[The physicochemical characteristics of ribosomal fractions from Pseudomonas aeruginosa obtained by precipitation with polyethylene glycol 6000]. / Fiziko-khimicheskaia kharakteristika ribosomal'nykh fraktsii iz Pseudomonas aeruginosa, poluchennykh osazhdeniem poliétilenglikolem 6000.

Ribosomal fractions containing up to 72% of ribosomal material and 25% of sugars (among them, about 6% of hexose) were isolated from P.aeruginosa cells, immunotypes F-1, 2, 6 and 7, by precipitation with polyethylene glycol 6000. Lipopolysaccharide, determined in the test for ketodesoxyoctanoic acid, was not detected in these fractions, but, as determined in the passive hemagglutination test, the content of O-antigen in the preparations was 3-25%. O-antigen and ribosome present in the fractions formed a complex, disintegrating after treatment with trilon B.

Protective ribosomal preparation from Shigella sonnei as a parenteral candidate vaccine.

A parenteral Shigella ribosomal vaccine (SRV) was investigated in animals for safety, antibody-inducing capacity, and protective activity. Ribosomal preparations from a Shigella sonnei phase I avirulent strain were obtained and shown to possess chemical, sedimentation, and other properties typical of bacterial ribosomes. No endotoxin contamination was revealed by a ketodeoxyoctonate assay, although the presence of some kind of O antigen was evidenced by serological findings and the high activity of SRV in inducing the O-antibody response and immunological memory in animals. SRV was nontoxic in mice, guinea pigs, and monkeys and induced no local reactions when injected subcutaneously in reasonable doses. Significant protection against a local Shigella infection (Sereny test) was seen in guinea pigs injected with SRV (efficiency index, about 60%) and the specificity of the protection was evident from cross-challenge experiments. The protective efficiency of SRV was especially high in rhesus monkeys challenged orally with virulent Shigella cells (89%, as calculated from the summarized data of several experiments in 71 animals). Protection in monkeys was long lasting and could be demonstrated several months after injection of SRV. An inexpensive technique can be used for the production of SRV on a large scale. The high immunogenicity of SRV is discussed in terms of the amplifying effect of the ribosome, which serves as a delivery system for polysaccharide O antigen. Further study of SRV as a candidate vaccine for humans seems justified by the data obtained.

[The immunogenic properties of the endotoxin protein: serum antibodies in animals and man]. / Immunogennye svoistva belka éndotoksina: syvorotochnye antitela u zhivotnykh i cheloveka.

Endotoxin protein or lipid A-associated protein (LAP) from Shigella sonnei was isolated and characterized earlier (Zh. mikrobiol. epidemiol. immunobiol., 1991, No. 4, pp. 47-50). In this investigation serum antibodies against LAP were studied in ELISA Anti-LAP antibodies were detected in high titers in the sera of nonimmunized mice, guinea pigs, rabbits, monkeys and healthy adults. We suppose that normal anti-LAP antibodies resulted from interaction between the immune system and environmental endotoxin. Parenteral injections of LAP to different animals induced intensive antibody response with a 100- to 1000-fold increase in the serum anti-LAP antibody level and a significant rise in the serum O-antibody level. The latter is seemingly due to the contamination of LAP with minute amounts of O-antigen (0.12% or less) and to the amplification of its immunogenicity by LAP. Both antigenic and amplifying activity of LAP was destroyed by proteinase K. The biological function of LAP and its possible use as a component of bacterial vaccines are briefly discussed.

[Shigella endotoxin protein--its isolation and physicochemical characteristics]. / Belok éndotoksina shigell--vydelenie i fiziko-khimicheskaia kharakteristika.

The scheme of the isolation of endotoxic protein from S. sonnei 9090 is presented. The isolation procedure includes the 10-minute hot (at 68 degrees C) extraction of protein from endotoxin with 45% aqueous phenol, the precipitation of protein from phenolic extract with 9.5 volumes of 95% ethanol, the purification of protein from lipid material and pigments by multiple extraction with the mixture of chloroform and ethanol in the proportion 2:1 by volume. The yield of protein obtained with the use of this isolation scheme is about 3% of the initial endotoxin preparation. Protein preparations obtained in accordance with this scheme contain 92-95% of protein (determined by Lowry's method), 2.3-3.0% of saccharides (determined by the phenol-sulfate method) and 0.02% of hexose amine, its presence indicating that the preparations contain lipid A (or its fragments) which is firmly bound with endotoxic protein and cannot be extracted with chloroform. As shown in the passive hemagglutination inhibition test, the content of endotoxin in the preparations is less than 0.003%. Out of 7-11 bands revealed by electrophoresis in 15% polyacrylamide gel in the presence of sodium dodecyl sulfate, 3 main bands have molecular weights of 43, 38 and 18 KD. Three antigens differing in their electrophoretic mobility and diffusion rate in 1% agarose gel can be detected in the preparations by the method of immunoelectrophoresis with the use of antisera to both endotoxin and endotoxic protein.

[Shigella endotoxin protein--its electrophoretic and serological properties]. / Belok éndotoksina shigell--élektroforeticheskie i serologicheskie svoistva.

The electrophoretic analysis of lipid A-associated protein (LAP), obtained from S. sonnei, in polyacrylamide gel in the presence of sodium dodecyl sulfate and urea has revealed the heterogeneity of the preparation; it has found to contain three main components with molecular weights of 43, 38 and 18 KD and some minor components with molecular weights of 49, 45 35, 30, 29, 27, 5, 21 and 14 KD. The electrophoretic mobility of the main protein components in the isolated preparation of LAP coincides with that of endotoxin components. The dissociation of proteins and lipopolysaccharide in the process of boiling the endotoxin in 2% sodium dodecyl sulfate is indicative of the noncovalent binding of these components. LAP contained in the endotoxin, in contrast to isolated LAP, is resistant to trypsin and proteinase K. The enzyme immunoassay (EIA) system with the use of LAP as a component of its solid phase has been developed, which makes it possible to carry out the quantitative determination of antibodies to this protein. The EIA system shows high sensitivity in the determination of anti-LAP IgG antibodies: in hyperimmune rabbit sera their titer is 1:250,000-1:800,000. As shown by the method of competitive EIA, the antigenic affinity of LAP of different origin corresponds to the degree of taxonomic propinquity of microorganisms: the maximal degree of cross reactions is observed between LAP obtained from S. sonnei, S. flexneri and Escherichia coli, while their affinity to Salmonella typhi is considerably less; remote microbial species (Bacterium bifidum and Sarcina marcescens) give practically no cross reactions.

[Pressor-depressor factors in renal vein blood in patients with renovascular hypertension]. / Pressorno-depressornye faktory v krovi pochechnykh ven u bol'nykh vazorenal'noi arterial'noi gipertenziei.

Development of renovascular hypertension was investigated in 68 patients. Interrelated defects found in components of renal pressor-depressor system may be a contributing factor in the disease onset. Contralateral pressor-depressor system is the first to start regulating deranged regional and general hemodynamics in functional failure of the kidneys. In organic lesion occurring in the affected kidney later than in the contralateral one, there is compensatory depressor activation upon the distress of the pressor function. This may be aimed at maintenance of adequate microcirculatory blood flow and metabolism.

[O-specific L-hapten in the composition of Shigella sonnei]. / O-spetsificheskii L-gapten v sostave Shigella sonnei.

Along with classical lipopolysaccharide (LPS), O-specific material not precipitated by ultracentrifugation has been isolated from the water-phenol extract of S. sonnei avirulent strain 9090 possessing complete antigenic properties. The purification of O-antigen contained in the supernatant fluid has been carried out by the gel filtration of the fluid, previously treated with ribonuclease, in a column packed with Sephadex G-100. The polysaccharide nature of O-antigen thus obtained, the absence of lipid A and KDO and the low content of hexoses, or core-specific saccharides of S. sonnei LPS, in this antigen make it possible to classify this material with O-components of microbial cells, described by different authors as "native protoplasmic polysaccharide" or "L-hapten" and formed by polymers of LPS O-side chains. The content of this component in S. sonnei strains under study is, on the average, 2.5% of the weight of dry microbial substance. L-hapten preparations obtained in the course of our investigations have been found to contain two O-specific antigens detected by immunoelectrophoresis and immunodiffusion, as well as by sedimentation in saccharose gradient, where they form peaks corresponding to 4.3 S and 10.8 S. This polysaccharide O-antigen is supposed to be capable of interaction with ribosomal particles and suitable for use as a component of ribosomal dysentery vaccines.

[The isolation and immunologic study of ribosomal preparations from Shigella flexneri]. / Poluchenie i immunologicheskoe issledovanie ribosomal'nykh preparatov iz Shigella flexneri.

S. flexneri ribosomal preparations were isolated by differential centrifugation or by fractionation with polyethylene glycol-6000. Their chemical composition and spectrophotometric properties were characteristic of ribosomes, and, as shown by the results of the serological assay, the content of O-specific component was, on the average, 1.4%. The ribosomal preparations were nontoxic for mice when injected intraperitoneally and intravenously in large doses and induced systemic O-antibody response in mice and rabbits. The parenteral administration of ribosomes to guinea pigs led to the increase of resistance to Shigella keratoconjunctivitis. The results of different tests with the use of this model greatly varied. According to the summary data of several tests, the ribosomal vaccine enhanced the resistance of the eyes from 11.3% to 48.5% and the effectiveness coefficient of immunization was 42 +/- 6. Ribosomes isolated from S. flexneri avirulent strain 2a 51.6 M (Iu. A. Belaia's vaccine) showed the same activity as those isolated from virulent strains. The results obtained in this study suggest the expediency of further experimental study of ribosomal preparations obtained from S. flexneri as potential vaccine.

[A protective factor of ribosomal shigellosis vaccine]. / Protektivnyi faktor ribosomal'noi shigelleznoi vaktsiny.

Shigella ribosomal vaccine was shown previously to possess protective properties in the keratoconjunctival test on guinea pigs and to be capable of preventing experimental infection in 90% of challenged monkeys. The presence of the O-specific component (OSC) constituting about 0.5% of the ribosomal preparation by serological activity suggested its importance for the protective effect. This was studied in experiments with two O-specific immunosorbents prepared by coupling anti-O rabbit antibodies with Staphylococcus aureus cells or with CNBr-Sepharose. Ribosomes treated with immunosorbents proved to be lacking the serologically active OSC and lost their ability to induce O-antibody response in rabbits and mice. After the removal of this component ribosomal preparations were incapable of ensuring protection from Shigella kerato-conjunctival infection. The isolated OSC was also inactive in this test. The data obtained in this investigation confirm the hypothesis stating that the protective activity of Shigella ribosomal vaccine is based on the combined action of ribosomes and O-specific factor whose nature and properties require further study.

[Ribosomal dysentery vaccine. Study of response and antigenic activity in healthy volunteers]. / Ribosomal'naia dizenteriinaia vaktsina. Izuchenie reaktogennosti i antigennoi aktivnosti na zdorovykh dobrovol'tsakh.

In earlier studies Shigella sonnei ribosomal vaccine was shown to be highly protective for guinea pigs and monkeys. The object of the present study, carried out in 20 healthy volunteers, was the safety and the antigenic activity of this vaccine. The subcutaneous injection of the ribosomal vaccine in doses of 100 micrograms and 200 micrograms produced no febrile reactions nor biochemical and histological changes. The minimal local reaction was observed after injection into the subscapular region: in this case 200 micrograms of the vaccine produced neither severe, nor moderate reactions. A single injection of this dose led to a more than 4-fold rise in the levels of total and cysteine-resistant O-antibodies, as well as to the prolonged elevation of the complement level in the serum.

[Biological properties of a ribosomal vaccine from S. sonnei obtained by polyethylene glycol fractionation]. / Biologicheskie svoistva ribosomal'noi vaktsiny iz S. sonnei, poluchennoi metodom fraktsionirovaniia poliétilenglikolem.

Shigella ribosomal vaccine was prepared by fractionation with polyethylene glycol (PEG), recently proposed as an alternative of the more expensive and labor-consuming technique of differential centrifugation. In the present investigation the biological activity of ribosomal preparations isolated by these two methods was compared. Ribosomal vaccine obtained by PEG fractionation proved to be nontoxic for mice (LD50 greater than 2 mg) and produced no local and systemic reactions in monkeys when introduced subcutaneously in a dose of 600 micrograms. Ribosomes isolated by the two methods did not differ in the antigenic potency of their O-specific component responsible for inducing antibody formation in guinea pigs and monkeys. The protective potency of Shigella ribosomal vaccines prepared by PEG fractionation and ultracentrifugation was compared by tests in guinea pigs under the conditions of intraconjunctival challenge 2 weeks after a single injection of 40-200 micrograms of ribosomes. The mean resistance rate (percentage of protected eyes) was almost the same with both preparations, 67% and 65%, while in the control (nonimmunized) group only 13% of eyes were resistant to challenge. In monkey tests two injections of ribosomal vaccine obtained by PEG fractionation ensured a high protective effect against dysentery. No clinical signs of dysentery were observed in two groups of the test animals (totaling 10 monkeys) immunized 5 and 16 weeks before oral challenge with a dose of 75 X 10(9) virulent shigellae, which caused dysentery of moderate severity in all 5 control monkeys. The low toxicity and high protective potency of ribosomes isolated from S. sonnei by PEG fractionation makes it possible to use this method for the large-scale production of ribosomal vaccine.