Composition of Bacteriophages for Therapeutic and Prophylactic Use in Monkeys.

Keeping rhesus monkeys as laboratory animals requires timely prevention and treatment of infections, including diseases of bacterial etiology. Based on our own studies of the microflora of healthy and sick monkeys, as well as analysis of published reports, we identified clinically significant representatives of pathogenic and opportunistic bacteria: E. coli, Staphylococcus aureus, Klebsiella spp., Proteus spp. The isolates of these bacterial species and genera circulating in monkeys kept in the enclosure were isolated, four virulent bacteriophage strains with a wide spectrum of lytic activity against these isolates were selected and newly isolated. The composition based on virulent bacteriophage strains was tested on monkeys with assessment of its safety and its dynamics of detection of phage-specific DNA.

[Adiponectin in normal and atherosclerotic intima of human aorta]. / Adiponektin v normal'noi i ateroskleroticheski izmenennoi intime aorty cheloveka.

BACKGROUND: Adiponectin (AN) is a protein synthesized by adipocytes that has regulatory effects on lipid and lipoprotein metabolism, increases tissue sensitivity to insulin, and modulates endothelial functions and inflammatory response. However, its involvement in the processes of atherogenesis remains poorly understood. OBJECTIVE: To determine the localization and sources of AN in atherosclerotic and normal human aortic intima. MATERIAL AND METHODS: Immunohistochemical study was performed on sections of atherosclerotic and normal human aorta obtained during autopsy. Reverse transcription real-time PCR was performed using biopsies of para-aortic and abdominal adipose tissue, intima-media of the thoracic aorta, atherosclerotic plaques of the human carotid and femoral arteries, as well as on endothelial cells isolated from the human thoracic aorta. Transendothelial transport of AN was evaluated in a two-chamber model using a monolayer of human endothelial cell hybridoma EA.Hy926. RESULTS: It has been established that AN is present in atherosclerotic but not in normal human aortic intima. At the same time, AN ADIPOQ mRNA was not detected either in the intima media of the human aorta, nor in isolated endothelial cells of the aorta, nor in cells of atherosclerotic plaques of the carotid and femoral arteries. AN slowly penetrated the endothelial monolayer in vitro, but this transport was significantly enhanced by the action of tumor necrosis factor-alpha (TNFa). CONCLUSION: Obtained data indicate that AN is present in atherosclerotic but not in normal aortic intima. We assume that AN is not synthesized by the cells of normal and atherosclerotic arterial walls, but permeates from the plasma. Transendothelial transport of AN, like many other plasma proteins, is activated during the development of atherosclerotic lesions, apparently under the action of pro-inflammatory cytokines, in particular, TNFα.

[The Influence of Adiponectin on Production of Apolipoproteins A-l and E by Human Macrophages].

Adiponectin is an adipose tissue hormone affecting energy and lipoprotein metabolism and modulating inflammatory responses. However, the role of this adipokine in atherogenesis remains poorly understood. The aim of this study was to investigate the effect of adiponectin on the production of apolipoproteins (apo) A-l and E by human macrophages (MP). The study was conducted on macrophage-like cells of the THP-1 cell line of two differentiation terms, 3 and 5 days (3d and 5d). Adiponectin (10 µg/mL) stimulated the expression of apoA-1 gene at the mRNA level in 5d MP, but not in 3d MP. The level of apoE mRNA in MP under the action of adiponectin was not affected. Adiponectin suppressed macrophage TNF gene expression, while it induced the expression of IL-10 gene in 5d MP. The secreted levels of apoA-1 and apoE proteins under the action of adiponectin in macrophages of both periods of differentiation remained unchanged, while the level of the surface apoA-1 protein in 5d MP was decreasing. Incubation of 5d MP with the PPARα nuclear receptor antagonist MK-886 or with the nuclear receptor LXR agonist TO-901317 resulted in cancellation of the stimulating effect of adiponectin on apoA-1 gene expression. These data indicate that adiponectin, in addition to its anti-inflammatory action, has a modulating effect on production of apoA-1 by macrophages. The latter is probably one of the mechanisms of the influence of this adipokine on atherogenesis.

[Urinary miRNA expression in cynomolgus macaques (Macaca fascicularis) fed high salt rations].

High food intake of sodium chloride is associated with damage not only the cardiovascular system, but also the kidneys. The mechanisms of the potential negative effects of high-salt diets on the kidneys have not been established. The aim of the study was to trace the changes in relative expression of miRNA-21, 203 and 133 in urine of cynomolgus macaques (Macaca fascicularis) fed high-salt diet with and without isolated soy proteins. Material and methods. The object of the study was 18 male cynomolgus macaques (Macaca fascicularis) aged 6-8 years with a body weight of 5.1-9.7 kg. The animals were divided in 3 groups (6 individuals each). The animals of the first (control) group received a standard diet (2 g NaCl/kg feed). The animals of the second group were fed high-salt diet (8 g NaCl/kg feed), of the third - high-salt diet combined with SUPRO 760 isolated soy protein (200 g/kg feed; instead of milk and egg proteins, corn gluten). Access to water was free. The follow-up period in this study was 4 months. In animals blood pressure (BP) and relative level of microRNA (miRNA) expression in urine were measured. Results and discussion. Keeping monkeys on the studied diets for 4 months did not lead to significant changes in systolic or diastolic BP compared with the initial level. In the control group, there were no distinct changes in the expression of miRNA-21 in urine during observation. In the other two groups, there was a significant increase (approximately equally) of this parameter in comparison with the initial values. Both high-salt diets resulted in a significant increase in the relative level of expression of miRNA-133 and miRNA-203 in urine compared to basal values. However, the increase in these parameters in the group of animals fed a high-salt diet in combination with soy isolate was significantly less than in monkeys fed only a high-salt diet. Conclusion. Possible, potentially negative effects of high-salt diets on kidney may be mediated by epigenomic mechanisms and partially modulated by the inclusion of isolated soy proteins in the diet.

Hypoxia as a Factor Involved in the Regulation of the apoA-1, ABCA1, and Complement C3 Gene Expression in Human Macrophages.

Hypoxia plays a critical role in progression of atherosclerosis. Local oxygen deficiency in a plaque creates a specific microenvironment that alters the transcriptome of resident cells, particularly of macrophages. Reverse cholesterol transport from plaque to liver is considered a main mechanism for regression of atherosclerosis. Ubiquitously expressed ATP-binding cassette transporter A1 (ABCA1) and liver- and small intestine-derived apolipoprotein A-1 (ApoA-1) are two main actors in this process. We recently reported endogenous apoA-1 expression in human macrophages. While ABCA1 and ApoA-1 have antiatherogenic properties, the role of complement factor C3 is controversial. Plasma C3 level positively correlates with the risk of cardiovascular diseases. On the other hand, C3 gene knockout in a murine atherosclerosis model increases both plaque size and triglycerides level in blood. In the present study, we show for the first time that a hypoxia-mimicking agent, CoCl2, induces the upregulation of the apoA-1 and C3 genes and the accumulation of intracellular and membrane protein ApoA-1 in THP-1 macrophages. The MEK1/2-Erk1/2 and MKK4/7-JNK1/2/3 cascades are involved in upregulation of ABCA1 and C3 via activation of transcription factor NF-κB, which interacts with the HIF-1α subunit of hypoxia-inducible factor 1 (HIF-1). The three major MAP-kinase cascades (Erk1/2, JNK1/2/3, and p38) and the NF-κB transcription factor are involved in the hypoxia-induced expression of the apoA-1 gene in THP-1 macrophages.

[Characterization of Distal and Proximal Alternative Promoters of the Human ApoA-I Gene].

Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoprotein (HDL). ApoA-I constitutes ~75% of the protein content of HDL. The main sites of ApoA-I synthesis in humans are the liver and the small intestine. The mechanisms that govern tissue-specific apoA-I transcription in tissues and organs other than the liver and the small intestine are poorly understood. It is known that the human apoA-I has two additional promoters, the proximal and the distal one. In this work these two alternative apoA-I promoters are characterized, their transcription start sites are mapped and their competition for apoA-Itranscription is demonstrated; the role of the alternative promoters in apoA-I expression in human cells and tissues other than hepatocytes and enterocytes is discussed.

In Vivo Experimental Study of Biological Compatibility of Tissue Engineered Tracheal Construct in Laboratory Primates.

Biological compatibility of a tissue engineered construct of the trachea (synthetic scaffold) and allogenic mesenchymal stem cells was studied on laboratory Papio hamadryas primates. Subcutaneous implantation and orthotopic transplantations of tissue engineered constructs were carried out. Histological studies of the construct showed chaotically located filaments and mononuclear cells fixed to them. Development of a fine connective tissue capsule was found at the site of subcutaneous implantation of the tissue engineered construct. The intact structure of the scaffold populated by various cell types in orthotopic specimens was confirmed by expression of specific proteins. The results indicated biological compatibility of the tissue engineered construct with the mesenchymal stem cells; no tissue rejection reactions were recorded; simulation of respiratory disease therapy on Papio hamadryas proved to be an adequate model.

Evaluation of the influence of decellularization on the changes in biomechanical properties of primate lungs.

The effect of decellularization on the biomechanical properties of macaque lungs was studied. The quality of the biological scaffold was additionally assessed by morphological methods, and the contents of extracellular matrix (ECM) fibers were determined both qualitatively and quantitatively. Histological analysis revealed no damage of structural integrity of ECM components, but the scaffold elasticity significantly decreased, which was confirmed by the changes in the hysteresis loop without a concomitant decrease in peak loads, with the mechanical strength of the samples being retained. These changes require taking additional measures to prevent a decrease in the effective lung volume.

[Preclinical study of safety of the new pharmacological substance AV0012 to treat hepatitis C].

Pharmacological safety of a new type of HCV inhibitor, AV0012, was studied including acute, subchronic and chronic toxicity in mice, rats and monkeys. Genotoxicity was assessed using the Ames test and the chromosomal aberrations assay in the bone marrow cells of mice. It is established that AV0012 has low toxicity in SHK line mice, Wistar line rats, and monkey of Rhesus macaques species. Results obtained in the study of genetic toxicity showed that AV0012 exhibits no mutagenic activity. Data on general toxicity and mutagenicity discussed in this paper, together with data on 1 the pharmacological activity, pharmacokinetics, and metabolism published previously, allow us to consider AV0012 as a candidate drug for clinical research phase I.

[Preclinical study of AV0012 early stage inhibitor of hepatitis C virus infection: I. In vitro ADME and pharmacokinetics].

In vitro immunohistochemical investigations on the human hepatoma cell line (Huh7) infected with hepatitis C virus (HCV) strain JFH-1 showed that AV0012 compound blocks the early stages of viral infection. AV0012 also blocked viral infection spread in tissue culture through the secreted virus and through tight cell-to-cell contact. AV0012 is a specific inhibitor of HCV but not of related pestivirus, flaviviruses and other RNA-containing viruses such as bovine diarrhea (BVDV), Venezuelan equine encephalitis (strain TC-83), dengue type 2 (New Guinea), yellow fever (strain 17D), west Nile fever, parainfluenza (type 3) virus, RSV (strain A2), and Rhinovirus (type 2 strain HGP). It is established that human serum does not significantly affect the antiviral activity of AV0012 in vitro. The drug combination studies with AV0012 and interferon alpha 2a in vitro showed that the two inhibitors act additively, which makes possible the use of this combination in clinical tests. AV0012 is highly soluble and stable in aqueous solutions and murine blood plasma, has limited metabolic stability, low binding to human plasma proteins, high permeability through biological membranes, and only interacts with isoenzymes 2D6 and 3A4 of human cytochrome P450. In animal pharmacokinetic studies, AV0012 was rapidly absorbed into the blood stream upon oral administration, showed sufficiently long half-elimination times, and had high oral bioavailability that reached 92% in monkeys. Further preclinical development of AV0012 is in progress.

[Cerebrovascular diseases and metabolic syndrome].

AIM: To study various aspects of cerebrovascular diseases (CVD) in the presence of metabolic syndrome (MS). SUBJECTS AND METHODS: A comprehensive clinical, laboratory, ultrasound, and neuroimaging study was conducted in 514 patients with symptomatic and asymptomatic atherosclerotic lesion of the internal carotid artery and MS. RESULTS: MS was found and proven to affect the following factors: a) the course and progression of carotid artery (CA) atherosclerotic lesion with transformation of its asymptomatic to symptomatic state; b) the structure and instability of an atherosclerotic plaque; c) the magnitude of blood theological changes; d) endothelial dysfunction; e) white matter changes; f) the clinical features of both acute and chronic CVD and the development of cognitive impairments. CONCLUSION: The association of the atherogenic activity of major components of MS, such as hyperinsulinemia, hypertension, dyslipidemia, and obesity, in the presence of dysregulated hemostasis and blood rheology substantially increases the risk of a progressive CA atherosclerotic process even in its asymptomatic course and accordingly favors the development and progression of different manifestations of CVD.

[On the possibilities of correction of changes of the gastrointestinal tract microbiota in patients with lung cancer treated receiving chemotherapy].

UNLABELLED: The aim of the study was the improvement of technologies of treatment of patients with the lung cancer receiving chemotherapy on the basis of using with the complex therapy of the combined probiotic based on the Bacillus subtilis strain. MATERIALS AND METHODS: 30 patients with the lung cancer receiving the first and second line of the first cycle of chemotherapy were included. The age of patients varied from 49 to 73 years, the average duration of the disease was 1 year. Patients of the main group (n = 21) received the combined probiotic based on the Bacillus subtilis strain together with the chemotherapy course. Patients of control group (n = 9) received only chemotherapeutic preparations. All patients were observed before and after treatment: the standardized inquiry for detection of intestinal complaints, microbiological research of feces (definition of qualitative and quantitative characteristics of gut microbiota), the research of metabolites of intestinal microorganisms in blood by the method of the gas-liquid chromatography - mass-spectrometry by G. A. Osipov's method. The efficiency of probiotic therapy was evaluated by results of studied indicators dynamics. RESULTS: Main symptoms of the intestinal dyspepsia were observed in patients with the lung cancer receiving chemotherapy such as constipation and intestinal microflora violations (decreased quantity of Lactobacillus, Bifidobacterium, Bacteroides and increased quantity of different pathogenic microorganisms). It was noted decreased rate of intestinal dyspepsia symptoms and improvement of intestinal microflora composition after the treatment course by the combined probiotic based on the Bacillus subtilis strain. CONCLUSION: Using of probiotic medicines with the chemotherapy in lung cancer patients is promising to reduce the frequency of gastrointestinal complaints and prevent deterioration of the gut microflora.

Spectrally resolved analysis of fluorescence blinking of single dye molecules in polymers at low temperatures.

We present a method for the spectrally resolved analysis of fluorescence blinking of single quantum emitters. It is based on the well-known technique of repeated recording of single-molecule (SM) fluorescence excitation spectra. The potential of our approach is presented for the example of single tetra-tert-butylterrylene molecules in an amorphous polymer matrix (polyisobutylene), which exhibit fluorescence blinking at cryogenic temperatures. Measuring the spectral dependence of the blinking statistics improves the possibility to clarify the microscopic nature of the dark state(s) of the emitters. We demonstrate how the blinking statistics can be definitely attributed to conformational changes in the local environment of a SM and how the parameters of the corresponding elementary excitations can be measured. The analysis of the blinking statistics as a function of the optical excitation frequency allows us to discriminate between photo-induced and spontaneous transitions into a dark state.

[Synthesis of GnRH analogs and their application in targeted gene delivery systems].

A set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized using solid phase peptide synthesis and chemical ligation technique. Selective chemical ligation was achieved as a result of hydrazone formation in the course of interaction between NLS hydrazide and GnRH analog modified by pyruvic acid. The efficiency of synthesized peptide carriers was demonstrated in experiments with human cancer cells transfected by reporter luciferase and beta-galactosidase genes or suicide HSV-1 thymidine kinase gene. It was shown that selectivity of action on cancer cells can be achieved as a result of peptide/DNA complex penetration through the cell membrane by GnRH receptor-mediated endocytosis pathway.

[Assessment of surgical correction of parameters of the vertebral-motor segment in traumas and degenerative diseases of the vertebral column].

Retrospective clinico-radial data obtained in 163 patients were compared with traumas and degenerative diseases of the vertebral column of those who were treated by decompressive and decompressive-stabilizing interventions. Characteristic alterations were determined such as considerably decreased area of the spinal canal (in 44.2% of the patients), intervertebral foramen (in 57.7%) and the volume of intervertebral canal (in 66.3%), which allowed specification of the character and volume of the operative interventions and objective assessment of adequacy of surgical correction.

[Expression of the starfish complement component C3 gene homologue under the influence of bacterial lipopolysaccharide].

The fragment of a homologue of complement component C3 gene has been cloned and sequenced from the starfish, Asterias rubens. Phylogenetic analysis of ArC3-like gene demonstrates that ArC3-like gene has close similarity to C3 gene homologues of Deuterostomia invertebrate animals. High level of ArC3-like gene expression was identified in circulating cells (coelomocytes), in a gut's derivate (hepatopancreas) and in male gonada but not in stomach, female gonad and rectal gland of A. rubens starfish. ArC3-like gene expression was shown in all types of starfish coelomocytes: in lymphocyte-like cells, granular and nongranular amebocytes. Injection of bacterial lipopolysaccharide (LPS) solution into the coelomic cavity of starfish leads to the increase of ArC3-like gene expression in coelomocytes and hepatopancreas over the control level of sterile sea water injection. The level of ArC3-like gene expression increased in response to LPS reaching the maximum 6 h after the stimulation, and decreased to basal level 24 h after the stimulation. Injection of LPS solution stimulated the increase of ArC3-like gene expression level in hepatopancreas reaching the maximum 6-12 h after the stimulation, and the level of mRNA of ArC3-like gene had still been increased 48 h after LPS injection. The data demonstrates sustained positive regulation of ArC3-like gene expression under the influence of LPS.

[GnRH analogues containing SV-40 virus T-antigen nuclear localization sequence].

To improve the efficiency of anticancer drugs due to their delivery to intracellular targets a set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized. NLS was attached to the parent molecule via ε-amino group of D-Lysine in position 1 or 6 of peptide sequence using orthogonal protection strategy. The biological activity studies revealed that incorporation of NLS moiety significantly increases cytotoxic activity of palmitoyl-containing GnRH analogues in vitro. The influence of tested peptides on tumor cells does not accompanied by the destruction of cell membrane, as confirmed in experiments with normal fibroblasts, used as a control.

[Non-viral gene therapy approach for regenerative recovery of skin wounds in mammals].

The rate and character of skin tissue regeneration after wounds, burns and other traumas depend on the cell proliferation within damaged area. Acceleration of healing by stimulation of cell proliferation and extracellular matrix synthesis is one of the most important tasks of modern medicine. There are gene therapy approaches to wound treatment consisting in the transfer of genes encoding mitogenic growth factors to wound area. The most important step in the development of gene therapy approaches is the design of gene delivery tools. In spite of high efficacy of viral vectors, the non-viral means have some preferences (low toxicity, low immunogenity, safety and the absence of backside effects). Among non-viral gene delivery tools, molecular conjugates are the most popular because of their efficacy, simplicity, and the capacity to the targeted gene transfer. In the present work we have developed two molecular conjugates--NLS-TSF7 and NLS-TSF12 consisting of the modified signal of nuclear localization of T-antigen of SV40 virus (cationic part) and the peptide ligands of mammalian transferrin receptor (ligand part). These conjugates bind to plasmid DNA with formation of polyelectrolytic complexes and are capable to deliver plasmid DNA into cells expressing transferrin receptors by receptor-mediated endocytosis. Transfer of the expression vector of luciferase gene in the complex with molecular conjugate NLS-TSF7 to murine surface tissues led to about 100 fold increasing of luciferase activity in comparison with the transfer of free expression vector. Treatment of slash wounds in mice with the complexes of expression vector of synthetic human gene encoding insulin-like growth factor 1 with molecular conjugates NLS-TSF7 led to acceleration of healing in comparison with mice treated with free expression vector. The results obtained confirm the high efficiency of the developed regenerative gene therapy approach for the treatment of damaged skin tissues in mammals.

[Transfer of genetic constructions through the transplacental barrier into mice embryos].

Genetic modification of mammalian embryos is an important way to model various changes in human development; also, it is an instrument for studying the functions of certain genes in mammals. Using our own experience in developing modes of delivery of genetic constructions to mammals in a nonviral way, we present here data on the delivery of a eukaryotic expression vector to mice embryos through the transplacental barrier with the use of hydrodynamic intravenous injections of DNA-hybrid peptide complexes to pregnant females. The peptide has a cationic part for interaction with DNA and includes a ligand structure towards receptors of the releasing factor of luteinizing hormone (RFLH, luliberin). Advantages of the suggested method are simplicity, economy, nonimmunogenicity for females, and the ability to multiply repeat the procedure. On the basis of the method, systemic gene delivery into tissues of mammalian embryos may be developed.

[Chronic cerebrovascular diseases, metabolic syndrome and the status of hemorrheological and hemostatic systems].

AIM: to study an association between metabolic syndrome and endothelial dysfunction as a regulator of hemorrheological and hemostatic processes in patients with chronic forms of cerebral circulatory insufficiency. SUBJECTS AND METHODS: Forty-six patients with chronic cerebrovascular diseases (CCVD) were examined; of them 23 patients were diagnosed as having metabolic syndrome (MS). Clinical manifestations and major hemorrheological and hemostatic parameters, such as platelet and erythrocyte aggregation, fibrinogen, hematocrit, von Willebrand factor, antithrombin III, intercellular adhesion molecules (IAM), etc., were estimated. Endothelial dysfunction was studied from the data of cuff test (CT). RESULTS: MS promotes a higher degree of clinical symptomatology in patients with CCVD and more significant impairments in the hemorrheological and hemostatic systems. CT has shown that all the patients have an inadequate endothelial reaction - the antiaggregatory, fibrinolytic, and anticoagulant activities of the endothelium are lowered. There was endothelium-dependent hyperproduction of IAM. CONCLUSION: The found changes suggest that MS has a considerable impact on the formation of a significant procoagulant state of the hemorrheological and hemostatic systems in patients with CCVD.