Visnyk of Dnipropetrovsk University. Biology, medicine

93-96

Authors: V. L. Sokolenko, S. V. Sokolenko

The objective of this research is to assess the relation of radioactive contamination degree to immune system functioning, in the absence or presence of additional potential immunosuppressants. To achieve the objective, during the period of 1995–2015 we examined 250 people, students of Cherkasy State University, who lived in the areas of enhanced radiation monitoring before. Also we evaluated the additional impact of the emotional stress caused by examinations on examined students. Indicators of cellular immunity were determined by immunophenotyping and dyeing using Romanowsky-Giemsa method. The level of immunoglobulins in blood serum was determined by radial immunodiffusion (Mancini method). The level of cortisol in blood serum was determined by immunoenzyme method. We have found that in absence of the emotional stress among residents of the areas contaminated with radionuclides, cortisol level remained at the upper limit of homeostatic norm. There is an average positive correlation between the activity of radionuclides in the territories of residence and the level of cortisol. There are marked average positive correlations between the activity of radionuclides and the level of neutrophils, and low positive correlations with the levels of IgG and IgM in blood serum. Average negative correlations between the activity of radionuclides and the following parameters are also observed: absolute and relative number of functionally mature T-lymphocytes with phenotype CD3+, absolute and relative number of their helper subpopulation CD4+, absolute and relative number of natural killer cells with phenotype CD16+; and strong negative correlations with immunoregulatory index CD4+/CD8+. Cortisol level shows the similar correlation with the same parameters, but correlation coefficient is lower. Under conditions of additional stress, caused by emotional load during the examinations, cortisol level significantly increases. This enhanced previously discovered correlations and revealed new ones: average negative correlations between radionuclide activity / cortisol level and expression of pan-T cell marker CD5, medium positive correlations with absolute and relative number of suppressor T-lymphocytes with phenotype CD8+, correlation with the level of serum IgG changes from positive to negative. Additional short-term stress factor combined with chronic influence of low doses of radiation increases the dependence of immunosuppression, caused by radiation contamination of the areas where examined students live, and causes failures in the work of compensatory mechanisms, particularly at the level of serum immunoglobulin products.

103-107

Authors: H. N. Shiyntum, G. A. Ushakovа

Cadmiumclassified as a major carcinogen is considered a poisonous and unwanted heavy metal to a lot of tissues in many organisms. Of many publications already available, the general consensus is that the cadmium attenuating element is metallothionein (MT) through its interchangeable mechanism with Zn triggered by the presence of Cd, providing binding sites for Cd ions. MT was first discovered in the kidney cortex of the horse; it represents a low molecular weight protein, rich in cysteine residues which effectively bind with metals. Its functions consist in detoxification of heavy metals like mercury, arsenic, cadmium, homeostasis of essential metals including copper and zinc, anti-oxidation against reactive oxygen species, protection against DNA damage, oxidative stress, cell survival, angiogenesis, apoptosis, and increase of proliferation. In this work, we sought to highlight the protective function of MT in the brain and serum of rats by means of detoxification under induced effects of controlled Cd doses. We have done this by exposing Wistar rats to Cd at different doses in drinking water at different time intervals. In two independent experiments, 58 rats were subjected to 0.1 or 1.0 µg Cd2+/kg of body weight for 15 or 36 days under different conditions. The obtained data indicates the different functioning systems for the brain and the blood for MT metabolism under Cd effect. Our results indicate significant loss of metallothionein level in the brain and important increases in the amount of MT in serum proving that even minimal ingestion of toxic Cd is enough to trigger the release of MT protein in blood.

113-118

Authors: I. V. Prischepa, O. G. Prokushenkova, V. S. Nedzvetsky

Reactivation of glial cells, induced by metabolic disorders of glucose utilization and development of oxidative stress in retina under diabetes mellitus, is the key pathogenetic factor of diabetic retinopathy. Nanoparticles of C60 fullerene and some of their water-soluble derivates are known as one of the strongest antioxidants having neuroprotective effect in a number of pathologies and harmful influences. In the present study, for the first time, the effects of nanostructures of hydrated C60 fullerene (C60HyFn) on the expression and polypeptide composition of glial fibrillary acidic protein (GFAP) in retina of rats with streptozotocin (STZ)-induced diabetes have been evaluated. Using immunoblotting, 1.93-fold up-regulation of GFAP in diabetic rat retina as compared with control was shown, as a result of retinal glial cells reactivation induced by hyperglycemia. Increase in GFAP-immunolabeling associated with the reactive gliosis development in retina of diabetic rats was also confirmed by immuno-histochemical method. Consumption of C60HyFn solution (90 nM) as drinking water by diabetic rats for 12 weeks caused 1.51-fold decrease of GFAP level compared to untreated diabetic animals. In addition, C60HyFn caused statistically significant lowering of glycosylated hemoglobin concentration in blood serum of STZ-diabetic rats 1.58-fold. However, nanoparticles C60 did not affect neither insulin nor glucose levels in blood of diabetic rats. In conclusion, results obtained indicate that protective action of hydrated fullerene in the initial period of diabetic retinopathy of aged animals is realized through suppression of excessive activation of GFAP-positive retinal cells.

125-132

Authors: L. V. Altukhova, K. V. Kot, Y. G. Kot, K. S. Morozova, Y. E. Persky

Mechanisms of influence of volumetric autotransplantation of fibroblasts and of the mixture of fibroblasts and keratinocytes on the development of the local 3rd degree X-ray burn and the radiation skin ulcer in guinea pigs were investigated. We used deepadministration into the irradiation zone on its perimeter of 6 doses, which contained (150–160)×103 fibroblasts and (130–140)×103 keratinocytes in 100 µl. It is shown that this autotransplantation carried out 1 hour after the irradiation, and then every 24 hours, reduces the area of burn on the 35th day, compared to the control by 63%. Radiation ulcer appears on the 10th day after irradiation and is completely healed on the 25th day. With the same regimen of administration of only fibroblasts containing (200–210)×103 cells in 100 µl, these parameters of treatment were equal to 31% on 4th and 35th day, respectively. It is shown that as a result of radiation in the area of burn the level of gene expression of collagen types I and III, elastin, fibronectin, vinculin, decorin, hyaluronansynthases 1, 2, 3, matrix metalloproteinases 1, 2, 3, 7, 9 and hyaluronidase is reduced. Besides, in the burn area the level of gene expression of transforming growth factor α, fibroblast growth factors 1, 2, 8 and anti-inflammatory cytokines – interleukin 10 and transforming growth factor-β1 – is reduced, while the level of gene expression of proinflammatory cytokine (interleykin1β) increases. Both types of autotransplantation cause the growth of the expression level of all the structural genes and regulatory proteins of biopolymers and decrease in the expression level of interleukin 1β, which leads to activation of tissue regeneration and healing of the burn wound. Reasonsfor the higher efficiency of autotransplantation using the mixture of fibroblasts and keratinocytes compared to autotransplantation by fibroblasts only are both the larger total number of live cells regularly replacing dead cells in the burn area, and mutual stimulation of auto-fibroblasts and auto-keratinocytes to proliferate and to synthesize biologically active substances, i.e. cytokines and growth factors.

141-145

Authors: G. S. Maslak

Tumor growth progression of blood cells occurs due to changes in their genetic apparatus, which affects not only the cells morphological characteristics, but also their functional activity which to a greater extent depends on the membrane surface structures, a significant part of which is of glycoprotein nature. Complex type N-glycans are components of surface glycoproteins in the most of leukocytes. Thus, the study of changes in carbohydrate determinants of glycoproteins on the surface of leucocytes in tumorigenesis can help to reveal the mechanisms of this process. The aim of our study was to investigate the monocytes and granulocytes cytoplasmic membrane N-glycosylation in patients with chronic leukemia. The object of the study were blood cells of patients with chronic lymphocytic leukemia (n = 12) and polycythemia vera (n = 15) aged 58–66 years. Healthy hematologic volunteers (n = 15) aged 55 to 65 years were in the control group. N-glycan exposure on monocytes and granulocytes was investigated by flow cytometer Beckman Сoulter EPICS with Canavalia ensiformis lectin – Con A conjugated with fluorescent labels. The number of dead cells was monitored by means of binding them with propidium iodide. The result has been analyzed with FC Express. According to our data, levels of ConA-positive monocytes and granulocytes were 9,9 ± 1,0% and 32,7 ± 3,2%, respectively, in peripheral blood of healthy persons. The level of ConA-positive monocytes decreased to 31,0 ± 2,3% and the number of ConA-binding granulocytes increased to 66,7 ± 3,8% in patients with chronic lymphocytic leukemia compared with the norm. The number of ConA-positive monocytes decreased 3.3 times, and the level of granulocytes interacting with Canavalia ensiformis lectin slightly increased relative to control in polycythemia vera patients. There is significant increase in Con A-positive epitopes on granulocytes in patients with chronic lymphocytic leukemia and polycythemia vera compared with the control group, and absence of any difference in Con A-positive epitopes on monocytes. 100 times increase of the number of ConA-binding glycotopes is observed on the surface of granulocytes in peripheral blood of patients with polycythemia vera, and 3,3 times increase in patients with chronic lymphocytic leukemia, which may serve as an additional marker for the diagnosing these diseases.

151-155

Authors: D. Z. Vorobets

Methods of estimation of effectiveness of the open and laparoscopic pyeloplasty, as well as endo-urological palliative methods – laser resection, balloon dilatation and endopyelotomy, which determine the anatomical and functional peculiarities of renal pelvis and pyelo-ureteral junction with the help of ultrasound diagnostics during the forced diuresis, have been proposed. Changes of the area of renal pelvis, the velocity of post-furosemide increase of the scope of renal pelvis, rate of its drainage, changes in the diameter of pyeloureteral junction have been studied. This methodical approach is non-invasive, informative and simple in application. It is shown that dispersions of samples of patients after the open surgery do not differ from the dispersions of samples of the same patients before the operation on such parameters as areas of renal pelvis before the induction of furosemide, areas of renal pelvis after 15 minutes administration of furosemide, the rate of drainage after furosemide, the original diameter of pyeloureteral junction. This may indicate the stability of surgery results. For example, the larger renal pelvis by kidney size before the operation corresponded to the larger designed pelvis after the operation; renal pelvis drained faster before the operation, features faster drainage after the operation as well. Variation in the areas of renal pelvis which decreased in 40 minutes after furosemide, percent rate of longitudinal pelvis area, rate of after-furosemide increase in pelvis area, diameter of pyeloureteral junction in 15 minutes administration of furosemide after the open pyeloplasty was significantly different compared to the variation in the same parameter for the same patients before the operations. More substantial difference was observed in the same patients before and after Anderson-Hynes surgery by parameters of relative rate of after-furosemide drainage of pelvis and increase in diameter of pyeloureteral junction after 15 min administration of furosemide. That is, the same principle of operation provides similar results by anatomical parameters, such as size and diameter of pyeloureteral junction, but quite different results by functional parameters which reflect the possibility of draining of kidney in forced diuresis. Successful open pyeloplasty leads to a significant decrease in the pelvis area at different time intervals after furosemide administration, the relative increase in the pelvis area on the background of the induction of diuresis, rate of pelvis drainage, increase (normalization) in diameter of pyeloureteral junction, including the larger (better) gap of pyeloureteral junction after administration of diuretic. Concerning laparoscopic pyeloplasty, the dispersion of mean values of S, SPR, Vpr, V, VOT, D, DD after the operation was significantly different from those before the operation. This means that as in the case with open surgery, satisfactory clinical results such as reduction in renal pelvis and restoring the passage of urine through sufficient diameter pyeloureteral junction after laparoscopic pyeloplasty lead nevertheless to significant differences in the digital parameters during the objectification of operation effect by means of diuretic ultrasonography using furosemide-induced diuresis. Endoscopic surgery such as laser resection, endopyelotomy and balloon dilatation stably provides similar results (equal variances) by such parameters as pelvis area, which decreases in 40 min after furosemide administration, formation of the wide enough diameter of pyeloureteral junction and its minor fluctuations with the diuretic load. The decrease to normal parameters of all planes of renal pelvis (both before and after loading) and significant improvement of pelvis drainage (parameters responsible for the functional state of kidneys and pyeloureteral junction) indicate the success of palliative surgery in elimination of the narrowing of pyeloureteral junction.

161-164

Authors: Y. G. Kot, E. V. Kot, E. S. Morosova, E. E. Persky, M. A. Gritsenko, N. I. Bulankina

We studied gene expression of five metallothioneins (MT 1-5), ubiquitin and protein p53 and their products in fibroblasts culture of the skin and lungs of white rats of different ages (2 weeks, 1, 3, and 24 months) and determined its (metallothionein 1-5 types, ubiquitin, p53) product quantity. All these proteins are protective ones, but perform their functions by using different mechanisms. Metallothionein bind, transport and excrete ions of bivalent metals, ubiquitin controls the cleavage of the defective and short-lived proteins in the proteasome, protein p53 controls apoptosis, thus ensuring the genome stability. The similarity of age dynamics of gene expression of ubiquitin and MT of cells of both sources has been shown – maximum at 3 months. Expression of p53 gene has a difference: both in the skin and lungs expression increases up to 24 months. Product quantity of p53 has a minimum in the skin at 3 months and remains constant; in the lungs, this value has a maximum at 1 month.