A (1-->3)-beta-D-linked heptasaccharide is the unit ligand for glucan pattern recognition receptors on human monocytes.

Glucans are fungal cell wall polysaccharides which stimulate innate immune responses. We determined the minimum unit ligand that would bind to glucan receptors on human U937 cells using laminarin-derived pentaose, hexaose, and heptaose glucan polymers. When U937 membranes were pretreated with the oligosaccharides and passed over a glucan surface, only the heptasaccharide inhibited the interaction of glucan with membrane receptors at a K(d) of 31 microM (95% CI 20-48 microM) and 100% inhibition. However, the glucan heptasaccharide did not stimulate U937 monocyte NFkappaB signaling, nor did it increase survival in a murine model of polymicrobial sepsis. Laminarin, a larger and more complex glucan polymer (M(w) = 7700 g/mol), only partially inhibited binding (61 +/- 4%) at a K(d) of 2.6 microM (99% CI 1.7-4.2 microM) with characteristics of a single binding site. These results indicate that a heptasaccharide is the smallest unit ligand recognized by macrophage glucan receptors. The data also indicate the presence of at least two glucan-binding sites on U937 cells and that the binding sites on human monocyte/macrophages can discriminate between glucan polymers. The heptasaccharide and laminarin were receptor antagonists, but they were not receptor agonists with respect to activation of NFkappaB-dependent signaling pathways or protection against experimental sepsis.

Normal human fibroblasts express pattern recognition receptors for fungal (1-->3)-beta-D-glucans.

Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity. Glucans are thought to mediate their effects via interaction with membrane receptors on macrophages, neutrophils, and NK cells. There have been no reports of glucan receptors on nonimmune cells. We investigated the binding of a water-soluble glucan in primary cultures of normal human dermal fibroblasts (NHDF). Membranes from NHDF exhibited saturable binding with an apparent dissociation constant (K(D)) of 8.9 +/- 1.9 microg of protein per ml and a maximum binding of 100 +/- 8 resonance units. Competition studies demonstrated the presence of at least two glucan binding sites on NHDF. Glucan phosphate competed for all binding sites, with a K(D) of 5.6 microM (95% confidence interval [CI], 3.0 to 11 microM), while laminarin competed for 69% +/- 6% of binding sites, with a K(D) of 3.7 microM (95% CI, 1.9 to 7.3 microM). Glucan (1 microg/ml) stimulated fibroblast NF-kappaB nuclear binding activity and interleukin 6 (IL-6) gene expression in a time-dependent manner. NF-kappaB was activated at 4, 8, and 12 h, while IL-6 mRNA levels were increased by 48% at 8 h. This is the first report of pattern recognition receptors for glucan on human fibroblasts and the first demonstration of glucan binding sites on cells other than leukocytes. It also provides the first evidence that glucans can directly modulate the functional activity of NHDF. These results provide new insights into the mechanisms by which the host recognizes and responds to fungal (1-->3)-beta-D-glucans and suggests that the response to glucans may not be confined to cells of the immune system.

Glucans exhibit weak antioxidant activity, but stimulate macrophage free radical activity.

Polymeric carbohydrates have been reported to modulate inflammatory responses in vitro and in vivo. Previous reports suggest that certain carbohydrate polymers, such as (1-->3)-beta-D-glucans, may possess free radical scavenging activity. If glucans are free radical scavengers then it might explain, in part, the ability of these ligands to modulate inflammatory responses. The present study examined the free radical scavenging activity of a variety of carbohydrate polymers and the effect of the polymers on free radical levels in a murine macrophage cell line. All of the carbohydrates exhibited concentration dependent antioxidant effects (EC(50) range = 807 to 43 microg/ml). However, the antioxidant activity for the carbohydrates was modest in comparison with PDTC (EC(50) = 0.13 microg/ml) and the carbohydrate concentration required for antioxidant activity was high (x EC(50) = 283 microg/ml). The antioxidant ability of the polymers was greater (p < .05) than their monosaccharide constituents, i.e., dextrose EC(50) = 807 vs. glucan sulfate EC(50) = 43 microg/ml. Coincubation of glucans with murine J774a.1 cells increased free radical levels when compared to controls. Therefore, the weak free radical scavenging activity of glucan polymers cannot explain their modulatory effect on inflammatory responses in tissue culture and/or disease models of inflammation.

The influence of glucan polymer structure and solution conformation on binding to (1-->3)-beta-D-glucan receptors in a human monocyte-like cell line.

Glucans are (1-3)-beta-D-linked polymers of glucose that are produced as fungal cell wall constituents and are also released into the extracellular milieu. Glucans modulate immune function via macrophage participation. The first step in macrophage activation by (1-3)-beta-D-glucans is thought to be the binding of the polymer to specific macrophage receptors. We examined the binding/uptake of a variety of water soluble (1-3)-beta-D-glucans and control polymers with different physicochemical properties to investigate the relationship between polymer structure and receptor binding in the CR3- human promonocytic cell line, U937. We observed that the U937 receptors were specific for (1-->3)-beta-D-glucan binding, since mannan, dextran, or barley glucan did not bind. Scleroglucan exhibited the highest binding affinity with an IC(50)of 23 nM, three orders of magnitude greater than the other (1-->3)-beta-D-glucan polymers examined. The rank order competitive binding affinities for the glucan polymers were scleroglucan>>>schizophyllan > laminarin > glucan phosphate > glucan sulfate. Scleroglucan also exhibited a triple helical solution structure (nu = 1.82, beta = 0.8). There were two different binding/uptake sites on U937 cells. Glucan phosphate and schizophyllan interacted nonselectively with the two sites. Scleroglucan and glucan sulfate interacted preferentially with one site, while laminarin interacted preferentially with the other site. These data indicate that U937 cells have at least two non-CR3 receptor(s) which specifically interact with (1-->3)-beta-D-glucans and that the triple helical solution conformation, molecular weight and charge of the glucan polymer may be important determinants in receptor ligand interaction.

The application of various protic acids in the extraction of (1-->3)-beta-D-glucan from Saccharomyces cerevisiae.

Glucans are (1-->3)-beta-linked glucose polymers which have immune-stimulating capability. The extraction of water-insoluble (1-->3)-beta-D-glucan form Saccharomyces cerevisiae employs hydrochloric acid. Hydrochloric acid is difficult to employ in the large-scale pharmaceutical extraction of glucans due to its corrosive nature and toxicity. To address these concerns, we determined whether acetic, formic or phosphoric acid can be substituted for hydrochloric acid in the process for the isolation of (1-->3)-beta-D-glucan. The resulting microparticulate glucans were employed as the starting material for the production of (1-->3)-beta-D-glucan phosphate. 13C NMR analysis of the glucan phosphates derived from the acetic, formic or phosphoric acid-extracted microparticulate glucan show excellent correspondence to hydrochloric acid extracted glucan and laminarin, a (1-->3)-beta-D-glucan standard, indicating that the primary structure is not altered by the acid used for extraction. Glucan phosphate prepared from hydrochloric acid had a Mw of 7.2 x 10(4) g/mol, rmsz of 17.7 nm, of 1.50 and (eta) of 49.0 mL/g. Glucan phosphate prepared from acetic acid had a primary polymer peak with a Mw of 1.4 x 10(6) g/mol, rmsz of 23.6 nm, I of 1.93 and (eta) of 62.4 mL/g. Glucan phosphate prepared from formic acid had a main polymer peak with a Mw of 1.2 x 10(6) g/mol, rmsz 27.1 nm, I of 1.56 and (eta) of 89.0 mL/g. Glucan phosphate prepared from phosphoric acid had a primary polymer peak with a Mw of 6.6 x 10(5) g/mol, rmsz of 32.3 nm, I of 2.70 and (eta) of 91.3 mL/g. These data indicate that the molecular mass, size, polydispersity and intrinsic viscosity of the glucan phosphate obtained is influenced by the pKa of protic acid employed to extract the microparticulate glucan. However, the primary structure and side-chain branching are not substantially altered regardless of the acid employed.

Receptor binding and internalization of a water-soluble (1-->3)-beta-D-glucan biologic response modifier in two monocyte/macrophage cell lines.

Glucan phosphate, a water-soluble, chemically defined (1-->3)-beta-D-glucan biologic response modifier, has been reported to exert antisepsis activity and accelerate wound healing. In this study we describe the specific binding of glucan phosphate to human and murine monocyte/macrophage cell lines, U937 and J774A.1, respectively. At 37 degrees C, equilibrium binding was rapidly achieved, i.e., within 1 min. In U937 cells, binding occurred with an affinity (Kd) of 37 microM and a Bmax of 65 x 106 binding sites/cell at 37 degrees C. In J774A.1 cells, glucan phosphate bound with an affinity (Kd) of 24 microM and a Bmax of 53 x 106 binding sites/cell at 37 degrees C. In both cases there was insignificant nonspecific binding. We further demonstrated that bound glucan phosphate cannot be displaced by a 50-fold excess of unlabeled ligand, suggesting internalization of glucan phosphate. Transmission electron microscopy showed significantly increased cytoplasmic vacuolization and significantly decreased mitotic activity in glucan phosphate-treated U937 cells compared with that in untreated cells. Pullulan, a random coil alpha-(1-->4)-(1-->6)-linked glucose polymer that served as a control, did not compete for the same binding site as glucan phosphate in either cell line, indicating the specificity of the binding site for (1-->3)-beta-D-glucans. We conclude that water-soluble pharmaceutical grade (1-->3)-beta-D-glucan phosphate specifically binds to and is internalized by U937 and J774A.1 cells.

Isolation of a previously unidentified polysaccharide (MAR-10) from Hyssop officinalis that exhibits strong activity against human immunodeficiency virus type 1.

A polysaccharide (MAR-10) was isolated from the aqueous extract of the plant Hyssop officinalis and examined for its activity against HIV-1 (SF strain) in HUT78 T cell line and primary cultures of peripheral blood mononuclear cells. MAR-10, in a concentration-dependent manner, inhibited HIV-1 replication as demonstrated by the inhibition of HIV-1 p24 antigen and syncytia formation. Furthermore, MAR-10 had no significant direct toxicity or effect on lymphocyte functions or CD4+ and CD8+ T cell counts. In addition, MAR-10 has broad spectrum anti-glycosidase activity. Our study demonstrates that MAR-10 contains strong anti-HIV-1 activity that may be useful in the treatment of patients with HIV-1 infection.

Development of a water-soluble, sulfated (1-->3)-beta-D-glucan biological response modifier derived from Saccharomyces cerevisiae.

This report describes a method for the solubilization of micro-particulate (1-->3)-beta-D-glucan. Insoluble glucan is dissolved in methyl sulfoxide and urea (8 M) and partially sulfated at 100 degrees. The resulting water-soluble product is called glucan sulfate. The conversion rate is 98%, and the preparation is endotoxin free as determined by the Limulus lysate procedure. Glucan sulfate is composed of 34.06% C, 6.15% H, 50.30% O, 5.69% S and 3.23% N, and has a repeating unit empirical formula of (C6H10O5)8.3 SO3NH4+.4 H2O, suggesting that, on the average, a sulfate group is substituted on every third glucose subunit along the polymer. Molecular weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (HPSEC). Two polymer peaks were resolved. Peak 1 (Mw = 1.25 x 10(6) g/mol) represents < 1% of the total polymer mass. Peak 2 (Mw = 1.45 x 10(4) g/mol) comprises > 99% of polymers. 13C NMR spectroscopy confirmed the beta-(1-->3) interchain linkage. In solution, glucan sulfate polymers self-associate in a triple helix. Glucan sulfate stimulates murine bone marrow proliferation following intravenous administration. The ability to prepare a immunologically active, water-soluble (1-->3)-beta-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.

Development, physicochemical characterization and preclinical efficacy evaluation of a water soluble glucan sulfate derived from Saccharomyces cerevisiae.

This report describes the development, characterization and preclinical efficacy evaluation of water soluble glucan sulfate. Glucan sulfate was derived from insoluble beta-1,3-D-glucan isolated from Saccharomyces cerevisiae. The proposed repeating unit empirical formula of glucan sulfate is [(C6H10O5)5.3H2SO4]n. Two polymer peaks were resolved by aqueous high-performance size exclusion chromatography (HPSEC) with on-line multi-angle laser light scattering (MALLS) photometry and differential viscometry. Peak 1 (MW = 1219697 Da) represents approximately 1% of the total polymers, while peak 2 (MW = 8884 Da) accounts for approximately 99% of polymers. 13C-NMR spectroscopy suggests that glucan sulfate polymer strands may be partially cross-linked. Glucan sulfate (250 mg/kg, i.v.) increased (P less than 0.01) macrophage vascular clearance of 131I-reticuloendothelial emulsion by 42% (P less than 0.01) and in vitro bone marrow proliferation by 46% (P less than 0.05). Glucan sulfate (250 mg/kg, i.v.) increased (P less than 0.05) median survival time of C57B1/6J mice with syngeneic melanoma B16 or sarcoma M5076. In addition, glucan sulfate immunoprophylaxis increased resistance of mice to challenge with Escherichia coli, Candida albicans or Mouse Hepatitis Virus strain A-59. We concluded that: (1) insoluble beta-1,3-D-glucan can be converted to a water soluble sulfated form; (2) glucan sulfate activates macrophages and stimulates bone marrow; (3) glucan sulfate exerts antitumor therapeutic activity, and (4) glucan sulfate immunoprophylaxis will modify the course of experimental infectious disease.

A method for the solubilization of a (1----3)-beta-D-glucan isolated from Saccharomyces cerevisiae.

This report describes a method for the solubilization of a micro-particulate beta-D-glucan. Insoluble glucan is dissolved in methyl sulfoxide and urea (8M) and partially phosphorylated at 100 degrees. The resulting water-soluble product is called glucan phosphate. The conversion rate is 70%, and the preparation is endotoxin free as determined by the Limulus lysate procedure. Glucan phosphate is composed of 34.66% C, 6.29% H, 42.83% O, and 2.23% P and has a repeating-unit empirical formula of (C6H10O5)7.PO3H2, indicating a phosphate group substitution on every seventh glucose subunit. Molecular-weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (s.e.c.) with on-line, multi-angle laser light scattering (m.a.l.l.s.) photometry and differential viscometry (d.v.). Two polymer peaks were resolved. Peak 1 (Mw = 3.57 x 10(6) daltons), represents approximately 2% of the total polymers, while peak 2 (Mw = 1.10 x 10(5) daltons) comprises approximately 98% of polymers. 13C- and 31P-n.m.r. spectroscopy confirmed the beta-1,3 interchain linkage and the presence of a phosphate group. In solution, glucan phosphate polymers self-associate in a triple-helical arrangement. The ability to prepare a immunologically active, non-toxic, water-soluble beta-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.

Isolation, physicochemical characterization and preclinical efficacy evaluation of soluble scleroglucan.

Herein we describe the isolation, physicochemical characterization and preclinical evaluation of a water-soluble biologic response modifier extracted from Sclerotium glucanicum. Alkaline extraction of insoluble S. glucanicum exopolymers produced a soluble scleroglucan composed of a triple-helical beta-1,3-linked glucopyranose backbone with single beta-1,6-linked glucopyranosyl branches every third subunit. Scleroglucan has a weight average molecular mass of 1.56 x 10(6) Da, a weight average root mean square distance from the center of gravity of the molecule to its farthest elements of 51.8 nm, a polydispersity (weight-average molecular mass/number average molecular mass) of 1.83 and intrinsic viscosity of 3.081 dl/g. Scleroglucan (250 mg/kg, intravenously) stimulated in vivo murine macrophage phagocytic activity (66%, P less than .001) and increased in vitro macrophage tumor cytotoxicity against syngeneic tumor targets by 124% (P less than .05). Scleroglucan enhanced (P less than .001) murine bone marrow proliferation in a biphasic manner by up to 328%. Scleroglucan therapy increased survival of mice challenged with syngeneic lymphoma, melanoma or adenocarcinoma. AKR/J mice bearing syngeneic lymphoma (1 x 10(3) cells, intraperitoneally) demonstrated increased (P less than .001) long-term survival (100% vs. 0%, greater than 64 days). C57Bl/6J mice bearing syngeneic melanoma B16 (5 x 10(5) cells, subcutaneously) demonstrated increased long-term survival (64% vs. 0%, P less than .05). C57Bl/6J mice bearing syngeneic adenocarcinoma BW10232 (1 x 10(5) cells, subcutaneously) demonstrated increased (P less than .05) median survival time. In addition, scleroglucan prophylaxis increased resistance of mice to challenge with Staphylococcus aureus, Candida albicans and mouse hepatitis virus A-59. Scleroglucan did not induce toxicity or hepatomegaly. We conclude that: 1) a branched, water-soluble beta-1,3-linked scleroglucan biologic response modifier can be extracted from S. glucanicum; 2) scleroglucan will stimulate immunity, modify experimental neoplastic disease and increase resistance to microbial challenge; and 3) scleroglucan shows promise as an immunopotentiating drug.

External ocular inflammatory effects of lipoxygenase enzyme products.

We systematically observed the effects of lipoxygenase enzyme products (5-, 8-, 9-, 12-, and 15-HETE and leukotrienes (LT) C4, D4, and B4) on the external ocular inflammatory process in rabbits. Our results, using 1 and 10 micrograms enzymatic preparations topically applied to the conjunctiva, were consistent with the potent chemotactic activity of 12-HETE and LTB4. Modulation of the inflammatory process can be accomplished better, as a result of our findings, by inhibition of both the lipoxygenase and cyclooxygenase pathways.

Inhibition of soybean lipoxygenase by SKF 525-A and metyrapone.

To determine whether agents which inhibit cytochrome P-450 enzymes also inhibit lipoxygenase, the effects of metyrapone and SKF 525-A were assessed on soybean lipoxygenase using a spectrophotometric technique which allows for measurement of both the rate and magnitude of product formation. Both SKF 525-A and metyrapone inhibited the rate of product formation and the final amount of product formed in 5 min incubations SKF 525-A was 5 to 5 times more potent than metyrapone, with the IC50 for SKF 525-A 40 microM and for metyrapone between 150 and 200 microM as determined by the total product formation in 5 minutes. Analysis of the reduced product by HPLC confirmed that the substances monitored were those generated by the 15-lipoxygenase enzyme.

Reversible inhibitors of beta-glucosidase.

A variety of reversible inhibitors of sweet almond beta-glucosidase were examined. These included simple sugars and sugar derivatives, amines and phenols. With respect to the sugar inhibitors and, indeed, the various glycoside substrates, the enzyme has what can be considered a "relaxed specificity". No single substituent on glucose, for example, is essential for binding. Replacement of a hydroxyl group with an anionic substituent reduces the affinity while substitution with a cationic (amine) substituent enhances the affinity. Amines, in general, are good inhibitors, binding more tightly than the corresponding alcohols: pKiRNH3+ = 0.645pKiROH + 1.77 (n = 9, r = 0.97). The affinity of a series of 10 primary amines was found to be strongly influenced by substituent hydrophobicity: pKi = 0.52 pi + 1.32 (r = 0.95). The major binding determinant of the glycoside substrates is the aglycon moiety. Thus, the Ki values of phenols are similar in magnitude to the Ks values of the corresponding aryl beta-glucoside. The pH dependence for the inhibition by various phenols indicates that it is the un-ionized phenol which binds to the enzyme when an enzymic group of pKa = 6.8 (+/- 0.1) is protonated. The affinity of the phenol inhibitor is dependent on its basicity with a Brønsted coefficient for binding of beta = -0.26 (n = 14, r = 0.98). The pH dependence of the binding of two particularly potent beta-glucosidase inhibitors was also examined. 1-Deoxynojirimycin (1,5-dideoxy-1,5-imino-D-glucitol) has a pH-corrected Ki = 6.5 microM, and D-glucono-1,5-lactam has a pH-corrected Ki = 29 microM.(ABSTRACT TRUNCATED AT 250 WORDS)