Glucan Preparations Research Articles

Preparation of resistant glucan by high pressure processing (HPP) and enzymatic methods increases indigestibility

This study describes a new method for producing high-quality resistant glucan by characterizing the structural mechanism of indigestibility. The structures and properties of resistant glucans were characterized before and after in vitro simulated digestion. The results demonstrated that high-pressure processing (HPP) led to the complete disappearance of crystal peaks and increased the efficiency of the two enzymes (α-amylase and transglucosidase). Moreover, α-1,6 and β-linkages were abundant in the connecting parts of the long and branching chains in the resulting resistant glucans, thus hindering the ability of digestive enzymes to hydrolyze short chains with a degree of polymerization (DP) ≤ 6. In addition, transglucosidase activity led to a higher proportion of short chains (DP 3–6), further promoting indigestibility. We demonstrated that, without rectification and decolorization, the dietary fiber content was >75 %, and the degree of branching increased to 50.9 %, indicating higher indigestibility than that resistant glucans produced by traditional methods.

Candida utilis ATCC 9950 Cell Walls and β(1,3)/(1,6)-Glucan Preparations Produced Using Agro-Waste as a Mycotoxins Trap.

Mycotoxins are harmful contaminants of food and feed worldwide. Feed additives with the abilities to trap mycotoxins are considered substances which regulate toxin transfer from feed to tissue, reducing its absorption in animal digestive tract. Market analysis emphasizes growing interest of feed producers in mycotoxins binders obtained from yeast biomass. The aim of the study was to prescreen cell walls (CW) and β(1,3)/(1,6)-glucan (β-G) preparations isolated from Candida utilis ATCC 9950 cultivated on waste potato juice water with glycerol as adsorbents for aflatoxin B1 (AFB1), zearalenone (ZEN), ochratoxin A (OTA), deoxynivalenol (DON), nivalenol (NIV), T-2 toxin (T-2) and fumonisin B1 (FB1). The adsorption was studied in single concentration tests at pH 3.0 and 6.0 in the presence of 1% of the adsorbent and 500 ng/mL of individual toxin. Evaluated CW and β-G preparations had the potential to bind ZEN, OTA and AFB1 rather than DON, NIV, T-2 toxin and FB1. The highest percentage of adsorption (about 83%), adsorption capacity (approx. 41 µg/ g preparation) and distribution coefficient (458.7mL/g) was found for zearalenone when CW preparation was used under acidic conditions. Higher protein content in CW and smaller particles sizes of the formulation could influence more efficient binding of ZEN, OTA, DON and T-2 toxin at appropriate pH compared to purified β-G. Obtained results show the possibility to transform the waste potato juice water into valuable Candida utilis ATCC 9950 preparation with mycotoxins adsorption properties. Further research is important to improve the binding capacity of studied preparations by increasing the active surface of adsorption.

Valorization of Deproteinated Potato Juice Water into \u03b2-Glucan Preparation of C. utilis Origin: Comparative Study of Preparations Obtained by Two Isolation Methods

Purposeβ(1,3)/(1,6)-Glucan of yeast origin is a bioactive polysaccharide used in nutrition, healthcare, agriculture and wastewater treatment. Yeast cultivation focused on the biosynthesis of β-glucan with simultaneous utilization of industrial wastes as a culture medium is a new way of obtaining these functional polysaccharides. The aim of current study was to isolate and characterize the purified β(1,3)/(1,6)-glucan preparation from Candida utilis ATTC 9950 biomass propagated in waste deproteinated potato juice water with the addition of glycerol.MethodsThe production of biomass of tested C. utilis strain was carried on biofermentor scale. The isolation of cell wall β-glucan was tested with two different methods: the alkaline-acid procedure coupled with hot-water extraction (method 1) and isolation based on cell wall autoclaving, lipids extraction and proteolysis (method 2). The isolated fractions were further analyzed with infrared spectroscopy.ResultsObtained preparations contained approx. 82% of β(1,3)/(1,6)-glucan after shortened extraction comparing with procedures described in literature. Infrared spectra of examined preparations demonstrated highly similar pattern to β-glucan standard of Saccharomyces cerevisiae origin. The preparation produced by method 2 showed higher ratio of β(1,3)- to β(1,6)-glucan. The production efficiency for method 2 was 70%, while for the method 1 it was significantly lower, i.e. 49%, indicating substantial material losses during alkaline-acid purification.ConclusionsThe C. utilis ATCC 9950 yeast cultivated on waste deproteinated potato juice water supplemented with glycerol is a highly efficient system to obtain purified β-glucan preparation.Graphical

Immune-modulating activities of glucans extracted from Pleurotus ostreatus and Pleurotus eryngii

We compared the immune-modulating activity of glucans extracted from P. ostreatus and P. eryngii on phagocytosis of peripheral blood neutrophils, and superoxide release from HL-60 cells. The results suggest that the anti-inflammatory properties of these glucans are partially mediated through modulation of neutrophil effector functions (P. eryngii was more effective). Additionally, both glucans dose-dependently competed for the anti-Dectin-1 and anti-CR3 antibody binding. We then tested the putative anti-inflammatory effects of the extracted glucans in inflammatory bowel disease (IBD) using the dextran sulfate sodium (DSS)–induced model in mice. The clinical symptoms of IBD were efficiently relieved by the treatment with two different doses of the glucan from both fungi. Glucan fractions, from either P. ostreatus or P. eryngii, markedly prevented TNF-α mediated inflammation in the DSS–induced inflamed intestine. These results suggest that there are variations in glucan preparations from different fungi in their anti-inflammatory ability.

Immunomodulating Effects Exerted by Glucans Extracted from the King Oyster Culinary-Medicinal Mushroom Pleurotus eryngii (Agaricomycetes) Grown in Substrates Containing Various Concentrations of Olive Mill Waste

We have recently demonstrated that we could enhance glucan content in Pleurotus eryngii following cultivation of the mushrooms on a substrate containing different concentrations of olive mill solid waste (OMSW). These changes are directly related to the content of OMSW in the growing substrate. Using dextran sulfate sodium (DSS)-in-flammatory bowel disease (IBD) mice model, we measured the colonic inflammatory response to the different glucan preparations. We found that the histology damaging score (HDS) resulting from DSS treatment reach a value of 11.8 ± 2.3 were efficiently downregulated by treatment with the fungal extracted glucans. Glucans extracted from stalks cultivated at 20% OMSW downregulated to a HDS value of 6.4 ± 0.5 whereas those cultivated at 80% OMSW showed the strongest effects (5.5 ± 0.6). Similar downregulatory effects were obtained for expression of various intestinal cytokines. All tested glucans were equally effective in regulating the number of CD14/CD16 monocytes from 18.2 ± 2.7% for DSS to 6.4 ± 2.0 for DSS + glucans extracted from stalks cultivated at 50% OMSW. We tested the effect of glucans on lipopolysaccharide-induced production of TNF-α, which demonstrated that stalk-derived glucans were more effective than caps-derived glucans. Isolated glucans competed with anti-Dectin-1 and anti-CR3 antibodies, indicating that they contain β-glucans recognized by these receptors. In conclusion, the most effective glucans in ameliorating IBD-associated symptoms induced by DSS treatment in mice were glucan extracts prepared from the stalk of P. eryngii grown at higher concentrations of OMSW. We conclude that these stress-induced growing conditions may be helpful in selecting more effective glucans derived from edible mushrooms.

The scale-up cultivation of Candida utilis in waste potato juice water with glycerol affects biomass and \u03b2(1,3)/(1,6)-glucan characteristic and yield

New ideas on production of yeast origin β-glucan preparations for industrial application are attracting interest considering market development of that high-value functional polysaccharide. Sellecting an efficient yeast producer and designing culture conditions are a prerequisite for obtaining high yield of β-glucan. The aim of this study was to describe at the first time the influence of the mode of cultivation (shake-flasks and batch fermentation) and time of culture on characteristic and yield of biomass and β(1,3)/(1,6)-glucan preparations of Candida utilis ATCC 9950 after cultivation in medium based on waste potato juice water supplemented with 10% of glycerol. After shake-flask culture, the biomass was characterized by higher protein content (app. 26.5%) compared to 19% after batch fermentation while the cultivation on a biofermentor scale promoted polysaccharides biosynthesis. The highest output of purified β(1,3)/(1,6)-glucan preparation (5.3 gd.w./L), containing app. 85% of that polysaccharide, was found after 48 h cultivation in biofermentor. Batch fermentation promoted biosynthesis of alkali-insoluble β(1,3)/(1,6)-glucan fraction, decreasing the content of β(1,6)-glucan. The yield of β(1,3)/(1,6)-glucan synthesis was 0.063 (g/g glycerol), while the productivity of that polysaccharide reached 0.094 (g/L/h). Longer batch fermentation (72 h) resulted in reduction of production efficiency of β-glucan preparation under studied conditions. The results of the study provide a new efficient biotechnological solution to produce high-value β-glucan preparations of C. utilis origin based on valorization of agro-waste potato juice water with glycerol.

Preparation of a Strong Gelatin-Short Linear Glucan Nanocomposite Hydrogel by an in Situ Self-Assembly Process.

Gelatin hydrogels exhibit excellent biocompatibility, nonimmunogenicity, and biodegradability, but they have limited applications in the food and medical industries because of their poor mechanical properties. Herein, we first developed an in situ self-assembly process for the preparation of gelatin-short linear glucan (SLG) nanocomposite hydrogels with enhanced mechanical strength. The microstructure, dynamic viscoelasticity, compression behavior, and thermal characteristics of the gelatin-SLG nanocomposite hydrogels were determined using scanning electron microscopy (SEM), dynamic rheological experiments, compression tests, and texture profile analysis tests. The SEM images revealed that nanoparticles were formed by the in situ self-assembly of SLG in the gelatin matrix and that the size of these nanoparticles ranged between 200 and 600 nm. The pores of the nanocomposite hydrogels were smaller than those of the pure gelatin hydrogels. Transmission electron microscopy images and X-ray diffraction further confirmed the presence of SLG nanoparticles with spherical shapes and B-type structures. Compared with pure gelatin hydrogels, the nanocomposite hydrogels exhibited improved mechanical behavior. Notably, the hardness and maximum values of the compressive stress of gelatin-SLG nanocomposites containing 5% SLG increased by about 2-fold and 3-fold, respectively, compared to the corresponding values of pure gelatin hydrogels.

Generation of Recombinant Antibodies against the beta-(1,6)-Branched beta-(1,3)-D-Glucan Schizophyllan from Immunized Mice via Phage Display.

beta-(1,6)-Branched beta-(1,3)-D-glucans like schizophyllan from the basidiomycete Schizophyllum commune excite various immunostimulatory effects and have been clinically tested as adjuvants. Some of the glucans are also applicable in food or petrol industry due to their viscosity and temperature stability in aqueous solution. Antibodies against these glucans could be used as tool for analysis of glucan preparations or for further research of its bioactivity. Therefore, an immune phage display library was constructed from mice immunized with schizophyllan. Three recombinant monoclonal antibodies were isolated from this library by affinity selection (panning) on schizophyllan. The half-maximal effective concentration (EC50) values for those antibodies varied between 16.4 ng mL−1 and 21.3 ng mL−1. The clones showed binding specificity not only for schizophyllan but also for other beta-(1,6)-branched beta-(1,3)-D-glucans of similar macromolecular structure. Denaturation of the secondary structure led to a reduced antibody binding, indicating an epitope requiring the correct conformation of the triple helical structure of the glucans.

Facile fabrication of amphiphilic AIE active glucan via formation of dynamic bonds: self assembly, stimuli responsiveness and biological imaging.

Fluorescent organic nanoparticles (FONs) with aggregation induced emission (AIE) properties have recently emerged as one of the most promising luminescent nanomaterials for biomedical applications due to their unique AIE feature. In this study, we reported the preparation of AIE active FONs through mixing AIE dye (TPE-CHO), 3-aminobenzeneboronic acids (ABBA) and glucan in one-pot. ABBA acted as a molecular "bridge" to conjugate TPE-CHO with glucan via formation of a Schiff base and phenyl borate. The resultant products (Glu-TPE FONs) showed amphiphilic properties and could self-assemble into nanoparticles in an aqueous solution. Glu-TPE FONs showed strong luminescence intensity and high water dispersibility because of the AIE properties of TPE-CHO and hydrophilic nature of glucan. To examine the biomedical application potential of glucan-AIE FONs, the responsiveness, biocompatibility and cell uptake behavior of Glu-TPE FONs were subsequently examined. We demonstrated that Glu-TPE FONs possess good biocompatibility and can be potentially used for biological imaging applications. More importantly, it is well known that the Schiff base and phenyl borate can respond to pH and glucose. Therefore, Glu-TPE FONs can be used for the fabrication of multifunctional biomaterials with stimuli responsiveness.

ПИТАННЯ ЩОДО СУПРАМОЛЕКУЛЯРНОЇ СТРУКТУРИ β-ГЛЮКАНУ ДРІЖДЖІВ SACCHAROMYCES CEREVISIAE

The biological activity of β-glucan depends on several factors: polysaccharide water solubility, its molecular mass, type and configuration of glycoside bonds between residual monosaccharides, and macromolecular branching and conformations.The article shows that structure of the baker's yeast (Saccharomyces cerevisiae) glucan, isolated with peroxide method, contains sections with varying order strength. Hydrogen peroxide (H2O2) yeast treatment is supplemented by the destruction of amorphous formations and increase of glucan preparations crystallinity degree in 2.6...3.3 times compared to the reference specimen. It has been shown that water-soluble glucan has more ordered supramolecular structure (crystallinity index is 4.06) than the original insoluble glucan (crystallinity index is 2.55). The water-soluble glucan preparation obtained by partial enzimatic degradation of structural β-glucan contains molecular formations in the form of triple helices.Animal experiments showed the effectiveness of obtained β-glucan preparations as immunomodulators. The aggregate research findings of preparation supramolecular structure and its medical and biological assessment would be an argument, confirming the relation of β-glucan’s biological action to triple helix in its structure.

Use of water-soluble polysaccharides from mango (Mangifera indica L.) fruits as alternative source of mutanase inductors

The fruits of mango have been suggested as a new alternative to streptococcal mutan for the mutanase induction in Trichoderma harzianum. The water-soluble polysaccharide was obtained from the mango fruits with the yield of 16.3%. Structural analyses revealed that this polymer contained of (1→4)-α-, (1→3)-α-linked Glcp. When the strain T. harzianum CCM F-340 was grown on glucan preparation from mango, maximal mutanase productivity obtained after 3 days' cultivation was 33.7 mU/mL. Additionally, using “real-time” RT-PCR, a 6-fold higher expression for mutanase was determined in the presence of this stimulus. Mutanase in a mixture with commercial dextranase showed a high hydrolytic potential in decomposition of native mutan, where maximal degrees of saccharification and solubilization of this biopolymer (60 and 90%, respectively) were reached in 6h at 45oC. The mutanase preparation was also effective in mutan removal from oral biofilms, especially in a mixture with dextranase (100% hydrolysis in 6h).

Comparison of the potency of a variety of β-glucans to induce cytokine production in human whole blood

β-Glucans are components of fungal cell walls and potent stimulants of innate immunity. The majority of research on biological activities of glucans has focused on β-(1→3)-glucans, which have been implicated in relation to fungal exposure-associated respiratory symptoms and as important stimulatory agents in anti-fungal immune responses. Fungi-and bacteria and plants-produce a wide variety of glucans with vast differences in the proportion and arrangement of their β-(1→3)-, -(1→4)- and -(1→6)-glycosidic linkages. Thus far, the pro-inflammatory potential of different β-glucans has not been studied within the same experimental model. Therefore, we compared the potency of 13 different glucan preparations to induce in vitro production of IL-1β, IL-6, IL-8 and TNF-α in human, whole blood cultures. The strongest inducers of all cytokines were pustulan [β-(1→6)-glucan], lichenan [β-(1→3)-(1→4)-glucan], xyloglucan [β-(1→4)-glucan] and pullulan [α-(1→4)-(1→6)-glucan]. Moderate-to-strong cytokine production was observed for curdlan [β-(1→3)-glucan], baker's yeast glucan [β-(1→3)-(1→6)-glucan] and barley glucan [β-(1→3)-(1→4)-glucan], while all other glucan preparations induced very low, or no, detectable levels of cytokines. We therefore conclude that innate immunity reactions are not exclusively induced by β-(1→3)-glucans, but also by β-(1→6)- and β-(1→4)-structures. Thus, not only β-(1→3)-glucan, but also other β-glucans and particularly β-(1→6)-glucans should be considered in future research.

Preparation of Water Soluble Yeast Glucan by Four Kinds of Solubilizing Processes

(1→3)-β-D-glucan from the inner cell wall of Saccharomyces cerevisiae is considered a member of a class of drugs known as biological response modifiers (BRM). However the glucan was an insoluble polysaccharide, which could be the major barrier to the utilization of glucan. In this case, the insoluble glucan was convent into a soluble form by four kind of solubilizing processes. The yield, solubility, chemistry structure and immunoprophylaxis efficacy of the soluble products were compared. Our date suggest that urea has a significant effect on yield, and DMSO has a significant effect on solubility. FT-IR spectra, 13C NMR spectra and helix-coil transition analysis demonstrate that the chemistry structure of native and solubilizing glucans have no significant difference. They still have the triple helical structure. The solubility and immunoprophylaxis efficacy assay indicate that the introduction of phosphate group not only enhanced the solubility of glucan, but also improved the survival rate of mice challenged with E. coli.

The effect of β-glucans on porcine leukocytes

β-Glucans are conserved glucose polymers found in the cell walls of plants, fungi, yeasts and bacteria. They have the capacity to activate innate immunity, thereby enhancing defence barriers. Besides differences in type of linkage and branching, β-glucans can vary in solubility, molecular mass, tertiary structure, polymer charge and solution conformation. All these characteristics may influence their immunomodulating effects. In this study, the effect of seven β-glucans that differed in origin (fungi, yeast, seaweed, bacteria or algae) and structure (linear or branched; soluble, gel or particulate) were tested on peripheral blood mononuclear cells (PBMC) and neutrophils of the pig. We looked at lymphocyte proliferation, reactive oxygen species (ROS), production by neutrophils and monocytes and cytokine production. The soluble β-glucans Laminarin and Sleroglucan did not activate ROS-production of monocytes and neutrophils while the particulate β-glucans (β-glucan from algae ( Euglena gracilis)) and glucan preparations from baker's yeast (Macrogard, Saccharomyces cerevisiae and Zymosan) had a stimulating effect. The highest stimulation of lymphocyte proliferation occurred by Curdlan (bacteria), Zymosan and the β-glucan of E. gracilis, especially at high concentrations (200 μg/ml and 800 μg/ml). TNF-α was particularly stimulated by Macrogard and S. cerevisiae, while all β-glucans (except Laminarin) induced IL-1β. Furthermore, it was interesting that all β-glucans and in particular Curdlan, gave rise to IL-10 secretion, whereas any β-glucan induced the release of IL-8, IL-4, IL-12, IL-6 or IFN-γ.

Orally administered glucans from the edible mushroomPleurotus pulmonariusreduce acute inflammation in dextran sulfate sodium-induced experimental colitis

Polysaccharides are one of the most potent mushroom-derived substances exhibiting anti-inflammatory and immunomodulatory properties. The aims of the present study were to determine whether orally administered glucans from the edible mushroom Pleurotus pulmonarius could attenuate or prevent the development of experimental colitis in mice. Colonic inflammation was induced in mice by treatment with 3.5 % dextran sulfate sodium (DSS) for 18 d. Before or after DSS administration, mice were given hot water solubles (HWS) or mycelium extract (ME) (2 or 20 mg per mouse) daily in their food. Colonic damage was macroscopically and histologically evaluated. Inflammation was assessed by changes in colon length, TNF-alpha levels released by colonic samples in organ culture and myeloperoxidase (MPO) activity. mRNA levels of pro-inflammatory (IL-1beta) and anti-inflammatory (IL-10) cytokines in colonic samples were determined by quantitative real-time RT-PCR. P. pulmonarius glucans attenuated and prevented the development of symptoms associated with DSS-induced colitis. High doses of HWS and ME blocked colon shortening, suppressed MPO activity and improved macroscopic score in all treatment groups. In addition, histological damage from colitis was reduced by HWS and ME at all doses. The tissue levels of TNF-alpha protein were significantly decreased and correlated with degree of inflammation and macroscopic score. All treatments significantly attenuated the increased DSS-mediated expression levels of IL-1beta. We conclude that the different glucan preparations (HWS or ME) harvested from P. pulmonarius when orally administered to DSS-treated mice attenuate the development of colonic inflammation, suggesting putative clinical utility for these extracts in the treatment of colitis.

Induction of Metalloproteinase 9 Secretion from Human Keratinocytes by Pleuran (β-Glucan from Pleurotus ostreatus)

Glucan preparations, primarily modified water-soluble glucans, are involved in the activation of the body's natural defense mechanisms and in the acceleration of the skin's wound-healing processes. Pleuran, an insoluble beta-D-glucan in hydrogel form, offers a natural alternative to more common chemically derivated soluble beta-D-glucans. Pleuran was applied to human keratinocyte primary cultures, and after 24 h of incubation the release of matrix metalloproteinase 9 (MMP-9) and metalloproteinase 2 (MMP-2) by stimulated keratinocytes was detected using gelatine zymography. There was a concentration-dependent increase in pro-MMP-9 release after treatment with pleuran over the concentration range of 2 to 200 microg/ml, but pro-MMP-2 was detected at a constant level. Moreover, the active forms of both MMPs were not detectable, indicating that in vitro autoactivation of these zymogens did not occur. The results indicate that pleuran is a potent keratinocyte stimulator of proMMP-9 release, which implies its application in dermatological therapies.

Modulation of liver fibrosis and pathophysiological changes in mice infected with Mesocestoides corti (M. vogae) after administration of glucan and liposomized glucan in combination with vitamin C.

The effects of glucan and liposomized glucan, alone or co-administered with vitamin C, and empty liposomes on hepatic fibrosis in mice infected with Mesocestoides corti (M. vogae) tetrathyridia were studied. Preparations were administered every third day from day 7 to day 31 post-infection (p.i.), nine doses in total. Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and cholesterol levels were measured in sera collected on days 11, 15, 21, 28, 32, 42, 50 and 65 p.i. Liver fibrosis was studied on the same days by measuring hydroxyproline concentration, which is considered a marker for collagen content. Larvicidal effects of the glucan and liposome preparations were estimated on day 65 p.i. in the liver and peritoneal cavity. Glucan formulations significantly enhanced collagen content, most prominently after administration of liposomized glucan in combination with vitamin C. Activities of both enzymes and cholesterol levels were slightly modified after administration of glucan alone. Liposomized glucan with vitamin C significantly increased ALT and AST activity and cholesterol levels up to days 28-32 p.i., after which they plateaued or declined. The most pronounced decrease was after administration of liposomized glucan and vitamin C. The same pattern of biochemical parameters in serum was observed after administration of empty liposomes, however, collagen content was not modified significantly. Larval counts in the liver and the peritoneal cavity were significantly reduced after treatment with either glucan formulation, but were unaffected following treatment with empty liposomes. In summary, intense fibrosis in the liver of mice treated with liposomized glucan and vitamin C did not result in the most extensive parenchymal cell injury but, rather in the highest efficacy of treatment. Liposomal lipids were probably utilized in the reparation of the damaged parenchymal cells, while glucan stimulated phagocytic cells.

Preparation of a unique glucan with large intervals in molecular weight distribution. Controlled ring-opening polymerization of O-permethylcyclodextrin.

O-Permethylated cyclodextrins (MeCDs) were found to be polymerizable with the initiator-activator system of HI-I2 or HI-ZnX2, undergoing ring-cleavage to give linear 1-->4-glucan. Among several conditions investigated, HI-ZnCl2 in CH2Cl2 at 0 degree C proved most effective to control this cationic ring-opening polymerization (CROP). The MALDI-TOF-MS spectrum revealed that the molecular weight distribution of the glucan obtained from gamma-MeCD uniquely consisted of large, regular intervals, each of which were identical to the molecular weight of gamma-MeCD (1634).

Increase of hematopoietic responses by triple or single helical conformer of an antitumor (1-->3)-beta-D-glucan preparation, Sonifilan, in cyclophosphamide-induced leukopenic mice.

It has been suggested that the immunopharmacological activity of soluble (1-->3)-beta-D-glucan depends on its conformation in mice. In this study, we examined the relationship between the conformation of Sonifilan (SPG) and hematopietic responses in cyclophosphamide (Cy)-induced leukopenic mice. SPG, a high molecular weight (1-->3)-beta-D-glucan, has a triple helical conformation in water, and it was changed by treatment with aqueous sodium hydroxide to the single helical conformer (SPG-OH). The effects of SPG or SPG-OH on hematopoietic responses in cyclophosphamide induced leukopenic mice were investigated by monitoring i) gene expression of cytokines by RT-PCR, ii) protein synthesis of interleukin 6 (IL-6) by ELISA and iii) colony formation of bone marrow cells (BMC). The mice administered Cy and SPG or SPG-OH expressed and produced higher levels of IL-6 mRNA and protein than the mice administered only Cy. Gene expression of NK1.1 was also induced by Cy/SPG (or SPG-OH) treatment. Induced gene expression of stem cell factor (SCF) and macrophage-colony stimulating factor (M-CSF) by SPG/SPG-OH were also found in in vitro culture of BMC from Cy treated mice. These results strongly suggested that conformation of the glucans, single and triple helix, are independent of the hematopietic response.

Effects of In Vivo Administration of Adamantylamide Dipeptide on Bone Marrow Granulocyte-Macrophage Hemopoietic Progenitor Cells (GM-CFC) and on Ability of Serum of the Treated Mice to Stimulate GM-CFC Colony Formation in Vitro: Comparison with Muramyl Dipeptide and Glucan

Adamantylamide dipeptide (AdDP), muramyl dipeptide (MDP), and glucan were shown to increase significantly the numbers of granulocyte-macrophage hemopoietic progenitor cells (GM-CFC) in the bone marrow of mice. However, whereas the sera of mice given MDP or glucan were found to stimulate the growth of colonies from GM-CFC in vitro, i.e. to produce a colony-stimulating activity (CSA), administration of AdDP did not lead to this effect. Nevertheless, when serum of mice given AdDP was added to the cultures concomitantly with a suboptimal concentration of mouse interleukin-3 (mIL-3), a broad spectrum hemopoietic stimulator, counts of colonies from GM-CFC were significantly increased, and accelerated growth of the colonies was found as well. This property of AdDP, i.e. its ability to exhibit co-stimulating activity (CoSA) without being able to exhibit CSA, suggests that AdDP acts in hemopoietic tissues differently as compared with the two other immunomodulators studied. It can be hypothesized that the action of AdDP is more specific when compared with its natural related compound, MDP, as well as with glucan. Our findings prove the possibility to stimulate by AdDP the granulopoietic compartment of hemopoiesis and are in agreement with previous observations concerning the absence of systemic side effects of AdDP. Both these qualities of AdDP may be advantageous when pondering over contingent clinical utilization of AdDP as hemopoietic stimulator.This work was supported by a grant (PZ-Z2/25/97) from the Ministry of Industry and Trade of the Czech Republic. The research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals issued by the Czech Society for Laboratory Animal Science. The authors are indebted to Dr. J. Sandula, Institute of Chemistry, Slovak Academy of Sciences, for the soluble glucan preparation.