Analysis of the Diversity and Functional Potential of Bacterial Communities in Tuberculomas.

The dynamics of lung microbiota in tuberculosis remains poorly understood. Sequencing of variable regions of the 16S rRNA gene from surgically excised tuberculosis foci and biopsy specimens of normal lung tissue allowed characterization of the diversity and predictive potential of bacterial communities. Taxonomic diversity indices attested to differences in the structure of microbial communities between "healthy" lungs and tuberculomas. The microbial composition of "healthy" lungs varied in taxonomic diversity and was presented by both gram-positive and gram-negative bacteria with sufficiently similar metabolic potential. The microbiota of the examined tuberculomas consisted of Mycobacterium tuberculosis in 99.9% of cases. A significant part of the metabolic pathways predicted by PICRUSt2 included cholesterol catabolism, sulfate assimilation, and various pathways for the biosynthesis of cell wall components.

Analysis of Natural Antibodies during the Development of Phantom Pain Syndrome.

We determined natural antibodies (n-Abs) to the regulators of the main systems of biochemical homeostasis: ß-endorphin, serotonin, dopamine, histamine, orphanin, angiotensin, GABA, glutamate, bradykinin, vasopressin, thrombin, and α-2-macroglobulin in individuals with phantom pain syndrome (PPS), resulting from amputation after injury. It was established that each patient has an individual immunoprofile, but for all of them there was a significant increase in the level of antibodies to serotonin, histamine, and angiotensin, which reflect the chronicity of the pain syndrome and do not depend on the self-assessment of the severity of PPS. Determination of the role of regulators of biochemical homeostasis in the development of phantom pain showed that, at high, moderate, and weak severity of PPS, the biogenic amine and angiotensinergic systems are activated. A decrease in PPS intensity normalizes deviations in all immunological parameters. The levels of n-Abs for the pain (ß-endorphin) and analgesic (orphanin) systems are significant only at low PPS. Monitoring the individual profile of n-Abs to endogenous regulators allows us to obtain an objective picture of the pain status of the patient's body.

Analysis of Immunobiochemical Parameters in Overweight People in Assessing the Risk of Cardiovascular Diseases.

A comparative analysis of specific immunobiochemical parameters, including natural antibodies (NAb) to endogenous regulators of the cardiovascular system, adrenal and gastrointestinal hormones, was performed in students aged 18-22 years with normal and increased body weight (the body mass index from 18.5 to 24.9 kg/m2 and from 25 to 29.9 kg/m2, respectively). The serum content of NAb and hormones was determined by ELISA. The level of the studied indicators depended on the body mass index value. In overweight subjects, the main immune indicators of the biogenic amine system, renin-angiotensin system, and kinin system exceeded the normal. The cortisol level was higher than in subjects with normal body weight. Aldosterone secretion was less dependent on the ACTH content and was lower than in students with normal body weight. The content of cholecystokinin and gastrin corresponded to the values for overweight. These trends in hormone contents are a predisposing factor for further weight gain. Practical significance of the combined assessment of disturbances in the immunological and biochemical homeostasis has been established. Analysis of the adrenal and gastrointestinal hormones can predict the risk of weight gain, but at the same time, changes in the level of immunological indicators in subjects with increased body weight characterizes the possibility of developing cardiovascular pathologies.

Online Service with Automated Interpretation of Sequencing Data and Prediction of Pyrazinamide Resistance in Mycobacterium tuberculosis.

Pyrazinamide plays an important role in the treatment of tuberculosis. However, the microbiological test for pyrazinamide resistance is more complex and less reliable than testing of susceptibility to other anti-tuberculosis drugs due to the need to grow the pathogen at pH 5.5. Identification of mutations that cause resistance to anti-tuberculosis drugs can replace microbiological methods. Mutations in the pncA gene are responsible for the main mechanism of the resistance to pyrazinamide and are found in more than 90% of resistant strains. However, the genetic method for determining drug susceptibility is very complex, because mutations leading to pyrazinamide resistance are diverse and scattered throughout the gene. We have developed a software package for automatic data interpretation and prediction of the resistance to pyrazinamide based on Sanger sequencing results. The effectiveness of detection of pyrazinamide resistance in 16 clinical samples was compared using the BACTEC MGIT 960 automated system and pncA gene Sanger sequencing with automated analysis of the results. A significant advantage of the developed method over a single microbiological study was shown, due to greater reliability of the results irrespective of the purity of isolates.

Comparative Determination of Natural Antibodies for the Assessment of the Cardiovascular System and the Level of Human Difficulty.

A comparative analysis of natural antibodies to ß-endorphin, angiotensin, dopamine, serotonin, parameters of the cardiovascular system and anxiety levels was carried out for 241 athletes of various qualifications and sports. The obtained indicators of the cardiovascular system were compared with reference values. A significant increase in the level of natural antibodies to angiotensin was established for all groups of athletes. In the case of dopamine, serotonin, these differences are associated with the qualification of the athlete, for ß-endorphin, differences in the level of the indicator depending on the sport were found. A group of individuals with high levels of situational and personal anxiety was found among highly qualified athletes. An increase in blood pressure in athletes of cyclic sports and martial arts is adaptive, and in athletes of speed-strength sports it leads to a change in the walls of the myocardium. As a result of the study, the possibility of a comprehensive determination of natural antibodies and functional indicators as diagnostic markers for assessing the state of the human cardiovascular system has been shown.

The effect of Escherichia coli ZP strain with a conjugation-based colicin E7 delivery on growth performance, hematological, biochemical, and histological parameters, gut microbiota, and nonspecific immunity of broilers.

The Escherichia coli ZP strain (ZP) was constructed based on the known probiotic E. coli strain Nissle 1917. It was genetically modified to carry the colicin E7 synthesis gene encoding DNase on a conjugative plasmid and the colicin E7 immunity gene in the chromosome. The aim of this study was to evaluate the effects of the daily ZP per oral administration (5 × 108 or 5 × 1010 CFU per bird) on the growth performance, hematological, biochemical, histological parameters, gut microbiota, and nonspecific immunity of the 4-24 days old broilers. The ZP administration increased the abundance of genera Bacillus, Butyrivibrio, and Clostridium and did not influence the weight gain of 4-16 days old broilers. The biochemical parameters were within normal ranges for poultry in experimental and control groups. The ZP administration had no effect on the erythrocyte numbers, hemoglobin and immunoglobulin Y concentrations, but significantly increased the serum lysozyme concentration, leukocyte numbers, and reactive oxygen species production by phagocytes compared with the control group. It did not cause inflammatory changes in intestinal mucosa, Peyer's patches, and spleen. Thus, the ZP had no detrimental effects on broiler health and could be an efficient probiotic for the broiler colibacillosis prophylaxis.

Microembolic Signals in Arteries of the Base of the Brain after Ischemic Stroke.

Embolic strokes make up a significant proportion of acute cerebrovascular accidents. Doppler blood flow monitoring with microembolodetection allows suggesting the embolic nature of cerebral infarction. The aim of this study was to identify factors associated with the presence of microembolic signals in patients with ischemic stroke. The study included 515 patients, microembolic signals were detected in 48 (9.3%) of them. According to multispiral CT angiography, significant differences in patients with and without microembolic signals were found for wall thickness of both common carotid arteries and for left internal carotid artery (p<0.05). Factor analysis revealed a variable that reflects the severity of left carotid arteries atherosclerosis, which was a significant predictor of registration of the microembolic signals (p=0.016).

A Method to Determine Xenobiotic Acetylation Rate by Taq SNP rs1495741.

Drug acetylation plays an important role in the medical practice. Modern methods of acetylation phenotype prediction are based on genotyping of polymorphisms in the second exon of the gene NAT2. Some disadvantages of these methods limit their application in the clinical practice. We developed a method of human genotyping based on identification of NAT2 gene polymorphism rs1495741 by real-time PCR. This method of genotype determination has a number of advantages: high sensitivity, simplicity, possibility of automated interpretation of the results, and feasibility in clinical laboratories.

A Method for Evaluation of the Level of Circulating Mitochondrial DNA by ND1 and ND2 Genes.

The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.

[Non-specific prevention of COVID-19 during vaccination against a new coronavirus infection: results of a multicenter, double-blind, placebo-controlled, randomized clinical trial].

BACKGROUND: A multicenter, double-blind, placebo-controlled, randomized clinical trial (RCT) of the phase III efficacy and safety of Ergoferon® for the non-specific prevention of COVID-19 during vaccination against a new coronavirus infection was conducted (permission of the Ministry of Health of the Russian Federation №559 dated 22.09.2021; ClinicalTrials.gov Identifier: NCT05069649). AIM: To evaluate the efficacy and safety of the use of Ergoferon for the non-specific prevention of COVID-19 during vaccination against a new coronavirus infection. MATERIALS AND METHODS: From October 2021 to April 2022, 1,057 patients aged 18 to 92 years who received component I of the "Gam-COVID-Vac" vaccine were included. After screening, 1,050 patients were randomized into 2 groups: 526 people received Ergoferon according to the prophylactic scheme - 1 tablet per administration 2 times a day for 3 weeks, the drug is not allowed during the meal and should be kept in the mouth without swallowing, until completely dissolved; 524 patients received a placebo according to the Ergoferon® scheme. The total duration of participation in the study was 5 weeks + 3 days. The primary endpoint is the number of RT-PCR - confirmed cases of SARS-CoV-2 infection, regardless of the presence of symptoms during participation in the study. An additional criterion of effectiveness is the proportion of those hospitalized with COVID-19. The safety assessment included consideration of the presence and nature of adverse events (AEs), their severity, relationship with the drug intake, and outcome. Statistical data processing was carried out using SAS 9.4 with the calculation of the exact Fisher test, χ2 test, Cochrane-Mantel-Hensel test, Wilcoxon test and other parameters. RESULTS: The ITT (Intention-to-treat) and PP [Per Protocol] efficacy analysis included data from 1,050 [970] patients: 526 [489] people - Ergoferon® group and 524 [481] people - Placebo group. The primary endpoint - the number of laboratory-confirmed cases of SARS-CoV-2 infections was 3 times less compared to placebo - 7 (1.43%) vs 22 (4.57%), respectively (p=0.0046; [p=0.0041]). Taking Ergoferon® reduces the risk of SARS-CoV-2 infection by more than 3 times in vaccinated patients during 5 weeks of the vaccination and post-vaccination periods (p=0.0046 [p=0.0041]). Of the COVID-19 patients in the Ergoferon® group (1.33%) nobody was hospitalized. According to the Post hoc analysis, Ergoferon® reduces the risk of COVID-19 disease by 4 times in the period between the components I and II of the "Gam-COVID-Vac" vaccine (p=0.0066 [p=0.006]). The frequency of AEs in both groups did not differ. There were no registered AEs associated with the drug with a reliable degree. There was a high level of patient compliance and good tolerability. CONCLUSION: Ergoferon is an effective and safe drug for the prevention of COVID-19 in people vaccinated against a new coronavirus infection.

Measurement of Immunological Parameters to Assess Human Body Readiness for Physical Load.

We measured the level of natural antibodies (nAb) to glutamate and GABA reflecting the balance of excitation and inhibition systems and involved in the adaptation processes in athletes receiving normalized physical activity in the dynamics of training (figure skaters, football players, and people actively involved in sports). It was found that each subject has an individual immunological profile and its parameters change in accordance with the training load. The measured levels of nAbs to GABA and glutamate correlate the physical activity of a person. The surveyed football players were divided into 3 groups according to the results of the analysis. Subjects of the first group had reliably high immunological indices in comparison with the control and were at the peak of physical form; in the third group, low immunological indices relative to the control indicated exhaustion and fatigue. The indicators of the second group corresponded to normal and demonstrated the resource of adaptation to load. The developed method can be used for assessing person's readiness for physical activity.

[The prevalence of autoimmune endocrine diseases in vitiligo patients].

Vitiligo is a common polygenic autoimmune disease in which the foci of depigmentation are formed on the skin and/or mucous membranes as a result of the death of melanocytes. There are several hypotheses for the pathogenesis of the disease, the leading role among them is played the autoimmune hypothesis. This review summarizes the available literature data on the prevalence and structure of comorbid endocrine autoimmune pathology in vitiligo patients. In most studies conducted in Europe, America and Asia the prevalence of autoimmune thyroid diseases (including autoimmune thyroiditis and Graves disease), diabetes mellitus and autoimmune adrenal insufficiency was higher in vitiligo patients than in the general population. The results of some studies indicate a frequent association of vitiligo with autoimmune polyglandular syndromes. In the structure of comorbid pathology the highest prevalence was in autoimmune thyroid diseases. A number of studies have established a higher prevalence of autoimmune endocrine diseases in women, as well as in nonsegmental vitiligo patients and in cases of family history of vitiligo and/or other autoimmune diseases. In addition, it was shown that the prevalence of endocrine diseases increases with increasing area of depigmentation. The data obtained justify the advisability of conducting a timely examination of vitiligo patients with the aim of early detection of comorbid diseases and the appointment of appropriate treatment. Further studies are needed to investigate the effect of the identified associations on the course of vitiligo and comorbid endocrinopathies, as well as the effectiveness of therapy and the quality of life of patients.

Mechanisms of Leptin and Ghrelin Action on Maturation and Functions of Dendritic Cells.

Molecular mechanisms of the immunomodulatory effects of leptin and ghrelin in concentrations typical for pregnancy on the maturation and functional activity of dendritic cells (DCs) generated from the peripheral blood monocytes of women are investigated. The presence of leptin during DC maturation did not affect the levels of CD83+CD1c+, CD86+CD1c+, and HLA-DR+CD1c+ DCs, but increased the amount and the activity of the enzyme indoleamine 2,3-dioxygenase (IDO). Cell culturing in the presence of ghrelin or combination of leptin and ghrelin reduced the percentage of CD86+CD1c+ DCs but did not affect the levels of CD83+CD1c+ and HLA-DR+CD1c+ DCs. In addition, ghrelin reduced the number of IDO molecules without affecting its activity. Simultaneous presence of leptin and ghrelin increased induced IDO activity without affecting the amount of the enzyme in DCs. The effects of leptin and ghrelin on the investigated functions of DCs in some cases correlated with high levels of cAMP. New mechanisms for leptin and ghrelin regulation of tolerogenic functions of DCs in pregnancy are proposed.

Pilot GWAS of caries in African-Americans shows genetic heterogeneity.

BACKGROUND: Dental caries is the most common chronic disease in the US and disproportionately affects racial/ethnic minorities. Caries is heritable, and though genetic heterogeneity exists between ancestries for a substantial portion of loci associated with complex disease, a genome-wide association study (GWAS) of caries specifically in African Americans has not been performed previously. METHODS: We performed exploratory GWAS of dental caries in 109 African American adults (age > 18) and 96 children (age 3-12) from the Center for Oral Health Research in Appalachia (COHRA1 cohort). Caries phenotypes (DMFS, DMFT, dft, and dfs indices) assessed by dental exams were tested for association with 5 million genotyped or imputed single nucleotide polymorphisms (SNPs), separately in the two age groups. The GWAS was performed using linear regression with adjustment for age, sex, and two principal components of ancestry. A maximum of 1 million adaptive permutations were run to determine empirical significance. RESULTS: No loci met the threshold for genome-wide significance, though some of the strongest signals were near genes previously implicated in caries such as antimicrobial peptide DEFB1 (rs2515501; p = 4.54 × 10- 6) and TUFT1 (rs11805632; p = 5.15 × 10- 6). Effect estimates of lead SNPs at suggestive loci were compared between African Americans and Caucasians (adults N = 918; children N = 983). Significant (p < 5 × 10- 8) genetic heterogeneity for caries risk was found between racial groups for 50% of the suggestive loci in children, and 12-18% of the suggestive loci in adults. CONCLUSIONS: The genetic heterogeneity results suggest that there may be differences in the contributions of genetic variants to caries across racial groups, and highlight the critical need for the inclusion of minorities in subsequent and larger genetic studies of caries in order to meet the goals of precision medicine and to reduce oral health disparities.

Effect of Estriol, Chorionic Gonadotropin, and Oncostatin M on the Expression of Recombinase RAG-1 in Regulatory T Lymphocyte Subpopulations.

We studied the effect of estriol, chorionic gonadotropin, oncostatin M, and hormone-cytokine combinations on the expression of recombinase RAG-1 in T-regulatory (Treg) and T helper 17 (Th17) lymphocytes. It was found that estriol in a concentration corresponding to the first trimester of pregnancy increased the level of Treg (CD4+FoxP3+) cells and suppressed the formation of Th17 (CD4+RORC+) lymphocytes. This effect was nor observed after individual administration of chorionic gonadotropin and oncostatin M, but some combinations of the studied hormones with oncostatin M increased the percentage of CD4+FOXP3+ cells. In the presence of oncostatin M, the studied hormones enhanced the expression of RAG-1 in CD4+FoxP3+ cells, but not in CD4+RORC+ cells, thereby initiating of Treg T-cell receptor (TCR) revision. The mechanisms of hormone cytokine control of induction of the immune tolerance during pregnancy are discussed.

Regulation of Recombinase Rag-1 Expression by Female Sex Steroids in Treg and Th17 Lymphocytes: Role of Oncostatin M.

The effect of estradiol (E2), progesterone (P4), and oncostatin M (OSM) on the differentiation of CD4+ T cells to T regulatory (Treg) lymphocytes and T helpers 17 (Th17) was investigated. The possibility of revision of the T cell receptor in these subpopulations by evaluating the expression of RAG-1 recombinase was also studied. E2 at concentrations characteristic of pregnancy trimester I, but no P4 or OSM, increased the Treg level. Combination of sex steroids with OSM increased the percent of CD4+FOXP3+ cells and enhanced RAG-1 expression in these cells, thus promoting the development of immune tolerance during pregnancy. In the study of Th17, such effect of the hormones and OSM was not detected.

Hormonal Regulation of Dendritic Cell Differentiation in the Thymus.

We studied the effect of hormones estriol, ghrelin, kisspeptin, and chorionic gonadotropin in concentrations corresponding to their content in the peripheral blood in each trimester of pregnancy on the expression of membrane molecules on myeloid and plasmacytoid dendritic cells of the thymus. It was found that thymic myeloid dendritic cells are sensitive to the action of estriol and kisspeptin. Estriol in a concentration of the first trimester of pregnancy reduces the number of myeloid dendritic cells expressing receptor for thymic stromal lymphopoietin (CD11c+TSLP-R+) and inhibitory molecule B7-H3 (CD11c+CD276+). In contrast to estriol, kisspeptin regulates the processes of differentiation of thymic myeloid dendritic cells in concentrations typical of the second-third trimesters and reduced their total number (CD11c+) and the number of cells expressing TSLP-R (CD11c+TSLP-R+). Estriol and kisspeptin do not affect the total number of plasmacytoid dendritic cells (CD303+) and expression of TSLP-R and CD276 by these cells. Ghrelin and chorionic gonadotropin in the studied concentrations had no significant effect on the total number of thymic myeloid and plasmacytoid dendritic cells and on the expression of membrane molecules of TSLP-R and CD276.

[The Activities of Representatives of Companies-Manufacturers of Pharmaceuticals Related to Informing Specialists with Purpose of Efficient Application of Medications].

The article presents the role of representative of company-manufacturer of pharmaceuticals in informing health care specialists about medications according WHO documentation. The analysis of regulation of procedure and permitted modes of their interaction with specialists in the Russian Federation. Also, an opinion of medical specialists is presented concerning the role of representatives of pharmaceutical companies in the portion of informing about medications. The results of questionnaire survey of medical representatives from the position of their role and tasks are presented concerning information activity during interaction with specialists. The proposals are formulated concerning necessity of legislative regulation of activities of representatives of the companies-manufacturers of pharmaceuticals.

Role of PKA and PI3K in Leptin and Ghrelin Regulation of Adaptive Subpopulations of Regulatory CD4+ T-Lymphocyte Formation.

The role of phosphatidylinositol-3 kinase (PI3K) and protein kinase A (PKA) in leptin and ghrelin regulation of formation of adaptive (a) subpopulations of CD4+ T-lymphocytes (helper (h) cells producing interleukin-17A) (aTÒ»17) and of T-regulatory lymphocytes (aTreg) in the context of physiological pregnancy is established. It is shown that leptin at a concentration typical for the second half of pregnancy (trimesters II-III) enhances the differentiation of aTÒ»17 with a high level of interleukin-17A (IL-17A) production and the expression of the chemokine receptor CCR6 with the participation of PI3K. Simultaneously, leptin reduces formation of aTreg expressing the suppressor molecule CTLA-4, which determines the function of these cells. Ghrelin at a concentration characteristic of the first half of pregnancy (trimesters I-II), in contrast, enhances aTreg formation and, in parallel, reduces the level aTÒ»17 (that express CCR6) and the IL-17A production by aTh17. PKA, likewise PI3K, participates in regulatory effects of ghrelin on the formation of aTÒ»17 and aTreg. The combined action of leptin and ghrelin (via PKA participation) enhances formation of only aTreg, which determines the priority of this molecular mechanism and explains why the investigated hormones with reciprocal differentiating potential do not come into antagonism at the level of immune system cells during pregnancy.

Apoptotic Endonuclease EndoG Inhibits Telomerase Activity and Induces Malignant Transformation of Human CD4+ T Cells.

Telomerase activity is regulated by an alternative splicing of mRNA of the telomerase catalytic subunit hTERT (human telomerase reverse transcriptase). Increased expression of the inactive spliced hTERT results in inhibition of telomerase activity. Little is known about the mechanism of hTERT mRNA alternative splicing. This study was aimed at determining the effect of an apoptotic endonuclease G (EndoG) on alternative splicing of hTERT and telomerase activity in CD4+ human T lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of the active full-length hTERT variant and upregulated the inactive alternatively spliced variant. Reduction of full-length hTERT levels caused downregulation of the telomerase activity, critical telomere shortening during cell division that converted cells into the replicative senescence state, activation of apoptosis, and finally cell death. Some cells survive and undergo a malignant transformation. Transformed cells feature increased telomerase activity and proliferative potential compared to the original CD4+ T cells. These cells have phenotype of T lymphoblastic leukemia cells and can form tumors and cause death in experimental mice.