The study of NF-κB transcription factor activation by Pseudomonas aeruginosa recombinant proteins in eukaryotic cell culture.

The transcription factor NF-κB is a key factor in the activation of immune responses; it is in turn activated by pattern recognition receptors, such as TLR and NLR receptors. The search for ligands activating innate immunity receptors is an important scientific problem due to the possibility of their use as adjuvants and immunomodulators. In this study the effect of recombinant Pseudomonas aeruginosa OprF proteins and a toxoid (a deletion atoxic form of exotoxin A) on the activation of TLR4, TLR9, NOD1, and NOD2 receptors has been investigated. The study was carried out using free and co-adsorbed on Al(OH)3 P. aeruginosa proteins and eukaryotic cells encoding these receptors and having NF-κB-dependent reporter genes. The enzymes encoded by the reported genes are able to cleave the substrate with the formation of a colored product, the concentration of which indicates the degree of receptor activation. It was found that free and adsorbed forms of the toxoid were able to activate the TLR4 surface receptor for lipopolysaccharide. OprF and the toxoid activated the intracellular NOD1 receptor, but only in the free form. This may be due to the fact that the cell lines used were not able to phagocytize aluminum hydroxide particles with protein adsorbed on them.

[Morphofunctional changes of dendritic cells induced by sulfated polysaccharides of brown algae]. / Morfofunktsional'nye izmeneniia dendritnykh kletok pod deistviem sul'fatirovannykh polisakharidov burykh vodoroslei.

The effects of various sulfated polysaccharides of brown algae Fucus evanescens, Saccharina cichorioides and Saccharina japonica on the morphofunctional changes of dendritic cells have been investigated using flow cytometry and phase-contrast microscopy. The dendritic cells are characterized by larger sizes, vacuolated cytoplasm, eccentrically located nucleus, and also by the presence of numerous cytoplasmic pseudopodia of various shapes. They express surface markers, indicating their maturation (CD83, CD11c, HLA-DR, CD86). Increased production of immunoregulatory (IL-12) and proinflammatory TNF-a, IL-6) cytokines (by dendritic cells polarizes the development of the Th-1 type immune response.

[Antiviral action and pathogenetic targets for seaweed sulfated polysaccharides in herpesvirus infections]. / Antivirusnoe deistvie i patogeneticheskie misheni sul'fatirovannykh polisakharidov morskikh vodoroslei pri gerpesvirusnykh infektsiiakh.

The review summarizes results of studies of effects of sulfated polysaccharides from seaweed on herpesviruses and the course of herpesvirus infections. Importance of this problem is determined by the prevalence of herpesviruses that can persist in the human body and demonstrate a high degree of immune mimicry and resistance to antiviral agents. A wide range of physiological action of sulfated polysaccharides, receptor agonists of innate and adaptive immune cells, which possess potent antiviral, antioxidant and anti-inflammatory activities, open the possibility of their use for creation of new generation pharmacological substances and agents with associated activity for the treatment of herpesvirus infections.

Inhibitory Effect of Polysaccharides of Marine Hydrobionts on Biofilm Formation.

In the review there are considered the recent data on the perspectives of the use of polysaccharides (PS) from marine hydrobionts for nhibition of formation of bacterial biofilms, which play a significant role in the onset and process of different infections, as well as for design of antiadhesive coatings on medical produce. Particular attention is paid to antiadhesive properties of natural PS from marine microorganisms, algae and invertebrate animals, which prevent formation of biofilms. Antibiofilm PS possess such positive characteristics, as biocompatibility and biodegradability, that is of great interest for medical and industrial applications. The possibility of simultaneous use of complexes of compounds of different chemical nature and mechanisms of action in infectious diseases, involving biofilm formation is of special interest. It is believed that biologically active substances from marine hydrobionts could serve as the basis for development of new antibiofilm drugs, including complex ones.

[Effect of probiotic product containing bifidobacteria and biogel from brown algae on the intestinal microflora and parameters of innate immunity in mice with experimental drug dysbacteriosis].

The article represents the results of studying the effect of a new fermented product (FP) containing the probiotic strain Bifidobacterium bifidum 791 and Biogel from brown algae Laminariajaponica on the composition of intestinal microflora and parameters of innate immunity in mice with experimental dysbacteriosis, induced by administration of gentamicin in dose of 25 mg per kg body weight during 7 days. The experimental animals received for 6 weeks in addition to the diet FP, which was 2% of the average volume of feed intake. The FP influence was manifested by more rapid reduction of dyspepsia symptoms, restoration of body weight and balance the intestinal microbiocenosis (increasing of bifido- and lactobacteria, typical E. coli, reducing of the bacteria genus Proteus and Clostridium, elimination of S. aureus). As the results of FP administration we observed the statistically significant reduction of endogenous intoxication values and increasing of the phagocyte activity of neutrophils, related to effector cells of innate immunity, compared with animals not receiving FP. Identified effects of FP are due to both its probiotic properties through the presence of bifidobacteria and immunomodulating and enteral sorbtion activities of alginate component.

[Effect of sulfated polysaccharides from brown seaweed Laminaria japonica on the morfology of lymfoid organs and functional characteristics of immunocompetent cells].

The effect of sulfated polysaccharide fucoidan from the brown alga Laminaria japonica on morphological characteristics of mouse lymphoid organs, subpopulations of spleen mononuclear leukocytes, cytokine production and cytotoxic activity of splenocytes has been investigated. Fucoidan promoted activation and proliferation of lymphoid hematopoietic cells in primary and secondary immunogenesis bodies, increased expression of markers CD19, NK, NKT, CD25, MHC II, TCR, TLR2 and TLR4, the cytotoxic activity of splenocytes and production of immunoregulatory and proinflammatory cytokines (IL- 2, IL-12, IFN-g, TNF-a, IL-6). This suggests activation of effector mechanisms of innate immunity and adaptive immune responses via the Th-1 type.

Effects of S. officinalis L. radix triterpene glycosides on innate immunity factors.

The effects of triterpene glycosides (saponins) extracted from Saponaria officinalis L. radices, on the cellular and humoral innate immunity factors were studied. Saponins stimulated the phagocytic, bactericidal, and adhesion activities of polymorphonuclear leukocytes. Optimal conditions of saponin treatment (dose and duration) were determined for mice. Saponins promoted the maturation of human peripheral blood dendritic cells, which was proven by high expression of CD83 (terminal differentiation marker) and CD86 (bone-stimulating molecule) and of HLA-DR and HLA-ABC molecules on the cell membrane. Saponins modulated the production of TNF-α, IL-1ß, IL-4, IL-6, and IFN-γ in cultured peripheral blood intact cells. The results help to understand some mechanisms of the effects of saponins extracted from Saponaria officinalis L. radix on the cellular and humoral factors of innate immunity and demonstrate good prospects of their practical use.

[Sulfated polysaccharides of brown seaweeds--ligands of toll-like receptors].

The interaction of sulfated polysaccharides--fucoidans from brown seaweeds Laminaria japonica, Laminaria cichorioides and Fucus evanescens with Toll-like receptors (TLRs) expressed on membranes of embryonic human kidney epithelial cells (HEK293-null, HEK293-TLR2/CD14, HEK293-hTLR4/CD14-MD2 and HEK293-hTLR2/6) was investigated. In vitro fucoidans specifically interacted with TLR-2, TLR-4, and the heterodimer TLR-2/6 resultated in activation of transcription nuclear factor NF-kappaB. Analysis of composition the hydrolyzed fucoidan from F. evanescens was carried out by gas-liquid chromatography and chromatography-mass spectrometry. Results indicated the absence of 3-3-hydroxytetradecanoic acid (3-OHC14), the basic component of lipopolysaccharides in the preparation. Thus, the obtained results suggested that fucoidans from brown seaweeds possessing immunotropic activity are independent ligands for TLRs, and are able to induce genetically determined biochemical processes of protection organisms against pathogenic microorganisms.

[Effect of sulfated polysaccharides from brown algae on proliferative and cytotoxic activity of mice splenocytes].

AIM: Study effect of fucoidans from brown algae on proliferative and cytotoxic activity of mice splenocytes. MATERIALS AND METHODS: Proliferative and cytotoxic activity of mice splenocytes in vitro and ex vivo were studied in lymphocyte blast transformation reaction and in cytotoxic MTT-test on K562 human erythroblast leucosis cell line. Microphotography and microscopy were performed by using Axiocam HS photosystem and computer program AxioVision 4 (Germany). RESULTS: Fucoidans from brown algae Fucus evanescens, Laminaria cichorioides and Laminaria japonica in vitro and ex vivo systems were established to increase proliferative activity of mice splenocytes which is evidenced by an increase of stimulation index. Results of in vitro and ex vivo cytotoxic activity studies demonstrate that fucoidans which are various by chemical structure stimulate activity of NK-cells and facilitate an increase of splenocyte cytotoxic potential level against NK sensitive K562 cell line. CONCLUSION: The data obtained from the study demonstrate an ability of fucoidans to stimulate splenocyte proliferation and NK-cell killer activity, and studies of relation between structure and functions of sulfated polysaccharides facilitate a more detailed understanding of aspects of their mechanism of action on innate immunity system, thus providing the basis for development of new immunobiologic preparations - modifiers/agonists of innate immunity.

Interactions between sulfated polysaccharides from sea brown algae and Toll-like receptors on HEK293 eukaryotic cells in vitro.

We studied the interactions between sulfated polysaccharides, fucoidans from sea brown algae Laminaria japonica, Laminaria cichorioides, and Fucus evanescens, with human Toll-like receptors (TLR) expressed on membranes of cultured human embryonic kidney cells (HEK293-null, HEK293-TLR2/CD14, HEK293-hTLR4/CD14-MD2, and HEK293-hTLR5). Fucoidans interacted with TLR-2 and TLR-4, but not with TLR-5, and were nontoxic for the cell cultures. L. japonica fucoidan (1 mg/ml), L. cichorioides fucoidan (100 µg/ml and 1 mg/ml), and F. evanescens fucoidan (10 µg/ml-1 mg/ml) activated transcription nuclear factor NF-Ï°B by binding specifically to TLR-2. L. japonica fucoidan (100 µg/ml and 1 mg/ml), L. cichorioides fucoidan (10 µg/ml-1 mg/ml), and F. evanescens fucoidan (1 µg/ml-1 mg/ml) activated NF-Ï°B via binding to TLR-4. These results indicated that fucoidans could induce in vivo defense from pathogenic microorganisms of various classes.

[Production of cytokines by murine bone marrow dendritic cells in vitro mediated by sulfated polysaccharides obtained from sea brown algae].

AIM: To assess in vitro cytokine production by murine bone marrow dendritic cells (DC) matured under the effect of sulfated polysaccharides--fucoidanes from sea brown algae Laminaria cichorioides and Laminaria japonica. MATERIALS AND METHODS: CBA line mice were used to obtain bone marrow origin precursors of DC. Isolation and study of chemical composition and structure of fucoidanes were performed using modern research methods. Expression of surface markers was determined by flow cytometry (FACS-analysis) using monoclonal antibodies to respective antigens. Levels of cytokine production were measured by t-ELISA using kits manufactured by Biosource (Belgium). RESULTS: I was determined that fucoidans induce maturation of DC that was evident by expression of terminal differentiation marker CD83, activation marker CD38, enhanced expression of costimulating CD86, antigen-presenting MHC II and adhesive CD11c molecules. Fucoidanes stimulate DC to produce proinflammatory (TNF-alpha, IL-6, IL-1beta) and regulatory (IL-12) cytokines. Fucoidanes enhance expression of TLR-2 and TLR-4 but do not influence on expression of TLR-9. CONCLUSION: It was shown that fucoidanes from sea brown algae L. cichorioides and L. japonica activate innate immunity system that is evident by enhanced expression of surface molecules associated with DC maturation and increased production of proinflammatory and regulatory cytokines by DC. Enhanced expression of TLR-2 and TLR-4 allows to suppose that studied fucoidanes could have anti-infective effect in vivo.

[Inhibition of adherence of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases from marine hydrobiontes].

A possibility of adhesion inhibition of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases of marine hydrobiontes was investigated using alpha-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, total enzyme preparation and beta-1,3-glucanase from marine fungi Chaetomium, total enzyme preparation and beta-1,3-glucanase from marine mollusk Littorina kurila, and total enzyme preparation from crystalline style of marine mollusk Spisula sachalinensis were used. The enzymes were added to test-tubes containing buccal epithelial cells and/or the toxigenic bacterial strain C. diphtheriae No 1129, v. gravis. All the investigated enzymes were able to abort C. diphtheriae adherence, to human buccal epithelocytes. Inhibition of adhesion was more pronounced in the case of treatment of epithelocytes with highly purified enzymes of marine hydrobiontes in comparison with total enzyme preparations. The significant inhibition of C. diphtheriae adhesion was observed when the enzymes were added to the epithelocytes with the attached microorganisms. The results obtained show that glycoside hydrolases of marine hydrobiontes degrade any carbohydrates expressed on cell surface of bacterium or human buccal epithelocytes, impair unique lectin-carbohydrate interaction and prevent the adhesion.

[Antiviral activity of sulfated polysaccharide from the brown algae Laminaria japonica against avian influenza A (H5N1) virus infection in the cultured cells].

The antiviral activity of the sulfated polysaccharide fucoidan from the brown sea algae Laminaria japonica against infection caused by highly virulent avian influenza virus (Alduck/Novosibirsk/02/05, H5N1) in the sensitive pig embryo kidney cell cultures was studied. Fucoidan was ascertained to have no cytotoxic activity and to show virucidal activity against influenza A/H5N1 virus. When given at concentrations of 50 to 500 microg/ml, fucoidan protected the cell cultures from the cytopathogenic activity of influenza virus in a dose of 0.01 TCID50/1.0 ml and was able to suppress influenza A/H5N1 virus production within 24 hours of infection when prophylactic and therapeutic-and-prophylactic treatment regimens were used.

[Antiviral action of sulfated polysaccharides].

The literature data on the structure and biological spectrum of sulfated polysaccharides (fucoidans) from the sea brown algae are presented. The review includes the data on the experimental studies and the results of the author's researches on the sulfated polysaccharides inhibitory action on virus adsorption on eukaryotic cells. Mechanisms of the antiviral action of the fucoidans from the sea brown algae are discussed.

[Inhibiting effects of fucoidans on Hantaan virus adsorption on a model of peritoneal macrophages in vitro].

A model of peritoneal macrophages was used to study the effect of fucoidans from brown seaweeds on the adsorption of Hantaan virus. Fucoidans were found to have antiviral activity, but to differ in the inhibition of hantavirus adsorption, which was associated with their structural features. The mechanism of their action is assumed to be shown via ligand-receptor interaction with certain cell membrane receptors and via blockage of hantavirus glycoproteins (G1 and G2), resulting in adsorption inhibition and preventing viral penetration into the macrophages.

[Screening of biopolymers from sea hydrocoles affecting the adsorption of Hantaan virus].

The biopolymers extracted from sea hydrocoles were screened and a group of sulfated polysaccharides--fucoidans having a pronounced inhibitory effect on the adsorption of Hantaan virus in the cultured Vero E6 cells was identified. The mechanism of action of sulfated polysaccharides was realized through competitive carbohydrate-specific binding to cell membrane receptors and through ligand-receptor interaction and blocking of Hantaan virus glycoproteins (G1 and G2).

[Pathogenicity factors of Corynebacterium diphtheriae circulating in the Primorski region under conditions of mass immunization of population]. / Faktory patogennosti Corynebacterium diphtheriae, tsirkuliruiushchikh v primorskom krae v usloviiakh massovoi immunizatsii naseleniia.

The study of the main pathogenicity factors of C. diphtheriae (adhesive activity, toxigenicity, detection of tox+ gene) circulating in the Primorski Territory has been made. As revealed in this study, at the period of declined epidemic process due to mass immunization of the adult and child population against diphtheria the selection of C. diphtheriae strains with weak toxigenicity and low adhesiveness was observed. No strains having tox+ genes have been detected among C. diphtheriae nontoxigenic strains circulating in the Primorski Territory.

[The antidiphtheria and antitetanus immunities of children of the Maritime Territory after the use of adsorbed DTP vaccine and adsorbed DT-m anatoxin]. / Sostoianie protivodifteriinogo i protivostolbniachnogo immuniteta detei Primorskogo kraia pri ispol'zovanii AKDS-vaktsiny i ADS-M-anatoksina.

The state of collective immunity to diphtheria and tetanus in children of preschool age, depending on the kind of vaccine preparation used for immunization, was studied. The immunological potency of adsorbed DPT vaccine (i.e. its capacity of forming prolonged and stable basic immunity) was shown to be higher in comparison with the potency of adsorbed DT toxoid with reduced antigen content (DT-R). The study revealed that in all groups of children the level of antitetanus immunity was higher than the level of antidiphtheria immunity, and 3 years later its decrease was less pronounced. A stricter approach to giving medical grounds for the use of low-reactive adsorbed DT-R in the immunization of children belonging to groups of risk is recommended.

[Feasibility of decreasing the adhesive activity of Corynebacterium diphtheriae by biopolymers of natural origin]. / Izuchenie vozmozhnosti snizheniia adgezivnoi aktivnosti Corynebacterium diphtheriae biopolimerami prirodnogo proiskhozhdneiia.

Possible decreasing of the Corynebacterium diphtheriae adhesive activity by natural biopolymers was studied. It was shown that the strains of C.diphtheriae circulating on the Primorye Territory had middle, low or minimal adhesive activity. Natural biopolymers were found to decrease the adhesive properties of C.diphtheriae. The results of the study are promising for further investigation of natural biopolymers as agents preventing C.diphtheriae colonization on the stomatopharynx mucosa.