Effects of Combined Exposure to Carbon Ions and Protons on the Pool of MCF-7 Breast Cancer Stem Cells In Vitro.

We studied the effects of single and combined action of protons and carbon ions 12C6+ on the pool of MCF-7 human breast cancer stem cells. Single irradiation with a beam of protons or carbon ions had no significant effects on the relative number of cancer stem cells (CSC). The effects of combined irradiation in a total equieffective dose of 4 Gy depended on the sequence of exposure to ionizing radiations: the relative number of CSC did not change after irradiation with carbon ions and then with protons, but increased in the case of the reverse sequence. The most favorable result, i.e. a decrease in the CSC pool, was observed in the case of sequential irradiation with carbon ions and protons and their equal contribution to total equieffective dose. In this case, the absolute number of CSC decreased by on average 2.1 times in comparison with the control (p<0.05). The revealed regularities are of interest for the further development of new methods of radiation therapy.

Correlation of Radiation Response of Cervical Cancer Stem Cells with Their Initial Number before Treatment and Molecular Genetic Features of Papillomavirus Infection.

The proportion of CD44+CD24low cancer stem cells (CSC) was determined in cervical scrapings of 41 patients with squamous cell carcinoma of the uterine cervix before treatment and after irradiation in a total focal dose of 10 Gy. The relationship of quantitative changes in the CSC population with such parameters of papillomavirus infection as genotype, viral load, and physical status of HPV DNA (the absence or presence of HPV DNA integration into the cell genome and the degree of integration) was studied. Single- and multi-factor analysis revealed 2 independent indicators affecting the radiation response of CSC: initial number of these cells before treatment and physical status of HPV DNA. The increase in the CSC proportion after radiation exposure was observed 4.5-fold more often in patients with an initially low proportion of CSC (<3%) than that in other patients (p=0.001). The CSC proportion increased by on average 3% after irradiation in patients with complete integration of HPV 16/18 DNA and decreased by 3.8 % in patients with partial integration or no integration (p=0.03).

Quantitative Changes in the Population of Cancer Stem Cells after Radiation Exposure in a Dose of 10 Gy as a Prognostic Marker of Immediate Results of the Treatment of Squamous Cell Cervical Cancer.

Prognostic significance of the proportion of cancer stem cells in cervical scrapings from 38 patients with uterine cervical cancer before treatment and after irradiation in a total dose of 10 Gy was assessed for immediate results of radio- and combined chemoradiotherapy evaluated by the degree of tumor regression in 3-6 months after the treatment. Cancer stem cells were detected as cells with CD44+CD24low immunophenotype by flow cytometry. The proportion of cancer stem cells in patients with the complete tumor regression decreased by on average 2.2±1.1% after irradiation, while in patients with partial regression this indicator increased by on average 3.3±2.3% (p=0.03). Multiple regression analysis revealed two independent indicators affecting tumor regression: the stage of the disease (which is quite expected) and change in the proportion of cancer stem cells after the first irradiation sessions (R=0.60, p<0.002 for the model in the whole). The proportion of cancer stem cells before the treatment did not have prognostic significance.

Effect of Different Regimens of Co-Transplantation of Hemopoietic and Mesenchymal Hemopoietic Stem Cells on the Rates of Hemopoietic Recovery in Laboratory Mice Treated with Cyclophosphamide in Sublethal Doses.

We studied the effect of mesenchymal stem cells and hemopoietic stem cells co-transplanted in different regimens (sequence and intervals between the injections within 0-48 h) on the rate of the bone marrow hemopoiesis recovery in 468 inbred mice with cytostatic aplasia caused by a single injection of cyclophosphamide in a dose of 500 mg/kg. The efficacy of stem cells was evaluated by animal survival and general physical condition, body weight dynamics, peripheral blood counts (leucocytes, platelets, and reticulocytes), total cellularity and the presence of hemopoietic stem cells in the bone marrow on day 9 after cyclophosphamide injection. The relative content of hemopoietic stem cells in the total myelokaryocyte pool was assessed by flow cytofluorometry using specific monoclonal antibodies to CD117 and CD184. The stimulating effect of co-transplantation of mesenchymal stem cells and hemopoietic stem cells on hemopoiesis was demonstrated by the indices of total cellularity and the relative content of CD117+ hemopoietic stem cells. The schemes with injection of mesenchymal stem cells 24-48 h prior to injection of hemopoietic stem cells were most effective.

[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.

[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.

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 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.

Effects of iEW synthetic peptide isomers on bone marrow colony-forming capacity in vivo.

Studies of cooperative effects of D- or L-isomers of iEW dipeptide and neuraminidase on the number of splenic CFU, comparison of the peptide effects on CD34+ cells and splenic CFU, and evaluation of the effects of iEW D- or L-isomers on parameters of T-cell activation showed that interactions of L-(iEW) and D-(iEW) with CD34+ surface receptors are realized through the same mechanism, while their different biological effects on hemopoietic precursor cells in vivo can be explained by their different influence on T-component of the microenvironment.

[T-cell tumors with aplastic syndromes]. / T-kletochnye opukholi, protekaiushchie s aplasticheskimi sindromami.

AIM: To detect and verify the existence of a specific form of T-cell tumor accompanied by isolated lesions of bone marrow and aplastic syndromes. MATERIAL AND METHODS: Four patients with aplastic syndromes were examined using clinical, histological, cytological, cytogenetic, and immunophenotypic methods. RESULTS: Four cases of T-cell tumors of bone marrow with clinical and morphological manifestations of aplastic syndrome and scanty proliferation activity in bone marrow alone were diagnosed. The proliferation activity in bone marrow was observed as formation of small clusters composed of small-size lymphoid cells with dense nucleus. Dynamic monitoring of two patients revealed a trend toward an increase in the lymphoproliferation base against the remaining clinical picture of aplastic syndrome. The T-cell immunophenotype characterized by disappearance of some markers or decrease in their density, was observed only in some blood and bone marrow lymphocytes. The most significant changes of immunophenotype were observed in one of the patients (CD2+CD3-CD4-CD5-CD7-CD8-CD16-CD56-CD45RO++). The same patient had pronounced cytogenetic changes (47XY+Y[8], 47, XY, del(1)(p10) [23], 46 XY [3]) and resistance to routine therapy, including cyclosporin. In one patient the process transformed into lymphosarcoma. CONCLUSION: The results obtained in four patients allow their clinicomorphological characteristics to be regarded as particular forms of T-cell tumors accompanied by bone marrow damage and aplastic syndrome.

[Use of thymodepressin for treating autoimmune cytopenia]. / Primenenie timodepressinoa dlia lecheniia autoimmunnykh tsitopenii.

AIM: To study influence of thymodepressin on the course of autoimmune cytopenia. MATERIAL AND METHODS: Thymodepressin is a new synthetic hemoregulatory dipeptide (gamma-D-Glu-D-Trp). It was used for the treatment of 22 patients with autoimmune cytopenia. RESULTS: Hemoglobin levels were elevated in autoimmune hemolytic anemia and platelet levels were high in idiopathic thrombocytopenic purpura. A thymodepressin course resulted in a fall of total lymphocyte count and activated CD3+CD69+ lymphocytes. CONCLUSION: The above results, safety, absence of toxicity and allergenicity, parenteral and intranasal useability open perspectives for further studies of therapeutic action of thymodepressin as an immunodepressant in autoimmune processes.

Determination of somatic mutant frequencies at glycophorin A and T-cell receptor loci for biodosimetry of acute and prolonged irradiation.

Somatic mutant frequencies at glycophorin A (GPA) and T-cell receptor (TCR) loci were assessed. The dependence of the GPA mutant frequency on doses of acute and prolonged irradiation was shown. In the case of acute irradiation the GPA mutant frequency displayed a three-fold greater dose-related increase as compared to prolonged irradiation. A dose-dependent increase in the TCR variant frequency was found only in a group of subjects with recent exposures. In Chernobyl clean-up workers the TCR mutant frequency was significantly higher than in control non-irradiated individuals.

Determination of somatic mutant frequencies at glycophorin A and T-cell receptor loci for biodosimetry of prolonged irradiation.

PURPOSE: To determine the variant frequencies (VF) at glycophorin A (GPA) and T-cell receptor (TCR) loci in persons exposed to prolonged ionizing radiation at different doses and to assess the significance of the GPA and TCR assays for biodosimetry of prolonged irradiation. MATERIALS AND METHODS: The VF values were determined by means of flow cytometry in 120 persons exposed between 1968-1996. Most exposures were in Chernobyl clean-up workers in 1986-1987. RESULTS: A significant correlation was shown between the NO GPA variant cell frequency and dose (r = 0.61, p < 0.0001). The slope of the linear regression was 6.3 x 10(-6) NO mutant cells/Gy. Dose-dependent increase in the TCR VF was found in the group with recent exposures (slope 2.1 x 10(-4) variant cells/Gy, r = 0.75, p = 0.0002). In the Chernobyl clean up workers who received doses less than 0.25 Gy the TCR VF unlike the GPA VF was significantly higher than in the control non-irradiated individuals (p < 0.01 and p > 0.05 respectively). CONCLUSIONS: The GPA assay has limited potential to be used as a biodosimeter of prolonged irradiation, at least in dose interval up to 2.0 Gy. The TCR assay is likely to have greater potential in estimation of recent radiation exposure than the GPA assay.

[Determination of mutation frequency at loci of glycophorin A and T-cell receptors: informativeness for biological dosimetry of acute and prolonged irradiation]. / Opredelenie chastoty mutatsii po lokusam glikoforina A i T-kletochnogo retseptora: informativnost' dlia biologicheskoi dozimetrii ostrogo i prolongirovannogo oblucheniia.

The frequencies of somatic mutations at loci of glycophorin A (GPA) and T-cell receptor (TCR) were determined in persons exposed professionally to ionizing radiation or a result of accidents at nuclear power plants and in control donors. Dependence of glycophorin A mutant (NO) cell frequency on doses of acute (up to 3.5 Gy) and prolonged (up to 15.0 Gy) irradiation was shown. The slope of linear regression corresponded to increase of NO-mutant frequency by 31.1 x 10(-6)/Gy (r = 0.87, p < 0.0001) for acute irradiation and by 6.3 x 10(-6)/Gy (r = 0.61, p < 0.0001) for prolonged one. The 5-fold decrease of the linear regression slope in the case of prolonged irradiation makes significantly worse permissive ability of the GPA test. Therefore its use for biological dosimetry of prolonged irradiation is not expedient in dose interval up to 1 Gy. The frequency of mutations in genes of T-cell receptor significantly correlated with dose of irradiation only in group of donors with recent radiation exposure (r = 0.75, p = 0.0002). Meanwhile, the TCR method is more sensitive and informative for biological dosimetry of recent radiation, than the GPA test.

[Determination of mutation frequency at loci of glycophorin A and T-cell receptor: study of Chernobyl AES accident cleanup workers]. / Opredelenie chastoty mutatsii po lokusam glikoforina A i T-kletochnogo retseptora: obsledovanie likvidatorov avarii na CHAES.

The frequencies of somatic mutations at loci of glycophorin A (GPA) and T-cell receptor (TCR) were determined in control unexposed donors and Chernobyl clean up workers, who received low doses of irradiation up to 0.25 Gy. High variability of mutant rates for two investigated genes was shown in the clean up workers. No significant difference in the GPA (NO) mutant frequencies was observed between the clean up workers and control donors (p > 0.05), though there is a tendency for increasing the GPA mutation rate in the clean up workers. Meanwhile, the TCR mutation rate was significantly increased the clean up workers (p < 0.01), perhaps because of acceleration of spontaneous mutagenesis and possible genome instability. Persons with elevated levels of mutations at two loci can present a group at high risk in respect to oncological diseases.

[Activation of student cognitive activities in the process of teaching medical microbiology]. / Aktivatsiia poznavatel'noi deiatel'nosti studentov v protsesse prepodavaniia meditsinskoi mikrobiologii.

The main trends of methodical work conducted at the chair of microbiology of Orenburng Medical Institute are presented. For the purpose of activation of cognition activity of students during medical microbiology teaching the following methods were applied: presentation of the teaching material, creation of visual teaching methods in a single methodical plan in accordance with the logic structure graphs of the subject as a whole, its individual sections and themes; introduction of problem teaching method, solution of practical tasks of the II and III learning level; introduction of scientific achievements of the chair into the teaching process. Result of evaluation of the efficacy of the teaching-methodical work of the chair carried out demonstrated that knowledge of the principal microbiology problems in the students and interns persisted for long periods of time.