Angiogenic potential of circulating peripheral blood neutrophils in kidney cancer

The role of neutrophils in kidney cancer is currently being studied. Their role in carcinogenesis is ambiguous. As one of the most abundant blood leukocytes, neutrophils play an important role in cancer progression through multiple mechanisms, including promotion of angiogenesis, immunosuppression, and cancer metastasis. Neutrophils synthesize and release pro-angiogenic factors that are able to directly or indirectly stimulate the growth and migration of endothelial cells, which in turn causes the formation of new blood vessels from pre-existing ones. The production of various factors by neutrophils, including proangiogenic ones, is mediated by the expression of the genes of these molecules. Functional heterogeneity is characterized by differences in neutrophil gene expression patterns. The aim of this study was to evaluate the angiogenic potential of circulating neutrophils in kidney cancer. The object of the study were blood neutrophils of patients with verified clear cell kidney cancer at stage I (T1N0M0G1, n = 28, median age 60), stage II (T2N0M0G2, n = 15, median age 61) and stage III (T3N0M0G2, n = 15, median age 63) before surgery. The control group consisted of apparently healthy donors (n = 15, median age 54). Serum levels of IL-8 and VEGF-A were assessed by enzyme immunoassay. Expression of the CXCL8 and VEGF-A genes in circulating neutrophils was determined by reverse transcription quantitative PCR. As a result of our study, an increase in the level of IL-8 and VEGF-A in the blood serum of patients with kidney cancer in all studied groups compared with the control group was revealed. We observed a direct correlation between serum levels of IL-8 and VEGF-A in patients with kidney cancer (r = 0.429; p = 0.016), which confirms the relationship of these angiogenic factors. A significant increase in CXCL8 gene expression by circulating neutrophils was found in patients on II (2.91, Q0.25-Q0.75: (1.296-4.99), p = 0.02) and III (1.93, Q0.25-Q0.75: (0.755-11.36, p = 0.014) stages of kidney cancer compared with the control group (1.50, Q0.25-Q0.75: (0.80-4.05)). However, VEGF-A gene expression by circulating neutrophils did not differ from those in the control group. Blood neutrophils in kidney cancer exercise their angiogenic potential through the production of IL-8

IL-4 and its polymorphism (<i>IL4</i>-589C/T) in cervical neoplasia

The transition of cervical neoplasia (CIN) to cervical cancer occurs with the active participation of IL-4, for which both pro- and antitumor effects have been shown with tumors of various localizations. The expression of cytokines is regulated at the transcriptional level in the promoter region of the gene. It has been shown that the genotype IL4 (589C/T) (rs2243250) is associated with the development of gastric and breast cancer. The contribution of IL-4 genotypic variations to the development of CIN has not yet been studied. The aim of the study was to assess the risk of developing cervical neoplasia by the presence polymorphism of IL4 (589C/T) and the level of IL-4. The object of the study was circulating neutrophils, serum and genomic DNA of 36 patients with CIN and 20 women without dysplasia (comparison group). Using ELISA, the level of IL-4 was determined in neutrophil lysate and serum. Phagocytic activity and adhesive ability (CD11b) of neutrophils were assessed. Allele-specific real-time PCR using Taq-Man probes was used to analyze of the IL4 589C/T (rs2243250). Statistical processing was carried out using Statistica 13 and Jamovi 1.6.5.0. As a result of the study, it was found that the level of IL-4 in serum and circulating neutrophils in patients with CIN is significantly higher than in the comparison group. The -589C* allele of the IL4 gene and the TT genotype are more common in the group with CIN (55.5%) than in the control (25%). At the same time, a direct relationship was established between the presence of polymorphism and increased adhesive ability and with indicators of the phagocytic number of circulating neutrophils. Analysis of the incidence of IL4 C589T by the «case-control» method showed that the chances of CIN formation in carriers of the -589C allele and the TT genotype were 3.75 (95% CI: 1.013 - 13.880, Chi-square = 4.161, p = 0.042). The -589C* allele and TT IL4 genotype, neutrophil and serum IL-4 levels are associated with HPV infection. Using a binary logistic regression model, we demonstrated the possibility of using IL-4 levels in circulating neutrophils and IL-4 gene polymorphism (589C/T) for the differential diagnosis of patients with CIN (χ2 = 15.6, p = 0.001). Significant significance for their combination was assessed by ROC-curve analysis (IL-4 in neutrophils; IL4 (-589С*), 75% probability. Thus, the IL4 (589C/T) is associated with the adhesive and phagocytic activity of circulating neutrophils. In HPV-infected patients, IL4 gene polymorphism (589C/T) can serve as a marker for early detection and prognosis of CIN

Phenotype of circulating neutrophils at different stages of cervical neoplasia

At the present time, there is no common point of view to the role of circulating neutrophils (NP) in emergence and development of neoplasia. It is suggested that due to high functional plasticity, the neutrophils may exhibit both pro- and antitumor activity. In order to study the NP phenotype at different stages of cervical neoplasia (CN), we have evaluated their absolute and relative amounts, myeloperoxidase activity, spontaneous and induced NST-test markers, and the level of intracellular cationic proteins. Spontaneous production of elastase and active forms of matrix metalloproteinases, the levels of IL-2, IL-8, IL-18, IFNy, G-CSF were determined in the NP cell lysates and in blood serum. The formation of extracellular traps (NET) was evaluated using 1-day cultures of Saccharomyces cerevisiae as an inducer. We examined 31 patients with cervical intraepithelial neoplasia (CIN) and 21 primary patients with cervical cancer (CC, Ia stage according to FIGO scale), as well as 25 practically healthy women. We revealed increased spontaneous and inducible oxygen-dependent cytolytic and phagocytic activity and spontaneous production of NET if compared to normal values, along with decreased absolute NP numbers in patients with CIN, thus suggesting the antitumor activity of NP. The levels of “pro-tumor” cytokines (MMP-9, IL-2 and G-CSF) become increased over normal levels as early as at the CIN stage, both for the neutrophils and blood plasma. High levels of regulatory IFNy and neutrophil-priming IL-8 in blood plasma do not presume any use of exogenous NP-activating factors at the stage of cervical dysplasia. At the initial stage of cervical cancer, the absolute NP amounts are significantly increased compared to normal counts. However, despite increased spontaneous oxygen-dependent cytolytic activity, the NPs have a significantly reduced activity of phagocytosis and sharply increased spontaneous production of NET, thus, generally, being characteristic to the “pro-tumorous” NP phenotype. IL-2 levels are elevated, and MMP-9 values are still increased in NP and blood plasma of patients with CC (stage Ia). Hence, the obtained results suggest some changes of NP phenotype to a pro-tumorous pattern during transition from intraepithelial dysplasia to cervical cancer. These results allowed us to design an algorithm for examining women with suspected cervical cancer, including IL-2 measurement in blood serum, and MMP-9 amounts in the NP lysates

Параметры глутатионовой системы и тиоредоксина в плазме крови и асците и полиморфизм гена GSTP1 Ile105Val как факторы резистентности к платиносодержащей химиотерапии у больных раком яичников

Background. Chemotherapy is one of the main types of treatment in ovarian cancer. Standard first-line treatment includes platinum drugs. Every fifth patient develops chemoresistance after platinum-containing first line therapy. Glutathione detoxification systems play an important role in platinum drugs utilization.Purpose. To assess the redox status of blood plasma and ascitic fluid in ovarian cancer patients before and after neoadjuvant platinum-containing chemotherapy (NACT).Materials and methods. We determined the activity of the glutathione system and thioredoxin levels in bloodplasma before and after NACT and in the ascitic fluid before NACT, and the presence of GSTP1 gene polymorphism (Ile105Val (rs1695), Ala114Val (rs1138272) in 30 III–IV FIGO stage ovarian cancer patients. Patients were divided into 3 groups: NR – no relapse in 2 years after last chemotherapy course; R1 – relapse in less than 6 months; R2 – relapse in more than 6 months.Results. We established an increase of the glutathione-transferase activity and a decrease of the GSH level inplasma after chemotherapy in R1 patients, and an opposite dynamic of glutathione-transferase and GSH in the R2 group. Thioredoxin level in plasma of all patients was lower than in the control group; differences in levels between groups were not statistically significant. GSTP1 105Val allele was more frequently present in patients than in the control group, and more frequently in R2 than in R1.Conclusion. The increase in plasma glutathione-transferase and glutathione-reductase levels can be a prognostic marker of early relapse. Thioredoxine dynamics do not correlate with the chemotherapy response. The presence of the GSTP1 105Val allele is a risk factor for ovarian cancer development, but a protective factor against early relapse.Введение. Химиотерапия является одним из основных видов лечения распространенного рака яичников (РЯ). У каждой пятой пациентки развивается химиорезистентность после платиносодержащей терапии первой линии. Система детоксикации глутатиона играет важную роль в утилизации платиновых препаратов из  опухолевых клеток. Цель. Оценить окислительно-восстановительный статус плазмы крови и асцитической  жидкости у больных РЯ до и после неоадъювантной платиносодержащей химиотерапии  (НАХТ).Материалы и методы. Мы определили активность глутатионовой системы и уровень тиоредоксина в плазме крови до и после НАХТ и в асцитической жидкости до НАХТ у 30 пациентов на III–IV стадиях (по FIGO) рака яичников. Пациенты были разделены на три группы: БР – без рецидивов в течение 2 лет после завершения химиотерапии; Р1 – рецидив заболевания в течение 6 мес после завершения химиотерапии первой линии; Р2 – рецидив после 6 мес от момента завершения химиотерапии первой линии.Результаты. Установлено увеличение активности GT и снижение уровня GSH в плазме  после химиотерапии у пациентов с Р1, а также противоположная динамика GT и GSH в  группе Р2. Уровень тиоредоксина в плазме у всех пациентов был ниже, чем в контрольной группе; различия в уровнях между группами не были статистически значимыми. Аллельный вариант 105Val гена GSTP1 выявлялся с более высокой частотой у пациентов с РЯ, чем в контроле, и чаще в группе Р2, чем у Р1.Заключение. Повышение активности GST и GR в плазме больных РЯ может быть  прогностическим маркером раннего рецидива. Динамика тиоредоксина не коррелирует с  ответом на химиотерапию. Присутствие аллеля 105Val в гене GSTP1 является фактором риска развития рака яичников, но защитным фактором против раннего рецидива

ВЗАИМОСВЯЗЬ УРОВНЕЙ ВОСПАЛИТЕЛЬНЫХ ЦИТОКИНОВ КРОВИ И ЧИСЛА ЦИРКУЛИРУЮщИХ ОПУХОЛЕВЫХ КЛЕТОК С ОТВЕТОМ НА СТАНДАРТНУЮ ХИМИОТЕРАПИЮ У БОЛЬНЫХ РАКОМ ЯИЧНИКОВ

Introduction. Serum chemokines are inflammatory mediators, which role is shown in the occurrence and progression of a number of malignant tumors. Produced by white blood cells, stem cells, tumor and endothelial cells, chemokines control their movement and positioning. Chronic inflammation underlies the progression of ovarian cancer (OC ). This increases the likelihood of chemokines stimulating or blocking tumor progression.The aim of the study was to evaluate the relationship between the blood levels of inflammatory cytokines in blood and the number of circulating tumor cells (CTC s) with the response to standard chemotherapy (CT ) in patients with cancer.Material and Methods. In patients with primary OC before and after 2–4 courses of chemotherapy and in patients with benign ovarian tumors (as a control), serum levels of CCL 2, CCL 3, CCL 4, CXCL 8 and CX3CL 1 were evaluated by multiplex xMAP analysis. The amount of CTC s (population CD 45-/ Epcam+/CK+) was determined using a flow cytometer. Patients with ovarian cancer were divided into 3 groups according to the platinum sensitivity criterion of GC JG 4th, and progression-free interval (PFI) was determined. Results. It was found that the levels of CCL 2, CCL 3, CCL 4, CXCL 8, and CX3CL 1 in case of OC did not significantly differ from that in the control, strongly negatively correlated with age (except for the CCL 2 level). CT significantly increased the level of CCL 2 in the group of refractory OC ; of CCL 3 – in the group of sensitive OC , of CCL 4 – in the groups of resistant and sensitive OC , and C XCL 8 level increased in the groups with resistant and sensitive OC and decreased in the group of refractory OC . The number of CTC s in patients with OC was significantly higher than in the control. After CT , a decrease in the amount of CTC s strongly and significantly correlated with a decrease in the level of CX3CL 1 in the groups of refractory andsensitive OC . The maximum PFI occurred with an increase in serum levels of CCL 3, CXCL 8, a decrease in CCL 4 and a constant level of CX3CL 1.Conclusion. Thus, no significant differences in the levels of CCL 2, CCL 3, CCL 4, and IL -8 between patients with OC and control groups were found. The levels of chemokines studied and the amount of CTC s differed in the groups divided by the tumor sensitivity to CT . We observed significant correlations between the amount of CTC s and the level of CX3CL 1 in the group of platinumsensitive OC .Введение. Сывороточные хемокины – медиаторы воспаления, роль которых доказана в возникновении и прогрессировании злокачественных опухолей ряда локализаций.  Продуцируясь лейкоцитами, стволовыми, опухолевыми и эндотелиальными клетками, хемокины контролируют их движение и позиционирование. Хроническое воспаление лежит в основе прогрессирования рака яичников (РЯ), и это повышает вероятность стимулирования или блокирования хемокинами прогрессирования опухоли. Целью исследования было изучение взаимосвязи уровней воспалительных цитокинов крови и числа циркулирующих опухолевых клеток (ЦОК) с ответом на стандартную химиотерапию у больных РЯ. Материал и методы. У пациенток с первичным РЯ до и после 2–4 курсов химиотерапии (ХТ) и пациенток с доброкачественными опухолями яичников (контроль) в сыворотке крови уровни ccl2, ccl3, ccl4, cXcl8 и cX3cl1 оценивали методом мультиплексного анализа xmap. Количество ЦОК (популяция cd45-/ epcam+/cK+) определяли на проточном цитометре. Пациентки с РЯ были разделены на 3 группы по критерию  платиночувствительности согласно gcJg 4th, было определено время до прогрессирования (pFi). Результаты. Установлено, что уровень ccl2, ccl3, ccl4, cXcl8 и cX3cl1 при РЯ значимо не отличался от такового в контроле, отрицательно коррелировал с возрастом (за  исключением уровня ccl2). Химиотерапия значимо повышала уровень ccl2 в группе  рефрактерного РЯ; ccl3 – в группе чувствительного РЯ, ccl4 – в группах резистентного и чувствительного РЯ, cXcl8 – повышала в группах с резистентным и чувствительным РЯ и снижала в группе рефрактерного РЯ. Количество ЦОК у больных с РЯ значимо выше, чем в контроле. После ХТ снижение количества ЦОК достоверно коррелировало со снижением уровня cX3cl1 в группах рефрактерного и чувствительного РЯ. Максимальная продолжительность периода без прогрессирования отмечена при повышении уровней ccl3, cXcl8, снижении уровня ccl4 и неизменном уровне cX3cl1. Выводы. Уровни ccl2, ccl3, ccl4 и il-8 значимо не различаются у больных РЯ и группы контроля. Уровни изученных хемокинов и количество ЦОК изменялись в группах с  различной чувствительностью опухоли к ХТ. При платиночувствительном РЯ наблюдаются значимые корреляции количества ЦОК и уровня cX3cl1 в сыворотке крови до лечения

Development of the therapeutic regimen based on the synergistic activity of cyclophosphamide and double-stranded DNA preparation which results in complete cure of mice engrafted with Krebs-2 ascites

Cumulative evidence obtained in this series of studies has guided the logic behind the development of a novel composite dsDNA-based preparation whose therapeutic application according to the specific regimen completely cures the mice engrafted with otherwise lethal Krebs-2 ascites. The likely mechanism involves elimination of TAMRA+ tumor-inducing stem cells (TISCs) from Krebs-2 tumors. We performed quantitative analysis of TISC dynamics in Krebs-2  ascites following treatment with the cytostatic drug cyclophosphamide (CP) and untreated control cells. In intact ascites, TISC percentage oscillates around a certain value. Following CP treatment and massive apoptosis of committed cancer cell subpopulation, we observed relative increase in TISC percentage, which is consistent with reduced susceptibility of TISCs to CP. Nonetheless, this treatment apparently synchronizes TISCs in a cell cycle phase when they become sensitive to further drug treatments. We describe the regimen of synergistic DNA + CP activity against Krebs-2 ascites. This protocol results in a complete cure of 50 % of Krebs-2 engrafted mice and involves three metronomic injections of CP exactly at the timepoints when repair cycles are about to finish combined with dsDNA injections 18 hours following each CP injection. The “final shot” uses CP + DNA treatment, which targets the surviving yet highly synchronized and therefore treatmentsensitive cells. The first three CP/DNA injections appear to arrest Krebs-2 cells in late S-G2-M phase and result in their simultaneous progression into G1-S phase. The timing of the “final shot” is crucial for the successful treatment, which eradicates tumorigenic cell subpopulation from Krebs-2 ascites. Additionally, we quantified the changes in several biochemical, cellular and morphopathological parameters in mice throughout different treatment stages

Analysis of different therapeutic schemes combining cyclophosphamide and doublestranded DNA preparation for eradication of Krebs-2 primary ascites in mice

In the present paper, we report on the series of experiments where multiple regimens of CP and dsDNA injections were tested for targeting the ascites form of murine Krebs-2 cancer in situ. We show that combining CP with cross-linked human and salmon dsDNA results in a synergistic toxicity for ascites-bearing mice, an observation supported by the histopathology analysis of organs and tissues of experimental animals. In contrast, using a composite mixture of native and cross-linked human and salmon DNA after CP injections leads to a significant increase in average lifespan of the treated mice. Further, we demonstrate that repeated rounds of CP+dsDNA injections result in dramatic anticancer effect. The timing of injections is chosen so that they target the cells that were insensitive to the previous treatments as they were in the G2/M phase. 3-4 rounds of injections are needed to eliminate the subpopulation of tumor-initiating cancer stem cells. Our experiments identified the regimen when complete resorption of the primary Krebs-2 ascites occurs in all of the treated animals, followed by a remarkable remission period lasting 7-9 days. Yet, this regimen does not prevent secondary site metastases (either solid or ascites form) from developing, which is likely caused by the migration of ascites cells into adjacent tissues or by incomplete eradication of cancer stem cells. To address these and other questions, we expanded the study and performed histopathology analysis, which indicated that secondary metastases is not the only cause of death. In fact, many animals displayed unfolding systemic inflammatory reaction which was culminated by multiple organ failure. Thus, we developed the concept for treating ascites form of Krebs-2 cancer, which allows elimination of the primary ascites

Characteristic of the active substance of the Saccharomyces cerevisiae preparation having radioprotective properties

The paper describes some biological features of the radioprotective effect of double-stranded RNA preparation. It was found that yeast RNA preparation has a prolonged radioprotective effect after irradiation by a lethal dose of 9.4 Gy. 100 % of animals survive on the 70th day of observation when irradiated 1 hour or 4 days after 7 mg RNA preparation injection, 60 % animals survive when irradiated on day 8 or 12. Time parameters of repair of double-stranded breaks induced by gamma rays were estimated. It was found that the injection of the RNA preparation at the time of maximum number of double-stranded breaks, 1 hour after irradiation, reduces the efficacy of radioprotective action compared with the injection 1 hour before irradiation and 4 hours after irradiation. A comparison of the radioprotective effect of the standard radioprotector B-190 and the RNA preparation was made in one experiment. It has been established that the total RNA preparation is more efficacious than B-190. Survival on the 40th day after irradiation was 78 % for the group of mice treated with the RNA preparation and 67 % for those treated with B-190. In the course of analytical studies of the total yeast RNA preparation, it was found that the preparation is a mixture of single-stranded and double-stranded RNA. It was shown that only double-stranded RNA has radioprotective properties. Injection of 160 μg double-stranded RNA protects 100 % of the experimental animals from an absolutely lethal dose of gamma radiation, 9.4 Gy. It was established that the radioprotective effect of double-stranded RNA does not depend on sequence, but depends on its double-stranded form and the presence of “open” ends of the molecule. It is supposed that the radioprotective effect of double-stranded RNA is associated with the participation of RNA molecules in the correct repair of radiation-damaged chromatin in blood stem cells. The hematopoietic pluripotent cells that have survived migrate to the periphery, reach the spleen and actively proliferate. The newly formed cell population restores the hematopoietic and immune systems, which determines the survival of lethally irradiated animals

Risks of reproductive loss and placenta-mediated pregnancy complications in women with antiphospholipid antibodies

Introduction. Antiphospholipid syndrome (AРS) is a multisystem disease characterized by elevated levels of antiphospholipid antibodies (AРA), arterial and/or venous thrombosis, thrombocytopenia, and habitual miscarriage. Various AРA types have multifactorial and indirect effects on pregnancy course from the earliest stages, leading to termination of pregnancy and putting the prerequisites for further obstetric complications. Aim: determine rate and relative risk of pregnancy complications in AРA carriers. Materials and Methods. The retrospective study, conducted between 2017 and 2019, included 268 pregnant women. All women underwent a comprehensive study consisting of clinical examination and laboratory methods. AРA of IgM and IgG classes were measured by using ELISA, lupus anticoagulant (LA) was estimated by using a coagulometer. Results. APA were detected in 213 (79.48 %) women examined; 55 (20.52 %) patients (n = 55) lacked APA. Failed in vitro fertilization attempts were 2 times more common in women with AРA (10.33 vs. 5.45 %, respectively; p &lt; 0.001). Also, significant differences were found in this group in antenatal fetal death (4.23 vs. 1.82 %, respectively; p &lt; 0.001) and chronic placental insufficiency without fetal malnutrition (23.47 vs. 18.18 %, respectively; p &lt; 0.05). Of 165 women with early pregnancy loss syndrome, AРA were found in 130 (78.79 %), late pregnancy loss – in 29 (76.32 %) and antenatal fetal death – in 9 (90.0 %) women carrying serum AРA. The relative risks of unfavorable pregnancy outcomes have been identified for various AРA subtypes. Conclusion. Identifying clear-cut relative risks of adverse pregnancy outcomes for various subtypes of antiphospholipid antibodies will allow to determine risk groups and develop a special treatment algorithm for preventing pregnancy complications and perinatal losses