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Laboratory diagnostics as a basis for 5P medicine
стр.5-12
As public health systems are being modernized across the world, conventional medicine is undergoing a serious transformation and new medical models are emerging based on personalized, predictive, preventive, participatory, precision, mobile, and digital approaches. So far, there is no consensus in the literature and the medical community about the goals, objectives and applications of these models, particularly precision medicine, which is sometimes perceived as merely a fancier term for personalized medicine. The role of laboratory diagnostics in precision medicine is also a matter of intense debate. This review analyzes the currently available information about precision medicine and gives examples of how 5P approaches can be used in clinical practice.
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Assessment of diastolic function in patients with chest pain and angiographically normal coronary arteries using ECG-gated SPECT
стр.13-19
Khachirova EA, Samoylenko LE, Shevchenko OP
The diagnosis and treatment of patients with angiographically normal or near normal coronary arteries remains a clinically relevant problem. The aim of this study was to assess diastolic function in patients with chest pain and normal/near normal coronary arteries (NECA) using ECG-gated SPECT/CT. The study recruited 49 patients presenting with chest pain, a positive cardiac stress test and normal coronary arteries, as demonstrated by coronary angiography. All patients were ordered a myocardial SPECT/CT scan, which was performed according to a two-day protocol. After the scan, the patients were divided into 3 groups. Group 1 consisted of 17 patients with microvascular angina. Group 2 was composed of 22 patients with borderline-high blood pressure or stage I hypertensive heart disease associated with secondary microvascular dysfunction. Ten seemingly healthy individuals constituted the control group. According to coronary angiography, the controls had no cardiovascular pathologies accompanied by coronary artery disorders or impaired myocardial perfusion (SPECT). The majority of patients from groups 1 and 2 were found to have impaired diastolic function. The impairments were more pronounced in group 2 tended to exacerbate with stress. The most sensitive parameter of diastolic function, MFR/3, was outside the reference range in almost all patients in groups 1 and 2. MFR/3 characterizes the mean filling rate of the left ventricle in the first third of diastole. The control group showed no symptoms of diastolic dysfunction. Thus, the patients with chest pain, a positive stress test and NECA had signs of left ventricular diastolic dysfunction exacerbated with stress. Such patients are at risk for heart failure with preserved ejection fraction.
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Investigating a correlation between the levels of peripheral blood cytokines and the risk for cardiovascular complications in patients with stage II essential hypertension
стр.20-26
Radaeva OA, Simbirtsev AS
Essential hypertension (EH) is one of the most common modifiable risk factors for cardiovascular diseases and death. The aim of this study was to investigate a correlation between the levels of some cytokines (interleukins, adhesion molecules, tumor necrosis and growth factors, etc.) in the peripheral blood of patients with stage II EH and the rate of complications (myocardial infarction, acute cerebrovascular events, and transient ischemic attacks) occurring in a 5-year follow-up period. Twenty-eight cytokines were measured using ELISA, including IL1β, IL1α, IL1ra, IL18, IL18BP, IL37, IL6, sIL6r, LIF, sLIFr, IGF-1, IGFBP-1, TNFα, sTNF-RI, sVCAM-1, IL17, IL2, IL4, IL10, TGF-β1, IL8, CX3CL1, CXCL10, INFγ, M-CSF, IL34, VEGF-A, and erythropoietin, and a few vasoactive peptides, including NО, iNOS, eNOS, ADMA, SDMA, Nt-proСNP, and Nt-proBNP, in the peripheral blood samples of 200 patients with stage II EH who had been suffering from this condition for 10 to 14 years and were receiving comparable therapies to bring their blood pressure down. The patients were followed up for 5 years to keep track of complications. The retrospective analysis revealed that the group of patients who developed complications during the 5-year follow-up period exhibited a decline in the levels of IL1ra ( р < 0.001) and IL10 ( р < 0.001) and a rise in IL1β ( р < 0.001), TNFα ( р < 0.001) and M-CSF ( р < 0.001) in comparison with the group of those who did not develop any complications. The multivariate Cox regression analysis was applied to the following parameters: IL1β > 18.8 pg/ml; IL1ra < 511 pg/ml; IL6 > 23.8 pg/ml; IL10 < 26.3 pg/ml; 389 pg/ml < M-CSF < 453 pg/ml; ADMA > 0.86 µmol/L; total cholesterol > 4.9 mmol/L; LDL> 3.0 mmol/L; HDL in men < 1.0 mmol/L; HDL in women < 1.2 mmol/L. The analysis revealed that M-CSF in the range from 389 to 453 pg/ml ( р < 0.001) and LDL above 3.0 mmol/L ( р < 0.01) correlated with an increase in the risk for end-organ damage in stage II EH. Changes in the cytokine levels can be regarded as a predictor of myocardial and cerebral damage in patients with stage II EH. Measurement of peripheral blood M-CSF can be included into the classic risk assessment schemes for the cardiovascular complications in the studied cohort of patients.
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Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments
стр.27-33
Kravchenko YE, Ivanov SV, Kravchenko DS, Frolova EI, Chumakov SP
Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.
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Detection of ser450leu mutation in rpoB gene of mycobacterium tuberculosis by allele-specific loop-mediated isothermal dna amplification method
стр.34-40
Filipenko ML, Oscorbin IP, Khrapov EA, Shamovskaya DV, Cherednichenko AG, Shvartz YaSh
To identify genetic mutations a rather time-consuming and expensive method of polymerase chain reaction (PCR) is widely used. The aim of the present work was to evaluate the possibility of using the two schemes of the method of allele-specific isothermal loop amplification (LAMP) to detect the TCG/TTG (S450L) mutation in the rpo B gene of Mycobacterium tuberculosis . 48 clinical isolates of M. tuberculosis and 11 samples of sputum were used, randomized and obtained in the microbiological laboratory of the city of Novosibirsk from incident patients. It is shown that the use of an analysis scheme using the allele-specific primer FIP compared to F3 has the best resolution: the difference between the amplification time of the mutation and the wild type allele was 22 ± 2,4 versus 13 ± 4,1 minutes ( p = 0,0011). When using 100 DNA genomic equivalents a true positive signal (amplification of the rpo B gene with a mutation using the corresponding allele-specific primer) was detected after 29,4 ± 3,4 minutes. A positive signal was visualized after adding SYBR Green I to the reaction, both when illuminated with daylight and when using a UV transilluminator. Using the developed method the DNA sample of 20 RIFR isolates from M. tuberculosis was analyzed containing the Ser450Leu mutation in the rpo B gene, 10 RIFR isolates containing other mutations in the rpo B gene and 18 RIFs isolates without any mutations; the presence of mutations in the samples was determined using classical Sanger sequencing. The sensitivity and specificity of LAMP for detecting a Ser450Leu mutation in the rpo B gene was 100%. This approach allows the use of crude lysates of mycobacteria as DNA, which reduces the total analysis time to 1,5 hour.
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Evaluation of the ejaculate microbiota by real-time pcr and culture-based technique
стр.41-45
Voroshilina ES, Zornikov DL, Panacheva EA
Among other things male sterility can be caused by inflammatory diseases of the urogenital tract, often associated with opportunistic microorganisms. Thus, it is necessary to implement modern methods for the detection and identification of opportunistic microorganisms in the urogenital tract. The aim of the work was to conduct comparative analysis of the ejaculate microbiota from men of the reproductive age and studied using quantitative polymerase chain reaction (PCR) and culture method. 86 samples of ejaculate collected from men aged 18-57 years after observing sexual abstinence for 3-5 days were examined. With culture study in 50% of samples we observed growth of gram positive facultative anaerobic bacteria in the amount less than 103 CFU/ml; in 16.3% of samples - the growth of bacteria was not observed. With real-time PCR in each sample 8-15 groups of microorganisms were detected (including the prevailing groups) in the amount of 102-106 GE/ml. In all 86 samples obligate anaerobes that cannot not be cultured in vitro were detected. The predominant groups of microorganisms, as determined by real-time PCR, were detected by the culture metod only in 24.4% of cases.
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Lipid metabolic changes in rat brain during permanent cerebral ischemia
стр.46-52
Kostyuk AI, Kotova DA, Demidovich AD, Panova AS, Kelmanson IV, Belousov VV, Bilan DS
With each year, millions of people remain targeted by brain stroke, it still is by all means a global concern of the mankind. Despite all efforts to understand this disease better, there is still a lack of information on pathophysiology of ischemic stroke. Scrutinized data on biochemical changes at early stages of ischemia may help understand the mechanisms of the disorder and possibly reveal ways to finding the cure. The key role in the pathogenesis of stroke belongs to lipids as well as to the molecules associated with their biosynthesis and functionality. On the one hand, stroke evokes a deep oxidative stress leading to damage to biomolecules including lipids while on the other hand, due to the lack of reducing equivalents, the cellular biosynthesis processes are interrupted. The focus of this work was to study the changes taking place in the tissues of rat brain as a result of ischemia including estimation of levels of total cholesterol, FFA, MDA, GSH, and NADP(H). It was shown that in 24 hours from the onset of ischemia, there was a significant decrease in levels of FFA, total cholesterol and GSH, and an increase in the level of MDA, a marker of lipid peroxidation. NADP(H) pool level decreased twice in 6 hours from MCAO.
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Heart injuries: main clinical symptoms
стр.53-56
Maslyakov VV, Krjukov EV, Barsukov VG, Kurkin KG, Dorzhiev PA, Gorbelik VR
Injuries to the heart are uncommon in peacetime, yet they result in life-threatening conditions, which makes timely diagnostics a crucial factor in saving patients' lives. In this connection, it is important to define the main signs of heart injuries. This study aimed to analyze the basic clinical symptoms associated with various wounds to the heart. We have retrospectively analyzed such symptoms registered in 86 patients with varying chest injuries that affect the heart. All patients were treated in the emergency surgery unit of the Engels Town Hospital from 1991 to 2017. 41 (47.6%) patient had stab wounds, and there were 45 (52.3%) cases of gunshot wounds. 23 (26.7%) patients had chest injuries affecting heart exclusively, while for 63 (73.2%) the consequences were wounds to other organs. We found that the clinical picture depends on the kind of injury to the heart: stab and slash wounds translate into more pronounced symptoms, while gunshot wounds do not produce such an effect. Accepting patients, practitioners should take this fact into account. The misdiagnosis rate for stab and slash heart wounds is 9.7%, that for gunshot wounds - 17.7%, the latter being the result of vagueness of the clinical picture. The clinical signs are most pronounced in the cases of stab and slash wounds to the heart.
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Humoral response to Epstein-Barr viral infection in patients with allergies
стр.57-64
Svirshchevskaya EV, Simonova MA, Matushevskaya EV, Fattakhova GV, Khlgatian SV, Ryazantsev DYu, Chudakov DB, Zavriev SK
Type I hypersensitivity is mediated by the production of IgE antibodies in response to normally harmless substances. Debate still continues about the mechanisms underlying allergic reactions. Reduced barrier tissue function can be one of the risk factors for allergies. The aim of the present work was to compare the humoral immune response to Epstein-Barr virus in patients allergic to the A. alternata fungus or D. farinae house dust mites and healthy donors. It is known that up to 90% of the world population are infected with EBV. This infection occurs at early age when a child develops allergy. The antibodies were analyzed using immuno-PCR and the recombinant EBV protein rEBNA. We were able to demonstrate that infection occurs at early age in both allergic patients and healthy donors. The proportion of EBP-seropositive individuals was comparable between the groups (75 and 74%). The proportion of patients with high IgG titers among patients with allergies was lower (7%) than in healthy donors (18%), suggesting a lower viral load. In patients with allergies (but not in healthy donors) IgG titers declined as children grew older ( р = 0.037). Besides, IgA titers were increased in patients with allergies in comparison with healthy donors, but differed between patients allergic to alternata and house dust mites. In allergic individuals, production of IgM against EBV was triggered earlier than in healthy donors. We conclude that IgM production and the IgA-mediated humoral response occur earlier in patients with allergies, causing a decline in IgG titers over time.
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A correlation between the fluctuations of cytokine concentrations measured in the morning and evening and the circadian blood pressure rhythm in patients with stage II essential hypertension
стр.65-70
Radaeva OA, Simbirtsev AS, Khovryakov AV
Today, increasing attention is being paid to the role of circadian rhythms in pathology. There are time-of-day-dependent immune markers that provide valuable information about disease progression. The aim of this study was to measure evening and morning concentrations of a few cytokines (interleukins, adhesion molecules, tumor necrosis/growth factors, etc.) in the peripheral blood of patients with stage II essential hypertension and to investigate how they correlate with a nocturnal blood pressure decline. Blood samples were collected from 90 patients with stage II EH at 7:00 a.m. and 8:00 p.m. Cytokine concentrations were measured using immunoassays. Based on 24-h blood pressure monitoring, the patients were distributed into 3 groups: dippers, non-dippers and night-peakers. The morning to evening ratios of cytokine concentrations in patients with EH differed from those in healthy controls due to an increase in the evening concentrations of somnogenic cytokines (IL1β, IL1α) and LIF, sLIFr, and M-CSF whose daily fluctuations patterns remain understudied. On the whole, the fluctuation patterns of the measured cytokines in patients with stage II EH who had had the condition for 10 to 14 years and were receiving no antihypertensive treatment at the time of our study differed from those displayed by healthy controls. A twenty percent rise in the evening concentrations of IL1α, LIF, sLIFr, M-CSF, and erythropoietin contributes significantly to pathological blood pressure rhythms (as demonstrated by the groups of non-dippers and night-peakers) in patients with stage II EH receiving no antihypertensive therapy. Understanding the pathophysiological role of cytokine levels and their fluctuations over a 24-h cycle could inspire new methods for EH prevention and reduce end-organ damage.
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Injectable collagen in correction of age-related skin changes: experimental and clinical parallels
стр.71-77
Manturova NE, Stenko AG, Petinati YaA, Chaikovskaya EA, Bolgarina AA
To a large extent, age-related facial skin changes, wrinkles and flabbiness, are attributed to the structural alterations in dermis, including of collagen fibers fragmentation and disorganization. There are various cosmetological correction methods that aim to activate neocollagenesis and dermal remodeling. From this perspective, intradermal injections of exogenous collagen preparations seem logical. This study aimed to investigate the efficacy and safety of Collost 7% collagen complex applied to correct the age-related facial skin changes, as well as clarify the possible mechanisms of skin rejuvenation resulting from a course of intradermal injections. 35 participants entered the study, 30 of them finished it. A set of indicators describing age-related skin changes was assessed with the help of clinical scales; the assessment revealed a pronounced improvement in the quality of the patients' skin, including smoothed relief in the area of localization of fine wrinkles. The therapy resulted in a statistically significant improvement of the skin's elasticity, which, combined with the changes discovered through US scanning (greater dermis thickness and echodensity), is an indirect indication of skin restructuring associated with accumulation of fibrous protein structures. These results allow parallels with the experimental data that shows activation of neocollagenesis in the skin of laboratory animals after a course of Collost 7% gel. The research revealed no serious adverse events. A course of collagen administered intradermally can be recommended as an aesthetic correction procedure, as well as means of prevention of atrophy that has a significant effect on skin's appearance and health status.
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Glycoprotein GP as a basis for the universal vaccine against Ebola virus disease
стр.78-85
Dolzhikova IV, Tukhvatulin AI, Gromova AS, Grousova DM, Tukhvatulina NM, Tokarskaya EA, Logunov DY, Naroditskiy BS, Gintsburg AL
Ebola virus disease (EVD) is one of the deadliest viral infections affecting humans and nonhuman primates. Of 6 known representatives of the Ebolavirus genus responsible for the disease, 3 can infect humans, causing acute highly contagious fever characterized by up to 90% fatality. These include Bundibugyo ebolavirus (BDBV), Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SUDV). The majority of the reported EVD cases are caused by ZEBOV. Vaccine development against the virus started in 1976, immediately after the causative agent of the infection was identified. So far, 4 vaccines have been approved. All of them are based on the protective epitope of the ZEBOV glycoprotein GP. Because SUDV and BDBV can also cause outbreaks and epidemics, it is vital to design a vaccine capable of conferring protection against all known ebolaviruses posing a threat to the human population. This article presents systematized data on the structure, immunogenicity and protective properties of ebolavirus glycoprotein GP, looks closely at the immunodominant epitopes of ZEBOV, SUDV and BDBV glycoprotein GP required to elicit a protective immune response, and offers a rational perspective on the development of a universal vaccine against EVD that relies on the use of vectors expressing two variants of GP represented by ZEBOV and SUDV.
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A genetically encoded biosensor roKate for monitoring the redox state of the glutathione pool
стр.86-92
Shokhina AG, Belousov VV, Bilan DS
Genetically encoded fluorescent sensors are exploited to study a variety of biological processes in living organisms in real time. In recent years, a whole family of biosensors has been developed, serving to visualize changes in the glutathione redox state. The aim of our experiment was to design a biosensor based on the red fluorescent protein mKate2 for measuring the 2GSH/GSSG ratio. A pair of cysteine amino acid residues were introduced into the structure of the fluorescent protein using site-directed mutagenesis. These residues form a disulfide bridge when the surrounding glutathione pool is oxidized, affecting the spectral characteristics of the protein. Our biosensor, which we called roKate, was tested in vitro on an isolated protein. Specifically, we examined the spectral characteristics, pH and the redox potential of the sensor. Additionally, the performance of roKate was evaluated using the culture of living mammalian cells. The fluorescent signal emitted by the sensor was very bright and remarkably stable under pH conditions varying in the physiological range. Irreversibly oxidized in mammalian cells, roKate stands out from other members of this biosensor family. This biosensor should be preferred in the experiments when the time between the manipulations with the biological object and the subsequent analysis of the induced effect is substantial, as is the case with long sample preparation.
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Takayasu's Arteritis: the retrospective analysis of patients from the Ural population
стр.93-101
Borodina IE, Popov AA, Salavatova GG, Shardina LA
Takayasu's arteritis (TA) is a rare disease that can be overlooked during the first visit to a GP, rheumatologist, or any other medical specialist due to a variety of its symptoms. The aim of this study was to describe the clinical presentation and the course of patients with TA residing in the Middle Ural. A retrospective analysis was conducted using the medical records of 183 patients treated at the Sverdlovsk Regional Clinical Hospital 1 from 1979 through 2018. The male to female ratio was 1:3. The mean age was 33.5 years for women and 35.2 for men. The most frequently involved arteries were subclavian (101 cases; 55%), carotid (98 cases; 53%) and renal (77 cases; 42%). Type V was the most common angiographic type. Arterial stenosis was present in 94 (51%) patients. Sixty-six patients received surgical interventions. Of all patients included in the analysis, 31 died. The observed 5-year survival was 92%, 10-year survival, 90% and 15-year survival, 80%. Seventy-two patients (39%) developed major adverse cardiovascular events (MACE), including myocardial infarction, ischemic stroke, and thrombosis of large arteries/veins. The clinical presentation of TA may vary in different geographical regions.
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Frequency of carbohydrate metabolism disorders in day-care patients with borderline fasting blood sugar levels and at least one risk factor for diabetes mellitus
стр.102-107
Boeva VV, Boeva TA, Zavyalov AN
In order to assess the diagnosis of carbohydrate metabolism disorders, day care patients from Tambov central regional hospital were investigated. The study was conducted during 6 months in 2018. The study included 91 patients and allowed the diagnosis of type 2 diabetes mellitus (DM) in 31 (34.0%) cases, 6 (6.5%) impaired fasting glucose and 22 (24.1%) impaired glucose tolerance. This survey highlighted the necessity to expand the screening populations at risk for developing type 2 diabetes. The rational for the 75-gram oral glucose tolerance test for all individuals with fasting plasma glucose ≥ 5.6 ≤ 6.0 mmol/l and having one or more risk factors for developing type 2 diabetes and / or metabolic syndrome is shown. Among these categories diabetes was detected in 4.3%, and prediabetes in 14.4% of cases.
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