A Comparative Assessment of Effects of Major Mediators of Acute Phase Response (IL-1, TNF-α, IL-6) on Breathing Pattern and Survival Rate in Rats with Acute Progressive Hypoxia.

A pressing issue of the day is the identification of therapeutic targets to suppress the "cytokine storm" in COVID-19 complicated by acute respiratory distress syndrome (ARDS) with concomitant hypoxemia. However, the key cytokine and its relative contribution to the pathogenesis of ARDS, which leads to high mortality, are unknown. A comparative assessment of the effect of elevated systemic levels of pro-inflammatory cytokines IL-1ß, TNF-1α and IL-6 on the respiratory patterns and survival rate in rats was carried out under progressively increasing acute hypoxia. Increasing hypoxia was simulated by a rebreathing method (from normoxia to apnea). The recorded parameters were the breathing pattern components (tidal volume and respiratory rate), minute ventilation (MV), oxygen saturation, apnea onset time, and posthypoxic survival rate. A comparative analysis was carried out under mild, moderate and severe hypoxia (at FIO2 = 15, 12 and 8%, respectively). It was shown that increasing hypoxia was accompanied by an acute suppression of the compensatory elevation of MV in rats with increased systemic levels of IL-1ß and TNF-1α. By contrast, IL-6 caused an intensive elevation of MV with increasing hypoxia. Acute hypoxia (FIO2 < 8%), in all experimental series, was accompanied by an impairment of the respiratory rhythm up to the development of apnea. Posthypoxic breathing restoration (survival rate) was 50% with IL-1ß and TNF-1α and only 10% with IL-6. The obtained results indicate that the elevated IL-6 level, despite the absence of respiratory disorders at the initial stage of the developing pathologic process, leads to a higher mortality in rats compared to IL-1ß and TNF-1α. This allows considering IL-6 as an early prognostic biomarker of a high risk of mortality under severe hypoxemia.

Participation of NO-Dependent Mechanisms in the Effects of Increased Systemic Level of Interleukin-1ß on Pial Microvessels under Conditions of Acute Hypoxia.

We studied the effect of increased systemic levels of the proinflammatory cytokine IL-1ß on the vasomotor reactions of pial microvessels in anesthetized rats under conditions of experimentally simulated progressively increasing acute normobaric hypoxia. Vital microscopy showed that more pronounced dilatation of pial vessels in response to IL-1ß under hypoxic conditions was almost completely prevented by pretreatment with non-specific NO synthase blocker L-NAME. These findings indicate the involvement of NO-dependent mechanisms in the vasodilator effect of proinflammatory cytokines under conditions of acute hypoxic exposure.

[The use of synthetic glycoconjugates as components of the immunochromatographic test for rapid serological diagnosis of leprosy.]

The glycoconjugates with BSA (bovine serum albumin) were synthesized using a next saccharide: disaccharide derivative M.leprae PGL-1 (phenolic glycolipid-1); a complex of the disaccharide fragment and the branched hexasaccharide fragment LAM (lipoarabinomannan); diarabinofuranose fragment LAM. These glycoconjugates were used as antigenic components for leprosy rapid serotest construction in immunochromatographic format (leprosy LF serotest). The data obtained with sera of leprosy patients, patients who have been in contact with leprosy, and healthy donors indicate that the most promising antigenic component is a BSA conjugate with two synthetic epitopes - a disaccharide derivative of PGL-1 and a branched hexasaccharide fragment of LAM. The leprosy LF serotest with such glycoconjugate demonstrated the greatest diagnostic sensitivity for main forms of leprosy - paucibacillary (PB) and multibacillary (MB).

[Construction of recombinant strain Brevibacillus choshinensis for chimeric borrelia Dbpa antigen production.]

The aim of this work was creation of recombinant chimeric protein using TAKARA expression system Brevibacillus choshinensis with fused gene dbpAAG, which include the parts of dbpAA and dbpAG genes coding the major antigenic determinants of decorinbinding proteins А (DbpA) from two species of borreliosis agents - Borrelia afzelii and Borrelia gаrinii. Such plasmid should be able to support the synthesis of recombinant chimeric polypeptide consisting immunogenic domains of DbpA Borrelia afzelii and Borrelia gаrinii in the stable and soluble forms, that important for effective using in Lyme diseases serodiagnosis. We chose the TAKARA expression system based on the strain Brevibacillus choshinensis and plasmid pNCMO2. It give us possibilities to obtain the scale quantity of the secreted soluble target proteins with native conformation in particular with conserve antigenic determinants. As results, the plasmid pNCMO2 with a fusion gene dbpAAG was constructed. Recombinante plasmide DNA pNCMO2/dbpAAG was used for Brevibacillus choshinensis trasformation. We were able to show that during cultivation in a liquid medium recombinant cells of В. choshinensis/pNCMO2/dbpAAG produced secreted chimeric 30кD protein with high immunoreactivity to Lyme borreliosis patient's serum.

[A comparative study of experimental and commercial serological tests for detection of antibodies in humans with tularemia.]

Experimental serological tests were developed to determine anti-tularensis antibodies in humans in immunochromatography formats (LF-test LPS Ft15) and enzyme immunoassay (ELISA LPS Ft15) using as an antigen highly purified LPS F. tularensis 15 NIIEG. Analysis was conducted of the sensitivity and specificity of the developed tests and commercial tularemia antigen «RNGA-Tul-AG¼ (production Stavropol research anti-plague Institute) in comparison with the commercial reference antigen, registered in the Russian Federation for the quantitative determination of human IgG tularemia - «ELISA classic Francisella tularensis IgG¼ SERION, Germany (IgG SERION ELISA). A study of human blood serum vaccinated against tularemia showed that the sensitivity and specificity of detection of anti-tularemia antibodies by «ELISA LPS Ft15¼ were 97.7 and 100%, compared with «ELISA IgG series¼. When determining antitularemia antibodies with the diagnosis «LF-test LPS Ft15¼, these parameters were compared to «ELISA IgG series¼ 94.3 and 100%. The sensitivity and specificity of «RNGA-Tul-AG¼ made compared to the «IgG ELISA, SERION¼ of 59.1% and 80%.

Dosing algorithms for vitamin K antagonists across VKORC1 and CYP2C9 genotypes.

Essentials Prospective studies of pharmacogenetic-guided (PG) coumarin dosing produced varying results. EU-PACT acenocoumarol and phenprocoumon trials compared PG and non-PG dosing algorithms. Sub-analysis of EU-PACT identified differences between trial arms across VKORC1-CYP2C9 groups. Adjustment of the PG algorithm might lead to a higher benefit of genotyping. SUMMARY: Background The multicenter, single-blind, randomized EU-PACT trial compared the safety and efficacy of genotype-guided and non-genetic dosing algorithms for acenocoumarol and phenprocoumon in patients with atrial fibrillation or deep vein thrombosis. The trial showed no differences in the primary outcome between the two dosing strategies. Objectives To explore possible reasons for the lack of differences between trial arms by performing a secondary analysis of EU-PACT data in order to evaluate the performance of both dosing algorithms across VKORC1-CYP2C9 genetic subgroups. Patients/Methods Anticoagulation control measured according to an International Normalized Ratio (INR) below (INR of < 2), within (INR of 2-3) and above (INR of > 3) the therapeutic range was compared across VKORC1-CYP2C9 subgroups. Owing to a low number of patients in each subgroup, trials for acenocoumarol and phenprocoumon were combined for analysis. Results Four weeks after therapy initiation, genotype-guided dosing increased the mean percentage of time in the therapeutic INR range (PTIR) in the VKORC1 GG-CYP2C9*1*1 subgroup as compared with the non-genetic dosing (difference of 14.68%, 95% confidence interval [CI] 5.38-23.98). For the VKORC1 AA-CYP2C9*1*1 subgroup, there was a higher risk of under-anticoagulation with the genotype-guided algorithm (difference of 19.9%; 95% CI 11.6-28.2). Twelve weeks after therapy initiation, no statistically significant differences in anticoagulation control between trial arms were noted across the VKORC1-CYP2C9 genetic subgroups. Conclusions EU-PACT genetic-guided dose initiation algorithms for acenocoumarol and phenprocoumon could have predicted the dose overcautiously in the VKORC1 AA-CYP2C9*1*1 subgroup. Adjustment of the genotype-guided algorithm could lead to a higher benefit of genotyping.

Effect of Simulated Microgravity and Lunar Gravity on Human Inspiratory Muscle Function: 'Selena-T' 2015 Study.

As a part of the multi-disciplinary "SELENA-T"-2015 Bed Rest Study, we investigated the pattern of inspiratory muscles fatigue in 22 healthy male subjects during incremental exercise test to exhaustion before and after 21 days of hypokinesia evoked by bed rest. Hypokinesia consisted of head-down bed rest (HDBR) at a minus 6° angle, simulating microgravity present on orbiting spacecraft, in 10 subjects. The remaining 12 subjects spent the first 5 days of hypokinesia in HDBR position and the subsequent 16 days in head-up bed rest (HUBR) at a plus 9.6° angle, as a presumed analog of lunar gravity that is six times less than Earth's gravity. Maximal inspiratory pressure (MIP) and electromyograms (EMG) of the diaphragm (D), parasternal (PS), sternocleidomastoid (SCM), and scalene (S) muscles served as indices of inspiratory muscle function. Before both HDBR and HUBR, exercise decreased MIP and centroid frequency (fc) of EMG (D, PS, SCM, and S) power spectrum (p < 0.05). After 3 weeks of HDBR, but not HUBR, inspiratory muscles fatigue was more expressed compared with control (p < 0.05). We conclude that HDBR lowers inspiratory muscles resistance to fatigue during high-intensity exercise while HUBR has no such effect. These changes may limit maximal ventilation and may contribute to exercise intolerance observed after prolonged simulated microgravity. The physiological mechanisms of respiratory muscle dysfunction after HDBR consist primarily of postural effects, and are not due only to hypokinesia.

[EFFECTS OF PROINFLAMMATORY CYTOKINE INTERLEUKIN-1P ON RESISTANCE TO ACUTE HYPOXIA].

Under a progressive growth of acute hypoxia the effect of high systemic levels of proinflammatory cytokine interleukin-1 Р (IL-1Р) were studied the reactions of the cardiorespiratory system of anesthetized Wistar rats. The results suggest a negative effect of IL-1 Р on the control mechanisms the respiratory and cardiovascular system, which was reflected in the reduction of resistance to acute hypoxia and the ability to spontaneous autoresuscitation after apnea in posthypoxic period, as well as in the development of circulatory collapse. It is assumed that the basis of the resistance mechanisms of the body to reduce the hypoxic exposure is multifactorial effects of increased levels of IL-1 Р, activation of HIF-1 a and NO production in the operation of the systems responsible for maintaining oxygen homeostasis.

[ASSOCIATED RESPIRATORY AND HEMODYNAMICS RESPONSE TO ACUTE NORMOBARIC PROGRESSIVE HYPOXIA IN ANESTHETIZED RATS].

The interdependent reactions of the cardiorespiratory system during experimental simulation of progressive acute hypoxia were studied in anesthetized Wistar rats. The results indicate that the extremely low oxygen content in the inhaled gas mixture to less than 6% lead to terminal sedation and apnea. After the cessation of hypoxic exposure were observed spontaneous autoresuscitation. Effects of progressive hypoxia, is an example of a multi-component interdependent reactions of the cardiorespiratory system, which are based on the respiratory and vasomotor center function disturbance and the predominance of parasympathetic influences on the heart. The obtained data can be used as a model of hypoxic apnea to examine the influence of physiologically active substances on the cardiorespiratory system at disease pathology.

[Genetic and epigenetic news in gerontology].

The overview represents the recent most conspicuous findings in aging studies. It includes new data on the whole genome association studies (GWAS) in big cohort of centenaries, recently found mutation protecting from Alzheimer disease, discovery of hypothalamus as a command center of human aging, very important data on the negative effect of common antioxidants in the treatment of lung cancer as well as new data concerning antiaging and anticancer effects of common drugs such as rapamycine and metformin. Substantial part of the review is devoted to the epigenetic problems of senescence and feasible impact of basic epigenetic mechanisms (methylation of DNA and histone proteins, DNA heterochromatization) in regulation of gene expression, long-term genome reprogramming during early childhood, and transgeneration transmission of epigenetic traits. The necessity of transition from molecular studies of dormant human genome (anatomy of human genome) to genome in action (dynamic genome) and thus with special emphasis to epigenetic medicine is stressed.

The COAG and EU-PACT trials: what is the clinical benefit of pharmacogenetic-guided coumarin dosing during therapy initiation?

Coumarin derivates are oral anticoagulants commonly prescribed for treatment and prevention of thromboembolism. Due to a small therapeutic index and large inter- and intrapatient differences in dose requirements, treatment with coumarins is challenging, particularly in its starting phase. Extensive evidence suggests that common genetic variants in CYP2C9 and VKORC1 genes together with a number of clinical factors are important determinants of the coumarin dose variability. Pharmacogenetic algorithms comprising both genetic and non-genetic factors were developed to improve the safety of coumarin therapy initiation. Recently, three randomized controlled trials (the COAG and the EU-PACT trials) on pharmacogenetic dosing of warfarin, acenocoumarol and phenprocoumon were published. In these trials different coumarin dosing strategies were compared to investigate whether or not pharmacogenetic testing could be beneficial for coumarin management. The purpose of this review was to present and discuss the design and results of these studies within the context of previously published randomized controlled trials and to address the issues surrounding the incorporation of coumarin pharmacogenetic testing into clinical practice.

Phenotype standardization of angioedema in the head and neck region caused by agents acting on the angiotensin system.

Angioedema is a potentially life-threatening adverse reaction to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. To study the genetic etiology of this rare adverse event, international consortia and multicenter recruitment of patients are needed. To reduce patient heterogeneity, we have standardized the phenotype. In brief, it comprises swelling in the head and neck region that first occurs during treatment. It should not coincide with urticaria or have another likely cause such as hereditary angioedema.

Three-dimensional structure of α-crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6.

Small heat shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N- and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly. This domain mediates dimer formation of individual protomers, which then may assemble into larger oligomers. In vertebrate sHSPs, the dimer interface is formed by the symmetrical antiparallel pairing of two ß-strands (ß7), generating an extended ß-sheet on one face of the ACD dimer. Recent structural studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the ß7/ß7 strand interface, which may, in part, give rise to the polydispersity often associated with the full-length proteins. To further analyze the structure of ACD dimers, we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 (HSP27) and HSPB6 (HSP20). Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical ß7/ß7 dimers but, rather, hexamers formed by an asymmetric contact between the ß4 and the ß7 strands from adjacent ACDs. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of ß7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the ß7/ß7 interface, with the results indicating that, under physiological conditions, there is only a single register of the strands for both proteins.

[Genomic of aging and predictive medicine].

New molecular-genetic methods stimulate substantial advances in complex diseases studies, speed up identification of new candidate genes participating in functional genetic modules (gene nets) associated with many common diseases. Decisive impact of predictive genetic studies in efficient implementation of genomic technology advances into presymptomatic identification of the subjects of high risk groups prone to various common complex diseases is reviewed. Substantial progress of genomic studies in genetic of aging processes, including complex metabolic processes and gene regulation is outlined. Advances of predictive medicine in pharmacogenomic, nutrigenomic, sport genomic as well as in genomic of aging substantiate real and soon progress in promotion active healthy longevity.

[Genetic passport--basic contribution to active longevity and maximal life-span duration].

The review was made on gene pass conception as individual DNA data bank reflecting unique genetic peculiarities of each human, its major potential contribution in achievements of active longevity and creation most favorable conditions for maximal duration of individual life-span. Participation of major age-regulated genes such as biological clock genes and the weak chain genes in aging processes of humans is briefly outlined. The significance of genetic testing of allelic polymorphisms and marker-genes implicated in common multifactorial disorders is stressed. The problems evoked by genetic results interpretation are mentioned. Special attention is paid to Genome Wide Association Studies (GWAS) technology implemented for analysis of genetic profiles and candidate genes associated with common diseases. Scientific problems and social interests in creation of individually oriented DNA-data banks (Gene Passes) amenable for the pregnant women, children, sportsmen, etc. are discussed. The relationship of Gene Pass idea to the current international genetic program "Personificated Genome" is highlighted. Feasible perspectives for genetic testing and basic contribution of Gene Pass into gerontology practical medical service are reviewed.

The taming of small heat-shock proteins: crystallization of the alpha-crystallin domain from human Hsp27.

Small heat-shock proteins (sHsps) are ubiquitous molecular chaperones. sHsps function as homooligomers or heterooligomers that are prone to subunit exchange and structural plasticity. Here, a procedure for obtaining diffraction-quality crystals of the alpha-crystallin domain of human Hsp27 is presented. Initially, limited proteolysis was used to delineate the corresponding stable fragment (residues 90-171). This fragment could be crystallized, but examination of the crystals using X-rays indicated partial disorder. The surface-entropy reduction approach was applied to ameliorate the crystal quality. Consequently, a double mutant E125A/E126A of the 90-171 fragment yielded well ordered crystals that diffracted to 2.0 A resolution.

[Genetic aspects of aging].

State of art in genetic of ageing is reviewed. Deciphering of human genome and recent advances in functional genomics contributed a lot to significant achievements in molecular medicine as well as in understanding of ageing mechanisms. Progressive transcriptome degeneration caused by expression modulations of specific aging genes underlies visible physiological, biochemical and hormonal changes in aging human bodies. Each human subject should be considered as physiological mosaic composed of the tissues and organs of different age. The existence of weak genetic chain confined to particular organ or tissue provides potential substrate for severe chronic disorders in aging people. Two main groups of the aging genes are highlighted: 1. Longevity genes identified in population studies of old people, and 2. The genes identified and proved in aging studies in experimental species. Already existing and feasible molecular approaches for extending of active human longevity are reviewed. They include targeted modulation of aging gene activity, as well as presymptomatic diagnostics and relevant preventive measures of severe and frequent multifactorial diseases. Combining of already known empirical methods of anti-aging medicine with unique genetic profile of each human (gene-pass) renders new awarding opportunities for the further advancements of human longevity programs.

[Comparative analysis of use of two strains of various genotypes of Borrelia burgdorferi sensu lato as antigens for antibody identification in Ixodes tick borreliosis by indirect immunofluorescence]. / Sravnitel'nyi analiz rezul'tatov ispol'zovaniia dvukh shtammov razlichnykh genovidov Borrelia burgdorferi sensu lato v kachestve antigenov dlia opredeleniia antitel pri iksodovykh kleshchevykh borreliozakh metodom nepriamoi immunofluorestsentsii.

The results of the indirect reaction of immune-fluorescence (IRIF) was studied in testing 49 sera of 19 patients with Lyme-borreliosis with antigens of genotypes Borrelia afzeli (strain Jp-21) and Borrelia burgdorferi sensu stricto (strain No. 17); rabbit fluorescini-sothiocyanate-marked conjugates to human immunoglobulins M and G as well as polyvalent conjugate were used of. No reliable differences were found between all positive and all negative results. The biggest portion of positive results was registered in tests with anti-G-conjugate (up to 92% with strain No 17).

[Expression analysis of proteins encoded by genes of the tag7/tagL (PGRP-S,L) family in human peripheral blood cells]. / Analiz ékspressii belkov, kodiruemykh semeistvom genov tag7/tagL(PGRP-S,L), v kletkakh perifericheskoi krovi cheloveka.

Various types of human blood cells were tested for expression of the Tag7/PGRP-SA and TagL/PGRP-L proteins, which belong to the family of proteins possessing the lysozyme-like peptidoglycan recognition protein (PGRP) domain. Expression regulation by several factors was demonstrated.

The differentially spliced mouse tagL gene, homolog of tag7/PGRP gene family in mammals and Drosophila, can recognize Gram-positive and Gram-negative bacterial cell wall independently of T phage lysozyme homology domain.

Tag7/PGRP, a recently characterized antimicrobial protein, is conserved from insects to mammals. Recently its involvement in Toll signalling in Drosophila was demonstrated. A number of genes representing a new family homologous to PGRP were identified in Drosophila and human. Here we describe a splicing pattern of the tagL gene, mouse member of tag7/PGRP family. Some of the identified splice variants lacked characteristics for the family T phage lysozyme homology domain (also known as PGRP domain). Accordingly to the predicted transmembrane domains, mouse TagL may be secreted as inducible proteins or retained on intracellular membranes. All detected splice variant isoforms of TagL bound Gram-positive, Gram-negative bacteria and peptidoglycan. This binding did not depend on the presence of T phage lysozyme homology domain but was associated with the C-terminal portion of the polypeptides. Thus, this variety of isoforms of a single gene may play a role in circulating bacteria recognition in mammals.