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.

[Digital image in the practice of Cytology: a pilot study.]

Cytological study is a highly specialized type of laboratory analysis of the cellular composition of biological material and is to assess the morphological characteristics of cellular elements. The modern development of digital technologies is increasingly forming the interest of specialists to such a section as telepathology (digital pathology), which is a process of virtual microscopy with the transformation of classical cytological preparations into digital. Most morphologists currently use some forms of digital imaging, such as static images obtained by optical cameras mounted under a microscope. The development of more high quality image and resolution in the digital pathology promotes the use of telepathology, including telecitology in their daily work for training specialists, counselling of medications, monitoring the quality of diagnosis.

[Possibilities of cytological diagnosis of the nature of the exudate at the stage of emergency.]

Cytological diagnosis by effusions is currently the only reliable method of morphological verification of the diagnosis, it has prognostic significance and determines the choice of treatment strategy. At the same time, the variability of normal mesothelial cells causes significant difficulties in its differential diagnosis with reactive mesothelium, malignant mesothelioma and cancer metastasis, which requires additional analytical methods. A retrospective study of cytological preparations for 2017 was conducted, as well as the effectiveness of the use of fluorescent immunocytochemistry (FITZ) on the test system "biochip" in combination with a traditional cytological study was evaluated. During the period of November 2017 - July 2018, 46 exudates of serous cavities were studied, which showed that 9 patients (19.6%) were diagnosed with metastatic effusion, 31 (66.7%) patients had reactive exudate, suspicion of the malignant nature of serous fluid was expressed in 4 patients (8.7%), and 4.8% of persons (2 samples) failed to make an accurate diagnosis. After an additional FITZ study using the "Biochip" test system, the number of patients diagnosed with metastatic effusion increased to 7 (25.9%) due to a decrease in the percentage of cases of unspecified effusion. The combined use of traditional cytology and fluorescent immunocytochemistry in the diagnosis of effusion fluids at the stage of emergency medical care to the patient complements each other and contributes to a faster and more reliable diagnosis, as it allows to confirm the malignancy of the test material, and to assume the primary focus.

[Strategy of probe selection for studying mRNAs that participate in receptor-mediated apoptosis signaling].

Death receptors (DRs) and the participants of DR-mediated signaling are characterized by a large number of mRNA isoforms generated by alternative splicing. Due to their high labor intensity and high cost, conventional methods (RT-PCR and RT-PCR in real time) are ineffective when the simultaneous detection of a plurality of mRNA isoforms is needed. In this regard, the use of DNA biochips is has prospective applications in analyzing the expression of many genes simultaneously. In this paper, we suggest an optimal strategy of probes selection aimed at detecting the maximum number of mRNA splice variants generated by major participants of DR-signaling. The objects of the study were 185 genes that form 1134 mRNA isoforms. As a result, a biochip design was developed that enables the detection of 499 mRNA isoforms (44% of total mRNA splice variants). The proposed strategy combines a high degree of modularity, the use of modern high-performance computers, and broad opportunities for setting up the selection criteria in accordance with the objectives of the study.

[Frequency of the occurrence of spliced variants of the messenger RNA DR3/LARD in herpesviral infection].

Analysis of frequency of the occurrence of membrane and soluble forms of the mRNA DR3/LARD in blood in herpesviral infection of various etiology was studied. Four forms of the mRNA DR3/LARD were detected with various frequencies in blood cells of healthy volunteers. Patients with herpesviral infection of various etiology were studied using RT-PCR. Two forms encoded membrane molecules (mRNA LARD 1a, mRNA DR3beta) and two other forms accorded soluble forms of receptor (mRNA LARD 3, mRNA soluble DR3beta). It was revealed that the frequency of the occurrence of mRNA soluble DR3beta form decreased in patients with the varicella zoster virus (VZV) infection in comparison with healthy volunteers. However, the patients with the Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection did not display significant change in occurrence of mRNA soluble DR3beta form. As a whole, changes in frequency of occurrence of spliced variants of mRNA DR3/LARD are directed toward modulation of apoptosis and restraint antiviral immune response.

[DR3/LARD mRNA spliced variants' frequency at colorectal cancer].

There are a lot of "death receptor" DR3/LARD mRNA variants that are formed during the alternative splicing. Membrane and soluble forms of the receptor are encoded with various types of the DR3/LARD mRNA and perform different functions. Using RT-PCR, the DR3/LARD mRNA spliced variants' frequency was measured in colon cancer patients' samples and cancer cell lines. In samples under investigation, four forms of the DR3/LARD mRNA were found with various frequencies. Two of them encoded the membrane receptors (LARD la mRNA and DR3beta mRNA) and other two expressed the soluble molecules (LARD 3 mRNA and soluble DR3beta mRNA). In blood of healthy volunteers 11 combinations (spectrums) of the DR3/LARD mRNA forms were revealed, and the "full" spectrum including all four variants of DR3/LARD mRNA dominated. In blood of colon cancer patients and tumour tissue samples, 6 DR3/LARD mRNA spectrums were found. The diversity of the DR3/LARD mRNA spectrums was decreased in colon cancer patients because of the frequency reduction of soluble DR3beta mRNA. Reduction of a variety of spectrums in cells of the patients was caused by decrease in occurrence of mRNA of the soluble DR3beta form. In samples of the tumor centers the spectrum with absence only mRNA of the soluble DR3beta form dominated. In blood of patients two spectrums prevailed: "full" range and presented mRNA LARD la and mRNA LARD 3. Only these two spectrums of mRNA DR3/LARD were also found in the tumor cell lines. Distinctions in occurrence of spectrums of DR3/LARD mRNA at healthy volunteers and colon cancer patients can define a different susceptibility of immunocompetent and tumor cells for apoptosis signals.

[Heterogeneous expression of CD38 gene in tumor tissue in patients with colorectal cancer].

The CD38 gene codes a membrane protein which takes part in cell adhesion and catalyzes the formation of cyclic ADP-ribose. Using RT-PCR method we tested the presence of full-size and alternative forms of mRNA CD38 in samples of tumor tissue of patients with colorectal cancer and in tumor cell lines. It was shown that there are the cells in the tumor tissue which expressed CD38 gene. In tumor tissue of patients the alternative form of mRNA CD38 was detected less frequently than full-size form. Cells of lines Colo-205, T-84, HCT15 and HCT116 contained mRNA CD38, in cells of lines Caco-2 and SW-620 mRNA CD38 was absent. In cells of tumor tissue on the first stage of colorectal cancer CD38 gene was expressed in 100% of cases. On the second, third and fourth stages of the disease gene expression was observed less often. The frequency of mRNA CD38 detection not depend on tumor localization, tumor grade and presence of metastases. Using method of restriction analysis CpG methylation was detected in binding sites of transcription factor Sp1 and receptor of retinoic acid (RARE) in all tested samples of tumor tissue independently of the presence or absence of mRNA CD38. The obtained data suggest that in the tumor cells of patients with colorectal cancer the expression of the CD38 gene is heterogeneous.