The Use of Microencapsulated Autologous Thrombi for Modelling Chronic Thromboembolic Pulmonary Hypertension in Rats.

Here we developed a model of chronic thromboembolic pulmonary hypertension (CTEPH) using repeated intravenous administration of microencapsulated thrombi with a controlled rate of biodegradation. Autologous thrombi encapsulated in alginate microspheres with a diameter of 190±48 µm were intravenously injected to rats 8 times every 4 days. In the comparison group, nonmodified thrombi were injected. After 6 weeks, a significant increase in systolic pressure in the right ventricle, a decrease in exercise tolerance, and an increase in the index of vascular wall hypertrophy were revealed in the group receiving injections of microencapsulated thrombi in comparison with the group receiving nonmodified thrombi and healthy animals. Thus, the developed representative CTEPH model can be used to test promising pharmacological substances.

[Bone mineral density in vegetarians and vegans].

The number of vegetarians and vegans is increasing each year. In this regard, studies of the quality of diets that exclude slaughter foods, as well as their impact on human health, are becoming more and more relevant. The main purpose of the study was to assess the bone mineral density (BMD) in Russian vegetarians and vegans, as well as in omnivores. Material and methods. Design - cross-sectional study. On an outpatient basis, we examined 103 conditionally healthy people aged 18 to 77 years with different diets: 36 vegans, 38 vegetarians and 29 omnivores. X-ray two energy absorptiometry was used to assess BMD. The density of the lumbar vertebrae (LI-LIV) and femoral neck was measured. Results. Osteopenia in the lumbar spine was diagnosed in 27.8% of vegans, 39.5% of vegetarians, and 31.0% of omnivores. In the femoral neck, BMD corresponding to osteopenia was detected in 19.4, 26.3, and 17.2% of cases, respectively. 18.4% of vegetarians and 6.9% of omnivores had BMD corresponding to osteoporosis in the lumbar spine. Osteoporosis was not diagnosed in the femoral neck. No significant differences were observed after exclusion of people over 50 years of age. This was probably due primarily to the fact that the largest number of peri and postmenopausal women were in the vegetarian group. Excluding people who had took vitamin D supplements regularly did not drastically change the results of the study. When taking into account both exclusion criteria, no significant differences were observed. Conclusion. The findings suggest that BMD in vegans and vegetarians in Russia does not differ from that in omnivores. However, further larger studies are required.

The influence of vegetarian and vegan diets on the state of bone mineral density in humans.

There are so many literatures about vegetarians being less prone to chronic, noninfectious diseases, which are, however, the main cause of the decline in quality of life and mortality in developed countries.However, according to various scientific sources, vegetarian and especially vegan diets often contain less saturated fats, protein, calcium, vitamins D and B12, or long-chain ω-3 PUFAs. One of the most common pathology associated with a predominantly plant diet is osteopenia and osteoporosis. An analysis of 13 studies has shown that vegetarians and vegans are at a higher risk of reducing of bone mineral density, thereby increasing the incidence of fractures.At the same time, plant-based diets are usually richer in many other micronutrients important for bone health: vitamins C and K, carotenoids, potassium, magnesium, manganese, copper, or silicon. Moreover, with the deepening of our knowledge about the role of nutrients in the body and the features of the nutritional status of the population, the quality of vegetarian and vegan diets also increases. They are less and less prone to micronutrient deficiencies. Recent studies show that BMD, as well as the risk of osteoporotic fractures, at least in vegetarians, equaled these indicators in omnivores.

[Analysis of complete sequence of cryptic plasmid pTP33 from Yersinia pestis isolated in Tuva natural focus of plague].

This paper studies a full nucleotide sequence of cryptic plasmid pTP33, which was isolated from the typical plague strain of the Tuvinian natural focus, Yersinia pestis I-2638. Sequencing was carried out using the 454 GS Junior platform (Roche). In analysis using the software package GS De Novo Assembler v. 2.7 (Roche) and the algorithm Newbler v. 2.7, 1855 nucleotide reads, which contained 1101246 nucleotides, were assembled to a contig of 33 978 bp. The GC content of the obtained nucleotide sequence was 50.25%. During annotation, we found 56 open reading frames. Homologs of the predicted reading frames were sought in the BLAST databases. We detected 22 reading frames coding hypothetical proteins, 23 frames coding phagerelated proteins, and 11 frames coding proteins with known functions, including toxin­antitoxin system YefM-YoeB, nucleic acids and polysaccharides metabolism proteins (exopolysaccharide production protein ExoZ, exodeoxyribonuclease VIII), and replication proteins (ParA). Some predicted pTP33 proteins were found to be homologs (from 45 to 75%) with sequences of phage-related proteins of certain microorganisms­endosymbionts of insects (Sodalis glossinidius) and endosymbionts of entomopathogenic nematodes (Photorhabdus luminescens, P. asymbiotica, Xenorhabdus bovienii).

Whole-Genome Sequencing of a Vibrio cholerae El Tor Strain Isolated in the Imported Cholera Focus in Siberia.

The draft genome sequence of Vibrio cholerae O1 strain I-1263, isolated from a patient in the imported focus in Siberia, was determined. The established structural features of the mobile genetic elements indicate stage-by-stage formation of a highly pathogenic V. cholerae clone and promote understanding of the mechanisms of evolutionary pathogen transformations.

[Polytypic strains in the genofund of tick-borne encephalitis virus].

Eighteen polytypic tick-borne encephalitis virus (TBEV) strains containing the fragments of E and NS1 protein genes of Siberian and Far Eastern, occasionally Siberian and European subtypes were isolated in the European and Asian parts of the tick-borne encephalitis (TBE) area. They were identified using real-time polymerase chain reaction, hybridization-fluorescence detection with genotype-specific probes, restriction fragment length polymorphism analysis, and E protein sequencing. The polytypic strains were isolated from individual Ixodes persulcatus ticks, their pools, from the blood of patients and the brain of dead patients. The isolation rates of the polytypic strains in the sympathry area of different TBEV subtypes ranged from 4.4% (the Irkutsk Region) to 15.1% (the Yaroslavl Region). In addition to 2 polytypic strains, a strain similar to the TBEV 886-84 strain was isolated. The TBEV subtypes entering into the composition of the polytypic strains show nongenetic interactions, such as neutral replication or competition. The polytypic strains are stable during passages in the cultured pig embryo kidney epithelial cells and on cloning. Mouse brain passage promotes dissociation of polytypic strains. The conditions for the formation of polytypic strains and their role in the etiology of TBE are discussed.

Mitochondrial dysfunction precedes other sub-cellular abnormalities in an in vitro model linked with cell death in Parkinson's disease.

Dysfunction of mitochondria, the ubiquitin proteasome system (UPS), and lysosomes are believed to contribute to the pathogenesis of Parkinson's disease (PD). If it were possible to rescue functionally compromised, but still viable neurons early in the disease process, this would slow the rate of neurodegeneration. Here, we used a catecholaminergic neuroblastoma cell line (SH-SY5Y) as a model of susceptible neurons in PD. To identify a target early in the cell death process that was common to all neurodegenerative processes linked with PD, cells were exposed to toxins that mimic cell death mechanisms associated with PD. The sub-cellular abnormalities that occur shortly after toxin exposure were determined. 3 h of exposure to either naphthazarin, to inhibit lysosomal function, Z-Ile-Glu(OBu(t))-Ala-Leu-H (PSI), to inhibit the UPS, or rotenone, to inhibit mitochondrial complex I, caused depolarisation of the mitochondrial membrane potential (2.5-fold, twofold, and 4.6-fold change, respectively compared to vehicle), suggesting impaired mitochondrial function. Following 24 h exposure to the same toxins, UPS and lysosomal function were also impaired, and ubiquitin levels were increased. Thus, following exposure to toxins that mimic three important, but disparate cell death mechanisms associated with PD, catecholaminergic cells initially experience mitochondrial dysfunction, which is then followed by abnormalities in UPS and lysosomal function. Thus, mitochondrial dysfunction is an early event in cell stress. We suggest that, in patients with PD, the surviving cells of the substantia nigra pars compacta are most susceptible to mitochondrial impairment. Thus, targeting the mitochondria may be useful for slowing the progression of neurodegeneration in PD.

[Deformation properties of human erythrocytes in the presence of x-ray contrast agents].

It is established that the x-ray contrast agents urografin (76 %), omnipaque-300, optiray-350, ultravist-300, and visipaque-270 produce a dose-dependent decrease in the deformation properties of human erythrocytes in vitro. The degree of manifestation of this negative influence increases with the osmotic activity of agents in the following order: visipaque = ultravist < or = omnipaque < or = optiray < urografin. The effect of these x-ray contrast agents on the deformation of erythrocytes is reversible. Erythrocytes restore their normal deformation properties upon removal of the osmotic pressure induced by the x-ray contrast agents.

[A morphological study of regeneration of the goldfish elasmoid scales].

Studies were carried out on the model of regeneration of mechanically removed elasmoid scales. Regenerating scales were morphologically analyzed using light and electron microscopy. It was found that the cells responsible for regeneration of the elasmoid scale plates could be classified as osteogenic elements. Little differentiated preosteoblasts were detected in the connective tissue of dermis on day 3 of regeneration, while partially calcified plates underlaid osteoblasts on day 7. The scale cover was fully restored on day 14 and it took two days for each bone plate to be formed. Osteocytes, fully differentiated osteogenic elements, were found in the deepest regions of newly formed scales.

Thrombin receptor agonist Peptide immobilized in microspheres stimulates reparative processes in rats with gastric ulcer.

The effect of synthetic thrombin receptor (PAR1) agonist peptide encapsulated in microspheres made of lactic and glycolic acid copolymer on tissue reparation was studied in rats with acetate-induced ulcer. PAR1 agonist peptide was immobilized in biodegraded lactic and glycolic acid microspheres by double emulgation, the kinetics of peptide release was analyzed, and the dynamics of ulcer healing was studied in experimental (administration of microspheres with the peptide into the stomach) and two control groups (administration of saline or spheres without peptide). Thrombin receptor agonist peptide gradually released from lactic and glycolic acid microspheres into the stomach shortened the inflammation phase and shifted the proliferation phase to the earlier period, thus accelerating healing of experimental ulcers in rats.

[Bishofit in the treatment of rheumatoid arthritis]. / Bishofit v lechenii bol'nykh revmatoidnym artritom.

AIM: To evaluate effectiveness of a natural mineral bishofit in combined treatment of rheumatoid arthritis (RA). MATERIAL AND METHODS: The study included 71 RA patients with minimal activity of the pathological process and articular disorders of stage II. Bishofit was used in baths and compresses. 33 AR patients of group 1 took bishofit baths (14 procedures) and non-steroid anti-inflammatory drugs (NSAID), 27 RA patients of group 2 were treated with bishofit compresses (14 procesures) and NSAID, 11 patients of group 3 (controls) received NSAID alone. The effect of the treatment was assessed on the basis of clinical data, the length of the affected joint circumference, antitryptic blood activity, activity of malate dehydrogenase, lactate dehydrogenase isoenzymes, plasmin, plasminogen, ESR, sialic acids. RESULTS: Bishofit-treated patients showed earlier (by 5-6 days) and greater (by 25-35%) decrease of the pain, joint and inflammatory indices as well as duration of morning stiffness, length of the affected joint circumference and joint mobility, improvement of the tests' results. A response and an appreciable response to combined therapy with bishofit were achieved in 62.9-63.6% of cases vs 45.5% in the control group. Bishofit baths and compresses did not differ considerably by therapeutic efficacy.

Role of different B-cell subsets in the specific and polyclonal immune response to T-independent antigens type 2.

Role of different B-cell subsets in the immune response to T-independent antigen type 2 (TI-2) was studied. BALB/c and C57BL/6 mice were immunized by polyvinylpyrrolidon (PVP), and the numbers of antibody- and Ig-forming cells (AFC and IFC, respectively) were determined by ELISPOT method. The number of cells producing non-specific Ig (nIFC) was calculated as the difference between the number of IFC and AFC; the number of nIFC induced by PVP was calculated as the difference between the number of nIFC in immune and control splenocytes. Immunization by PVP induced not only the AFC appearance, but also the increase in the number of the antigen-induced nIFC. The treatment of splenocytes by anti-CD5 antibodies and guinea pig complement reduced the increase in the numbers of newly formed AFC and nIFC to approximately 40% of control level. It means that CD5+ cells play an important role not only in the specific, but also in polyclonal immune response to non-self TI-2. To be sure that the decrease of AFC and nIFC numbers is due to depletion for CD5+ B-cells, but not CD5+ T-cells, splenocytes were separated to B-1 and B-2 subsets, and the numbers of AFC, IFC and nIFC were determined in each B-cell subpopulation separately. The overwhelming majority of newly formed AFC and nIFC was detected in B-1 subset. The numbers of AFC and nIFC in B-1 compartment was approximately 10-fold greater than in B-2 cells. A close parallelism between AFC and nIFC formation was observed. It is concluded that specific and polyclonal immune response to non-self TI-2-PVP-depends mainly on CD5+ B-1 subset.

Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS).

Monoclonal expansion of B cells and plasma cells, producing antibodies against 'self' molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström's macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P0, using beads coated with P0. P0-coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein--Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5+ positive, although the expression declined in vitro over time. The anti-P0 antibodies were of IgM-lambda type. The antibodies belonged to the VH3 gene family with presence of somatic mutations. The IgM reacted with P0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5+ clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P0-specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation.

[Production of monoclonal antibodies to T-independent type 2 antigens with OptiMem-Iscove medium]. / Poluchenie monoklonal'nykh antitel k T-nezavisimym antigenam 2 tipa s ispol'zovaniem credy OptiMem-Iscove .

To obtain monoclonal antibodies to T-independent antigens of type 2, having low immunogenicity and incapable of inducing the appearance of memory cells, the use of the medium OptiMem-Iscove (1:1) with 10% of fetal serum, glutamine (50 mM) and antibiotics (100 units/ml) is proposed. The main advantage of this medium is the possibility of cloning cells without the use of feeders. The method has been approved in the process of obtaining monoclonal antibodies (McAb) to TI-2 antigens, both bacterial (S3) and synthetic (PVP), as well as to McAb to T-dependent antigen (alpha-chains of human IgA).

[Human monoclonal antibodies to synthetic viral peptides]. / Monoklonal'nye antitela cheloveka k sinteticheskim peptidam virusa.

Cells producing human antibodies to synthetic viral peptides MH-24 (corresponding to 302-325 residues of HIV-1 gp120) and p107 (main peptide of EBV antigen EBNA-1) were fused with human x mouse heteromyeloma B6B11. The efficacy of specific hybridization was 40%. Hybrid cells H1F2 (MN-specific) and HF4 (p107-specific) were cloned by the limiting dilutions method. Monoclonal antibodies (MAb) were tested in enzyme immunoassay on a panel of synthetic peptides. All resultant MAbs were polyspecific for tested synthetic peptides. The data permit a conclusion that previously detected polyspecificity of human cultural antibodies to virus peptides is due to the true polyspecificity of antigen-binding centers of MAb, but not to the presence of strange cells in the culture.

Human monoclonal antibodies to MN-24 peptide of gp 120 HIV-1.

Human monoclonal IgM antibodies to synthetic peptide MN-24 corresponding to 303-325 residues of gp 120 HIV were obtained. Antibodies are made synthetic by CD5+B lymphocytes and are polyspecific: they react not only with different short peptides but with some macromolecular antigens as well. Anti-MN-24 antibodies possess antigen-binding activity but do not possess virus neutralizing activity. It is suggested that anti-MN-24 antibodies belong to the natural antibodies.

Role of different lymphocyte subpopulations in the formation of non-specific immunoglobulins induced by antigen injection.

The formation of antibody and non-specific immunoglobulin under the influence of T-dependent (TD) and type 2 T-independent (TI-2) antigens in mice of two congenic strains CBA (Lyb5-, Lyb5+) and CBA/N (Lyb5-) was studied. TD antigens induced in mice of both strains not only the appearance of antibody-forming cells (AFC), but also a great increase in the number of cells producing non-specific immunoglobulins (nIFC). TI-2 antigens induced the AFC and antigen-dependent nIFC formation in CBA mice only. It is concluded that during immune response to TI-2 antigens not only the AFC appearance but the increase in nIFC formation (polyclonal activation) is due mainly to the mature Lyb5+ B cells.

A new approach to obtain Lyb5-specific antiserum.

A new approach to obtain antiserum specific for Lyb5.2 allotype, was developed. Mice of DBA/2 inbred strain, bearing Lyb5.1 allotype, were made unresponsive to cellular antigens of Lyb5-CBA/N mice by specific tolerance induction. These tolerant recipients were further immunized with suspensions of splenocytes from CBA mice which contain Lyb5.2 + B lymphocytes. Thus obtained Lyb5.2-specific antiserum was cytotoxic for 25-30% of CBA or BALB/c (both Lyb5.2 + allotype) but not for CBA/N spleen cells. When splenocytes of BALB/c mice preimmunized with polyvinylpyrrolidone (T-independent type 2 antigen) were treated with this antiserum, 50% decrease in numbers of antibody-forming and antigen-induced non-specific immunoglobulin-forming cells was documented. We conclude that, during humoral immune response to T-independent type 2 antigen, not only antibody-forming cells but also non-specific immunoglobulin-forming cells are recruited from the Lyb5 + B cell pool.

Human monoclonal antibodies to viral peptides.

A new approach to the developing of human monoclonal antibodies to viral peptides is described. The method is based on the positive selection of B cells specific to viral peptides from normal human tissues. To isolate B cells bearing immunoglobulin receptors specific to viral epitopes magnetic beads coated by viral peptides were used. Human B lymphocytes specific to the neutralizing epitope of gp120 of human immunodeficiency virus and to the major epitope of Epstein-Barr nuclear antigen-I were isolated from freshly removed human tonsils. Preselected cells were transformed by Epstein-Barr virus and cultivated on irradiated human embryonic lung fibroblasts in RPMI 1640-OptiMEM medium with 10% fetal calf serum and all necessary supplements. The cultures of B cells producing antibodies to viral epitopes used for selection were obtained. Two cultures secreting IgM antibodies to the peptide MN-24 (neutralizing epitope of immunodeficiency virus) and to the peptide p107 of Epstein-Barr nuclear antigen-1 were expanded, and the specificity of the antibodies and antibody-producing cells was analyzed. The method of positive selection of B cells specific to viral peptides may be used for the preparation of human monoclonal antibodies to viral antigens.