Visnyk of Dnipropetrovsk University. Biology, medicine

90-93

Authors: O. S. Voronkova, O. A. Sіrokvasha, T. N. Shevchenko, A. І. Vіnnіkov

Today film-forming strains of bacteria play very important role in clinical pathology. Staphylococci are ones of most dangerous of them. This bacteria can determine different pathological processes, for example, complication of airway mucosa. The ability to form a biofilm is one of the main properties of nosocomial strains. These strains should be monitored and their carriers are to be properly treated. To determine the origin of staphylococci strains we used bacteriophages from the International kit. The aim of research was to determine the phage type of staphylococci film-forming strains, that were isolated from naso-pharingial mucosa. Phagetyping has been carried out for 16 film-forming strains of S. aureus. To solve this problem, we used the International phage kit by Fisher’s method. As a result, sensitivity to phages from the International kit showed 53.8% of studied strains of S. aureus. 64.3% of sensitivity strains were lysed by one of the phage, 21.4% – were by two of the phages, 14.3% – by three of the phages. Isolates were sensitive to phages: 81 – 42.9%, 75 – 35.7%, 28.6%were sensitive to phages 47 and 53. All cases of detection of sensitivity to phage 47 coincided with the ability to form biofilm. Among non-film-forming strains there was no sensitive strains for this phage. Film-forming strains resist to erythromycin (62.5%), ciprofloxacin (43.8%), gentamicin (56.3%), tetracycline (87.5%), amoxicillin (93.8%), and cefuroxime (37.5%). All cases of sensitivity to phage 47 coincided with resistance to erythromycin, amoxicillin and tetracycline. For two of these strains,wealso defined resistance to gentamicin and for one of them – to ciprofloxacin. Results of research allowed to relate the bacterial cultures for determining the type. This may have implications for studying of film-forming ability, because surface structures of bacterial cell take place in this process. Belonging of an isolate to specific phage type may indicate possible differences in the structure of the cell wall, composition of the cytoplasmic membrane, receptors, etc. So, phage typing may be used as one of teststo study film-forming processes. Besides, phage typing can be helpful in finding of the source and modes of transmission of nosocomial strains of bacteria. Given the increased level of antibioticresistance among film-forming strains of staphylococci, the importance of this monitoring is indisputable, because spreading of these strains may cause the failure of therapy.

99-103

Authors: G. S. Maslak

Tn-antigen (Thomsen-nouvelle antigen) is tumor-associated carbohydrate antigen with only one GalNAc residue attached to serine or threonine of polypeptide chain. There is not enough data about the expression of this glycotope in hematologic processes. But the correlations between increasing Tn-antigen expression on the cell surface and tumor growth progression, invasion, and activation of cell migration are well known. Therefore, the currently important area of modern research is studying of the impact of anticancer therapy by expression of this carbohydrate antigen in the onco-proliferative process. There are two types of cytostatic therapies in clinical hospitals of Ukraine: COP-therapy (cyclophosphamide, vincristine, prednisone) and FC-therapy (fludarabine, cyclophosphamide), which are the most popular due to their effectiveness and low price. The aim of our study was to investigate Tn-antigen exposure on the surface of lymphocytes, monocytes and granulocytes in polycythemia vera and subleukemic myelosis; to examine the influence of COP- and FC-therapies on Tn-antigen exponation in patients with chronic lymphocytic leukemia. The objects of the study were blood cells of patients with chronic lymphocytic leukemia (n = 25), polycythemia vera (n = 15) and subleukemic myelosis (n = 15) aged 58–66 years. Healthy hematologic volunteers (n = 15) aged 55 to 65 years were in the control group. Lymphocytes of patients with chronic lymphocytic leukemia (n = 25) were also studied after the chemotherapy treatment of patients divided into two groups: those who took COP-therapy (n = 13); and those who treated with FC-therapy (n = 12). Tn-antigen exposure on lymphocytes, monocytes and granulocytes was investigated by Beckman Сoulter EPICS flow cytometer with primary monoclonal Tn-antigen anybodies (Institute of Immunology, Moscow, Russia) and secondary fluorescein isothiocyanate labeled antybodies (Millipore, USA). The number of dead cells was monitored by binding them with propidium iodide. The result was analyzed with FC Express. According to our data, Tn-antigen exposure was not detected on the surface of blood cells (lymphocytes, monocytes and granulocytes) in the control group and in patients with polycythemia vera and subleukemic myelosis. Nevertheless, Tn-antigen was identified on the surface of more than 80% of lymphocytes in chronic lymphocytic leukemia patients. The intensity of this tumor-associated antigen exposure on lymphocytes membrane was 100 times higher compared with that in normal lymphocytes. In chronic lymphocytic leukemia patients after COP-treatment the number of lymphocytes with surface Tn-antigen was equal to 28,1 ± 0,8%, and after FC-treatment it decreased to 9,5 ± 0,5%. Moreover, positive effect of cytotoxic therapy used in treatment of patients with chronic lymphocytic leukemia on intensity of Tn-antigen exposure on the surface of lymphocytes was shown. FC-therapy (fludarabine, cyclophosphamide) is more effective; compared with the data prior to this treatment it 40 times reduced the relevant index. Therefore, it can be applied in Ukraine for chemotherapeutic treatment schemes effective against Tn-antigen.

110-114

Authors: A. H. Yaderna, L. P. Golodok, A. I. Vinnikov

The method of polymerase chain reaction (PCR) in real-time was used to analyze the quantitative characteristics of normal and potentially pathogenic aerobic/facultative-anaerobic and anaerobic biota in the urethra, cervical channel and vagina in healthy women aged 10–40. The biota of all the women under 40 years and some of women older than 40 was mostly represented by lactobacilli. Microbialcomposition of the biocenosis in some women older than 40 is characterized by reduction in quantity of lactobacilli and their replacement by anaerobic microorganisms, mainly, such as Atopobium vaginae (16%), Gardnerella vaginalis (12%), Megasphaera spp. (8%), Dialister spp. (8%), Eubacterium spp. (8%) and Porphyromonas spp. (4%). Rarely, a wide range of other pathogens plays its role, including inter alia: Trichomonas vaginalis (8%), Mycoplasma genitalium (4%), Neisseria gonorrhoeae (2%) andChlamydia trachomatis (1%). The most frequent are the following strain associations: A. vaginae andG. vaginalis,Eubacterium spp. andPorphyromonas spp.,U. (urealyticum + parvum) andM. genitalium,N. gonorrhoeae andCh. trachomatis,U. (urealyticum + parvum) andCandida spp. This is connected with anatomical and physiological characteristics of genitals, hormonal and immune system action. Quantitative study of the biota of urogenital tract in Dnipropetrovsk women with the use of real-time PCR is the sensitive method for diagnosing both physiological and pathological changes, and dysbiotic disorders at early stages and preventing their further development into more serious forms.

121-124

Authors: E. Tkach, M. Vakerich, V. Nikolaychuk

Specificneed in manganese is a characteristic feature of all plants. At present time, participation of this element in functioning in a number of processes and structures in the plant organism is proved. Indispensabilityof manganese as a component of plant metabolism is evidenced by death of plants aged 2–4 weeks at absolute exclusion of manganese nutrient medium, and symptoms of disorders at all levels of metabolism due to lack of this element. As opposed to symptoms of deficiency which are specific for every element, the signs of excess are more or less the same. In conditions of gradual increase of the concentration of ions as factors of influence, gradual manifestation of plant organism depression is observed: growth inhibition, root dying and leaf chlorosis, etc. Foliar feeding of grain crops including winter wheat with manganese is very important because they have a great need for such elements as manganese and copper. Efficiencyof the types of foliar feeding is connected with the mechanisms of penetration of manganese into the plant cells. Unlike other metals, manganese can be mobilized by sprouts from seed integument: metal absorption from wheat seeds lasts till the seventh day. That’s why, the aim of our work was to investigate the influence of pre-sowing processing of winter wheat seeds with solutions of different concentrations of manganese sulfate for detection of the optimal concentration and studying of phytotoxic influence of the excess manganese action. Vegetation tests were carried out in the laboratory conditions for performing the studies. Winter wheat seeds were steeped into solutions of manganese sulfate in such concentrations as 0.001%, 0.01%, 0.1%, 0.5% and 1%. After that they were couched in multi-component environment (sod-podzol soil). As a control variant, an experiment without treatment was used. The influence of different concentrations on the growth processes such as seed germination, length of the sprout, weight of the plants and their root system was estimated. It was determined that the optimal concentration of manganese sulfate for the pre-sowing processing was equal to 0.001–0.01%. The critical one, at which the phytotoxic action is manifested in growth depression and decrease of sprout biomass, is the use of 0.5–1.0% solution of manganese sulfate for the pre-sowing seed treatment.

131-137

Authors: V. Borisenko, S. Belov, I. Sorokina, N. Gorgol

The research is aimed at the study of dynamic pathomorphological changes of choledoch and acute cholangitis development factors determined during the experiment. 36 rats of Wistar line were under trial. The main group consisted of 30 animals undergoing the open laparotomy, choledoch ligation and puncture modeling of acute cholangitis by E. coli culture in 1 х 108 CFU/ml concentration under general anesthesia. 6 healthy rats were included in the control group. Samples of general biliary duct under autopsy for pathomorphological study were taken on the 3rd, 7th, 14th, 21st and 30th day. In panoramic samples colored by hematoxilin and eozin the degree of dystrophic, necrobiotic, hemodynamic, inflammatory and atrophic manifestations’ changes were studied. Average depth of choledoch wall and height of its epithelial lining were morphometrically estimated. Collagen of the IV type as well as expressing receptors to CD34 were defined with the help of monoclonal antibodies in choledoch epithelial cells of basal membranes and choledoch vessels endotheliocytes. In choledoch, enhancement of edema and inflammatory infiltration by lymphoplasmocytic elements with the admixture of neutrofils with granulation tissue was detected from the 3rd up to the 30th day of the experiment. From the 14th day formation of bile clots of blood was detected in choledoch clearance, part of which was locked to its de-epitheliolized internal surface. According to morphometrical study data, choledoch wall depth increased from 261.1 ± 3.13 µm on the 3rd day to 572.5 ± 3.42 µm on the 30th day of the experiment. Mucosa membrane has lost its folding on the 14th day, epitheliocytes flattening was replaced by their destruction with fragments rejection into the duct lumen by the 30th day of the experiment. The epithelium height index decreased from 14.8 ± 0.09 µm on the 3rd day to 11.7 ± 0.15 µm on the 30 day of the experiment. Collagen of the IV type fluorescence intensity of vessel basal membranes from the 3rd day closely matched the control, its enhancement was detected by the 30th day. Choledoch epitheliocytes fluorescence, expressing the receptors to CD34 reliably decreased by the 30th day of the study. In its turn, content of collagen of the IV tyre, as well as vessels endothelium expression in preparations treated by PQA to CD34 reliably increased by the 30th day of the experiment. Thus, in pathogenesis of acute cholangitis apart from well-known factors such as cholestasis and infection the third factor was detected, in the way of biliary ducts mucosa membrane lesion. In morphogenesis of choledoch mucosa membrane lesion the decrease in role of epitheliocytes adhesive properties was stated as well as deficit of collagen of the IV type in the structure of epithelial basal membranes. During observations where epithelial covering consistency was preserved, inflammatory changes in choledoch were insignificant, which is proved by sufficient resistance of biliary ducts epithelium to infection in the presence of cholestasis and bacteriocholia, but in cases when mucosa membrane de-epitheliolization took place the development of severe purulent-destructive cholangitis and pericholangitis was detected.

143-147

Authors: S. Kyrychenko, I. Prishchepa, V. Lagoda, M. Velika, V. Nedzvetsky

The aim of this study was to examine whether the antioxidant alpha-lipoic acid protects neurons from diabetic-reperfusion injury. The streptozotocin (STZ) rat model was used to study the glial reactivity and prevention of gliosis by alpha-lipoic acid (alpha-LA) administration. The expression of glial fibrillary acidic protein (GFAP) was determined, as well as lipid peroxidation (LPO) and glu-tathione (GSH) levels in some brain tissues. We observed significant increasing of lipid peroxidation products in both hippocampus and cortex. Changesof polypeptide GFAP were observed in hippocampus and cortex. Both soluble and filamentous forms of GFAP featured the increase in hippocampus of rat with hyperthyreosis. In the filamentfractions, increase in the intensity of 49 kDa polypeptide band was found. In the same fraction of insoluble cytoskeleton proteins degraded HFKB polypeptides with molecular weight in the range of 46–41 kDa appeared. Markedincrease of degraded polypeptides was found in the soluble fraction of the brain stem. The intensity of the intact polypeptide – 49 kDa, as well as in the filament fraction, significantly increased. It is possible that increasing concentrations of soluble subunits glial filaments may be due to dissociation of own filaments during the reorganization of cytoskeleton structures. Given the results of Western blotting for filament fraction, increased content of soluble intact 49 kDa polypeptide is primarily the result of increased expression of HFKB and only partly due to redistribution of existing filament structures. Calculation and analysis of indicators showed high correlation between the increase in content and peroxidation products of HFKB.These results indicate the important role of oxidative stress in the induction of astroglial response under conditions of diabet encefalopathia. Administration of alpha-LA reduced the expression both of glial and neuronal markers. In addition, alpha-LA significantly prevented the increase in LPO levels found in diabetic rats. GSH levels increased by the administration of alpha-LA. This study suggests that alpha-LA prevents neural injury by inhibiting oxidative stress and suppressing reactive gliosis. All these changes were clearly counteracted by alpha-lipoic acid. The results of this study demonstrate that alpha-lipoic acid provides for protection to the GFAP, as a whole, from diabet -reperfusion injuries.