[Levilimab and baricitinib prescribing experience in outpatient COVID-19 patients' treatment].

AIM: To study the effect of levilimab or baricitinib in combination with standard therapy (ST) on the incidence of severe viral pneumonia associated with a new coronavirus infection COVID-19. MATERIALS AND METHODS: A multicenter, open-label observational study of the efficacy and safety of levilimab in combination with ST (group 1, n=100), baricitinib in combination with ST (group 2, n=139), or in comparison with ST (group 3, n=200) in outpatients with verified CT-1 pneumonia. RESULTS: According to the results of laboratory tests, patients treated with levilimab in combination with ST had the best dynamics of changes in CRP from reliably the highest level (mg/L) to the lowest in comparison with other groups. In the group of patients with ST, in contrast to the other groups, no dynamics of CRP was observed by day 5 of therapy. In group of hospitalized patients initially receiving levilimab in addition to ST, the rate of transfer to the intensive care unit (2 patients, 9.52%) and length of stay (4 days) was significantly lower compared to the values in patients in both the baricitinib group in combination with ST (7 patients, 15.56%; 5 days [interquartile range 36.5]) and in patients receiving ST alone (7 patients, 15.56%; 5 days [interquartile range 36.5]). Also in hospitalized patients we observed no statistically significant intergroup differences in the incidence of infectious complications and thromboembolic events, which confirms the safety of including levilimab or baricitinib in COVID-19 pathogenetic therapy regimens. Observational results support the hypothesis that the initial inclusion of levilimab or baricitinib in addition to ST is accompanied by a reduced risk of viral pneumonia progression. CONCLUSION: The addition of levilimab or baricitinib to the therapy regimen for coronavirus infection during the outpatient phase has demonstrated a preemptive anti-inflammatory effect and reduced the probability of lung tissue damage progression.

[A comparative characteristic of antigenic properties of recombinant and native hbs-antigens with G145R mutation and evaluation of their immunogenicity].

BACKGROUND: One of the important reasons for spreading of hepatitis B virus (HBV) under conditions of vaccinepressure is emergence of escape mutations. Prevalent G145R mutation in S-gene leads to the most expressed changes of serological properties of HBV. Consequently, HBsAg is modifed so thoroughly that it cannot be recognized by the majority of anti-HBs. Mutant G145R also differs from a wild type HBsAg by its immunogenic properties. At present, the relevance of enhancement of hepatitis B vaccine in view of mutant virus variants has been recognized. OBJECTIVES: a comparative study of antigenic and immunogenic properties of native and recombinant G145R mutants and an estimation of possibility for developing antigenic component of hepatitis B vaccine with G145R mutation in HBsAg. METHODS: antigenic properties of recombinant HBsAg with G145R mutation were compared with each other and with native mutants by serological fngerprinting method. Then, BALB/c mice and sheep were immunized with selected recombinant antigen under different protocols. Titers of antibodies specifc to wild type or mutant G145R type of HBsAg in sera of immunized animals were measured. RESULTS: it was found that not all the recombinant HBsAg variants with G145R substitution have the same antigenic properties as native HBsAg with similar mutation. Recombinant HBsAg selected according to the principle of antigenic similarity possesses immunogenicity in mice and sheep causing the production of antibodies reacting with native wild and mutant type HBsAg. It was shown that mutant antigen is less immunogenic, requires larger doses and more time for the development of immune response; however, it is capable of causing an antibody level comparable with wild type antigen. CONCLUSIONS: preliminary selection of recombinant HBsAg containing G145R mutation with antigenic and immunogenic properties similar to the native analogue creates the basis for development of a specifc component of hepatitis B vaccine with escape mutation G145R in HBsAg.

[The detection of immunoglobulins G to agents of TORCH-infections using technique of multiplex immunoassay on the basis of FOSFANtm technology.]

The immune chip is developed for detecting immunoglobulins G to agents of four infections of TORCH-complex (toxoplasmosis, German measles, cytomegalovirus and herpes viral infections) on the basis of FOSFANtm technology. The sensitivity and specificity of simultaneous detection of IgG on immune chip were comparable with indices of commercial immunoenzyme test-systems, including under analysis of standard panels of serums. This permits considering derived results as a basis for development of commercial multiplex test intended for highly productive screening of TORCH-infections in clinical laboratory.

[THE SELECTION OF ANTIGENS FOR DETECTING OF IMMUNOGLOBULIN G TO CYTOMEGALOVIRUS ON THE BASIS OF FOSFAN TECHNOLOGY].

The results of selection of composition of antigens to cytomegalovirus in the structure of multiplex test on the basis of FOSFAN™ technique are presented. In the process of detection of immunoglobulin G (IgG) to this virus the best indicators of sensitivity were registered with mosaic antigen containing immunodominant sequences of proteins pp150, gB, pp28 and pp52; reliably lower indicators of sensitivity was registered with phosphoprotein pp150; the lowest indicators of sensitivity were registered with proteins gB and pp65. The specificity made up from 93.5% to 96.8% independently of type of antigen. The mosaic antigen ensured the best ratio between sensitivity and specificity of immunoassay and is considered as the main component of immunochip for detecting IgG to cytomegalovirus.

[Predictors of cardiac arrhythmias in patients with arterial hypertension during exercise stress testing].

Arterial hypertension is an important risk factor for atrial and ventricular arrhythmias. 203 male patients were examined in order to identify predictors of cardiac arrhythmias in patients with arterial hypertension during exercise stress testing. All participants were studied by 24-hour ambulatory blood pressure monitoring, transthoracic echocardiography, an ultrasound scan of the carotid arteries and treadmill test. 47,3% of patients presented cardiac arrhythmias during exercise stress testing. The left ventricular mass, diastolic function and carotid intima-media thickness were found to be independent predictors of exercise-induced arrhythmias. The use of the exercise stress testing may be reasonable for additional risk stratification in hypertensive patients.

MORPHOLOGICAL ANALYSIS OF HEPATITIS B VIRUS WITH ESCAPE MUTATIONS IN S-gene G145R AND S143L.

BACKGROUND: In terms of serological properties and immunization, the wild type of HBsAg HBV and its G145R mutant behave as different antigens. This testifies to serious structural changes, which presumably could have a significant impact on the morphogenesis of virions and subviral particles. Nevertheless, morphological and ultrastructural investigations of HBV with G145R mutation have not been carried yet. OBJECTIVES: Research of structural and morphological organization of HBV in the presence of the G145R escape mutation. METHODS: Studies of sera, purified viruses and recombinant HBsAg were carried out by transmission electron microscopy by the method of negative staining and indirect reaction of immunelabeling using monoclonal antibodies of different specificity. Specimens of wild type HBV and HBV with S143L mutation obtained in an identical manner were used as the control. RESULTS: The presence of typical virus particles of HBV was shown in the specimens of wild strain and HBV with S143L mutation. Specimens of HBV with G145R mutation were characterized by expressed morphological heterogeneity. In the initial serum and in the specimen of purified virus containing G145R mutant, large oval particles 60-70 nm and up to 200 nm in size, respectively, were found. The presence of antigen structures of HBV in all heterogeneous forms was confirmed. It was shown that forming of subviral particles in the process of expression of the recombinant HBsAg with G145R mutation depends on conditions of expression and purification of the protein. They can vary from well-formed circular and oval particles to practically unstructured fine-grained masses. CONCLUSION: Direct data on the impact of G145R escape-mutation in S-gene, in contrast to S143L mutation, on the morphogenesis of virions and subviral particles of HBV were obtained.

[Molecular diagnosis and frequencies of primary hypolactasia in populations of RUSSIA and neighboring countries].

The digestion of the milk sugar (lactose) is observed every normal child but not in every adult. The decreased lactase synthesis in some adults results in problems with digestion of the whole milk (primary hypolactasy). An association of lactase activity in adults with carrying of the allele T within the polymorphism C/T-13910 located upstream of the lactase gene and 100% association of hypolactasy with the genotype C/C has recently been shown for a Finnish sample. In the present work we determined the LCT* C/T_13910 genotypes and allele frequencies in populations from Russia. The genotype C/C frequencies varied from 36.6% for Russians to 88.2% for Chukchi and were close to the published medical and epidemiological data on hypolactasy frequencies in respective populations. Genotyping was performed by three different methods to identify the optimal one. Our results have shown that the studied locus is the key determinant for the primary hypolactasy development in various human populations. Consequently, the DNA diagnostics of the C/C genotype carrying is a promising predictive test to detect the primary hypolactasy long before its clinical development. Practical application of this type of diagnostics would be a step towards the individual-oriented medicine.

The influence of the isolation technique of influenza virus nucleoprotein on its antigenic properties.

ELISA has been used to study the antigenic properties 1. of influenza virus nucleoprotein (NP-1) isolated from virions with the help of preparative polyacrylamide gel electrophoresis (PAGE); 2. of virion ribonucleoprotein (NP-2), and 3. of NP structures prepared by dissociation of ribonucleoprotein into RNA and protein in sucrose gradient containing NaCl (NP-3). The investigation of immunologic cross-reactivity has shown complete identity of NP-2 and NP-3 and their striking difference from NP-1. In contrast to NP-2, NP-3 was not contaminated by other virus antigens, it was a good immunogen and could be used for preparation of monospecific antisera of high titre. NP-1 did not induce a high antibody response,however, like NP-2 and NP-3, it retained its capacity to react with antisera to native virus. Owing to its simple production and high yield, this protein can be used in serodiagnosis for testing the antibody level against NP-protein in convalescent sera.

Antigenic reactivity of matrix protein and nucleoprotein of influenza virus as detected by EIA after dissociation with different detergents.

Solid phase enzyme-immunoassay (EIA) was employed to assess the antigenic reactivity of matrix protein (M) and nucleoprotein (NP) of influenza A virus adsorbed to polystyrene in the presence of different detergents such as beta-octaglucoside (OG), Triton X-100, Tween-20, sodium dodecylsulphate (SDS), sodium deoxycholate (Doch-Na), Nonidet P-40 (NP-40), and sarcosyl at concentrations ranging from 0 to 2%. The antigenic reactivity of NP was the highest in the absence of detergents. For M protein, Doch-Na, SDS, NP-40 and sarcosyl of 0.05-0.1% enhanced the chromatophoric response in EIA 1.5-2 times. In contrast, the antigenic reactivity of M protein remained unchanged after OG or Triton X-100 treatments, and it decreased in the presence of Tween-20.

Detection of tick-borne encephalitis virus in ixodid ticks collected in natural foci by time-resolved fluoroimmunoassay.

Time-resolved fluoroimmunoassay (TR-FIA) was used for the first time for evaluation of infestation of ixodid ticks with tick-borne encephalitis virus. Comparison of TR-FIA results with those obtained in enzyme immunoassay and by virus isolation confirmed the high efficacy of the method in question. Positive results of TR-FIA coincided with the data of virus isolation in 83.6% cases, the level of false-negative results did not exceed 1.2%, the overall time consumption amounted to about 1.2 hr.

[Circular dichroism spectra study on the ribonucleoprotein structure of influenza virus]. / Issledovanie struktury RNP virusa grippa metodom krugovogo dikhroizma.

The CD spectra of Influenza virus RNA and RNP were examined in the ultraviolet region (220--320 nm) at different temperatures, NaCl and urea concentrations. The magnitude of the positive band for RNP decreases gradually upon increasing the temperature, indicating that rather weak interaction between RNA and protein occurs. However the temperature drastically affects the intensity of the negative band about 222 nm. In the temperature range where the protein melts the positive band at longer wavelengths shows a temperature dependence that is similar for RNP and free RNA. Addition of NaCl results in an increase in intensity and blue shift of the positive CD band of RNP. In the presence of 1 M NaCl the CD spectrum of RNP is very close to that of protein-free RNA. Urea concentrations up to 10 M has little effect on the CD spectrum of RNP. These results suggest that only ionic but not hydrophobic and hydrogen bondings are involved in the RNA-protein interaction in Influenza virus RNP.

[Changes in the rate of protein synthesis in parenchymatous liver cells during stimulation of the mononuclear phagocyte system]. / Izmenenie skorosti sinteza belka v parenkhimnykh kletkakh pecheni pri stimuliatsii sistemy mononuklearnykh fagotsitov.

In vivo stimulation of mononuclear phagocyte system (MPS) by zymosan, dextrane sulfate, and prodigiosan caused almost a two-fold increase in hepatic protein synthesis. The rate of 14C-leucine incorporation increased both into total and soluble proteins. To define the cellular locus of these changes, preparations of hepatic parenchymal and nonparenchymal cells were obtained from the control and LPS-stimulated rats. The results indicate that the treatment of rats with prodigiosan stimulate protein synthesis in hepatocytes. No effect on protein synthesis of non-parenchymal cells was observed. Stimulation of MPS also caused a significant increase in 14C-leucine incorporation into serum lipoproteins. The results suggest that MPS may be involved in regulation of protein synthesis in hepatic parenchymal cells.

[Analysis of the DRD4 gene polymorphism in populations of Russia and neighboring countries]. / Issledovanie polimorfizma gena DRD4 v populiatsiiakh Rossia i sopredel'nykh stran.

Allele and genotype frequencies of the VNTR polymorphism in the third exon of human DRD4 gene were determined in 544 individuals living in Russia (Russians, Bashkirs, Tatars, and Mordovians) and in the neighboring countries (Kazakhs and Ukrainians). The data obtained were compared with the allele frequency distribution patterns reported for the populations of Eurasia. Similarly to other Eurasian populations, in our population samples R4 allele was prevalent (64 to 87%). The frequency of this allele in the populations of Western Europe constitute 61 to 71%, while in the populations of Asia it varies from 74 to 96%. In this respect, the populations studied occupied the intermediate position. In the samples examined the R7 allele frequency decreased from 7% in Ukrainians to 1% in Bashkirs, while in Kazakhs and Mordovians the allele was absent. This finding was consistent with the R7 allele distribution pattern in the populations of Eurasia, characterized by higher frequency in the West and lower frequency or absence of the allele in the East. In the group of 22 Eurasian populations, the R7 allele frequency negatively correlated with the frequency of the R4 allele (r = -0.86 at P < 0.001). Unlike the R4 and R7 alleles, the frequency of which changed in the eastward direction, the R2 allele frequency distribution displayed slightly expressed latitudinal increase southwards. The DRD4 genotype distribution deviated from the equilibrium in most of the samples examined. In some samples, statistically significant increase of the R2/R2 homozygotes frequency was demonstrated. One of the possible explanations of this phenomenon is assortative mating with respect to phenotypic (behavioral) allele manifestation. The data obtained can serve as the basis for the investigation of the possible role of the DRD4 alleles as the risk factors for the development of alcoholism and other types of addictions.

[Designing and clinical testing of immune-enzyme and immunofluorescence test systems for serodiagnosis of ixodes borreliosis]. / Razrabotka i klinicheskaia aprobatsiia immunofermentnykh i immunoliuminestsentnykh test-sistem dlia serodiagnostiki iksodovykh kleshchevykh borreliozov.

The methods of immune enzyme assay (MIEA) and of lanthanide immunofluorescence analysis (LIFA) were used to work out the test systems for the detection (in blood serum of patients) of specific IgM IgG antibodies to the B. burgdorferi spirochete--a causative agent of ixodic borrelioses. The test system was clinically tested versus the indirect immunofluorescence reaction (IIFR) and commercial immune enzyme test system (CIET). The results of antibodies' detection were shown to correlate with the analysis data for the same sera in IIFR and to be in line with a real presence or absence of the disease. Test systems based on LIFA were proven to be most sensitive and specific.

[The use of lanthanide immunofluorescence analysis for demonstrating the antigen of viral hepatitis A]. / Primenenie lantanidnogo immunofliuorestsentnogo analiza dlia indikatsii antigena virusnogo gepatita A.

The study dealt with the development of an express method for detection of hepatitis A virus (HAV) antigen employing lantanides, especially europium. The new time-resolved fluoroimmunoassay (TRFIA), alongside with a significant shortening of the time for reaction, was also 8 times as sensitive as the analogous enzyme immunoassay. The TRFIA may be used effectively both for control of the antigen amplification in HAV-infected cell culture and for early diagnosis of acute forms of viral hepatitis A.

[Isolation and characteristics of monoclonal antibodies to influenza virus types A and B]. / Poluchenie i kharakteristika monoklonal'nykh antitel k virusam grippa tipov A i B.

Monoclonal antibodies (MCA) to influenza type A (10F) and B (5H and 6H) viruses have been prepared. By immunoblotting method, MCA 10F were found to be specific for NP-protein of influenza A virus, and MCA 5H and 6H to be specific for hemagglutinin of influenza B virus. It was established that the 10F clone interacted with all the investigated influenza A virus strains with different antigenic formulae (H1N1, H2N2, H3N2) and could be used for typing of this virus type. Clones 5H and 6H react specifically with hemagglutinins of influenza B viruses isolated in 1940, 1979, and 1983 which makes them useful for diagnosis of influenza B.

[Immunoenzyme test system based on antibody F(ab)2-fragments for detecting the NP protein of the influenza virus]. / Immunofermentnaia test-sistema na osnove F(ab)2-fragmentov antitel dlia vyiavleniia belka NP virusa grippa.

A test system has been proposed for the detection of influenza A virus NP protein by solid-phase enzyme immunoassay in a modified system of "double" antibodies: the primary antibodies were F(ab)2-fragments of immunoglobulin class G isolated from rabbit antiserum specific for NP protein which was also used as the source of secondary antibodies. The antigen-bound antibodies were detected by protein A (from St. aureus) conjugate with horseradish peroxidase. The high specificity and sensitivity of the test system (0.3 ng/ml NP protein) and a minimal level of nonspecific reactions were demonstrated. The advantages of this method are discussed.

[Proteolytic cleavage of influenza virus matrix protein and nucleoprotein in an in vitro system]. / Proteoliticheskoe rasshcheplenie matriksnogo belka i belka nukleoproteida virusa grippa v sisteme in vitro.

Proteolytic modification of NP-protein which is a part of influenza virus ribonucleoprotein (RNP) was studied in vitro. NP-protein of influenza virus (mol. weight 56 000) was shown upon RNP treatment with bromeline to undergo proteolytic cleavage revealing 2 more polypeptides with molecular weights of 53 000 and 45 000 daltons. The same classes of NP-proteins were present in RNPs recovered from subviral units obtained by virus treatment with bromeline at buffer pH below 7. Electrophoresis in 12% polyacrylamide gel under reducing conditions of subvirus units obtained by treatment of virions with bromeline at the buffer pH below 7 also demonstrated proteolytic cleavage of M-protein present in them into fragments with molecular weights of 8 500-12 000 daltons. Similar results were obtained upon direct treatment with bromeline of M-protein isolated from virions.

[Reversible dissociation of influenza virus ribonucleoprotein]. / Obratimaia dissotsiatsiia ribonukleoproteida virusa grippa.

Dissociation of influenza virus RNP under the effect of salt was studied. Separation of RNA and protein components of influenza virus RNP was shown to occur in a linear 15-30% sucrose concentration gradient containing 1.1 M NaCl. Upon RNP dissociation, protein-protein interactions between individual molecules of the structural protein were retained. The sedimentation coefficient of the protein component was 52S. The possibility of reassociation of the RNA-protein complex was studied. More complete reassociation was observed to occur in the presence of 0.1 M NaCl. The resulting RNA-protein complex morphologically is similar to the native RNP of influenza virus.

[Use of the cation detergent, cetyltrimethylammonium bromide, in isolating the RNA of the influenza virus]. / Ispol'zovanie kationnogo detergenta tsetil-trimetil-ammonium bromida pri vydelenii RNK virusa grippa.

Isolation of virion RNA of influenza virus by the SDS-phenol method from subviral particles obtained from purified virions by treating them with a cation detergent, cetyl-methyl-ammonium bromide, increased RNA yields approximately 2-fold as compared with isolation of this RNA from whole virions.