ROC-Analysis of the Results as regards <I>B. pseudomallei</I> and <I>B. mallei</I> Agents Detection Using Solid-Phase ELISA

Analyzed are operational characteristic curves (ROC-curves) of solid-phase ELISA variants with immune sera to cellular and extracellular Burkholderia antigens with the aim to determine the most effective ones for B. pseudomallei and B. mallei antigen detection. It is shown that among solid-phase ELISA variants with immune sera to cellular and extracellular antigens, those with sera to homologous antigens are the most effective. Demonstrated is the possibility of application of solid-phase ELISA variants with immune sera to antigens of B. thailandensis and B. cepacia strains for identification and typing of glanders and melioidosis agents. Assessed are prospects of extracellular antigen application for differentiation between closely related species of “ pseudomallei ” group

Application of Latex-Agglutination for Rapid Detection of Pathogenic Burkholderia

The aim. Development of a drug for the identification of Burkholderia pseudomallei and Burkholderia mallei grown in solid nutrient medium at the stage of rapid diagnosis of pathogenic bulkholderia. Materials and methods. Latex agglutination method, in which suspensions of polymers are used as a carrier of agglutinins. The agent recognizing the antigen of the target cells is a monoclonal antibody directed to the epitopes of the glycoprotein capsule of melioidosis causative agent. A liquid latex diagnosticum was prepared from a suspension of polymer polystyrene with a microsphere diameter of 0.8-1.1 pm, the surface of which was loaded with a pre-selected concentration of antibodies. We used typical strains of melioidosis and glanders agents with a full antigenic structure, as well as closely related and heterologous microorganisms. Suspensions with a concentration of 1.0·109 m.c./ml were prepared from bacterial cultures. The reaction was carried out on glass Petri dishes heated to 37 °C with visual recording of the results using four cross system. Results and discussion. The latex agglutination reaction is based on agglutination of microbial cells (B. pseudomallei and B. mallei) with monoclonal antibodies that recognize the epitopes of the glycoprotein capsule of pathogenic burkholderia. 14 monoclonal antibodies of different class and epitope orientation were checked. The determining factors for antibody selection was their specificity in agglutination reaction with strains of melioidosis and glanders agents, as well as preservation of agglutinating activity after immobilization on a polymer carrier. As a result, monoclonal antibodies of class G immunoglobulin to capsule glycoprotein epitopes (AG 8) of the melioidosis agent were selected as an agent recognizing the target cell antigen. As a result, after mixing the samples with the diagnosticum, in samples containing B. pseudomallei and B. mallei bacteria, visible agglutinate generates in 10-15 min. Samples with closely related and heterologous microorganisms lacked agglutinate and were registered as negative. The obtained diagnosticum is characterized by high specificity

Application of the Rapid Linear Immune-Electrophoresis for Differentiation between Burkholderia

Objective of the study was to develop a method that would allow for rapid linear immune-electrophoresis to differentiate between pathogenic agents of glanders and melioidosis and non-pathogenic closely-related Burkholderia . The put-forward modification of the technique made it possible to detect the antigens of pathogenic B. pseudomallei and B. mallei due to the presence of precipitation lines in between the sample gel and the one with immune sera. B. thailandensis, B. cepacia, and B. gladioli did not form such precipitation lines, which in its turn provided for the possibility to differentiate between the mentioned ones and pathogenic Burkholderia. The rapid enhanced linear immune-electrophoresis is easy to perform and compelling, and takes little time. It is qualified for identification of heterogenic and specific antigens in Burkholderia , selection of immune sera containing antibodies to the existing antigens, and can be used as a supportive alternative analytical means for the detection of antigens of pathogenic Burkholderia

Utilization of Immunoblotting in Studies of Epitope Targeting in Monoclonal Antibodies to Melioidosis Agent Antigen 200 kDa

Objective of the research was to use immunoblotting for studies of epitope targeting in monoclonal antibodies to 200 kDa Burkholderia pseudomallei antigen, which are synthesized by hybridomas-producers from the two collections in the laboratory of immunodiagnostics and biotechnology at the premises of Volgograd Research Anti-Plague Institute. Employed were 8 typical strains of melioidosis agent with the complete antigenic structure. Antigen preparations were separated by means of denaturating vertical electrophoresis in 12 % polyacrylamide gel with 0.1 % sodium dodecylsulfate. During the process of cell-replication, 12 hybridomas-producers were given preparative amounts of monoclonal antibodies to 200 kDa Burkholderia pseudomallei glycoprotein. Following that, immunoperoxidase conjugates were manufactured. Epitope targeting of monoclonal antibodies was evaluated using immunoblotting. With the help of vertical electrophoresis identified was the presence of several mandatory major components contained in the antigen complexes of the salt-water and formamid B. pseudomallei extracts . Differential staining substantiated glycoprotein origin of certain antigen components. Immunoblotting with the stated above antigen preparations revealed epitope targeting of a number of monoclonal antibodies to 200 kDa antigen of melioidosis agent; demonstrated were the differences in their specific interaction with biopolymers which form part of the antigen specter. Those differences were characteristic of hybridomas-producers belonging to different collections, as well as of particular strains of B. pseudomallei

Application of Cytokines and Synthetic Peptides for Increase in Immunogenicity of Melioidosis Antigens

B. pseudomallei 100 resistance (61.5 % of survived animals). It was concluded that it is a good practice to include bestim in the schedule of complex immunization against melioidosis

Comparative Analysis of Immunochemical Methods Applied for Studies of Pathogenic Burkholderia Antigens

Studied are immunochemical properties of antigen preparations, the spectrum and molar masses of the components contained. Demonstrated is the significance of vertical SDS-polyacrylamide gel electrophoresis, immunodiffusion test, immunoelectrophoresis assay, rocket immunoelectrophoresis with specific sera for identification and differentiation of Burkholderia. Rocket immunoelectrophoresis should be viewed as the most informative method which allows for differentiation between pathogenic and non-pathogenic for humans Burkholderia under usual terms

Effect of new water-soluble phenolic antioxidants on the activity of Nrf2-driven enzymes, glutathione system, and Nrf2 translocation into the nucleus

Understanding the role of reactive oxygen and nitrogen species in eustress (redox balance) and distress (oxidative stress) development poses new challenges for biomedical scientists and pharmacologists in the search for compounds that can not only have a direct antioxidant (antiradical) effect, but also affect redox-sensitive signaling pathways, primarily Keap1/Nrf2/ARE system. Aim of the study was to investigate the influence of novel water-soluble structurally related monophenols on key elements of Keap1/Nrf2/ARE system induction (activity of Nrf2-driven enzymes, the state of the glutathione system, and intracellular redistribution of transcription factor Nrf2).Material and methods. Five original hydrophilic structurally related monophenols, differing in the number of tert-butyl ortho-substituents, the length of the para-alkyl substituent, and the presence of a divalent sulfur or selenium atom in it were investigated (phenoxane, the potassium salt of phenosan acid, was used as a reference compound). Cell lines U937 and J774 were cultured for 24 h in the presence of tested compounds, and comparative analysis was performed of its ability to induce the synthesis of Nrf2-driven enzymes of phase II xenobiotic detoxification pathway and antioxidant enzymes (NAD(P)H: quinone oxidoreductase 1 (NQO1), glutathione S-transferases (GST), glutathione peroxidases, glutathione reductase (biochemical spectrophotometric methods were used to study their activity), as well as to influence the state of glutathione system (spectrophotometry) and translocation of transcription factor Nrf2 into the nucleus (immunofluorescent staining, confocal microscopy) (key events of Keap1/Nrf2/ARE signaling system activation).Results and discussion. Monophenol TS-13 have found to be the most effective inducer of tested enzymes in U937 cells among the structural analogs, while the structure of the para-alkyl substituent and the degree of OH group hindrance are important for the implementation of this effect; TS-13 also effectively enhanced Nrf2 import into J774 cell nucleus. The NQO1- and GST-inducing abilities of structurally related monophenols are closely interrelated, which indicates the possibility of coordinated induction of these enzymes and the presence of a common regulatory system that ensures their activation in response to cell treatment with phenolic antioxidants

EXPRESSION OF PROTEIN GENES PARTICIPATING IN FIBROPLASTIC PROCESSES IN MICE LUNG DURING THE DEVELOPMENT OF TUBERCULOUS INFLAMMATION

The study analyzes the expression of protein genes involved in intracellular signaling pathways associated with a profibrotic response, activation of epithelial-mesenchymal and endothelial-mesenchymal transitions, in modeling tuberculous granulomatosis and pharmaceutical effects. Material and methods. The study was performed on male BALB/c mice aged two months and weighing 18–22 g. Generalized tuberculous granulomatosis was simulated by a single intravenous (retro-orbital) injection of 0.5 mg BCG vaccine in 0.2 ml of isotonic aqueous NaCl solution (SS), after 4 months a part of mice started to receive treatment drugs, after 2 months the animals were sacrificed by decapitation (6 months after BCG vaccine administration) and their lung tissues were collected. Mice were divided into 6 groups, 5 males in each: intact, which were intravenously into the retro-orbital sinus injected with 0.2 ml SS (C); infected with BCG and receiving SS intraperitoneal injections (BS); infected with BCG and received intraperitoneal injections of isonicotinic acid hydrazide solution (INAH) (BI); infected with BCG and received intraperitoneal injections of dextrazide solution (conjugate of 40 kDa oxidized dextran and INAH) (BD); infected with BCG and received intraperitoneal or inhalation injections of solution of molecular-nanosomal pharmaceutical compositions of oxidized dextran (MNPC) (BMP and BMH, respectively). Finally, the expression of mRNA of matrix metalloproteinase 9, type III procollagen, TGF-β, and transcription factors ZEB1 and Snai1 was determined by real-time PCR in mice lung tissue. Results. The modeling of generalized tuberculous granulomatosis was found to be accompanied by the induction of epithelial-mesenchymal transition and the activation of profibrotic processes, which after 6 months was manifested in increase of the type III collagen α1-chain and TGF-β mRNA expression. Administration of traditional (INAH) and original (dextrazide, MNPC) preparations with anti-tuberculosis activity within two months has inhibitory activity of varying severity in relation to various markers of these processes

Toxicity of new monophenolic synthetic activator of Keap1/Nrf2/ARE redox-sensitive signaling system <i>in vitro</i> and <i>in vivo</i>

One of the promising areas of modern pharmacology is the development of «indirect antioxidants» capable of activating redox-sensitive signaling systems, primarily the Keap1/Nrf2/ARE system. Among its chemical inductors is the hydrophilic monosubstituted monophenol (3’-tert-butyl-4’-hydroxyphenyl)sodium propylthiosulfonate (TS-13) in development. The aim of the study was to investigate TS-13 antiproliferative activity against tumor cell line BT-474 in vitro and acute oral toxicity in mice in vivo. Material and methods. The relationship between TS-13 concentration and proliferative activity of human breast ductal carcinoma cell line BT-474 was determined using the MTT test, the IC&lt;sub&gt;50&lt;/sub&gt; was calculated and compared to the previously obtained for MCF-7 line; results were correlated with the functional properties of cells based on gene expression (in silico GSEA). In vivo acute toxicity was studied in 50 female C57Bl/6J mice, who received a TS-13 solution in distilled water at various doses by intragastric gavage. LD&lt;sub&gt;50&lt;/sub&gt; obtained experimentally and predicted in silico using the GUSAR web service were compared. Results and discussion. TS-13 inhibited the proliferation of BT-474 cells in a concentration-dependent manner (exponential approximation, IC&lt;sub&gt;50&lt;/sub&gt; = 59.5 μM) and was 2.2 times more toxic than for MCF-7 cells. This may be due to functional differences between the BT-474 and MCF-7 lines, as evidenced by the GSEA results. The LD&lt;sub&gt;50&lt;/sub&gt; value established in the in vivo experiment was 936 mg/kg body weight, the obtained value satisfactorily corresponds to the predicted in silico (561 mg/kg), although in reality the compound turned out to be somewhat less toxic than could be expected based on its structure. Conclusions. A study of the acute toxicity of the new water-soluble monophenol TC-13 allows the classification of it as slightly toxic (toxicity rating level 4) according to the Hodge – Sterner scale) or as moderately hazardous (hazard class 3) according to GOST 12.1.007-76