[Options for the detection and identifications of agents causing viral hemorrhagic fevers]. / Moznosti detekce a identifikace puvodcu virových hemoragických.

Viral hemorrhagic fevers (VHFs) belong to the most deadly diseases. Therefore rapid detection and identification of causal agent is very important for epidemiological investigation and countermeasures. The review present all published information about VHF detection and identification, with focus on polymerase chain reaction (PCR) methods. In the first part, basic information about microbiological diagnostic technique is provided. The second part is concerned with detection and identification methods for individual agents causingt VHFs.

Proteome alterations in gamma-irradiated human T-lymphocyte leukemia cells.

Analyses of the protein expression profiles of irradiated cells may be beneficial for identification of new biomolecules of radiation-induced cell damage. Therefore, in this study we exploited the proteomic approach to identify proteins whose expression is significantly altered in gamma-irradiated human T-lymphocyte leukemia cells. MOLT-4 cells were irradiated with 7.5 Gy and the cell lysates were collected at different times after irradiation (2, 5 and 12 h). The proteins were separated by two-dimensional electrophoresis and quantified using an image evaluation system. Proteins exhibiting significant radiation-induced alterations in abundance were identified by peptide mass fingerprinting. We identified 14 proteins that were either up- or down-regulated. Cellular levels of four of the proteins (Rho GDP dissociation inhibitor 1 and 2, Ran binding protein 1, serine/threonine protein kinase PAK2) were further analyzed by two-dimensional immunoblotting to confirm the data obtained from proteome analysis. All identified proteins were classified according to their cellular function, including their participation in biochemical and signaling pathways. Taken together, our results suggest the feasibility of the proteome method for monitoring of cellular radiation responses.

Towards proteome database of Francisella tularensis.

The accessibility of the partial genome sequence of Francisella tularensis strain Schu 4 was the starting point for a comprehensive proteome analysis of the intracellular pathogen F. tularensis. The main goal of this study is identification of protein candidates of value for the development of diagnostics, therapeutics and vaccines. In this review, the current status of 2-DE F. tularensis database building, approaches used for identification of biologically important subsets of F. tularensis proteins, and functional and topological assignments of identified proteins using various prediction programs and database homology searches are presented.

A real-time PCR array for hierarchical identification of Francisella isolates.

A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis.

The alteration of immune reactions in inbred BALB/c mice following low-level sarin inhalation exposure.

To study the influence of low-level sarin inhalation exposure on immune functions, inbred BALB/c mice were exposed to low concentrations of sarin for 60 min in the inhalation chamber. The evaluation of immune functions was carried out using phenotyping of CD3 (T lymphocytes), CD4 (helper T lymphocytes), CD8 (cytotoxic T lymphocytes), and CD19 cells (B lymphocytes) in the lungs, blood, and spleen, lymphoproliferation of spleen cells stimulated in vitro by various mitogens (concanavalin A, lipopolysaccharides), phagocyte activity of peritoneal and alveolar macrophages, production of N-oxides by peritoneal macrophages, and the measurement of the natural killer cell activity at 1 wk following sarin exposure. The results were compared to the values obtained from control mice exposed to pure air instead of sarin. The results indicate that low doses of sarin are able to alter the reaction of immune system at one week following exposure to sarin. While the numbers of CD3 cells in the lungs, blood, and spleen were slightly decreased, an increase in CD19 cells was observed, especially in the lungs and blood. The reduced proportion of T lymphocytes is caused by decay of CD4-positive T cells. Lymphoproliferation was significantly decreased regardless of the mitogen and sarin concentration used. The production of N-oxides by peritoneal macrophages was stimulated after exposure to the highest dose of sarin, whereas their ability to phagocytize the microbes was increased after exposure to the lowest dose of sarin. The natural killer cell activity was significantly higher in the case of inhalation exposure of mice to the highest level of sarin. Thus, not only organophosphorus insecticides but also nerve agents such as sarin are able to alter immune functions even at a dose that does not cause clinically manifested disruption of cholinergic nervous system in the case of inhalation exposure. Nevertheless, the alteration of immune functions following the inhalation exposure to a symptomatic concentration of sarin seems to be more pronounced.