Rapid Assessment of Neutralizing Antibodies Using Influenza Viruses with a Luciferase Reporter.

Influenza viruses cause acute respiratory infections, especially in the autumn-winter period. They are characterized by a high mutation frequency and cause annual seasonal epidemics. The detection of antibodies that neutralize the virus is an important criterion in the assessment of population immunity and the influenza vaccine effectiveness. In this study, a method for determining the titer of virus-neutralizing antibodies in blood serum has been developed. A new test called the luciferase neutralization assay uses a bioluminescent signal for detection. The assay is based on engineered influenza reporter viruses with various surface antigens and a nanoluciferase reporter protein in the NS1 reading frame. Using the developed method, we studied paired sera of volunteers obtained before and after vaccination. The proposed assay was compared with the conventional antibody assessment methods (microneutralization and hemagglutination inhibition assay); a high degree of correlation was observed. At the same time, the use of the luciferase neutralization assay made it possible to reduce the time required for the analysis and to simplify the detection procedure. Supplementary Information: The online version contains supplementary material available at 10.1134/S0003683822070067.

Deuterated polyunsaturated fatty acids inhibit photoirradiation-induced lipid peroxidation in lipid bilayers.

Lipid peroxidation (LPO) plays a key role in many age-related neurodegenerative conditions and other disorders. Light irradiation can initiate LPO through various mechanisms and is of importance in retinal and dermatological pathologies. The introduction of deuterated polyunsaturated fatty acids (D-PUFA) into membrane lipids is a promising approach for protection against LPO. Here, we report the protective effects of D-PUFA against the photodynamically induced LPO, using illumination in the presence of the photosensitizer trisulfonated aluminum phthalocyanine (AlPcS3) in liposomes and giant unilamellar vesicles (GUV), as assessed in four experimental models: 1) sulforhodamine B leakage from liposomes, detected with fluorescence correlation spectroscopy (FCS); 2) formation of diene conjugates in liposomal membranes, measured by absorbance at 234 nm; 3) membrane leakage in GUV assessed by optical phase-contrast intensity observations; 4) UPLC-MS/MS method to detect oxidized linoleic acid (Lin)-derived metabolites. Specifically, in liposomes or GUV containing H-PUFA (dilinoleyl-sn-glycero-3-phosphatidylcholine), light irradiation led to an extensive oxidative damage to bilayers. By contrast, no damage was observed in lipid bilayers containing 20% or more D-PUFA (D2-Lin or D10-docosahexanenoic acid). Remarkably, addition of tocopherol increased the dye leakage from liposomes in H-PUFA bilayers compared to photoirradiation alone, signifying tocopherol's pro-oxidant properties. However, in the presence of D-PUFA the opposite effect was observed, whereby adding tocopherol increased the resistance to LPO. These findings suggest a method to augment the protective effects of D-PUFA, which are currently undergoing clinical trials in several neurological and retinal diseases that involve LPO.

Old dog, new tricks: Influenza A virus NS1 and in vitro fibrillogenesis.

The influenza NS1 protein is involved in suppression of the host immune response. Recently, there is growing evidence that prion-like protein aggregation plays an important role in cellular signaling and immune responses. In this work, we obtained a recombinant, influenza A NS1 protein and showed that it is able to form amyloid-like fibrils in vitro. Using proteolysis and subsequent mass spectrometry, we showed that regions resistant to protease hydrolysis highly differ between the native NS1 form (NS1-N) and fibrillar form (NS1-F); this indicates that significant structural changes occur during fibril formation. We also found a protein fragment that is capable of inducing the process of fibrillogenesis at 37 °C. The discovery of the ability of NS1 to form amyloid-like fibrils may be relevant to uncovering relationships between influenza A infection and modulation of the immune response.

Prediction of response to Certolizumab-Pegol in rheumatoid arthritis (PreCePRA) by functional MRI of the brain - Study protocol for a randomized double-blind controlled study.

BACKGROUND: Tumor necrosis factor inhibitors (TNFi) signify a major advance in the treatment of rheumatoid arthritis (RA). However, treatment success initially remains uncertain as approximately half of the patients do not respond adequately to TNFi. Thus, an unmet need exists to better predict therapeutic outcome of biologicals. OBJECTIVES: We investigated whether brain activity associated with arthritis measured by functional magnetic resonance imaging (fMRI) of the brain can serve as a predictor of response to TNFi in RA patients. METHODS: PreCePRA is a multi-center, randomized, double-blind, placebo-controlled fMRI trial on patients with RA [1] [2]. Active RA patients failing csDMARDs therapy with a DAS28 > 3.2 and at least three tender and/or swollen joints underwent a brain BOLD (blood-oxygen-level dependent) fMRI scan upon joint compression at screening. Patients were then randomized into a 12-week double-blinded treatment phase with 200 mg Certolizumab Pegol (CZP) every two weeks (arm 1: fMRI BOLD signal activated volume > 2000 voxel, i.e. 2 cm3; arm 2: fMRI BOLD signal activated volume <2000 voxel) or placebo (arm 3). DAS28 low disease activity at 12 weeks was assigned as primary endpoint. A 12-week follow-up phase in which patients were switched from the placebo to the treatment arm followed the blinded phase. fMRI was carried out at screening as well as after 12 and 24 weeks of receiving CZP or placebo. CONCLUSION: We hypothesize that high-level central nervous representation of pain in patients with rheumatoid arthritis predicts response to the TNFi CZP which we further investigate in the PreCePRA trial.

Cold and distant: structural features of the nucleoprotein complex of a cold-adapted influenza A virus strain.

Two influenza A nucleoprotein variants (wild-type: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance between nucleoprotein complex strands. The isolated complexes of two ribonucleoprotein variants were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDFEREGYS) is prone to temperature-dependent self-association, unlike the peptide corresponding to E292G substitution (SGYDFGREGYS). It was also shown that the SGYDFEREGYS peptide is capable of interacting with a monomeric nucleoprotein (wild type); this interaction's equilibrium dissociation constant is five orders of magnitude lower than for the SGYDFGREGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins were studied at temperatures of 15, 32, and 37 °C. SANS data show that the structures of the studied complexes at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.Communicated by Ramaswamy H. Sarma.

[Inflammatory metabolites of arahidonic acid in tear fluid in UV-induced corneal damage]. / Vospalitel'nye metabolity arakhidonovoi kisloty v sleznoi zhidkosti pri indutsirovannom ul'trafioletovym izlucheniem povrezhdenii rogovitsy.

The ultraviolet (UV) B-induced damage of the eye surface of experimental animals (rabbits) includes loss of corneal epithelium, apoptosis of keratocytes and stromal edema. These changes are accompanied by clinically and histologically manifested corneal inflammation, neutrophil infiltration, and exudation of the anterior chamber of the eye. According to mass spectrometric analysis, UV-induced corneal damage is associated with pronounced changes in the lipid composition of tears, including a decrease in the amount of arachidonic acid and prostaglandin E2 and an increase in the concentrations of prostaglandin D2 and its derivative 15d-PGJ2. In addition, it is accompanied by an alteration in the levels of hydroxyeicosate tetraenic acid derivatives, namely upregulation of 12-HETE and downregulation of 5-HETE. The revealed changes indicate the activation of metabolic pathways involving 5-lipoxygenase, 12-lipoxygenase, cyclooxygenase 1 and 2, and prostaglandin-D-synthase. These findings contribute to understanding mechanisms of UV-induced keratitis and point on feasibility of selective anti-inflammatory therapy for improving corneal regeneration after iatrogenic UV damage.

Influence of the Linking Order of Fragments of HA2 and M2e of the influenza A Virus to Flagellin on the Properties of Recombinant Proteins.

The ectodomain of the M2 protein (M2e) and the conserved fragment of the second subunit of hemagglutinin (HA2) are promising candidates for broadly protective vaccines. In this paper, we report on the design of chimeric constructs with differing orders of linkage of four tandem copies of M2e and the conserved fragment of HA2 (76-130) from phylogenetic group II influenza A viruses to the C-terminus of flagellin. The 3D-structure of two chimeric proteins showed that interior location of the M2e tandem copies (Flg-4M2e-HA2) provides partial α-helix formation nontypical of native M2e on the virion surface. The C-terminal position of the M2e tandem copies (Flg-HA2-4M2e) largely retained its native M2e conformation. These conformational differences in the structure of the two chimeric proteins were shown to affect their immunogenic properties. Different antibody levels induced by the chimeric proteins were detected. The protein Flg-HA2-4M2e was more immunogenic as compared to Flg-4M2e-HA2, with the former offering full protection to mice against a lethal challenge. We obtained evidence suggesting that the order of linkage of target antigens in a fusion protein may influence the 3D conformation of the chimeric construct, which leads to changes in immunogenicity and protective potency.

Antiinflammatory Effect of Rosiglitazone via Modulation of mRNA Stability of Interleukin 10 and Cyclooxygenase 2 in Astrocytes.

Investigation of molecular mechanisms of proinflammatory stimuli signaling in astrocytes is important for understanding their role in pathogenesis of central nervous system diseases as well as in functioning of the innate immunity system in non-immune cells. Here we show that lipopolysaccharide (LPS) stimulation of primary rat astrocytes led to conventional inflammatory response: increase in both proinflammatory (tumor necrosis factor, TNFα; prostaglandin E2, PGE2) and antiinflammatory marker (interleukin 10, IL-10) levels. The protein level of cyclooxygenase 2 (COX-2) was also increased. Rosiglitazone strengthened LPS-induced mRNA expression of COX-2 and IL-10 but not TNFα. Rosiglitazone is an agonist of nuclear receptor PPARγ, but its impact on IL-10 expression was not influenced by a PPARγ antagonist, GW9662, suggesting PPARγ-independent effect of rosiglitazone. The degradation of mRNA is one of the steps of inflammation regulation and might be affected by small molecules. In experiments with actinomycin D, we found that mRNA half-lives of IL-10, COX-2, and TNFα in naive astrocytes were 70, 44, and 19 min, respectively. LPS stimulation caused 2-fold increase in IL-10 and COX-2 mRNA decay rates, whereas addition of rosiglitazone restored them to the initial level. TNFα decay rate was not changed by these stimulations. This suggests that mRNA decay rate could be regulated by small molecules. Moreover, rosiglitazone could be used as a substance stimulating the resolution of inflammation without influence on proinflammatory signals. These results open new perspectives in the search for inflammation resolution modulators.

[In vitro Antiviral Activity of Recombinant Antibodies of IgG and IgA Isotypes to Hemagglutinin of the Influenza A Virus].

Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.

[Melatonin - known problems and perspectives of clinical usage]. / Melatonin - izvestnye i novye oblasti klinicheskogo primeneniia.

The article discusses well-known and ongoing studies of mechanisms of action of melatonin. The main clinical effects of melatonin are discussed. The emphasis on the chronobiological effect of melatonin, its adaptogenic and anti-carcinogenic properties has been done in the article. The most frequent manifestations of epiphyseal melatonin deficiency are various functional disorders in the form of insomnia, anxiety or depressive disorders. Recommendations on the effective use of melatonin in its deficiency due to pathology are given.

[Vaccination in rheumatology: Evolution of views on the problem]. / Vaktsinatsiia v revmatologii: évoliutsiia vzgliadov na problemu.

The problem of coinfections that are due to both a rheumatic disease (RD) itself and the need to use immunosuppressive drugs deserves apparent attention in modern rheumatology. Coinfections substantially affect morbidity and mortality rates, especially in diffuse connective tissue diseases. The data available in the literature on the above subject matter suggest that vaccination is a powerful method for prevention of infectious diseases that are the most important problem for patients with RD.

[Impact of mutations in nucleoprotein on replication of influenza virus A/Hong Kong/1/68/162/35 reassortants at different temperatures].

The nucleoprotein (NP) of influenza virus is a multifunctional RNA binding protein. The role of NP in the adaptation of influenza viruses to a host has been experimentally proved. Ambiguous data are available on the role of nucleoprotein in the attenuation of influenza A viruses, which is characterized by ability to replicate at low temperature (26°C) and inability to replicate at high temperature (39°C). Influenza virus donor strain A/Hong Kong/1/68/162/35 (H3N2), adapted to growth at low temperature, differs from the wild type virus by 14 amino acid mutations in the internal and non-structural proteins. Two mutations occurred in the NP: Gly102Arg and Glu292Gly. We have obtained viruses with point reverse-mutations in these positions and compared their replication at different temperatures by measuring infectious activity in chicken embryos. It has been shown that reverse mutation Gly292Glu in the NP reduced virus ability to replicate at low temperature, the introduction of the second reverse mutation Arg102Gly completely abolished virus cold adaptation.

SAFETY AND IMMUNOGENICITY OF COLD-ADAPTED RECOMBINANT INFLUENZA VECTOR EXPRESSING ESAT-6 AND AG85А ANTIGENS OF M. TUBERCULOSIS.

Recombinant viral vectors represent one of the most promising platforms for creating a new generation of vaccines against tuberculosis. We constructed a vaccine candidate based on a cold-adapted influenza vector with a truncated NS1 protein containing an insert of tuberculosis ESAT-6 and Ag85A antigens. The recombinant virus possessed a cold-adapted and temperature-sensitive phenotype and was attenuated for mice when administered intranasally. Immunofluorescent staining and Western blot showed the expression of ESAT-6 protein in MDCK cells infected by recombinant virus. After intranasal administration to mice, the recombinant virus stimulated a specific anti-tuberculosis CD4 + Th1-type response with the formation of polyfunctional antigen-specific T cells.

A Fusion Protein Based on the Second Subunit of Hemagglutinin of Influenza A/H2N2 Viruses Provides Cross Immunity.

Conserved fragments of the second subunit of hemagglutinin (HA2) are of great interest for the design of vaccine constructs that can provide protective immunity against influenza A viruses of different subtypes. A recombinant fusion protein, FlgMH, was constructed on the basis of flagellin and a highly conserved HA2 fragment (35-107) of influenza viruses of the subtype A/H2N2, containing B cell, CD4+ T cell, and CD8+ T cell epitopes. The native conformation of the HA2 fragment was partially preserved upon its attachment to the C-terminus of flagellin within the recombinant fusion protein FlgMH. FlgMH was shown to stimulate a mixed Th1/Th2 response of cross-reactive antibodies, which bind to influenza viruses of the first phylogenetic group (H1, H2, H5), to the target sequence as well as the induction of specific cytotoxic T cells (CD3+CD8+IFNγ+). Immunization with the recombinant protein protected animals from a lethal influenza infection. The developed FlgMH protein is a promising agent that may be included in an influenza vaccine with a wide spectrum of action which will be able to stimulate the T and B cell immune responses.

Vestibular disorders following different types of head and neck trauma.

This review focuses on the published literature on vestibular disorders following different types of head and neck trauma. Current knowledge of the different causes and underlying mechanisms of vestibular disorders, as well as the sites of organic damage, is presented. Non-organic mechanisms are also surveyed. The frequency of occurrence of vestibular symptoms, and of other accompanying subjective complaints, associated with different types of trauma is presented and related to the specific causes. Hypotheses about the pathogenesis of traumatic vestibular disorders are presented, and the knowledge derived from animal experiments is also discussed. We believe this to be a very important topic, since vestibular complaints in traumatic patients often remain undiagnosed or underestimated in clinical practice. This review article aims to suggest directions for additional research and to provide guidance to both the scientific and clinical practice communities.

[Influence of D1, D2 Receptor Blockade in Basolateral Amygdala on Behavior of Rats with High or Low Levels of Anxiety and Fear].

The influence of bilateral D1 or D2 receptors antagonists infusions into the rat basolateral amygdala on anxiety, as well as the expression, extinction and re-learning of conditioned fear was studied. Subjects were the male Wistar rats with high and low anxiety behavior in elevated plus maze, and also rats with low and high freezing responses during fear conditioning. The infusion of D1 receptor antagonist (SCH23390, 1 µg/0.5 µL in each side) reduced the expression of the conditioned fear to sound in rats with low freezing level, accelerated fear extinction and impaired re-learning in all animals. The injection of D2 receptor antagonist (raclopride, 1 µg/0.5 µL in each side) accelerated the extinction of conditioned fear to contextual cues in all rats and had a weak anxiolytic-like effect on behavior of high anxiety rats in elevated plus maze. These findings testify to the role of D1 receptors in the acquisition, expression and extinction of conditioned fear to stimuli, and D2 receptors in the occurrence of anxiety and fear to the contextual cues. There was discovered different sensitivity of animals with different levels of anxiety and fear to the infusion of dopamine receptors antagonists in the amygdala that suggested the inequality of dopaminergic transmission in the amygdala of animals with individual differences.

Regulation of cyclooxygenase 2 expression by agonists of PPAR nuclear receptors in the model of endotoxin tolerance in astrocytes.

Endotoxin tolerance (ET) represents a state of an altered immune response induced by multiple stimulations of a cell, a tissue, or an organism with lipopolysaccharide. Characteristics of ET include downregulation of induction of proinflammatory genes (TNFα, IL6, and others) and enhancement of induction of antiinflammatory genes (IL10, TGFß). ET generally has protective functions; nevertheless, it might result in a state of innate immune deficiency and cause negative outcomes. A current issue is the search for the mechanisms controlling the level of inflammation in the course of endotoxin tolerance. In this work, we investigated the change in cyclooxygenase 2 (Cox2) expression in the model of endotoxin tolerance in astrocytes and analyzed the possibility of regulating this process applying nuclear receptor PPAR agonists. Our results indicate that: 1) endotoxin tolerance can be induced in astrocytes and results in TNFα and Cox2 mRNA induction decrease upon secondary stimulation; 2) tolerance is revealed on the level of TNFα release and Cox2 protein expression; 3) PPAR agonists GW7647, L-165041, and rosiglitazone control Cox2 mRNA expression levels under conditions of endotoxin tolerance. In particular, rosiglitazone (a PPARγ agonist) induces Cox2 mRNA expression, while GW7647 (a PPARα agonist) and L-165041 (a PPARß agonist) suppress the expression. Our results demonstrate that Cox2 can be up- and downregulated during endotoxin tolerance in astrocytes, and PPAR agonists might be effective for controlling this target under conditions of multiple proinflammatory stimulations of brain tissues with endotoxin.

[Scientific rationale for basic directions of the optimization of the population health in the development of municipal environmental programs].

For the solution of ecological problems in the framework of the preparation of the municipal ecological program in the city of Verkhnyaya Pyshma (Sverdlovskaya Oblast) there was peiformed the assessment of the state of population health, the evaluation of carcinogenic and non-carcinogenic health risk from chemicals that pollute the air and drinking water Atmospheric air was established to be the main environment cause for carcinogenic and non-carcinogenic risks. The obtained results served as the basis for the development of technological, sanitary and hygienical measures of the program aimed at optimizing of the population health.

[Vaccines against highly pathogenic influenza viruses of the avian origin].

Worldwide spreading of H5 and H7 highly pathogenic influenza viruses of the avian origin, which periodically infect and kill humans without prior adaptation, poses a constant threat of the new pandemic. The effectiveness of the pandemic prevention completely depends on the quality of the existing influenza vaccines. Typical methods of the vaccine production from the antigenically relevant strains are problematic in case of high virulent H5 and H7 viruses. Therefore, new approaches to the construction of the vaccine strains and production technologies are required in order to protect the population.

[Immunization with live attenuated A/H5N1 vaccine protects guinea pigs from reinfection].

Cold-adapted influenza virus A/HK/1/68/162/35(H3N2) was developed as unified donor of attenuation and high reproductive capacity forvaccine strains. The reassortant of this donor with surface antigens of highly pathogenic strain Alchicken/Astana/6/05 (H5N1) was tested in guinea pigs as a live or inactivated preparation. Immunization with both formulations induced equal levels of serum virus specific antibodies, while the level of mucosal antibodies was significantly higher in animals immunized with live virus. The challenge with the homologous virus demonstrated complete virus clearance only in this group, thereby indicating a direct correlation of the protection level with the level of mucosal antibodies.