Strategies for Using Muramyl Peptides - Modulators of Innate Immunity of Bacterial Origin - in Medicine.

The spread of infectious diseases is rampant. The emergence of new infections, the irrational use of antibiotics in medicine and their widespread use in agriculture contribute to the emergence of microorganisms that are resistant to antimicrobial drugs. By 2050, mortality from antibiotic-resistant strains of bacteria is projected to increase up to 10 million people per year, which will exceed mortality from cancer. Mutations in bacteria and viruses are occurring faster than new drugs and vaccines are being introduced to the market. In search of effective protection against infections, new strategies and approaches are being developed, one of which is the use of innate immunity activators in combination with etiotropic chemotherapy drugs. Muramyl peptides, which are part of peptidoglycan of cell walls of all known bacteria, regularly formed in the body during the breakdown of microflora and considered to be natural regulators of immunity. Their interaction with intracellular receptors launches a sequence of processes that ultimately leads to the increased expression of genes of MHC molecules, pro-inflammatory mediators, cytokines and their soluble and membrane-associated receptors. As a result, all subpopulations of immunocompetent cells are activated: macrophages and dendritic cells, neutrophils, T-, B- lymphocytes and natural killer cells for an adequate response to foreign or transformed antigens, manifested both in the regulation of the inflammatory response and in providing immunological tolerance. Muramyl peptides take part in the process of hematopoiesis, stimulating production of colony-stimulating factors, which is the basis for their use in the treatment of oncological diseases. In this review we highlight clinical trials of drugs based on muramyl peptides, as well as clinical efficacy of drugs mifamurtide, lycopid, liasten and polimuramil. Such a multifactorial effect of muramyl peptides and a well-known mechanism of activity make them promising drugs in the treatment and preventing of infectious, allergic and oncological diseases, and in the composition of vaccines.

Structural characteristics of immunostimulating polysaccharides from lentinus edodes.

There is a significant amount of experimental evidence suggesting that polysaccharides from mushrooms enhance the host immune system by activating various mechanisms in immune cells, including macrophages. In this study, polysaccharides from Lentinus edodes were found to stimulate the functional activation of macrophages to secrete inflammatory mediators and cytokines and increase the phagocytotic uptake. The chemical properties of the stimulatory polysaccharides, CPFN-G-I, CPBN-G, and CPBA-G, were determined based on their monosaccharide composition, which mainly consisted of glucose and mannose. According to FT-IR and GC/MS, the structure of CPFN-G-I, purified from the fruiting body of L. edodes, was found to consist of a beta-1,6-branched-beta-1,4-glucan, whereas CPBN-G and CPBA-G, purified from the liquid.

Post-translational modification by O-GlcNAc: another way to change protein function.

Modification of intracellular proteins by the beta-linkage of the monosaccharide, N-acetylglucosamine to serine or threonine hydroxyls (O-GlcNAc) is abundant and reversible. Although many proteins bear this post-translational covalent modification, the changes in function of the proteins as a result of this modification are only starting to be understood. In this article, we describe how aspects of the flux from the glucose backbone to this modification are modified and how the cellular activity and content of the GC-box binding transcription factor, Sp1, is altered by O-glycosylation. The association of the enzyme that puts on the O-GlcNAc modification with the bi-functional enzyme that removes this modification is discussed relative to the transition between transcriptional repression and activation.

Synthesis and biological evaluation of an anticancer vaccine containing the C-glycoside analogue of the Tn epitope.

The C-saccharide analogue of the GalNAc (Tn epitope) has been covalently linked to the T cell epitope peptide (328)(-)(340)OVA using a chemoselective convergent synthetic approach. In this way, a non-hydrolyzable synthetic vaccine was obtained composed by a B epitope conjugated to a T cell epitope. This compound was tested in a proliferation assay with spleen cells from DO11.10 mice. The molecule was recognized by transgenic T cells although at a slightly lower efficiency if compared with the reference peptide OVA. An additional experiment with dendritic cells fixed with glutaraldehyde shows that the glycopeptide can bind to extracellular MHC molecules without need of internalization and processing and that the C-glycoside part does not interfere with TCR recognition. These observations constitute an important starting point for the use of this molecule as vaccine against the Tn-expressing TA3-Ha mouse mammary carcinoma.

Purification and characterization of two mannan-binding lectins from mouse serum.

Mannan-binding lectin (MBL) is a serum protein that activates the complement system after binding to glycoconjugates found on the surface of microorganisms. By molecular cloning two forms of MBL have been identified in the mouse (mMBL-A and mMBL-C), but only mMBL-A has been purified and characterized at the protein level. MBL-C has been termed the liver form of MBL. The present report describes the purification and characterization of mMBL-A and mMBL-C from serum. The two forms of mMBL could be separated both by ion-exchange and carbohydrate-affinity chromatography. The initial identification by immunochemical technique was confirmed by N-terminal amino-acid sequencing. Both proteins give bands corresponding to polypeptide chains of 28 kDa on SDS-PAGE in the reduced state, but mMBL-A migrated more rapidly than mMBL-C in acid/urea-PAGE, in accordance with the calculated pIs. Both forms mediated activation of complement component C4 in mannan-coated microtiter wells. MBL-A showed a higher affinity for d -glucose and alpha-methyl-d -glucose then did MBL-C. Serum concentrations of mMBL-A in laboratory strains and wild mice were found to vary from 5 to 80 microg/ml, with wild mice tending to show higher levels than laboratory strains.

Interleukin-6 production induced in peripheral blood mononuclear cells by a serum factor from IgA nephropathy patients is inhibited in vitro by specific sugars.

The basal production of IL-6 by cultured peripheral blood mononuclear cells (PBMC) from patients with IgAN, is markedly higher (A, 109 pg/ml) as compared to that of PBMC of either patients without clinical signs (I, 39 pg/ml) or appropriate controls (C, 44 pg/ml). When PBMC from healthy subjects were incubated in the presence of sera from patients A, the IL-6 production was strongly enhanced. No such an effect was observed by stimulating PBMC with sera from the other two groups of subjects (I, C). In another experiment we observed that the IL-6 production stimulated by serum from patients A could be inhibited by addition of specific monosaccharides. The inhibitory effect was rapidly abolished when the sugar-containing medium was substituted with the original one. Finally molecular components from serum of A were grossly separated by gel column chromatography. Individual fractions were incubated with PBMC of C: fractions with Mr > 30,000 highly stimulated the release of IL-6 (up to 1320 pg/ml); fractions with lower molecular weight were inactive. The data suggest the presence of an IL-6 releasing factor in the serum of IgAN patients. Although the chemical nature of such a factor is not yet established, the observations reported focus our attention to the lectins family. Since this factor seems potentially important in the understanding of the pathogenesis of IgAN, both its isolation and structural/functional characterization deserve further efforts.

Specificity analysis of antibodies formed in rabbits to a mannosyl trisaccharide: similarity with lectin binding activity. Para-aminophenyl O-alpha-D-mannopyranosyl-(1-->2)-alpha-D-mannopyranosyl- (1-->6)-alpha-D-mannopyranoside linked to bovine serum albumin as an antigen.

The para-aminophenyl derivative of Man alpha 1-->2Man alpha 1-->6Man alpha 1-->was coupled via a diazotization reaction to bovine serum albumin, and the resulting glycoconjugate was used to immunize two rabbits. The resultant antisera were tested for reactivity with a number of related mono, di- and trisaccharides to determine the immunodominant portion of this trisaccharide. Two populations of antibody resulted, one of which required the reducing end mannose, and could react with either an N-acetylglucosamine or a mannose as the penultimate sugar. The other population reacted with the Man alpha 1-->2Man alpha 1-->6Man alpha 1-->. The aglycone moiety and its configurations play an important role in determining the specificity of antibodies to this synthetic antigen. The similarity of this reactivity to the reactivity of mannose binding lectins is discussed.

Plant lectins--histochemical and cytochemical applications in oncology.

Lectins are sugar binding proteins or glycoproteins of non-immune origin derived from various plants or animals with specific sugar binding capacity. This property of lectins can be used to identify structural differences between normal and malignant cells. Malignant transformation is accompanied by several changes in cell membrane. Studies have shown that the lectin binding pattern of these cells may indicate the invasive potential of tumours. Lectins can also be used as carriers. Lectins conjugated to chemotherapeutic agents has been found to be more useful in the treatment of tumours induced in animals.

[Localization of the antigenic determinants in carcinoembryonic antigen: the role of the carbohydrate component]. / Lokalizatsiia antigennykh determinant v rakovo-émbrional'nom antigene, rol' uglevodnogo komponenta.

The majority of topographic antigenic determinants of the carcinoembryonic antigen (CEA) representing a glycoprotein were shown to contain sugar residues of the CEA carbohydrate chains. Periodate oxidation of CEA followed by reduction afforded a corresponding CEA derivate (CEA-POR), which retained 3% antigenic activity of CEA. In secondary and tertiary structures CEA-POR was proved to be close to CEA pre heated to 80 degrees C. Formation of borate complexes with sugar residues of CEA decreased the CEA antigenic activity to 5% while a but did not affect the spatial structure of its protein core.

A human lymphokine released by mononuclear blood cells upon contact with CEA.

As revealed by the macrophage electrophoretic mobility (MEM) technique mononuclear blood cells from certain cancer patients respond to carcinoembryonic antigen (CEA). This phenomenon appeared to be due to a specific lymphokine release. In this study, the lymphokine activity of supernatant pools was stepwise enriched by gel filtration on Sephadex. The mediator activity was recovered within a molecular mass region less than 47 kDA. The lymphokine was highly enriched during further gel filtration steps and showed a single activity peak in the molecular mass region of 23.5 kDa. Gel filtrations of appropriate control supernatants resulted in biologically inactive fractions. The lymphokine was heat-labile at 56 degrees C, showed a clear-cut, dose-dependent effect on macrophages, and could be blocked by fucose. Preparative gel electrophoresis of radiolabeled and unlabeled lymphokine resulted in two corresponding peaks of biological activity and radioactivity.

Reactivity of antisera to endogenous primate retrovirus with a human T cell membrane protein: recognition of a nonviral glycoprotein by antibodies directed only against carbohydrate components.

A glycosylated protein of approximately 70,000 daltons (gp70) from the surface of human peripheral blood T cells was immunoprecipitated by antisera to the baboon endogenous retrovirus (BEV-M7) and the serologically related feline endogenous retrovirus (RD114) but not by antisera to other retroviruses. Whereas preliminary absorption experiments were consistent with a possible viral specificity for this reaction, detailed biochemical and serologic characterization of the purified cellular protein suggested that it was not related to the gp70 of either M7 or RD114 viruses. The specificity of the reaction was further analyzed by assays of cellular gp70 antigenicity after exposure to exo- and endoglycosidases or trypsin and carbohydrate hapten inhibition studies. The results of these experiments were consistent with the interpretation that the glycoprotein was being recognized by antibody binding to the carbohydrate moiety of the molecule. These results provide an example of an antibody activity that could lead to inappropriate conclusions when sensitive radioimmunoprecipitation techniques are used for the biochemical analysis of antigenic systems. They emphasize the necessity of purifying cellular antigens as a prerequisite to determining the exact basis for a serologic reaction.

Epidermal surface saccharides reactive with phytohemagglutinins and pemphigus antigen.

Inorder to study the relationship between epidermal surface saccharides and pemphigus antigen(s), fluorescein-labelled Concanavalin A (Con A) and Phytohemagglutinin-P (PHA-P) were used. These phytohemagglutinins were found to bind with the intercellular areas of human epidermis. Alpha-methyl-D-mannoside and alpha-methyl-D-glucoside inhibited the epidermal intercellular staining pattern produced by ConA-FITC, while N-acetyl-D-galactosamine blocked the same staining pattern produced by PHA-P-FITC. Normal human skin reacted with pemphigus antibody and pemphigus skin with the deposition of IgG both gave a positive intercellular staining pattern with fluorescein labelled phytohemagglutinins. Our data indicated the non-identity of the binding of Con A and PHA-P, and pemphigus antigen(s).