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

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

Study on Hemostimulating Properties of Granulocyte-Macrophage Colony Stimulating Factor

The hemostimulating properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) make possible its clinical use in alleviating side effects of anti-cancer radio- and chemotherapy, in bone marrow transplantation, and in the treatment of some primary immunodeficiency conditions associated with leukopenia. The State Research Center of Virology and Biotechnology “Vector” of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing has developed a high-performance technology for production of recombinant human GM-CSF (rhGM-CSF) based on a recombinant E. coli strain. The aim of the study was to assess hemostimulating activity of the rhGM-CSF preparation obtained using the new developed technology, as observed in cell culture and in the mice model of myelosuppression induced by cyclophosphamide administration. Materials and methods: in vitro evaluation of rhGM-CSF hemostimulating activity was performed by MTT assay in the commercial HL-60 promyelocytic leukemia cell culture with preliminary suppression of cell growth rate by adding a low concentration of dimethyl sulfoxide to the medium. In vivo studies were carried out in CBA/CaLac mice with cyclophosphamide-induced myelosuppression. The hemostimulating properties of the drug were evaluated after subcutaneous administration of 1–175 µg/kg doses for 4–5 days, following administration of a cytostatic agent. The total number of leukocytes and the content of their morphological forms were determined in blood samples taken at different time points after the drug administration. The statistical processing of the experimental data was based on analysis of variance using Statgraphics v. 5.0 software. Results: the proliferative activity of HL-60 cells incubated with the rhGM-CSF preparation for 72 hours was shown to be dose-dependent. The highest values of the increase in proliferative activity associated with an increase in the drug dose were observed in the concentration range from 0.04 to 0.64 ng/mL (proliferative activity increased by 11–18% when the dose was increased twofold). The experiments in mice demonstrated a two-phase pattern of the dose-dependent effect. The drug showed the highest hemostimulating effect at the dose of 90 µg/kg. Conclusions: the rhGM-CSF preparation obtained using the new developed technology has a pronounced hemostimulating activity confirmed by both in vitro and in vivo test systems

Pharmacokinetics of Recombinant Human Tumor Necrosis Factor Alpha in the Delivery System

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

Исследование гемостимулирующих свойств рекомбинантного гранулоцитарно-макрофагального колониестимулирующего фактора человека

The hemostimulating properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) make possible its clinical use in alleviating side effects of anti-cancer radio- and chemotherapy, in bone marrow transplantation, and in the treatment of some primary immunodeficiency conditions associated with leukopenia. The State Research Center of Virology and Biotechnology “Vector” of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing has developed a high-performance technology for production of recombinant human GM-CSF (rhGM-CSF) based on a recombinant E. coli strain. The aim of the study was to assess hemostimulating activity of the rhGM-CSF preparation obtained using the new developed technology, as observed in cell culture and in the mice model of myelosuppression induced by cyclophosphamide administration. Materials and methods: in vitro evaluation of rhGM-CSF hemostimulating activity was performed by MTT assay in the commercial HL-60 promyelocytic leukemia cell culture with preliminary suppression of cell growth rate by adding a low concentration of dimethyl sulfoxide to the medium. In vivo studies were carried out in CBA/CaLac mice with cyclophosphamide-induced myelosuppression. The hemostimulating properties of the drug were evaluated after subcutaneous administration of 1–175 µg/kg doses for 4–5 days, following administration of a cytostatic agent. The total number of leukocytes and the content of their morphological forms were determined in blood samples taken at different time points after the drug administration. The statistical processing of the experimental data was based on analysis of variance using Statgraphics v. 5.0 software. Results: the proliferative activity of HL-60 cells incubated with the rhGM-CSF preparation for 72 hours was shown to be dose-dependent. The highest values of the increase in proliferative activity associated with an increase in the drug dose were observed in the concentration range from 0.04 to 0.64 ng/mL (proliferative activity increased by 11–18% when the dose was increased twofold). The experiments in mice demonstrated a two-phase pattern of the dose-dependent effect. The drug showed the highest hemostimulating effect at the dose of 90 µg/kg. Conclusions: the rhGM-CSF preparation obtained using the new developed technology has a pronounced hemostimulating activity confirmed by both in vitro and in vivo test systems. Гемостимулирующие свойства гранулоцитарно-макрофагального колониестимулирующего фактора (ГМ-КСФ) позволяют использовать его в клинике для снижения побочных эффектов радио- и химиотерапии онкологических заболеваний, при трансплантации костного мозга, для лечения некоторых первичных иммунодефицитных состояний, связанных с лейкопенией. В ФБУН ГНЦ ВБ «Вектор» Роспотребнадзора разработана высокопроизводительная технология получения рекомбинантного ГМ-КСФ человека (рчГМ-КСФ) на основе рекомбинантного штамма E. coli. Цель работы: изучение гемостимулирующей активности препарата рекомбинантного ГМ-КСФ человека, полученного по разработанной технологии, в культуре клеток и на модели миелосупрессии у мышей, вызванной введением циклофосфана. Материалы и методы: оценку гемостимулирующей активности рчГМ-КСФ in vitro проводили с использованием МТТ-теста на коммерческой культуре клеток промиелоцитарной лейкемии HL-60, скорость роста которых предварительно подавляли добавлением в среду низкой концентрации диметилсульфоксида. Исследования in vivo проводили на мышах линии СВА/Calaс в условиях миелосупрессии, вызванной введением циклофосфана. Гемостимулирующие свойства препарата оценивали при его подкожном введении в диапазоне доз от 1 до 175 мкг/кг в течение 4–5 суток после введения цитостатика. В образцах крови, взятых в разные сроки после введения, определяли общее количество лейкоцитов и содержание их морфологических форм. Данные эксперимента обрабатывали методами вариационной статистики с помощью пакета программ Statgraphics, v. 5.0. Результаты: пролиферативная активность клеток HL-60, инкубированных с препаратом рчГМ-КСФ в течение 72 ч, носила дозозависимый характер. Наиболее высокие значения повышения пролиферативной активности при увеличении дозы препарата наблюдались в диапазоне концентраций от 0,04 до 0,64 нг/мл (прирост значений показателя при двукратном увеличении дозы составлял 11–18%). В экспериментах на мышах продемонстрирован двухфазный характер дозовой зависимости эффекта. Наибольшую гемостимулирующую активность препарат проявлял в дозе 90 мкг/кг. Выводы: препарат рчГМ-КСФ, полученный по разработанной технологии, обладает выраженной гемостимулирующей активностью, подтвержденной в системах in vitro и in vivo