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A 10-amino-acid repeating sequence of the hemagglutinating portion of Clostridium difficile toxin A has been synthesized and used to produce antisera in rabbits. Antipeptide antibody inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. Immunoblot analysis with the antipeptide antibody revealed cross-reactivity with native toxin, a recombinant protein containing the toxin A repeats, and a glucan-binding protein from Streptococcus mutans whose primary structure has repeating amino acid motifs similar to those of the synthetic peptide. A polyclonal antibody against the glucan-binding protein, which cross-reacted with purified toxin A, also inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. We recently identified toxin A and the glucan-binding protein as members of a novel family of clostridial and streptococcal binding proteins based on conserved repeating amino acid motifs at the C-terminal region of the molecules. This study provides immunological and functional evidence of the predicted relationship between toxin A and the glucan-binding protein and further implicates the repeating subunits as ligand-binding domains in this family of proteins.

The phagocytosis of beta-glucan particles by human neutrophils and the associated activation of NADPH O2- forming oxidase were accompanied by an increased hydrolysis of phosphoinositides by phospholipase C, hydrolysis of phosphatidylcholine by phospholipase D, accumulation of diglyceride (DG) mass, and [Ca2+]i rise. The reaction of phospholipid hydrolysis played a minor role in the formation of DG, which was mainly formed by de novo synthesis from glucose. The activation of this pathway was shown by the stimulation of the incorporation of [U-14C]glucose into DG, which occurred very rapidly after the challenge of neutrophils with beta-glucan particles. This DG derived from glucose was found almost completely as 1-acyl-2-acyl-glycerol (DAG). On the basis of the finding that phosphatidic acid was the precursor of DAG, an increase in the incorporation of [U-14C]acetate into DAG did not occur, and the [14C]radioactivity was in the glycerol backbone, the synthesis of DAG from [U-14C]glucose occurred very likely via dihydroxyacetone phosphate and glycerol 3-phosphate, stepwise acylation to phosphatidic acid, and dephosphorylation by phosphatidate phosphatase.

Intraperitoneal injection of beta-1,3-D-glucan coupled to the surface of monodisperse methacrylate microbeads improves the resistance against bacterial infections in mice, while methacrylate microbeads alone do not. The effect of the glucan-derivatized microbeads (GDM) is considered to be mediated through peritoneal macrophages. We show that both GDM and the underivatized methacrylate microbeads (UDM) treated with normal serum were rapidly bound and phagocytized by mouse peritoneal macrophages in vitro. We found that both complement and fibronectin opsonized the beads and were responsible for the uptake. Treatment of microbeads with serum lacking fibronectin and complement activity still gave some uptake of GDM, but not uptake of UDM. The uptake of GDM was similar to the uptake of untreated GDM and was inhibited by pretreatment of macrophages with soluble beta-1,3-D-glucan. Our conclusion is that GDM and UDM intraperitoneally bind fibronectin and C3 through activation of the alternative pathway of complement. This leads to their phagocytosis by macrophages through fibronectin and complement receptors. GDM are also internalized via beta-glucan receptors. We present the hypothesis that the beta-glucan receptors on peritoneal macrophages account for the protective effect of GDM in intraperitoneal bacterial infections.

We examined the effect of soluble derivatives of yeast glucan on the humoral immune response of various strains of inbred mice after administration of different doses according to various schedules. Glucan was injected i.v. or s.c. in a single dose or repeatedly. The immune response was examined by determining the titres of serum hemagglutinins against sheep erythrocytes (SRBC-Ab). The immunoadjuvant effect of glucan derivatives depends on the inbred strain used, on the dose of glucan, mode and time of administration with respect to antigen injection. The results have shown that the stimulatory effect of glucan derivatives occurred already after a single injection, the optimum dose being 10-20 mg/kg. Intravenous injection was more efficient than the subcutaneous one. In some cases, a slight increase of the spleen mass was observed.

Antibodies against Schizophyllan (SPG) or SPG conjugated with bovine serum albumin (BSA) were raised in rabbits by multiple subcutaneous immunization. The titer of SPG-reactive antibodies in the antiserum to SPG-BSA conjugate was significantly higher than in the antiserum to SPG as assessed by enzyme-linked immunosorbent assay (ELISA). The SPG-reactive antibodies purified by affinity chromatography using a Protein A-Sepharose CL-4B column interacted with the SPG-BSA conjugate spotted on a nitrocellulose membrane filter in a dose-dependent manner, but the anti-SPG antibodies did not interact with BSA. Immunocytochemical staining has also shown that the anti-SPG antibodies reacted with SPG taken up by mouse peritoneal macrophages.

Mice immunized with a glucan-killed parasite vaccine exhibited enhanced resistance to Leishmania donovani infection as evidenced by decreased hepatic parasite burdens when compared to unvaccinated controls. This resistance was not seen in mice immunized with killed parasites alone. Glucan vaccination resulted in increased resistance at day 6, but this effect was no longer present by day 20 of the experiment. Treatment of vaccinated and control mice with antithymocyte sera abrogated protection against infection, whether such resistance was vaccine induced or the result of acquired immunity.

With the use of monoclonal antibodies, raised against the human polyglucosan, positive staining of polyglucosan bodies (PGB) was detected in the brain, spinal cord and cecum of aged dogs. PGB in feline brain were also positively stained with these antibodies. These findings indicate that animal PGB share common antigenicity with human PGB.

Ability of Sugi basic protein (SBP)-pullulan conjugate to elicit the Arthus reaction was found to be markedly reduced, about 100 times lower than that of native SBP. To analyze this reduced ability, activation of complement by immune complex consisting of SBP-pullulan and anti-SBP antibodies was studied. Tests for complement consumption, C3 conversion and cleavage of factor B revealed that immune complex formed with SBP-pullulan is incapable of supporting efficient activation of the complement system. Previously, we have shown data suggesting that SBP-pullulan conjugate would be a good candidate for desensitization therapy against cedar pollinosis. The results presented in this paper provide additional support for the suggestion.

The binding of the iC3b receptor (CR3) to unopsonized zymosan was shown to result from CR3 attachment to cell wall beta-glucans. A specificity of neutrophil responses for beta-glucan was first suggested by a comparison of yeast (Saccharomyces cerevisiae) cell wall components for stimulation of a neutrophil superoxide burst. Neutrophils responded poorly to heat-killed yeast, but gave increasingly better responses to cell wall polysaccharides devoid of proteins (zymosan) and nearly pure beta-glucan particles derived from zymosan. Zymosan triggered a burst that was 29% as great as that stimulated by phorbol myristate acetate (PMA), and beta-glucan particles stimulated a burst that was 72% as great as that produced by PMA. Phagocytic responses to yeast were also inhibited by soluble glucans but not by soluble mannans. Three types of experiments demonstrated a role for CR3 in these responses. First, neutrophil ingestion of either yeast or yeast-derived beta-glucan particles was blocked by monoclonal anti-CR3, fluid-phase iC3b, or soluble beta-glucan from barley. Monocyte ingestion of beta-glucan particles was also blocked by anti-CR3, but not by anti-CR1 or anti-C3. Second, the neutrophil superoxide burst response to either zymosan or beta-glucan particles was blocked by anti-CR3 or fluid-phase iC3b, and was completely absent with neutrophils from 3 patients with an inherited deficiency of CR3. Third, CR3 was isolated from solubilized neutrophils by affinity chromatography on beta-glucan-Sepharose.

A vaccination protocol against murine Babesia microti infection, using glucan, a beta-1,3-glucopyranose derivative of yeast cell walls, and glutaraldehyde-fixed infected erythrocytes was evaluated. BALB/c mice were immunized intravenously four times at 2-day intervals with 2 X 10(8) fixed infected erythrocytes with and without glucan (0.45 mg). The mice were challenged 2 weeks after the last immunization with 10(8) viable infected erythrocytes. Peak parasitemia was significantly reduced (8.9 +/- 1.0%; P less than 0.001) in glucan-immunized mice as compared with nonimmunized controls (41.2 +/- 1.4%), glucan-treated controls (31.7 +/- 2.5%; P less than 0.05), or mice which received fixed infected erythrocytes without glucan (21.0 +/- 1.2%; P less than 0.01). Humoral and cellular immunity to B. microti was evaluated before challenge by measuring antibody titers and splenocyte blastogenic responses to B. microti antigens. The in vitro cellular response was inversely correlated with mean peak parasitemia (P less than 0.05). These observations demonstrate that glucan is an effective adjuvant in enhancing immunity to murine babesiosis.

Pretreatment of rats with the RES stimulant, glucan, markedly increases their sensitivity to endotoxic shock. Intravenous administration of S. enteritidis endotoxin (10 micrograms/100 gm B.W.) produced a more severe shock in glucan-pretreated rats than IV injection of 1 mg/100 gm B.W. of endotoxin in normal rats. Endotoxic shock in glucan-sensitized rats was associated with a precipitous increase in serum activity of lysosomal enzymes and a more severe hypoglycemic response. The shocked glucan-pretreated rats died before marked increases in plasma hepatocyte enzyme activity were apparent. Assessment of RE-phagocytic function with the 131I RE test-lipid emulsion revealed a rapid clearance and hepatic uptake of the emulsion in nonshocked glucan-control animals. This hyperphagocytic function, however, was abolished at two hours after injection of endotoxin. Hepatic ultrastructure in shocked glucan and normal rats revealed extensive sinusoidal changes. Hepatocytes, with the exception of a depletion of glycogen granules, were comparatively intact in the shocked-glucan group as opposed to the shocked control group. Pretreatment with methylprednisolone (60 mg/kg) protected the glucan-treated rats from endotoxin and was relatively more effective in diminishing the hypoglycemic response than preventing a loss of lysosomal integrity. Thus, despite the absence of overt ultrastructural changes in hepatocytes, altered glucoregulation appears to be a significant factor in the enhanced sensitivity of glucan-pretreated rats to endotoxin.

Intravenous injections of glucan simultaneously with Formalin-killed erythrocytic stages of Plasmodium berghei elicited a greater degree of resistance in mice against subsequent infection with viable parasites than injections of killed erythrocytic stages alone. In two experiments with P. berghei strain NK 65, 100% of mice immunized with the glucan-dead parasite preparation survived challenge, whereas only 28.6% of mice receiving dead parasites alone survived. In the third experiment, using P. berghei strain NYU-2, the same proportion of mice survived after immunization with glucan and dead parasites as with dead parasites alone (i.e., 10 of 11 in each group), but mice immunized with the glucan-dead parasite preparation experienced parasitemias of significantly less intensity and shorter duration than mice which received only dead parasites before infection. Inoculation of glucan alone or with normal erythrocytes conferred no protection against challenge.

Immunoglobulin A (IgA) antibodies reactive with Streptococcus mutans MT3 cells (serotype c) were sought, using an indirect enzyme-linked immunosorbent assay, in the saliva of humans who either harbored or did not harbor detectable levels of this organism. Samples of unstimulated whole saliva from three adults and one child who were infected with S. mutans contained IgA which bound to MT3 cells. Saliva samples of two adults studied also contained IgA which reacted with S. mutans strains of serotypes e, g, a, and b, the latter two of which are rarely isolated from humans. The saliva of three children who did not harbor detectable levels of S. mutans and of three of five predentate infants also contained IgA reactive with MT3 cells. The latter observation is of special interest since S. mutans does not colonize the mouth before eruption of teeth. Thus, the presence of salivary IgA reactive with S. mutans cells is not necessarily related to present or past infection by this organism. Absorption with MT3 cells markedly reduced the reactivity of adult saliva without greatly altering the total concentration of IgA present; this suggests that the IgA was not binding to S. mutans MT3 cells via Fc receptors. The possibility that the antibodies which reacted with S. mutans MT3 may have been induced to other bacteria with cross-reactive antigens was supported by the finding that absorption of saliva with mixed bacterial growth derived from common dairy products significantly reduced its reactivity. Absorption experiments further suggested that a significant portion of the salivary IgA antibodies was binding to glucans on the cell surface.

We conducted studies with mice, rats, and monkeys which demonstrated the ability of glucan to induce either nonspecific or specific enhancement of host resistance to infectious diseases. Intravenous pretreatment of mice with glucan significantly enhanced the survival of mice challenged with either Venezuelan equine encephalomyelitis (VEE) virus or Rift Valley fever virus. Pretreatment was beneficial when initiated 3 days before challenge with VEE virus and 7 days before challenge with Rift Valley fever virus. Treatment of mice after VEE challenge did not increase their survival compared with controls. Glucan pretreatment of rats provided increased resistance to both intraperitoneal and low-dose aerosol challenges with virulent Francisella tularensis when the glucan was given intravenously, but not when it was administered intranasally. In contrast, intranasal glucan pretreatment enhanced the survival of mice when they were challenged by aerosol with Pseudomonas pseudomallei, whereas intravenous glucan pretreatment did not increase survival. mice given glucan combined with a marginally immunogenic dose of VEE vaccine were more resistant to homologous virus challenge than were mice given either Freund complete adjuvant plus vaccine or vaccine alone. Similarly, both primary and secondary VEE antibody titers in cynomolgus monkeys given glucan with VEE vaccine were significantly greater than titers in vaccine controls.

Binding constants of monomers of seven BALB/c IgM, four BALB/c IgA, and one C57BL/6 IgA anti-alpha (1 leads to 6) dextran hybridoma antibodies with dextran B512 and with isomaltoheptaose were determined by affinity electrophoresis. Bindings constants to dextran range from 1.52 X 10(5) to 4.43 X 10(5) ml/g for the five IgA monomers and from 1.70 X 10(3) to 6.10 X 10(4) ml/g for the seven IgM monomers. Antibody monomers containing both specific and nonspecific (derived from the myeloma cell that was used to generate the hybridomas) light chains are shown to have association constants with dextran 6 to 30-fold lower than monomers containing only specific light chain, suggesting that the association of specific heavy chain with nonspecific light chain does not result in an anti-dextran combining site. Binding constants with isomaltoheptaose range from 1.45 X 10(4) to 7.01 X 10(4)/M for the IgA proteins and from 6.46 X 10(3) to 7.70 X 10(4)/M for the IgM proteins. The binding constants with dextran and with isomaltoheptaose, and the electrophoretic, immunochemical and idiotypic characteristics of the hybridoma proteins are discussed.

The effect of pre- or posttreatment with glucan, a reticuloendothelial stimulant, on the course of Leishmania donovani infection was assessed in highly susceptible hamsters. Intravenous administration of glucan before or after L. donovani infection significantly suppressed proliferation of amastigote-stage parasites in liver and spleen. Glucan-activated peritoneal macrophages in vitro also significantly reduced multiplication of the intracellular parasite. Ultrastructural studies revealed a well-defined hepatic granulomatous response to glucan, with hypertrophic Kupffer cells and reduced numbers of intracellular parasites compared to the control group. In additional studies, groups of hamsters were immunized by intravenous injections of glucan with Formalin-killed promastigote-stage L. donovani cells and challenged 60 days after the last immunizing injection. This treatment regimen significantly prolonged the mean survival time of those hamsters which died after infection, relative to untreated control groups. Hamsters stimulated with the glucan-killed promastigote preparation also exhibited significant reductions in splenic amastigotes on days 10 and 21 postinfection compared with all other control groups, but on day 35, splenic amastigotes did not differ significantly from those of control animals. Our composite observations provide evidence for glucan-enhanced nonspecific resistance of hamsters to visceral leishmaniasis.

The effect of yeast beta-1, 3-glucan as an immunopotentiator of delayed type-hypersensitivity reactions (DHR) was studied. Delayed-type-hypersensitivity reactions in mice sensitized intraperitoneally (IP) with sheep red blood cells (SRBC) and pretreated three days previously with glucan given IP were significantly increased. However, mice sensitized IP with SRBC three days after the subcutaneous (SC) administration of glucan showed depressed DHR. Glucan given at the same site but not at distance strongly potentiated the DHR induced by SC sensitization with SRBC. Subcutaneous injection of glucan and SRBC given together also resulted in a sustained DHR which persisted twelve days after sensitization when DHR of control mice had waned.

The trypsin-sensitive receptor that mediates phagocytosis of unopsonized zymosan particles by human monocytes has been designated as a beta-glucan receptor because of its functional inhibition by specific algal and plant beta-glucans. Soluble ligands that are chemically and structurally identical to beta-glucan constituents of zymosan were isolated from a carbohydrate-enriched fraction of yeast extract by sequential chromatography on DE-cellulose, SP-Sephadex, and Con A-Sepharose. Preincubation of adherent human monocytes with 278, 210, and 2.5 micrograms/ml hexose equivalents in pooled chromatographic fractions from DE-cellulose, SP-Sephadex, and Con A-Sepharose, respectively, effected 50% reductions in subsequent phagocytosis of zymosan particles without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (ESIgG). The purified yeast extract-derived beta-glucans, which contained 92% glucose and 8% mannose by gas chromatographic analysis and eluted from a Sephacryl S-200 column as a broad peak with a Kav of 0.39 and estimated molecular sizes of from 20,000 to 70,000 m.w., required only 3.5 +/- 0.9 micrograms/ml (mean +/- SD, n = 6), as compared with 31.5 micrograms/ml of the algal beta-glucan laminarin to achieve 50% decreases in zymosan ingestion. Alternatively, soluble yeast beta-glucans with estimated molecular sizes of from 2 X 10(5) to 2 X 10(6) were prepared from yeast glucan particles, which contained 98% glucose and 0% mannose, by sonication and sequential centrifugation at 15,000 and 100,000 X G for 30 and 60 min, respectively. Monocyte ingestion of zymosan was reduced by 50% by pretreatment with 60 ng/ml of the soluble beta-glucans in 15,000 X G supernatants, whereas ingestion of ESIgG was unaffected by as much as 50 micrograms/ml of this material. Partial acid hydrolysis of soluble glucan-derived beta-glucans in 15,000 X G supernatants followed by gel filtration on Bio-Gel P-4 revealed two well-defined peaks within the inclusion volume of the column with phagocytosis-inhibiting activity. Oligoglucosides that eluted at a Kav of 0.46 had an estimated molecular size of 2,000 m.w. and effected a 48% reduction in zymosan ingestion at inputs of 2 to 5 micrograms/ml, and smaller oligoglucosides with a Kav of 0.82 and an estimated molecular size of 1,000 m.w. effected a 50% reduction at inputs of 25 micrograms/ml. Preincubation of monocytes for 2 min with 25 micrograms/ml of the oligoglucosides with estimated molecular size of 1,000 m.w. and with 50 ng/ml of soluble glucan-derived beta-glucans in 100,000 X G supernatants reduced zymosan ingestion by 41% +/- 4 and 44% +/- 3 (mean +/- SD, n = 3), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

GTF activity was separated into water-insoluble (GTF-I) and water-soluble (GTF-S) polyglucan-synthesizing enzymes. Each preparation demonstrated a single band on 6% SDS PAGE. Only water-insoluble or water-soluble polyglucan was synthesized by the respective enzyme preparation. Rats were immunized, on Days 1 and 14, with either GTF-I or GTF-S in adjuvant. Animals were bled 13, 35 and 54 days after the initial immunization. Individual antisera were tested against either the GTF-I or the GTF-S for inhibition of radioactive glucose incorporation into glucan, and in gel diffusion, and by Western transfer analyses. The respective antisera reacted with the homologous, but not the heterologous enzyme in gel diffusion and Western transfer. GTF-I activity was not inhibited by antibody to GTF-S, but antibody to GTF-I inhibited GTF-I by 68%. GTF-S was inhibited by more than 60% by each of 3 anti-GTF-S sera. Only one anti-GTF-I serum inhibited GTF-S at as much as a modest 30% level. These data support the antigenic and functional distinctiveness of the GTF enzymes of S. sobrinus 6715.

Human monocytes phagocytose particulate activators of the alternative complement pathway through beta-glucan receptors in the absence of opsonins. Recognition of soluble beta-glucans by monocytes selectively inhibits ingestion of particulate activators and has no effect on responses mediated by monocyte receptors for Fc-IgG, complement, or fibronectin. The smallest ligand unit recognized by monocyte beta-glucan receptors is an acid-resistant heptaglucoside present in yeast cell walls. Mouse monoclonal anti-beta-glucan antibodies have been prepared, one of which completely neutralizes the inhibitory capacity of the HPLC-purified heptaglucoside. This antibody has been used as immunogen for the preparation of an anti-Id. The pretreatment of monocytes with low concentrations of anti-Id inhibits monocyte ingestion of zymosan particles but not EsIgG, suggesting that this antibody has specificity for monocyte beta-glucan receptors and is a powerful probe for further receptor studies. Other receptors with specificities for carbohydrates are also present on mononuclear phagocytes. Receptors for mannose/fucose and those for galactose have been isolated and cloned. The development of probes, such as structural analogs of the active heptaglucoside and the anti-Id, will bring the beta-glucan receptors to a similar stage of definition. A major factor that adds a considerable degree of difficulty to studies of the beta-glucan receptors and is not shared by the other receptors for carbohydrates is the requirement for structural conformations provided by the alignment of several glucose units rather than the recognition of a hexose residue in, for example, a glycoconjugate. The designation of these receptors as beta-glucan receptors has inadvertently taken us into a new area of nonimmune defense. Animal studies indicate that beta-glucans with 1,3- and/or 1,6-linkages are active pharmacologic agents that rapidly confer protection to a normal host against a variety of biologic insults. The beta-glucan receptors provide a mechanism by which a heightened state of host responsiveness is initiated.

Mice immunized by a series of intravenous, intraperitoneal or subcutaneous injections of formalin-killed Leishmania donovani promastigotes with glucan, a beta 1, 3 polyglucose, exhibited a significant degree of resistance against subsequent infection with viable promastigotes. Intramuscular immunization was not effective. Immunization via subcutaneous injections of dead promastigotes simultaneously with glucan elicited protective resistance, positive skin test responsiveness before and after challenge and increased antipromastigote antibody levels. Injections of glucan alone induced a lesser degree of resistance against infection without significant skin test or humoral responsiveness. Injections of dead promastigotes alone elicited increased antibody levels but no skin test responsiveness or resistance against infection.

The dose response for elicitation of the hypersensitive reaction in potato tuber discs by arachidonic acid (AA) suggested saturation at higher concentrations. Glucans from Phytophthora infestans, inactive themselves as elicitors of the hypersensitive reaction, enhanced sesquiterpene accumulation and hypersensitive browning elicited by AA. Significant activity (seven times control values) was observed with 33 pmol AA/3.0-cm potato disc in the presence of glucans. Glucans did not affect accumulation of steroid glycoalkaloids, influence the timing or relative amounts of sesquiterpenes which accumulate, or affect recovery of AA added to potato discs. Glucans enhanced activity whether added to potato discs 18 h prior to AA, at the same time as AA, or 18 h after AA. Elicitor activity in the presence of glucans was evident with 20-carbon unsaturated fatty acids that had little or no elicitor activity in the absence of glucans. The position of double bonds had considerable influence on the specific activity of unsaturated fatty acids. The most active had a minimum of three double bonds in a methylene-interrupted series beginning with delta 5, e.g., delta 5,8,11. A delta 5 double bond conferred significant activity even if it was not part of a methylene-interrupted series. The 20-carbon chain length appeared optimal for elicitor activity. The 22-carbon chain acids had low activity, and 16- and 18-carbon acids were inactive. A free carboxyl group or easily transesterified group appeared necessary for activity. Arachidonyl alcohol had very low activity and arachidonyl cyanide was inactive. AA-containing phosphatidylcholine, lysophosphatidylcholine and monoacylglycerol were at least as active as free AA, AA-containing diacylglycerols were slightly less active than free AA, and triarachidonyl glycerol was inactive.

The early cellular responses to antitumor immunomodulators and conventional inducers, especially the polymorphonuclear leukocyte (PMN) responses, were examined in the peritoneal cavity of mice to investigate their effect on primary defense mechanisms. Immunomodulators were classified into 5 groups in terms of PMN response on the basis of its duration (declining or persistent) and extent (high or low induction): 1) TAK (beta-1,3-glucan)-type (high, persistent), 2) lentinan-type (high, declining), 3) yeast mannan-type (low, declining), 4) LPS (lipopolysaccharide)-type (low, persistent), 5) others (no effect). Since the general PMN response is of the declining type, the persistence of PMN with TAK- and LPS-type immunomodulators is a characteristic of the PMN-inducing activity. With respect to the extent, TAK- and lentinan-type immunomodulators induced larger numbers of PMN and macrophages than conventional inducers. These results suggest that some types of immunomodulators have effects on the early host-defense mechanism. From the viewpoint of the general self-defense mechanism we also compared these PMN responses with those to bacteria and to tumor inoculation, and the properties of substances inducing high PMN response, i.e., those with the quality of "foreignness," are discussed.

Golden hamsters were immunized with various antigen fractions of Leishmania donovani promastigotes. Beta 1,3-glucan was used as an adjuvant in these vaccination experiments. The results indicate that immunization of animals with the microsomal fraction (subcellular fraction III) in combination with glucan confers considerable immune protection against L. donovani infection. The immune protection was confirmed by correspondingly lower parasite burden in the livers and spleens of test animals compared to controls. Additionally, the vaccinated animals showed positive skin test responsiveness after challenge, along with increased antibody titres. Immunization of animals with whole and particulate antigen fractions was also found to afford a high degree of resistance. The other subcellular and soluble antigen fractions conferred very little protection. In these experiments, glucan was found to be a potent adjuvant when injected, intraperitoneally, with Leishmania antigens. Similar doses of parasite extracts given without an adjuvant were able to confer only very little or no protection.

Some antitumor immunomodulators, such as a linear beta-1,3-D-glucan from Alcaligenes faecalis var. myxogenes IFO 13140 (TAK), induce potent tumoricidal activity of polymorphonuclear leukocytes (PMNs). In the present study we investigated the role of calcium on the tumoricidal activity of PMNs induced by immunomodulators, especially TAK. The calcium chelator ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) almost completely inhibited TAK-induced PMN cytotoxicity and this inhibition was restored by Ca2+ but not by Mg2+. In Ca2+- and Mg2+-free medium, PMN cytotoxicity induced by TAK was recovered by the addition of Ca2+ provided that Mg2+ was also present. By scopoletin assay, hydrogen peroxide released from PMNs by TAK was also observed in the presence of Ca2+ but not in its absence. The PMN cytotoxicities induced by the other immunomodulators, Propionibacterium acnes, Bacillus Calmette-Guérin, zymosan A, and Nocardia cell wall skeletons were also Ca2+ dependent, judging from studies with EGTA and measurement of hydrogen peroxide release in the presence and absence of Ca2+. The Ca2+ dependency of these PMN cytotoxicities suggests that Ca2+ influx is involved in the cytolytic process, but PMN cytotoxicity was not induced by simple addition of the calcium ionophore A23187. Like TAK, phorbol myristate acetate induced PMN cytotoxicity but this cytotoxicity was not Ca2+ dependent. The present report demonstrates the difference in Ca2+ dependency of these PMN cytotoxicities; i.e., extracellular calcium was required for immunomodulator-induced PMN cytotoxicity, but not for phorbol myristate acetate-induced PMN cytotoxicity. This suggests that the processes of induction of PMN cytotoxicity by the two types of activators are not identical.

Anti-dextran B1375 antibodies were raised in rabbits by injecting formalin-killed Leuconostoc mesenteroides strain NRRL B1375, and the anti-dextran serum was used to examine native dextran B1375, and synthetic linear and four alpha-(1----3)-branched alpha-(1----6)-D-glucopyranans for similarities. The antiserum reacted with the homologous dextran B1375 and also with all the synthetic linear and branched glucans. Precipitation and precipitation-inhibition studies indicated that the antiserum contained at least three groups of antibodies with different specificities, the first specific for linear alpha-(1----6)-D-glucopyranan structure, the second specific for alpha-D-glycopyranosyl-(1----3)-branching and the last specific for another, unknown structure present in the dextran B1375 molecule. Two samples of the synthetic branched glucans were shown to be immunochemically the most similar to natural dextran B1375 by inhibition experiments.

Previously, we reported that one of the factors that determines whether or not an animal will be prepared for hapten help after priming is the type of adjuvant used. The present work was undertaken, therefore, to determine which of a diverse variety of adjuvants or biological response modifiers would be effective. They included Freund's complete (CFA) and incomplete (FICA) adjuvants, particulate glucan, muramyl dipeptide (MDP), and its L-ala-glycerol-mycolate derivative. Help by the azobenzenearsonate (ABA) hapten was measured as the augmentation of the anti-bovine gamma-globulin (BGG) plaque-forming cell (PFC) response to ABA-BGG of mice that had been hapten-primed with ABA conjugated to ovalbumin (OVA). The results showed that FICA was ineffective. MDP was effective but only if administered with FICA during hapten-priming. MDP-L-ala-glycerol-mycolate was effective without any adjuvant but only within a narrow dose range. Particulate glucan was as effective as CFA in preparing mice for hapten help. As the macrophage is the primary cellular target of those biological response modifiers that were effective, we conclude that it plays an important role in the cellular interaction involved in the mediation of hapten help.

Well defined cell-mediated immune responses were detectable following experimental immunization of hamsters with Entamoeba histolytica antigen, using glucan as an adjuvant. Peritoneal cells from amoeba antigen-glucan sensitized animals, upon incubation with specific antigen in vitro, were found to release into the supernatant a macrophage migration inhibition factor (MIF). Such supernatant fluids inhibited the migration of macrophages from non-sensitized hamsters. The production of MIF was found to be greatly increased if glucan is added to amoeba antigen when sensitizing animals. The optimal concentration for maximum inhibition was recorded at 10(-8) dilution of the supernatant.

Candida albicans is an opportunistic human pathogen with a cell wall that is qualitatively similar in carbohydrate composition to zymosan. Monolayers of human peripheral blood monocytes ingested 2.5 x 10(6)-2.5 x 10(7) heat-killed C. albicans blastospores in a dose-related manner. The percentage of monocytes ingesting at least one C. albicans reached a near plateau level of 68 +/- 6% (mean +/- SD, n = 3) when 2.5 x 10(7) particles per ml were incubated with monocytes for 30 min. Pretreatment of monocytes with 10 ng/ml to 100 micrograms/ml of soluble yeast beta-glucan inhibited C. albicans ingestion in a dose-dependent manner without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (EsIgG). Pretreatment of monocytes with 100 micrograms/ml of soluble yeast beta-glucan inhibited the ingestion of 1 or more C. albicans by 80 +/- 11% (mean +/- SD, n = 4), with 50% inhibition occurring with approximately 7 micrograms/ml of soluble beta-glucan. Incubation of monocytes with 100 micrograms/ml to 1000 micrograms/ml of soluble yeast mannan resulted in no significant inhibition of C. albicans ingestion. The pretreatment of monolayers of monocytes for 30 min with 1 microgram/ml to 50 micrograms/ml of affinity-purified trypsin resulted in a dose-dependent inhibition of C. albicans ingestion with 10 micrograms/ml of trypsin inhibiting the percentage of monocytes ingesting 1 or more organisms by 71 +/- 6%. The inhibitory effects of soluble yeast beta-glucan and trypsin pretreatment on the ingestion of heat-killed C. albicans are comparable to the inhibitory effect of these agents on monocyte phagocytosis of zymosan and glucan particles. This relationship indicates that C. albicans contain a ligand that is recognized by the monocyte beta-glucan receptor of human monocytes.

Immunoglobulin E (IgE) antibody response was found to be suppressed selectively and antigen specifically in mice given an antigen conjugated with pullulan, a linear copolymer of maltotriose, whereas IgM and IgG antibody responses were enhanced. On the basis of this finding, tetanus toxin was conjugated with pullulan by cyanuric chloride in the hope that the toxin would be detoxified by the conjugation procedure and could be used as an IgE-suppressing and IgG-enhancing toxoid without the aid of an aluminum adjuvant. This procedure of tetanus toxoid-pullulan conjugation apparently detoxified the toxin. Administration of the resulting tetanus toxoid, tetanus toxin-pullulan conjugate, to mice induced strong suppression of IgE antibody response with fairly good IgG response, whereas the alum-precipitated toxoid or plain toxoid, customarily used for vaccination, elicited high IgE antibody formation. The IgE antibody response was minimal, but the IgG antibody response was maximal in the conjugate-primed mice even after a booster injection with an IgE antibody-inducing dose of the alum-precipitated toxoid.

Several yeast species in the genera Candida, Saccharomyces and Cryptococcus showed powerful immunoadjuvant, chemotherapy-synergic effects against a histocompatible, virus-induced murine lymphoma. Sensitizing and booster intraperitoneal injections of 2 x 10(7) yeast cells on days -14 and +1 (with respect to tumor challenge on day 0) followed by treatment with antiblastic drugs (on day +5) were required to elicit optimum activity. The antitumor effect was not markedly influenced by the morphological growth form of merthiolate-inactivated C. albicans nor by the nature of the carbon source in the growth medium, except for C. albicans cells grown in a medium containing stearic acid, which were not effective. These cells had a higher ratio of soluble to insoluble cell wall components, as compared to glucose-grown cells, but this finding alone could hardly explain the lack of antitumor effects. Previous observations, suggesting that the alkali-acid insoluble beta-glucan (in the form of cell wall ghosts) is the only component of yeast cell walls endowed with antitumor activity comparable to that of whole cells, were confirmed and extended; the soluble mannan and glucan-protein fractions were unable to replace whole cells and glucan ghosts even as sensitizers or as boosting agents.

Beta 1-3 polyglucose or glucan, an extract of cell wall of Saccharomyces cerevisiae, has been successfully employed in this laboratory as an effective immunopotentiator in experimental studies on amoebiasis. An antigen extract from Entamoeba histolytica was combined with beta, 1-3 glucan for immunizing guinea pigs. In order to study the effectiveness of such vaccine preparations, several batches of guinea pigs were immunized with amoeba antigen alone, and in combination with various immunoadjuvants. Antigen inoculations were carried out via intraperitoneal route. Protective immune responses were obtained against amoeba antigen by using glucan as an adjuvant partner. The study showed that glucan can be safely used as an effective immune enhancer.

Both dextran B-1355 and nigerosyl-KLH (N-KLH) contain alpha(1----3) diglucosyl moieties and both induce lambda class serum antibodies which recognize the alpha(1----3) linkage. However, as shown here, some of the antibodies induced by the thymus-dependent form, N-KLH, have a distinct fine specificity. It is known that B-1355 induces antibodies which resemble the anti-alpha(1----3) myeloma proteins MOPC 104E and J558. The new fine specificity which does not resemble such antibodies is found in the serum of N-KLH primed mice challenged with N-KLH. The two fine specificity types are distinguished by their sensitivity to inhibition by nigerose in ELISA using B-1355 or N-BSA as bound antigen. Twelve hybridomas were produced from N-KLH primed mice boosted with either N-KLH or B-1355. Six of the 12 had the new fine specificity; only two out of the 12 expressed the IdX determinant commonly associated with the B-1355 response and neither of these possessed the new reactivity pattern. Comparative inhibition with the disaccharide nigerose and the tetrasaccharide nigerantetraose indicated that 11 out of the 12 hybridoma proteins have combining sites larger than a disaccharide. Southern and Northern blot analysis of eight hybridomas revealed that all expressed VH genes from the J558 family, but that at least two distinct VH genes from this family were used. The data support the hypothesis that immunization with an alpha(1----3) diglucosyl-protein conjugate alters the composition of the B cell pool capable of producing lambda class antibodies from that ordinarily observed following immunization with B-1355.

Macrophages stimulated by an insoluble beta-1,3-D-glucan from yeast cell walls were able to destroy tumour cells as measured by the release of radioactive label from prelabelled 14C-thymidine cells. Target cells were B-16 melanoma, P-815 mastocytoma, and the L-929 cell line. A significant target cell killing by macrophages stimulated by glucan was observed after 72-96 h. The cytolysis of L-929 cells was investigated in some detail. No stable soluble cytolytic factor appeared to be released into the medium during the stimulation of macrophages by glucan, since cell-free spent medium had no cytotoxic effect on L-929 cells. The densities of the macrophage monolayers were critical for an effective target cell killing; dense cultures showed more cytotoxicity than less dense cultures. The kinetics of the development of macrophage-mediated cytotoxicity suggests a minimum stimulation period of 4 days for maximal cytolysis.

Cross-reactions of four synthetic branched glucans (3-O-alpha-D-glucopyranosyl-(1----6)-alpha-D-glucopyranans: V39, V17, V37, and V32, each containing one unit glucose branches amounting to 11-12%, 33-43%, 50-54%, and 71-100%, respectively) with rabbit anti-N4 dextran were examined. All four samples precipitated antibodies raised in rabbits by injecting N4 dextran-concanavalin A conjugate. The ability of glucans to precipitate antibody depended on the quantity of branches, samples with more branches precipitating less antibody nitrogen under the same conditions. This may indicate an inhibitory effect of the branches on precipitation. Oligosaccharide inhibition assay showed that the precipitation reactions were specific for (1----6)-alpha-D-glucopyranosyl linkages, and the maximum size of the alpha-(1----6)-specific antibody combining site corresponded to isomaltopentaose. Determination of antibody nitrogen and glucan in the precipitates indicated that the ratios of one combining site of antibody to numbers of glucose residues were 1:9 (V39), 1:11 (V17), and 1:16 (V37) in the extreme antibody excess region. A synthetic sample of manno-glucan ((1----6)-alpha-D-glucopyranan containing about 27% of randomly linked 3-O-alpha-D-mannopyranosyl side chains) also reacted with the same antibody.

Kupffer cells compose 80-90% of fixed tissue macrophages and have been suggested to play an important role in hepatic antitumor resistance. In the present study, the ability of resting and activated Kupffer cells to lyse syngeneic mammary adenocarcinoma BW10232 cells was evaluated. Activated Kupffer cells were isolated from C57Bl/6J mice following single of multiple intravenous (IV) injections of glucan (0.45 mg/mouse), a potent macrophage-activating agent. Mice receiving 5% (w/v) dextrose served as control. Resting Kupffer cells induced significant (P less than .05) 4% and 12% specific lysis of adenocarcinoma cells at target:effector ratios of 1:10 and 1:50, respectively. Kupffer-cell-mediated tumoricidal activity was depressed on day 1 following a single IV injection of glucan. By day 3 postglucan, the antitumor activity of Kupffer cells returned to control levels and was enhanced on days 5 and 10. Following multiple IV injections of glucan on days -5, -3, and -1, Kupffer-cell-mediated cytotoxicity was elevated on days 1 and 4. These observations demonstrate that resting Kupffer cells are significantly cytotoxic to adenocarcinoma cells at T:E ratios of 1:10 and 1:50 and following a transient inhibition of Kupffer-cell-mediated tumoricidal activity, glucan was effective in significantly enhancing the antitumor activity of Kupffer cells.

The protective efficacy of glucan as an adjuvant with killed promastigotes of Leishmania donovani was compared with that of soluble or particulate fractions of the parasite. When these vaccine preparations were injected either intravenously or subcutaneously in CF-1 mice, glucan potentiated resistance against L. donovani infections as reflected by significant reductions in hepatic amastigote counts relative to infected control mice. The leishmanial antigens alone afforded no protection. Serum direct agglutination titers to leishmanial antigens were highest in all groups given the vaccine intravenously, whereas the delayed-type hypersensitivity response to the antigen was positive only in groups immunized subcutaneously with glucan as an adjuvant. Some index of protection and immune response against visceral infection with the parasite was seen in groups vaccinated with glucan and soluble antigens. However, the protection afforded by glucan and particulate antigens of L. donovani more closely paralleled the resistance of mice treated with glucan and unfractionated killed promastigotes. Further antigenic analysis of particulate fractions of L. donovani may optimize effective immunization when used with appropriate adjuvants, e.g., glucan.

In an attempt to protect mice against experimental infection with P. berghei, mice were immunized against soluble extract of P. berghei in combination with beta-1,3 glucan or FCA and also independently. Mice immunized against P. berghei antigen-glucan developed well defined cell mediated and humoral immune responses, while mice injected with antigen FCA or antigen alone developed only an antibody response. Antigen-glucan immunization afforded a high degree of immune protection to the host against the challenge with live parasites.

Phagocytosis of heat-killed yeast (HK-yeast), zymosan, and glucan particles by thioglycollate-elicited mouse peritoneal macrophages (Tg-macrophages) was inhibited by soluble glucan polymers/oligomers. The inhibitory capacity of soluble glucans decreased steeply with the decrease in the degree of polymerization (DPn); i.e., the concentration at which 50% inhibition of phagocytosis was attained was 0.23 microgram/ml for glucan 1 (DPn 24.8), 0.8 microgram/ml for glucan 2 (DPn 21.9), and greater than 40 micrograms/ml for glucan 3 (DPn 13.8). The glucan polymers were obtained by partial hydrolysis of glucan particles with formic acid (90%, 95 degrees C, 20 min) and fractionation according to solubility in ethanol water mixtures. A short preincubation (5 min, 4 or 37 degrees C) of Tg-macrophages with glucan 1 led to a subsequent inhibition of HK-yeast phagocytosis. Recovery of the phagocytic function was slow (27% in 3 h; 68% in 5 h) and required protein synthesis. beta-Glucan receptor expression was also suppressed by dexamethasone treatment. Mannan exerted at high concentrations (5 mg/ml) a partial inhibitory activity which was totally abrogated by beta-glucanase treatment. Treatment of macrophages with glucan together with mannan did not enhance the inhibitory capacity of glucan beyond the component abrogated by enzyme treatment. Contribution of local opsonization of HK-yeast to the phagocytic response (involvement of complement receptors) was indirectly negated; (a) glucan 1 which inhibits HK-yeast phagocytosis by up to 95% is not an activator of complement and therefore could not compete for the opsonizing proteins; (b) cycloheximide treatment in itself inhibited only partially HK-yeast phagocytosis whereas it inhibited the reexpression of the glucan receptors; (c) glucan 1 did not affect the phagocytosis of serum opsonized HK-yeast. Thus under the experimental conditions described, phagocytosis of HK-yeast by murine macrophages is mediated by and large by the beta-glucan receptors, while the mannose receptors and complement receptors do not contribute to the process.

The immunopotentiating activity and mechanisms of PSAT and scleroglucan, two beta 1-3, beta 1-6 glucan, were investigated in mice: these polysaccharides increase the chemiluminescence of peritoneal phagocytes and the serum C3 level. Augmentation of DNA synthesis of spleen cells was induced in vivo by injecting PSAT or scleroglucan. An additional proliferative effect of these polysaccharides was observed when spleen cells were incubated with mitogens. Moreover, PSAT enhances the number of Thy1 lymphocytes and increases the ratio of lymphocytes L3T4/Lyt2 in mice infected with Toxoplasma gondii. No significant changes in immunoglobulin levels were found. These data indicate that PSAT and scleroglucan favorably affect the non-specific host defense and cellular immune response in mice.

Unopsonized particulate zymosan and its major carbohydrate component glucan were phagocytosed under serum-free conditions by adherent polymorphonuclear leucocytes (PMN) in a dose- and time-dependent manner. Preincubation of PMN monolayers with mannan did not cause a reduction in the phagocytosis of either particle. The phagocytic response was inhibited by preincubation of the cells with trypsin at a concentration that did not inhibit the phagocytosis of sheep erythrocytes coated with IgG or of latex particles. Homology of the recognition mechanisms for glucan and zymosan was confirmed when cells cultured on fixed glucan or on fixed zymosan failed to ingest either particle to more than 40% of control phagocytosis. Similarly, zymosan and glucan activated PMN in suspension, in a dose- and time-dependent manner, to generate reactive oxygen species which were measured as luminol-dependent chemiluminescence (CL). There was, however, a four-fold greater CL response to zymosan. Preincubation of PMN with mannan resulted in a significantly decreased CL response to zymosan, while the response to glucan was unaffected. The CL response was also sensitive to a range of concentrations of trypsin. In contrast, two other complex polysaccharide particles (barley-derived beta-glucan and algae-derived laminarin) were not phagocytosed by PMN, nor did they cause the generation of CL, despite the fact that they possessed the capacity, in common with zymosan and glucan, to activate the alternative pathway of complement. The identification of a trypsin-sensitive recognition mechanism on the surface of human PMN for unopsonized zymosan and glucan represents a response not hitherto characterized. Furthermore, our data indicate that the phagocytosis of unopsonized zymosan by human PMN is dependent primarily on its glucan content, but that its capacity to activate the respiratory burst may involve mannan and the recruitment of a second cell surface recognition mechanism.