Immunomodulatory properties of recombinant human granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a myelopoietic growth factor that exerts pleiotropic effect not only on the differentiation of immature progenitor cells into polymorphonuclear neutrophils, monocytes/macrophages and dendritic cells, but also controls the functioning of differentiated cells. GM-CSF is currently being investigated in clinical trials as an immunomodulator and adjuvant. However, a wide range of biological activities and, sometimes, paradoxical effects of this cytokine require more thorough studies of its action, in order to predict its efficacy under different conditions of immunotherapy. In this work, we have studied the effect of recombinant human GM-CSF on metabolic activity of mouse peritoneal exudate cells in primary cell cultures. Metabolic (redox) activity of the cells was assessed by their ability to reduce nitroblue tetrazolium (NBT) in the course of MF- and Fc-dependent phagocytosis triggered by addition of opsonized zymosan, or sheep erythrocytes to the culture medium. We have shown the dose-dependent stimulatory effect of GM-CSF on the oxidative metabolism of phagocytic peritoneal macrophages and neutrophils. Upon culturing the pepton-elicited cells at wide range of GM-CSF concentrations (5 to 40,000 ng/mL) for 2 and 24 hours, a more pronounced effect of the substance was observed for neutrophils. The GM-CSF preparation caused a significant increase (by 13-17%) in the redox activity of neutrophils induced by opsonized zymosan that persisted at a low dose range, and was retained after 24 hours. The stimulatory effect of GM-CSF on macrophages with NBT index increase by 16% was observed in the short-term cultures. In general, the elicited cells of both types showed a more pronounced response to lower concentrations of GM-CSF (5-125 ng/mL), and weaker effect at higher doses of the preparation. A similar dependence was found when studying the resident macrophages. Culturing of resident cells with GM-CSF at the doses of 5,000 to 40,000 ng/mL for 24 hours caused a significantly increased redox activity of the cells induced by zymosan, or sheep erythrocytes (by 33-52%). In both cases, the maximal response was detected at a dose of 5,000 ng/mL and decreased with increasing dose. The stimulatory effect of GM-CSF upon resident macrophages was more pronounced as compared to elicited cells, which was characterized by the prolonged period of cell activation (up to 24 hours of culture). The data obtained are of interest, in view of prospective usage of GM-CSF as a component of immunomodulatory and adjuvant therapy for various infectious diseases

Monopoly of legal provisions

The existing defect of the legal field in the form of the inconsistency of the norms that make it up, gives rise to inconsistency, imbalance of the main social regulator of social relations. Building the normative material without analysing the need to create a separate regulator, as well as the obligation to establish a relationship with existing norms, determined the significance of such a study. The definition of competition of norms is an object of scientific research quite often. However, the content of this category and its law enforcement significance, in our opinion, have not been established quite correctly. The presence of competition of norms indicates the inconsistency of legal regulators with the principles of certainty of law, fairness and stability of legal regulation, which affects the effectiveness of laws. The research patterns are due to the need for a systematic analysis of this institution in order to identify uncovered aspects in the construction of a clear, systemic and interdependent system of legal regulation. Purpose of the research. The importance of developing fundamentally new approaches to the institution of uncertainty mediated by the phenomenon of competition to overcome the imbalance of the existing legal system, in connection with the existing need to present the normative material unambiguously and clearly. Methods. In the course of the study, in combination with a complex and systemic analysis, the following general scientific methods of cognition were used: dialectical, hermeneutic, and synthesis methods, the method of ascent from the abstract to the concrete, and the method of generalization and comparison. Results and novelty. Competition of norms is the result of an incorrect construction of the system of legal norms indicating a certain artificiality of this definition regarding the possible flexibility of the legal system. In our opinion, an increase in monopoly and individualized norms will lead to a systematic nature of the normative material, as well as an increase in confidence in it on the part of all participants in legal relations. The certainty of law must become a legal axiom

Double-stranded RNAs are promising adjuvants for enhancing immunogenicity of vaccines

Background. The most effective way to prevent infectious diseases is vaccination. Adjuvants contribute to the optimization of the immune response of vaccines. Double-stranded ribonucleic acids (dsRNAs) from natural sources are promising, but insufficiently studied adjuvants. The aim of the work was to study the adjuvant activity of dsRNA obtained from the killer strain of Saccharomyces cerevisiae using two models of induction of a specific immune response. Materials and methods. In the experiments, the substance of the drug Ridostin containing dsRNA, 21.72% (produced by Institute of Medical Biotechnology of the State Research Center of Virology and Biotechnology Vector), was used. A specific immune response was modeled using ovalbumin (OVA) or the substance of the EpiVacCorona vaccine (EVC). The experiments were carried out in 200 female BALB/c mice. Mice of the experimental groups were injected twice with antigen and adjuvant together with a 28-day interval, mice of the comparison group with antigen only. On the 10th day after the second immunization, blood samples were collected to determine the level of specific antibodies using enzyme immunoassay. The results were evaluated by calculation of the average geometric titers of specific antibodies against OVA or EVC. Results. OVA or EVC administered twice induced the specific antibodies in mice in dose-dependent titers. The combined administration of antigen and dsRNA increased the strength of the immune response. The highest stimulating effect of dsRNA was observed in the dose of 100 g/mouse administered into mice immunized with OVA (1 g/mouse) or in the dose of 50 g/mouse in mice immunized with EVC substance (0.25 of a human dose per mouse). Conclusion. The data obtained indicate that the substance of dsRNA exerts adjuvant properties, which gives reason to consider dsRNA as a promising adjuvant for peptide vaccines

Stimulating effect of double-stranded yeast RNA on the activity of interferon system genes

Influence of double-stranded RNA (dsRNA) from Saccharomyces cerevisiae yeast upon expression levels of the macrophage genes encoding TLR3 receptor, interferons alpha and beta (IFNα, IFNβ), 2’,5’-oligoadenylate synthetase (OAS) and protein kinase R (PKR) enzymes has been studied in the J774 mouse histiocytic cell culture and in vivo in Balb/c mice. It has been shown that dsRNA exerts a selective activating effect on genes of TLR3 receptor, antiviral proteins IFNα, IFNβ, and OAS, both in vitro and in vivo. With J774 cell culture, the highest induction capacity was observed for the IFNβ gene: 365 to 802-fold. The stimulatory effect was dependent on the dose of dsRNA in the range of 16.9 to 125 μg/ml. The preparation enhanced IFNα gene activity to lesser degree (more than 10-fold), TLR3 and OAS (3 to 4-fold), while the expression levels for these genes were not significantly dependent on the dose of dsRNA. The stimulating effect of dsRNA was dosedependent in murine peritoneal macrophages. The maximum activating effect of the preparation was shown upon administration of the effective antiviral dose (0.5 mg of dsRNA/kg). Five hours after intraperitoneal injection of dsRNA, the highest level of mRNA synthesis was observed for IFNα (54-fold), OAS (43-fold) and TLR3 (28-fold) genes. Expression of the IFNβ gene increased to a lesser degree (9-fold). An increase in the dose of preparation to 1.5 mg/kg led to decrease of the stimulatory effect. Expression levels of the IFNα, TLR3, and OAS genes in that case decreased by 2-4-fold as compared to a lower dose, and the PKR gene expression was 5-fold lower compared to the control. One day after dsRNA administration, a tendency was observed for both experimental groups towards a decreased transcription of macrophage genes, if compared with the 5-hour term. The weakening of gene activity was less pronounced in animals treated with dsRNA at the dose of 1.5 mg/kg. The transcription indices for IFNβ, OAS, and TLR3 genes were much higher during this period (5-10-fold higher than the control values). The dynamics of PKR gene transcription in both experimental systems was significantly different from the expression of other studied genes. The dsRNA preparation at this dose range did not have a pronounced stimulatory effect upon expression of this gene. A moderate increase in PKR gene activity in macrophages of mice was observed only a day following intraperitoneal administration of dsRNA. Concentrations and length of dsRNA molecules are known to be critical factors to the PKR gene activation. An ability to increase the expression of the gene is shown at low dsRNA concentrations (10-7 g/ml and below), while highly polymeric dsRNAs weaken the gene activity. Since the doses and concentrations of dsRNA used in our experiments were significantly different from those mentioned above, it could, in general, affect regulation of PKR gene transcription towards reduction of the stimulatory effect

MEDICINAL FORM OF TNF-α FOR LOCAL ADMINISTRATION

Composite preparation of tumor necrosis factor alpha and rheopolyglukin and polyethylene glycol (TNF-α+PG+PEG) was obtained. The specific activity of the samples was 4,13 х 107 IU/mg. The cytolytic activity of drugs TNF-α+PG+PEG and rhTNF-α did not change after 4 months when stored at 6 °С. Preparation TNF-α+PG+PEG provided a moderately prolonged elevation of TNF-alpha in blood of laboratory mice in contrast to TNF-α when they applied to the skin. The composite preparation did not have toxic, allergic and locally irritating action in experiments on laboratory animals

Фармакокинетика рекомбинантного человеческого фактора некроза опухоли альфа в составе средства доставки

The main problems of using TNF-alpha in antitumor therapy are its rapid degradation in the bloodstream and the limited selectivity of accumulation in the tumor tissue. The SRC VB «Vector» developed a biodegradable molecular construct that provides protection against proteases and ensures targeted delivery of proteins to the tumor tissue. This construct was used to create an antitumor drug containing recombinant human TNF-alpha (rhTNF-alpha).The aim of the study was to analyse rhTNF-alpha pharmacokinetics in the delivery system after a single administration.Materials and methods: the rhTNF-alpha drug carried by the delivery system was intravenously administered to female outbred ICR (СD-1) mice only once at two effective antitumor doses, 2.55 μg and 5.1 μg / 20 g of body weight. The concentration of TNF-alpha in the serum and supernatants of organ homogenates, obtained at different time points after administration, was analysed by immunoenzyme assay.Results: the obtained curves of TNF-alpha concentration in the blood were satisfactorily described by the equation for the twocompartment model without absorption. The rapid phase of elimination from the blood took 0–4 h, the slow one — 4–24 h. The highest specific content of protein was observed in the skin, spleen, and kidneys tissue. The calculation of pharmacokinetic parameters demonstrated that the highest values of tissue availability fT were obtained for the kidneys and skin; the drug was retained for longer periods of time in the kidneys, liver and skin (according to the MRT data). As a rule, complete elimination of the drug was observed by the end of the first day after administration.Conclusions: rhTNF-alpha carried by the delivery system was quickly eliminated from the blood and distributed in the internal organ tissues after a single intravenous administration to mice in the effective doses range. The main organs in which rhTNF-alpha was distributed were skin, kidneys, and spleen. The elimination of the drug from the blood was a two-phase process which was generally over by the end of the first day.Основными проблемами использования фактора некроза опухоли альфа (ФНО-альфа) в противоопухолевой терапии являются его быстрая деградация в кровеносном русле и ограниченная селективность накопления в ткани опухоли. В ФБУН ГНЦ ВБ «Вектор» Роспотребнадзора создана биодеградируемая молекулярная конструкция, обеспечивающая защиту от протеаз и адресную доставку белков в ткань опухоли. На основе этой конструкции разработан противоопухолевый препарат, содержащий рекомбинантный ФНО-альфа человека (рчФНО-альфа). Цель работы: изучить фармакокинетику рчФНО-альфа в средстве доставки при его однократном введении. Материалы и методы: препарат рчФНО-альфа в средстве доставки вводили самкам аутбредных мышей ICR (CD-1) однократно внутривенно в двух эффективных противоопухолевых дозах 2,55 мкг и 5,1 мкг на 20 г массы тела. Концентрацию ФНО-альфа в сыворотке крови и супернатантах гомогенатов органов, взятых в разные сроки после введения, определяли иммуноферментным методом. Результаты: полученные кривые изменения содержания в крови ФНО-альфа удовлетворительно описывались уравнением для двухчастевой модели без всасывания. Быстрая фаза процесса выведения из крови приходилась на период 0–4 ч, медленная — 4–24 ч. Наиболее высоким удельное содержание белка было в ткани кожи, селезенке и почках. Расчет фармакокинетических параметров указал на то, что наиболее высокие значения тканевой доступности fT были установлены для почек и кожи; более длительно (в соответствии с данными MRT) препарат удерживался в почках, печени и коже. Процесс элиминации препарата в основном завершался к концу первых суток после введения. Выводы: рчФНО-альфа в средстве доставки при однократном внутривенном введении мышам в диапазоне эффективных доз быстро элиминировался из крови и распределялся по тканям внутренних органов. Основными органами распределения препарата являлись кожа, почки и селезенка. Процесс элиминации препарата из крови носил двухфазный характер и в основном завершался к концу первых суток

Повышение точности прогнозирования генерации фотоэлектрических станций на основе алгоритмов k-средних и k-ближайших соседей

Renewable energy sources (RES) are seen as a means of the fuel and energy complex carbon footprint reduction but the stochastic nature of generation complicates RES integration with electric power systems. Therefore, it is necessary to develop and improve methods for forecasting of the power plants generation using the energy of the sun, wind and water flows. One of the ways to improve the accuracy of forecast models is a deep analysis of meteorological conditions as the main factor affecting the power generation. In this paper, a method for adapting of forecast models to the meteorological conditions of photovoltaic stations operation based on machine learning algorithms was proposed and studied. In this case, unsupervised learning is first performed using the k-means method to form clusters. For this, it is also proposed to use studied the feature space dimensionality reduction algorithm to visualize and estimate the clustering accuracy. Then, for each cluster, its own machine learning model was trained for generation forecasting and the k-nearest neighbours algorithm was built to attribute the current conditions at the model operation stage to one of the formed clusters. The study was conducted on hourly meteorological data for the period from 1985 to 2021. A feature of the approach is the clustering of weather conditions on hourly rather than daily intervals. As a result, the mean absolute percentage error of forecasting is reduced significantly, depending on the prediction model used. For the best case, the error in forecasting of a photovoltaic plant generation an hour ahead was 9 %.Возобновляемые источники энергии рассматриваются как средство снижения углеродного следа топливно-энергетического комплекса, при этом стохастический характер генерации осложняет их интеграцию с электроэнергетическими системами. Эта существенная трудность обусловливает необходимость создавать и совершенствовать методы прогнозирования генерации электрических станций, использующих энергию солнца, ветра и водных потоков. Наиболее важным направлением, обеспечивающим повышение точности прогнозных моделей, является глубокий анализ метеорологических условий как главного фактора, влияющего на выработку электроэнергии. В данной работе предложен и исследован метод адаптации прогнозных моделей под метеорологические условия работы фотоэлектрических станций на базе алгоритмов машинного обучения. При этом вначале выполняется обучение без учителя методом k-средних для формирования кластеров. Для этой задачи также предложено и исследовано использование алгоритма понижения размерности пространства признаков для визуализации оценки точности кластеризации. Затем для каждого кластера построена своя модель машинного обучения для формирования прогнозов и алгоритм k-ближайших соседей для отнесения текущих условий на этапе эксплуатации модели к одному из сформированных кластеров. Исследование было проведено на почасовых метеорологических данных за период с 1985 по 2021 г. Одной из особенностей этого подхода является кластеризация метеоусловий на часовых, а не суточных интервалах. В результате средний модуль относительной ошибки прогнозирования существенно снижается в зависимости от используемой модели прогнозирования. Для наилучшего варианта ошибка прогнозирования генерации фотоэлектрической станции на час вперед составила 9 %

Public Transport in Almaty

ústav dopravních systém

Monopoly of legal provisions

The existing defect of the legal field in the form of the inconsistency of the norms that make it up, gives rise to inconsistency, imbalance of the main social regulator of social relations. Building the normative material without analysing the need to create a separate regulator, as well as the obligation to establish a relationship with existing norms, determined the significance of such a study. The definition of competition of norms is an object of scientific research quite often. However, the content of this category and its law enforcement significance, in our opinion, have not been established quite correctly. The presence of competition of norms indicates the inconsistency of legal regulators with the principles of certainty of law, fairness and stability of legal regulation, which affects the effectiveness of laws. The research patterns are due to the need for a systematic analysis of this institution in order to identify uncovered aspects in the construction of a clear, systemic and interdependent system of legal regulation. Purpose of the research. The importance of developing fundamentally new approaches to the institution of uncertainty mediated by the phenomenon of competition to overcome the imbalance of the existing legal system, in connection with the existing need to present the normative material unambiguously and clearly. Methods. In the course of the study, in combination with a complex and systemic analysis, the following general scientific methods of cognition were used: dialectical, hermeneutic, and synthesis methods, the method of ascent from the abstract to the concrete, and the method of generalization and comparison. Results and novelty. Competition of norms is the result of an incorrect construction of the system of legal norms indicating a certain artificiality of this definition regarding the possible flexibility of the legal system. In our opinion, an increase in monopoly and individualized norms will lead to a systematic nature of the normative material, as well as an increase in confidence in it on the part of all participants in legal relations. The certainty of law must become a legal axiom