Application of Basophil Activation Test with Anthraxin for Laboratory ( <I>in vitro</I>) Diagnostics of Anthrax

Demonstrated is the possibility to use in vitro basophil activation test with anthraxin, with registration of the results by means of flow cytometry, for anthrax diagnostics. This approach seems promising as it provides quantitative assessment of sensitization of the organism and does not cause its additional allergization. The duration of the analysis is 1h. The test is suggested for application, as an express one, for early and retrospective laboratory diagnostics of anthrax, estimation of post-vaccinal immunity

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 &gt; 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

STUDYING DEVELOPMENT OF POST-VACCINAL CELLULAR IMMUNITY AGAINST BRUCELLOSIS BY MEANS OF LYMPHOCYTE <i>IN VITRO</i> TESTS USING AN EXPERIMENTAL ANTIGENIC COMPLEX

Regulatory framework and methodological approaches to evaluation of immunological effects of vaccination against brucellosis are not established, and the degree of immunological post-vaccinal rearrangement is not yet developed. Due to leading role of cellular immunity in formation of immune protection against brucellosis, evaluation the cellular response in response to antigenic stimulation may be considered the most informative and objective approach to analysis of immune changes in the body during vaccination. In order to develop the most diagnostically informative methods for design of antigen-stimulation cell tests in vitro, a careful selection of a stimulating agent (antigen) is required, which should have a sufficient activating potential, thus providing specificity of reaction under in vitro conditions. The aim of the present study is to study the in vitro specific activity of a protein-polysaccharide antigenic complex from the Brucella abortus 19 BA strain (BrAg), and an opportunity of its application in order to assess the formation of post-vaccinal cellular immunity against brucellosis.The study was performed with white laboratory mice (n = 50) immunized with the Brucella abortus 19 BA strain. The control group (n = 50) consisted of laboratory mice that received a sterile saline solution in a volume of 0.5 ml. Blood samples were taken from immunized and control animals before vaccination, and 7, 14, 21, and 30 days after immunization. By means of flow cytometry, the activation molecules CD25, CD69, MHC II and CD95, expressed on T lymphocytes (CD3+CD69+, CD3+CD25+, CD3+CD95+, CD3+MHC+) were determined. To observe the development of immunity, the intensity of expression of T lymphocyte activation markers was calculated using the stimulation quotient. BrAg was used for specific in vitro stimulation of T lymphocytes. The liquid brucellosis allergen (brucellin) was used as an antigen for comparison, when studying opportunity of BrAg usage for assessing the postvaccinal immunity development.The following results were obtained: BrAg has pronounced specific activity, it did not cause non-specific in vitro reactions (activation) of T lymphocytes, thus enabling its application as a test antigen when evaluating development of adaptive vaccine immunity against brucella.Experimental testing of brucellosis antigen for carrying out the in vitro antigen-stimulated cellular reactions, aiming for evaluation of post-vaccinal immunity development against brucellosis, showed that the usage of BrAg promotes increase in diagnostic sensitivity of cellular reactions under in vitro experimental conditions. The applied experimental antigen is a quite promising tool for development of laboratory algorithms for brucellosis diagnostics, and assessment of actual vaccination efficiency in cohorts previously vaccinated against brucellosis

Analysis of Epizootiological-Epidemiological Situation on Brucellosis in the Russian Federation in 2018 and Forecast for 2019

Presented is the analysis of brucellosis incidence among humans and animals in the Russian Federation in 2018. Epizootiological situation in the regions of developed animal husbandry remains reasonably tense. In 2018, as in previous years, the foci of bovine cattle and small ruminant brucellosis were registered in the North Caucasian, Southern Federal Districts, Volga and Siberian Federal Districts, the share of which made up to more than 90% of all registered in Russia potentially hazardous as regards brucellosis areas and cases of the disease in animals. Against the background of long-term unfavorable epizootic condition, the incidence of brucellosis over the past three years was, on average, 14 % lower than the average long-term indicators. The greatest number of cases (94.1 % of the overall Russian incidence) is registered in the administrative subjects of the North Caucasus Federal District, Southern Federal District and Siberian Federal District, which have the maximum levels of brucellosis incidence in cattle (88.9 %) and small ruminants (95 %). In 2019, persistence of epidemiological problems in regard to brucellosis in the subjects of the North Caucasus Federal District (primarily the Republic of Dagestan, Stavropol Territory), the Southern Federal District (the Republic of Kalmykia, Volgograd and Astrakhan Regions), and the Siberian Federal District (the Tuva Republic, the Omsk and Tyumen Regions) is predicted. The number of human cases of brucellosis may be within the range of 290–310 cases (intensive incidence rate per 100 thousand population – 0.21)

Brucellosis: Trends in the Development of Situation in the World and Forecast for 2022 in the Russian Federation

An analysis of trends in the development of situation on brucellosis in the world over past decade and the data on the main risk factors for the occurrence of epidemiological complications regarding this infection in various regions of the world are provided in the paper. An expert assessment of the current epizootiological and epidemiological situation on brucellosis, the coverage of population and animals with immunization in the Russian Federation is given. Over 9 months of 2021, 210 potentially hazardous as regards brucellosis in cattle areas and 24 sites – as regards brucellosis in small ruminants – were registered in Russia. Compared to the same period in 2020, there was a decrease in the number of newly identified hazardous sites for bovine brucellosis by 35.8 % (117 areas). However, long-term upward trend in epizootiological adversity for bovine brucellosis in Russia persists. The epidemiological situation on brucellosis in the country for the period of 2012–2021 is characterized as unfavorable. Decrease in the number of newly detected human brucellosis cases (by 25.1 % of long-term average values) is observed against the background of persistent unfavorable epizootic conditions for brucellosis among epidemiologically significant species of small ruminants and cattle in regions with developed animal husbandry. In 2021, clusters of human cases were registered in the Republic of Dagestan and Penza Region. In the Republic of Dagestan, against the background of aggravation of epizootiological and epidemiological situation on brucellosis, there was also an alarming trend towards prevalence of a relatively high incidence among minors. The proportion of cases of brucellosis among children under the age of 17 in the Republic amounted to 60.3 % of the total number of minors with newly diagnosed brucellosis in Russia over the past 10 years. Taking into account current epizootic, epidemic situations and the long-term dynamics of the development of situation on  brucellosis in the Russian Federation, the incidence of brucellosis among the population  is predicted to be 10–15 % lower than the average long-term values – 0.18–0.20 per 100000 of the population – in 2022. The number of human cases of brucellosis can range from 250 to 300

ORGANIZATION OF LABORATORY RESEARCH OF CLINICAL AND ENVIRONMENTAL SAMPLES DURING THE ANTHRAX OUTBREAK IN THE YAMALO-NENETS AUTONOMOUS DISTRICT IN 2016

Presented are the order and features of organization of operative diagnostic and monitoring studies of the material during the anthrax outbreak in the Yamalo-Nenets Autonomous District in 2016. Characterized are the main directions of the laboratory base work. Represented are the results of laboratory studies of various materials (clinical samples, material from animals, samples of environmental objects) for the presence of Bacillus anthracis

Molecular mechanisms of action and prediction of response to oxaliplatin in colorectal cancer cells

The platinum compound oxaliplatin has been shown to be an effective chemotherapeutic agent for the treatment of colorectal cancer. In this study, we investigate the molecular mechanisms of action of oxaliplatin to identify means of predicting response to this agent. Exposure of colon cancer cells to oxaliplatin resulted in G2/M arrest and apoptosis. Immunofluorescent staining demonstrated that the apoptotic cascade initiated by oxaliplatin is characterised by translocation of Bax to the mitochondria and cytochrome c release into the cytosol. Oxaliplatin treatment resulted in caspase 3 activation and oxaliplatin-induced apoptosis was abrogated by inhibition of caspase activity with z-VAD-fmk, but was independent of Fas/FasL association. Targeted inactivation of Bax or p53 in HCT116 cells resulted in significantly increased resistance to oxaliplatin. However, the mutational status of p53 was unable to predict response to oxaliplatin in a panel of 30 different colorectal cancer cell lines. In contrast, the expression profile of these 30 cell lines, assessed using a 9216-sequence cDNA microarray, successfully predicted the apoptotic response to oxaliplatin. A leave-one-out cross-validation approach was used to demonstrate a significant correlation between experimentally observed and expression profile predicted apoptosis in response to clinically achievable doses of oxaliplatin (R=0.53; P=0.002). In addition, these microarray experiments identified several genes involved in control of apoptosis and DNA damage repair that were significantly correlated with response to oxaliplatin

Analysis of Brucellosis Incidence and Molecular-Genetic Characteristics of Brucella Population in the Territory of the Russian Federation

An analysis of brucellosis incidence in Russia in 2013–2022 and the data on genetic diversity of Brucella melitensis and Brucella abortus populations isolated in Russia in 1939–2022 are provided in the review. Over the past decade, the epidemiological situation in Russia has been characterized as unstable against the background of persistent unfavorable conditions for brucellosis in cattle and small ruminants. During the period of 2013–2022 (9 months), 4298 epizootic foci as regards brucellosis in cattle (89164 sick animals) and 371 as regards brucellosis in small ruminants (13569) were registered. The largest number of epizootic brucellosis foci was recorded in the North-Caucasian and Southern Federal Districts. In 2013–2022, on average, 327 cases of brucellosis among people were registered annually, the incidence rate per 100 000 of the population was 0.24. Up to 70–90 % of brucellosis cases were detected in the south of the European part of the country. A trend towards deterioration of the situation on brucellosis in Volga (Penza and Samara Regions) and Central (Smolensk, Voronezh and Tula Regions) Federal Districts is observed. There is a connection between the intensity of epidemic manifestations of brucellosis and the level of anthropurgic enzooty of territories. In 2022, 467 cases of brucellosis were reported (0.32 per 100 000 of the population), which is 42.8 % higher than annual average values over 10 years. In 2023, a measure of stability of incidence rates, by 20–25 % above average long-term values, is to be expected. Incidence of brucellosis in humans will be within the range of 380–410 cases (0.26–0.28 per 100 000 of the population). The results of genotyping of B. melitensis strains point to an increase in the proportion of isolates with an MLVA-profile characteristic of strains from enzootic as regards brucellosis countries of Middle East and North Africa over past 20–25 years, which can indicate importation (introduction) of the infection from these territories to Russia through small ruminants and/or biomaterial from them

High-Content, High-Throughput Analysis of Cell Cycle Perturbations Induced by the HSP90 Inhibitor XL888

BACKGROUND: Many proteins that are dysregulated or mutated in cancer cells rely on the molecular chaperone HSP90 for their proper folding and activity, which has led to considerable interest in HSP90 as a cancer drug target. The diverse array of HSP90 client proteins encompasses oncogenic drivers, cell cycle components, and a variety of regulatory factors, so inhibition of HSP90 perturbs multiple cellular processes, including mitogenic signaling and cell cycle control. Although many reports have investigated HSP90 inhibition in the context of the cell cycle, no large-scale studies have examined potential correlations between cell genotype and the cell cycle phenotypes of HSP90 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: To address this question, we developed a novel high-content, high-throughput cell cycle assay and profiled the effects of two distinct small molecule HSP90 inhibitors (XL888 and 17-AAG [17-allylamino-17-demethoxygeldanamycin]) in a large, genetically diverse panel of cancer cell lines. The cell cycle phenotypes of both inhibitors were strikingly similar and fell into three classes: accumulation in M-phase, G2-phase, or G1-phase. Accumulation in M-phase was the most prominent phenotype and notably, was also correlated with TP53 mutant status. We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines. CONCLUSIONS/SIGNIFICANCE: Our analysis of the cell cycle phenotypes induced by HSP90 inhibition in 25 cancer cell lines revealed that the phenotypic response was highly dependent on cellular genotype as well as on the concentration of HSP90 inhibitor and the time of treatment. M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53. We draw upon previous literature to suggest an integrated model that accounts for these varying observations