Description and Genomic Analysis of the First Facultatively Lithoautotrophic, Thermophilic Bacteria of the Genus Thermaerobacter Isolated from Low-temperature Sediments of Lake Baikal.

Members of the genus Thermaerobacter belong to the phylum Firmicutes and all isolates characterised to date are strictly aerobic and thermophilic. They were isolated from a mud sample of the Challenger Deep in the Mariana Trench, hydrothermal vents, and silt compost. A novel thermophilic, facultatively lithoautotrophic bacteria of the genus Thermaerobacter, strain PB12/4term (=VKM B-3151T), with a metabolism that is uncharacteristic of the type species, was isolated from low-temperature surface sediments near the Posolsk Bank methane seep, Lake Baikal, Russia. The new strain grows with molecular hydrogen as electron donor, elemental sulfur, and thiosulfate as electron acceptors, and CO2/[Formula: see text] as carbon source. The genome of strain PB12/4term consists of one chromosome with a total length of 2.820.915 bp and the G+C content of the genomic DNA was 72.2%. The phylogenomic reconstruction based on 120 conserved bacterial single-copy proteins revealed that strain PB12/4term belongs to the genus Thermaerobacter within in the class Thermaerobacteria, phylum Firmicutes_E. The strain PB12/4term is closely related to Thermaerobacter subterraneus DSM 13965 (ANI=95.08%, AF=0.91) and Thermaerobacter marianensis DSM 12885 (ANI=84.98%, AF=0.77). Genomic and experimental data confirm the ability of the Thermaerobacter PB12/4term pure culture to facultatively lithotrophic growth, which is provided by the presence of [NiFe]hydrogenase enzymes that are absent in T. marianensis DSM 12885 and T. subterraneus DSM 13965. The data obtained on the physiological and biochemical differences of strain PB12/4term provide a deeper insight into the species diversity and functional activity of the genus Thermaerobacter.

Wound healing activity of aqueous dispersion of fullerene C60 produced by "green technology".

In addition to exhibited antioxidant and anti-inflammatory activity, fullerene C60 is a promising wound healing agent. An important stage in the production of fullerene-based ointments is the stability of the aqueous fullerene dispersion (AFD) with minimum size of colloidal fullerene aggregates and sufficiently high concentration. To achieve these parameters tangential flow filtration of fullerene C60 was used ("green technology"). As estimated by small-angle neutron scattering and dynamic light scattering purified AFDs with narrow-size distribution nanoclusters have a size of 6 nm and are assembled into agglomerates which reach a size of 150 nm. The ability of the AFD to exhibit regenerative activity was studied using the animal wound model. This study shows for the first time that the fullerene-based composition stimulates the healing of wounds of various origins. We assume that the mechanism of the AFD wound-healing activity is associated with the aryl hydrocarbon receptor and macrophages activity.

The potential of anti-infectives and immunomodulators as therapies for asthma and asthma exacerbations.

Asthma is responsible for approximately 25,000 deaths annually in Europe despite available medicines that maintain asthma control and reduce asthma exacerbations. Better treatments are urgently needed for the control of chronic asthma and reduction in asthma exacerbations, the major cause of asthma mortality. Much research spanning >20 years shows a strong association between microorganisms including pathogens in asthma onset, severity and exacerbation, yet with the exception of antibiotics, few treatments are available that specifically target the offending pathogens. Recent insights into the microbiome suggest that modulating commensal organisms within the gut or lung may also be a possible way to treat/prevent asthma. The European Academy of Allergy & Clinical Immunology Task Force on Anti-infectives in Asthma was initiated to investigate the potential of anti-infectives and immunomodulators in asthma. This review provides a concise summary of the current literature and aimed to identify and address key questions that concern the use of anti-infectives and both microbe- and host-based immunomodulators and their feasibility for use in asthma.

Influenza burden, prevention, and treatment in asthma-A scoping review by the EAACI Influenza in asthma task force.

To address uncertainties in the prevention and management of influenza in people with asthma, we performed a scoping review of the published literature on influenza burden; current vaccine recommendations; vaccination coverage; immunogenicity, efficacy, effectiveness, and safety of influenza vaccines; and the benefits of antiviral drugs in people with asthma. We found significant variation in the reported rates of influenza detection in individuals with acute asthma exacerbations making it unclear to what degree influenza causes exacerbations of underlying asthma. The strongest evidence of an association was seen in studies of children. Countries in the European Union currently recommend influenza vaccination of adults with asthma; however, coverage varied between regions. Coverage was lower among children with asthma. Limited data suggest that good seroprotection and seroconversion can be achieved in both children and adults with asthma and that vaccination confers a degree of protection against influenza illness and asthma-related morbidity to children with asthma. There were insufficient data to determine efficacy in adults. Overall, influenza vaccines appeared to be safe for people with asthma. We identify knowledge gaps and make recommendations on future research needs in relation to influenza in patients with asthma.

The Role of Interleukin-33 in Pathogenesis of Bronchial Asthma. New Experimental Data.

Interleukin-33 (IL-33) belongs to the IL-1 cytokine family and plays an important role in modulating immune system by inducing Th2 immune response via the ST2 membrane receptor. Epithelial cells are the major producers of IL-33. However, IL-33 is also secreted by other cells, e.g., bone marrow cells, dendritic cells, macrophages, and mast cells. IL-33 targets a broad range of cell types bearing the ST2 surface receptor. Many ST2-positive cells, such as Th2 cells, mast cells, basophils, and eosinophils, are involved in the development of allergic bronchial asthma (BA). This suggests that IL-33 directly participates in BA pathogenesis. Currently, the role of IL-33 in pathogenesis of inflammatory disorders, including BA, has been extensively investigated using clinical samples collected from patients, as well as asthma animal models. In particular, numerous studies on blocking IL-33 and its receptor by monoclonal antibodies in asthma mouse model have been performed over the last several years; IL-33- and ST2-deficient transgenic mice have also been generated. In this review, we summarized and analyzed the data on the role of IL-33 in BA pathogenesis and the prospects for creating new treatments for BA.

[The multidirectional effects of thrombin and the possibility of their control in neurology]. / Raznonapravlennye effekty trombina na protsessy gemokoagulyatsii i vozmozhnost' ikh kontrolya v nevrologii.

The review presents the main physiological functions of thrombin. The procoagulant and anticoagulant activities of the key serine protease are discussed in both physiological and pathological conditions of hemostasis. The involvement of thrombin in atherogenesis, as well as its role as a mediator of vascular dysfunction and inflammation in both the peripheral and central nervous system, is highlighted. A pronounced imbalance between the pro- and anticoagulant systems leads to an increase in thrombin formation and creates conditions for the development of thrombosis. Tests that allow direct or indirect assessment of thrombin's functional activity are presented. The potential applications of direct thrombin inhibitors and direct blockers of thrombin PAR receptors in vascular neurology are also considered.

[Toxoplasmosis in the practice of a neurologist]. / Toksoplazmoz v praktike nevrologa.

Toxoplasmosis is a zoonotic protozoal disease characterized by a chronic course, polymorphism of clinical manifestations, predominant damage to the central nervous system, organs of vision, liver and lungs. The causative agent of the disease is the obligate intracellular parasite Toxoplasma gondii, which circulates widely in the external environment and has a large circle of intermediate hosts. Toxoplasmosis is classified by the method of infection (congenital or acquired), by pathogenesis (acute or chronic), by manifestation (latent or with the manifestation of symptoms). According to the state of the human immune system, the disease can occur without immunodeficiency, while the patient has a chronic lifelong carrier, and with immunodeficiency. People with HIV most commonly present with cerebral toxoplasmosis. The article presents a case of the development of toxoplasmosis in a patient in the absence of a burdened history.

RSV infection modulates IL-15 production and MICA levels in respiratory epithelial cells.

The cytokine interleukin (IL)-15, major histocompatibility complex (MHC) class I molecules and MHC class I chain-related proteins (MIC) A and B are involved in cellular immune responses to virus infections but their role in respiratory syncytial virus (RSV) infection has not been studied. We aimed to determine how RSV infection modulates IL-15 production, MHC class I and MICA expression in respiratory epithelial cells, the molecular pathways implicated in virus-induced IL-15 production and how interferon (IFN)-γ alters RSV-induced IL-15 production and MHC class I and MICA expression. We infected respiratory epithelial cell lines (A549 and BEAS-2B cells) and primary bronchial epithelial cells with RSV and measured production of IL-15, expression of MHC I and MICA and the role of the transcription factor nuclear factor (NF)-κB. We report here that RSV increases IL-15 in respiratory epithelial cells via virus replication and NF-κB-dependent mechanisms. Furthermore, RSV infection of epithelial cells upregulated cell surface expression of MICA and levels of soluble MICA. IFN-γ upregulated RSV induction of soluble IL-15 but inhibited induction of MICA. Upregulation of IL-15, MHC I and MICA are likely to be important mechanisms in activating immune responses to RSV by epithelial cells.

[Polymorphic variants of genes encoding interleukin-6 and fibrinogen, the risk of ischemic stroke and fibrinogen levels].

Carriage frequencies of alleles and genotypes of polymorphous locus of -174G>C IL6 (rs1800795) were analyzed in the patients with ischemic stroke (IS) of Russian ethnic descent (200 cases) and in the control group of the same ethnic descent with similar sex and age (140 controls). Significant differences were identified in frequencies of carriage (in homo- or heterozygous form) of allele IL6*-174G (p = 0.0029, OR = 2.9, 95% CI: 1.4-5.8), which can be considered as risk factor for IS and in frequencies of IL6*-174C/C genotype carriage, correspondingly (p = 0.0029, OR = 0.35, 95% CI: 0.17-0.69). After sex stratification of patients and controls similar significant differences were observed only between female patients and controls, after age stratification the difference was observed only for the age group older 60 years. Complex analysis of association of SNP -174G>C IL6 alleles and genotypes carriage in combination with SNP 4266A>G (Thr312Ala) FGA (rs6050) (see symbol) -249C>T FGB (rs1800788) with IS revealed protective combinations IL6*-174C/C + FGA* 4266A (see symbol) IL6*-174C/C + FGB*-249C, which were slightly more significant than single protective genotype IL6*-174C/C associated with IS and their ORs didn't differ substantially from the single genotypes's OR value. At the same time the combinations of alternative allele IL6*-174G with the same FGB*-249C or FGA* 4266A alleles were revealed and their association significance levels as well as OR values were lower than the values for the single risk allele IL6*-174G. In case of the mutual carriage of IL6*-174G allele with FGA*4266A/A, FGB*-249C/C genotypes or with combinations of these alleles/genotypes the "neutralized" effect became stronger. In other words, we observed association of IS with allele/genotype combinations of genes IL6, FGA and FGB, in which IL6 plays key role and FGA and FGB have modulating function. In analysis of association of fibrinogen plasma levels with three analyzed polymorphous loci significant differences were not revealed.

[Evaluation of the efficiency of differential diagnosis of influenza by multiplex real-time polymerase chain reaction].

The paper gives the results of a comparative analysis of the detection of influenza viruses in clinical samples, by using multiplex real-time polymerase chain reaction (RT-PCR) and by virus isolation in MDCK cell cultures. The investigation employed 267 nasopharyngeal swab specimens obtained from patients with influenza symptoms during two epidemic seasons (2008-2009 and 2009-2010). Influenza viruses were found in 104 samples (48 with influenza A virus (IAV) and 56 with influenza B virus (IBV)) by multiplex RT-RCR and in 84 samples (35 with IAV and 49 with IBV) by a cultural technique. The results of detection of influenza viruses by the two methods showed 89.4% agreement. The diagnostic sensitivity of multiplex RT-PCR testing a panel of the clinical samples in question was estimated to be 94.3% for IAV and 95.9% for IBV. The diagnostic sensitivity of multiplex RT-PCR in virus detection was demonstrated to be not only highly competitive with virus isolation, but also superior to the latter.

Nonspecif ic response of Lake Baikal phytoplankton to anthropogenic impact.

In this study, we present the first results on oxidation stress in Lake Baikal phytoplankton and its adaptation to environmental changes under anthropogenic impact. As was shown, the changing of the dominant species of phytoplankton collected from the surface water layer (~0.3 m) took place from February to June 2021. Phytoplankton were collected at a nearshore station (a littoral station at a distance of ~0.01 km from the shoreline, depth to bottom is ~5 m) and an offshore station (a pelagic station at a distance of ~1 km from the shoreline, depth to bottom is ~543 m). In February, dinoflagellates were dominant (~40 %) as well as diatoms (≤33 %) and green algae (≤12 %). Their biomass was 100 mg·m-3. In March, chrysophytes were dominant (up to 50 %) as well as cryptophytes (≤43 %) and dinoflagellates (≤30 %). Their biomass was 160-270 mg·m-3. In April, biomass increased up to 700-3100 mg·m-3 with the dominance of large cell dinoflagellates (up to 99 %), chrysophytes (up to 50 %), and cryptophytes (up to 35 %). By the end of the first decade of May, the percentage of dinoflagellates decreased and that of cryptophytes increased. In the second decade of May, the percentage of diatoms increased up to ~26-38 % but phytoplankton biomass was minimal (13-30 mg·m-3). By June, the percentage of diatoms in the samples reached 44-75 % at 60-550 mg·m-3. The oxidation stress of phytoplankton as a nonspecific adaptive response to a prolonged, intensive, or recurrent effect of a stress factor was estimated from the content of thiobarbituric acid reactive substances (TBARS). The mean content of these substances (markers of the lipid peroxidation) was determined spectrophotometrically. The oxidation stress of phytoplankton was revealed only when diatom algae dominated. It can be explained by adaptation of algae of other classes to the stress factor. The content of the lipid peroxidation markers in the coastal phytoplankton collected close to the settlement of Listvyanka known as a large touristic center was estimated from 100 to 500 µg·g-1 of dry weight of sample. During the period of diatom blooming in 2016 and 2018, oxidation stress of phytoplankton collected near large settlements was found. In phytoplankton from deep-water pelagic stations most remote from settlements, stress was not revealed. Using the method of gas chromatography, we showed a lower (up to 15 %) content of polyunsaturated fatty acids in phytoplankton characterized by stress occurrence. This confirms cell membrane damages. In Lake Baikal surface water, we found a higher content of synthetic anionic surfactants (sodium alkylbenzene sulfonates), which are components of detergents and cause oxidation stress of hydrobionts (up to 30 ± 4 µg·L-1). The presence of these substances in a water ecosystem can result in exhausting of phytoplankton cell resources, homeostasis imbalance, stress, pathological changes, and rearrangements in phytoplankton assemblage.

[Nonimmunogenic staphylokinase in the treatment of acute ischemic stroke (FRIDA trial results)]. / Neimmunogennaya stafilokinaza ­ novyi tromboliticheskii preparat v lechenii ishemicheskogo insul'ta (rezul'taty issledovaniya FRIDA).

AIM OF THE STUDY: To investigate the efficacy and safety of non-immunogenic staphylokinase (NS) compared with alteplase (A) in patients with acute ischemic stroke (AIS) within 4.5 h after symptom onset. MATERIAL AND METHODS: 336 patients with IS within 4.5 h after symptom onset were included in a randomized, open-label, multicenter, parallel-group, non-inferiority comparative trial of NS vs A (168 patients in each group). NS was administered as an intravenous bolus in a dose of 10 mg, regardless of body weight, over 10 s, A was administered as a bolus infusion in a dose of 0.9 mg/kg, maximum 90 mg over 1 hour. The primary efficacy endpoint was a favorable outcome, defined as a modified Rankin scale (mRS) score of 0-1 on day 90. Safety endpoints included all-cause mortality on day 90, symptomatic intracranial haemorrhage, and other serious adverse events (SAEs). RESULTS: At day 90, 84 (50%) patients reached the primary endpoint (mRS 0-1) in the NS group, 68 (41%) patients - in the A group (p=0.10, OR=1.47, 95% CI=0.93-2.32). The difference between groups NS and A was 9.5% (95% CI= -1.7-20.7) and the lower limit of the 95% CI did not cross the margin of non-inferiority (pnon-inferiority<0.0001). There were no significant differences in the frequency of deaths between the groups: on day 90, 17 (10%) patients in the NS group and 24 (14%) in the A group had died (p=0.32). There was a trend towards significant differences in the frequency of symptomatic intracranial haemorrhage: NS group - 5 (3%) patients, A group - 13 (8%) patients (p=0.087, OR=0.37, 95% CI=0.1-1.13). There were significant differences in the number of patients with SAEs: in the NS group - 22 (13%) patients, in the A group - 37 (22%) patients (p=0.044, OR=0.53, 95% CI=0.28-0.98). CONCLUSION: The presented results of the FRIDA trial are the first in the world to use a drug based on NS in patients with IS. It has been shown that a single bolus (within 10 s) administration of NS at a standard dose of 10 mg, regardless of body weight, allows to conduct fast, effective and safe thrombolytic therapy in patients with IS within 4.5 h after symptom onset. In further clinical tials of NS, it is planned to expand the therapeutic window beyond 4.5 h after symptom onset in patients with IS.

[Recognition receptors of innate immunity (NLR, RLr, and CLR)].

Current concepts of recognition receptors of innate immunity (pattern-recognition receptors, PRR) are discussed in the review. Structural and functional features of receptors from the families NOD-like receptors (NLRs), RIG-like receptors (RLRs), and C-type lectin-like receptors (CLRs) are described. These receptors are found on cell surface or in cytoplasm, and also could be presented in organism in secretory form. Data on exogenous and endogenous ligands, signal transduction mechanisms that induce production of proinflammatory cytokines, chemokines, and antimicrobial petides are summarized in the review. Special attention is paid to family of NLR receptors, which are involved in generation and activation of multimeric protein complex called an inflammasome. Activation of inflammasome leads to generation of active forms of proinflammatory cytokines belonging to IL-1 family (IL-1beta, IL-18, and IL-33) from their precursor peptides due to effect of caspase-1. Data regarding involvement of innate immunity receptors in development and pathogenesis of various diseases are presented.

Genetic Diversity and Evolution of the Biological Features of the Pandemic SARS-CoV-2.

The new coronavirus infection (COVID-19) represents a challenge for global health. Since the outbreak began, the number of confirmed cases has exceeded 117 million, with more than 2.6 million deaths worldwide. With public health measures aimed at containing the spread of the disease, several countries have faced a crisis in the availability of intensive care units. Currently, a large-scale effort is underway to identify the nucleotide sequences of the SARS-CoV-2 coronavirus that is an etiological agent of COVID-19. Global sequencing of thousands of viral genomes has revealed many common genetic variants, which enables the monitoring of the evolution of SARS-CoV-2 and the tracking of its spread over time. Understanding the current evolution of SARS-CoV-2 is necessary not only for a retrospective analysis of the new coronavirus infection spread, but also for the development of approaches to the therapy and prophylaxis of COVID-19. In this review, we have focused on the general characteristics of SARS-CoV-2 and COVID-19. Also, we have analyzed available publications on the genetic diversity of the virus and the relationship between the diversity and the biological properties of SARS-CoV-2, such as virulence and contagiousness.

[Quick test for measurement of rubella virus titer in virus-containing fluid using RT-PCR].

AIM: To develop method of rubella virus titer measurement in virus-containing fluid using real-time PCR (RT-PCR) with fluorescent detection. MATERIALS AND METHODS: Measurement of infectious titer of rubella virus (Wistar RA 27/3 strain) cultivated on Vero cells was performed simultaneously by RT-PCR and cytopathic effect assay (CEA) on PK-13 cell culture and then results obtained by each method were compared. RESULTS: Time interval after inoculation, in which difference between virus titer measured by both methods did not exceed 0.3 1gTCD50/ml (value acceptable by WHO), was 2 - 7 days. Pearson correlation coefficient between two values for the mentioned interval was close to 1, which point to good agreement of results. In control sample--international vaccine standard of rubella virus--difference in virus titer determined by RT-PCR and CEA was within 0.2 1gTCD50/ml that lower than value acceptable by WHO. CONCLUSION: Method for measurement of rubella virus titer in virus-containing fluid using RT-PCR was developed, which characterized by high specificity, sensitivity, standard performing, shorter time needed for procedure compared with classic methods and, at the same time, high correlation of its results with results obtained by the latter methods during defined time interval.

[Toll-like receptor-mediated functional activity of mononuclear cells in children with neutropenia].

AIM: To study the Toll-like receptor (TLR)-mediated functional activity of peripheral blood mononuclear cells (PBMC) from children with different forms of neutropenia. MATERIALS AND METHODS: TLR-mediated functional activity of PBMC was evaluated by production of proinflammatory cytokines--TNF alpha and IFN alpha. Ligands for TLR1/2, TLR 2/6, TLR4, TLR5, and TLR9 were used to stimulate TNF alpha production by PBMC from healthy children and children with neutropenia. Ligands for TLR3, TLR4, TLR7, TLR7/8, TLR8, and TLR9 were used to induce production of IFN alpha. Levels of TNF alpha and IFN alpha were measured in PBMC supernatants by ELISA. The group of patients with neutropenia included 9 children with immune neutropenia and 3 children with congenital neutropenia. Control group consisted of 12 healthy children of the same age range. RESULTS: It was revealed that TLR2, TLR4, and TLR5 ligands have enhanced stimulating effect on TNF alpha production by PBMC of children with congenital neutropenia and had no effect on PBMC of children with immune neutropenia. Children with immune neutropenia are characterized by significantly increased IFN alpha production induced by ligands of TLR3, TLR8, and TLR9. CONCLUSION: Revealed changes of TLR-mediated functional activity of PBMC from children with various forms of neutropenia may play significant role in development and course of infections in these patients.

[Corrective effect of cyclooxygenase inhibitor on functional status of mononuclear cells expressing Toll-like receptors].

AIM: To study the influence of the COX inhibitor--lornoxicam (LX)--on Toll-like receptor (TLR)-mediated production of proinflammatory and anti-inflammatory cytokines by peripheral blood mononuclear cells (PBMC) from healthy subjects and patients with acute pancreatitis (AP) in vitro. MATERIALS AND METHODS: Cytokine production by PBMC of healthy donors was stimulated by TLR1/2 ligand peptidoglycan (PG) and TLR4 ligand lypopolysaccharide (LPS) in presence of LX. Levels of cyotokines (IL-1beta, IL-6, IL-8, IL-10, IL-12, and TNFalpha) were measured by ELISA. Group of patients with acute pancreatitis of toxic etiology included 11 subjects: patients from main group received combined therapy supplemented with NSAID from the oxicam class--LX; patients who received only standard basic treatment formed comparison group. RESULTS: It was found that in vitro LX inhibits production of both proinflammatory and anti-inflammatory cytokines by PBMC of healthy subjects mediated by ligands of TLR1/2 and TLR4. Maximal inhibitory effect of LX was observed when cytokine production was induced through TLR1/2. Patients with AP demonstrated increased production of TNFalpha induced by TLR1/2 and TLR4 ligands. CONCLUSION: LX inhibits TLR-mediated production of both proinflammatory (IL-1, IL-6, IL-8, IL-12, TNFalpha) and anti-inflammatory (IL-10) cytokinesby PBMC of healthy subjects in vitro. Treatment with LX in patients with AP results in diminished effector function of TLR1/2 and TLR4 already during 1st day of the illness and normalization of these indices by 6th day.

[Use of multiplex PCR method with real-time detection for differential diagnosis of respiratory viral infections].

Multiplex real-time polymerase chain reaction test-system with fluorescent detection (RT-PCR) for simultaneous identification of main agents of acute respiratory viral infections: influenza A (IAV) and B viruses (IBV), parainfluenza viruses types 1, 2, 3, 4 (PIV 1 - 4), adenoviruses (ADV), respiratory syncitial virus (RSV), rhinoviruses (RV) and enteroviruses (EV), in presence internal positive control (IPC) represented by vaccine strain of rubella virus RA 27/3. Using multiplex RT-PCR method, respiratory viruses were detected in 116 out of 226 clinical samples (nasal swabs) obtained from patients with symptoms of acute respiratory infection: in 68 (58.6%) samples--IBV; in 21 (18.1%)--IAV; in 12 (10.3%) --RV; in 6 (5.2%)--PIV 2; in 4--(3.4%) ADV; in 3 (2.6%)--RSV; in 2 (1.7%)--EV; in 2 (1.7%)--PIV 4; in 1 (0.9%)--PIV 3; in 1 (0.9%)--PIV 1. Mixed infection was observed in 4 (3.4%) patients. PCR assay allowed to reveal various respiratory viruses in 51.3% of samples. At the same time samples were tested for the presence of 12 respiratory viruses--IAV, IBV, PIV 1 - 4, RSV, RV, metapneumoviruses, and coronaviruses NL63, 229E and OC43--in the presence of IPC represented by equine arteritis virus using analogous PCR test-system provided by medical center of Leiden university. Results of tests for detection of IAV, IBV, RSV, PIV 1 - 4, and RV, analyzed by both systems, agreed in 94%. Multiplex format of RT-PCR performing significantly reduces time and cost of the test, which make it suitable and effective instrument of epidemiological studies.

Diversity of PEGylation methods of liposomes and their influence on RNA delivery.

Gene therapy is a promising approach for personalized medicine, but its application in humans requires development of efficient and safe vehicles. PEGylated liposomes are some of the most suitable delivery systems for nucleic acids because of their stability under physiological conditions and prolonged circulation time, compared to conventional and other types of "stealth" liposomes. In vitro/in vivo activity of PEGylated liposomes is highly dependent on PEG motif abundance. The process of "stealth" coverage formation is a very important parameter for efficient transfection assays and further fate determination of the PEG layer after tissue penetration. In this review, we discuss the latest methods of PEGylated liposome preparation.

Experimental protocol for development of adjuvant-free murine chronic model of allergic asthma.

BACKGROUND: Mouse models of allergic asthma play a crucial role in exploring of asthma pathogenesis and testing of novel anti-inflammatory drugs. Widely used acute asthma models usually developed with adjuvant (aluminum hydroxide (alum)) do not reproduce one of the main asthma feature - airway remodeling while chronic asthma model mimic the pathophysiology of human disease. Moreover, the use of alum causes distress in experimental animals and impedes the test of adjuvant-containing drugs. In this study, we aimed to develop a chronic adjuvant-free asthma model with pronounced asthmatic phenotype. METHODS: Female BALB/c mice were divided into 3 groups. The first group was sensitized with intraperitoneal injections of ovalbumin (OVA) emulsified in aluminum hydroxide on days 0, 14, 28 followed by two stages of intranasally challenge with OVA on days 41-43 and 62-64. The second group was subcutaneously sensitized with the same dose of OVA without adjuvant and challenged on the same days. The third group (negative control) included mice which did not received any kind of treatment (i.e. sensitization and challenge). Serum levels of OVA-specific IgE, IgG2a and IgG1 antibodies were detected by ELISA. Airway hyper-responsiveness was measured by non-invasive plethysmography on days 44 and 65. Bronchoalveolar lavage fluids (BALF) sampled in all groups on days 45 and 66 were analyzed by light microscopy. The left lung was removed for histological analysis. The IL-4 and IFNγ mRNA expression in BALF cells was evaluated by RT-PCR. RESULTS: The OVA-specific IgE antibody response was two-fold increased in mice from adjuvant-free group compared to the adjuvant group that reflects reorientation of immune response towards Th2 phenotype. At the same time, the level of OVA-specific IgG1 and IgG2a antibodies was increased in the adjuvant group. Airway hyperresponsiveness to methacholine in mice of both experimental groups was two-fold higher than in control. Analysis of cell composition in BAL has shown a significant increase in eosinophil count in both experimental groups that indicate the development of allergic inflammation. Lung histology revealed airway remodeling in both experimental groups including goblet cell hyperplasia/metaplasia, thickening of airway walls, collagen deposition in the wall of distal airways. Additionally, the tendency to develop hypertrophy of bronchial smooth muscle layer was observed. Study of gene expression in BAL cells revealed the increase of IL-4 level in both adjuvant and adjuvant-free groups while IFNγ expression in both experimental groups was similar to control group. CONCLUSION: We have developed a chronic adjuvant-free mouse asthma model which possesses all necessary features of the disease including airway remodeling and is more suitable for pre-clinical evaluation of novel therapeutic approaches including adjuvant-containing drugs.