Enhancement of hematopoietic recovery in gamma-irradiated mice by the joint use of diclofenac, an inhibitor of prostaglandin production, and glucan, a macrophage activator.

The effects of diclofenac (inhibitor of prostaglandin production) and carboxymethylglucan (immunomodulator and an agent stimulating hematopoiesis), when given to mice 1 day before gamma-irradiation, were studied. Both of the agents were administered either alone or in combination. The investigations included the assessment of post-irradiation hematopoietic recovery in terms of bone marrow and spleen cellularity and endogenous spleen colony formation, as well as the determination of the survival of lethally irradiated mice. The results demonstrated at least additive radioprotective effects when mice were given diclofenac and carboxymethylglucan in combination. Experimental evidence provided by the increased 125iodo-deoxyuridine incorporation into the spleen and elevated hydroxyurea kill of endogenous spleen colony-forming units indicated that the beneficial action of the combined treatment could be a consequence of increased cell proliferation in the hematopoietic tissue. It is likely that the inhibition of prostaglandin production (diclofenac action) and the concomitant increased release of growth factors (glucan action) shift the regulatory balance towards the predominance of positive hematopoietic control.

Effect of carboxymethyl-chitin-glucan on cyclophosphamide induced mutagenicity.

The effect of high molecular carboxymethyl-chitin-glucan (CMCG), administered either intraperitoneally, intravenously or orally prior to cyclophosphamide injection, on the frequency of micronucleated reticulocytes was evaluated in peripheral blood of female ICR mice. Both intraperitoneal and intravenous administration of CMCG decreased the clastogenic effect of cyclophosphamide. The protective effect of CMCG was concentration dependent, with a higher decrease achieved by 100 mg/kg than by 50 mg/kg body weight. On the other hand, not even five peroral pretreatments with CMCG in the dose of 200 mg/kg body weight during the week prior to simultaneous administration of CMCG and cyclophosphamide induced a decrease of micronucleated reticulocytes in peripheral blood. It is therefore conceivable that CMCG failed to pass through the gastrointestinal tract, probably due to its high molecular weight. The antimutagenic effect of CMCG against cyclophosphamide was manifested by its intraperitoneal and intravenous administration to female ICR mice.

Effect of ultrasonicated carboxymethylglucan on cyclophosphamide induced mutagenicity.

Carboxymethylglucan (CMG) with ultrasonically lowered molecular weight (0.89 x 10(5)) was administered either intraperitoneally, intravenously or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei in mouse bone marrow was evaluated. Both parenteral (intraperitoneal and intravenous) and oral administration of CMG decreased the clastogenic effect of CP. The protective effect induced by intravenous and intraperitoneal administration was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg body weight. With the lower dose of CMG a 2-h interval was necessary between intravenous CMG administration and CP injection. Oral pretreatment of mice with CMG decreased statistically significantly the frequency of micronuclei in polychromatic erythrocytes of the bone marrow. The fact that ultrasonically depolymerized CMG was effective also on oral administration is indicative of the passage of smaller CMG molecules through the wall of the gastrointestinal tract.

Ultrasonication: the way to achieve antimutagenic effect of carboxymethyl-chitin-glucan by oral administration.

Carboxymethyl-chitin-glucan (CMCG) isolated from Aspergillus niger was ultrasonicated to decrease its molecular weight. Ultrasonicated CMCG with molecular weight 0.19 x 10(-5) was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei in polychromatic erythrocytes of mouse bone marrow was evaluated. Both ways of CMCG administration significantly decreased the clastogenic effect of CP. The protective effect of CMCG was concentration dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b.wt. Ultrasonic depolymerization of high molecular CMCG resulted in its anticlastogenic effect against CP not only on intraperitoneal, but also on oral administration, achieved by decreasing its molecular weight. Ultrasonication proved to be an efficient way to obtain molecules of CMCG able to pass through the cell walls of the gastrointestinal tract.

Protective effect of sulfoethylglucan against hexavalent chromium.

The effect of pretreatment with sulfoethylglucan (SEG) on the frequency of micronuclei and the liver alkaline phosphatase activity induced by potassium bichromate (Cr(VI)) in mice was evaluated. Simultaneous application of SEG and Cr(VI) decreased the frequency of micronuclei in bone marrow cells (P < 0.01) and the level of liver alkaline phosphatase activity in comparison to the Cr(VI) group. Pretreatment with SEG 24 h prior to the first Cr(VI) application resulted in a more pronounced decrease in the Cr(VI)-induced frequency of micronuclei. The mechanisms of the protective effects of sulfoethylglucan could be explained either by the formation of Cr ion complexes with sulfoethyl groups of glucan or by the scavenging ability of SEG to trap hydroxyl radicals.

Protective effect of the yeast glucomannan against cyclophosphamide-induced mutagenicity.

Glucomannan (GM) isolated from Candida utilis with molecular weight 30 kDa was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei was evaluated in polychromatic erythrocytes of mouse bone marrow. GM administration by either route decreased significantly (p<0.002) the clastogenic effect of CP. The protective effect was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b. wt. (body weight). The fact that GM was effective also at oral administration is indicative of the passage of GM molecules through the wall of the gastrointestinal tract. The important characteristics of GM isolated from C. utilis, such as good water solubility, relatively small molecular weight (30 kDa), and antimutagenic effect exerted also at oral administration, appear to be promising features for its prospective use as a natural protective agent.

Antioxidative and antimutagenic activity of yeast cell wall mannans in vitro.

Antioxidative and antimutagenic effect of yeast cell wall mannans, in particular, extracellular glucomannan (EC-GM) and glucomannan (GM-C.u.) both from Candida utilis, mannan from Saccharomyces cerevisiae (M-S.c.) and mannan from Candida albicans (M-C.a.) was evaluated. Luminol-dependent photochemical method using trolox as a standard showed that EC-GM, GM-C.u., M-S.c. and M-C.a. have relatively good antioxidative properties. EC-GM exhibited the highest antioxidative activity, followed by GM-C.u. and M-S.c. M-C.a. showed the least antioxidative activity. These mannans were experimentally confirmed to exhibit different, statistically significant antimutagenic activity in reducing damage of chloroplast DNA of the flagellate Euglena gracilis induced by ofloxacin and acridine orange (AO). We suggest that the antimutagenic effect of EC-GM, GM-C.u., M-S.c. and M-C.a. against ofloxacin is based on their ability to scavenge reactive oxygen radicals. With AO, the reduction of the chloroplast DNA lession could be a result of the absorptive capacity of the mannans. The important characteristics of mannans isolated from the yeast cell walls, such as good water solubility, relatively small molecular weight (15-30kDa), and antimutagenic effect exerted through different mode of action, appear to be a promising features for their prospective use as a natural protective (antimutagenic) agents.

Novel structure of the cellular mannan of the pathogenic yeast Candida krusei.

On the basis of methylation analysis, 13C- and 1H-n.m.r. spectroscopic data, and the results of immunological methods, a new structure for the cell-wall alpha-D-mannan of the pathogenic yeast Candida krusei is proposed. In contrast to the alpha-D-mannans of other pathogenic yeasts of the Candida species, which have (1----6)-linked main chains and many (1----2)- and/or (1----3)-linked side chains, the C. krusei mannan is lightly branched and contains (1----2) and (1----6) linkages in the ratio 3:1.

Hemopoiesis stimulating and radioprotective effects of carboxymethylglucan.

Carboxymethylglucan, a novel soluble derivative of beta-1,3-glucan, was found to enhance hemopoietic recovery in sublethally gamma-irradiated mice and to increase survival in lethally irradiated animals when given 24 hours prior to irradiation. Postirradiation treatment with carboxymethylglucan also induced favourable effects in terms of survival when used in combination with preirradiation cystamine administration.

Ultrasonic depolymerization of the chitin-glucan complex from Aspergillus niger and antimutagenic activity of its product.

Sonication was effective for the depolymerization of carboxymethylated chitin-glucan complex (CM-CG) isolated from the cell wall of Aspergillus niger. After 10 min of sonication and subsequent gel filtration, two samples (CM-CG(I) and CM CG(II)) with significantly distinct molecular weights (660 kDa and 19 kDa, respectively) and different nitrogen contents (3.02 and 1.69%) were obtained. CM-CG(II) with lower Mw was also effective against cyclophosphamide mutagenicity by oral administration in mice.

Effect of ultrasonic treatment on the molecular weight of carboxymethylated chitin-glucan complex from Aspergillus niger.

Different factors affecting the efficiency of the ultrasonic depolymerization of the high-molecular-weight carboxymethylated chitin-glucan prepared from the fungal mycelium of Aspergillus niger have been investigated. The influence of the following parameters was examined: concentration of the chitin-glucan complex, duration of ultrasonic irradiation, reaction temperature and volume of the ultrasonicated solution. The optimized conditions for the efficient ultrasonic depolymerization include: polysaccharide concentration--0.2 mg ml-1; volume of the sonicated solution--25 ml; duration of the sonication--10 min; and constant cooling of the sonicated sample in an ice-water bath.

Mitogenic activity of particulate yeast beta-(1-->3)-D-glucan and its water-soluble derivatives.

Particulate beta-D-glucan was isolated from baker's yeast using autolysis and delipidization of the cells, followed by alkaline and acid treatment. The residual water-insoluble glucan termed cerevan has a beta-(1-->3)-linked backbone with beta-(1-->6)-linked short side chains. In order to achieve water solubility of the glucan, various derivatives were prepared (carboxymethyl-,carboxyethyl-,hydroxyethyl-,sulfoethyl-), and the beta-glucan was oxidized to glucuronoglucan. Their solubility, degree of substitution (DS), and molecular weight distribution (Mw) were compared. The immunomodulatory activity of these preparations was investigated in mitogenic and co-mitogenic tests on rat thymocytes. Cerevan showed higher stimulation indices compared with the known immunomodulator zymosan. Of the water-soluble derivatives, sulfoethylglucan was found to be the most active. Of the carboxymethyl derivatives of various DS, the preparation with DS = 0.75 exhibited the highest activity. Water-soluble carboxymethyl preparations with DS > 1.0 and low-molecular-weight glucuronoglucan were inactive.

Chitotriosidase as a new marker of macrophage stimulation in a tumor model treated with cyclophosphamide and Ukrain.

Ukrain has previously been demonstrated to exert a malignotoxic effect in vivo. This antitumor drug has been effective in the treatment of some malignancies in experimental animals as a result of immunostimulation (macrophage stimulation). In the present study, serum chitotriosidase activity was measured as a biochemical marker of macrophage stimulation in several murine and rat models of macrophage stimulation. It was shown that zymosan, carboxymethylated glucan and Triton WR 1339 administration to CBA mice or Wistar rats was followed by a considerable increase in serum chitotriosidase activity. Murine LS lymphosarcoma development decreased serum chitotriosidase activity. Antitumor treatment by Ukrain or cyclophosphamide did not restore this index to the normal value.

Cysteine proteinase inhibitor level in tumor and normal tissues in control and cured mice.

Cystatin C is the best known extracellular endogenous cysteine proteinase inhibitor and has been studied as a possible index of tumor growth and as a marker of the effectiveness of antitumor therapy. The aim of this study was to evaluate cystatin C concentrations in murine tumor tissues (compared with other organs not directly involved with tumor development, such as the liver and spleen) during treatment with several antitumor drugs (Ukrain and/or cyclophosphane). Cystatin C concentrations in murine tissues and biological fluids was determined by enzyme-linked immunosorbent (ELISA) assay. The cystatin C ELISA test is a sandwich immunoassay, which uses immobilized rabbit antihuman cystatin C Pab and mouse antihuman cystatin C Mab-HRP (monoclonal antibodies, conjugated with horseradish peroxidase). We observed decreased serum cystatin C concentrations compared with controls in all nontreated tumor models: HA-1 hepatoma (solid and ascitic forms), lung adenocarcinoma (solid and ascitic forms) and LS lymphosarcoma. In the ascitic fluid of mice with HA-1 hepatoma the cystatin C concentration was much lower than in the serum of the same mice (about 20-fold lower). In the HA-1 model of hepatoma cells cystatin C concentration decreased about 2-3-fold compared with the control (intact liver) and Ukrain significantly increased the cystatin C concentration. Cyclophosphane treatment of LS lymphosarcoma significantly increased the cystatin C concentration in serum. Cyclophosphane treatment (50 mg/kg, single injection) increased cystatin C by up to 8-fold more in tumor issue. Ukrain treatment of LS lymphosarcoma was also followed by increased levels of cystatin C in tumor tissue (4-fold); cyclophosphane plus Ukrain had a similar positive effect. In the group with LS lymphosarcoma Ukrain or cyclophosphane plus Ukrain treatment induced a significant increase in cystatin C concentration in liver. Liver cystatin C concentration decreased in the HA-1 hepatoma group and treatment with Ukrain or carboxymethylated beta-1, 3-glucan (CMG) increased this index in both groups. Spleen cystatin C concentrations decreased about 5-fold in LS lymphosarcoma compared with controls and combined treatment with cyclophosphane plus Ukrain restored the index to the normal value. We can conclude that both murine tumors studied were characterized by low cystatin C concentrations in tumor tissues and decreased cystatin C concentrations (to a lesser degree) were also observed in liver and spleen as a result of the "toxic" effect of tumor bearing. Effective treatment in all cases (especially with Ukrain or a combination of cyclophosphane plus Ukrain) induced a significant increase in cystatin C. Obviously, the decrease in cystatin C concentration predominantly in tumor tissue was connected with tumor development and restoration of cystatin C level may be used as a marker of efficacy of antitumor therapy.

Haemopoiesis-enhancing effects of repeatedly administered carboxymethylglucan in mice exposed to fractionated irradiation.

Carboxymethylglucan (CMG), a water-soluble glucan derivative, enhanced the number of granulocytes in the peripheral blood as well as other indices of haemopoietic recovery (total cellularity and the number of granulocyte-macrophage progenitor cells in femoral marrow, spleen weight) investigated after fractionated gamma-irradiation in mice (five doses of 2 Gy each, or three, four and five doses of 3 Gy each given at 24-h intervals). An increased liver weight and a more pronounced anaemia found in the CMG-treated mice suggested, however, that also inflammatory side effects were evoked by the repeated administration of CMG. On the other hand, the development of tolerance, i.e., a decreased effectiveness of the treatment with CMG upon its repeated administration, did not seem to play any major role under the experimental conditions studied, because the protective effects of CMG increased with the increasing number of CMG injections.

Enhancement by carboxymethylglucan of early cellular damage in 1 Gy-irradiated mice.

Our results describe a novel carboxymethylglucan (CMG) activity, namely its radiosensitizing effect on early cellular damage in mice gamma-irradiated with a dose of 1 Gy. An increase of thymidine levels in blood plasma, determined 4 h after irradiation, was used as an indicator of the early cell death. The radiosensitizing effect was observed when administering CMG at time intervals close to irradiation time (1 h before to 1 h after irradiation). Diclofenac (an inhibitor of prostaglandin production) had no modifying effects on elevation of plasma thymidine levels induced by radiation or radiation + CMG. Pentoxifylline (an inhibitor of synthesis of tumour necrosis factor and of phosphodiesterase) administration elevated plasma thymidine to similar levels as CMG alone, combined pentoxifylline + CMG treatment had not additive effects.

Hyporesponsiveness of murine myeloid progenitor cells to glucan following its repeated administration.

A moderate marrow granulocytic hyperplasia developed after 4 injections of glucan (soluble derivative carboxymethylglucan) administered to mice at 3-4-day intervals. However, when evaluating the response of the marrow granulocyte-macrophage colony-forming cells (GM-CFC) to the fifth glucan injection given in the repetitive treatment schedule, the development of hyporesponsiveness of these cells was found, contrary to the stimulatory action of a single glucan injection. In addition, the prompt increase in serum granulocyte-macrophage colony-stimulating activity occurring after a single glucan injection, and proposed to upregulate myelopoiesis, was absent in mice treated with glucan repeatedly. A joint administration of glucan with diclofenac, an inhibitor of prostaglandin synthesis, was able to enhance the GM-CFC response to a single glucan injection, probably due to the removal of the downregulation action of prostaglandins on these progenitor cells. However, a repeated administration of diclofenac with glucan did not reduce substantially the development of GM-CFC hyporesponsiveness. Thus, the results suggest that the GM-CFC hyporesponsiveness or tolerance to repeated glucan injections was induced by weakening the mechanisms of positive control mediated by the serum colony-stimulating activity.

Modulatory effect of glucans on the functional and biochemical activities of guinea-pig macrophages.

The particulate and soluble glucan preparations isolated from Saccharomyces cerevisiae and Candida albicans affected various activities of guinea-pig macrophages. The stimulation of INT reduction and superoxide production were observed in the presence of all insoluble and some soluble glucans in vitro, but most of the preparations under study had an inhibitory effect on phagocytic activity. On the other hand, the stimulation of both metabolic and functional activities was obtained in vivo. Macrophages from guinea-pigs treated with glucans exerted an increased ability to reduce INT and to produce superoxide. Their candidacidal capacity was rapidly elevated, and peritoneal macrophages had raised phagocytic activity as well. A more pronounced stimulatory effect was observed in the presence of insoluble rather than soluble glucans, and this was more expressive in vitro than in vivo.

Glucomannan from Candida utilis. Structural investigation.

Structure of the glucomannan isolated from the cell walls of Candida utilis has been investigated using acetolysis fragmentation, methylation analysis and NMR spectroscopy. The structure of the glucomannan resembles that of the cellular mannans of other Candida species, except that the longer tetra- and pentassacharide side-chains are terminated with a glucosyl residue. Presence of the nonreducing glucosyl groups at the ends of the side-chains caused the C. utilis not to cross-react in a double immunodiffusion test with other Candida species that possess mannan antigens and cross-react with Hansenula species with glucomannan antigens.

[Modulatory effect of glucans on the function of murine macrophages, NK-cells and lymphocytes]. / Modulacní efekt glukanu na funkcní aktivitu mysích makrofágu, NK-bunek a lymfocytu.

The particulate glucan (G1), soluble glucan preparations (G2 to G5, and G7) isolated from Saccharomyces cerevisiae, and glucomanan prepared from culture fluid after cultivation of Candida utilis (G6) were tested for their immunomodulatory activity in vivo and in vitro. In tests in vivo three soluble glucans (G3, G4, and G7) injected s.c. to mice in the dose of 10 mg/kg increased the cytotoxic activity of peritoneal macrophages. The influence of glucans on natural killer cells was without significance. The lymphoproliferative reaction of spleen cells to polyclonal mitogens was inhibited by all the preparations used with the exception of soluble glucan G2. The mitogenic effect of the preparations, co-stimulatory tests and direct cytotoxicity to cells of cell lines used in cytotoxicity assays were assessed in vitro. The transformation index of glucans in the study was increased according to the glucan and dose tested. Inhibition of the lymphoproliferative reaction measured by the co-stimulatory test for optimal concentration of Concanavalin A occurred in a wide range of doses for the preparations G1 to G6. The preparation G7 increased the incorporation of 3HTdR under the same conditions. The use of a suboptimal concentration of Concanavalin A revealed co-stimulatory activity of all the preparations tested. Assessment of the cytotoxic activity of peritoneal macrophages and of the activity of natural killer cells induced in vitro was complicated by the direct cytotoxicity of particulate glucan and soluble glucan G5 (carboxymethylglucan) for target cells (YAC 1, and YAC 1 and K 562 resp.).