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—We performed a comparative analysis of the ability of a number of pharmacologically active natural compounds that are often considered as antioxidants to affect the redox status of the tumor (Ehrlich adenocarcinoma) and immune (splenocytes) cells. The following substances were studied: flavone luteolin and its sulfated derivative, 7,3'-luteolin disulfate from the eelgrass Zostera asiatica; oxycarotenoid astaxanthin from microalgae Haematococcus pluvialis, and a medicinal agent, Gistokhrom, whose active ingredient is hydroxy-1,4-naphthoquinone, echinochrome A from the flat sea urchin Scaphechinus mirabilis. The redox properties of the test compounds were evaluated in vitro by measuring the intracellular content of reactive oxygen species (ROS) using a selective fluorescent indicator, 2',7'-dihydrochlorofluorescein diacetate. The effect of the test substances on the intracellular level of ROS was determined at low (1 μg/mL) and high (10 μg/mL) doses, and in the presence or absence of a strong ROS inducer, the known antitumor agent doxorubicin, at a 10 μg/mL dose in an incubation medium. It has been shown that luteolin possesses the greatest antioxidant activity against both tumor and immune cells; luteolin disulfate and astaxanthin, which at the above doses reduce the ROS level both in the presence and absence of an inducer, have a lower antioxidant activity. Interestingly, unlike them, the drug Gistokhrom has a slight antioxidant effect on tumor cells only at a high dose and a weak effect on the redox status of splenocytes. In this work, we analyzed the possible role of ROS in biological activity and the mechanisms of action of the studied substances.

A comparative study of the effects of natural compounds with different biological activity spectra and mechanisms of action on the dynamics of the change in the redox-status of tumor and immune cells was carried out by measuring the intracellular level of reactive oxygen species depending on the dose and incubation time. The quinazoline alkaloid tryptanthrin, phenol propanoid rosmarinic acid, and the anticancer agent doxorubicin were tested. This study was performed on Ehrlich adenocarcinoma tumor cells and splenocytes after loading with the oxidant sensing fluorescent probe 2′,7′-dichlorofluorescein diacetate. It was shown that when rosmarinic acid influences tumor cells it has a pronounced antioxidant activity at a low dose (1 mg/mL), while a high dose of rosmarinic acid (10 mg/mL) exhibits prooxidant activity. Interestingly, in a splenocyte cell culture, rosmarinic acid reduced the level of reactive oxygen species at low and high doses. The combined application of doxorubicin with rosmarinic acid at a low dose reduced the prooxidant effect of doxorubicin, which is a potent inducer of reactive oxygen species in tumor cells. Tryptanthrin is also a potent inducer of reactive oxygen species with respect to tumor and immune cells; it is a more potent prooxidant than doxorubicin. In addition, tryptanthrin enhanced the doxorubicin-induced formation of reactive oxygen species by tumor cells in the combined use of doxorubicin with tryptanthrin. However, the prooxidant effect of tryptanthrin is short-term and decreases after a prolonged incubation. The effect of reactive oxygen species on the potent mechanisms of the biological activities of the individual and combined substances under study is discussed.

To study the adjuvant activity and to optimize tubular immunostimulating complexes (TI complexes) with respect to the soluble antigen, an investigation was performed on cucumariosides and their fractions, i.e., individual triterpene glycosides that were obtained from the holothurian Cucumaria japonica, as well as on phospholipids from the sea urchin Strongylocentrotus intermedius and the starfish Distolasterias nippon included in the composition of TI complexes in order to assess their effects on the immunogenicity of human serum albumin (HSA). Generally, TI complexes showed a moderate adjuvant activity with respect to HSA; this activity can be adjusted by substitution of a cucumarioside and/or a polar lipid in the composition of the TI complex. TI complexes based on monogalactosyldyacylglycerol (MGDG) from the sea alga Ulva lactuca and cucumariosides A2-2 and A4-4 maximally stimulated anti-HSA antibody production in mice. Substitution of MGDG for phosphatidylcholine (PC) from the starfish D. nippon with an increased (compared with other investigated phospholipids) ratio of n-3/n-6 polyunsaturated fatty acids did not result in a change in the immunostimulating activity of the TI complex. However, the use of other phospholipids led to a decrease in the adjuvant activity of the TI complex, compared with that of the TI complex based on PC from D. nippon and cucumarioside A2-2. The greatest fluctuations in the contents of cytokines IL-6, IL-12, IFN-γ and GM-CSF depending on the composition of the TI complexes indicate the possibility of regulation of the T-cell immune response. The values of hematological parameters were normal.

The tubular immunostimulating complex (TI-complex) consisting of cucumarioside A2-2, cholesterol and monogalactosyldiacylglycerol (MGDG) from marine macrophytes is the perspective antigen delivery system for subunit vaccines. MGDG is a lipid matrix for the protein antigen incorporated in the TI-complex. The aim of the present work was to study the influence of MGDGs from different macrophytes on conformation and immunogenicity of the secreted recombinant uncleaved hemagglutinin monomer (HA0S) of influenza A virus H1/N1. Differential scanning calorimetry, fluorescence spectroscopy and circular dichroism showed a dependence of the conformational changes of HA0S on the microviscosity of MGDG. The most viscous MGDG from Zostera marina induced the strongest rearrangements in protein conformation. Immunization of mice with HA0S within TI-complexes comprising different MGDGs resulted in an approximately 2-fold increase of the levels of anti-HA0S antibodies and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared with those induced by HA0S alone. TI-complexes based on MGDG from Z. marina stimulated the maximal production of GM-CSF. However, humoral immune response (anti-HA0S antibodies), unlike cell-mediated immune response (GM-CSF), did not depend on the physicochemical properties of MGDGs. It is assumed that this is due to the different localization and conformational lipid sensitivity of the HA0S regions, which are responsible for these types of immune responses.

Pharmacological activity of echinochrome A (EchA) alone and in the biologically active additives (BAA) Timarin, administered per os has been investigated in volunteers. Blood hematological, immunological, and biochemical parameters were investigated before and after administration of the substances used. EchA decreased serum glutatione (GSH) and increased catalase activity 1 h after treatment. Later (3 h after administration) catalase activity normalized, while GSH exceeded the initial level. Changes in blood lipids suggest decreased risk of atherogenesis. Changes found in blood sex hormone levels indicate that Timarin may influence sex gland functioning. Changes of hematological and immunological parameters have been interpreted as the result of a mild stressor effect of both EchA alone and in the BAA Timarin increasing adaptation reactivity of the body.

Immunostimulating complexes (ISCOM) have been modified by replacement of structural components of ISCOM; phosphatidylcholine has been replaced, by glycolipid of monogalactosyldiacylglycerol from sea macrophytes, saponin QuilA has been replaced by triterpene glycoside, Cucumarioside A2-2. The resultant complex includes morphological structures of two types: the ISCOM-like structures with the characteristic morphology and sizes and also the tubular structures with diameter of about 40 nm and length of 150–400 nm. We have defined these structures as TI-complexes. These TI-complexes exhibit considerably lower toxicity than ISCOM. They may include an amphiphilic protein antigen and provide immunoadjuvant effect during experimental vaccination. Under conditions of the experimental immunization of mice with a weak immunogen (subunit membrane pore protein from Y. pseudotuberculosis), TI-complexes with antigen provided stronger humoral immune response to antigen than the complexes of porin with classical ISCOM, liposomes and Freund’s adjuvant. Thus, the TI-complexes represent a new type of adjuvant carriers for antigens.

The tubular immunostimulating complex (TI-complex) is a novel nanoparticulate antigen delivery system consisting of cholesterol, triterpene glycoside cucumarioside A(2)-2, and glycolipid monogalactosyldiacylglycerol (MGDG) isolated from marine macrophytes. MGDG is crucial for the formation of a lipid matrix for the protein antigen incorporated in TI-complexes. Fatty acid composition and the physical state of this glycolipid depend on the taxonomic position of marine macrophytes. Therefore, the aim of the present work was to study the capacity of MGDGs, isolated from five species of marine macrophytes, to influence conformation and to enhance immunogenicity of porin from Yersinia pseudotuberculosis (YOmpF) as a model antigen of subunit vaccine based on TI-complexes. The trimeric porin was chosen for these experiments, because it was approximately two times more immunogenic than monomeric porin incorporated in TI-complexes. Immunization of mice with YOmpF within TI-complexes, comprised of different MGDGs, revealed a dependence of the immunostimulating effect of TI-complexes on the microvicosity of this glycolipid. TI-complexes comprising MGDGs from Sargassum pallidum and Ulva fenestrata with medium microviscosity induced maximal levels of anti-porin antibodies (four times higher when compared with those induced by pure porin). The adjuvant effect of TI-complexes based on other MGDGs varied by 2.8, 2.3 and 1.3 times for TI-complexes comprised of MGDGs from Zostera marina, Ahnfeltia tobuchiensis, and Laminaria japonica, respectively. MGDGs are also able to influence cytokine mechanisms of immunological regulation. DSC and spectroscopic studies showed that maximal immunostimulating effect of TI-complexes correlated with a moderate stabilizing influence of MGDGs from S. pallidum and U. fenestrata on the conformation of porin. The results obtained suggest lipid "nanofluidics" as a novel strategy for optimizing the immune response to protein antigens within lipid particulate systems.

Background There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity. Results The cucumarioside A2-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times. Conclusions We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.