[USE OF VIBRIO CHOLERAE SURFACE STRUCTURES FOR SPECIFIC PROPHY- LAXIS AND DIAGNOSTICS OF CHOLERA].

The need for efficient and cost-effective cholera vaccine hasn't lost its actuality in view of the emergence of new strains leading to severe clinical forms of cholera and capable to replace strains of the seventh.cholera pandemic, and in connection with the threat of cholera spreading beyond the borders of endemic countries. In this review data from literature sources are presented about the use of outer membrane proteins, vesicles, cell ghosts of the cholera causative agent in specific prophylaxis and diagnostics of the disease.

[SOME ASPECTS OF NON-SPECIFIC PROPHYLAXIS AND THERAPY OF ESPECIALLY DANGEROUS INFECTIONS].

Recently, due to spread of dangerous and especially dangerous infections much attention is given to development of complex approaches to their prophylaxis and therapy. Data on use of immune modulators, cytokines, probiotics, preparations of plant origin for non-specific prophylaxis of especially dangerous infections are analyzed in the review, and expediency of their combined use with specific and emergency prophlaxis of these diseases is evaluated.

[Endotoxin tolerance of mice to the effect of lipopolysaccharide and lipopolysaccharide-mice toxin complex of virulent strain Yersinia pestis 231].

AIM: Comparative study of the effect of endotoxin tolerance of mice to the effect of lipopolysaccharide (LPS37) and complex of lipopolysaccharide with mice toxin (LPS37-MT) of a virulent Yersinia pestis 231 strain. MATERIALS AND METHODS: Preparations of LPS of highly virulent strain Y. pestis 231 obtained by phenol method from cells cultivated at 37 degrees C as well as commercial preparations of S-LPS and R-LPS of Escherichia coli were used. Mice toxin was isolated from vaccine strain Y. pestis EV76. Effect of endotoxin tolerance was determined in mice treated with aminosugar D-galactosamine. RESULTS: The effect of initial LPS37 and modified form LPS37-MT of Y. pestis 231 was established to significantly differ from each other. When Y. pestis LPS37 is combined with heterologous forms--E. coli LPS or Y. pestis LPS37-MT, the inflammatory response of the organism differs and varies from complete or partial tolerance to complete lack thereof. For LPS37-MT complex only the sequence of administration to bioassay animals of LPS preparations is principal. In the case when primary activation is carried out by LPS37-MT and secondary--by Y. pestis LPS37 or S- and R- forms of E. coli LPS--the tolerance effect is absent. On the contrary, if LPS37-MT is used for recurrent activation against the background of all the other LPS forms including Y. pestis 231 LPS37 the inflammatory response is completely suppressed. CONCLUSION: Tolerance of mice to effect of LPS and LPS-MT complex of virulent Y. pestis 231 strain was shown to be different.

[Toxicity of Yersinia pestis EV76 lipopolysaccharides for mice sensitized by D-galactosamine].

AIM: To study toxicity of lipopolysaccharides (LPS28 and LPS 37) of Yersinia pestis for mice sensitized by D-galactosamine (D-GalN). MATERIALS AND METHODS: LPS were obtained by the Westphal method from Y. pestis EV76 strain grown at temperatures of 28 and 37 degrees C. Dexamethasone and pentoxifylline were used as immunodepressants. Uridine was used for interruption of D-GalN effect. RESULTS: It was revealed that administration of D-GalN to mice increased their sensitivity to LPS of Y. pestis. Maximal increase in LPS toxicity was observed after simultaneous administration of D-GalN and LPS. D-GalN in dose 20 mg per mouse determined 100% lethality of animals during 24 h after administration of 10 mcg of LPS28 and 25 mcg of LPS37. Uridine in dose of 20 mcg per mouse administered 1 h after LPS and D-GalN neutralized effect of LPS in the presence of D-GalN. Dexamethasone and pentoxifylline did not protect animals sensitized by D-GalN against lethal effect of Y. pestis LPS. CONCLUSION: It was found experimentally that D-GalN enhances toxic effect of LPS28 in hundreds of times, and non-toxic LPS37 of Y. pestis EV76 demonstrated toxicity comparable to LPS28. Thus the D-GalN model could be used for enhancement of laboratory animals sensitivity to effect of Y. pestis LPS.

[Toxicity and cytokine-inducing activity of lipopolysaccharide of virulent Yersinia pestis 231 strain].

AIM: Determine correlation between toxicity and cytokine inducing activity of parent and conformation modified forms of lipopolysaccharides (LPS) of virulent Yersinia pestis strain. MATERIALS AND METHODS: LPS was isolated by phenol method from Y. pestis 231 cells grown at 37 degrees C (LPS37). LPS37 was modified by "mice" toxin (MT) Y. pestis. Toxicity was controlled in mice. TNFalpha and IFNgamma cytokine production was determined by enzyme immunoassay. The study was performed in human monocytes U-937 cell line. TLR4 re-stimulation was performed after activation of monocytes by S-LPS and R-LPS of Escherichia coli. RESULTS: LPS37 conformation change of virulent Y. pestis 231 strain during formation of complex with "mice" toxin increases its toxicity for animals by 2 times. LPS37 and LPS37-MT induce TNFalpha and IFNgamma synthesis by human monocytes. LPS37 simultaneously activates MyD88-dependent as well as MyD88-independent signal pathways. Modified LPS37-MT form is a strong activator only of MyD88-dependent pathway and thereafter induces synthesis of predominately one of the cytokines--TNFalpha. Monocyte response to primary and recurrent activation by LPS37 and LPS37-MT corresponds to R- and S-LPS E. coli cytokine response profile. CONCLUSION: A direct correlation between toxicity of LPS37 and LPS37-MT and their TNFalpha-inducing activity was demonstrated in the study. LPS37 and LPS37-MT of Y. pestis 231 differentially activates TLR4 signal pathways of human monocytes.

[Effect of Yersinia pestis EV 76 lypopolysaccharides with different levels of toxicity on dynamics of TNF-alpha and INF-gamma synthesis by human monocytes].

UNLABELLED: AIM. To study dynamics of synthesis of TNF-alpha and INF-gamma by cell line U-937 human monocytes under the effect of Yersinia pestis EV 76 lypopolysaccharides (LPS) with different levels of toxicity: original LPS28 and LPS37 as well as their conformationally--changed variants with enhanced toxicity--complex of LPS with murine toxin (MT) of Y. pestis, and LPS modified by biologicall active compound (BAC) obtained from human erythrocytes. MATERIALS AND METHODS: Using phenol method, LPS were obtained from Y. pestis EV 76 cells grown at 28 and 37 degrees C. Production of cytokines was measured by ELISA. RESULTS: It was shown that original and modified forms of LPS28 and LPS37 induce synthesis of both TNF-alpha and INF-gamma by human monocytes. Expression of genes for two ways of synthesis of these cytokines points to activation and transmission of signal induced by all studied forms of Y. pestis EV 76 LPS through TLR4. Levels of activity of MyD88-dependent and MyD88-independent signaling pathways are different and depend from chemical structure of LPS28 and LPS37, conformation of their modified forms and duration of their exposition with monocytes. Dynamics ofcytokine synthesis corresponds to response of synergized TLR on activation with profound agonistic/antagonistic effect. CONCLUSION: It was determined that conformational modifications of Y. pestis EV76 LPS occurring due to effect of MT and BAC accompanied by quantitative, qualitative and temporal changes of TNF-alpha and INF-gamma synthesis by human monocytes and correlate with increase of their toxic properties.

[Effect of neutrophilokines on functional activity of macrophages during formation of immunity against cholera].

AIM: To study effect of neutrophilokines on functional activity of macrophages (Mph) during formation of immunity against cholera. MATERIALS AND METHODS: In order to obtain peritoneal neutrophils (Nph), 2 ml of 0.1% glycogen solution in buffered with phosphates sodium chloride solution was administered intraperitoneally to 100 outbred mice. Vibrio cholerae 1130 in dose 10 microbial cells/Nph and cholera toxin (CT) in dose 1 or 10 mcg/ ml were used as inducers of neutrophilokines synthesis. Obtained neutrophilokines were assessed on their effect on phagocytic activity of Mph, resistance of these cells to cytotoxic and apoptogenic effects of Vibrio cholerae and CT as well as effect on lysosomal apparatus of Mph. RESULTS: It was established that neutrophilokines induced by Vibrio cholerae and CT stimulate killer activity of Mph and lability of their lysosomal membranes, and suppress programmed death of these cells. CONCLUSION: Results of studies revealed immunoregulatory activity of neutrophilokines relative to Mph and demonstrated ability for cooperation between mono- and polynuclear phagocytes mediated by cytokines and, in particular, neutrophilokines.

[Evaluation of the immunoregulatory activity of neutrophilokine fractions in a macrophage model].

A procedure was proposed to evaluate the immunoregulatory activity of neutrophilokine fractions on a model of macrophages. It was established that all the fractions studied did not affect the absorptive capacity of these cells in both primary and secondary immune responses. At the same time, the majority of neutrophilokine fractions modulated the killer activity of macrophages: they potentiated or inhibited it. The proposed procedure for evaluating the regulatory effect of individual neutrophilokine fractions on a model of studying the killer activity makes it possible not only to characterize their activity, but also to identify helper and suppressor fractions, which discloses approaches to correcting an immune response by means of these fractions.

[Influence of neutrophilokines on population and subpopulation repertoire of lymphocytes and their functional activity during immune response against plague].

Results of experimental study of regulatory effect of nutrophilokines induced by Yersinia pestis EV strain on population and subpopulation repertoire of lymphocytes and their functional activity during immune response against plague infection are presented. It was established that these neutrophilokines stimulate CD4+ and suppress CD8+ lymphocytes. Helper effect of neutrophilokines on functional activity of lymphocytes was more pronounced during secondary than during primary immune response.

[Role of cholera toxin-induced apoptosis in alteration of macrophages in mice].

AIM: To study the role of active programmed cell death induced by Vibrio cholerae antigens in alteration of peritoneal macrophages of experimental animals. MATERIALS AND METHODS: Apoptosis was assessed by cytofluorometric analysis with propidium iodide using cytofluorometer "Coulter" as well as on characteristic morphological changes of cells in stained histological preparations. RESULTS: Performed experiments carried out by both methods provide evidence that V. cholerae and its antigens (cholera toxin, neuraminidase, chitinase, and lypopolysaccharide) cause apoptosis of mice peritoneal macrophages, which leads to their alteration. CONCLUSION: Our results demonstrate that programmed cell death of phagocytes is one of the causes of cytotoxic effect of V.cholerae and its antigens. Performed experiments show the role of apoptosis of macrophages in formation of postimmunization immunosuppression after vaccination against cholera.

[Preparation of fractions and characteristic of neutrophilokines complex induced by Yersinia pestis EV].

Biochemical and immunobiologic characteristics of fractions of neutrophilokines during primary and secondary immune response against plague infection are presented. Fractions were obtained using gel chromatography from neutrophilokines complex induced by vaccine strain of Yersinia pestis. It was revealed that fractions of neutrophilokines regulate IL-2 synthesis by Th1-helpers, IL-4 and IL-5 synthesis by Th2-helpers and also expression of IL-2 receptors by immunocompetent cells. Helper effect of neutrophilokines' fractions was more pronounced during secondary immune response.

[Influence of neutrophilokines on the synthesis of lymphokines in the process of the formation of immunity to plague].

The evaluation of the complex of neutrophilokines whose synthesis was induced by Yersinia pestis vaccine strain EV on the production of lymphokines in the process of the formation of primary and secondary immunity to plague is presented. As revealed in this study, neutrophilokines regulate the synthesis of IL-2 by T helpers of type 1, IL-4 and IL-5 by T helpers of type 2, IL-1 by B lymphocytes, as well as the expression of receptors IL-2 by immunocompetent cells. The helper effect of neutrophilokines is more pronounced in the secondary immune response.

[Comparative evaluation of mono- and neutrophilokins inducing activity of Yersinia pestis antigens]. / Sravnitel'naia otsenka mono- i neitrofilokinindutsiruiushchei aktivnosti antigenov Yersinia pestis.

The results of the comparative analysis of the cytokine inducing activity of Yersinia pestis EV antigens are presented. Y. pestis fraction 1A (F1A) and lipopolysaccharide (LPS) were shown to induce mono- and neutrophilokines, regulating cooperative interaction of phagocytes in the process of immunity formation to plague. Neutrophilokines and monokines exceed in their capacity for inducing F1A such acknowledged inductor as Escherichia coli LPS. As revealed by the comparative evaluation of Y. pestis EV LPS and E. coli LPS, neutrophilokines synthesized under the action of the former preparation, have greater influence on the inhibition of the macrophage migration from the infection focus as well as on digestive activity of these cells (in secondary immune response) and on the labilization of the lysosome membranes of macrophages than neutrophilokines induced by E. coli LPS. At the same time they produce a lesser modulating effect on the killer and chemotactic activity of neutrophils, as well as on the expression of FC receptors (FcR) on their surface in comparison with monokines, synthesized under the influence of E. coli LPS.

[Comparative evaluation of the neutrophilokine-inducing activity of Yersinia pestis EV and Escherichia coli lipopolysaccharides]. / Sravnetel'naia otsenka neitrofilokinindutsiruiushchei aktivnosti lipopolisakkharidov Yersinia pestis EV i Escherichia coli.

As shown in this study, neutrophilokine-inducing capacity of Y. pestis EV lipopolysaccharide (LPS) was not inferior to, and in secondary immune response even exceeded, that of E. coli LPS. Neutrophilokines synthesized under the action of the former preparation produced greater influence on the inhibition of macrophage migration from the focus of infection, the phagocytic activity of these cells (in secondary immune response) and the labilization of the lysosomic membranes of macrophages than neutrophilokines induced by E. coli LPS. Only in primary immune response the digestive capacity of macrophages was more actively stimulated by neutrophilokines induced by E. coli LPS. Both preparations did not induce the secretion of neutrophilokines regulating the expression of Fc-receptors on the surface of macrophages.

[Monokine-producing activity of human monocyte-like cell line U937 induced by Yersinia pestis EV antigens]. / Monokinprodutsiruiushchaia aktivnost' monotsitopodobnoi linii kletok cheloveka U937, indutsirovannaia antigenami Yersinia pestis EV.

The data on a study of the monokine-producing ability of human monocyte-like cell line U937 are presented. Antigens of Yersinia pestis EV (lipopolysaccharide and fraction 1A) induce monokine production by cell line U937. The obtained monokines essentially enhance neutrophil killer and chemotactic activities, stimulate FcR expression, increase the number of lysosomes, and the lability of lysosomal membranes in neutrophils. F1A significantly suppresses LPS in respect to the ability to induce monokine production, which stimulate neutrophil functional activity.

[A method of assessing immunogenicity of preparations for plague prevention]. / Sposob opredeleniia immunogennosti preparatov dlia profilaktiki chumy.

A rapid, economic, and simple method for assessing the immunogenic properties of preparations for plague prevention is proposed. It is based on amplification of the bactericidal activity of peritoneal macrophages of experimental animals in the course of forming antiplague immunity. The increase in intracellular killing was assessed by the index of macrophage activation, which permits a tentative assessment of the immunogenic properties of the agent. This method is 6 times more rapid and requires 5 times less animals than routine methods and involves no manipulations with virulent strains of Yersinia pestis.

[The effect of a modification to the lipopolysaccharide of Yersinia pestis on its neutrophilokine-inducing activity]. / Vliianie modifikatsii lipopolisakharida chumnogo mikroba na neitrofilokin-indutsiruiushchuiu aktivnost'.

The ability of the isolated and modified Yersinia pestis LPS to induce the synthesis of the neutrophilokines that regulate the macrophage functional activity was studied. It is established that the Y. pestis LPS detoxication, especially by the method of deacylation, does not lead to the decrease in biological, in particular neutrophilokine-inducing activity of these preparations, but actually even increases it. These results are in agreement with many reports showing the possibility of decreasing the LPS toxicity without reducing immunostimulatory activity of this important component of the outer membrane of gram-negative bacteria.

[Increased effectiveness of etiotropic therapy of experimental plague in albino mice at the stage of generalized infection]. / Povyshenie éffectivnosti étiotropnoi terapii éksperimental'noi chymy belykh myshei na stadii generalizatsii infektsii.

Therapeutic efficacies of various drugs were studied comparatively in the treatment of experimental plague in albino mice at the stage of the infection generalization. It was shown that out of the tested drugs such as ciprofloxacin, amikacin, gentamicin, rifampicin and polymyxin B only ciprofloxacin provided a rather high therapeutic effect in the treatment of the plaque septic form. The in vitro and in vivo experiments demonstrated that ciprofloxacin had an antitoxic action on lipopolysaccharide (LPS) and the plague microbe toxin. In comparison to ciprofloxacin, polymyxin B had a higher neutralizing activity. It was found that the efficacy of the experimental plague treatment at the stage of the infection generalization increased with the use of combinations of the drugs with antitoxic and antibacterial activities (ciprofloxacin and polymyxin B).

[The activation of Yersinia pestis "murine" toxin and endotoxin by hemolyzed erythrocytes from mammalian blood]. / Aktivatsiia "myshinogo" toksina i éndotoksina Yersinia pestis gemolizirovannymi éritrotsitami krovi mlekopitaiushchikh.

Y. pestis "mouse" toxin and endotoxin have been found to be capable of being activated with hemolyzed mammalian red blood cells. The LD50 of the activated endotoxin decreases 5-10 times in comparison with the initial preparation. The LD50 of the activated "mouse" toxin decreases 5 times. As revealed in this study, the joint introduction of nonlethal doses of "mouse" toxin and endotoxin is highly toxic for white mice and guinea pigs. The presence of both "mouse" toxin and endotoxin in the toxic mixture is an essential factor for these two species of animals.

[Immune electron microscopy in the study of biological matter]. / Immunoélektronno-mikroskopicheskii metod v issledovanii biologicheskikh ob''ektov.

A brief review of literature data and our investigations on the antibodies used for specific labeling in electron microscopy is presented. Considered are the problems connected with structure and function of separate components of bacterial viruses revealed by means of specific antibodies. The results of fine differentiation of antigenic components in the case of phages of the colidysentery group allowed to elucidate the functional role of the adsorption apparatus in the course of phage interaction with the bacterial cell. The topology of structural proteins (gene-products 35, 36, 37) of the tail's long strands for phages T4, DDVI+h and DDVIh is determined. Antigenic properties of proteins that are found in the composition of two forms of Bacillus mycoides are demonstrated immune-electronmicroscopically. On the basis of this finding, a conclusion was made that one of these phages acted as precursor, the other--as satellite during the simultaneous development of these phages in the bacterial cell. It was also established that temperate and virulent phages are related antigenically, which proves that lysogenic bacteria can be one of the phage sources on the environmental conditions. Visualization of non-ribosomal genes of procaryots that code for structural proteins of a defective phage proves the efficiency of the immune-electronmicroscopic method for investigating of biological objects.