An Algorithm for the Selection of Probes for Specific Detection of Human Disease Pathogens Using the DNA Microarray Technology.

The aim of the study was to develop an algorithm for the selection of discriminating probes to identify a wide range of causative agents of human infectious diseases. Materials and Methods: The algorithm for selecting the probes was implemented in the form of the disprose (DIScrimination PRObe SElection) computer program written in the R language. Additionally, third-party software was used: the BLAST+ and ViennaRNA Package programs. The developed algorithm was tested by selecting specific probes for detecting Chlamydophila (Chlamydia) pneumoniae - an atypical bacterial pathogen causing community-acquired pneumonia (CAP). Nucleotide sequences for analysis were downloaded from the NCBI databank. Results: An algorithm for the selection of specific probes capable of detecting human infectious pathogens has been developed. The algorithm is implemented in the form of the disprose modular program, which allows for performing all stages of the probe selection process: loading the nucleotide sequences and their metadata from available databanks, creating local databases, forming a pool of probes, calculating their physicochemical parameters, aligning the probes and sequences contained in local databases, processing and evaluating the alignment results. The algorithm was successfully tested and its performance was confirmed by selecting a set of probes for the specific detection of Chlamydophila pneumoniae. The specificity of the selected probes calculated in silico indicated a low risk of their nonspecific binding and a high potential of using them as molecular genetic diagnostic tools (DNA microarrays, PCR). Conclusion: An algorithm for the selection of specific probes detecting a wide range of human pathogens in clinical biomaterial has been developed and implemented in the form of the disprose modular program. The probes selected using this program can serve as the functional basis of DNA-oriented microarrays able to identify causative agents of polyetiological diseases, such as CAP. Due to the flexibility and openness of the program, the scope of its application can be expanded.

The fluorescent immunocytochemical diagnostics of precancerous diseases and cancer of the oral mucosa.

Purpose of the study improving the diagnosis of precancerous diseases and cancer of the oral mucosa using fluorescent immunocytochemical studies by direct immunofluorescence. A clinical laboratory examination of 111 patients was carried out: 46 patients with squamous cell carcinoma of the oral mucosa, 35 people with precancerous lesions (17 leukoplakia, 18 - oral lichen planus) and 30 healthy people. All patients underwent a traditional cytological examination and an additional immunocytochemical examination by direct immunofluorescence, the expression levels of tumor markers P53, P16 and Ki67 were determined. The data were compared with the results of histological analysis. As a result of the study, it was revealed that in patients with cancer, the expression of oncomarker P53 was four times higher than in patients with precancerous pathology. In 6.52% of cases, co-expression of markers Ki67 and P16 was found. Thus, the advantages of fluorescent immunocytochemical diagnostics were the absence of invasive traumatic intake of the biomaterial in the patient, reduction in the timing of obtaining the result, high sensitivity, and the possibility of remote evaluation of the results. Therefore, that increases the accessibility of the method, and the possibility of using this method for a screening study of population.

Cytokine Profile of CCR6+ T-Helpers Isolated from the Blood of Patients with Peptic Ulcer Associated with Helicobacter pylori Infection.

We previously found that the number of CCR6+ T-helpers with the phenotype of effector/effector memory T cells increases in the blood of patients with H. pylori-associated peptic ulcer. The mature phenotype and the expression of the chemokine receptor CCR6, which is involved in migration of lymphocytes to the inflamed mucous membrane of the gastrointestinal tract, suggests that these cells are involved in the immune response observed in this clinical condition. To better understand the pathogenetic role of these cells, it is necessary to study their functional activity, specifically, the production of pro-inflammatory cytokines involved in the pathogenesis of the disease. The aim of the study was to evaluate changes in the blood level of pro-inflammatory types of mature CCR6+ T-helpers in H. pylori-associated peptic ulcer disease. MATERIALS AND METHODS: CCR6+ T-helpers were isolated from the blood by using immuno-magnetic separation adapted to this study. The number of T-helpers of types 1 and 17 (Th1 and Th17) and cells with mixed properties of Th1 and Th17 (Th1/Th17) was determined by intracellular cytokine assay. RESULTS: Initially, we planned to activate unseparated peripheral blood mononuclear cells ex vivo and evaluate the number of cytokine producers among mature CCR6+ T-helper cells by gating them during the flow cytometry. However, dramatic changes in the phenotype of T-helpers upon activation did not allow us to reliably identify the cells of interest. Subsequently, we used a two-stage immunomagnetic separation procedure to obtain functionally active mature CCR6+ T-helpers with a purity of >90%. The quantitative yield of these cells from the blood of patients with gastric and duodenal peptic ulcer associated with H. pylori was 9 times higher than that from the blood of healthy donors. Activation of CCR6+ T-helpers purified from blood of ulcer patients revealed an increased content of Th1, Th17, and Th1/Th17. One ml of the patient's blood yielded 18.1 times more CCR6+ Th1, 19.4 times more CCR6+ Th17, and 21.1 times more CCR6+ Th1/Th17 compared with the blood of healthy subjects. CONCLUSION: The content of mature CCR6+ T-helper cells with pro-inflammatory activity significantly increases in the blood of patients with peptic ulcer associated with H. pylori infection.