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.

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.

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.

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.

SNP rs657152 Is Not Associated with the Level of Viral Load in COVID-19 or the Probability of Disease in the Population of Caucasians in Eastern Siberia.

Cross-replicating associations with rs657152 at the 9q34.2c locus and rs11385942 at the 3p21.31 locus found in patients with severe COVID-19 in the Caucasian population require the study of the discovered phenomenon in various populations, including as an independent biological marker. Primers and TaqMan probes for PCR discrimination of the A and C alleles in single nucleotide polymorphism (SNP) rs657152 have been developed. The polymorphism of the rs657152 A/C locus was determined in 129 patients with COVID-19 and in a control group of 466 healthy individuals. There were no significant differences in the frequency of distribution of the A and C alleles, 0.47/0.53 and 0.45/0.55, between patients and healthy subjects, respectively. Also, no differences were found in the distribution of alleles in patients with a high viral load in the smear (Ct in the range of 16-25) in comparison with an average and low viral load (Ct in the range of 26-40).

[Predictors of epilepsy in children after ischemic stroke]. / Prediktory formirovaniya epilepsii u perenesshikh ishemicheskii insul't detei.

AIM: To determine clinical/instrumental predictors of symptomatic epilepsy after ischemic stroke in children. MATERIAL AND METHODS: One hundred and thirty-six patients, aged 0-15 years, with the diagnosis of ischemic stroke (ICD-10 I63.0-I63.9) were examined. The duration of the study was 18 months - 12 years. Patients were stratified into post-stroke (n=22) and control (n=114) groups, the latter included patients without epilepsy regardless of the presence of convulsive seizures in the acute stage of stroke. Predictors were determined based on EEG and characteristics of convulsive syndrome in the acute stage of stroke. RESULTS AND CONCLUSION: The following prognostic criteria were found: generalized type of seizures, focal type of seizures with secondary generalization, epileptiform (peak and/or peak-wave) activity, focal character of epileptiform activity, generalized type of seizures in the combination with slow wave background activity on EEG, generalized type of seizures in the combination with slow wave activity and disorganized activity on EEG.

[Role of NO-synthase in stimulation of opiate receptors and kidney oxidative stress resistance]. / Rol' NO-sintazy v stimuliatsii opiatnykh retseptorov i ustoichivosti pochek k oksidativnomu stressu.

It was established that dalarginum injection before ARI (acute renal insufficiency) formation prevented an increases of proteolysis, decrease of SOD (superoxide dismutase), increase of NO2-/NO3- content in kidney tissue. Antioxidant effect of opiate receptor agonist was completely abolished by preliminary injection of OR antagonist--naloxone. Aminoguanidine nitrate (inducible NO-synthase inhibitor) injection removed positive effect of OR stimulation too. Thus OR stimulation increases kidney oxidative stress resistance due to NO-synthase and SOD activation.

[Rehabilitation of patients with chronic bronchitis and spine diseases]. / Vosstanovitel'naia terapiia bol'nykh khronicheskim bronkhitom s zabolevaniiami pozvonochnika.

Correction of disturbed respiratory function and inhibition of progression of pulmonary inflammatory and obstructive processes is the aim of respiration rehabilitation in patients with chronic bronchitis (CB). It is known that rehabilitation in CB patients with spinal diseases meets with difficulties as dystrophic lesions of the spine entail its functional-anatomic disorders at the corresponding level. By supporting progression of dystrophic spinal and osteovertebral junction disorders, chronic bronchopulmonary inflammation participates in formation of interrelated respiratory affections. New approaches to rehabilitation of CB patients with spinal lesions lie in use of pharmacopuncture, acupuncture, deep reflex-muscular massage and therapeutic exercise in consensual sequence.

[Effect of dalarginum on the level of nitric oxide stable metabolites in rat kidneys during apoptosis activation]. / Vliianie dalargina na soderzhanie metabolitov oksida azota v pochkakh krys pri aktivatsii apoptoza.

The influence of dalarginum on the level of nitric oxide stable metabolites in the rat kidneys was investigated at various models of apoptosis activation (formed by two methods of acute renal failure). Dalarginum (synthetic leu-enkephaline) was entered in a dose of 100 micrograms/kg a day. Detection of apoptosis was carried out by determination of DNA fragmentation. Administration of dalarginum resulted in the significant decrease of NO--nitrite- and nitrate anions stable metabolites content, the latter is capable to be one of the mechanisms of the preparation antiapoptotic effect.

[Clinico-diagnostic role of ferritin and lactoferrin assays in benign and malignant affections of lungs and pleura]. / Kliniko-diagnosticheskoe znachenie issledovanii ferritina i laktoferrina pri dobrokachestvennykh i zlokachestvennykh porazheniiakh legkikh i plevry.

Immunochemical tests of blood sera from 160 healthy residents of the town of Astrakhan were performed to determine standard quantities of ferritin and lactoferrin in the serum. Examinations of the saliva from 280 healthy subjects provided the frequency of occurrence and levels of excretory lactoferrin. These iron-containing proteins were also assessed in different biosubstrates (blood serum, sputum, saliva, pleural fluid, lung tissues) from 550 patients with benign and malignant lesions of the lungs and pleura. Tests for ferritin and lactoferrin proved useful in evaluation of bronchopulmonary inflammation activity, early detection of pulmonary and intrapleural supputation, differential diagnosis of chronic nonspecific pulmonary diseases and lung cancer. The excretory salivary lactoferrin provided sufficient information on the condition of adaptive mechanisms of local defense of the lungs in premorbid state in 420 workers of the gas-processing plant exposed to aggressive occupational pollutants.

[Use of piperacillin/tazobactam as empirical monotherapy in the treatment of bacterial infections in a resuscitation department]. / Primenenie piperatsillin/tazobaktama v kachestve émpiricheskoi monoterapii pri lechenii bakterial'nykh infektsii v reanimatsionnom otdelenii.

Fifteen patients with severe bacterial infection (12 with pneumonia) that developed in the resuscitation unit were subjected to the empirical monotherapy with piperacillin/tazobactam (P/T) or tazocin under an open randomized controlled experiment. P/T was administered intravenously in a dose of 4.5 g every 8 hours for 5 to 12 days (9.3 days on the average). When the monotherapy was not sufficiently efficient the patients were additionally treated with amikacin administered intravenously in a dose of 0.5 g every 8-12 hours. The favourable effect was observed in 14 patients (93 per cent). 7 of them were treated with P/T alone and 7 were treated with P/T in combination with amikacin. The primary pathogens were eradicated in 8 (73 per cent) out of the 11 patients treated with P/T alone. Before the treatment 34 microbial strains were isolated from the patients. 77 per cent of them were susceptible to P/T. The treatment with P/T resulted in eradication of 27 bacterial strains (79 per cent) including 67 per cent of gram-positive organisms and 86 per cent of gram-negative organisms. The adverse effects were recorded in 1 patient on the 6th day of the treatment: skin eruption and pruritus that required the treatment discontinuation. The results showed that the use of P/T in the initial empirical monotherapy of infections in patients under resuscitation conditions could be efficient.

[Action of biologically active peptides and steroids on nuclear magnetic resonance relaxation of protons of kidney tissue water in vitro]. / Deistvie biologicheski aktivnykh peptidov i steroidov na iaderno-magnitno-rezonansnuiu relaksatsiiu protonov tkanevoi vody pochek in vitro.

The effect of biologically active peptides (adrenocorticotropin, parathyroid hormone, calcitonin, prolactin) and steroids (aldosterone, progesterone, testosterone) hormones on NMR-relaxation proton of tissue water of kidney have been investigated in vitro. Results of this study suggest that peptide hormones caused dehydration of tissues via sodium regulation. Steroids, quite on the contrary, caused the hydration of kidney tissue independent of the movement of osmotic active electrolyte.

An Alu element in the myeloperoxidase promoter contains a composite SP1-thyroid hormone-retinoic acid response element.

An Alu element preceding the myeloperoxidase gene (MPO) contains four hexamer motifs related to the consensus recognition sequence for nuclear hormone receptors (AGGTCA), arranged as direct repeats with spacing of 2, 4, and 2 nucleotides (DR-2-4-2). Gel shift experiments and transient transfection assays demonstrate that these sequences include binding sites for retinoic acid and thyroid hormone receptors and function in vivo to activate transcription of a chloramphenicol acetyltransferase reporter gene. The first DR-2 elements of the series do not bind known receptors but do bind the SP1 transcription factor. Two alleles of the MPO gene exist that differ at one position within this element, resulting in one allele with and one without a strong SP1 binding site. The element with the SP1 site activates transcription by 25-fold in transient transfection assays, while the alternative allele confers severalfold less transcriptional activity. Most cases of acute myelocytic leukemia are homozygous for the allele with the SP1 binding site, suggesting this element plays an important role in regulating the MPO gene in myeloid leukemias. This MPO-Alu is a representative of an Alu subclass numbering approximately 400,000 copies, suggesting many genes may be regulated by such elements.

[Expression of the human alpha-1-antitrypsin gene in Escherichia coli]. / Ekspressiia gena alpha-1-antitripsina cheloveka v kletkakh Escherichia coli.

The cDNA, containing the complete human alpha-1-antitrypsin (AT) sequence starting from codon 2, was used to construct bacterial strains producing AT. The fusion protein was obtained by junction of the AT cDNA to the fragment of an Escherichia coli ompF gene. We have also modified the AT cDNA's 5'-terminal part to construct DNAs containing ATG-codon and cDNA sequences starting from codons 1 or 2. These DNAs were inserted into E. coli expression vectors pBR322/trpII-8 and pKK223-3 that allow transcription from efficient trp- and tac-promoters. This construction resulted in the induction of a 46 kDa protein. The polypeptide produced was recognized by an antiserum raised against human alpha-1-antitrypsin protein. Truncation of the gene at its 5'-end or synthesis as a fusion OmpF-AT protein increases expression up to 10-fold, to a level of approximately 1%. On the contrary, no dependence on the promoter type has been observed. Physical properties of the recombinant proteins are discussed.

[The pharmacological characteristics, immunochemical identification and study of the location of quisqualate receptors in the rat and human brains]. / Farmakologicheskaia kharakteristika, immunokhimicheskaia identifikatsiia i issledovanie lokalizatsii kviskvalatnykh retseptorov golovnogo mozga krysy i cheloveka.

The kinetics of [3H]-L-glutamate binding to brain synaptic membranes (SM) and to glutamate-binding proteins (GBP) was determined with agonist and monoclonal antibodies (MAbs). It was revealed, that rat and human brain GBP have individual protein components with M(r) from 14 to 92 kDa. Quisqualate inhibited [3H]-L-glutamate binding to solubilized and to purified 68 kDa protein component. MAbs have the most activity, and NMDA was failure. It has been shown that 68 kDa component antigen determinants are similar to those of bovine, frog and rat brain synaptic membranes. Anti-GBP monoclonal antibodies blocked functional non-NMDA receptors in isolated frog spinal cord. Immunocytochemistry was done on rat and human brain sections. Distribution of quisqualate receptors was determined with light and electron microscopy. Some properties of vertebrate CNS non-NMDA receptors are discussed.