Sequence analysis of the non-coding control region of John Cunningham virus isolates from patients with multiple sclerosis treated with natalizumab

Introduction. The John Cunningham virus (JCPyV) causes a fatal demyelinating disease of the central nervous system known as progressive multifocal leukoencephalopathy (PML). In healthy people, the JCPyV non-coding control region (NCCR) is not rearranged, while NCCRs in immunocompromised patients are characterized by frequent rearrangements and can be associated with PML development. Therefore, patients treated with natalizumab, which decreases the migration of leukocytes and monocytes through the blood-brain barrier to inflammatory foci, are at increased risk of developing PML. The purpose of the study was to analyze NCCR sequences of JCPyV isolates from patients with multiple sclerosis (MS) treated with natalizumab. Materials and methods. A total of 26 blood plasma samples and 8 cerebrospinal fluid samples were analyzed using nested PCR to study the JCPyV NCCR structure in Russian MS patients treated with natalizumab. The NCCRs present in the samples were cloned and sequenced by Sanger sequencing. All the JCPyV NCCR sequences were compared with the archetype sequence and mapped. The NCCR sequences were also examined for presence of putative transcription factor binding sites. Results. A total of 48 NCCR sequences were found. The analysis showed that up to 55% of NCCRs were identified as rearranged NCCRs, while the other were archetype-like NCCRs. All the sequences can be divided into 6 types with one dominant rearrangement pattern. This rearranged NCCR was also found in a patient with the confirmed PML diagnosis and a poor prognosis. All the rearranged NCCRs were characterized by the presence of additional transcription factor binding sites. Conclusion. The study has helped identify previously unknown NCCR patterns typical of MS patients treated with natalizumab in Russia, thus confirming the need for the further research on NCCR rearrangements in MS patients undergoing natalizumab treatment to gain better understanding of the origin of neurovirulent JCPyV variants

Development of a protective lyophilisation medium and conditions to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin

Liquid erythrocyte diagnostic preparations have a practical disadvantage; i.e., long-distance transportation involving possible non-compliance with cold-chain requirements may result in a complete loss of biological activity. A lyophilisation technology is necessary to ensure that the preparations retain their original properties for a long time. The aim of the work was to develop a protective medium and conditions for lyophilisation to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin. Materials and methods: Gelatin, thiourea, trehalose, sucrose, dextran, and Tween 80 were used as excipients for protective media. The authors used nine strains of homologous and heterologous microorganisms of different genera and species to control the lyophilised diagnostic preparation sensitivity and specificity. Evaluation of the main stability-related quality attributes (appearance of the dried preparation, loss on drying, solubility, appearance after reconstitution, appearance after settling, sensitivity, specificity) considered the temperatures specific to the climatic zones where the in vitro diagnostics is intended to be marketed and used. Results: The authors developed protective stabilising media with different compositions, used them in freeze-drying of the preparation and carried out control testing. The most promising was the lyophilisation medium containing a smaller amount of ingredients —6% of dextran, 0.06% of Tween 80 and up to 0.01% of sodium azide—as it was the simplest one to prepare and ensured complete preservation of the quality attributes. The authors carried out practical evaluation of lyophilisation procedures, and the 12–14-hour procedure proved to be the most cost-effective. Conclusions: The results of long-term, or real time, and accelerated stability testing of the lyophilised diagnostic preparation demonstrated the possibility of two-year storage at a labelled temperature of 2–8 °C, as well as at elevated and low temperatures of 30±2 °С and –18 °С, respectively. The tests showed no negative effects of the temperatures on the controlled quality attributes

Construction and Approval of the Test-System for the Detection of Antibodies to Anthrax Agent Using Indirect Fluorescent Immunoassay

Constructed and validated has been the test-system for the detection of antibodies to anthrax agent using indirect fluorescent immunoassay. The panel consists of antigen preparation, positive control sample, negative control, and FITS-labeled rabbit antibodies. Studied have been 10 acapsular B. anthracis strains with different plasmid content and colony morphology. B. anthracis vaccine strain STI-1 is demonstrated to be the optimal one as antigen preparation. Sensitivity of this test-system is not less than 1:40, the working dilution of FITC-conjugate being 1:16. Specificity of the panel has been studied using 100 sera of healthy donors. Approved is 100 % reproducibility of the technique at different time intervals, as well as when carried by different specialists. The test-system allows for confirmation of anthrax diagnosis in humans

METHODOLOGICAL ASPECTS OF CONVENIENT MICROTOOLS MANUFACTURING FOR CELL CULTURES MANIPULATING

This paper presents a step-by-step plan for the manufacture of retention and injection microtools for conducting micropanipulations on various cell lines.Работа выполнена в рамках госзадания ИИФ УрО РАН (тема № АААА-А18-118020590108-7)

Using CAST-test to investigate human specific hypersensitivity to the anthrax pathogen

We present the results of applying functional cytometric test of antigen-stimulated activation basophils to assess specific immunological reactivity in the people with anthrax, and immunized with anthrax vaccine. As a criterion for antigen-specific basophil activation, we measured expression of the CD63 membrane receptor, which reflects the process of anaphylactic basophil degranulation. To determine spontaneous and antigen-induced activation of basophils (CCR3+CD63+), a FlowCAST reagent kit (Buhlmann laboratories AG, Switzerland) was used. Anthraxin, an experimental anthrax allergen (a hydrolysate the Bacillus anthracis STI-1 strain), manufactured by the Stavropol Anti-Plague Institute, was used as a specific antigen. As based on clinical and experimental data, a threshold value of > 10% of anthraxin-activated (CCR3+CD63+) basophils was accepted for the in vitro immunodiagnostic CAST test, as a laboratory criterion for the subjects exhibiting specific immune response, i.e., IgE-mediated sensitization. It was shown that, in anthrax patients within one week after onset of the disease (3-7 days), a positive CAST result was obtained in 92.3% cases; the levels of specific basophil activation with anthraxin averaged 37.9% (12.01 ÷ 78.9%). Immunological examination of individuals three weeks (21 days) after vaccination against anthrax revealed CAST-positivity in all the vaccinated persons. Intensity of anthraxin-induced basophil activation the vaccinated subjects was ranged from 10.87 to 30.03%, averaging 17.86%. The overall values of spontaneous and specific activation ranged within 12.39 ÷ 41.46%. The study opens prospectives for implementation of basophil antigenic activation test in the Flow CAST format in diagnostics of anthrax and to identify specific immune rearrangements after vaccination in humans, as an index of actual vaccination rates. Usage of CAST test with anthraxin makes it possible to identify anthrax patients at the early stages (2-4 days after onset of the disease) including, among patients with an increased CCR3+CD63+ background values, evaluation of immunological efficiency in the cohorts at risk for vaccination. At the same time, it was found that a significant decrease in diagnostic sensitivity of CAST test could be observed in the patients immune to anthrax pathogen who received intensive antibacterial and pathogenetic therapy at the early stages of infection, including glucocorticosteroids (anti-inflammatory drugs) and desensitizing agents that inhibit the degree of hypersensitivity development and its expression

Научно-методические разработки биотехнологий производства иммунобиологических препаратов для экспресс-диагностики инфекционных заболеваний и детекции их возбудителе

The present article describes the scientific and methodological development of biotechnological manufacture of test-system components (diagnostic preparations) for instant diagnosis of plague, brucellosis, tularemia, anthrax, cholera. In this regard, in the first place the effective methods for obtaining complete antigenic complexes used for immunizing animals for the purpose of developing highly potent immune sera have been established. These antisera were used in determining optimum parameters of manufacture on the basis of their diagnosticums. Methodical basis of developing magnetic immunosorbents for selective concentration of infectious agents and their instant diagnosis methods has been mentioned. Moreover, the article describes the development of piezoelectric quartz crystal biosensors to detect plague, brucellosis and tularemia pathogens by gravimetric flow injection analysis, allowing to quickly implement the process of reliable identification of a test pathogen in antigen-antibody complex.Представлены научно-методические разработки биотехнологий производства компонентов тест-систем (диагностических препаратов) для экспресс-диагностики чумы, бруцеллеза, туляремии, сибирской язвы, холеры. Для этого, прежде всего, были отработаны эффективные методики получения полноценных антигенных комплексов, применяемых для иммунизации животных в целях получения высокоактивных иммунных сывороток. Полученные иммунные сыворотки были использованы при определении оптимальных параметров производства на их основе диагностикумов. Приведены методические основы конструирования магноиммуносорбентов для селективного концентрирования возбудителей инфекционных заболеваний и их индикации экспресс-методами. Кроме того, приведена разработка пьезокварцевых биосенсорных устройств для выявления возбудителей чумы, бруцеллеза, туляремии в гравиметрическом проточно-инжекционном анализе, позволяющих быстро реализовать процесс достоверного распознавания исследуемого патогена в образовавшемся комплексе антиген-антител

Improvement of Lyme Borreliosis Agent Indication Methods

On the basis of silica - aluminosilicate, modified by carboxymethylated lignin and carbodiimide, obtained are the composite microgranulated magnetic immunoadsorbents (MIA) with high adsorption activity, which are characterized by the standardized structural characteristics and mechanical strength. Application of MIAs makes it possible, at the stage of tick samples preparation, to eliminate various admixtures via reiterative irrigations of the sorbent with the infectious agent fixed on it. Therefore negative influence of admixtures on the performed analysis is excluded, and the target agent is concentrated to the maximum limit. Thus the specificity and sensitivity of PCR-analysis enhances

Разработка защитной среды высушивания и режима лиофилизации для стабилизации диагностикума эритроцитарного туляремийного иммуноглобулинового

Liquid erythrocyte diagnostic preparations have a practical disadvantage; i.e., long-distance transportation involving possible non-compliance with cold-chain requirements may result in a complete loss of biological activity. A lyophilisation technology is necessary to ensure that the preparations retain their original properties for a long time. The aim of the work was to develop a protective medium and conditions for lyophilisation to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin. Materials and methods: Gelatin, thiourea, trehalose, sucrose, dextran, and Tween 80 were used as excipients for protective media. The authors used nine strains of homologous and heterologous microorganisms of different genera and species to control the lyophilised diagnostic preparation sensitivity and specificity. Evaluation of the main stability-related quality attributes (appearance of the dried preparation, loss on drying, solubility, appearance after reconstitution, appearance after settling, sensitivity, specificity) considered the temperatures specific to the climatic zones where the in vitro diagnostics is intended to be marketed and used. Results: The authors developed protective stabilising media with different compositions, used them in freeze-drying of the preparation and carried out control testing. The most promising was the lyophilisation medium containing a smaller amount of ingredients —6% of dextran, 0.06% of Tween 80 and up to 0.01% of sodium azide—as it was the simplest one to prepare and ensured complete preservation of the quality attributes. The authors carried out practical evaluation of lyophilisation procedures, and the 12–14-hour procedure proved to be the most cost-effective. Conclusions: The results of long-term, or real time, and accelerated stability testing of the lyophilised diagnostic preparation demonstrated the possibility of two-year storage at a labelled temperature of 2–8 °C, as well as at elevated and low temperatures of 30±2 °С and –18 °С, respectively. The tests showed no negative effects of the temperatures on the controlled quality attributes.Практическое применение эритроцитарных диагностических препаратов выявило недостатки, связанные с их транспортировкой на значительные расстояния с возможным несоблюдением режимов холодовой цепи, что может привести к полной потере их биологической активности. Для стабилизации основных свойств жидких форм эритроцитарных диагностических препаратов в настоящее время необходима разработка технологии получения лиофилизированных форм диагностикумов, которая позволит сохранить первоначальные свойства препарата в течение длительного времени. Цель работы: разработка защитной среды высушивания и режима лиофилизации для стабилизации диагностикума эритроцитарного туляремийного иммуноглобулинового. Материалы и методы: были использованы вспомогательные вещества для подготовки защитных сред (желатин, тиомочевина, трегалоза, сахароза, декстран, твин 80). Для контроля чувствительности и специфичности лиофилизированных диагностикумов использовали 9 штаммов гомологичных и гетерологичных микроорганизмов разных родов и видов. При изучении стабильности основных показателей качества препаратов для диагностики in vitro (внешний вид высушенного препарата; потеря в массе при высушивании; растворимость, внешний вид восстановленного препарата; внешний вид препарата после отстаивания; чувствительность; специфичность) учитывали температуры различных климатических зон, в которых предполагается их реализация и использование. Результаты: разработаны и использованы стабилизирующие защитные среды с различным составом, с последующей сублимационной сушкой препарата и постановкой контрольных исследований. Наиболее перспективной признана среда высушивания для эритроцитарных диагностикумов, содержащая в своем составе меньшее количество ингредиентов — 6% декстрана, 0,06% твин 80 и азид натрия до 0,01%, как наиболее простая в исполнении и обеспечивающая полное сохранение качественных показателей препарата. Отработан рентабельный 12–14-часовой режим лиофилизации. Выводы: по совокупности полученных результатов изучения стабильности в реальном времени (долговременная стабильность) и ускоренном исследовании стабильности лиофилизированных форм диагностикума показана возможность хранения в течение двух лет при регламентированной температуре от 2 до 8 °С, а также в условиях повышенных и пониженных температур при 30±2 °С и минус 18 °С соответственно. Отрицательного влияния указанных температур на результаты контролируемых показателей не выявлено

Scientific and methodical development of biotechnological production of immunobiological preparations for instant diagnosis of infectious diseases and detection of pathogens

The present article describes the scientific and methodological development of biotechnological manufacture of test-system components (diagnostic preparations) for instant diagnosis of plague, brucellosis, tularemia, anthrax, cholera. In this regard, in the first place the effective methods for obtaining complete antigenic complexes used for immunizing animals for the purpose of developing highly potent immune sera have been established. These antisera were used in determining optimum parameters of manufacture on the basis of their diagnosticums. Methodical basis of developing magnetic immunosorbents for selective concentration of infectious agents and their instant diagnosis methods has been mentioned. Moreover, the article describes the development of piezoelectric quartz crystal biosensors to detect plague, brucellosis and tularemia pathogens by gravimetric flow injection analysis, allowing to quickly implement the process of reliable identification of a test pathogen in antigen-antibody complex