Combination of Muramylpeptides from Gram-Negative Bacteria Corrects Cyclophosphamide-Induced Disorders of Hematopoiesis and Spleen Cell Composition in Mice with B16 Melanoma.

We studied the effect of a combination of three muramylpeptides from gram-negative bacteria (Polymuramyl) on hematological parameters, morphology of the spleen, and serum cytokine level in mice with B16 melanoma treated with cyclophosphamide. Intraperitoneal administration of cyclophosphamide to tumor-bearing animals sharply reduced the number of leukocytes, especially neutrophils, in the blood and depleted the cellular composition of the spleen white pulp. Subsequent three daily intramuscular injections of Polymuramyl in doses of 70 and 860 ng/mouse for 3 days, as well as subcutaneous injection of the reference drug filgrastim (granulocytic CSF) in a dose of 12 µg/mouse partially corrected hematopoietic disorders and restored the cellular composition of the spleen. Granulocytic CSF more effectively replenished the content of mature neutrophils in the blood, while Polymuramyl restored the content of stab neutrophils. In contrast to granulocytic CSF, administration of Polymuramyl was followed by an increase in the level of granulocyte-macrophage CSF and a tendency to an increase in the serum content of IL-6, which indicates the involvement of these cytokines in the hematopoietic activity of Polymuramyl.

A Combination of Muramylpeptides from Gram-Negative Bacteria Corrects Hematological and Immunological Disorders Induced by Cyclophosphamide.

We have studied the effect of a combination of three natural muramylpeptides containing a meso-diaminopimelic acid residue (polyramyl) on the subpopulations of circulating T cells, spleen morphology, and leukocyte level in the blood of C57Bl/6 mice with cyclophosphamideinduced immunosuppression. Intraperitoneal injections of cyclophosphamide in a dose of 100 mg/kg on days 1, 3, 5, and 7 of the experiment reduced leukocyte count and the relative number of CD4+ T cells in the blood, and also depleted the cellular composition of splenic white pulp on day 10. Subcutaneous injections of polyramyl in a dose of 200 µg/mouse on days 8 and 9 practically completely restored blood leukocytes count and morphology of the splenic white pulp. Moreover, administration of polyramyl induced marked tendency to increase in the relative number of CD4+ T cells and CD4/CD8 ratio in mice with cyclophosphamideinduced immunosuppression.

[Induction and protective properties of antibodies against muramyl peptides of gram-negative bacteria].

AIM: Characterize the role of humoral immune response in mechanisms of action of muramyl dipeptide immune stimulators. MATERIALS AND METHODS: Mice were immunized by a complex of muramyl peptides (CMP) obtained from Salmonella typhi peptidoglycan and consisting of 3 components: 1) N-acetyl-D-glucoasminyl-(beta1- > 4)-N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid (GMtri); 2) N-acetyl-D-glucosaminyl-(beta1- > 4)-N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine (GMtetra) and 3) GMtetra dimer (diGMtetra), in which monomeric residues of GMtetra are linked by an amid bond between carboxyl group of terminal D-alanine of one of GMtetra residues and omega-amino group of meso-diaminopimelic acid of the other GMtetra residue. RESULTS: Immunization resulted in a multifold increase of IgM, IgG1 and IgG2a titers against CMP. Antibodies were directed against the whole molecule of diGMtetra and did not recognize its fragments. Sera of mice immunized with CMP protected the mice from lethal infection with Gram-negative (S. typhimurium) but not Gram-positive (Staphylococcus aureus) microorganisms. CONCLUSION: Induction of protective antibodies may present a novel mechanism of action of muramyl dipeptide immune stimulators.

[Study of cross-activity of Streptococcus pneumoniae antigen preparations].

AIM: Study cross-activity of S. pneumoniae antigen preparations. MATERIALS AND METHODS: Antigen preparations were obtained by ultrasound disintegration (from bacteria in R-form), extraction with water (from serotype 3 bacteria), cetavlon and trichloroacetic acid (from serotype 6A bacteria). Chemical composition and immunochemic properties of preparations were studied by contemporary methods as well as in experiments with direct and cross-protection of mice from infection. RESULTS: 3 of 4 preparations (except ultrasound disintegrate) had approximately 30% of protein. In immunodiffusion reaction they interacted with hyper immune rabbit sera obtained against 12 various pneumococcus serotypes--1, 3, 4, 6A, 6B, 9V, 9N, 14, 18C, 19A, 19F and 23F. In animal experiments 30 - 70% of mice were protected from subsequent infection with knowingly high dose of homologous and 3 heterologous pneumococcus strains. In immunoblotting the highest number of components serologically active with heterologous sera was formed by cetavlon extract (12 - 23). Addition of capsule polysaccharides to the preparation increased its cross-protective activity. CONCLUSION: By data set and the highest yield, water extract is reasonable for isolation of cross-reactive proteins of pneumococcus. Development of another method of extraction from cultural fluid is necessary for obtaining extracellular protein antigens. Generation of vaccines containing cross-reactive proteins of pneumococcus and capsule polysaccharides is a promising direction.

[Experimental study of candidate vaccines against variable or quasi-species pathogenes: multiepitopic synthetic peptide antigenes and new receptor-guiding adjuvants].

The short multiepitopic synthetic peptides from the sequences of hypervariable area of V3-loope of gp120 of HIV don't induce anti-peptides antibodies production in mice themselves. We prepared the potent immunogen by noncovalent conjugations of the multitude peptides with pure peptidoglycans from cell wall of Salmonella typhi. The sera from immunized mice have the anti-peptides antibody titers (3-5) x 10(5) in ELISA, as high as Freund's adjuvant is of use.

[Structure of the oligosaccharide region (core) of the lipopolysaccharides of Shigella flexneri types 2a and 5b].

The full structure of the lipopolysaccharide core of bacteria Shigella flexneri types 2a and 5b, the causative agents of bacillary dysentery (shigellosis), was established by chemical methods, high-resolution electrospray ionization mass spectrometry, and two-dimensional NMR spectroscopy. The structure of the O-antigen repeating unit and the configuration and position of the linkage between the O-antigen and the core were determined in the lipopolysaccharide of S. flexneri type 2a.

[Changes in functional activity of macrophages caused by bacterial muramylpeptides].

Muramylpeptides from bacteria cell wall are strong stimulators of immune system and phagocytic cells are main effectors. Dimer containing glucoseaminylmuramylpentapeptide (di-GMPP) was obtained from cell wall of Salmonella typhi bacteria. Di-GMPP decrease the phagocytic activity of macrophages obtained from peripheral blood of healthy donors and increase intracellular killing. Also di-GMPP resulted in decrease of expression of macrophages' receptors which play role in phagocytosis (CD16, CD64, CD11b) and detection of bacterial molecular patterns (TLR2, TLR4, CD206), as well as in increase of expression of antigen-presenting (HLA-DR) and costimulatory molecules (CD86, CD40) which involved in formation of immunological synapse and presentation of antigens to T- and B-lymphocytes.

FITC-labeled lipopolysaccharide: use as a probe for liposomal membrane incorporation studies.

FITC-labeled LPS from Neisseria meningitidis can be used as a probe to follow the process of LPS incorporation into liposomal membrane and to study its interaction with a bilayer. The incorporation of FITC-LPS into the bilayer was proved by physicochemical methods as well as by liposomal LPS toxicity decrease in actinomycin D-sensitized mice. Fluorescence intensity increase was observed upon the insertion of FITC-LPS into the membrane of dehydration/rehydration vesicles and vesicles obtained by co-sonication of lipid suspension and FITC-LPS. Following FITC-LPS fluorescence polarization it was shown that the substance seems to be clusterized in the liposomal membrane starting from FITC-LPS/lipid molar ratio 1:800.

Structure of the repeating unit of the O-specific polysaccharide of the lipopolysaccharide of Yersinia kristensenii strain 490 (O:12,25).

The O-specific polysaccharide isolated by mild acid degradation of the lipopolysaccharide of Y. kristensenii strain 490 (O:12,25) contained D-glucose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, 2-acetamido-2,6-dideoxy-L-galactose, glycerol, and phosphate in the ratios 2:2:1:1:1:1. On the basis of 31P- and 13C-n.m.r. data, methylation analysis, dephosphorylation, solvolysis with anhydrous hydrogen fluoride, and Smith degradation, it was concluded that the repeating unit of the polysaccharide was a branched hexaosylglycerol phosphate with the following structure. [formula: see text]

Somatic antigens of Shigella: the structure of the polysaccharide chain of Shigella boydii type 2 lipopolysaccharide.

The structure of the polysaccharide chain of Shigella boydii type 2 lipopolysaccharide was established using mainly 13C-n.m.r. spectroscopy, partial hydrolysis, Smith degradation, and methylation analysis. The repeating unit of the polysaccharide was concluded to be a branched hexasaccharide, as follows: (formula in text). Acetaldehyde was detected in the hyrolysate of the lipopolysaccharide, but no evidence was obtained to indicate that acetaldehyde is located in the polysaccharide moiety.

Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7.

The structure of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O:7 has been investigated, using n.m.r. spectroscopy, methylation analysis, partial hydrolysis, and Smith degradation as the principal methods. It is concluded that the polysaccharide is constructed of repeating pentasaccharide units having the structure (formula; see text) where D-QuipNAc stands for 4-acetamido-4,6-dideoxy-D-glucopyranose. The 13C-n.m.r. spectrum of the polysaccharide has been interpreted completely.

Structure of the O-specific polysaccharide chain of Shigella boydii type 5 lipopolysaccharide: a repeated study.

An acidic, partially O-acetylated O-specific polysaccharide was obtained by mild acid degradation of Shigella boydii type 5 lipopolysaccharide and studied by 1H and 13C NMR spectroscopy, including 2D COSY, 13C-1H heteronuclear COSY, 1D NOE, and 2D ROESY experiments, and chemical methods (sugar and methylation analysis, O-deacetylation, carboxyl reduction, solvolysis with anhydrous HF, partial acid hydrolysis. Smith degradation). It was concluded that the polysaccharide has a hexasaccharide repeating unit of the following structure: [Formula: See Text] with the degree of O-acetylation varying over 30-50%. The established structure differs from that proposed recently for the O-specific polysaccharide of the same S. boydii serotype [M.J. Albert et al., Carbohydr. Res., 265 (1994) 121-127].

The structure of the cyclic enterobacterial common antigen (ECA) from Yersinia pestis.

Two antigenic acidic polysaccharides related to enterobacterial common antigen (ECA) were isolated from a vaccine strain of a pathogenic microorganism Yersinia pestis. The low molecular weight polysaccharide (LMP) is composed of equal amounts of 2-acetamido-2-deoxy-D-mannuronic acid, 4-acetamido-4,6-dideoxy-D-galactose (Fuc4NAc), and 2-amino-2-deoxy-D-glucose which is partially N- and partially 6-O-acetylated. The structure of the trisaccharide repeating unit was established by analyses of LMP and the completely N-acetylated LMP (LMP-NAc) using 1H and 13C NMR spectroscopy, including 2D COSY and 1D NOE spectroscopy. Deamination of LMP with nitrous acid gave a set of oligomers terminated with 2,5-anhydromannose and ranging from tri- to dodeca-saccharides, thus indicating a random distribution of free amino groups. FABMS analyses of LMP and LMP-NAc showed that LMP consists mainly of the cyclic tetramer of the trisaccharide repeating unit together with a small amount of the cyclic trimer and a very small amount of the cyclic pentamer and has, thus, the following structure: [formula: see text] where R is Ac or H (approximately 1:1), R' is Ac or H (approximately 1:4), and n = 4 (major), 3, 5 (minor). Small proportions of the linear trimer and the linear tetramer were also detected in the preparations. The high molecular weight polysaccharide is linear and has the same (or a very similar) repeating unit as LMP.

[Isolation of a highly purified capsular polysaccharide from Salmonella enterica serovar Typhi (Salmonella typhi)--Vi-antigen and its use in serological diagnosis of typhoid fever]. / Kapsul'nyi polisakharid Salmonella enterica serovara Typhi (Salmonella typhi)--Vi-antigen vysokoi stepeni ochistki dlia serologicheskoi diagnostiki briushnogo tifa.

For use in differential diagnostics of typhoid fever, samples of the capsular polysaccharide from Salmonella enterica serovar Typhi (usually named Vi-antigen) were isolated and characterized by physicochemical and serological methods. It was shown that only the sample of Vi-antigen with the minimal (0.57%) admixture of the corresponding lipopolysaccharide (LPS) from S. typhi retained a high serological activity in the tests with monoreceptor anti-Vi sera. However, it exhibited a substantially weaker reaction with sera from normal donors and patients with acute nontyphoid salmonelloses, than Vi-antigen preparations with a higher (0.8-1.2%) LPS content. The chromatographically pure Vi-antigen was purified by triple reprecipitation with hexadecyltrimethylammonium bromide. The content of the LPS admixture in the resulting Vi-antigen samples was quantitatively determined by GC. A high purification level of the Vi-antigen from the LPS admixture allows us to hope that this preparation could serve as a basic component of the test system for the diagnostics of typhoid fever. The English version of the paper.

[Study of the structure of O-specific polysaccharide of Salmonella anatum using 1H- and 13C-NMR-spectroscopy]. / Izuchenie stroeniia O-spetsificheskogo polisakharida Salmonella anatum s primeneniem 1H- i 13C-IaMR-spektroskopii.

The Salmonella anatum O-specific polysaccharide structure was fully confirmed by means of complete interpretation of its 13C NMR spectrum, using the selective double resonances and NOE experiments: [-3(6Ac)GaIp alpha 1-6Manp beta 1-4Rhap alpha 1-]n.

[Antigenic determinants of bacteria. The structure of the repeating unit of a specific polysaccharide from Shigella boydii type 7]. / Antigennye polisakharidy bakterii. Stroenie povtoriaiushchegosia zvena spetsificheskogo polisakharida Shigella boydii, tip 7.

Acid hydrolysis of the antigenic lipopolysaccharide from Shigella boydii type 7 afforded a specific polysaccharide composed of 2-acetamido-2-deoxy-D-glucose, D-glucose, D-galactose, 5-acetamido-3,5,7,9-tetradeoxy-7-[(3R)-3-hydroxybutyramido]-L- glycero-L-manno-nonulosonic acid (NonN2A) and acetic acid residues in the 1:1:2:1:1 ratio. From the results of methylation analysis, hydrogen fluoride solvolysis and Smith degradation, the structure of the repeating unit of the specific polysaccharide was dedused as: -2) Galf (beta 1-3)GlcNAcp (alpha 1-8)NonN2A (beta 2-6) Galp (alpha 1-6) Glcp (alpha 1-4 increases Ac. The 13C NMR spectrum of the polysaccharide was interpreted, and the spectral data fully confirmed the structure of the polysaccharide repeating unit.

[Somatic antigens of Shigella and Escherichia coli. Determination of the structure of O-specific polysaccharide from Shigella boydii type 12 and its serological properties compared with the O-specific polysaccharide from Escherichia coli]. / Somaticheskie antigeny Shigella i Escherichia coli. Ustanovlenie stroeniia O-spetsificheskogo polisakharida iz Shigella boydii, tip 12, i izuchenie ego serologicheskikh svoistv v sravnenii s O-spetsificheskim polisakharidom iz Escherichia coli.

The structure of the O-specific polysaccharide of the somatic antigen (lipopolysaccharide) of Shigella boydii, type 12, was established by 1H- and 13C-NMR, methylation analysis and partial acid hydrolysis methods. The polysaccharide consists of pentasaccharide repeating units of the following structure: (formula; see text) The amount of O-acetyl groups was far less than stoichiometric, only about 2 for 3-4 repeating units. Nevertheless, the results of serological studies revealed 3-O-acetyl-alpha-L-rhamnose residue to be the major immunodominant group. In spite of the presence of similar trisaccharide fragments, the lipopolysaccharide and polysaccharide from Shigella boydii type 12 gave no crossreaction with lipopolysaccharide and polysaccharide from Escherichia coli 07. The possible reasons of the absence of serological relatedness between the Sh. boydii, type 12, and E. coli 07 cells were discussed.

[Somatic antigens of the Brucella genus. The structure of the O-specific polysaccharide chain of Brucella melitensis lipopolysaccharide]. / Somaticheskie antigeny roda Brucella. Stroenie O-spetsificheskoi polisakharidnoi tsepi lipopolisakharida Brucella melitensis.

The phenol-phase soluble antigenic lipopolysaccharide was isolated from Brucella melitensis, strain 565, by the routine phenol/water procedure followed by chromatography on Sepharose 4B. After mild acid hydrolysis and chromatography on Sephadex G-50, the lipopolysaccharide yielded a linear O-specific polysaccharide built up from 1,2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl units. The structure of the polysaccharide was deduced mainly from the nuclear magnetic resonance and methylation analyses. The phenol-soluble lipopolysaccharide, isolated from commercial vaccine strain B. abortus 19-BA, on mild hydrolysis afforded material, 13C and 1H-NMR spectra of which were identical to those of the O-specific polysaccharide from B. melitensis 565.

[Antigenic polysaccharides of Shigella bacteria. Structure of the polysaccharide chain of the lipopolysaccharide from Shigella boydii, type 11]. / Antigennye polisakharidy bakterii roda Shigella. Struktura polisakharidnoi tsepi lipopolisakharida Shigella boydii, tip 11.

On mild acid degradation of the Shigella boydii, type 11 lipopolysaccharide, the corresponding O-specific polysaccharide composed of D-glucuronic acid, 2-acetylamino-2-deoxy-D-glucose, D-ribose and L-rhamnose residues in the ratio 1:1:1:3 was obtained. Methylation, partial acid hydrolysis and 13C-NMR spectral data for the polysaccharide led to the structure of the oligosaccharide repeating unit as a branched hexasaccharide: [formula: see text]. Numerous O-acetyl groups attached non-stoichiometrically to the residues of D-glucuronic acid, L-rhamnose and 2-acetylamino-2-deoxy-D-glucose were located with the use of 13C-NMR spectroscopy.

[Bacterial antigenic polysaccharides. 12. Structure and 13C NMR spectrum of the polysaccharide chain of Shigella boydii type 8 lipopolysaccharide]. / Antigennye polisakharidy bakterii. 12. Ustanovlenie struktury i interpretatsiia spektra 13C-IaMR spetsificheskoi polisakharidnoi tsepi lipopolisakharida Shigella boydii tip 8.

A specific acidic polysaccharide was isolated from Sh. boydii type 8 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucuronic acid, D-galacturonic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose and 2-amino-1,3-propanediol residues in 1:1:1:1:1 ratio. From the results of methylation analysis, partial acid hydrolysis and Smith degradation, the structure of the repeating unit of the specific polysaccharide was deduced as: (Formula: see text). The 13C NMR spectra of native, O-deacetylated and carboxyl-reduced polysaccharides, as well as the spectrum of oligosaccharide produced by Smith degradation were interpreted. The 13C NMR data fully confirmed the structure of the polysaccharide repeating unit.