The Influence of Metabolites of Microorganisms of the Genus Bacillus from Permafrost Rocks on T Lymphocyte Differentiation.

We studied the influence of metabolites of permafrost microorganisms obtained at different temperature incubation conditions on activity of differentiation of regulatory (Treg) and effector T lymphocytes. It was found that the effect of metabolites is largely regulated by their type that depends on the temperature of production ("cold" at 5°C, "medium temperature" at 22°C, and "warm" at 37°C). The studied metabolites influenced the differentiation of Tregs (CD4+CD25hiCD127-) and the expression of markers of early (CD69), middle (CD25), and late (HLA DR) activation of CD4+ and CD8+ T lymphocytes. In the case of "cold" metabolites, the increase in Treg levels was associated with a decrease in the intensity of CD4+ T lymphocyte differentiation, and under the influence of "warm" metabolites - with a decrease in the activity of CD8+ T lymphocyte differentiation. Under the influence of "medium-temperature" metabolites, Tregs had approximately the same effect on the intensity of CD4+ and CD8+ T lymphocyte differentiation.

Influence of Paleobacteria on the Proliferative Activity of Human Lymphocytes In Vitro.

When testing the proliferative activity of 14 strains of permafrost microorganisms in the reaction of blast transformation of human lymphocytes in vitro, a strain (Alcaligenes sp.) with mitogen properties was isolated (20-fold increase in the rate of lymphocyte proliferation in comparison with the control). Four strains activated lymphocyte proliferation by 3-9 times in comparison with the control. Three strains produced substances with cytostatic properties and reduced proliferation activity by 33-43% and one strain (Bacillus sp.) almost completely suppressed phytohemagglutinin-induced lymphocyte proliferation. These data indicate that strains with a unique immunobiological potential are concentrated in the population of permafrost microorganisms that have undergone rigorous evolutionary selection.

The Influence of Permafrost Microorganisms on Monocytes Differentiation In Vitro.

Metabolites of fossil microorganisms of dispersed watered rocks that have passed into a frozen state (Bacillus sp. strains 2/09 and 9/08, Bacillus megaterium 8-75) can modulate the differentiation activity of human peripheral blood monocytes into phenotypically different subpopulations when cultured in vitro for 7 days. This effect is largely determined by the type of metabolites that depends on the temperature of their production: cold (collected after bacterium culturing at 5°C), medium (at 22°C), and warm temperature (at 37°C). All three types of metabolites had a weak negative influence on the level of classical (CD14hiCD16-) monocytes and stimulated the differentiation of intermediate (CD14+CD16+) and non-classical (CD14loCD16+) monocytes. The monocytes differentiation into the subpopulation of intermediate (CD14+CD16+) was stimulated to a greater extent by medium-temperature metabolites of the strain 8/75 and into the subpopulation of non-classical (CD14loCD16+) monocytes by warm metabolites of the strains 8/75 and 2/09 throughout culturing of mononuclear cells (days 1, 3, 7). Bearing in mind the anti-infection activity of intermediate and non-classical monocytes, we can consider strains 8/75 Bacillus megaterium and 2/09 Bacillus sp. promising for their in-depth testing.

Reparative and Immunomodulatory Potential of Low-Molecular-Weight Fractions of Secondary Metabolites of Bacillus sp.

The experiment was performed on male BALB/c mice with modeled skin wound. Two chromatographic fractions of secondary metabolites of permafrost bacteria Bacillus sp., that represent a mixture of polyethylene-polypropylene glycols oligomers with a molecular weight from 900 to 1350 Da were used as therapeutic agents. Application of the test substances on the wound surface and their parenteral administration significantly accelerated wound epithelialization in comparison with untreated control, activated metabolic processes, and improved humoral immunity. In in vitro experiments, the fractions activated the synthesis of IFNγ and IL-4 by human peripheral blood mononuclear cells. We conclude that polyethylene-polypropylene glycol oligomers can be a part of effector molecules that determine the repair and immunomodulatory potential of secondary metabolites of permafrost microorganisms Bacillus sp.

Dose-Dependent Effect of Bacillus sp. Metabolites from Permafrost on Lymphocyte Differentiation in the Thymus.

We studied the effect of a single intraperitoneal injection of metabolites from Bacillus sp. M3 strain isolated from permafrost (from 5×103 to 50×103 microbial bodies) on differentiation of T cells in the thymus of F1(CBA/Black-6) mice. On day 21 after the injection of metabolites, a dose-dependent decrease in the level of CD34+CD44+ and an increase in the number of CD34+CD44-, CD25-TCR+, CD25+TCR+max, CD4+CD8-, CD4-CD8+, and CD44+TCR+ lymphocytes were observed in the thymus. The increase in thymus level of mature (CD25+TCR+max) and migration-ready (CD44+TCR+) T cells in combination with a moderate decrease in the level of T cell precursors entering the thymus from the bone marrow (CD34+CD44+) can indicate a modulating influence of Bacillus sp. metabolites on functional activity of the thymus aimed at maintenance of the T cell balance in the body.

Effects of Temperature on the Ability of Metabolites from Permafrost Microorganisms to Activate the Synthesis of Systemic Cytokines by Mononuclear Cells.

The effects of metabolites of Bacillus sp. strain M3 and Bacillus megaterium strain 8/75-1 isolated from permafrost formations, as well as a medicinal Bacillus cereus strain IP5832 on the secretion of TNFα, IL-1ß, and IL-10 by mononuclear cells of human peripheral blood depending on the temperature of bacteria culturing (-16oC, -5oC, and 42oC) were studied. Metabolites of 8/75-1 and M3 strains cultured at 42oC produced more potent stimulating effect on TNFα and IL-10 synthesis than metabolites from these bacteria cultured at -16oC and -5oC (p<0.01). Temperature did not affect the ability of metabolites to modulate IL-1ß synthesis in all bacterial strains and the ability of IP5832 strain metabolites to modulate TNFα production. IL-10 secretion was minimum under the effect of metabolites of IP5832 strain cultured at 42oC. The TNFα/IL-10 and IL-1ß/IL-10 ratios indicated that metabolites of permafrost bacteria cultured at 42oC can shift the balance towards the synthesis of proinflammatory cytokines.

Effects of Secondary Metabolites of Permafrost Bacillus sp. on Cytokine Synthesis by Human Peripheral Blood Mononuclear Cells.

We studied the effects of secondary metabolites of Bacillus sp. isolated from late Neogene permafrost on secretion of proinflammatory (TNF-α, IL-1ß, IL-8, IL-2, and IFNγ) and antiinflammatory (IL-4 and IL-10) cytokines by human peripheral blood mononuclear cells. It was found that metabolites of Bacillus sp. produced more potent effect on cytokine secretion than mitogen phytohemagglutinin and metabolites of Bacillus cereus, medicinal strain IP5832. Activity of metabolites depended on the temperature of bacteria incubation. "Cold" metabolites of Bacillus sp. (isolated at -5°C) primarily induced Th1-mediated secretion of IFNγ, while "warm" metabolites (obtained at 37°C) induced Th2-mediated secretion of IL-4. The results suggest that Bacillus sp. metabolites are promising material for the development of immunomodulating drugs.

Reparation and Immunomodulating Properties of Bacillus sp. Metabolites from Permafrost.

An ointment containing metabolites of Bacillus sp. microorganisms isolated from permafrost samples was applied onto the skin wound of BALB/c mice. Metabolites isolated during culturing of Bacillus sp. at 37°C produced a potent therapeutic effect and promoted wound epithelialization by 30% in comparison with the control (ointment base) and by 20% in comparison with Solcoseryl. Treatment with Bacillus sp. metabolites stimulated predominantly humoral immunity, reduced the time of wound contraction and the volume of scar tissue, and promoted complete hair recovery. These metabolites can be considered as modulators of the wound process with predominance of regeneration mechanisms.

Effects of permafrost microorganisms on skin wound reparation.

Local application of ointment with Bacillus spp. strain MG8 (15,000-20,000 living bacterial cells), isolated from permafrost specimens, on the skin wound of about 60 mm(2) stimulated the reparation processes in experimental mice. A possible mechanism stimulating the regeneration of the damaged tissues under the effect of MG8 could be modulation of the immune system reactivity with more rapid switchover to humoral immunity anti-inflammatory mechanisms aimed at de novo synthesis of protein.

Experimental study of the effects of permafrost microorganisms on the morphofunctional activity of the immune system.

The immunobiological potential of a new microorganism species isolated from permafrost specimens (PMO strain 3M) collected from the Mammoth Mountain in Yakutia was studied in laboratory mice. PMO injected intraperitoneally in doses of 2500 to 50 × 10(6) microbial bodies caused characteristic dose-dependent effects on the structure and functions of the immune system (thymus and spleen indexes, functional activity of splenic macrophages, cellular and humoral immunity). Doses of PMO stimulating functional activities of both cellular and humoral immunity were detected.

[Relict microorganisms of cryolithozone as possible objects of gerontology].

Permafrost is widely distributed in the Northern Hemisphere, and its age reaches hundreds of thousands and millions of years. Permafrost contains alive microorganisms which are not frozen due to relatively high temperature of the environment (-2...-8 degrees C), but the microorganisms are immobilized and therefore aged probably similar to the age of permafrost. Longevity of the relict microbial cells is related obviously to their mechanism of protection against heat, radiation, free radicals and other damaging agents. A strain of Bacillus sp. was isolated from permafrost aged of about 3 million years, 16S rDNA sequence was identified and preliminary testing of bacterial culture on Drosophila melanogaster and mice was made. Immune stimulation and improvement of physical condition were observed, and that, together with the age of the microbial cells, presents the relict microorganisms as objects of gerontology.

Effects of short-term hypothermal and contrast exposure on immunophysiological parameters of laboratory animals.

Experiments on inbred animals showed that short-term exposure in cold water significantly modified structural and functional parameters of the immune system at different levels of its organization, from bone marrow hemopoiesis to effector stage of the immune response to antigen. The thermal factor caused changes in nonspecific and specific mechanisms of the immune system. Hypothermal exposure (7-9 degrees C, 5 sec) increased the thymic index and bone marrow lymphocyte count, reduced absorption capacity and stimulated metabolic activity of phagocytes, stimulated cell-mediated and suppressed humoral immunity. Contrast exposure in cold and hot water (7-9 degrees C, 5 sec/40-42 degrees C, 30 sec) increased monocyte count in bone marrow and reduced it in the their peripheral blood, reduced metabolic activity of phagocytes, stimulated cell-mediated and suppressed humoral immunity. These data demonstrate physiological mechanisms of interactions between the thermoregulatory and immune systems.

[The formation of host-parasite relationships in an experimental mixed pathology of opisthorchiasis-tuberculosis as dependent on the phase of the Opisthorchis infestation]. / Formirovanie otnoshenii khoziain--parazit pri éksperimental'noi mikstpatologii opistorkhoz--tuberkulez v zavisimosti ot fazy opistorkhoznoi invazii.

Superinfection of animals having Opisthorchis invasion with Mycobacterium tuberculosis leads to destabilization of host-parasite relationships in opisthorchiasis and formation of new host-parasitocenotic interrelations whose manifestation depends on the phase of the invasive process. At the acute invasive phase of mixed pathology (2 weeks) the activity of the host's immune system increases, while the biological activity of helminths and the number of M. tuberculosis colonies decrease. And on the contrary, at the subacute phase of mixed pathology (2.5 months) the activity of the host's immune system decreases, while the reproductive activity of helminths and the isolation rate of M. tuberculosis from pulmonary tissue increases in comparison with the groups of animals with monoinvasion and monoinfection.

[The pathomorphological changes in the liver and immune system organs of golden hamsters with experimental opisthorchiasis and tuberculosis infections]. / Patomorfologicheskie izmeneniia v pecheni i organakh immunnoi sistemy zolotistykh khomiakov pri éksperimental'nom zarazhenii opistorkhozom i tuberkulezom.

It has been established that lymphoid tissue involution caused by mixed experimental tuberculosis-opisthorchiasis infection was accompanied by a considerable attenuation of fibrosis and less intensive inflammatory destructive changes and liver fibrosis, as compared to monoinvasion with opisthorchis. In mixed infection there was a decrease in immunoreactivity of animals as assessed by the number of antibody-forming spleen cells per immunization with sheep red blood cells, compared to opisthorchiasis monoinvasion and a slight increase in this parameter compared to tuberculosis monoinfection.

[Influence of permafrost microorganisms on the quality and duration of life of laboratory animals].

Microorganisms of genus Bacillus strain 3M (permafrost microorganisms--PM), allocated from the samples of permafrost soils from Mamontova Mountain (Yakutia) are influenced by the quality and duration of life of elderly mice. PMs were injected intraperitoneally to CBA mice at the age of 17 months. Motion activity, muscular force (lifting of cargo test), food instinct activity (dosed starvation test) and cellular immunity activity (reaction of delayed-type hypersensitivity in vivo) of animals have increased, and psychoemotional condition of animals has improved under influence of PM. Average duration of life has increased by 9.19%, minimal duration of life--by 41.93%, maximal duration of life--by 7.68%. Duration of survival (the remaining life after injection of microorganisms) has increased more significantly: minimal--by 141.9%, on the average--by 28.0%, maximal--by 20.4%. Mechanisms of PM influence on increase of laboratory mice duration of life up to the top specific limit through the positive influence on quality of their life are discussed.

Specific and nonspecific reactions of mouse immune system under the effect of short-term exposure in warm and/or cold water.

Transient changes in environmental temperature produce a short-term, but significant effect on the immune system reactions in laboratory mice. Activities of nonspecific resistance factors (peritoneal macrophages) in mice exposed in warm or cold water were characterized by similar reactions, while the reactions of cellular and humoral immunity were opposite. Exposure to cold water activated cellular immunity, while warm water activated humoral immune system. The most significant changes in the immune system reactions were observed during the first 3 days of thermal exposure. Temperature alteration from cold to warm leads to activation of cellular and suppression of humoral components of the immune system. Alteration of water temperature from warm to cold leads to activation of nonspecific resistance factors, cellular and humoral immunity.