Anti-Inflammatory Effect of Technologically Processed Antibodies to the Molecules of the Major Histocompatibility Complex in a Model of Acute Inflammation In Vivo.

The anti-inflammatory effect of technologically processed antibodies (TPA) to immune targets (MHC I and MHC II) was assessed in the carrageenan-induced rat paw edema model. The parameters "increase in edema" and "suppression of edema" significantly decreased (p<0.05) against the background of treatment with TPA and the reference drug indomethacin compared to the placebo group. The tested TPA produced an anti-inflammatory effect.

Antiallergic Effects of Technologically Processed Antibodies to MHC II.

The risk of developing anaphylactic reactions to medications introduces additional difficulties for effective pharmacotherapy. Using a model of systemic anaphylaxis in mice, we showed that preventive administration of a preparation containing technologically processed antibodies (TPA) to MHC II induces an anti-anaphylactic effect comparable to that of dexamethasone (when assessing the severity of systemic anaphylaxis 30 and 60 min after challenge injection of the model antigen ovalbumin). The revealed activity may be related to the ability of TPA to MHC II to regulate the antigen presentation system and shift the immune response towards the production of IgG instead of IgE typical of anaphylactic reaction.

Efficacy of Raphamin against Pneumococcal Infection: a Preclinical Study.

The aim of the study was to evaluate the activity of Raphamin in a model of non-lethal pneumococcal infection caused by Streptococcus pneumoniae 3 in BALB/c mice. The drug or placebo was administered intragastrically 3 days prior to infection, 2 h before and 2 h post infection, and then for 3 full days, alone or in combination with antibiotic (amoxicil-lin/clavulanic acid). Raphamin monotherapy significantly decreased bacterial load in the lungs in comparison with placebo (p<0.05) which was comparable to the effect in antibiotic alone or combined with Raphamin. Raphamin prevented reproduction of Streptococcus pneumoniae in the lower respiratory tract and its combination with the antibiotic was safe and did not reduce the efficacy of amoxicillin/clavulanic acid.

Combined Drug with Antibacterial Effect Supports the Normal Intestinal Microflora.

Widespread use of antibiotics leads to an imbalance of normal intestinal microflora and to the development of multidrug resistance. The problem can be solved by administration of the antibiotics in combination with the drugs that have an immunotropic effect. We studied the effect of the drug containing technologically processed affinity purified antibodies to IFNγ, CD4 receptor, ß2-microglobulin of MHC class I, and ß2-domain of MHC II combined with antibiotics on the composition of intestinal microflora of pigs and the total number of microbiome resistance genes. Using the methods of NGS sequencing and quantitative PCR, we found that the drug contributes to the maintenance of normal microflora and, consequently, to the symbiotic relationship of the host with microflora, and prevents the reproduction of pathogenic bacterial species. Analysis for the presence of the resistance genes of gastrointestinal microorganisms showed that the drug does not affect the qualitative and quantitative composition of these genes of the intestinal microbiome.

Nootropic Activity of Prospekta in a Blind Placebo-Controlled Study in a Model of Focal Cerebral Ischemia in Rats.

Analysis of the pharmacological activity of the original drug Prospekta in a rat model of focal cerebral ischemia revealed its nootropic effect: course treatment in the post-ischemic period led to recovery of the neurological status of animals at the peak of neurological deficit. Evaluation of the therapeutic potential of the drug in morphological and functional CNS disorders allowed us to conclude that it is advisable to carry out further studies of its biological activity at the preclinical stage (the results obtained in animals were successfully confirmed in a clinical trial of drug efficacy in the treatment of moderate cognitive disorders in the early recovery period after ischemic stroke). Studies of the nootropic activity in other pathologies of the nervous system are also promising.

[Molecular genetics in diagnosis of Coats disease: combination of oligogenic variants associated with different forms of hereditary retinal dystrophy]. / Molekulyarno-geneticheskie nakhodki pri diagnostike bolezni Koatsa: sochetanie oligolokusnykh izmenenii, svyazannykh s raznymi nozologicheskimi formami nasledstvennoi retinopatii.

Coats disease (OMIM 300216) is a form of hereditary retinal dystrophy, which occurs due to congenital abnormality of retinal vessels and features unilateral exudative vitreoretinopathy. Coats disease mostly occurs sporadically; its genetic cause is still undetermined. Molecular genetic research including whole exome sequencing by the NGS method was used to define a genetic cause of the observed phenotype. Two heterozygous variants in different genomic loci associated with other forms of hereditary retinal dystrophy were detected, a rare variant in the HMCN1 gene c.9571C>T, p.(Arg3191Cys), and a known pathogenic variant in the NPHP4 gene c.2930C>T, p.(Thr977Met). The HMCN1 gene is responsible for dominant age-related macular degeneration (OMIM 603075), pathogenic variants in the NPHP4 gene cause recessive Senior-Løken syndrome 4 (OMIM 266900). These genes encode the proteins that are involved in the regulation of integrity of the blood-retinal barrier in the vascular endothelium (NPHP4) and retinal pigment epithelium (HMCN1). The identified mutation in the NPHP4 gene could lead to decreased function of the NPHP4 protein and contribute to the development of retinal degeneration, potentially of oligogenic nature.

Antiviral Activity of Technologically Processed Antibodies to CD4 Receptor against Influenza Infection.

The antiviral activity of technologically processed antibodies to CD4 receptor was evaluated a model of sublethal A/California/04/2009 (H1N1)pdm09-induced influenza infection in female BALB/c mice. The technologically processed antibodies increased animal survival rate by 50% in comparison with the placebo group (p<0.05), which correlated with significant inhibition of virus replication in the lungs (p<0.05). The reference drug Tamiflu increased mouse survival rate (by 47%), decreased the virus titer in the lungs, and prevented body weight loss (p<0.05 in comparison with the placebo group by all parameters). The intrinsic protective activity of technologically processed antibodies to CD4 receptor was demonstrated, which manifested in a decrease in viral load in the lower respiratory tract and an increase in the survival rate.

[Susceptibility of animal species to experimental SARS-CoV-2 (Coronaviridae: Coronavirinae: Betacoronavirus; Sarbecovirus) infection].

Due to the new coronavirus infection pandemic, the global scientific community has been forced to change the direction of the most research, focusing on vaccine development as well as the search for new antiviral drugs to treat COVID-19. The choice of experimental models, timeframe and approaches for evaluating drugs and vaccines under development is crucial for the development of effective measures to prevent and control this disease.The purpose of this review was to summarize the relevant data concerning the susceptibility of laboratory animals to SARS-CoV-2. This paper describes the most virus-susceptible animal species that can be used to reproduce coronavirus infection, stressing the main advantages and disadvantages of each of them.According to the latest data, small rodents (Rodentia) and non-human primates (Strepsirrhini) are commonly used in the scientific community to model coronavirus infection. The viral load in the upper and lower parts of the respiratory system, clinical symptoms of infection (weight loss, body temperature and general health status), pathomorphological picture in target organs and the production of antibodies after infection are considered to the main markers of pathology. Despite the vast amount of data, none of the described models of SARS-CoV-2 infection may be considered a gold standard, since they do not reproduce all spectrum of morphological and pathogenetic mechanisms of infection, and do not fully reflect the clinical picture observed in patients in human population.Based on the analyzed literature data, we suppose that Syrian hamster (Mesocricetus auratus) and mice (Muridae) expressing the angiotensin converting enzyme receptor 2 (ACE2) are the most suitable animal species for their use in experiments with SARS-CoV-2 infection. The development of neutralizing antibodies makes it possible to evaluate the efficacy of vaccines, while the course and severity of symptoms infection makes the use of mice and hamsters especially popular for screening pharmacological substances with antiviral mechanism of action, when their administration can prevent or slow the disease progression.

Human Umbilical Cord Tissue-Derived Multipotent Mesenchymal Stromal Cells Exhibit Maximum Secretory Activity in the Presence of Umbilical Cord Blood Serum.

Using multiplex analysis, we performed a comparative study of cytokine and growth factor production by human umbilical cord tissue-derived multipotent mesenchymal stromal cells (UC-MSC) cultured under standard conditions and in the presence of human umbilical cord blood serum (UCBS). It was found that the secretion of most studied molecules, including well-known inductors of regeneration HGF, G-CSF, GM-CSF, and VEGF by UCMSC considerably increased in the presence of 5% UCBS. The use of UCBS allows not only obtaining xenogenic-free cellular and cell-free therapeutic products, but also increasing the secretion of most biologically active molecules capable of stimulating repair processes.

L-Ascorbic Acid in the Epigenetic Regulation of Cancer Development and Stem Cell Reprogramming.

Recent studies have significantly expanded our understanding of the mechanisms of L-ascorbic acid (ASC, vitamin C) action, leading to the emergence of several hypotheses that validate the possibility of using ASC in clinical practice. ASC may be considered an epigenetic drug capable of reducing aberrant DNA and histone hypermethylation, which could be helpful in the treatment of some cancers and neurodegenerative diseases. The clinical potency of ASC is also associated with regenerative medicine; in particular with the production of iPSCs. The effect of ASC on somatic cell reprogramming is most convincingly explained by a combined enhancement of the activity of the enzymes involved in the active demethylation of DNA and histones. This review describes how ASC can affect the epigenetic status of a cell and how it can be used in anticancer therapy and stem cell reprogramming.

Human Umbilical Cord Blood Serum/Plasma: Cytokine Profile and Prospective Application in Regenerative Medicine.

The concentrations of cytokines and growth factors in human umbilical cord blood serum and plasma samples were measured by multiplex analysis. It was found that in comparison with peripheral blood serum of adult donors, umbilical cord blood serum and plasma contain significantly higher concentrations of the most studied molecules including IL-4, 5, 6, 7, 10 and 15, MCP-1, SCF, and SDF, as well as growth factors directly involved in the processes of regeneration (G-CSF, HGF, PDGF-BB, and VEGF). Thus, umbilical cord blood plasma and especially serum are a rich source of cytokines and growth factors with anti-inflammatory, anti-apoptotic, and angiogenic effects and can be used in various fields of regenerative medicine.

Spectrum of CFTR mutations in Chechen cystic fibrosis patients: high frequency of c.1545_1546delTA (p.Tyr515X; 1677delTA) and c.274G>A (p.Glu92Lys, E92K) mutations in North Caucasus.

BACKGROUND: Cystic fibrosis (CF; OMIM #219700) is a common autosomal recessive disease caused by pathogenic variants (henceforward mutations) in the cystic fibrosis transmembrane conductance regulator gene (CFTR). The spectrum and frequencies of CFTR mutations vary among different populations. Characterization of the specific distribution of CFTR mutations can be used to optimize genetic counseling, foster reproductive choices, and facilitate the introduction of mutation-specific therapies. Chechens are a distinct Caucasian ethnic group of the Nakh peoples that originated from the North Caucasus. Chechens are one of the oldest ethnic groups in the Caucasus, the sixth largest ethnic group in the Russian Federation (RF), and constitute the majority population of the Chechen Republic (Chechnya). The spectrum of CFTR mutations in a representative cohort of Chechen CF patients and healthy individuals was analyzed. METHODS: Molecular genetic analysis of 34 CFTR mutations (representing approx. 80-85% of mutations in multiethnic CF populations of the RF) was performed in 32 CF patients from 31 unrelated Chechen families living in Chechnya. One hundred randomly chosen healthy Chechens were analyzed for the 15 most common "Russian" mutations. The clinical symptoms in Chechen CF patients with different CFTR genotypes were investigated. RESULTS: High frequencies of c.1545_1546delTA (p.Tyr515X; 1677delTA) (52 out of 64 CFTR alleles tested; 81.3%) and c.274G > A (p.Glu92Lys, E92K) (8/64, 12.5%) mutations were found. Twenty patients were homozygous for the c.1545_1546delTA mutation, and eight were compound heterozygous for the c.1545_1546delTA and c.274G > A mutations. Three carriers of the c.1545_1546delTA mutation were also found in the cohort of 100 apparently healthy Chechens (frequency - 0.015). The c.1545_1546delTA and c.274G > A mutations are linked to the same haplotype (22-7-16-13) of intragenic Short Tandem Repeat markers, i.e., IVS1CA, IVS6aGATT, IVS8CA, and IVS17bCA. CONCLUSIONS: The distribution of CFTR mutations in the Chechen CF population is unique regarding the high frequency of mutations c.1545_1546delTA and c.274G > A (more than 90% of the mutant alleles). The c.274G > A mutation is associated with a less severe course of CF than that observed in c.1545_1546delTA homozygotes. Testing for these two variants can be proposed as the first step of CF DNA diagnosis in the Chechen population.

Genetic and Epigenetic Mechanisms of ß-Globin Gene Switching.

Vertebrates have multiple forms of hemoglobin that differ in the composition of their polypeptide chains. During ontogenesis, the composition of these subunits changes. Genes encoding different α- and ß-polypeptide chains are located in two multigene clusters on different chromosomes. Each cluster contains several genes that are expressed at different stages of ontogenesis. The phenomenon of stage-specific transcription of globin genes is referred to as globin gene switching. Mechanisms of expression switching, stage-specific activation, and repression of transcription of α- and ß-globin genes are of interest from both theoretical and practical points of view. Alteration of balanced expression of globin genes, which usually occurs due to damage to adult ß-globin genes, leads to development of severe diseases - hemoglobinopathies. In most cases, reactivation of the fetal hemoglobin gene in patients with ß-thalassemia and sickle cell disease can reduce negative consequences of irreversible alterations of expression of the ß-globin genes. This review focuses on the current state of research on genetic and epigenetic mechanisms underlying stage-specific switching of ß-globin genes.

[Comparative analysis of the synchronization methods of normal and transformed human cells].

Reactions of genetically identical cells to various exogenous and endogenous stimuli can vary significantly. One of the main factors of this non-genetic cellular heterogeneity is the cell cycle. The most convenient way to study the subcellular processes depending on the cell cycle stage is the synchronization of the cells. Toxic inhibitors of DNA replication and/or mitotic spindle assembly are typically used to synchronize cells. It is important to accurately select the synchronization method for a particular experiment. In this study, we performed a comparative analysis of the synchronization methods of normal and transformed human cells, paying special attention to the accuracy of synchronization and toxicity of the methods used.

Effects of Release-Active Antibodies to CD4 Receptor on the Level of lck-Kinase in Cultured Mononuclear Cells from Human Peripheral Blood.

For evaluation of effects of release-active antibodies to CD4 on cultured lymphocytes from human peripheral blood, we measured intracellular content of lck-kinase cell-based ELISA. In cells treated with release-active antibodies to CD4, the content of intracellular lck-kinase significantly (p<0.01) decreased in comparison with the control (purified water processed in a similar way). Phytohemagglutinin had no effect on the concentration of lck-kinase in cells. The decrease in the content of CD4-associated lck protein suggests that the preparation enhanced intracellular coupling of lck-kinase with T-cell receptor and potentiated T-cell immune response.

[The experience of application of target sequencing in molecular diagnostic of mucoviscidosis.]

The mucoviscidosis is one of frequent monogenic diseases. In Russia, in case of mucoviscidosis carrying out of DNA-diagnostic is optional. However, its application permits shortening time of diagnosing, increasing efficiency of of therapeutic treatment and preventing secondary manifestation of disease in family. The DNA-diagnostic using panels on frequent mutations in gene CFTR is recommended in cases of uncertain clinical picture and under borderline values of specific laboratory indices. In Russia, application of such panels permit detecting up to 90% of pathological alleles in gene CFTR. To detect more rare alleles the Sanger sequencing is traditionally applied. Lately, highly productive sequencing techniques became available to detect rare mutations. The actual article presents evaluation of efficiency of application of test-system based on technology of target sequencing for detecting mutations unidentified at primary DNA-diagnostic. Besides, in two patients with mucoviscidosis the application of highly productive sequencing techniques permitted to identify previously unknown nonsense mutations Q1038X (c.3112C>T) и W1310X (c.3930G>A).

Light-induced antibacterial activity of electrospun chitosan-based material containing photosensitizer.

Increasing antimicrobial resistance requires the development of novel materials and approaches for treatment of various infections. Utilization of photodynamic therapy represents an advanced alternative to antibiotics and metal-based agents. Here, we report the fabrication of electrospun material that possesses benefits of both topical antimicrobial and photodynamic therapies. This material combines chitosan, as a biocompatible polymer, and a second generation photosensitizer. The incorporation of photosensitizer doesn't affect the material morphology and its nearly uniform distribution in fibers structure was observed by confocal Raman microscopy. Owing to photosensitizer the prepared material exhibits the light-induced and spatially limited antimicrobial activity that was demonstrated against Staphylococcus aureus, an important etiological infectious agent. Such material can be potentially used in antibacterial therapy of chronic wounds, infections of diabetic ulcers, and burns, as well as rapidly spreading and intractable soft-tissue infections caused by resistant bacteria.

Photosensitizer-loaded electrospun chitosan-based scaffolds for photodynamic therapy and tissue engineering.

Novel chitosan-based nanofibrous composite materials containing different amounts of the photosensitizer Photosens were obtained by electrospinning and were characterized by scanning electron microscopy and by confocal laser scanning microscopy. The release of Photosens from the materials was investigated in water and in phosphate-buffered saline. A noncancerous (MC3T3-E1 murine osteoblasts) and a cancerous [T-47D (mammary gland)] cell line were cultivated on Photosens-containing scaffolds, and cell growth and metabolic activity were examined by confocal laser scanning microscopy and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide assay, respectively. The viability of both cell lines on Photosens-containing fibers decreased in a spatial manner upon laser irradiation of an appropriate wavelength and power density. Interestingly, the noncancerous MC3T3-E1 cells grown on Photosens -containing scaffolds were less affected by the irradiation. We conclude that the Photosens-containing electrospun chitosan nanofibers described here are of potential interest for biomedical applications, particularly topical photodynamic therapy and tissue engineering.

Binding of Protein Factor CTCF within Chicken Genome Alpha-Globin Locus.

A systematic search for DNA fragments containing potential CTCF transcription factor binding sites in the chicken alpha-globin domain and its flanking regions was performed by means of the two-dimension electrophoretic mobility shift assay. For the alpha-globin domain fragments selected, the occupancy by the CTCF in erythroid and lymphoid chicken cells was tested by chromatin immunoprecipitation. Only one of 13 DNA fragments capable of CTCF binding in vitro was efficiently bound to this protein in vivo in erythroid cells, and somewhat less efficiently - in lymphoid cells. So, binding of CTCF to the DNA fragment in vitro in most cases does not mean that this fragment will be occupied by CTCF in the cell nucleus. Yet, CTCF binding in vivo, as a rule, is accompanied by the binding of the protein to this DNA region in vitro. During the erythroid differentiation, no significant changes in CTCF binding to the DNA fragments studied were detected.