Особенности иммунного ответа на опухолевые штаммы EL-4 у мышей линий C57BL/6, В10.D2 (R101) и BALB/c 1 Фонд перспективных исследований, 121059, г.Москва, Российская Федерация 2 Федеральное государственное бюджетное учреждение «Государственный научный центр «Институт иммунологии» Федерального медико-

Aim. To study cellular and humoral immunological parameters of the employees of production sites located at the former mining and uranium ore processing territories. Materials and methods. In this work the clinical laboratory and immunological methods of examination were used. Statistical processing of the results was performed by using nonparametric analysis methods in the StatSoft Statistica 6 software package. Results. 101 employees of Chepetskiy mechanical plant were examined at the age of 19-62. 76 (75.2%) of them contacted with industrial factor, 64 - with radiation and 12 - with chemical. 25 (24.8%) employees had no contact with industrial factor. The immunological examination of the employees in contact with industrial factor revealed laboratory signs of imbalance in the immune system with IgE-overproduction. The risk group for secondary immunodeficiency with the leading infectious syndrome was 15.8%. Conclusion. Changes of cellular and humoral parameters on average values were not revealed. Infectious syndrome - recurrent ETN viral infections (13.9%) and herpes virus infection (13.9%) prevailed in the structure of the employees of Chepetskiy mechanical plant. A statistically significant difference in the content (percentage and absolute) of CD16+ lymphocytes was found in the employees of Chepetskiy mechanical plant contacted with industrial factor. These results had no clinical significance, what can be considered as an adaptive response to the chemical factor.

The indices of T-cell immunity are decreased in children of early age with megalothymus: content of T-cells (CD3+), Т-helpers (СD4+ Т-cells), cytotoxic Т-lymphocytes (СD8+ Т-cells), regulatory Т-cells (CD4+CD25hi) and also activated Т-lymphocytes (CD4+CD25lo и CD3+HLA-DR+). The increasing of expression of alterations was observed as degree of megalothymus was progressing. The only exclusion were regulatory T-cells. Their level was decreased under megalothymus degree I and II and was closer to indices of age standard under megalothymus degree III. Also, it was demonstrated that capacity of T-cells to differentiation to cytokine-producing cells type Th1 and Th2 increases as far as increases expression of megalothymus. However, this process remains within framework inherent to comparison group. The T-lymphopenia under megalothymus is conjugated with decreasing of emigration of T-cells from thymus to peripheral section of immune system. These alterations bring on deficiency of T-cell chain of immune system and can favor manifestation of its incapacity especially in conditions of increased load under effect of pathogens.

The ROS concentration and proliferation activity (Ki67 expression.marker) have been studied in the periphery blood lymphocytes of Moscow and Obninsk citizens, donors, Chernobyl disaster liquidators, inhabitants of the region contaminated after Chernobyl disaster (Klincy) and individuals with prostate cancer. It was shown that ROS concentration in lymphocytes of the liquidators and residents of the polluted region was lowered. But in lymphocytes of patients with tumors the ROS content was higher in comparison with the control. The cell content with Ki67 expression in lymphocytes of the individuals resided in the polluted region and tumor patients was lowered.

The article demonstrates that in children with thymomegalia the process of T-lymphopoesis in thymus is depressed. The functional condition of thymus was evaluated by detection of T-receptor excisional rings using polymerase chain reaction technique in real time. The T-receptor excisional rings are circular molecular structures developing in the process of rearrangement of V-genes of T-cell receptor. The most intensive and statistically reliable decreasing of content of T-receptor excisional rings in lymphocytes of blood is detected during first two years of life: 29.6 (18.1-41.0) copies per 1000 lymphocytes against 73.3 (54.0-83.8) copies in control. Hereinafter, (after two years) difference in level of T-receptor excisional rings inc children with thymomegalia in comparison with children without enlargement of thymus is less expressed i.e. it amounts to 1.8 times at the age before 10 years. In individuals of older age thymomegalia is a rather rare pathology. The examination of single patient aged of 40 years and with thymomegalia factually total absence of T-receptor excisional rings in cells of his blood was established.

The children of early age with thymomegalia and acute obstructive bronchitis suffer from lower T-lymphopoiesis in thymus gland evaluated on content of T-receptor excision rings. The T-receptor excision rings represent ring-shaped DNA molecules detecting only in T-cells recently emigrated from thymus gland. They serve as a measure of intensity of maturation of T-lymphocytes and their migration from this gland i.e. functional activity of this organ. The addition of immune modulating preparation tactivin to the complex treatment of acute obstructive bronchitis promotes repairing of affected T-lymphopoiesis. Besides, content of T-receptor excision rings practically reaches the level detected in children without thymomegalia.

The genome damage (frequency of cells with micronuclei and chromosome aberrations), concentration of reactive oxygen forms (ROS), markers of lymphocytes activation, expression of proliferation (CD69, Ki67) and proapoptotic antigen (CD95), as well as the ability to adaptive response have been investigated in blood lymphocytes of healthy donors and patients with prostate gland cancer. The influence of hormone-therapy on lymphocytes properties and connection between the parameters studied with the effectiveness of treatment, which was estimated by the level of prostate specific antigen (PSA), have been investigated. It was discovered that the genome damage to the patients with prostate gland cancer lymphocytes does not differ from control. The increase of the ROS level and decrease of radiosensitivity (irradiation of isolated lymphocytes in vitro at a dose of 1 Gy) are observed but they are insignificant. The content of the cells expressing CD69 and CD95 markers doesn't change but the expression of proliferative activity marker Ki67 in cells decreases. Radiosensitivity of lymphocytes in patients with prostate gland cancer correlates with the CD95 markers expression--a higher radio sensitivity points to their predisposition to apoptotic death. The expression of the markers studied depends on the oxidative status--a high ROS level suppresses their expression. The hormone therapy applied before radiotherapy leads to the increase in radiosensitivity and decrease in ROS. As the MN test shows, the ability to adaptive response of the lymphocytes in patients with prostate gland cancer is increased as compared with lymphocytes of healthy donors but it is insignificant; moreover, hormones do not influence the ability to the adaptive response. The high oxidative status further the formation of the adaptive response. We suppose that the discovered correlation between the initial, before treatment, frequency of lymphocytes with micronuclei and treatment effectiveness, namely, the decreased number of damaged cells associated with the treatment efficiency, is very important for the treatment prognosis. The results obtained can be very important for the experimental justification and understanding a possible use of blood lymphocytes for the additional diagnostics of prostate gland cancer and prognosis for its successful treatment.

Recovery and migration of T-cells from the thymus to the secondary lymphoid organs in mice after sublethal gamma irradiation were investigated by measuring T-cell receptor excision circles (TRECs). The TRECs level practically represents the cellularity of thymus, in particular it correlates with the quantity of T-cells which have rearranged TCR genes and express the receptor complex CD3-TCR. So, TRECs can be considered as one of the markers of these cells. TREC-containing cells form a subset of recent thymic emigrants in the secondary lymphoid organs. After a significant TREC decrease in the lymph nodes within the early phase (4 days) after irradiation, we registered the increase of their number during urgent organ recovery due to T-cell migration from the thymus (the maximum is on the 10th day). The secondary thymic atrophy is accompanied by a weakening migration of the T-cells containing TRECs to lymph nodes. A significant TREC increase in the spleen was registered on the 4th day after irradiation. The rest of the recovery period. (up to 60 days) is characterized by the low TREC level. Thus, determination of TREC level allows obtaining additional information about recovery and migratory processes in lymphoid organs during post-radiation regeneration.

В лимфоцитах периферической крови здоровых людей и больных раком предстательной железы (РПЖ) изучали поврежденность генома (частоту клеток с микроядрами и аберрациями хромосом), концентрацию активных форм кислорода (АФК), экспрессию маркеров активации (CD69), пролиферации (Ki67) и проапоптотического антигена CD95 Т-лимфоцитов, адаптивную способность. Исследовали также влияние гормонотерапии на свойства лимфоцитов и связь изученных показателей с эффективностью лечения, которую оценивали по уровню содержания в крови простатспецифического антигена (ПСА). Было обнаружено, что поврежденность генома при РПЖ не отличается от контроля, наблюдаются снижение радиочувствительности (облучение лимфоцитов в условиях in vitro в дозе 1 Гр) и повышение концентрации АФК, но эти изменения статистически незначимы. Содержание клеток, экспрессирующих маркеры CD69 и CD95, при РПЖ не изменяется, снижается экспрессия маркера пролиферативной активности Ki67. Радиочувствительность лимфоцитов при РПЖ коррелирует с экспрессией маркера CD95: при более высокой радиочувствительности этих клеток можно предполагать предрасположенность их к апоптотической гибели. Введение гормонов, курс которых проводят перед лучевой терапией, приводит к увеличению радиочувствительности лимфоцитов при РПЖ, снижению содержания АФК. Адаптивная способность лимфоцитов крови при РПЖ возрастает статистически незначимо, на нее гормоны не влияют, но высокий оксидативный статус способствует формированию адаптивного ответа. Для прогноза лечения представляется очень важным обнаруженная нами корреляционная связь между исходной (до начала лечения) частотой клеток с МЯ и эффективностью лечения: чем меньше поврежденных клеток, тем выше эффективность лечения. Полученные результаты могут иметь значение для экспериментального обоснования и понимания возможности использования лимфоцитов крови для дополнительной диагностики РПЖ и прогноза успеха его лечения.

The evidence of weakening of T cells emigration from thymus to peripheral immune system in children with thymomegaly is presented. It means the thymopoiesis is reduced in children with thymomegaly. The thymic emigration was investigated by two methods: real time PCR (the TREC level was measured) and flow cytometry (the number of CD3 +CD4 +CD45RA +CD31 + cells was measured). It was shown that the number of TREC copies per 1000 lymphocytes is significantly decreased in children with thymomegaly till 10 years. The TREC level in children with thymomegaly till 2 years approximately fits to the normal level of 8 years old children at which functional thymic activity is reduced in comparison with smaller children. In parallel with TREC level decrease with age the percentage and absolute content of CD3 +CD4 +CD45RA +CD31 + cells in blood is also decreased. However the TREC reduce is more intensive than CD3 +CD4 +CD45RA +CD31 + cells number decrease. The investigation of CD3 +CD4 +CD45RA +CD31 + cells number in blood of 2-10 years children with thymomegaly and without it showed that the number of cells of this subpopulation in thymomegaly is not changed.

У больных ревматоидным артритом (РА), различными формами ИБС и здоровых лиц разного возраста проводилось исследование колец реаранжировки альфа-цепи Т-клеточного рецептора (TREC) и фенотипа лимфоцитов с помощью проточной цитометрии, в том числе с применением метода магнитной сепарации с целью выявления признаком генотипической и фенотипической незрелости и определения количества клеток, несущих маркеры ранних тимических эмигрантов (РТЭ). В ходе исследования были выявлены отрицательная корреляция между уровнем TREC у доноров и больных инфарктом миокарда, у больных ревматоидным артритом подобной взаимосвязи не выявлено, что может говорить об изменении динамики тимической активности при данном заболевании. При исследовании фенотипа у больных было выявлено увеличение содержания клеток CD3 -4 + при высокой степени активности заболевания и внесуставных проявлениях. Метод магнитной сепарации лимфоцитов может быть использован для получения обогащенной суспензии клеток CD4 + и дальнейшего изучения еубпопуляции РТЭ.

The T-cell receptor excision circles (TREC) content in peripheral blood lymphocytes is considerably decreased in patients with autoimmune rheumatoid arthritis. TREC are circles which are excised from the genomic DNA in the thymus during rearrangement of TCR genes. High TREC level is peculiar to T-cells which have recently emigrated from the thymus (Recent thymic emigrants RTE). The degree of TREC-contaning lymphocytes decrease with age is significantly higher in patients with rheumatoid arthritis in comparison with age-matched healthy donors. So we can state premature thymus aging in this autoimmune disease. Expressiveness of distinctions of the TREC content (in comparison with TREC level in healthy donors) is increased with age: it 2-fold for age of 41-50 years, almost 4-fold for age of 51-60 years, 45-fold for persons older than 60 years. The obtained data testifies that thymic T-lymphopoiesis is reduced in autoimmune processes; at the same time intensive proliferation of peripheral T-cells and RTE redistribution in the organism with rapid migration to the tissue damaged by autoimmune process may also contribute to TREC level decrease.

Expression of activation (CD69) and proliferation (Ki67) markers, their connection with each other, with the oxidative status (reactive oxygen species--ROS) and with radiosensitivity (determined by micronucleus test) have been studied on stimulated blood lymphocytes from Moscow inhabitants. It was shown that the content of T-lymphocytes with the expressed CD69 and the content of T-lymphocytes with the expressed Ki67 markers correlate (r = 0.571; p = 0.0004). We can suppose that expression of the CD69 marker (24 h after PHA stimulation) is needed for the cell cycle progression, but it is not enough for the high expression of Ki67 markers 48 h after stimulation (DNA synthesis phase). It was discovered that T-lymphocytes with the CD69 marker or T-lymphocytes with the Ki67 marker are connected by the negative correlation with the frequency of irradiated cell with micronucleus (MN) r = -0.487; p = 0.010; r = -0.440; p = 0.008, respectively. So we can suppose that lymphocyte radiosensitivity decreased with the increase of expression activation and proliferation markers. It was shown that radiosensitivity determined by MN test is not connected with the oxidative status determined by the reactive oxygen species content including superoxide anion radicals. It is possible to explain by the fact that the ROS concentration has been determined in non-stimulated lymphocytes, but frequencies of cells with MN - in the stimulated cells 48 h after stimulation. Using separate analysis of individual differences by the studied parameters that were determined in the same people, it was shown that individual differences are high enough in the same cases. For example, the radiosensitivity when cells were irradiated 48 h after stimulation, ROS concentration, cell content with activation and proliferation markers. In conclusion, we can say that we failed to find important correlation between the parameters studied. However, the presence of individual differences in the marker expression, the frequency of MN cells, the oxidative status in the usual inhabitants, typical donors in Moscow, is very important.