Кафедра лабораторної діагностики біологічних систем

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Alternative routes for HB-EGF mitogenic effects inhibition
(2023) Siromolot, Andriy; Kolybo, Denys
Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathophysiological functions. Also, HB-EGF plays a pivotal role in distinct tumors progression. HB-EGF is highly expressed in certain cancer cells, such as ovarian and breast cancers, that makes it beneficial for the development of antitumor therapeutic agents. Neutralizing agents against HB-EGF can act on two strategies: sterically blocking the binding of HB-EGF with receptors (monoclonal or polyclonal antibodies, diphtheria toxin derivatives) or capable preventing the formation of soluble HB-EGF from proHB-EGF (ectodomain shedding) with MMPs or ADAMs inhibitors. Recombinant analogs of Corynebacterium diphtheriae toxin fragments (subunit B) and it toxoid CRM197 are able to block the binding of fluorescent derivative HB-EGF (HB-EGF-mCherry) with receptors HER1 and HER4 on the surface of A431 and Vero cell lines. It has been shown that immune serum-containing polyclonal antibodies against HB-EGF is able to inhibit HBEGF binding to surface cellular receptors and block the mitogenic activation with HB-EGF of cells by deactivating Ras-MAPK/ERK1/2. Mitogenic activity of sHB-EGF is significantly enhanced by membrane-associated heparan sulfate proteoglycans (HSPG). Interaction of the N-terminal heparin-binding region of HB-EGF with HSPG leads to stabilization of the EGF-like domain of the molecule in complex with the receptor. The ability of heparin to block proliferation of A431 and Vero cells by exogenous HB-EGF has been demonstrated, probably, due to binding with heparin-binding domain of HB-EGF. Moreover, leupeptin and a broad-spectrum metalloproteinase blocker GM6001, and EDTA significantly decrease paracrine activation of cells by HB-EGF. Thus, searching and development of effective neutralizing agents against sHB-EGF is important and perspective research direction for establishing of a new targeted antitumor therapy.
Analysis of polyamine levels in blood serum of patients with breast cancer using optical SPR-based immune biosensor
(2019) Prylutskyi, Maksym; Starodub, Nickolaj
The aim of the study was to develop a method for qualitative and quantitative analysis of polyamines as potential tumour markers in blood serum of patients diseased with breast cancer with use of newly created immune biosensor based on effect of surface plasmon resonance.
Assessment of hematopoietic stem cells in norma and pathology in cell culture in vitro and in vivo
(2019) Bilko, Denys; Sviezhentseva, Ilona; Bilko, Nadiia
The aim of the current study was to to determine the role of erythroid lineage of differentiation side by side with granulocyte-macrophage progenitor cell in the mechanism of resistance for TICs therapy.
Assessment of Hemopoietic Progenitor Cells in Cell Culture Correlates With Cytogenetic Analysis of Patients with Chronic Myeloid Leukemia : [preprint]
(2010) Bilko, Nadiia; Diachenko, M.; Borbuliak, Iryna; Dyagil, Iryna
Abstracts of the XVIII. Wilsede Meeting "Modern Trends in Human Leukemia and Cancer", June 19-23, 2010.
Assessment of hemopoietic progenitor cells in patients affected by Chernobyl accident and risk of oncohematological diseases
(2009) Bilko, Nadiia
Chromosomal segregation in sperm of Robertsonian translocation carriers
(2013) Pylyp, Larysa; Zukin, Valery; Bilko, Nadiia
Purpose To study meiotic segregation patterns of Robertsonian translocations in sperm of male carriers and to assess the frequencies of unbalanced sperm formation. Methods FISH with combination of probes to detect all the variants of meiotic segregation was performed on decondensed sperm nuclei of 5 carriers of der(13;14), 3 carriers of der(14;21) and one carrier of a rare der(13;21) translocation. Results The frequency of sperm with alternate segregation and normal/balanced chromosomal complement ranged from 68 % to 94.4 % (mean 79.2±8.4). Adjacent segregation was detected in 17.9±7.3 % of sperm (from 5.6 % to 29 %). No significant differences in frequencies of gametes with nullisomies and disomies of chromosomes involved in translocations were observed. The mean frequency of 3:0 segregation products was 2.5±1.4 %. Conclusions All analyzed patients showed homogenous segregation pattern with clear predominance of alternate segregation resulting in normal/balanced sperm production. Still, from 5.8–32% (mean 20.4±8.3 %) of sperm was unbalanced, which is the evidence of the increased risk of unbalanced offspring in carriers of Robertsonian translocations. Our results highlight the importance of genetic counseling of Robertsonian translocation carriers prior to ICSI or IVF.
Circulating hematopoietic progenitor cells in patients affected by chornobyl accident
(2016) Bilko, Nadiia; Dyagil, Iryna; Russu, Iryna; Bilko, Denys
High radiation sensitivity of stem cells and their ability to accumulate sublethal radiation damage provides the basis for investigation of hematopoietic progenitors using in vivo culture methodology. Unique samples of peripheral blood and bone marrow were derived from the patients affected by Chomobyl accident during liquidation campaign. Aim: To investigate functional activity of circulating hematopoietic progenitor cells from peripheral blood and bone marrow of cleanup workers in early and remote periods after the accident at Chornobyl nuclear power plant (CNPP). Materials and Methods: The assessment of the functional activity of circulating hematopoietic progenitor cells was performed in samples of peripheral blood and bone marrow of 46 cleanup workers, who were treated in the National Scientific Center for Radiation Medicine of the Academy of Medical Sciences of Ukraine alongside with 35 non radiated patients, who served as a control. Work was performed by culturing peripheral blood and bone marrow mononuclear cells in the original gel diffusion capsules, implanted into the peritoneal cavity of CBA mice. Results: It was shown that hematopoietic progenitor cells could be identified in the peripheral blood of liquidators of CNPP accident. At the same time the number of functionally active progenitor cells of the bone marrow was significantly decreased and during the next 10 years after the accident, counts of circulating progenitor cells in the peripheral blood as well as functionally active hematopoietic cells in bone marrow returned to normal levels. Conclusion: It was shown that hematopoietic progenitor cells are detected not only in the bone marrow but also in the peripheral blood of liquidators as a consequence of radiation exposure associated with CNPP accident. This article is a part of a Special Issue entitled “The Chornobyl Nuclear Accident: Thirty Years After”.
Comparison of Methods for Cardiomyocyte Differetiation of Murine Embryonic Stem and Induced Pluripotent Cells
(2011) Budash, Galyna; Saric, Tomo; Hescheler, Juergen; Malysheva, Svetlana; Bilko, Denys; Bilko, Nadiia
Detection of air-borne mycotoxin levels by immunobiosensor
(2018) Starodub, Nickolaj; Kluchevich, M.; Székács, A.; Vigera, S.
The main purpose of this article is to investigate the possibility of detection of the level of mycotoxins in air samples by a surface plasmon resonance (SPR) immunobiosensor. Air samples of 0.25-0.5 m3 volume collected using a special miniature air pump device were passed through 20% acetonitrile as an absorption liquid, and 20 μl aliquots of the extract were analyzed by an SPR immunobiosensor. Comparative analyses by the determination of T2 mycotoxin content were carried out in air and in grain samples upon dispersion in a phytotron. Moreover, mycotoxin content was investigated also under field conditions in air and in vegetable matrix, in the absence and presence of Fusarium fungi, and results were shown to have similar characteristics: the level of mycotoxins detected in air samples correlated with that in grain samples. Thus, results of air-borne mycotoxin detection were correlate with mycotoxin levels detected in environmental samples and may used for screening purposes.
Detection of biogenic polyamines in blood of patients with breast cancer
(2019) Prylutskyi, Maksym; Bilko, Nadiia; Starodub, Nickolaj
The main threat of cancer diseases is their spreading throughout the population of many countries of the world and the complexity of their diagnostics at the early stages. Because of that, search and development of the latest diagnostic methods for oncological diseases which would allow them to be diagnosed more precisely and rapidly is going on. The objective of the study was developing a method for qualitative and quantitative analysis of polyamines as potential tumour markers in blood serum of patients with breast cancer using a newly created immune biosensor based on the effect of surface plasmon resonance (SPR). In the process of the study we used basics of immune analysis, methods of biosensor analysis with preliminary modification of biosensor analytical surface with certain reagents, such as protein A and BSA, for better orientation of the sensitive layer made from antibodies. During the study 30 samples of blood serum were analyzed, 21 of which were obtained from the patients with breast cancer and 9 samples were normal, taken from healthy people and used as controls. Analysis of blood serum samples was made, using previously created calibration curve, based on polyamine solutionsin concentrations from 5 ng/mL to 1 μg/mL. It allowed determination of the presence of polyamines in blood samples and approximate concentration of polyamines comparing resonance angle shift in calibration curve and blood samples. According to the obtained results, the concentration of polyamines exceeded their physiological levels and was in the range of 21.3–125.1 ng/mL. The proposed approach allows one to determine the presence and approximate concentrations of polyamines in range from 5 ng/mL to 1 μg/mL in samples of blood serum of patients with breast cancer which correlates with tumour size and the age of the patients.
Detection of polyamines using immunobiosensor based on zinc oxide nanoparticles
(2019) Prylutskyi, Maksym; Starodub, Mykola
Aim: To analyze the performance of biosensor based on nanoparticles of zinc oxide for the detection of spermine and spermidine in solution and in cell culture. Materials and Methods: Zinc oxide nanoparticles were used for preparing biosensor containing antibodies to spermine and spermidine. Polyamine concentration is solutions of spermine and spermidine as well as in lyophilisate of MCF-7 cells was measured by luminescence of the samples excited by laser beam at 380 nm. Results: The minimum concentration for the detection of polyamines in model solutions is 10 ng/ml, and maximum one is 100 ng/ml. A higher level of luminescence intensity of nanoparticles was found during analysis the polyamines in MCF-7 lyophilisate allowing for detecting polyamines at concentrations from 100 cells/ml to 100,000 cells/ml. Conclusions: The proposed biosensor system for determining the level of biogenic polyamines in cell lyophilisate using the optical properties of zinc oxide nanoparticles is promising for further improvement of the methodology and its implementation for detection and measurement of polyamines in biological systems.
Determination of the Concentration of Polyamines with SPR-based Immune Biosensor for Early Diagnostics of Breast Cancer
(2016) Prylutskyi, Maksym; Starodub, Nickolaj; Bilko, Nadiia
The paper presents the results of research on the development of immune biosensor test system for express detection of polyamines in cells of breast cancer. Determination of polyamines was performed by using an analytical device - immune biosensor based on surface plasmon resonance (SPR), where "antigen-antibody" reaction is performed in real time on the surface of transducer, resulting in formation of immune complexes and recording the shift of resonance angle. The method can detect the studied polyamines spermine and spermidine in concentration less than ’0 ng in ’ mL, and with increasing concentrations a statistical probability of the analysis' result rise sharply. Moreover, the dependence of sensitivity of biosensor response from the concentration of polyamines is in the range of ’0-’00 ng /mL.
Determination of the optimal chemotherapy drugs pretreatment time through cultivation of hemopoietic cells in cml-patients treated with tyrosine kinase inhibitors
(2014) Zhaleiko, I.; Perekhrestenko, T.; Bilko, Denys; Dyagil, Iryna; Bilko, Nadiia
Targeted therapy drugs, including imatinib, are used for inhibiting the marker oncoprotein of chronic myeloid leukemia — BCR-ABL tyrosine kinase. However, in some patients the drug resistance can emerge too rapidly and a previous treatment with chemotherapy drugs can lead to formation of resistance. Aim: To evaluate the influence of drugs that were used prior to the imatinib on the performance of the functional activity of bone marrow cells from chronic myeloid leukemia patients and their individual responses to therapy. Methods: Bone marrow aspirate from 57 patients, who were getting busulfan (19 patients) or hydroxy- carbamide (38 patients) prior to imatinib was studied with cytogenetic and tissue culture methods in vitro. Results: Obtained data suggested that pretreatment with busulfan, regardless of duration, negatively affects the response to further therapy with imatinib. Instead, after using hydroxycarbamide as a previous therapy for six month, there was optimal response to imatinib. In those cases when duration of pretreatment with hydroxycarbamide was increased to a year or more, there was a suboptimal response and a resistance to imatinib therapy. In addition, there was a positive correlation between the number of cell aggregates (colonies and clusters) in semisolid agar and the duration of a prior treatment with hydroxycarbamide, if previous therapy did not exceed 20 months. With an increase of pretreatment terms to 21 months or more, such a correlation was not observed. Conclusions: These results suggest that chemotherapeutic agents (busulfan and hydroxycarbamide) may additionally contribute to the accumulation of mutations in the genome of leukemic cell clone affecting the behavior of these cells in vitro.
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies
(2016) Budash, Galyna; Bilko, Denys; Bilko, Nadiia
Мета. Знайти зв’язок між розміром ембріоїдних тілець та ефективністю диференціювання плюрипотентних стовбурових клітин в кардіоміоцити. Методи. Трансгенні клітинні лінії індукованих плюрипотентних клітин AT25 та ембріональних стовбурових клітин D3 aPIG44 диференціювали в кардіоміоцити в AggreWell планшетах. Вище згадані планшети містять мікролунки, які дають можливість сформувати ембріоїдні тільця з плюрипотентних стовбурових клітини певного заданого розміру. Обидві клітинні лінії були генетично модифіковані і експресували IRES-фланкирований зелений флуоресцентний білок ^GFP), під контролем кардіоспецифічного a-MHC про- мотера. Для перевірки ефективність процесів диференціювання були застосовані методи проточної цитометрії та флуоресцентної мікроскопії. Результати. Було встановлено, що ефективність диференціювання ембріоїдних тілець отриманих з лінії індукованих плюрипотентних клітин лінії AT25 розміром 250 і 1000 клітин менша в порівнянні з ембріональними тільцями сформованими з 500 і 750 клітин. Кількість eGFP+ клітин, отриманих з ембріоїдних тілець розміром 500 клітин була 8,5 разів більшою ніж в порівнянні з ембріональними тільцями розміром 250 клітин (що становило 2,86 ± 0,30 % кардіоміоци- тів для ембріоїдних тілець розміром 500 клітин, і лише 0,34 % eGFP+ клітин для ембріоїдних тілець розміром 250 клітин). Висновки. Впливати на ефективність диференціювання плю- рипотентних стовбурових клітин в кардіоміоцити можна змінюючи початковий розмір ембріоїдних тілець. Серед ембріоїд- них тілець, утворених в діапазоні від 250 до 2000 клітин, найвищий відсоток eGFP + клітин отримували з ембріоїдних тілець, утворених 500 клітинами.
Dynamics of Telomere Length and Telomerase Activity in the Human Fetal Liver at 5-12 Weeks of Gestation
(2018) Sorochynska, Khrystyna; Sych, Nataliia; Duda, Alla; Kulebyakina, Kateryna; Krasnienkov, Dmytro; Vaiserman, Alexander; Vatlitsov, Denys
Fetal stem cell- (FSC-) based therapy is a promising treatment option for many diseases. The differentiation potential of FSCs is greater than that in adult stem cells, and they are more tissue-specific and have lower immunogenicity and better intrinsic homing than embryonic ones. Embryonic stem cells have higher proliferative potential than FSCs but can cause teratomas. Therefore, an evaluation of this potential represents an important biomedical challenge. Since regulation of telomere length (TL) is one mechanism governing cellular proliferation, TL is a useful surrogate marker for cell replicative potential. The prenatal dynamics of TL, however, has never been comprehensively studied. In the present study, dynamics of TL and telomerase activity in the human fetal liver during 5–12 weeks of gestation is examined. Both TL and telomerase activity were positively correlated with week of gestation. For both parameters studied, the trend to increase was evident up to 10th week of gestation. After that, they reached a plateau and remained stable. These findings indicate that telomerase activity remains high during the fetal stage, suggesting high replicative capacity of FSCs and their considerable potential for transplantation therapies. These findings, however, are preliminary only due to small sample size and require further evaluation.
Effects of fetal stem cells on life quality of patients with ulcerative colitis
(2018) Demchuk, Mariya; Ivankova, Olena; Klunnyk, Mariya; Matiyashchuk, Iryna; Sych, Nataliia; Sinelnyk, Andriy; Karayev, T.; Shulak, M.; Skalozub, Marina; Sorochynska, Khrystyna
In this article we briefly summarized on the main characteristic features of ulcerative colitis (UC) and accentuated on reduced life quality among the patients with UC, in particular, effect of clinical manifestations of the disease on everyday life was described in such patients. Unambiguously, the principal direction to better life quality in patients with UC consists in decreasing activity of inflammatory process. Significant aspect in solution of this problem for present day suggests use of fetal stem cells (FSCs) in complex treatment of the patients suffering from UC. Specifically, by virtue of stem cell therapy use, rapid lowering of clinical signs of UC was recorded, patients revealed stabilization of hemoglobin and total protein levels in blood. Changes of such parameters in the CG patients tended to be much slower. Within the whole period under study, we recorded a consistent pattern between decreased evidence of underlying disease manifestation that is coupled with higher life quality among the UC patients. In conformity with IBDQ international questionnaire, describing that bowel symptoms, systemic symptoms, emotional and social function, life quality among UC patients of the MG were increased (51.33% vs. 96.43%), whereas the patients of the CG reported the value 61.16% that was higher only within the range of 86.16%. Administration of suspensions with FCSs for complex therapy in UC patients proved to be one of the ways for refining social and psychological wellbeing of the individuals under study.
Efficacy of Fetal Stem Cells Use in Complex Treatment of Patients With Insulin-resistant Type 2 Diabetes Mellitus
(2016) Demchuk, Mariya; Ivankova, Olena; Klunnyk, Mariya; Matiyashchuk, Iryna; Sych, Nataliia; Sinelnyk, Andriy; Novytska, Dr. Alla; Sorochynska, Khrystyna
Objective: To identify efficacy of fetal stem cells in treatment of the patients suffering from Type 2 diabetes mellitus in presence of insulin resistance and to summarize all characteristics of post-transplantation disease course. Materials and methods: 42 patients suffering from Type 2 diabetes mellitus (T2DM) in presence of insulin resistance were examined including 27 men and 15 women. Glycaemia ranges were controlled by diet therapy in 7 patients (16.7%), whereas the rest of the patients were recommended antihyperglycemic medicines (biguanides, thiazolidinediones, sulfonylurea derivatives and a-reductase inhibitors). Fetal stem cells (FSCs) were administered in complex therapy for the patients with T2DM and the data of clinical, laboratory and anthropometric investigations were analyzed over the study period. Results: Positive effects to values of carbohydrate and lipid metabolism, as well as arterial blood pressure (ABP) in T2DM have been justified. FSCs capacity to decreasing insulin resistance (in accordance with the HOMA-IR scale) which results in stable continuous diabetes mellitus compensation was established. Conclusion: FSCs use in complex treatment of patients with T2DM stabilizes disease compensation and leads to decrease in hyperinsulinemia and lower insulin resistance.
Entrapment modification of the polyacrylamide gel with chitosan polymers for the purposes of surface activation for tissue engineering
(2016) Bilko, Denys; Borodulin, Y.; Antoniuk, N.; Kolesnyk, Iryna; Burban, Anatoliy
In recent years hydrogels have become attractive targets for development of the controlled release rate substances that are characterized by suitable chemical and physical properties. Recent advances in the tissue engineering and cell therapy technologies have introduced additional value to the biologically compatible hydrogels that could be chemically modified with specific factors, such as cell microenvironment proteins, allowing for shaping and controlled growth of the cells in the 3D formations, suitable for further tissue engineering and cell therapy. This paper reports a mechanism of the entrapment modification of the polyacrylamide based gels with chitosan polymer. Such modification allows for the further covalent crosslinking of the protein-based growth factors and other extracellular matrix proteins that fully support proliferation and maintenance of the adhesion dependent and adhesion independent cells, including hematopoietic and mesenchymal stem cells.
Expression of SSEA-1 in different clones of reprogrammed murine embryonic fibrodlasts
(2011) Malysheva, Svetlana; Saric, Tomo; Hescheler, Juergen; Budash, Galyna; Bilko, Denys; Bilko, Nadiia
Features of damage and recovery of rats hematopoietic system following the exposure to sublethal doses of ionizing radiation
(2019) Sniezhkova, Yelyzaveta; Rodionova, Natalia; Pakharenko, Marharyta; Russu, Iryna; Kavertseva, Mariia; Dubova, Tetiana; Bilko, Nadiia
The aim of the current study was to determine colony-forming activity of granulocytic-macrophage and erythroid progenitor cells in non-irradiated animals and after irradiation in sublethal doses and to investigate the process of hematopoietic recovery using cell culture in vivo.

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