Ultrastructure and Morphological Variability of Non-Culturable Forms of Yersinia pseudotuberculosis Bacteria.

One of the mechanisms underlying the appearance of chronic infections is transition of pathogens into a non-culturable state, which is largely associated with the use of antibiotics. We studied ultrastructure of dormant bacteria Yersinia pseudotuberculosis obtained from the vegetative form of strain 512 by inhibition with kanamycin. On the model of the causative agent of pseudotuberculosis we showed that transition of prokaryotes to a dormant state occurs through apoptosis of bacteria. Fragmentation and condensation of chromatin with the formation of electron-dense fibrils, clumps and large conglomerates characteristic of apoptosis were found in the nucleoid zone of the cytoplasm of inhibited bacterial cells. These results are of great importance for understanding the mechanisms of the existence of pathogens in different conditions, as well as for identifying the causative agents of infectious diseases.

Biosensor Technologies in Medicine: from Detection of Biochemical Markers to Research into Molecular Targets (Review).

Infections are a major cause of premature death. Fast and accurate laboratory diagnostics of infectious diseases is a key condition for the timely initiation and success of treatment. Potentially, it can reduce morbidity, as well as prevent the outbreak and spread of dangerous epidemics. The traditional methods of laboratory diagnostics of infectious diseases are quite time- and labour-consuming, require expensive equipment and trained personnel, which is crucial within limited resources. The fast biosensor-based methods that combine the diagnostic capabilities of biomedicine with modern technological advances in microelectronics, optoelectronics, and nanotechnology make an alternative. The modern achievements in the development of label-free biosensors make them promising diagnostic tools that combine rapid detection of specific molecular markers, simplicity, ease-of-use, efficiency, accuracy, and cost-effectiveness with the tendency to the development of portable platforms. These qualities exceed the generally accepted standards of microbiological and immunological diagnostics and open up broad prospects for using these analytical systems in clinical practice directly at the site of medical care provision (point-of-care, POC concept). A wide variety of modern biosensor designs are based on the use of diverse formats of analytical and technological strategies, identification of various regulatory and functional molecular markers associated with infectious pathogens. The solution to the existing problems in biosensing will open up great prospects for these rapidly developing diagnostic biotechnologies.

Ultrastructural Changes of Bacteria in Static Cultures of Yersinia pseudotuberculosis under Long Storage under Conditions of Low Temperature.

Electron microscopy study revealed changes in the ultrastructure of bacteria of Yersinia pseudotuberculosis strains characterized by significantly reduced reproductive ability and virulence potential after long-term storage at low temperature of 4-8°C. Most bacterial cells contained dark cytosol with reduced cellular material or empty cytosol, while the cell wall was preserved. The revealed ultrastructural changes in the bacterial cells of the static culture of Y. pseudotuberculosis suggest that storage of strains under low positive temperatures could induce the transition of the majority of bacterial cell population to a dormant, non-cultivated state with a decrease in their virulence. This fact is of great scientific and applied importance in studies of causative agents of saprozoonoses, including pseudotuberculosis, which has the etiopathogenetic background of persistent infection.

THE STRUCTURAL CHANGES OF MACROPHAGES INFECTED WITH TICK-BORNE ENCEPHALITIS VIRUS.

Macrophages belong to the innate immune cells and play a key role in the pathogenesis of viral infections. The results of ultrastructural study of macrophages infected with tick-borne encephalitis virus (TBEV), the Flavivirus family, pathogens of human infections, affecting the nervous system, were presented. With the assistance of virological methods was found that the TBEV are absorbed by macrophages and replication in them. An ultrastructural study has shown that the virus enters into the cytoplasm by local destruction of plasmalemma and newly synthesized virus particles exited from the cell by same. Simultaneously there is a seal of perinuclear cytoplasm space, where found in a large number of ribosomes, microfilaments, ribonucleoprotein fibers and viral special structure: nucleocapsids, tubular formations and viral layers (fabrics). On the surface of last structures the newly synthesized virus particles were visualized. Thus, the evidence shows that macrophages play a role in the spread of TBEV, being for their the target cell. As active antigen presenting cells the macrophages can modulate the protective response of the body and influence on the pathogenesis of tick-borne encephalitis.

Morphogenesis of Experimental Infection Caused by Plasmid Variants of Yersinia pseudotuberculosis.

The dynamics of pathomorphological changes in response to infection with plasmid variants of Yersinia pseudotuberculosis was studied in experimental animals. Variability of cell injuries in pseudotuberculosis histopathology depended on the plasmid-associated virulence of the infection agent. Infection with highly virulent two-plasmid strain pYV48:pVM82 MDa and Y. pseudotuberculosis strain with low virulence with the only plasmid pVM82 MDa led to the development of cell destruction (necrosis and apoptosis) in the target organs. Apoptosis predominated in response to infection by plasmid variant pVM82 MDa with low virulence.

[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.

[Pathomorphism of Hantavirus infection under prolonged heat stress]. / Patomorfoz khantavirusnoi infektsii na fone prolongirovannogo teplovogo stressa.

AIM: to provide the morphological characteristics of experimental Hantavirus infection under heat stress conditions to identify the possibilities of its modeling in resistant laboratory animals. MATERIAL AND METHODS: Experiments were carried out on outbred albino mice that were divided into 4 groups: 1) intact mice unexposed to heating; 2) those exposed to heating; 3) Hantavirus-infected animals unexposed to preheating; 4) those exposed to preheating. The animals in Groups 2-4 were long exposed to heat stress at a temperature of 30 °C for 3 hours during 3 days. Strain Aa 60343 (PM-79-95) of the Far East genovariant of Hantaan virus (the genus Hantavirus, family Bunyaviridae) from the collection of the G.P. Somov Research Institute of Epidemiology and Microbiology was used to induce infection. The animals in Groups 3 and 4 were intraperitoneally injected with 700 FFU of Hantavirus per mouse. Materials (lung, liver, kidney, and spleen) taken from Groups 2-4 animals were collected for histological examination on days 1, 3, 7 and 14 of observation. RESULTS: The intact albino mice in Group 1 showed no histopathological changes in the organs. After heat exposure, Group 2 animals were found to have an immunomorphological response in the interstitial tissues of the lung, liver and kidney in partial lymphoid hypoplasia of the spleen. There were no signs of inapparent infection in the presence of marked immunomorphological changes in the organs in Group 3 of hantavirus-infected animals unexposed to preheating. In Group 4, those exposed to preheating exhibited dystrophic and destructive changes in the target organs (lungs, kidneys) in the presence of immunodeficiency manifestations of manifestations that were more pronounced in dead animals. CONCLUSION: In an unresponsive model (adult albino mice), Hantavirus infection caused only obvious immunomorphological changes in the organs. Prolonged preheat stress in the hantavirus-infected animals promoted inapparent infection and morphological manifestations of induced secondary immunodeficiency that was responsible for the manifestation of an infectious process in some animals.

[PLASMID-ASSOCIATED VIRULENCE OF YERSINIA PSEUDOTUBERCULOSIS AND INFECTIOUS PROCESS].

Literature data regarding genetically-determined pathogenicity factors of Y pseudotuberculo- sis and associated manifestations of this infection caused by various plasmid types of the causative agent are generalized. Principal attention is given to features of cell-tissue alterations mediated by virulence plasmid pYV, as well as effects of pathogenicity of an understudied pVM82 plasmid present only in Y pseudotuberculosis sttains causing clinical-epidemic manifestation of the infec- tions as Far East scarlet-like fever (FESLF). The data obtained on the ability of far-eastern strains to produceYPMa super-antigenj Ypseudotuberculosis-derivative mitogenA, probablygive evidence on its key role in FESLF pathogenesis. Variability of damage of innate immunity cells and target- organs caused by various plasmid types of Y pseudotuberculosis by virulence could determine polymorphism of clinical-morphological manifestations of this infection. In-depth understanding of dependency of immune pathogenesis mechanisms of the disease on molecular characteristics of the causative agent opens up-perspectives of enhancement of diagnostics and prognosis of the severity of the course of pseudotuberculosis and yersiniosis in human in general.

[The antimicrobial strategies of neutrophils under infection pathology.]

The neutrophilic granulocytes were traditionally considered exclusively as phagocytes - killer cells of microorganisms invaded human organism. The discoveries of last decade permitted to significantly reconsider this role and importance of neutrophils in implementation of affect mechanisms of inherent and adaptive immunity. The modern achievements expanded our conceptions about anti-microbial strategies of neutrophilic granulocytes under infection pathology: phagocytosis, degranulation and development of neutrophil extracellular traps. These strategies also play a key role in damage of tissues, providing cytotoxic functions. The article presents actual data concerning the role of interaction between neutrophils and adaptive immune cells in development of mechanism of destructive pathological activation of immune response that results in an auto-aggression, induction of chronic inflammation and development of oncologic and auto-immune diseases.

[The metabolism of the innate immunity cells in bacterial infections].

Metabolic activity of innate immunity cells infected by various doses of Gram-negative (Yersinia pseudotuberculosis, Salmonella enteritidis) and Gram-positive (Staphylococcus aureus, Listeria monocytogenes) bacteria has been investigated. Using various animal models we found that during the initial period (up to 2 days) changes of infection in cellular responses depend on the type of the pathogen. In response to infection caused by Gram-negative bacteria predominant neutrophil accumulation in the foci of inflammation was observed, while Gram-positive bacteria induced preferential accumulation of macrophages. The study of metabolism of these cells showed that the response of terminally differentiated primed phagocytes to pathogen appearance was higher than in cells circulating in blood. In addition to the priming state the phagocyte reactivity is influenced by the bacterial load. At a low phagocyte/microbe ratio the cells reaction is almost undetectable, while an excess of microorganisms causes (despite of the increase of the phagocytic parameters) the hyperactivation of cell metabolism and production of maximal amounts of bactericide agents, which exhibit a damaging effect on the cell itself.

Effects of immunomodulators on functional activity of innate immunity cells infected with Streptococcus pneumoniae.

Low activity of bactericidal enzymes was found in innate immunity cells infected with S. pneumonia. The death of these cells was fastened under these conditions. On the contrary, treatment with antibiotic maxifloxacin was followed by an increase in activity of bactericidal enzymes in phagocytes and induced their death via necrosis. Analysis of the therapeutic properties of immunomodulators tinrostim and licopid in combination with maxifloxacin showed that these combinations correct functional activity of cells infected with S. pneumonia.

Effect of thermolabile toxin from Yersinia pseudotuberculosis on functions of innate immunity cells.

The thermolabile toxin of Yersinia pseudotuberculosis produces a selective dose-dependent stimulating effect on functional activity of innate immunity cells. Prolonged apoptosis-inducing action of the toxin was associated with activation of enzymes of the oxygen-dependent system (LDH and myeloperoxidase) at the early terms of observation (up to 3 h). In turn, increased number of macrophages with apoptotic changes was noted at the early stages of contact with the thermolabile toxin (5 h), and its further growth was observed against the background of activation of mitochondrial enzymes and production of NO metabolites.

Morphological validation of hydroxyethylstarch use during the acute period of severe brain injury.

The effects of 10% hydroxyethylstarch solution on the cerebral microcirculatory bed and the blood-brain barrier were studied in Wistar rats during the acute period of severe brain injury. Positive changes in the morphometric values of the cerebral capillaries were observed in animals receiving intravenous injections of the drug, which promoted reduction of vascular permeability and cerebral tissue edema. These results confirmed the efficiency of hydroxyethylstarch as a component of infusion therapy in patients with severe brain injury.

Pathomorphosis of experimental infection in mice, infected by Streptococcus pneumoniae, under the effect of immunotropic drugs.

Pathomorphological changes in the organs of animals intranasally infected with Streptococcus pneumoniae were studied under conditions of immunotropic therapy added to antibiotic therapy. The pathomorphosis in the lungs, spleen, and thymus in animals treated with likopid, tinrostim, and roncoleukin was described. A positive time course of the pathological process in experimental animals in comparison with intact animals and animals receiving no immunotropic drugs was demonstrated.

[Neuroprotective effects of sulfated polysaccharides from seaweed].

Currently, neurodegenerative diseases (NDD) occupy a significant place in the structure of disease of the elderly, which dictates the need to find new and effective treatment and prevention of this pathology. At the heart of NDD development is a violation of the metabolism and the conformational change of cellular proteins with subsequent accumulation and aggregation of their in certain groups of neurons. The immediate cause of the death of the affected neurons in NDD is initiated by intracellular proteins apoptosis, during which a large number ofneurotransmitter glutamate is released. The consequence of an imbalance in the synthesis and release of neurotransmitters are related the memory impairment, motor coordination and cognitive abilities of human. Based on the analysis of the extensive literature domestic and predominantly foreign authors of the last decade the modern data on the effect of sulfated polysaccharides (SPS) of algae in vivo and in vitro in degenerative processes of the nervous system. Found that due to its multi-point impact, SPS have on the body antioxidant, anti-inflammatory, antiapoptotic, antihyperlipidemic, anti-toxic effects. Consequently, SPS can arrest a number of secondary pathological effects observed in neurodegenerative diseases (oxidative stress, inflammation, the phenomenon of increased neuronal apoptosis, toxic effects etc.). Varieties of pathogenic mechanisms underlying NDD makes possible the combined use of neuroprotective compounds acting sequentially in different stages of a pathological process. Accumulated a lot of experimental evidence to assume that the SPS may be the basis for the creation of next-generation drugs for the treatment of neurodegenerative diseases.

[Biochemical markers of virus cytopathogenicity in macrophages].

The results of macrophage metabolism studies at their infection by viruses differing in the level of virulence are presented. With the purpose of optimizing the estimation of viral cytopathogenic effects on macrophages, an index of cell reactions, which allows one to reveal the degree of virus influence in standard units, is offered. Generally, the application of high-sensitivity methods for functional activity determination and identification of the correlative communication between its changes and morphological features of cells can be prescribed to objective identification methods of not only viral reproduction, but also differentiation of types and the degree of their cytopathogenic effects.

[The physiological role of nitric oxide in infectious process].

This review presents the recent data on the physiological role of the molecule nitric oxide (NO) and its derivatives in the pathogenesis of bacterial and viral infection. Indicated that the effect of NO on individual cells involved in inflammation and immune regulation may be ambiguous, and depends on the cellular environment on the concentration NO and other factors. Thus, the interaction between reactive oxygen and NO derivatives provides a molecular basis for synergy between the respiratory burst and synthesis of NO, which leads to the formation of peroxynitrite, which has powerful bactericidal potential. At the same time production of NO can counteract the oxidant damage to cell membranes by blocking the oxidation of lipids and protect cells from damage, for example, by inhibiting the respiratory burst. Thus, nitrate and oxidative stress observed in pathophysiological states, has a modulating effect on the function of cells and tissues of various organs.

[The new views to pseudotuberculosis pathology].

For the first time the nature of cell damages under pseudotuberculosis as a generalized infection has been analyzed by positions of the modern knowledge about types of the cell death. The own and literature data have pointed to presence the apoptosis-induced effect of Yersinia pseudotuberculosis, a causative agent of this infection. In conclusion, the typical pathological changes for pseudotuberculosis infection such as granuloma formation with the central karyorrhexis could be appearance of apoptotic and secondary necrotic changes.

Neutrophil apoptosis induction by tick-borne encephalitis virus.

Tick-borne encephalitis virus infects neutrophils and induces their apoptosis, judging from moderate increase of succinate dehydrogenase activity and a trend to anaerobic energy production in neutrophils infected with the virus (shown by an increase of lactate dehydrogenase activity).

[The metabolic activity of neutrophils and monocytes as the model for the study of the organism biocompatibility with different materials].

In stimulated status neutrophils and monocytes in the foci of introduction the different materials are capable to excrete of biologically active substances by means of which if influence on processes of reparation. The express changes of blood leukocytes activity in response on the introduction of stimulated agents are assume as a basis of the proposed model of the organism biocompatibility with different materials. On example of the influence of different materials (ceramics, titanium, latex, surgical steel and copper), from which can be made implant and surgical instruments it was showed that the leukocytes were showed the different reaction of enzyme systems. This model allows the completely and objectively to study the influence of different materials on the neutrophyls and monocytes--the effected cells of the inflammatory process. Moreover, the activity of cation proteins and plasma membrane enzymes of leukocytes is the most expressed factor.