Evaluation of Regenerative Potential of Cryopreserved Adenosine-Modified Monocytes on the Model of Burn Wounds.

We studied viability of adenosine-modified monocytes after cryopreservation and their regenerative potential. The optimal method of cell cryopreservation was the use of serum-free medium followed by storage in liquid nitrogen. Injections of adenosine-modified monocytes after 12-week cryopreservation around grade IIIB burn wound accelerated healing of the skin defect and significantly improved the histological pattern of the damage area at delayed terms.

Analysis of Gut Microbiota in Patients with Parkinson's Disease.

Gut microbiota of patients with Parkinson's disease and healthy volunteers was analyzed by the method of high throughput 16S rRNA sequencing of bacterial genomes. In patients with Parkinson's diseases, changes in the content of 9 genera and 15 species of microorganisms were revealed: reduced content of Dorea, Bacteroides, Prevotella, Faecalibacterium, Bacteroides massiliensis, Stoquefichus massiliensis, Bacteroides coprocola, Blautia glucerasea, Dorea longicatena, Bacteroides dorei, Bacteroides plebeus, Prevotella copri, Coprococcus eutactus, and Ruminococcus callidus, and increased content of Christensenella, Catabacter, Lactobacillus, Oscillospira, Bifidobacterium, Christensenella minuta, Catabacter hongkongensis, Lactobacillus mucosae, Ruminococcus bromii, and Papillibacter cinnamivorans. This microbiological pattern of gut microflora can trigger local inflammation followed by aggregation of α-synuclein and generation of Lewy bodies.

Biological Collections: Chasing the Ideal.

This article is based on the results of an analysis of existing biological collections in Russia and abroad set up in the framework of the project "Scientific Basis of the National Biobank -Depository of Living Systems" by M.V. Lomonosov Moscow State University [1].

[Application of GLAD-PCR analysis for the methylation sites detection in the regulatory areas of tumor-suppressor genes ELMo1 and EsR1 in colorectal cancer].

Aberrant methylation of regulation regions of tumorsuppressor genes is showed for many cancer diseases. In course of this modification an enzyme DNMT3 methylates RCGY sites in CpG-islands of regulation regions producing R(5mC)GY sites. Earlier we developed GLAD-PCR assay to determine R(5mC)GY site in a definite position of human genome. In this work we have applied GLAD-PCR assay to determine R(5mC)GY sites in regulation regions of ESR1 and ELMO1 tumor-suppressor genes. We have studied a fragment of first exon of ELMO1 gene and a part of ESR1 promoter region in DNA preparations from malignant cell line SW837 and colorectal tumor samples. We have checked four sites in each region and found two highly methylated sites: GCGC in first exon of ELMO1 gene and GCGT in promoter region of ESR1 gene. Site GCGT is weakly methylated in healthy tissues and more methylated in the most of colorectal samples. Site GCGC is not methylated in healthy tissues and significantly methylated in 60% of colorectal samples. A possibility to use GLAD-PCR assay for cancer diagnostics is discussed.

[Correlation Between Emotional-Affective Disorders and Gut Microbiota Composition in Patients with Parkinson's Disease].

Background: Despite the efforts of scientific community the data available on the correlation between emotional-affective symptoms of Parkinson's disease and changes in microbiome is still scarce. Deeper studies of nonmotor symptoms evident in premotor stages of the disease and the reciprocal influence of microbiota may help to understand the etiology and pathogenesis of PD neurodegeneration better. Aim of the Study: Discover the relations between emotional-affective disorders prevalent in PD population and changes in gut microbiota composition. Methods: 51 patient diagnosed with PD participated in the study. Every participant's emotional-affective state was examined using Beck's Depression Inventory (BDI) and Hospital Anxiety and Depression Scale (HADS). Taxonomic richness of microbiome was studied using 16S ribosomal RNA gene sequencing, bioinformatics, and statistical analysis. Results: Anxiety and depression are prevalent affective disorders in patients with PD. In our study, most of the subjects demonstrated certain anxiety and depression. Taxonomic diversity of gut microbiota in BP was increasing with the increase in anxiety levels, reaching the maximum in the group with subclinical anxiety, and decreasing in the group with clinically significant anxiety disorder. At the species level, patients with clinically significant anxiety had higher abundance of Clostridium clariflavum compared to the anxiety-free patients. Patients with moderate depression were characterized by the higher prevalence of Christensenella minuta, Clostridium disporicum, and Oscillibacter valericigenes compared to subjects without depression or with mild depression. Conclusion: The data we received in our study allow better understanding of PD pathogenesis.

[Clinical and Functional Phenotypes of Chronic Obstructive Pulmonary Disease, the Effect on the Severity of the Disease and Prognostic Value in Assessing the Risk of Disease Progression].

Background: Chronic obstructive pulmonary disease (COPD) is characterized by progressive limitation of airflow rate, hyperergic inflammatory response of the respiratory tract, and systemic manifestations. Prognosis of the disease depends on the severity of these pathogenetic components. FEV1 which characterizes the speed limit airflow do not allow predicting the rate of COPD progression. Aims: Comparison of the prognostic significance of such clinical parameters as frequency of exacerbations and the development of comorbid diseases to assess the nature of COPD progression by using different classification approaches. Materials and Methods: The prospective comparative study included 98 patients with COPD. In the framework of the study protocol, 2 visits were required when a practitioner recruited patients who met inclusion/exclusion criteria, obtained the signed informed consent, collected the anamnestic data, and performed basic procedures of the study: spirometry, 6-minute stepper test, assessment of dyspnea on questionnaire mMRC, body plethysmography, lung diffusion capacity study, dopplerechocardiography, tomography of the chest. Visit 2 was conducted in 12 months after the first one to assess the dynamics of the disease. The dynamics of the disease was considered negative if, upon repeated examination, the patient was referred to the group with more severe COPD. Results: Our study demonstrates that comprehensive assessment of such factors as the frequency of COPD exacerbations in the preceding 12 months and the presence of comorbid diseases in a patient is reasonable for assessment of disease severity and determination of disease prognosis. At the same time the frequency of COPD exacerbations as one of the evaluated factors is most strongly associated with disease progression. Conclusions: Thus, a practitioner is recommended to use the proposed additional clinical criteria to assess the severity and degree of progression of COPD.

[Clinico-functional phenotypes of chronic obstructive pulmonary disease, their influence on the severity of the disease, and prognostic value for risk evaluation].

Attempts at optimizing classification of chronic obstructive pulmonary disease (COPD) reflect clinical heterogeneity of this pathology and provide a basis for the search of new phenotypic markers (especially at the early stages of the disease) that could be useful for prognostication of its severity in individual patients. One of the potential makers is phenotyping of COPD with distinguishing bronchitic, emphysemic, and mixed phenotypes. This paper presents results of analysis of functional characteristics of the patients with these phenotypes. They are shown to reflect clinical and functional features of the disease that may be of value for diagnostic purposes, the choice of the treatment strategy and prognosis of the outcome in individual patients.

[THE CHARACTERISTICS OF SYSTEMIC AND LOCAL IMMUNE REACTIONS UNDER HABITUAL NON-CARRYING OF PREGNANCY].

The study was carried out to evaluate clinical informativeness ofstudies of immunological parameters of structural and functional elements of endometrium in complex with profile of embritropic autoimmune antibodies under non-carrying of pregnancy The sampling consisted of 19 women of reproductive age with episodes of non-carrying in first semester. They were examined for inherited thrombophilia and profiles of immune reactivity of natural regulatory auto-antibodies (ELI-P-Complex). The biopsy material of endometrium was subjected to histological and immune histochemical (CD16, CD56, CD68, CB138, estrogen and progesterone receptors) analyses. In women with non-carrying of pregnancy polyclonal immunosuppression, increased level of embriotoxic auto antibodies to Fc- fragment of immunoglobulin, insulin and thyroglobulin are detected. The immune histochemical analysis established expressed decreasing of amount of progesterone receptors of stromal elements of endometrium after spontaneous abortion. The activation of killer activity of decidual lymphocytes under habitual non-carrying of pregnancy was detected too. The positive dependency was established between: expression of progesterone receptors in glandular cells of endometrium and level of anti-spermatic antibodies in blood serum; expression of progesterone receptors in stroma and level of antibodies to human chorial gonadotropin; expression of receptors of estrogens in stroma and level of antibodies to Fc-fragment of immmunoglobulines. The study data permits affirming that evaluation of level of regulatory auto-antibodies is informative at examination of woman after episode of non-carrying of pregnancy. The production of antibodies has direct relationship with pathological processes in endometrium and it can be one of criteria of completeness of pre-conceptional preparation.

[Stimulation of adenosine receptors on myeloid cells enhance leukocyte migration at the site of burn injury].

Adenosine, endogenous purine nucleoside, is an ATP metabolite that also acts as an extracellular signaling molecule. The concentration of extracellular adenosine rises during hypoxia and cell damage leading to numerous pleiotropic effects. Although a high concentration of adenosine was found at burn injury, the effect has not been well elucidated. We have studied human peripheral blood myeloid cell, due to their expression of specific adenosine receptors and capacity to migrate to the site of burn injury. We have shown that myeloid cells after 72 hours of stimulation of adenosine receptors develop altered expression of surface antigens: preserved monocyte's marker CD14 with already expressed dendritic cell markers (CD209, CD1a). Whereas untreated cells have already lost monocyte marker in 72 hours, and express CD1a more abundantly. Adenosine modified myeloid cells express also higher levels of mRNA of proinflammatory cytokines and chemoattractants (IL-6, IL-8, IL-1 b). Using mouse model of the burn injury we have shown, that adenosine modified bone marrow derived myeloid cells injected in the site of the injury promote migration of granulocytes, monocytes, macrophages, and fibroblasts on the 7th day after burn. Thus, stimulation of adenosine receptors alters differentiation and function of myeloid cells. In the site of burn injury adenosine modified myeloid cells augment cell migration due to paracrine factors.

[The estimate of association between gene polymorphisms and the frequency and spectrum of cytogenetic abnormalities in the cohort of Siberian Group of Chemical Enterprises employees exposed to professional irradiation (microarray studies)].

The results from the research into the association between polymorphisms of genes-candidates for individual radiosensitivity and the frequency and spectrum of cytogenetic abnormalities are analyzed. The study was conducted among Siberian Group of Chemical Enterprises healthy employees exposed to professional irradiation in a dose range of 100-300 mSv. Genotyping of DNA samples from 96 employees was carried out by oligonucleotide microarray: "Cancer_SNP_Panel GT-17-211" ("Illumina") containing 1, 421 SNP-markers (Single Nucleotide Polymorphisms) of 406 genes. The standard cytogenetic analysis was performed in the entire examined group. We have also analyzed the association of these SNP with the frequencies of aberrant cells and following chromosomal aberrations: single chromatid fragments, chromatid exchanges, paired fragments, dicentric, ring and frequencies, translocations. We have found that 40 SNP (rs1800389, rs1051690, rs2392221, rs1041163, rs2114443, rs6083, rs1760904, rs4986894, rs488133, rs7462102, rs11249938, rs34206126, rs33945943, rs34324628, rs5742694, rs978458, rs5742667, rs2373721, rs2162679, rs889162, rs2233679, rs2010457, rs2873950, rs1574154, rs10934500, rs4688046, rs10934503, rs4624596, rs2288729, rs4227, rs1367696, rs751087, rs1269486, rs1149901, rs1800404, rs887477, rs696405, rs751087, rs81 92284, rs312016) are associated with the frequency of different types of chromosomal abnormalities (p-value with FDR of Benjamini-Hochberg--equal less than 0.05). 24 SNP (underlined) are associated with more than one type of chromosome abnormalities. In the future, we are going to confirm the results in further studies on the cohort of more than 600 persons.

Study of DNA interaction with cobalt ferrite nanoparticles.

Interaction of cobalt ferrite nanopowder and nucleic acid was investigated. Superparamagnetic cobalt ferrite nanoparticles (6-12 nm) were prepared by mechanochemical synthesis. Structure of the nanopowder was characterized using X-ray diffraction. It was shown that cobalt ferrite nanoparticles were associated with ssDNA and dsDNA in Tris-buffer resulting in bionanocomposite formation with mass weight relation nanoparticles: DNA 1:(0.083 +/- 0.003) and 1:(0.075 +/- 0.003) respectively. The mechanism of interaction between a DNA and cobalt ferrite nanoparticles was considered basing on the whole set of obtained data: FTIR-spectroscopy, analyzing desorption of DNA from the surface of the particles while changing the chemical content of the medium, and on the modeling interaction of specific biomolecule fragments with surface of a inorganic material. It was supposed that the linkage was based on coordination interaction of the phosphate groups and oxygen atoms heterocyclic bases of DNA with metal ions on the particle surface. These data can be used to design specific magnetic DNA-nanoparticles hybrid structures.

Evaluation of the resistance of DNA immobilized on ferrimagnetic particles of cobalt ferrite nanopowder against nuclease cleavage.

DNA was immobilized on ferrimagnetic particles of cobalt ferrite nanopowder (CoFe(2)O(4)) and its resistance to endonuclease (DNase I) hydrolysis was studied. Immobilization on cobalt ferrite nanoparticles prevented enzymatic cleavage of DNA. This process was not associated with enzyme inactivation under the effect of nanosize cobalt ferrite and was presumably determined by lesser availability of the DNA molecule as a result of its interaction with nanoparticles.

[DNA diagnosis of mixed invasions of Opisthorchis felineus and Metorchis bilis by polymerase chain reaction].

Opisthorchiasis is one of the significant naturofocal diseases in Russia. The diagnosis of opisthorchiasis may mask some diseases, such as opisthorchiasis, metorchiasis, and clonorchiasis - biohelminthoses that are induced by various representatives of the family Opisthorchiidae-Opisthorchis felineus/O.viverrini, Metorchis bilis, and Clonorchis sinensis, respectively. Coproovoscopy and serologic methods fail to accurately define the species-specific affiliation; in this connection the identification of opisthorchids, by using DNA diagnostic techniques, becomes urgent. The present paper gives the results of development of DNA diagnosticum, which differentiates parasitic diseases induced by O. felineus and M. bills. The ribosomal RNA gene cluster fragment incorporating internal transcribed spacer 2 (ITS2) was used as a diagnostic marker. A system for diagnosing opisthorchiasis was developed as a multiplex polymerase chain analysis and tested on 37 patients infected with various species of opisthorchids.

[Investigation of the interaction between DNA and cobalt ferrite nanoparticles by FTIR spectroscopy].

The interaction of DNA with nanoparticles of cobalt ferrite powder prepared by the mechano-chemical method was studied. It was shown that CoFe(2)O(4) nanoparticles efficiently bind DNA in aqueous solutions (Tris-HCl), forming a bionanocomposite. The adsorption capacity of CoFe(2)O(4) nanoparticles for DNA was evaluated to be 5.25 x 10(-3) mol/m(2). The desorption of DNA from the surface of the particles was analyzed while changing the pH, the ionic strength, and the chemical content of the medium. The DNA-CoFe(2)O(4) nanocomposite was investigated by FTIR spectroscopy. The block of the data allowed one to consider the mechanism of the interaction between a polynucleotide and CoFe(2)O(4) nanoparticles and to make the assumption that the binding occurred due to the coordination interaction of the phosphate groups and heterocyclic bases of DNA (oxygen atoms of thymine and guanine) with metal ions on the particle surface. The analysis of the IR spectra showed that binding can lead to the partial destabilization of the DNA structure, with the B conformation of a polynucleotide being preserved.

[Genetic diagnostic technique for opisthorchiasis].

OBJECTIVE: To develop a test system for polymerase chain reaction (PCR) diagnosis of Opisthorchis felineus in human feces. Based on the Internet databases, the authors have developed oligonucleotide primers, as well as TaqMan tests (fluorescence label probes) specific to O. felineus genome to perform PCR with the real-time detection of a result. Conditions for PCR, including temperatures, the number of components of amplification, etc., were selected. Test systems on fecal samples from 254 subjects randomly selected and on those from 100 patients with verified opisthorchiasis, and on those from 100 healthy individuals were studies. The significance of the test systems was ascertained to be about 98%. Their sensitivity in determining O. felineus is about 10(3) copies of target DNA per ml. The efficiency of various techniques (coproovoscopy, enzyme immunoassay, PCR) was compared to detect Opisthorchis invasion.

Specific adrenergic responses of smooth muscles in the vascular wall of guinea pig pulmonary arteries during ovalbumin sensitization.

The adrenergic contractile responses of smooth muscles in the vascular wall of guinea pig pulmonary arteries were studied during ovalbumin sensitization. Sensitization was followed by inhibition of contractile responses to an alpha-adrenoceptor agonist mesatone, prevented endothelium-derived relaxation, and potentiated the contractile response to isoproterenol. Administration of a beta-adrenoceptor agonist isoproterenol potentiated the increase in mechanical strain of smooth muscles in the pulmonary artery precontracted with high-potassium Krebs solution. Removal of the endothelium had no effect on the contractile response of smooth muscle segments from the pulmonary artery of intact and sensitized guinea pigs to b-adrenergic influences. The contractile responses of smooth muscles of the pulmonary artery are associated with activity of the cAMP-dependent signal system and play a role in the pathogenesis of ventilation-perfusion disturbances during atopic inflammation.

[Nitric oxide metabolites and their significance in pathogenesis of bronchial asthma].

To study the possible potential of nitric oxide (NO) in the processes of atopic inflammation, the authors evaluated the intensity of nitrosyling and oxidative stresses in the bronchoalvelar lavage fluid and expired air condensate of patients with bronchial asthma (BA). Chronic inflammation was shown to result in an increase in the processes of nitration and oxidation in worsening BA and to reliably correlate with airway NO-producing functions, by explaining the pathological effects of NO due to the formation of 3-nitrotyrosine and malonic dialdehyde with the existing imbalance in NO metabolism, by intensifying nitrosylating stress. In the authors' opinion, nitrosothiols that are required as a NO donor may be rapidly destroyed or virtually do not form so the peroxynitrite-nitrosothiol ratio may predetermine the final effects of NO.

A pilot screening of prevalence of atopic states and opisthorchosis and their relationship in people of Tomsk Oblast.

The study was aimed to estimate the relationship between the prevalence of allergic disease and helminth invasion by the trematode Opisthorchis felineus in rural and urban populations of Tomsk Oblast (West Siberia, Russia). Two hundred and one people from Kargasok village of Tomsk Oblast and 196 from the city of Tomsk were screened for the presence of atopy and O. felineus invasion. Opisthorchosis was found in 66 participants (32.8%) from Kargasok and in 22 people (11.2%) from Tomsk. Atopic diseases were more common in the urban population than in the rural: 52.8 and 31.4%, respectively. Positive skin-prick tests were significantly higher in the urban population than in rural people: 83.2 vs 24.4%, respectively. It was found that in the city, the presence of antibodies to O. felineus negatively correlates with the atopic sensitization by skin-prick tests. However, in the village, opisthorchosis was positively associated with atopic diseases. The data obtained confirm the negative association of rural lifestyle and atopic diseases prevalence and indicate that O. felineus invasion might be a modifying factor of this relationship in Tomsk Oblast.

Role of respiratory epithelium in the development of hyperreactivity of bronchial smooth muscles.

In guinea pigs sensitized with ovalbumin the respiratory epithelium lost its ability to modulate the responses of airway smooth muscles to histaminergic stimuli. Incubation of bronchial segments with IL-5 potentiated the contractile responses of bronchial smooth muscles to histamine in both intact and sensitized animals. Incubation of bronchial segments with IL-5 receptors moderated contractile activity of segments from sensitized pigs, but not in the segments from intact controls.

[Contractile properties of smooth muscles of bronchial tubes at formation of hyperreactance of airways].

Contractile properties of smooth muscles of bronchial tubes of porpoises in formation of hyperreactance airways, were studied. Sensitization by ovalbumin results in formation of hyperresponsiveness of the airways as shown in increase in amplitude of contractile responses to histaminergic influences and oppression of adrenergic relaxations of segments. Respiratory epithelium oppresses contractile responses of airways in intact animals to histaminergic influences. Influence of epithelium on adrenergic responses depends on precontractile factor: they increase in prereduction by histamine. In formation of hyperresponse, the epithelium loses ability to modulate responses of smooth muscles of the airways. Incubation segments of smooth muscles of the airways in interleukin 5 increases contractile responses to histaminergic influences. Receptor of interleukin 5 (the soluble fraction) neutralizes the effect of exogenous interleukin 5, and incubation of receptor of sensitized animals oppresses histaminergic contractile responses.