Chemical polysialylation of recombinant human proteins

© Springer Science+Business Media New York 2015. All right reserved. Design of drug with prolonged therapeutic action is one of the rapid developing fields of modern medical science and required implementation of different methods of protein chemistry and molecular biology. There are several therapeutic proteins needing increasing of their stability, pharmacokinetic, and pharmacodynamics parameters. To make long-live DNA-encoded drug PEGylation was proposed. Alternatively polysialic (colominic) acid, extracted from the cell wall of E. coli, fractionated to the desired size by anionexchange chromatography and chemically activated to the amine-reactive aldehyde form, may be chemically attached to the polypeptide chain. Conjugates of proteins and polysialic acid generally resemble properties of protein- PEG conjugates, but possess significant negative net charge and are thought to be fully degradable after endocytosis due to the presence of intracellular enzymes, hydrolyzing the polysialic acid. Complete biodegradation of the polysialic acid moiety makes this kind of conjugates preferable for creation of drugs, intended for chronic use. Here, we describe two different protocols of chemical polysialylation. First protocol was employed for the CHO-derived human butyrylcholinesterase with optimized for recovery of specific enzyme activity. Polysialic acid moieties are attached at various lysine residues. Another protocol was developed for high-yield conjugation of human insulin; major conjugation point is the N-terminal residue of the insulin's light chain. These methods may allow to produce polysialylated conjugates of various proteins or polypeptides with reasonable yield and without significant loss of functional activity

Влияние полиморфизма гена индуцибельной NO-синтазы на достижение контроля бронхиальной астмы у детей на фоне терапии ингаляционными кортикостероидами

The polymorphism of the gene of inducible NO-synthase (iNOS) was studied in 65 children with bronchial asthma (BA). The goal was to determine the probability to achieve the BA control in children against the background of inhalation glucocorticosteroid (IGCS) therapy depending on the polymorphism of the iNOS gene. The genotype of polymorphism of the iNOS gene was studied by the method of polymerase chain reaction. It has been found that the achievement of BA control in children against the background of the IGCS treatment is associated with the carriage of homozygote of «wild» allele G of polymorphism G954C of the iNOS gene. The absence of BA control is related to the presence of pathological allele . The result obtained can favor the pre С - diction of the uncontrollable BA course in children.Проведено исследование полиморфизма гена индуцибельной NO-синтазы (iNOS) у 65 детей, страдающих бронхиальной астмой (БА). Цель исследования - установить вероятность достижения контроля БА у детей на фоне лечения ингаляционными глюкокортикостероидами (ИГКС) в зависимости от полиморфизма гена iNOS. Исследование генотипа полиморфизма гена iNOSосуществлялось методом полимеразной цепной реакции. Установлено, что достижение контроля болезни у детей на фоне лечения ИГКС ассоциировано с носительством гомозиготы «дикого» аллеля G полиморфизма G954C гена iNOS. Отсутствие контроля болезни сопряжено с наличием патологического аллелея С. Полученный результат может способствовать прогнозированию неконтролируемого течения БА у детей

ТЕХНОЛОГИЧЕСКАЯ ПЛАТФОРМА «ПЕДИАТРИЯ». ПРЕДЛОЖЕНИЯ К КОНЦЕПЦИИ ПРОГРАММНЫХ МЕРОПРИЯТИЙ

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Genetically engineered CD80–pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide–MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols

ВОЗМОЖНОСТИ ТЕЛЕМЕДИЦИНСКИХ ТЕХНОЛОГИЙ В ЛУЧЕВОЙ ДИАГНОСТИКЕ

The paper reviewed the definition of terminology and the history of the development of telemedicine. The main directions of development of telemedicine technologies in Russia and abroad. Provides information about the different areas of information and communication technologies in health care. Presented as a variety of telemedicine services for medical purposes. The main problem hindering the development of Telemedicine and Medical Radiology in particular.В работе рассмотрены терминология и история развития телемедицины в России и за рубежом. Указаны сведения о различных направлениях применения информационно-телекоммуникационных технологий в здравоохранении. Представлены разновидности телемедицины как услуги медицинского назначения. Приведены основные проблемы, сдерживающие развитие телемедицины и телерадиологии в частности

Новые данные о согласованности результатов кожного аллерготестирования к пищевым аллергенам с уровнем специфического иммуноглобулина Е и симптомами пищевой аллергии у детей в эндемичных по описторхозу очагах

Aim: to investigate the consistency of skin prick-tests results with specific IgE level and symptoms of food allergy. The criteria of food allergy in children of opisthorchiasis endemic areas were developed: symptoms of food allergy within 2 hours, skin pricktests weal size ≥1 mm and (or) specific IgE ≥0,35 kUA/l.Представлен анализ согласованности результатов кожного аллерготестирования к пищевым аллергенам с оценкой уровня специфического иммуноглобулина Е (IgE) и симптомами пищевой аллергии у детей. На основании результатов эпидемиологического исследования предложены следующие критерии диагноза пищевой аллергии у детей, проживающих в регионах с высокой распространенностью описторхоза: наличие клинических симптомов, возникающих в течение 2 ч после употребления продукта питания, положительные результаты кожного аллерготестирования — средний диаметр папулы не менее 1 мм и (или) повышение уровня специфического IgE сыворотки крови не менее 0,35 кЕдА/л

Possibilities of radionuclide visualization of HER2/neu-positive breast cancer using a radiopharmaceutical based on recombinant targeting molecules DARPin9_29

Epidermal growth receptor HER2/neu is still of great interest, the overexpression of which is most often observed in patients with breast cancer and accounts for 15–20 % of cases. Present methods of HER2/neu determination have a number of significant drawbacks. In recent years, alternative framework proteins are used for the targeted radionuclide imaging. Molecules of DARPin (Design Ankyrin Repeat Protein) are one of representatives of scaffolds. Material and methods. The study included 4 breast cancer patients (T1-2N0-1M0) who were not receiving systemic therapy at the time of the study: in 2 patients, HER2/neu overexpression was noted, in 2 patients – not detected. HER2/neu status was determined using an immunohistochemical method and a FISH assay. At the preclinical stage, radiopharmaceutical 99mTc-DARPin9_29 was injected intravenously to all patients, «WholeBody» scintigraphy and single-photon emission computed tomography were performed 2 hours after injection. Results. The distribution of radiopharmaceuticals in organs 2 hours after injection revealed the greatest accumulation in the liver and kidneys. In studying of tumor/background indicator it was revealed that values of the studied parameter in patients with overexpression of HER2 receptors are more than 3 times higher than the values in the subgroup of patients with negative expression of this marker. Conclusion. According to the results of preliminary studies, the 99mTc-DARPin9_29 demonstrated significant differences between tumors with and without HER2/neu overexpression

Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs

© 2016 Springer Science+Business Media DordrechtObjective: Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment. Results: Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT. Conclusion: Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS

Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs

OBJECTIVE: Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment.RESULTS: Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT.CONCLUSION: Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS