and sex who did not have contact with sources of radiation. Indicators of the cellular immunity were determined by flow cytometry. Comparison of a cell-mediated immunity was conducted separately in the two age groups (20-40 and 41-70 years). The significant reduction inthe relative content of CD4+CD8 T-helper cells and the increase in the relative content of CD3-CD16, CD56+ NK-cells were found in both age groups of the PPEI employees in comparison with the age-matched control groups (p < 0.05). Separate analysis of the results in the low dose group (up to 50 mSv) demonstrated reducing the relative content of T-helper cells and increasing the proportion of NK-cells (as in the analysis of whole groups without taking into account the cumulative dose), as well as reducing the proportion of CD8+CD25+ activated lymphocytes in PPEI employees as compared to the age-matched control. Multiple regression analysis of the immunological parameters dependence on age and dose established a significant correlation of the relative content of CD3-CD19+ B-cells (r = -0.284, p = 2.9 x 10(-4)) and CD19+CD5+ B1-lymphocytes (r = -0.241, p = 0.002) with the dose of employees regardless of age, indicating the relationship of the changes in the B-cell component of immune system with the radiation factor.

[Increase in the number of cancer stem cells after exposure to low-LET radiation].

Radioresistance of cancer stem cells (CSCs) is regarded as one of the possible causes of cancer recurrence after radiotherapy. Since the regularities and mechanisms of radiation effects on this population of cells have not been sufficiently studied, the aim of this work is to elucidate the changes in the CSC number after γ-irradiation in stable cultures of tumor cells in vitro and tumor tissue in vivo (in the course of radiation therapy of patients with cancers of the upper respiratory tract). CSCs were identified in the cell lines B16, MCF-7, HeLa by the ability to exclude the fluorescent dye Hoechst 33342 (SP method) 48-72 h after irradiation at the doses of 1-20 Gy and in biopsy material by immunophenotype CD44+CD24(-/low) before and 24 h after irradiation at the total dose of 10 Gy. The essential differences in the response of CSCs and other cancer cells were found after exposure to low-LET radiation. The absolute number of CSCs increased after a single exposure at the doses ranging from 1 to 5-10 Gy in different cell cultures, but a further dose increase maintained the current number of CSCs or decreased it. At the same time, the number of non CSCs significantly decreased with increasing doses of radiation exposure, as expected. Fractionated irradiation in vivo at a total dose of 10 Gy increased the relative amount of CSCs in most patients. The registered changes are an integral indicator of cell death, cell division delay immediately after irradiation, proliferation at a later time, possible dedifferentiation of non CSCs, etc. The exact contribution of each of them to the radiation-induced increase of the CSCs number is of considerable interest and requires further research.

[Influence of systemic photodynamic therapy on circulating tumor cells in peripheral blood of cancer patients].

The aim of this investigation is to study the number of circulating tumor cells (CTCs) in the blood of cancer patients after systemic photodynamic therapy (PDT) at different times and to assess apoptosis of these cells. The study group consisted of 19 patients with malignant tumors of epithelial origin at various stages (II-IV). CTC identification was performed with flow cytometry by immunophenotype Ep-CAM (CD326)+ CD45-. CTC apoptosis was identified by criteria of plasma membrane integrity and phosphatidylserine translocation on the outer surface of the membrane. Negative correlation between the CTC frequency and apoptotic death rate of these cells was found in patients before the treatment (R = -0.51, p = 0.03). CTC frequency gradually reduced during the first three days after PDT, and then it was maintained at the same level until the end of the follow-up (7 days). At the individual level, the effect of PDT depended on the frequency of CTCs before the treatment: the decrease in these cell frequency occurred significantly more often in the patients with an initially high frequency of CTCs than in other patients (p = 0.05). With the decrease in the CTC frequency, apoptotic death increased within 6 hours after the treatment and remained at the same level until the end of the follow-up period. The results demonstrate the efficacy of systemic PDT for elimination of tumor cells circulating in the peripheral blood of cancer patients with different localization of primary tumor and stage of disease.

[Radioresistance mechanisms of side population cells in mouse melanoma cell line B16].

As it was shown by us earlier, side population (SP) cells are more resistant to the low-LET radiation than the other part of mouse melanoma B16 cells (Matchuk et al., 2012). The aim of our research was finding some mechanisms of radioresistance, therefore we analyzed SP and nonSP cell cycle distribution, spontaneous and radiation induced DNA double-strand breaks (number of γH2AX foci) and intracellular NO concentration. The results indicate that SP cells have significantly less DNA double-strand breaks after irradiation at dose of 3 Gy than nonSP cells (24.4 vs 40.3, accordingly, P < 0.05 Mann-Whitney Ucriterion). SP cells are more quiescent compared to nonSP G1/G0 fraction is 85 vs 39%, accordingly, P < 0.01 Mann-Whitney U criterion). Most nonSP cells reside in S, G2/M phases (61%), believed to be rather radiosensitive. Thus, the difference of SP and nonSP cells radiosensitivity can be partly explained by peculiarities of cell cycle distribution. NO concentration is 1.5 times higher in SP than nonSP cells (P < 0.05 Mann-Whitney U criterion); since it is known that NO inhibits apoptosis, being one of the mechanisms of genetic stability maintenance, greater number of spontaneous DNA double-strand breaks in SP cells is unsurprising (P < 0.05 Mann-Whitney U criterion). The above-listed results explain considerably the higher resistance of SP cells to the action of low-LET radiation in comparison with other melanoma B16 cells. Further study of this question can become the basis for development of tools to target SP cells and, ultimately, more effective cancer treatment.

[The significance of physical status of human papillomavirus type 16 for predicting the effectiveness of invasive cervical cancer treatment].

For the 83 patients with HPV 16-cancer of the cervix (cervical cancer) I-III stages it was performed a comparative analysis of primary tumor response to therapy, the clinical outcome of the disease for 3-5 years after radical treatment and an evaluation of the possible contribution in these rates of the physical status of the virus. It was shown that total tumors regression in the early stages of the observation predominate in patients with "high-integrated" virus DNA (the degree of integration > 50%) of compared with a group of patients with episomal and "low-integrated" form of the virus, but in a distant periods (3-5 years) in the first group predominate an adverse outcome of disease. This pattern is true for tumors of stage I-III, and for less common--I-II stages. It is assumed that the integration of HPV16 DNA into the cellular genome may serve as an independent predictor of clinical outcome of cervical cancer.

[Sensitivity of melanoma B16 side population to low- and high-LET radiation].

Cancer stem cells (CSC) found in multiple tumor types and cancer cell lines were shown to be more resistant to low-LET radiation in comparison to other cancer cells. Therefore, CSC are supposed to determine the long-term effect of cancer therapy. Research into the CSC sensitivity to high-LET radiation is of great interest because of the advances in hadron therapy. The aim of this investigation is to compare CSC and other cancer cell sensitivity to the low- (60Co gamma-rays) and high-LET (neutron) radiation. To identify CSC, we used the low cytometry-based side population (SP) technique based on the CSC capacity to produce the efflux of the vital dye Hoechst 33342. SP and non SP cells were sorted and exposed to gamma and neutron radiation at doses of 1-10 Gy and 0.1-4.7 Gy, correspondingly. We applied the colony-formation test to examine the SP and non SP survival rate after irradiation. It was shown that the sensitivity of SP to gamma-irradiation was lower than that of other cells: D0 average values (+/- SE) made up 2.3 +/- 0.3 Gy and 1.4 +/- 0.2 Gy, correspondingly (p = 0.047). The survival rate of SP and non SP did not differ after neutron irradiation. The values of relative biological effectiveness of neutron radiation relative to gamma-radiation at the D10 level were 2.6 for SP and 2.1 for other cells. The obtained results justify for the first time a high efficiency of application of neutrons in radiotherapy from the point of view of CSC elimination.

[Effect of synthetic tripeptides on hemopoietic stem cells in norm and after gamma-irradiation].

Our work was aimed at researching into the influence of dipeptide (gamma-dGlu-dTrp) "Timodepressin" and this dipeptide-based tripeptides on the colony-forming ability of the irradiated in vitro bone marrow and hemopoietic stem cells of the normal organism. Also studied was the effect of various doses (1-1000 microg/kg) of one oftripeptides (dAla-gammadGlu-dTrp) on the output of exogenous splenic colonies in the case of its introduction 48 hours before irradiation. It is shown that the mode of influence of the preparations produced on the basis ofdipeptides dGlu-dTrp and gamma-dGlu-dTrp on the initial stages ofa hemopoiesis in the normal and irradiated organism depends on the nature of the additional amino-acid residue and its optical orientation.

Formation of potential radiation risk groups to render timely targeted medical care: lessons of Chernobyl.

The paper discusses technology for establishing potential cancer risk groups, based on methods of molecular and radiation epidemiology. Assay of gene mutations at the T-cell receptor (TCR) locus as the method of molecular epidemiology was used for measuring the frequency of TCR-mutations in 320 nuclear workers of the Institute of Physics and Power Engineering (IPPE). The method of radiation epidemiology was applied to the estimation of attributable risk fraction (ARF) for solid cancers in these groups. The main estimates of radiation risk after the Chernobyl accident are in close agreement with the International Commission on Radiological Protection (ICRP) Publication, 103 models published in 2007. In nuclear workers of the IPPE with ARF ≥ 10%, the increased level of TCR-mutations occurs more often (risk ratio=9.7; 95% CI: 2.9; 32.1).

[Variability in the frequency of TCR-mutant lymphocytes associated with gene polymorphisms in women living in radiation-polluted areas].

The paper presents the results of an association study of a predisposition to increased somatic mutagenesis detected by the test for TCR-mutant lymphocytes (CD3-CD4+ phenotype). A study group consisted of 251 women who lived in the towns polluted by radionuclides after the Chernobyl accident and had estrogen-dependent reproductive system diseases (uterine myoma, fibrocystic mastopathy). The carriage of minor alleles in the genes (CYP1A1, GSTM1, and ABCB1) of all three stages of detoxification of xenobiotics was associated with the rise in the spontaneous frequency of TCR-mutant cells. Overweight modified the genotype (at CYP1A1 and GSTT1 loci) - environment interaction. When background radiation became higher, the contribution of minor alleles in the CYP1A1 genes to the instability recorded as the elevated frequency of TCR-mutant cells increased.

[Influence of peptide derivatives of diketopiperazines on hemopoietic stem cells of an intact organism and irradiated bone marrow cells in vitro].

We have performed structural and functional studies of the hemotropic activity for a number of novel 2,5-diketopiperazine peptidomimetic derivatives. We employed a mouse model of hemopoietic stem cells cloning in the spleen of lethally irradiated animals. Biologic activity of synthetic products was studied in two experimental models: 1) in vitro irradiated bone marrow SFU-S was used for studying the radio modifying activity; 2) the biological effect of peptidomimetics on the intact non-irradiated bone marrow was evaluated in vivo. Various ways of administration of the peptidomimetics studied were used in the in vivo experiments: intravenous, intraperitoneal or subcutaneous injections and oral administration in the dose range of 10-10000 microg/kg. As a result of our work, we have discovered 2,5-diketopiperazine peptidomimetic derivatives with the dual activity: stimulation of intact committed SFU-S and radiomodifying activity.

[Infection with high-risk human papillomavirus and prognosis of cervical carcinoma].

Real-time polymerase chain reaction procedure was used to evaluate bioptic tumor samples from patients suffering cervical carcinoma (CC) stages I-IV. Out of 110 patients, high-risk human papillomavirus (HPV) infection was identified in 98 (89.1%), HPV type 16--63, HPV type 18--10 and HPV type 45--5. One of genotypes 31.33, 35, 39, 52, 58, 59 was established in 8 and a combination of several genotypes of the virus--12 patients. Frequency of remission in CC patients associated with HPV type 16 who had survived 3 years was significantly higher than in the same category associated with HPV type 18 (p=0.03). Relapse frequency and mortality rates in patients with tumors associated with one of viruses 31.33, 35, 39, 52, 58 or 59 were higher as compared with HPV type 16--associated cases 2 years (p=0.03) or 3 years on (p=0.11), respectively. A similar trend was established for squamous-cell tumors stages 1 and 2 (p=0.07) (p=0.12), respectively. No difference was observed in efficacy of therapy for infection with one or a combination of several genotypes of high-risk HPV. Hence, the genotype of virus is believed to be a factor of prognosis in CC early cancers. However, a definitive conclusion cannot be reached until results of a larger body of evidence and longer follow-up are available.

[Some features of immunological status in young peoples irradiated in utero or in age up to 4 years as result of Chernobyl accident].

Some characteristics of immune system, namely quantities of serum immunoglobulins A, G, M and activity of free and hidden autoantibodies to DNA, cardiolipin and microsomal thyroid antigen were studied in young people irradiated in utero or in age up to 4 years through Chernobyl accident. The hallmarker of observed immunological changes is low content of immunoglobulin A. Degree of reduction was in back proportion with level 137Cs contamination inhabit territory. A lowering of the content of IgA in persons irradiated in utero depends on period of pregnancy at a moment of the accident: the most reduction was observed in young people irradiated in the first trimester of gestation. It was shown elevation of activity autoantibodies to cardiolipin. Both deficit of IgA and elevation activity the autoantibodies were observed only in proportion of young people irradiated in utero or in early period of life.

[The association of TCR-mutant cells with DNA polymorphism in women residing in radiation polluted regions of the Russian Federation].

Using flow-cytometric method the frequency of lymphocytes beaming mutations at T-cell receptor (TCR) locus was assessed in women residing in radiation polluted regions of Bryansk and Tula Districts. Simultaneously genotyping of the 8 polymorph loci for genes involved in detoxication of xenobiotics and oestrogen metabolism was carried out. The increased TCR-mutant cell frequency was found to be characteristic of homozygotes of the low activity appropriated enzymes for 3 loci (HFE187, GSTM1 and MTHFR) at least. This tendency was statistically significant in case of deletion polymorphism of the GSTM1 gene: TCR-mutant cell frequency of the homozygous carriers of a deletion at the GSTM1 locus was (4.63 +/- 0.18) x 10(-4) while it was (4.05 +/- 0.15) x 10(-4) in other groups of persons. The greatest mutant cell frequency was observed in carriers of the minor allele 4889G of the locus CYP1A. More often the increased values of the TCR-mutant cells (outside range "3sigma") were determined in women with genotypes A/G or G/G of the locus CYP1A1 (25%) than in carries of the normal genotype A/A (1.6%) (OR = 20.6; p = 0.0002). The comparison of the groups of women with reproductive system diseases reveals significant elevation in the mean TCR-mutant cell frequency in inhabitants of the most radiation polluted region among others.

[The changing of cell-free DNA properties of peripheral blood and TCR-mutant cell frequency in individuals exposed to ionizing radiation].

Some properties of the cell-free DNA (cfDNA) of peripheral blood plasma were assessed in 153 employees of atomic industry enterprises. The contents of ribosomal repeat (rDNA) and its concentration in plasma increased in cfDNA of the group of persons in comparison with non-irradiated individuals. The contents of satellite III in cfDNA of donors and of irradiated persons do not differ and less than in DNA nucleus. The correlation between cumulative dose of radiation, contents of rDNA in cfDNA and the frequency of lymphocytes bearing mutations at T-cell receptor (TCR) locus was obtained. The definition of three indications in irradiated persons: the contents of ribosomal genes in cfDNA, TCR-mutant cell frequency and concentration of ribosomal genes in blood plasma--may be useful for revealing individuals in organism of which an intensive cell apoptosis takes place and there is an increased probability of carcinogenesis and of progress of disease of immune system.

Dipeptide gamma-d-Glu-d-Trp (thymodepressin) inhibits migration of CD34+ cells from the bone marrow into peripheral blood during tumor growth.

We studied the effect of dipeptide gamma-d-Glu-d-Trp (thymodepressin) on migration of CD34+ hemopoietic precursors and their direct adhesion to fibronectin in tumor-bearing mice on days 8, 11, 15, and 17 of tumor growth and on expression of CXCR-4 (CD184+) to SDF-1 and integrin beta1 (CD29+) by bone marrow cells. In tumor-bearing mice treated with gamma-d-Glu-d-Trp, the percent of CD34+ hemopoietic precursors in the peripheral blood considerably decreased throughout the observation period; the content of CD34+ hemopoietic precursors in the tumor tissue was 2-3-fold below the control against the background of increased content of CD34+ cells in the bone marrow. In animals treated with the peptide, the content of cells expressing CXCR-4 in the peripheral blood, bone marrow, and tumor tissue significantly decreased, while the percent of cells expressing integrin beta1 receptor (CD29+) in the bone marrow increased 2-fold, which was paralleled by an almost 2-fold increase in the percent of cells binding to fibronectin. We hypothesized that dipeptide gamma-d-Glu-d-Trp suppressed mobilization/migration of CD34+ hemopoietic precursor cells from the bone marrow to the peripheral blood of tumor-bearing mice.

[The recovery parameters of DNA strand breaks of Ehrlich ascites cells after the combined action of ionizing radiation and hyperthermia].

A mathematical model of DNA strand breaks postirradiation repair and the methodology allowing to differentiate the mechanism of inhibition of DNA strand breaks recovery after combined actions of ionizing radiation and hyperthermia have been described in this paper. Using this model and the results published by other authors for DNA strand breaks of Ehrlich ascites cells, there have been obtained the data showing that the portion of DNA-damages that the cell incapable to recover after consecutive thermoradiation action was risen with an increase in thermal load under insignificant change of repair constant. It means the mechanism of DNA strand breaks recovery inhibition is realized in a greater extent through the formation of irreversible damages but not through the damage of repair process itself.

[The correlation between intracellular level of nitric oxide and frequency of gene somatic mutations after low dose radiation exposure].

The purpose of this work was the study of possible relationship between intracellular NO level and somatic mutagenesis after irradiation with low doses. The level of NO in peripheral blood lymphocytes and frequency of the TCR-mutant cells were assessed by flow cytometry in 64 workers of atomic industry with mean dose (+/- SE) 114.9 +/- 10.8 MSV, accumulated within 21.4 +/- 1.1 years, and 66 age- matched control donors. The mean frequency of the TCR-mutant cells in this groups was (6.1 +/- 1.0) x 10(-4) and (4.1 +/- 0.2) x 10(-4) respectively (p = 0.06). 14% of workers of atomic industry had the TCR-mutant cell frequencies exceeding the 95% confidence interval in control donors. It was found the positive correlation between the intracellular NO level and the TCR-mutant frequency (R = 0.36, p < 0.01). The mean level of NO in individuals with the elevated TCR-mutant frequency was significantly higher than in others: 1619 +/- 57 vs 1340 +/- 40 relative units (p = 0.01). The results suggest that nitric oxide may come into elevating frequency of the mutant cells in some proportion of individuals exposed to low doses of ionizing radiation not excepting formation of genome instability.

The effects of the EW dipeptide optical and chemical isomers on the CFU-S population in intact and irradiated mice.

The influence of Glu-Trp (EW) synthetic dipeptide isomers on hemopoietic progenitor cells and certain immune response reactions is determined by their optical and chemical properties. Thus, the all L-amino acid containing dipeptides L-Glu-L-Trp and L-gammaGlu-L-Trp have no effect on proliferation of committed and pluripotent CFU-S in intact bone marrow. The optical isomers of the Glu residue are an essential determinant of the EW dipeptide biological activity. The inversion of the amino acid optical form imparts suppressor properties: D-Glu-D-Trp,D--gammaGlu-D-Trp, D-Glu-L-Trp and D-gammaGlu-L-Trp inhibit proliferation of hemopoietic progenitors in intact bone marrow. The type of the peptide bond between L-Glu and Trp is another important factor for the biological activity of the L-Glu-containing peptides. Unlike L-Glu-D-Trp with alpha-peptide bond, the dipeptide L-gammaGlu-D-Trp with gamma-peptide bond stimulates CFU-S-8 proliferation in intact bone marrow. The diverse effects of the EW optical isomers on hemopoietic progenitors underlie the radioprotective properties of the D-Glu-containing dipeptides and the radiotherapeutic ones of the L-Glu dipeptides. In animals, pre-irradiation injection of D-Glu-D-Trp, D-gammaGlu-D-Trp, D-Glu-L-Trp, D-gammaGlu-L-Trp, or post-irradiation injection of L-Glu-L-Trp, L-gammaGlu-L-Trp promoted regeneration of the hemopoietic progenitor population.

Thymodepressin inhibits migration of CD34+ cells from bone marrow in normal and granulocyte CSF-stimulated hemopoiesis.

We studied the effect of thymodepressin on migration and adhesion of mouse hemopoietic CD34+ cells under normal conditions and under the effect of granulocytic CSF. It was found that the peptide reduced the absolute number of CD34+ hemopoietic cells in the peripheral blood, increased the percent of cells bound to fibronectin and expressing receptor for integrin beta1 (CD29+) in the bone marrow of mice under normal conditions and after stimulation with granulocytic CSF, and reduced the relative number of cells carrying CXCR4 receptor for stromal factor-1 (CD184+) in the bone marrow (CD34+CD184+) and blood (CD184+) of mice stimulated with granulocytic CSF. The results suggest that thymodepressin can inhibit migration of CD34+ cells from bone marrow into peripheral blood under conditions of normal and granulocytic CSF-stimulated hemopoiesis.

Effects of structural and "mixed" isomers of Glu-Trp dipeptide on normal hemopoietic stem cells.

We studied the effects of optical (dd-, ll-, dl-, and ld-dipeptides with alpha-bond, EW) structural isomers and cyclic (dd-, ll-, dl-, and ld-dipeptides with gamma-bond, iEW) analogs of Glu-Trp synthetic dipeptide on the population of normal hemopoietic stem cells. Dipeptides containing lGlu (lGlu-lTrp, lGlu-dTrp) injected to mice were inert towards committed bone marrow CFU-S; dGlu-containing dipeptides (dGlu-dTrp, dGlu-lTrp) inhibited the growth of CFU-S-8; and LiGlu-dTrp stimulated these cells. Inhibitory or stimulatory effects of optical and chemical isomers of Glu-Trp dipeptide are determined by optical orientation and nature of peptide bond of Glu residue. The effects of cyclic and mixed peptides towards colony formation are similar to those of the corresponding linear dipeptides.