Interaction of Neutrophils and Biofilm Formed by Uropathogenic Escherichia coli Strains with Different Pathogenic Potential.

The biofilm formation by uropathogenic E. coli (UPEC) allows bacteria to avoid the influence of the host immune system that determines the pathogenesis of persistent urinary tract infections. The purpose of this work was to evaluate the mutual influence of neutrophils and biofilms formed by UPEC with different set of virulence-associated genes (VAGs). E. coli R11 and R32 strains with a wide range of virulence factors were characterized by low biofilm biomass that did not change after interaction with neutrophils. The biomass index decreased after interaction with neutrophils for strains with a limited set of pathogenicity factors (R33, R36, R45, and R44) and a "thick" biofilm. Bacterial cells and biofilm supernatants of all UPEC strains reduced viability (DiOC6(3)+/PI-) and stimulated early apoptosis (DiOC6(3)-/PI-) of neutrophils. The number of viable neutrophils was higher, while the number of apoptotic and necrotic (DiOC6(3)-/PI+) cells was lower under the action of supernatants of strains R44, R36, R45 in comparison with bacterial cells. Thus, modulation of the innate cell functions depends on the realization of the pathogenic potential of UPEC bacteria in urinary tract biofilms that determines the development of recurrent urinary tract infections.

[Packaging of Subchromosomal-Size DNA Molecules in Chromatin Bodies in the Ciliate Macronucleus].

A fundamental difference between somatic nuclei (macronuclei) of ciliates and cell nuclei of higher eukaryotes is that the macronuclear genome is a huge number (up to tens or hundreds of thousands) of gene-sized (0.5-25 kb) or subchromosomal (up to 2000 kb) minichromosomes. Electron microscopy shows that macronuclear chromatin usually looks like chromatin bodies or fibrils 200-300 nm thick in the interphase. However, the question of how many DNA molecules are contained in an individual chromatin body remains open. The organization of chromatin in macronuclei was studied in the ciliates Didinium nasutum and three Paramecium sp, which differ in pulsed-field gel electrophoresis (PFGE) karyotype, and compared with the model of topologically associated domains (TADs) of higher eukaryotic nuclei. PFGE showed that the sizes of macronuclear DNAs ranged from 50 to 1700 kb, while the majority of the molecules were less than 500 kb in length. A comparative quantitative analysis of the PFGE and electron microscopic data showed that each chromatin body contained one minichromosome in P. multimicronucleatum in the logarithmic growth phase, while bodies in the D. nasutum macronucleus contained two or more DNA molecules each. Chromatin bodies aggregated during starvation, when activity of the macronuclei decreased, leading to an increase of chromatin body size or the formation of 200- to 300-nm fibrils of several chromatin bodies. A model was proposed to explain the formation of such structures. In terms of topological characteristics, macronuclear chromatin bodies with subchromosomal DNA molecules were found to correspond to higher eukaryotic TADs.

[Relapsing-remitting multiple sclerosis: clinical and immunological aspects of the pathology on the example of molecules Sema4D and CD72]. / Remittiruyushchii rasseyannyi skleroz: kliniko-immunologicheskie aspekty patologii na primere molekul Sema4D i CD72.

OBJECTIVE: To study the expression of Sema4D (CD100), receptor CD72 and a role of Sema4D-CD72-dependent signal in the control of the functions of immunocompetent cells in relapsing-remitting multiple sclerosis (RRMS). MATERIAL AND METHODS: We studied 76 patients, including 52 with RRMS (41 in remission and 11 in exacerbation), 35 women (67.3%) and 17 men (32.7%) aged 18-55 years, who did not receive disease-modifying drugs, and 24 healthy donors. A controlled clinical and immunological examination of patients with RRMS was carried out proving the involvement of the Sema4D molecule and its CD72 receptor in pathological reactions in this autoimmune disease. RESULTS AND CONCLUSION: The use of SemaD as a target in the treatment of RRMS is scientifically substantiated. In case of a positive decision on the use of anti-Sema4D drugs, it will be necessary to take into account the effects of semaphorin not only in the central nervous system, but also in the immune system of patients with RRMS.

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.

Molecular Mechanisms of Control of Differentiation of Regulatory T-Lymphocytes by Exogenous Melatonin.

We investigated the role of epiphyseal hormone melatonin in the differentiation of naive CD4+ T cells into regulatory T cells (Treg). The hormone at physiological and pharmacological concentrations inhibited Treg differentiation, decreasing both the proportion of CD4+FOXP3+ cells in the culture and the level of TGF-ß, the key cytokine for this T cell subpopulation. The inhibitory effect of exogenous melatonin was due to its interaction with the membrane receptors MT1 and MT2. At the same time, the signals realized through RORα-the nuclear receptor for melatonin-stimulated Treg formation; however, they were considerably weaker than the signals from the membrane receptors and were overlapped by the latter. Since the Treg subpopulation plays an important role in physiological and pathological processes in the body, the revealed effects of melatonin should be taken into account in its therapeutic use.

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.

[Relapsing-remitting multiple sclerosis: clinical and immunological aspects of the pathology on the example Sema4D and CD72]. / Remittiruiushchii rasseiannyi skleroz: kliniko-immunologicheskie aspekty patologii na primere molekul Sema4D i CD72.

AIM: To explore the expression of Sema4D, CD72 receptor and a role of Sema4D-CD72 signal in the control of immunocompetent cell function in remitting-relapsing multiple sclerosis (RRMS). MATERIAL AND METHODS: Fifty-two patients with RRMS diagnosis according to 2010 revised McDonald's criteria were studied. The control group included 24 healthy people. A flow cytometry method was used to measure the expression of semaphorin Sema4D by T-lymphocytes of peripheral blood, expression of CD72 receptor by B-lymphocytes, percentage of cells containing pro- and anti-inflammatory cytokines. The level of soluble Sema4D (sSema4D) was evaluated by ELISA. RESULTS: The level of Sema4D expression on T-lymphocytes (Mean Fluorescence Intensity - MFI) prevailed in cell subpopulations in patients with RRMS compared with the control group. Characteristics of membrane and sSema4D correlate with clinical presentations of the autoimmune disease. An increase in sSema4D level during cell cultivation was identified in RRMS patients. The results show the involvement of Sema4D in the hyperactivation of B-cell-mediated immunity through CD72 receptor and induction of proinflammatory cytokine synthesis. CONCLUSION: RRMS is associated with elevated expression of Sema4D in the immune system. Membrane and sSema4D involved in the pathological process in RRMS. The authors suggest several mechanisms of the involvement of semaphorin and its receptor in the pathogenesis of RRMS: the direct damage of nervous tissues by sSema4D penetrated through the blood brain barrier disrupted in RRMS or by membrane Sema4D due to the infiltration of the central nervous system by T-lymphocytes and hyperactivation of B-cell-mediated immunity.

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.

Estimation of the bacteriocin ColE7 conjugation-based "kill" - "anti-kill" antimicrobial system by real-time PCR, fluorescence staining and bioluminescence assays.

The efficiency of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system, was assessed using real-time PCR, flow cytometry and bioluminescence. The ColE7 antimicrobial system consists of the genetically modified Escherichia coli strain Nissle 1917 harbouring a conjugative plasmid (derivative of the F-plasmid) encoding the "kill" gene (ColE7 activity gene) and a chromosomally encoded "anti-kill" gene (ColE7 immunity gene). On the basis of traJ gene expression in the killer donor cells, our results showed that the efficiency of the here studied antimicrobial system against target E. coli was higher at 4 than at 24 h. Flow cytometry was used to indirectly estimate DNase activity of the antimicrobial system, as lysis of target E. coli cells in the conjugative mixture with the killer donor strain led to reduction in cell cytosol fluorescence. According to a lux assay, E. coli TG1 (pXen lux+ Apr ) with constitutive luminescence were killed already after 2 h of treatment. Target sensor E. coli C600 with DNA damage SOS-inducible luminescence showed significantly lower SOS induction 6 and 24 h following treatment with the killer donor strain. Our results thus showed that bioluminescent techniques are quick and suitable for estimation of the ColE7 bacterial conjugation-based antimicrobial system antibacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial antimicrobial resistance is worldwide rising and causing deaths of thousands of patients infected with multi-drug resistant bacterial strains. In addition, there is a lack of efficient alternative antimicrobial agents. The significance of our research is the use of a number of methods (real-time PCR, flow cytometry and bioluminescence-based technique) to assess the antibacterial activity of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system. Bioluminescent techniques proved to be rapid and suitable for estimation of antibacterial activity of ColE7 bacterial conjugation-based antimicrobial system and possibly other related systems.

Role of Endogenous Melatonin in the Regulation of Th17/Treg Balance during Pregnancy.

We studied the role of endogenous melatonin in the development and functioning of T cells that produce IL-17 (Th17) and regulatory T cells (Treg) during pregnancy. The study was performed ex vivo and in vitro with auto-serum as the source of endogenous melatonin under conditions of blockade of melatonin-dependent signaling. Participation of the hormone in the regulation of differentiation of both CD4+RORγt+ and CD4+FoxP3+T cells and their key products IL-17A and TGF-ß was demonstrated. It is known that the normal gestational process is accompanied by a decrease in Th17/Treg ratio due to hormonal changes. The sensitivity of the studied subpopulations to melatonin during pregnancy can affect its outcome.

[The course and development of epilepsy in patients with typical variant of Rett syndrome and mutations]. / Osobennosti épilepsii u detei s tipichnym variantom sindroma Retta, vyzvannym mutatsiei.

AIM: To study the anamnesis, clinical state, electro-encephalographic and brain MRI characteristics in patients with Rett syndrome (МЕСР2) and epilepsy. MATERIAL AND METHODS: Eleven female patients, aged from 3 to 23 years, with Rett syndrome and MeCP2 mutations were studied. The study continued for 10 years (2006-2015). Assessment of neurological and mental status, night sleep video-EEG monitoring, MRI were performed. RESULTS AND CONCLUSION: Epilepsy was diagnosed in six cases (54.5%). Mean age at onset of epileptic seizures was 3 years 9 month. The following types of seizures were described: generalized, myoclonic, myotonic, tonic, versive, focal motor, atypical absences. Status epilepticus developed in one patient. Generalized seizures were identified in 56.25%, focal seizures in 43.75%. EEG changes were found in 9 patients (81.8%): slowing of the activity, episodes of periodic regional slowing, regional epileptiform activity and diffuse epileptiform activity, benign focal epileptiform discharges (BFED) of childhood, multiregional epileptiform activity. Five patients were treated with antiepileptic drugs. All of them had improved during treatment: a reduction of frequency of seizures was up to 50% in 4 cases (80%). One patient with resistant epilepsy was treated with the combination of drugs (levetiracetam, topiramate, zonisamide, benzodiazepine) that led to stopping of seizures during night sleep and decrease in the frequency of daytime seizures by 50%. Further research of epilepsy and efficacy of antiepileptic drugs in Rett syndrome is required.

Involvement of Semaphorin (Sema4D) in T-Dependent Activation of B Cells.

The involvement of endogenous semaphorin (Sema4D) into the key stage of T-dependent differentiation of B cells, formation of plasmoblasts, was demonstrated in vitro in T/B cell co-culture under conditions of polyclonal activation of T cells. The effect of semaphorin was not associated with activation of high-affinity Sema4D receptor plexin B1, but involves lowaffinity receptor CD72. These data indicate that Sema4D-dependent signal regulates not only the initial stage of B-cell activation, proliferative response to the antigen, but also further differentiation of B cells into plasma cells.

[Differential diagnosis of paroxysms of tonic muscle tension in children of early age with delay of psychomotor development and abnormal neurologic status]. / Differentsial'naia diagnostika paroksizmov tonicheskogo napriazheniia myshts épilepticheskoi i neépilepticheskoi prirody u detei rannego vozrasta s zaderzhkoi psikhomotornogo razvitiia i ochagovymi nevrologicheskimi simptomami.

AIM: To study neurologic status, results of video-EEG monitoring and magnetic resonance imaging in children under 3 years old with paroxysms of tonic muscle tension. MATERIAL AND METHODS: One hundred and forty-six infants and young children with motor disturbances and different variants of clinically similar epileptic seizures, hyperkinesis and stereotypes were examined. RESULTS AND CONCLUSION: Cerebral palsy (91%), genetic and chromosomal abnormalities (6%), brain malformations (2%) were identified. Neurological status was characterized by pseudobulbar syndrome (100% of cases), hemiparesis (1%), tetraparesis (81%), diffuse muscular hypotonia (18%), intellectual and speech development delay (76%), autistic behavior (16%). During the prolong video-EEG monitoring, paroxysmal tonic muscle tensions were recorded in all patients: epileptic seizures were observed in 113 patients (77.40%), non-epileptic paroxysms in 51 (34.93%). The combination of epileptic and non-epileptic paroxysms was observed in 18 patients (12.33%). In 4 patients (2.75%), it was not possible to determine the genesis of paroxysms even during the prolong video-EEG-monitoring because of myographic artefacts. Five clinical and electroencephalographic combinations of dystonic attacks, epileptic seizures and epileptiform activity were identified. These data allow improving the diagnosis of epilepsy and avoiding unnecessary treatment with antiepileptic drugs. Our study has shown a high diagnostic value of video-EEG monitoring with the inclusion of sleep in patients with paroxysmal conditions in infancy and early childhood.

MicroRNA in hormonal mechanisms of regulation of NK cell function.

We investigated the effect of human chorionic gonadotropin, estriol, leptin, ghrelin, and kisspeptin on the microRNA expression in separated NK cells. All of these hormones are able to effectively modulate the expression of microRNAs both stimulating and suppressing the cytotoxic potential of NK cells and thereby indirectly regulate the functions of these lymphocytes.

New aspects of the Seam4D-dependent control of lymphocyte activation.

Novel targets for action of the class IV semaphorin Seam4D have been identified in the immune system. The low-affinity CD72 receptor for Seam4D was detected not only on B lymphocytes, but also in a proportion of T cells, whereas the high-affinity semaphorin receptor, plexin B1, originally considered to belong to non-immune cells, proved to be in a great proportion of intact T and B cells. Seam4D is constitutively expressed in B cells, which, along with T cells, can serve as a source of both membrane and soluble semaphorin. The results obtained make significant adjustments in understanding of Seam4D effects in lymphoid cells.

Hormonal regulation of NK cell cytotoxic activity.

The effects of chorionic gonadotropin, estriol (E3), leptin, ghrelin, and kisspeptin on the intracellular expression of perforin, granzyme A, and granzyme B was studied in separated NK cells. All studied hormones except E3 are could modulate the expression of cytotoxic enzymes in NK cells by suppression of the expression of the most active proapoptotic agents, resulting in increased expression of granzyme A, which is typical of the decidual subpopulation of these lymphocytes.

[The course and the development of epilepsy in patients with typical variant of Rett syndrome and mutations]. / Osobennosti techeniia i razvitiia épilepsii u detei s tipichnym variantom sindroma Retta, vyzvannogo mutatsiei.

AIM: Studying data of anamnesis, clinical state, electro-encephalographic, brain MRI in patients with Rett syndrome (МЕСР2). MATERIAL AND METHODS: We studied 11 patients (female) from three to 23 years old with Rett syndrome and MeCP2 mutations. Observation continued 10 years (2006-2015). We analyzed the results of the neurological status, night sleep video-EEG monitoring, MRI. RESULTS AND CONCLUSION: Epilepsy diagnosed in six cases (54, 5%). The overage age of debut of epileptic seizures was 3 years 9 months. There are some types of seizures: generalized, myoclonic, myotonic, tonic, versive, focal motor, atypical absences. Status epilepticus evolved in one patient. Generalized seizures were 56, 25%, focal seizures - 43, 75%. EEG changing marked in nine patients (81, 8%): slowdown back activity, episodes of periodic regional slowdown, regional epileptiform activity, and diffuse epileptiform activity like benign focal epileptiform discharges (BFED). five patients took antiepileptic drugs. All of them had improved during treatment. There were reducing of frequency of the seizures up 50% - 4 cases (80%). one patients with resistant epilepsy was taken combination of drugs (levetirecetam, topiromat, zonisamide, benzodiazepine) with stopping of seizures in the night sleep and decreasing of frequency of daytime seizures to 50%. We believe there is very important of study epilepsy in patients with Rett syndrome and improvement of its treatment.

Roles of leptin and ghrelin in the regulation of the phenotype and cytokine production by NK cells from peripheral blood.

Both leptin and ghrelin used separately at the concentrations corresponding to trimesters II-III of pregnancy increase the number of CD56bright NK cells in mononuclear cell suspension; their combination also enhances the L-selectin expression on the surface of these cells in the culture. These hormones do not affect the production of TGF-ß1, IL-17А, or IFN-γ by NK cells, and they inhibit the production of IL-10. Leptin decreses the IL-4 production by NKp46+ cells, but the presence of ghrelin abrogates this effect.

Role of Melatonin in the Regulation of Differentiation of T Cells Producing Interleukin-17 (Th17).

We studied the ability of melatonin in physiological and pharmacological concentrations to induce and/or regulate differentiation of T cells producing IL-17 (Th17). This hormone produced the opposite effect on CD4+T cells, which depended on their activation status. Melatonin induced the synthesis of IL-17A by intact T cells, but had little effect on activated cells. Melatonin in high (pharmacological) concentration decreased the intracellular expression of this cytokine under conditions of polyclonal activation. Melatonin had a dose-depended effect. Taking into the fact that Th17 cells play an important role in the immune defense, it can be suggested that the regulation of their activity by melatonin contributes to this process.

[The Influence of Chorionic Gonadotropin and Estriol on NK Cells Phenotype and Functional Activity.]

The influence of chorionic gonadotropin (CG) and estriol (E3) at concentrations typical of pregnancy on the expression of phenotypic markers and cytokine production by separated NK cells were studied. It is found that these hormones increase the percentage of CD56brightL-selectin+ NK-cells, but also stimulate the expression of the inhibitory molecule NKG2A in the lymphocytes. In addition, E3 and CG stimulate the production of TGF-B, inhibiting the secretion of all other cytokines by separated NK cells. In general, these hor- mones contribute to the formation of the phenotype and cytokine spectrum characteristic of the regulatory NK3 subpopulation of NK cells during pregnancy.