Bulletin of RSMU

ISSN Print 2500–1094 ISSN Online 2542–1204

Bulletin of RSMU

BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)

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Shubina J, Jankevic T, Goltsov AYu, Mukosey IS, Kochetkova TO, Bystritsky AA, Barkov IYu, Tetruashvili NK, Kim LV, Trofimov DYu

Introduced into clinical practice in 2011, non-invasive prenatal testing (NIPT) allows detection of chromosomal aneuploidies in the fetus using maternal blood samples. Multiple studies have shown that one of the key factors affecting the result of this test is the fetal DNA fraction. The aim of this work was to develop a method capable of measuring the fetal DNA fraction based on targeted SNP sequencing. We selected polymorphisms with high frequency of heterozygous genotype from the international HapMap database. To estimate the frequency of these polymorphisms in the Russian population, we used 827 DNA donor samples. Fetal DNA fraction was measured in 87 plasma samples of pregnant women. Sequencing was performed on Ion Proton and Ion S5. We determined the frequencies of the studied polymorphisms in the pooled samples and compared the data on 53 SNPs in the pooled and 87 individual samples. The median difference was 3.4%. The correlation between the results obtained by targeted SNP sequencing and Y chromosome read count was 0.7. Thus, the proposed method can be used to estimate the fetal DNA fraction using SNP genotyping regardless of the fetus’s sex.

Received: 2018-04-15

Published online: 2018-08-11

DOI: 10.24075/brsmu.2018.031

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VIEWS 4439

Ivashchenko AV, Fedyaev IM, Yablokov AE, Kolganov IN, Balandin EI, Tlustenko VP

One of the alternatives to permanent tooth removal is intentional reimplantation. A tooth saved by reimplantation can later serve as a support for various types of dental prostheses. Tooth replantation is indicated if there is an infection in the periapical area unresponsive to conservative treatment or apicoectomy is not possible, etc. The female patient S. presented with missing teeth and significant coronal decay both on the upper and lower jaws. Three months after the teeth had been extracted and reimplanted, the patient underwent another panoramic radiography scan. On the post-op orthopantomogram the dental cement appeared evenly distributed in the root canals and the trabecular bone tissue was rebuilding in the periapical area of the replanted teeth. Later, we installed fixed dental porcelain-fused-to-metal prostheses supported by the replanted teeth. The patient also received removable dentures. A 2-year follow-up showed no signs of pathologic mobility in the replanted teeth, robust trabecular tissue regeneration in the periapical area and stability of dental prostheses supported by the replanted teeth.

Received: 2018-04-04

Published online: 2018-08-02

DOI: 10.24075/brsmu.2018.030

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VIEWS 4357

Shakhanov AV, Uryasev OM

Nitric oxide has a significant role in the pathogenesis of bronchial asthma and hypertension. Its synthesis is catalyzed by NO synthases. The nucleotide composition of genes coding for these enzymes can affect their activity; therefore, it is important to understand the effect of the NOS3 786C/T polymorphism (rs2070744) on the blood levels of nitric oxide in patients with bronchial asthma and hypertension. Our study recruited 71 individuals. The main group consisted of 24 asthmatic hypertensive patients. Two comparison groups included patients with isolated asthma and isolated hypertension. All patients were genotyped for the NOS3 786C/T polymorphism. We measured total nitric oxide metabolites in their blood using a photocolorimetric technique and the Griess reagent. The levels of nitric oxide in the exhaled air were determined electrochemically using a portable NObreath monitor. The blood levels of nitric oxide metabolites amounted to 69.7 (60.0; 70.4) μmol/l in the CC genotype carriers, 68.9 (57.7; 77.4) μmol/l in the CT genotype carriers and 67.7 (59.7; 79.3) μmol/l in the patients with the TT genotype (p = 0.843). Individually, the groups demonstrated a clear association between the NOS3 786C/T polymorphism and the blood levels of nitric oxide metabolites. The patients with bronchial asthma and hypertension demonstrated a tendency to increasing nitric oxide levels following the pattern CC < CT < TT (p = 0.033 and p = 0.024, respectively). Thus, the C allele of the NOS3 786C/T polymorphism is associated with lower blood levels of nitric oxide metabolites in patients with bronchial asthma and hypertension.

Received: 2018-04-20

Published online: 2018-07-29

DOI: 10.24075/brsmu.2018.029

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VIEWS 4603

Smelova IV, Golovneva ES

Hypothyroidism remains a pressing concern. Laser irradiation is a widely used treatment option for patients with thyroid pathologies. Its efficacy depends on the applied dose. Changes in the form and volume of the structural components of the glands, such as thyrocytes and follicles, are dose-dependent and signal their functional state, which affects production, accumulation and secretion of thyroid hormones. The aim of our study was to explore the effect of infrared medium-power laser with total energy densities of 112 J/cm2 and 450 J/cm2 on the morphology and function of the thyroid and its follicles in health and hypothyroidism. The experiment was conducted in male rats. It was demonstrated that laser radiation affects the morphological state of thyrocytes and follicles of both intact animals and animals with experimentally induced hypothyroidism. Comparison of two laser regimens revealed that 112 J/cm2 energies stimulated tissue regeneration and thyroid activity in general, whereas 450 J/cm2 energies suppressed those processes. Our findings can be used to study hypothyroidism treatment options in the experimental setting.

Received: 2018-02-15

Published online: 2018-07-28

DOI: 10.24075/brsmu.2018.028

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VIEWS 4622

Zhulai GА, Churov AV, Oleinik EK, Romanov AA, Semakova PN, Oleinik VM

Pathogenesis of colorectal cancer (CRC) is accompanied by significant changes in the immune system. However, the role of the adenosine-A2AR-mediated immunosuppressive pathway in oncogenesis and more specifically, the expression of ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1, also known as CD39) remains unclear. The aim of this work was to study the role of СD4+ Т cells, most importantly CD39-expressing regulatory T cells (Tregs) in the formation of immune suppression in CRC and in patients with acute pancreatitis (AP). Expression of CD39 by peripheral blood lymphocytes and tumor-infiltrating lymphocytes (TILs) was measured by flow cytometry. The levels of CD39 messenger RNA (mRNA) in the peripheral blood leukocytes were determined by real-time PCR. Our study reveals that patients with CRC accumulate peripheral CD4+CD39+ cells in the advanced stages of the disease. The proportion of CD39-expressing CD4+ Т cells in the total pool of TILs was higher than in the peripheral blood of the same patients. Tregs of both peripheral blood and tumor specimens of CRC patients showed increased CD39 expression. We have found reliable correlations between the levels of CD4+CD39+ T cells and the parameters of cell-mediated immunity in CRC patients. Also, CD39 mRNA levels gradually increased during CRC progression. In contrast, patients with AP have the same levels of CD39 mRNA and peripheral blood CD4+CD39+ Т cells as the controls. Finally, we conclude that activation of CD4+CD39+ Т cells has an important role in oncogenesis and needs to be studied further.

Received: 2018-01-25

Published online: 2018-07-25

DOI: 10.24075/brsmu.2018.027

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VIEWS 5078

Zheltukhin AO, Soboleva AV, Sosnovtseva AO, Le TH, Ilyinskaya GV, Kochetkov DV, Lipatova AV, Chumakov PM

Stem cells that penetrated deeply into the brain tissue are the main reason behind the relapses of glioblastoma multiforme after surgery. Finding new approaches to counter such relapses, including those that make use of oncolytic viruses, is a pressing issue. This study aimed to determine the sensitivity of cells of human glioblastoma multiforme to non-pathogenic enteroviruses, in vitro and in vivo (mice xenografts model). Glioblastoma tumor cells were exposed to type 1 poliovirus (Sabin vaccine strain), Coxsackie virus A7 (strain LEV8), Coxsackie virus A9 (strain LEV9) and Coxsackie virus B5 (strain LEV14). The virus reproduction intensity and cytolytic activity were assessed through infection of monolayered glioblastoma cell cultures. The ability of glialoblastoma cell cultures (enriched with tumor stem cells) to build subcutaneous tumors in immunodeficient mice after those cultures were exposed to viruses signaled the effectiveness of glioblastoma stem cells destruction. The study revealed that Coxsackie virus A7 and type 1 poliovirus possess the most pronounced oncolytic and replicative properties when tested on gliblastoma cells infected with viruses in vitro and on subcutaneous tumor xenografts in immunodeficient mice (in vivo). Type 1 poliovirus and Coxsackie virus A7 virus prevented development of tumors when glioblastoma neurospheric cell cultures were preincubated with viruses before subcutaneous implantation. Coxsackie virus B5 only managed to reduce the number of tumors developed, and Coxsackie virus A9 did not affect the tumor development at all. Thus, a number of non-pathogenic enteroviruses strains can destroy glioblastoma's stem cells, i.e. they show promise in the context of development of therapeutic agents for relapse-free treatment of glioblastomas. Ключевые

Received: 2018-06-26

Published online: 2018-07-13

DOI: 10.24075/brsmu.2018.026

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VIEWS 5161

Soboleva AV, Lipatova AV, Kochetkov DV, Chumakov PM

Existing therapies for glioblastoma multiforme do not ensure patient’s recovery. Oncolytic viruses (OV) represent a promising alternative as they can destroy glioblastoma-initiating stem cells, which the major cause of relapses. However, while individual OV strains are effective for some patients, they could be ineffective for others. To achieve a predictable therapeutic effect, live tumor cells of the patient need to be tested for the sensitivity to different viruses. The aim of this study was to assess how the sensitivity of tumor cells to viruses changes with passaging in cell culture. Primary glioblastoma cell cultures were prepared from excised tumors. We compared sensitivity of cells to four non-pathogenic enteroviruses (type 1 poliovirus, Coxsackie virus A7, Echoviruses 1 and 12), for freshly-explanted primary tumor cell cultures and for those that had undergone 700 divisions during the passaging. The sensitivity was assessed based on the proportion of viable cells by the MTT assay 72 hours after the cells were inoculated with serial 10-fold dilutions of virus preparations. Cells isolated from the tumors of 3 patients exhibited varying sensitivity to the used viral strains. Differences in the lowest virus dose required for the successful infection of cell cultures were as high as 105. Passaging induced sensitivity shifts, such as increased or decreased sensitivity to individual viruses. Differences in the sensitivity correlated with the ability of the infected cells to produce the virus. Based on our findings, we conclude that the sensitivity of cancer cells to viruses should be tested at very early stages of passaging, preferably in primary cultures.

Received: 2018-06-16

Published online: 2018-07-08

DOI: 10.24075/brsmu.2018.025

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VIEWS 5139

Karakozova MV, Nazarov PA

Multidrug resistance pumps (MDR pumps) of bacteria confer protection against aggressive environmental factors. The genes coding for MDR pumps are thought to be variable. They belong to the group of the so-called contingency genes, i.e are necessary for bacterial adaptation to the changing environment. The aim of the present work was to establish how conserved are the sequences of genes coding for MDR pumps. We analyzed the sequences of AcrA, AcrB and TolC proteins of different Escherichia coli strains. Using sequence alignment tools, we demonstrated that strains originating in different countries and cultured in the labs for a long time are amazingly conserved in terms of AcrAB-TolC sequences. They resemble housekeeping genes, suggesting the involvement of the AcrAB-TolC pump into permanent “cleaning” of various biotic and abiotic agents.

Received: 2018-02-19

Published online: 2018-07-06

DOI: 10.24075/brsmu.2018.024

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VIEWS 5129