NOVEL GLUCAN PREPARATION

Abstract: The present invention relates to neutral, aqueous soluble beta -glucans which exert potent and specific immunological effects without stimulating the production of certain cytokines, to preparations containing the novel beta -glucans, and to a novel manufacturing process therefor. The neutral, aqueous soluble beta -glucan preparation has a high affinity for the beta -glucan receptor of human monocytes and retains two primary biological (or immunological) activities, (1) the enhancement of microbicidal activity of phagocytic cells, and (2) monocyte, neutrophil and platelet hemopoietic activity. Unlike soluble glucans described in the prior art, the neutral, aqueous soluble beta -glucan of this invention neither induces nor primes IL-1 beta and TNF alpha production in vitro and in vivo. Safe and efficacious preparations of neutral, aqueous soluble beta -glucan of the present invention can be used in therapeutic and/or prophylactic treatment regimens of humans and animals to enhance their immune response, without stimulating the production of certain biochemical mediators (e.g., IL-1 beta , TNF alpha ) that can cause detrimental side effects, such as fever and inflammation.

Description: NOVEL GLUCAN PREPARATION Background of the Invention In the early 1960's, zymosan, a crude insoluble yeast extract prepared by boiling yeast before and after trypsin treatment, was noted to produce marked hyperplasia and functional stimulation of the reticuloendothelial system in rodents. In animal studies, zymosan preparations were shown to inactivate complement component C3, to enhance antibody formation, to promote survival following irradiation, to increase resistance to bacterial infections, to inhibit tumor development, to promote graft rejection, and to inhibit dietary-induced hypercholesterolemia and cholesterosis. Zymosan was shown to consist of polysaccharides, proteins, fats, and inorganic elements; however, subsequent studies identified the active components of the yeast cell wall as a pure polysaccharide, specifically 0-glucan. In conventional nomenclature, the polysaccharide 3-glucan is known as poly-(1-6)-3-O-glucopyranosy1-(1-3)-S-D-glucopyranose (PGG) . Repetition of biological assays with j8-glucan indicated that most of the above functional activities identified with zymosan were retained by the purified β- glucan preparation. The properties of 0-glucan are quite similar to those of endotoxin in increasing nonspecific immunity and resis¬ tance to infection. The activities of S-glucan as an immune adjuvant and hemopoietic stimulator compare to those of more complex biological response modifiers (BRMs) , such as bacillus Calmette-Guerin (BCG) and Corvnebacterium parvuro. The functional activities of yeast 0-glucan are also comparable to those structurally similar carbohydrate polymers isolated from fungi and plants. These higher molecular weight (l-3)-/3-D-glucans such as schizophyllan, lentinan, krestin, grifolan, and pachy an exhibit similar immunomodulatory activities. A common mechanism shared by all these /S-glucan preparations is their stimulation of cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) . Lentinan has been extensively investigated for its antitumor properties, both in animal models at 1 mg/kg for 10 days and in clinical trials since the late 1970s in Japan for advanced or recurrent malignant lymphoma and colorectal, mammary, lung and gastric cancers. In cancer chemotherapy, lentinan has been administered at 0.5-5 mg/day, intramuscularly (I.M.) or intravenously (I.V.), two or three times per week alone, or in combination with antineoplastic drugs. In addition to the activities ascribed to yeast glucans, studies suggest lentinan acts as a T-cell immunopotentiator, inducing cytotoxic activities, including production of interleukins 1 and 3 and colony-stimulating factors (CSF) . (Chihara et al. , 1989, Int♦ J. Immunotherapy. 4.:145-154; Hamuro and Chihara, In Lentinan. An Immunopotentiator) Various preparations of both particulate and soluble /S-glucans have been tested in animal models to evaluate biological activities. The use of soluble and insoluble |8-glucans alone or as vaccine adjuvants for viral and bacterial antigens has been shown in animal models to markedly increase resistance to a variety of bacterial, fungal, protozoan and viral infections. The hemopoietic effects of /S-glucan have been correlated with increased peripheral blood leukocyte counts and bone marrow and splenic cellularity, reflecting increased numbers of granulocyte-macrophage progenitor cells, splenic pluripotent stem cells, and erythroid progenitor cells, aε well as, increased serum levels of granulocyte-monocyte colony-stimulating factor (GM-CSF) . Furthermore, the hemopoietic and anti-infective effects of /S-glucan were active in cyclophosphamide-treated immunosuppressed animals. /S-glucan was shown to be beneficial in animal models of trauma, wound healing and tumorigenesis. However, various insoluble and soluble preparations of β- glucan differed significantly in biological specificity and potency, with effective dosages varying from 25 to 500 mg/kg intravenously or intraperitoneally (I.P.) in models for protection against infection and for hemopoiesis. Insoluble preparations demonstrated undesirable toxicological properties manifested by hepatosplenomegaly and granuloma formation. Clinical interest was focused on a soluble glucan preparation which would retain biological activity yet yield negligible toxicity when administered systemically. Chronic systemic administration of a soluble phosphorylated glucan over a wide range of doses (40-1000 mg/kg) yielded negligible toxicity in animals (DiLuzio et al« . 1979, Int. J. of Cancer. 24;773-779; DiLuzio, U.S. Patent 4,739,046). The molecular mechanism of action of /S-glucan has been elucidated by the demonstration of specific /3-glucan receptor binding sites on the cell membranes of human neutrophils and acrophages. Mannans, galactans, α(l-4)- linked glucose polymers and /S(1-4)-linked glucose polymers have no avidity for this receptor. These /S-glucan binding sites are opso in-independent phagocytic receptors for particulate activators of the alternate complement pathway, similar to Escherichia coli lipopolysaccharide (LPS) and some animal red blood cells. Ligand binding to the /S-glucan receptor, in the absence of antibody, results in complement activation, phagocytosis, lysosomal enzyme release, and prostaglandin, thromboxane and leukotriene generation; thereby increasing nonspecific resistance to infection. However, soluble /S-glucan preparations described in the prior art demonstrated stimulation of cytokines. Increases in plasma and splenic levels of interleukins 1 and 2 (IL-1, IL-2) in addition to TNF were observed in vivo and corresponded to induction of the synthesis of these cytokines in vitro. (See Sherwood et al.. 1987, Int. J. Immunopharmac.. £:261-267 (enhancement of IL-1 and IL-2 levels in rats injected with soluble glucan); Williams et al- . 1988, Int. J. Immunopharmac. .10:405-414 (systemic administration of soluble glucan to AIDS patients increased IL-1 and IL-2 levels which were accompanied by chills and fever); Browder et al* , 1990, Ann. Surα.. 211:605-613 (glucan administration to trauma patients increased serum IL-1 levels, but not TNF levels) ; Adachi _gt al . , 1990, Chem. Phar . Bull.. 38:988-992 (chemically cross-linked /S(l-3) glucans induced IL-1 production in mice) .) Interleukin-1 is a primary immunologic mediator involved in cellular defense mechanisms. Numerous studies have been carried out on the application of IL-1 to enhance non-specific resistance to infection in a variety of clinical states. Pomposelli et al * . J. Parent. Ent. Nutr. 12111:212-218, (1988). The major problem associated with the excessive stimulation or exogenous administration of IL-1 and other cellular mediators in humans is toxicity and side effects resulting from the disruption of the gentle balance of the immunoregulatory network. Fauci et al., Ann. Int. Med.. 106:421-433 (1987). IL-1 is an inflammatory cytokine that has been shown to adversely affect a variety of tissues and organs. For instance, recombinant IL-1 has been shown to cause death, hypotensive shock, leukopenia, thrombocytopenia, anemia and lactic acidosis. In addition, IL-1 induces sodium excretion, anorexia, slow wave sleep, bone resorption, decreased pain threshold and expression of many inflammatory-associated cytokines. It is also toxic to the insulin secreting beta cells of the pancreas. Patients suffering from a number of inflammatory diseases have elevated levels of IL-1 in their systems. Administration of agents that enhance further IL-1 production only exacerbate these inflammatory conditions. Tumor necrosis factor is also involved in infection, inflammation and cancer. Small amounts of TNF release growth factors while in larger amounts, TNF can cause septic shock, aches, pains, fever, clotting of blood, degradation of bone and stimulation of white blood cells and other immune defenses. Summary of the Invention The present invention relates to neutral soluble /S- glucans which enhance a host's immune defense mechanisms to infection but do not induce an inflammatory response, to preparations containing the neutral soluble /S-glucans, and to a novel manufacturing process therefor. In the present method, soluble glucan which induces cytokine production is processed through a unique series of acid, alkaline and neutral treatment steps to yield a conforma- tionally pure neutral soluble glucan preparation with unique biological properties. The neutral soluble glucan preparation retains a specific subset of immunological properties common to /S-glucans but uniquely does not induce the production of IL-1 and TNF in vitro or in vivo. Throughout this specification, unless otherwise indicated, the expressions "neutral soluble glucan" and "neutral soluble )S-glucan" refer to the composition prepared as described in Example 1. The neutral soluble glucan preparation is produced by treating insoluble glucan with acid to produce a water soluble glucan, dissociating the native conformations of the soluble glucan at alkaline pH, purifying the desired molecular weight fraction at alkaline pH, re-annealing the dissociated glucan fraction under controlled conditions of time, temperature and pH to form a unique triple helical conformation, and further purifying under neutral pH to remove single helix and aggregated materials to yield a conformationally pure, neutral, water soluble, underivatized glucan which has a unique biological profile. The neutral soluble glucan preparation has a high affinity for the /S-glucan receptor of human monocytes and retains two primary biological activities, (1) the enhancement of microbicidal activity of phagocytic cells, and (2) monocyte, neutrophil and platelet hemopoietic activity. Unlike soluble glucans described in the prior art, the neutral soluble glucan of this invention neither induces nor primes mononuclear cellε to increase IL-1 and TNF production in vitro and in vivo. The neutral soluble glucan preparation iε appropriate for parenteral (e.g., intravenouε, intraperitoneal, subcutaneous, intramuscular), topical, oral or intranasal administration to humans and animals as an anti-infective to combat infection associated with burns, surgery, chemotherapy, bone marrow disorders and other conditions in which the immune system may be compromised. Neutral soluble glucan produced by the present method can be maintained in a clear solution and equilibrated in a pharmaceutically acceptable carrier. Safe and efficacious preparations of the neutral soluble glucan of the present invention can be used in therapeutic and/or prophylactic treatment regimens of humans and animals to enhance their immune response, without stimulating the production of certain biochemical mediators (e.g., IL-1 and TNF) that can cause detrimental side effects, such as fever and inflammation. Brief Description of the Figures Fig. 1 shows the general structure of neutral soluble glucan as being a linear /S(1-3)-linked glucose polymer having periodic branching via a single /S(1-6)-linked glucose moiety. Fig. 2 shows a gel permeation chromatogram (pH 7) of soluble glucan which has not been purified by alkali dissociation and re-annealing. The chromatogram shows three species, referred to herein as high molecular weight aggregate (Ag) , Peak A and Peak B (single helix glucan) . Fig. 3 is a chromatogram obtained for the neutral soluble glucan by gel permeation chromatography. The solid line represents the neutral soluble glucan at pH 7 and the broken line represents the neutral soluble glucan at pH 13. Fig. 4 is a chromatogram obtained for the single helix /S-glucan (Peak B) by gel permeation chromatography. The solid line represents Peak B at pH 7 and the broken line represents Peak B at pH 13. Fig. 5 shows the change in serum IL-1 levels, over time, taken from patients intravenously infused with placebo (broken line) or neutral soluble glucan (solid line) . Fig. 6 shows the change in serum TNF levels, over time, taken from patients intravenouεly infused with placebo (broken line) or neutral soluble glucan (solid line) . Fig. 7 is a diagram representing peripheral blood counts from irradiated mice following administration of neutral soluble glucan. Fig. 8 is a diagram representing platelet cell counts from cisplatin-treated mice following administration of neutral soluble glucan. Detailed Description of Invention The invention relates to a neutral soluble /S-glucan polymer that can bind to the /S-glucan receptor and activate only a desired subset of immune responses. The terms "neutral soluble /3-glucan" and "neutral soluble glucan", unless otherwise specified, refer to the composition prepared as described in Example 1. This neutral soluble /S-glucan has been shown to increase the number of neutrophils and monocytes as well as their direct infection fighting activity (phagocytosis and microbial killing) . However, the neutral soluble β- glucan does not stimulate the production of biochemical mediators, such as IL-1 and TNF, that can cauεe detrimen- tal side effects such as high fever, inflammation, wasting disease and organ failure. These advantageous properties make neutral soluble glucan preparations of this invention useful in the prevention and treatment of infection because they selectively activate only those components of the immune system responsible for the initial response to infection, without stimulating the release of certain biochemical mediators that can cauεe adverεe side effects. The solution containing the neutral soluble /S-glucan also lacks the toxicity common to many immunomodulators. The neutral soluble /S-glucans of this invention are composed of glucose monomers organized as a /S(l-3) linked glucopyranose backbone with periodic branching via /S(l-6) glycosidic linkages. The neutral soluble glucan prepara¬ tions contain glucans, which have not been substantially modified by substitution with functional (e.g., charged) groups or other covalent attachments. The general structure of the neutral soluble glucan is shown in Fig. 1. The biologically active preparation of this invention is a confor ationally purified form of /S-glucan produced by dissociating the native glucan conformations and re- annealing and purifying the resulting unique triple helical conformation. The unique conformation of the neutral soluble glucan contributes to the glucan , s ability to selectively activate the immune system without stimulating the production of detrimental biochemical mediators. The neutral soluble glucan preparations of this invention are prepared from insoluble glucan particleε, preferably derived from yeast organisms. See Manners et al..- Bioche . J.. 135:19-30. (1973) for a general procedure to make insoluble yeast glucans. Glucan particles which are particularly useful as starting materials in the present invention are whole glucan particles (WGP) described by Jamas et al.. in U.S. Patent NOS. 4,810,646, 4,992,540, 5,082,936 and 5,028,703, the teachings of all of which are hereby incorporated herein by reference. The source of the whole glucan particles can be the broad spectrum of glucan-containing yeast organisms which contain /S-glucans in their cell wallε. Whole glucan particles obtained from the strains Saccharomvces cerevisiae R4 (NRRL Y-15903; deposit made in connection with U.S. Patent No. 4,810,646) and R4 Ad (ATCC No. 74181) are particularly useful. Other strains of yeast that can be used include Saccharomvces delbrueckii. Saccharomvces rosei. Saccharomvces microellipεodes. Saccharo vces carlsbergenεis. Schizosacharomyces po be. Kluweromvceε lactis. Kluweromyces fragilis. Kluweromvces polvsporus. Candida albicanε. Candida cloacae f Candida tropicalis. Candida utilis. Hansenula wingeri. Hansenula ami. Hansenula henricii. Hansenula americana. A procedure for extraction of whole glucan particles is described by Jamas et al.. in U.S. Patent Nos. 4,810,646, 4,992,540, 5,082,936 and 5,028,703. For the purpose of this present invention, it is not necesεary to conduct the final organic extraction and waεh steps de¬ scribed by Jamas et al. In the present process, whole glucan particles are suspended in an acid solution under conditions sufficient to dissolve the acid-soluble glucan portion. For most glucans, an acid solution having a pH of from about 1 to about 5 and at a temperature of from about 20 to about 100°C is sufficient. Preferably, the acid used is an organic acid capable of dissolving the acid-soluble glucan portion. Acetic acid, at concentrations of from about 0.1 to about 5 M or formic acid at concentrations of from about 50% to 98% (w/v) are useful for this purpose. The treatment time may vary from about 10 minutes to about 20 hours depending on the acid concentration, temperature and source of whole glucan particles. For example, modified glucans having more /S(l-6) branching than naturally- occurring, or wild-type glucans, require more stringent conditions, i.e., longer exposure times and higher temperatures. This acid-treatment step can be repeated under similar or variable conditions. One preferred procesεing method iε described in the exemplification using glucan derived from S. cerevisiae strain R4 Ad. In another embodiment of the present method, whole glucan particles from the strain, S. cerevisiae R4, which have a higher level of /S(l-6) branching than naturally-occurring glucans, are used, and treatment is carried out with 90% (w/v) formic acid at 20 β C for about 20 minutes and then at 85°C for about 30 minutes. The insoluble glucan particles are then separated from the solution by an appropriate separation technique, for example, by centrifugation or filtration. The pH of the resulting slurry is adjuεted with an alkaline compound such as sodium hydroxide, to a pH of about 7 to about 14. The precipitate is collected by centrifugation and iε boiled in purified water (e.g., USP) for three hours. The slurry is then resuspended in hot alkali having a concentration sufficient to solubilize the glucan polymers. Alkaline compounds which can be used in this step include alkali-metal or alkali-earth metal hydroxides, such as sodium hydroxide or potassium hydroxide, having a concentration of from about 0.01 to about 10 N. This step can be conducted at a temperature of from about 4°C to about 121°C, preferably from about 20 β C to about 100 β C. In one embodiment of the process, the conditions utilized are a l M εolution of sodium hydroxide at a temperature of about 80-100°C and a contact time of approximately 1-2 hours. The reεulting mixture contains εolubilized glucan molecules and particulate glucan residue and generally has a dark brown color due to oxidation of contaminating proteinε and sugars. The particulate residue is removed from the mixture by an appropriate separation technique, e.g., centrifugation and/or filtration. In another embodiment of the procesε the acid-soluble glucans are precipitated after the preceding acid hydrolysiε reaction by the addition of about 1.5 volumeε of ethanol. The mixture iε chilled to about 4 β C for two (2) hours and the resulting precipitate is collected by centrifugation or filtration and washed with water. The pellet iε then reεuspended in water, and stirred for three (3) to twelve (12) hours at a temperature between about 20°C and 100°C. At this point the pH is adjuεted to approximately 10 to 13 with a baεe εuch as sodium hydroxide. The resulting solution contains dissociated soluble glucan molecules. This solution is now purified to remove traces of insoluble glucan and high molecular weight soluble glucans which can cause aggregation. This step can be carried out by an appropriate purification technique, for example, by ultrafiltration, utilizing membranes with nominal molecular weight (NMW) levels or cut-offs in the range of about 1,000 to 100,000 daltons. It was diεcovered that in order to prevent gradual aggregation or precipitation of the glucan polymers the preferred membrane for this step has a nominal molecular weight cut-off of about 100,000 daltons. The soluble glucan is then further purified at alkaline pH to remove low molecular weight materials. This step can be carried out by an appropriate purification technique, for example, by ultrafiltration, utilizing membranes with nominal molecular weight levels or cut-offs in the range of 1,000 to 30,000 daltons. The resulting dissociated soluble glucan is re- annealed under controlled conditions of time (e.g., from about 10 to about 120 minutes), temperature (e.g., from about 50 to about 70°C) and pH. The pH of the solution is adjusted to the range of about 3.5-11 (preferably 6-8) with an acid, such as hydrochloric acid. The purpose of this re-annealing step is to cause the soluble glucan to rearrange from a single helix conformation to a new ordered triple helical conformation. The re-annealed glucan solution iε then εize fractionated, for example by using 30,000-70,000 NMW and 100,000-500,000 NMW cut-off membrane ultrafilters to εelectively remove high and low molecular weight soluble glucans. Prior to sizing, the soluble glucans exist aε a mixture of conformations including random coils, gel matrices or aggregates, triple helices and single heliceε. The objective of the εizing step is to obtain an enriched fraction for the re-annealed conformation of specific molecular weight. The order in which the ultrafilters are used is a matter of preference. The concentrated fraction obtained is enriched in the soluble, biologically active neutral soluble glucan. The glucan concentrate is further purified, for example, by diafiltration using a 10,000 dalton membrane. The preferred concentration of the εoluble glucan after thiε εtep iε from about 2 to about 10 mg/ l. The neutralized solution can then be further purified, for example, by diafiltration, using a pharmaceutically acceptable medium (e.g., sterile water for injection, phosphate-buffered saline (PBS) , isotonic saline, dextrose) suitable for parenteral administration. The preferred membrane for this diafiltration step haε a nominal molecular weight cut-off of about 10,000 daltonε. The final concentration of the glucan solution is adjusted in the range of about 0.5 to 10 mg/ml. In accordance with pharmaceutical manufacturing standards for parenteral products, the solution can be terminally sterilized by filtration through a 0.22 μm filter. The neutral soluble glucan preparation obtained by this process is sterile, non-antigenic, esεentially pyrogen-free, and can be εtored at room temperature (e.g., 15-30°C) for extended periods of time without degradation. Thiε process is unique in that it results in a neutral aqueous solution of (pH 4.5 to 7.0) immunologically active glucans which is suitable for parenteral administration. For purposes of the present invention, the term "soluble" as used herein to describe glucans obtained by the present process, means a visually clear solution can be formed in an aqueous medium such as water, PBS, isotonic saline, or a dextrose solution having a neutral pH (e.g., from about pH 5 to about 7.5), at room temperature (about 20-25 β C) and at a concentration of up to about 10 mg/ml. The term "aqueous medium" referε to water and water-rich phases, particularly to pharmaceutically acceptable aqueous liquids, including PBS, saline and dextrose solutions. The expression "visually clear" means that at a concentration of 1 mg/ml, the absorption of the solution at 530 nm is less than OD 0.01 greater than the OD of an otherwise identical solution lacking the B-glucan component. The resulting solution is substantially free of protein contamination, is non-antigenic, non-pyrogenic and is pharmaceutically acceptable for parenteral administration to animals and humans. However, if desired, the soluble glucan can be dried by an appropriate drying method, such as lyophilization, and stored in dry form. The neutral soluble glucans of this invention can be used as safe, effective, therapeutic and/or prophylactic agents, either alone or aε adjuvantε, to enhance the immune response in humans and animalε. Soluble glucanε produced by the preεent method selectively activate only those components that are reεponεible for the initial response to infection, without stimulating or priming the immune system to release certain biochemical mediators (e.g., IL-1, TNF, IL-6, IL-8 and GM-CSF) that can cause adverse side effects. As such, the present soluble glucan composition can be used to prevent or treat infectious diseases in malnourished patients, patients undergoing surgery and bone marrow transplants, patients undergoing chemotherapy or radiotherapy, neutropenic patients, HIV- infected patients, trauma patients, burn patients, patients with chronic or resistant infections such as those resulting from myelodysplastic syndrome, and the elderly, all of who may have weakened immune systems. An immunocompromised individual iε generally defined as a person who exhibits an attenuated or reduced ability to mount a normal cellular and/or humoral defense to challenge by infectious agentε, e.g., viruses, bacteria, fungi and protozoa. A protein malnourished individual is generally defined as a person who has a serum albumin level of less than about 3.2 grams per deciliter (g/dl) and/or unintentional weight loss of greater than 10% of usual body weight. More particularly, the method of the invention can be used to therapeutically or prophylactically treat animals or humans who are at a heightened risk of infection due to imminent surgery, injury, illness, radiation or chemotherapy, or other condition which deleteriouεly affects the immune system. The method is useful to treat patients who have a disease or disorder which causeε the normal metabolic immune response to be reduced or depressed, such as HIV infection (AIDS) . For example, the method can be used to pre-initiate the metabolic immune response in patients who are undergoing chemotherapy or radiation therapy, or who are at a heightened risk for developing secondary infections or poεt-operative complicationε becauεe of a disease, disorder or treatment resulting in a reduced ability to mobilize the body's normal metabolic responses to infection. Treatment with the neutral soluble glucans has been shown to be particularly effective in mobilizing the host's normal immune defenseε, thereby engendering a measure of protection from infection in the treated host. The present composition is generally administered to an animal or a human in an amount sufficient to produce immune system enhancement. The mode of administration of the neutral soluble glucan can be oral, enteral, parenter¬ al, intravenous, subcutaneous, intraperitoneal, intramuscular, topical or intranaεal. The form in which the compoεition will be administered (e.g., powder, tablet, capsule, solution, emulsion) will depend upon the route by which it is administered. The quantity of the composition to be administered will be determined on an individual basis, and will be based at leaεt in part on consideration of the severity of infection or injury in the patient, the patient's condition or overall health, the patient's weight and the time available before surgery, chemotherapy or other high-risk treatment. In general, a single dose will preferably contain approximately 0.01 to approximately 10 mg of modified glucan per kilogram of body weight, and preferably from about 0.1 to 2.5 mg/kg. The dosage for topical application will depend upon the particular wound to be treated, the degree of infection and severity of the wound. A typical dosage for wounds will be from about 0.001 mg/ml to about 2 mg/ml, and preferably from about 0.01 to about 0.5 mg/ml. In general, the compositionε of the present invention can be administered to an individual periodically as neceεεary to stimulate the individual's immune response. An individual skilled in the medical arts will be able to determine the length of time during which the composition is administered and the dosage, depending upon the physical condition of the patient and the disease or disorder being treated. As stated above, the composition may also be used as a preventative treatment to pre- initiate the normal metabolic defenseε which the body obilizeε againεt infections. Neutral soluble /S-glucan can be used for the prevention and treatment of infectionε cauεed by a broad spectrum of bacterial, fungal, viral and protozoan pathogens. The prophylactic adminiεtration of neutral soluble /S-glucan to a person undergoing surgery, either preoperatively, intraoperatively and/or post-operatively, will reduce the incidence and severity of post-operative infections in both normal and high-risk patients. For example, in patients undergoing surgical procedures that are claεεified aε contaminated or potentially contaminated (e.g., gaεtrointeεtinal surgery, hysterectomy, ceεarean section, transurethral prostatectomy) and in patients in whom infection at the operative site would present a serious risk (e.g., proεthetic arthroplaεty, cardiovaεcular surgery) , concurrent initial therapy with an appropriate antibacterial agent and the present neutral soluble glucan preparation will reduce the incidence and severity of infectious complicationε. In patientε who are immunosuppresεed, not only by disease (e.g., cancer, AIDS) but by courseε of chemother¬ apy and/or radiotherapy, the prophylactic adminiεtration of the soluble glucan will reduce the incidence of infections caused by a broad spectrum of opportuniεtic pathogens including many unusual bacteria, fungi and viruses. Therapy using neutral soluble /3-glucan has demonstrated a significant radio-protective effect with its ability to enhance and prolong macrophage function and regeneration and, as a result enhance resistance to microbial invasion and infection. In high risk patients (e.g., over age 65, diabetics, patients having cancer, malnutrition, renal disease, emphysema, dehydration, restricted mobility, etc.) hospitalization frequently is associated with a high incidence of serious nosocomial infection. Treatment with neutral soluble /S-glucan may be started empirically before catheterization, use of respiratorε, drainage tubes, intenεive care unitε, prolonged hoεpitalizationε, etc. to help prevent the infectionε that are commonly associated with these procedureε. Concurrent therapy with antimi- crobial agents and the neutral soluble /S-glucan is indicated for the treatment of chronic, severe, refractory, complex and difficult to treat infections. The compoεitions administered in the method of the present invention can optionally include other components, in addition to the neutral soluble /S-glucan. The other components that can be included in a particular compoεition are determined primarily by the manner in which the compoεition iε to be administered. For example, a composition to be administered orally in tablet form can include, in addition to neutral soluble /S-glucan, a filler (e.g., lactose), a binder (e.g., carboxymethyl cellulose, gum arabic, gelatin) , an adjuvant, a flavoring agent, a coloring agent and a coating material (e.g., wax or plasticizer) . A composition to be administered in liquid form can include neutral soluble /S-glucan and, optionally, an emulsifying agent, a flavoring agent and/or a coloring agent. A composition for parenteral adminiεtration can be mixed, dissolved or emulsified in water, sterile saline. PBS, dextrose or other biologically acceptable carrier. A compoεition for topical administration can be formulated into a gel, ointment, lotion, cream or other form in which the composition is capable of coating the site to be treated, e.g., wound site. Compositions comprising neutral soluble glucan can also be administered topically to a wound site to stimulate and enhance wound healing and repair. Wounds due to ulcers, acne, viral infections, fungal infections or periodontal diseaεe, among otherε, can be treated according to the methodε of thiε invention to accelerate the healing process. Alternatively, the neutral soluble /S-glucan can be injected into the wound or afflicted area. In addition to wound repair, the composition can be used to treat infection asεociated therewith or the causative agents that reεult in the wound. A composition for topical administration can be formulated into a gel, ointment, lotion, cream or other form in which the composition iε capable of coating the site to be treated, e.g., wound site. The dosage for topical application will depend upon the particular wound to be treated, the degree of infection and severity of the wound. A typical dosage for wounds will be from about 0.01 mg/ml to about 2 mg/ml, and preferably from about 0.01 to about 0.5 mg/ml. Another particular uεe of the compositions of this invention is for the treatment of myelodyεplaεtic syndrome (MDS) . MDS, frequently referred to as preleukemia syndrome, is a group of clonal hematopoietic stem cell disorders characterized by abnormal bone marrow differentiation and maturation leading to peripheral cytopenia with high probability of eventual leukemic conversion. Recurrent infection, hemorrhaging and terminal infection reεulting in death typically accompany MDS. Thus, in order to reduce the severity of the diεease and the frequency of infection, compositions comprising modified glucan can be chronically administered to a patient diagnosed as having MDS according to the methods of thiε invention, in order to specifically increase the infection fighting activity of the patient's white blood cells. Other bone marrow disorders, such as aplaεtic anemia (a condition of quantitatively reduced and defective hematopoieεis) can be treated to reduce infection and hemorrhage that are associated with thiε disease state. Neutral soluble glucan produced by the present method enhances the non-specific defenses of mammalian mononuclear cellε and εignificantly increases their ability to respond to an infectiouε challenge. The unique property of neutral εoluble glucan macrophage activation iε that it does not result in increased body temperatures (i.e., fever) as has been reported with many non-specific stimulants of those defenses. This critical advantage of neutral εoluble glucan may lie in the natural profile of responses it mediates in white blood cells. It has been shown that the neutral soluble /3-glucan of the present invention selectively activates immune reεponses but does not directly stimulate or prime cytokine (e.g., IL-1 and TNF) release from mononuclear cells, thuε diεtinguishing the present neutral soluble glucan from other glucan preparations (e.g., lentinan, kresein) and immunoεtimulantε. In addition, it haε been demonεtrated herein that the neutral soluble glucan preparation of the present invention posεeεses an unexpected platelet εtimulating property. Although it waε known that glucanε have the ability to εtimulate white blood cell hematopoieεiε, the disclosed platelet stimulating property had not been reported or anticipated. This property can be exploited in a therapeutic regimen for use aε an adjuvant in parallel with radiation or chemotherapy treatment. Radiation and chemotherapy are known to result in neutropenia (reduced polymorphonuclear (PMN) leukocyte cell count) and thrombocytopenia (reduced platelet count) . At present, these conditionε are treated by the administration of colony-stimulating factors such as GM- CSF and granulocyte colony-stimulating factor (G-CSF) . Such factors are effective in overcoming neutropenia, but fail to impact upon thrombocytopenia. Thus, the platelet stimulating property of the neutral soluble glucan preparation of this invention can be used, for example, aε a therapeutic agent to prevent or minimize the development of thrombocytopenia which limitε the doεe of the radiation or chemotherapeutic agent which is used to treat cancer. The invention iε further illuεtrated by the following Examples. EXAMPLES EXAMPLE l: PREPARATION OF NEUTRAL SOLUBLE GLUCAN FROM S. CEREVISIAE Saccharomyces cerevisiae strain R4 Ad (a non-recombi- nant derivative of wild-type strain A364A) , was grown in a large-scale fermentation culture using a defined glucoεe, ammonium sulfate minimal medium. The production culture was maintained under glucoεe limitation in a feed-batch mode (New Brunεwick MPP80) . When the growing culture reached late logarithmic phaεe, the fermentation waε ended and the /S-glucan waε εtabilized by adjuεting the culture to pH 12 ± 0.5 uεing 10 M NaOH. The yeast cells containing 0-glucan were harvested by continuouε-flow centrifugation (Westfalia SA-1) . After centrifugation, the cells were collected into a stainleεε εteel extraction vessel. The first step in the extraction procesε waε an alkaline extraction accompliεhed by mixing the cellε with 1 M sodium hydroxide (NaOH) at 90 ± 5°C for 1 hour. Upon completion of this alkaline extraction, the /S-glucan remained in the solid phase, which waε collected by continuouε centrifugation (Weεtfalia SA-1) . The collected cell wall fraction waε extracted a second time using the same procedure and under the same conditions. Treatment with alkali hydrolyzed and solubilized the cellular proteins, nucleic acids, mannans, soluble glucans and polar lipidε into the εupernatant fraction, and deacety- lated chitin to chitosan in the cell wall. The second step in the extraction procesε waε a pH 4.5 ± 0.05 (adjusted with concentrated HCl) extraction at 75 ± 5°C for 1 hour. Thiε waε followed by a 0.1 M acetic acid extraction to complete the removal of glycogen, chitin, chitosan and remaining proteins. The solids were collected and rinsed twice with Purified Water USP to remove any residual acid as well as any yeast degradation products. The third step in the extraction process was a set of six organic extractions. The first four extractionε were carried out in isopropanol. The solids were collected by centrifugation and then subjected to two acetone extrac¬ tions. The two-stage organic extractions eliminated nonpolar lipids and hydrophobic proteins which may have co-purified with the drug substance. The resulting wet solids were dried in a vacuum oven at 65 ± 5°C for 48-96 hours to yield a free-flowing powder. At this stage the extraction procesε yielded a stable, insoluble intermediate consiεting of approximately 90% /S-glucan, called whole glucan particleε (WGPε) . The dry WGP intermediate was stored at 15-30°C until further use. The WGP powder was reεuεpended in 98% (w/v) formic acid, in a glaεε reaction veεεel at room temperature. The resulting mixture was heated to 85 ± 5°C for 20 minutes. Under these conditions, the WGPs were partially hydrolyzed and solubilized to provide the deεired molecular weight distribution of εoluble /S-glucan which was then precipitated by adding 1.5 volumes of ethanol. After complete mixing, the preparation was centrifuged to collect the /S-glucan precipitate. Any residual formic acid was removed by boiling the /S-glucan preparation in Purified Water USP for three hourε. Any unhydrolyzed WGPε were then removed from the /S- glucan solution by centrifugation. The /S-glucan solution was raised to pH 12.5 ± 0.5 by the addition of the concen- trated sodium hydroxide. The remaining purification steps were carried out by ultrafiltration. The soluble alkaline /S-glucan preparation was pasεed through a 100,000 nominal molecular weight (NMW) cut-off membrane ultrafilter (Amicon DC10) . Under alkaline conditionε thiε membrane ultrafilter removed insoluble and high molecular weight soluble /S-glucan. Trace low molecular weight degradation products were then removed by recirculation through a 10,000 NMW cut-off membrane ultrafilter. The ultrafiltration waε conducted aε a constant volume wash with 0.1 M NaOH. The /3-glucan solution was re-annealed under controlled conditionε by adjusting the pH to 7.0 ± 0.5 with concentrated hydrochloric acid, heating to 60 ± 10 β C, which was maintained for 20 minutes and then cooled. The neutral re-annealed εolution waε then concentrated and waεhed with Sodium Chloride Injection USP in a 70,000 NMW cut-off membrane ultrafilter (Filtron Minisep) to enrich for the re-annealed neutral soluble glucan. Next the material was filtered through a 300,000 NMW cut-off membrane ultrafilter (Filtron Minisep) to remove high molecular weight and aggregated glucan molecules. In the same ultrafilter, the neutral soluble glucan material was washed with Sodium Chloride Injection USP in a constant volume wash mode. The neutral soluble glucan was then concentrated in a 10,000 NMW cut-off membrane ultrafilter. The concentration proceεε continued until a concentration of at leaεt 1.0 mg/ml hexoεe equivalent was achieved. The resulting neutral soluble glucan was then subjected to filtration through a depyrogenating filter (0.1 micron Posidyne) and a sterile 0.2 micron filter (Millipak) to yield sterile, pyrogen-free neutral soluble glucan. The neutral soluble glucan solution was stored at controlled room temperature (15-30°C) until further use. The aqueous solubility of neutral soluble glucan in the pH range of 4 to 8 is approximately 100 mg/ml. The εolubil- ity increased with increasing pH and reached approx. 150 mg/ml at pH 13. EXAMPLE 2: ANALYSIS OF NEUTRAL SOLUBLE GLUCAN A. Glucose. Mannoεe and Glucosamine Monosaccharide analyεiε waε performed to quantitate the relative amountε of /S-glucan (aε glucoεe) , mannan or phoεphomannan (aε mannoεe) , and chitin (aε N-acetyl glucosamine) in the neutral soluble glucan. The sample was hydrolyzed to monoεaccharideε in 2 M trifluoroacetic acid for 4 hours at 110°C, evaporated to drynesε, and redissolved in water. Monoεaccharideε were separated on a Dionex HPLC system using a CarboPac PA100 column (4 x 250 mm) using 5 M NaOH at 1 ml/min and quantitated using a pulsed electrochemical detector (Dionex Model PED-1) . The sensitivity of this aεεay for monosaccharides iε 0.1% (w/w) . Glucose (retention time of 16.6 min) was identified as the only monosaccharide component of neutral soluble glucan along with traces of glucose degradation products (from hydrolysis) anhydroglucose at 2.5 min and 5- hydroxymethylfurfural at 4.3 min. The reεultε confirm that neutral soluble glucan consiεted of ≥98% glucoεe. B. FTIR Fourier tranεform infrared εpectroεcopy by diffuεe reflectance (FTIR, Matson Instruments, Polaris) of lyophi- lized neutral soluble glucan samples was used to determine the anomeric structure (α vs. β) , and linkage type (/3(1- 3), 0(1-6), /3(l-4)) present in neutral soluble glucan. Absorption maxima of 890 cm" 1 identified /S(l-3) linkages; 920 cm *1 identified /S(l-6) linkages. No presence of α- 1inked anomers (e.g., glycogen, 850 cm" 1 ) or /3(1-4)-linked polysaccharides (e.g., chitin, 930 cm" 1 ) were detected. EXAMPLE 3: CONFORMATIONAL ANALYSIS A solution of jS-glucan which waε not processed by alkali dissociation and re-annealing was analyzed for its compositional identity by gel permeation chromatography (pH 7) and found to contain multiple specieε, referred to herein as high molecular weight aggregate (Ag) , Peak A and Peak B (See Figure 2) . Neutral soluble glucan which was prepared by alkali dissociation and re-annealing as described in Example 1, is present as a single peak (see Figure 3) with an average molecular weight of 92,660 daltons at pH 7. The distinct conformationε of neutral soluble glucan and Peak B were demonstrated by gel permeation chromatography at pH 7 and pH 13 uεing a refractive index detector. Neutral εoluble glucan under- went a significant conformational transition from pH 7 to pH 13 which illustrates complete disεociation of the multiple helix at pH 7 to a single helical form at pH 13 (see Fig. 3) . In contrast. Peak B only underwent a slight shift in molecular weight from pH 7 to pH 13 (see Fig. 4) . The molecular weight of neutral soluble glucan and Peak B glucans as a function of pH iε shown below in Table 1. Table 1 Sample MW MW Ratio pH 7 pH 13 (pH 7/pH 13) Neutral soluble 92,666 18,693 4.96 glucan Peak B 8,317 7,168 1.16 The conformation of neutral soluble glucan and Peak B glucan waε also determined by aniline blue complexing (Evans et al * , 1984, Carb. Pol.. 4.:215-230; Adachi et al.. 1988, Carb. Res.. 177:91-100) . using curdlan, a linear /S(l-3) glucan, aε the triple helix control and pustulan, a /S(l-6) glucan, as a non-ordered conformational control. The results are discussed below and shown in Table 2. The curdlan triple helix control complexed with aniline blue resulting in high fluorescence. Increasing the NaOH concentration began to dissociate the curdlan triple helix slightly, but NaOH concentrations >0.25 M are required for complete disεociation of curdlan. The puεtulan non-ordered control only formed a weak complex with aniline blue resulting in low fluorescence measurementε which were not affected by NaOH concentration. The neutral εoluble glucan complexed effectively with aniline blue at low NaOH concentration (25mM NaOH) resulting in high fluorescence. However, the neutral soluble glucan conformation dissociated significantly (50%) at NaOH concentrations aε low aε 150 mM NaOH indicating that it exists as a unique conformation compared to naturally occurring /S-glucans, such as laminarin and curdlan, which require significantly higher NaOH concentrations for dissociation to occur. Peak B formed a weak complex with aniline blue due to its single helical conformation. Table 2 Conformational Analysis of Glucans by Aniline Blue Complexing Fluorescence Test Material 25 mM 100 mM 150 mM NaOH NaOH NaOH Blank 0 2 0 Curdlan 53.5 41.6 36 /3(l-3) glucan Pustulan 9.8 8.3 8.0 /S(l-6) glucan Neutral soluble glucan 40 25.6 20.2 Peak B 12.4 6.2 4.1 EXAMPLE 4: EFFECTS OF NEUTRAL SOLUBLE GLUCAN ON HUMAN MONOCYTE PRODUCTION OF TNFα Human peripheral blood mononuclear cells were iεolated (Januεz et al.. (1987) , J. Immunol.. 138: 3897- 3901) from normal citrated and dextran-treated blood, waεhed in Hank's balanced salt solution (HBSS) , lacking calcium, magnesium, and phenol red, and purified by gradient centrifugation on cuεhions of Ficoll-Paque (Pharmacia Fine Chemicals, Piscataway, NJ) . The mononuclear cellε were collected into HBSS, waεhed twice, resuspended in RPMI 1640 Medium (Gibco, Grand Iεland, NY) containing 1% heat-inactivated autologous serum (56°C for 30 min.), and counted on the Coulter counter. For the preparation of monocyte monolayers, 1 ml of 2.2 x 10 6 mononuclear cells/ml was plated into wells of 24-well tissue culture plates (CoStar, Cambridge, MA) , incubated for 1 hour at 37 β C in a humidified atmosphere of 5% C0 2 , and washed three times with RPMI to remove nonadherent cells. A second 1 ml aliquot of 2.2 x 10* mononuclear cells/ml was layered into each well and incubated for 2 hours described above before removal of the nonadherent cellε. By viεual enumeration at 4OX with an inverted phaεe microscope and a calibrated reticle, the number of adherent cells for 30 different donors was 0.77 ± 0.20 X 10 6 per well (mean ± SD) . By morphology and nonspecific esteraεe staining, >95% of the adherent cells were monocytes. Monocyte monolayers were incubated at 37°C in the C0 2 chamber for 0 to 8 hourε with 0.5 ml of RPMI, 1% heat- inactivated autologouε serum, 10 mM HEPES, and 5 mM MgCl 2 in the absence and presence of variouε glucan prepara- tionε. The culture supernatant was removed, clarified by centrifugation at 14,000 g for 5 min at 4°C, and εtored at -70 β C before aεεay of TNFα. The concentration of TNFα in the monocyte super- natantε waε meaεured by an enzyme-linked immunoadsorbent asεay (ELISA) with the BIOKINE TNF Test kit (T Cell Sciences, Cambridge, MA) , which had a lower limit of detectability of 40 pg/ml. The data are expressed as pg per 10 6 monocytes, which waε calculated by dividing the quantity of cytokine in 0.5 ml of εupernatant by the number of monocytes per well. For the determination of cell-asεociated levels of TNFα, the adherent monocytes were lysed in 0.25 ml PBS by three rounds of freezing and thawing, the lysates were cleared of debris by centrifugation at 14,000 g for 5 min at 4°C, and the resulting supernatants were stored at -70 β C. Newly prepared monocyte monolayers contained no detectable levels of intracellular TNFα. The results are shown in Tables 3 and 4 below. Table 3 TNFα Syntheεiε by Human Monocyteε Stimulated with Variouε Glucan Preparationε TNFα (pg/10 6 monocytes) Glucan Cone. 1 2 3 Mean±SD Buffer Control 36 39 26±21 Neutral soluble lmg/ml 44 51 33 43±9 glucan Laminarin lmg/ml 372 324 227 308±74 Whole Glucan particles 4X10 7 /ml 2129 1478 1683 1763±333 Table 4 TNFα Stimulation by Different Conformational Structures of Soluble /3-Glucan Table 3 shows that TNFα was stimulated by inεoluble glucan particleε and by laminarin, a εoluble /3(l-6) and 0(1-3) linked glucan. There was no stimulation of TNFα by neutral soluble glucan. Table 4 εhowε εimilar results, but further confirms that TNFα stimulation iε dependent upon conformational structure. The neutral soluble glucan did not stimulate TNFα while Peak B (single helical conformation) did stimulate TNFα. EXAMPLE 5: AVIDITY OF NEUTRAL SOLUBLE GLUCAN FOR THE GLUCAN RECEPTOR Monolayers of human monocytes, prepared on siliconized glaεε coverεlips (Czop et al. , 1978, J. Immunol.. 120:1132) . were incubated for 18 minutes at 37°C in a humidified 5% C0 2 incubator with either 0.25 ml of buffer (RPMI-Mg-HEPES) or a range of concentrationε (0.1- 50 μg/ml) of neutral εoluble glucan. The monocyte monolayers were then washed twice with 50 ml of RPMI 1640 medium and were layered with 0.25 ml of 4.8 x 10 6 /ml zymosan particles (Czop and Austen, 1985, J. Immunol.. 134:2588-2593) . After a 30 minute incubation at 37 β C, the monolayers were washed three times with 50 ml of Hank's balanced salt solution to remove noningested zymosan particles. The monolayerε were then fixed and stained with Giemsa. The ingestion of zymosan particles by at least 300 monocytes per monolayer was determined by visual observation under a 1000X light microscope. Monocyte monolayers pretreated with buffer, 50 or 500 μg/ml of neutral soluble glucan as described above were subεequently tested for their capacity to ingest IgG coated sheep erythrocytes (E'IgG) . After an 18 minute preincubation with the neutral soluble glucan, the monolayers were incubated with 0.25 ml of 1 x 10 7 /ml E'IgG for 30 inuteε at 37°C, waεhed three times with 50 ml of Hank's balanced salt solution, treated for 4 minutes with 0.84% NH 4 C1 to lyse noningested E'IgG, and fixed and stained as deεcribed above. The percentageε of monocyteε ingeεting > 1 and > 3 E'IgG were determined by counting at least 300 monocytes per monolayer. The percent inhibition of monocyte ingeεtion waε determined by εubtracting the percentage of monocyteε ingeεting targetε after pretreatment with the neutral soluble glucan from the percentage ingesting targetε after pretreatment with buffer, dividing thiε number by the percentage ingeεting targets after pretreatment with buffer and multiplying by 100. The data are expressed as the mean of two experiments and are reported in Table 5. Table 5 Glucan-receptor Binding Capacity of Distinct Conformations of Soluble /3-glucans Test Material Cone. % ynhifrj-tjpn Buffer - 0% Neutral soluble glucan 50 μg/ml 74% 500 μg/ml 86% Peak B 50 μg/ml 50% 500 μg/ml 56% Both /3-glucan preparations tested above inhibited monocyte ingestion of zymoεan particles demonstrating their capacity to competitively bind to the /3-glucan receptor on human monocyteε. Neutral εoluble glucan demonεtrated a higher receptor binding capacity than Peak B aε indicated by the greater level of inhibition achieved at both 50 μg/ml and 500 μg/ml. Thiε biological aεεay demonεtrateε that the neutral εoluble glucan iε a superior ligand for the /S-glucan receptor. EXAMPLE 6: LACK OF IN VITRO STIMULATION OF IL-lβ AND TNFα FROM HUMAN MONONUCLEAR CELLS Venous blood waε obtained from healthy male volun- teerε and mononuclear cellε were fractionated by Ficoll-Hypaque centrifugation. The mononuclear cellε were waεhed, reεuεpended in endotoxin-free RPMI-1640 culture medium - ultrafiltered to remove endotoxinε aε deεcribed elsewhere (Dinarello et al * * 1987, J. Clin. Microbiol. 2 :1233-8) - at a concentration of 5 x 10 6 cells/ml and were aliquoted into 96-well microtiter plates fEndres et al.. 1989, N.E. J. Med. 320:265-271). The cellε were then incubated with either 1 ng/ml endotoxin (lipopolysaccharide, E. coli 055:B5, Sigma, St. Louis), or 10 to 1000 ng/ml /S-glucan, at 37°C for 24 hours in 5% C0 2 and then lysed by three freeze-thaw cycles fEndreε et al.. 1989, N.E. J. Med. 320:265-271). Syntheεis of IL-13 and TNFα was determined by specific radioimmunoassays aε deεcribed elεewhere (Lisi et al.. 1987, Lv ph Res. 6 :229-244; Lonnemann et al.. 1988, Lvmph. Res. 7:75-84; Van der Meer et al.. 1988, J. Leukocycte Biol. 43:216-223. To determine if neutral soluble glucan could act as a priming agent for cytokine syntheεiε with endotoxin, a known cytokine εtimulant, mononuclear cellε were pre-incubated with 1, 10, and 1000 ng/ml of the neutral εoluble glucan for 3 hourε at 37°C in 5% C0 2 . The cellε were waεhed to remove neutral εoluble glucan and were then incubated with 1 ng/ml endotoxin aε deεcribed above. IL-1/3 and TNFα were determined aε deεcribed above. The reεultε are summarized in Table 6. Neutral soluble glucan used as a stimulant at doses of 10-1000 ng/ml alone did not induce increased levels of IL-l/S or TNFα εyntheεiε over the control buffer treated cellε. Endotoxin LPS, a known εtimulant, reεulted in εignifi- cantly increaεed levelε of both cytokineε. In a εecond phaεe of thiε experiment neutral εoluble glucan was tested for itε ability to act aε a priming agent for mononuclear cell cytokine εyntheεiε. The cells from the same donorε were pre-incubated with three doses of neutral soluble glucan (10-1000 ng/ml) and were then exposed to endotoxin as a co-stimulant. Neutral soluble glucan did not reεult in any amplification of the IL-1/3 and TNFα levelε compared to endotoxin alone. Table 6 In Vitro IL-l/S and TNFα Syntheεiε by Human Peripheral Blood Mononuclear Cells LPS 1 ng/ml 2.62 2.22 'Values are the mean of two donors. EXAMPLE 7: IN VIVO PROTECTION AGAINST INFECTION IN MICE A sepsis model was developed in rats to characterize the efficacy of 0-glucan in protecting an immunologically intact hoεt againεt serious infections, such as thoεe which commonly occur following abdominal surgery. The rat model for intra-abdominal sepsiε has been well described in the scientific literature (Onderdonk et al. , 1974, Infect. Immun.. 10:1256-1259) . Groups of rats received neutral εoluble glucan (100 μg/0.2 ml) or saline control (0.2 ml) intramuscularly 24 hours and 4 hourε prior to infectious challenge. A defined polymicrobic infectious challenge (cecal inoculum) was placed into a gelatin capεule which waε then surgically implanted into the peritoneal cavity of anesthetized rats through an anterior midline inciεion. The early peritonitis from this experimentally induced infection waε aεεociated with the presence of gram-negative organisms within the blood and peritoneal cavity culminating in mortality. The cecal inoculum contained an array of facultative specieε, εuch E A . co i. aε well as other obligate anaerobes (Streptococcus sp., Bacteroides sp. , Clostridiu perfringens. Clostridium ramosum, Peptostreptococuε magnuε and productus. Proteus mira- biliε) . The animalε were obεerved four times per day for the first 48h and twice per day thereafter. The results are reported in Table 7. Table 7 Effect of Neutral Soluble Glucan on Mortality in a Rat Model for Intra-abdominal Sepsis Group Mortality(%) P vs. Saline Saline 12/20 (60) Neutral soluble glucan 2/10 (10) < 0.01 These resultε demonεtrate that neutral soluble glucan which does not induce IL-l/S and TNFα protects mice from lethal bacterial challenge. EXAMPLE 8: DEMONSTRATION OF SAFETY FOR HUMAN ADMINISTRATION A randomized, double-blind, placebo-controlled clinical trial waε conducted on healthy maleε to evaluate the safety of neutral soluble glucan (2.25 mg/kg) injected by intravenous infuεion compared to a placebo control. No adverse effects were observed. There was also no observed elevation in IL-1, TNF, IL- 6, IL-8 and GM-CSF. Single intravenous administration of neutral soluble glucan resulted in an increase in monocyteε and neutrophilε and in the killing activity of theεe cells proving that neutral soluble glucan retains the desirable immunological activities in humans. See Tableε 8, 9 and 10 below. However, aε εhown in Figureε 5 and 6 no changeε occurred in εeru IL-1 and TNF and none of the patients experienced fever or inflammatory reactions. The reεultε are consistent with the in vitro data reported in the earlier examples. Table 8 Change In Absolute Neutrophil Counts (x 1000/μl) After Neutral Soluble Glucan Administration B = Baseline meaεurement p < 0.01 with reεpect to baseline Table 9 Change in Monocyte Countε (X 1000/μl) After Soluble Neutral Glucan Adminiεtration 10 15 B = Baεeline measurement * p < 0.01 with respect to baseline Table 10 Ex Vivo Microbicidal Activity of Normal Volunteers Receiving Neutral Soluble Glucan Mean Change in % Killing 1 Doεe Level Hour 3 Hour 6 Hour 24 Day 2 Day 3 Day 6 Saline 0 2.5 mg/kg Mean 42.86 Neutral N 6 Soluble p-Value 0.062 Glucan Normalized with reεpect to the saline control EXAMPLE 9: DEMONSTRATION OF EFFICACY IN VIVO AS HUMAN ANTI-INVECTIVE In thiε clinical εtudy, the safety, tolerance, and potential efficacy of the neutral soluble /S-glucan was evaluated in patientε undergoing major thoracoabdominal surgery with high riεk of post-operative infection. Thirty-four males and femaleε who underwent surgery received 0.5 mg/kg of the neutral soluble /S-glucan preparation or saline placebo, given as an intravenouε infuεion of 50 to 200 ml over one hour. Patientε received multiple εequential doεeε of the neutral εoluble /S-glucan or placebo at 12 to 24 hours prior to surgery, l to 4 hours prior to surgery, 48 hourε poεt- surgery, and 96 hours post-surgery. Hospitalization, infections, and usage of anti- infective medications were examined as potential clinical efficacy parameters. Compared to patients given saline placebo infusions, patients who received the neutral soluble /3-glucan spent an average of five fewer days in the hospital (12.3 + 6.1 days versus 17.3 + 15.5 days) and three fewer dayε in the Intenεive Care Unit (0.1 + 0.4 verεuε 3.3 + 6.3 dayε; p<0.03, one-way analyεiε of variance) . The number of anti-infective medication preεcrip- tionε written per εtudy day following εurgery was consistently higher for control patients than for β- glucan recipient patientε. Control patientε were preεcribed an average of three times the number of anti-infective medications as β-glucan recipients over the time period from εurgery to diεcharge (p<0.005). During the Treatment and Poεt-Treatment Follow-up Phaεeε, a total of 22 culture-confirmed infections in 5 control patients and 8 infectionε in 5 /S-glucan recipient patientε were identified (p<0.002). Neutrophilε (PMNs) and monocytes/macrophages (MOs) were purified from blood εampleε obtained at Baεeline, Day 1, and Day 5 and examined for baεal and phorbol myriεate acetate εtimulated microbicidal activity againεt Staphylococcuε aureuε. Eεcherichia coli and Candida albicanε. The neutral soluble 0-glucan treatment generally increased the basal and phorbol- induced microbicidal activity of MOs and PMNε. EXAMPLE 10: WOUND HEALING EFFECTS OF NEUTRAL SOLUBLE GLUCANS Wound healing εtudieε were performed in a hairless mouse model having full thicknesε wounds with and without Staphylococcuε aureuε infection. Hairleεε SKH- 1 inbred mice (6-8 weekε of age) were anesthetized with ether and a midline 3 cm full thickness longitudinal incision was made with a number 10 scalpel blade, producing a full thicknesε wound that did not penetrate the underlying faεcia. Inciεionε were closed using steel clipε placed at 1 cm intervalε. Formulations of neutral εoluble glucan in phoε- phate buffered εaline were applied 30 minutes following wounding and reapplied at 24 hour intervals during the seven day post-operative period. Two micrograms of neutral εoluble glucan/mouεe per day was topically applied. Wounds were examined daily and rank-ordered for effectiveness of formulation for enhancement of visual based wound healing. Woundε were scored for closure on a scale of 0-5, with 5 indicating the most healing. In one group of mice infected, the wound waε treated with a culture of IO 7 Staphylococcus aureus 30 minutes after wounding and 2 hrs prior to treatment with the neutral εoluble glucan formulation. Hiεtological evaluation of the wound εite of each teεt group was made. The dermis of the control group (untreated wound) waε heavily infiltrated with both lymphocyteε and monocyteε/macrophageε. However, re- epithelialization that occurred at the epidermal layer waε incomplete. The tissue section showed that the dermal tissue was weak, in that the tissue integrity was not maintained when it was sectioned. The histology of the wounded tiεεue isolated from mice treated for three days with phosphate buffered saline containing the neutral soluble glucan showed that there was a heavy infiltration of macrophages and lymphocytes. Tiεεue integrity was good. When topically applied to a wound, a composition of neutral soluble glucan stimulated white blood cell entry and activity at the wound site and accelerated wound healing within the dermal layer of the wound. Furthermore, the compoεition effectively eliminated infection produced by bacterial infection (S^ aureus) and prevented the progresεion to sepsis. Untreated wounds progressed to sepεiε. EXAMPLE 11: STIMULATION OF PLATELET PROLIFERATION BY NEUTRAL SOLUBLE GLUCAN - The platelet proliferation stimulatory effect of the neutral soluble glucan waε teεted in an animal model εyεtem following either irradiation or adminiεtration of the chemotherapeutic agent ciεplatin. Theεe experimentε demonεtrated the unexpected platelet εtimulatory effect. More specifically, saline or neutral soluble glucan prepared as deεcribed in Example 1 was administered to groupε of 10 mice aε a εingle IV bolus 20 hours prior to radiation exposure. Mice were bilaterally exposed to a total-body irradiation of 7.5- Gy. Fourteen days after irradiation the mice were sacrificed and whole blood samples were analyzed for peripheral blood counts. As shown in Figure 7, the platelet cell count from neutral soluble glucan-treated mice was increased nearly 3-fold relative to saline- treated control levels. In addition to tests on irradiated mice, cisplatin-treated mice were also tested for the effect of the neutral soluble glucan on platelet hematopoiesiε. Balb/c mice were injected intravenouεly with ciεplatin at a doεe of 9.3 mg/kg through the tail vein one hour before injecting either saline or the neutral soluble glucan, prepared as described in Example 1, intramuscularly in a single dose of 0 (saline) or 2 mg/kg on Day 0. Platelet counts were determined before treatment (Day 0) and at 2, 4, 6, 8, and 10 dayε poεt-treatment. The reεultε of thiε experiment are shown in Figure 8. Each data point represents the mean and standard error of platelet countε from five mice. The εtatiεtically εignificant differences (p<0.05) between the εaline and neutral εoluble glucan (2 mg/kg) are noted. Biological Depoεit Saccharomyceε cereviεiae εtrain R4 Ad waε depoεited on Auguεt 20, 1992 with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland, under the termε of the Budapeεt Treaty. The εtrain haε been aεεigned ATCC acceεεion number 74181. Upon iεεuance of a patent, thiε depoεit will be irrevocable. Eouivalents Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalentε to the specific materials and components deεcribed herein. Such equivalents are intended to be encompasεed in the scope of the following claims:

Claims: CLAIMS 1. A neutral, aqueous soluble 3-glucan preparation which enhances host defense mechaniεmε to infection and does not induce an inflammatory response. 2. A neutral, aqueous εoluble 3-glucan preparation of Claim 1 in which the hoεt iε a human. 3. A neutral, aqueous soluble 0-glucan preparation of Claim 1 which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5 X IO 6 cells/ml, reεultε in a leεs than 2- fold increase in interleukin-1/3 and tumor necrosis factor-α syntheεiε over levelε obtained following an otherwiεe identical incubation with a buffered εolution lacking the 3-glucan component. 4. A neutral, aqueous soluble 3-glucan preparation of Claim 1 which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with an endotoxin-stimulated human peripheral blood mononuclear cell culture of about 5 X IO 6 cellε/ml, reεultε in a less than 2-fold increase in interleukin-13 and tumor necrosis factor-α syntheεiε over levelε obtained with endotoxin stimulation alone. 5. A 0-glucan preparation of Claim 4 in which the human peripheral blood mononuclear cells are stimulated with Eεcherichia coli lipopolyεaccharide endotoxin at a concentration of about 1 ng/ml. 6. A neutral, aqueous soluble /9-glucan preparation consiεting eεεentially of a molecular εpecies which migrates as a εingle peak when analyzed by gel permeation chromatography, the molecular εpecieε being characterized by a triple helical conformation. 7. A neutral, aqueous soluble 3-glucan of Claim 6 wherein the molecular εpecies binds specifically to the /S-glucan receptor of human monocytes. 8. A neutral, aqueous soluble 0-glucan having a triple helical conformation which when mixed at a concentration of 1 mg/ml with aniline blue forms a fluorescent complex in 25 mM NaOH and which loses about 50% of its 25 mM NaOH fluorescence in 150 mM NaOH. 9. A method for producing a neutral, aqueous soluble 3-glucan preparation, comprising: a) treating a suspension of insoluble 3-glucan with an organic acid under conditions sufficient to dissolve the organic acid- soluble portion of the 0-glucan; b) treating the organic acid-soluble 3-glucan with alkali under conditions sufficient to denature the native conformation of the εoluble 9-glucan; c) neutralizing the denatured εoluble 3-glucan under conditionε εufficient to re-anneal the εoluble 3-glucan; and d) purifying the re-annealed εoluble 0-glucan to obtain a neutral, aqueouε εoluble 9-glucan having a triple helical conformation which, when incubated for greater than 3 hourε at a concentration of about 1 μg/ml with an endotoxin εtimulated human peripheral blood mononuclear cell culture of about 5 X IO 6 cellε/ml, results in a less than 2-fold increase in interleukin-1 / S and tumor necrosis factor-α synthesis over levels obtained with endotoxin stimulation alone. 10. A method of Claim 9 wherein the insoluble 3-glucan is a whole glucan particle. 11. A method of Claim 9 wherein step a) is performed at a pH of from about l to about 5 and a temperature of from about 20 to about 100° C. 12. A method of Claim 9 wherein the organic acid is acetic acid or formic acid. 13. The method of Claim 9 wherein step (b) is performed at a pH of from about 7 to about 14 and a temperature of from about 40 to about 121° C. 14. The method of Claim 9 further comprising the step of purifying the denatured 3-glucan prior to step (c) to remove insoluble 3-glucans and aggregated soluble 3-glucans therefrom. 15. The method of Claim 9 wherein the purification step is performed using 1,000 to 100,000 dalton nominal molecular weight cut-off ultrafilters. 16. The method of Claim 9 wherein step (c) is performed at a pH of about 3.5 to 11.0 and at a temperature of from about 50 to 70° C. 17. The method of Claim 9 wherein the step (d) is performed using a 30,000 to 70,000 nominal molecular weight cut-off ultrafilter and a 100,000 to 500,000 nominal molecular weight cut-off ultrafilter. 18. A neutral, aqueous soluble /3-glucan produced by the method of Claim 9. 19. A method for preventing infection in a mammal that is at risk for infection, the method comprising parenterally administering to the mammal a neutral, aqueous εoluble 0-glucan which, when incubated for greater than 3 hourε at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5 X IO 6 cellε/ml, reεultε in a less than 2-fold increase in interleukin-1/3 and tumor necrosis factor-α synthesis over levels obtained following an otherwiεe identical incubation with a buffered solution lacking the 3-glucan component. 20. A method of Claim 19 wherein the mammal is at risk for infection as a result of an invaεive εurgical procedure. 21. A method for stimulating repair and healing of a wound site on a mammal comprising adminiεtering to the wound εite, an effective amount of a neutral, aqueouε εoluble 3-glucan which, when incubated for greater than 3 hourε at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5 X IO 6 cells/ml, results in a less than 2-fold increase in interleukin-1/3 and tumor necrosis factor-α synthesis over levels obtained following an otherwise identical incubation with a buffered solution lacking the β- glucan component. 22. A method of Claim 21 wherein the 3-glucan is topically administered to the wound site or is injected into the wound site. 23. A method for εtimulating platelet proliferation, compriεing adminiεtering to a mammal a compoεition compriεing a neutral, aqueouε εoluble /3-glucan in a phyεiologically acceptable vehicle, the neutral, aqueouε εoluble 3-glucan being prepared by: a) treating a εuspension of insoluble 3-glucan with an organic acid under conditionε sufficient to disεolve the organic acid- εoluble portion of the 3-glucan; b) treating the organic acid-soluble 3-glucan with alkali under conditions sufficient to denature the native conformation of the soluble 3-glucan; c) neutralizing the denatured soluble 3-glucan under conditions sufficient to re-anneal the εoluble /3-glucan; and d) purifying the re-annealed soluble 3-glucan to obtain a neutral, aqueous εoluble 8-glucan having a triple helical conformation which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with an endotoxin stimulated human peripheral blood mononuclear cell culture of about 5 X IO 6 cells/ml, reεultε in a leεs than 2-fold increase in interleukin-1/3 and tumor necrosis factor-α syntheεiε over levels obtained with endotoxin stimulation alone. 24. A pharmaceutical compoεition compriεing a neutral, aqueous soluble /3-glucan preparation which enhances hoεt defense mechanisms to infection and does not induce an inflammatory responεe, the neutral, aqueouε εoluble 0-glucan preparation being solubilized in a pharmaceutically acceptable carrier. 25. A pharmaceutical composition of Claim 24 which, when incubated for greater than 3 hours at a concentration of about l μg/ml with a human peripheral blood mononuclear cell culture of about 5 X IO 6 cells/ml, resultε in a leεε than 2-fold increaεe in interleukin-13 and tumor necrosis factor-α syntheεiε over levelε obtained following an otherwise identical incubation with a pharmaceutically acceptable carrier lacking the β- glucan component. 26. A method for treating infection in a mammal that is at risk for infection, the method comprising parenterally administering to the mammal a neutral, aqueous soluble 3-glucan which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5 X IO 6 cells/ml, results in a less than 2-fold increase in interleukin-13 and tumor necroεiε factor-α εyntheεis over levels obtained following an otherwise identical incubation with a buffered solution lacking the J-glucan component. 27. A method of Claim 26 wherein the mammal is at riεk for infection aε a reεult of an invaεive surgical procedure. 28. The method of Claim 16 wherein step (c) iε performed at a pH of about 6.0 to 8.0.

Inventors: EASSON D DAVIDSON JR, JAMAS SPIROS, OSTROFF GARY R, EASSON, D., DAVIDSON, JR., JAMAS, SPIROS, OSTROFF, GARY, R.
Applicants: ALPHA BETA TECHNOLOGY, EASSON D DAVIDSON JR, JAMAS SPIROS, OSTROFF GARY R, ALPHA-BETA TECHNOLOGY, INC.
Application year: 1993
Main classification: A61K31
IPC codes: A61K9, A61K31, A61K36, A61P7, A61P17, A61P31, A61P37, A61P43, C08B37