Conformations of PPG-glucan

The present invention relates to soluble .beta.-glucan compositions. In one embodiment, the invention provides a soluble .beta.-glucan composition which is substantially in a triple helix aggregate conformation under physiological conditions, such as physiological temperature and pH. Such a composition is generally characterized by an aggregate number greater than about 6. The invention further provides methods of preparing .beta.-glucan compositions of this type, as well as methods of characterizing the conformational state of .beta.-glucans.

BACKGROUND OF THE INVENTION 
Underivatized water-soluble .beta.(1-3)-glucan (also referred to as 
PGG-glucan or BETAFECTIN.RTM.) is a unique soluble .beta.-glucan which is 
prepared via a proprietary process. The biological activity of this 
polysaccharide differs from that of particulate or other soluble 
.beta.-glucans. Several laboratories have reported direct induction of 
arachidonic acid metabolites (Czop et al., J. Immunol. 141: 3170-3176 
(1988)), cytokines (Abel and Czop, Intl. J. Immunopharmacol. 14: 1363-1373 
(1992); Doita et al., J. Leuk. Biol. 14: 173-183 (1992)) and oxidative 
burst (Cain et al., Complement 4: 75-86 (1987); Gallin et al., Int. J. 
Immunopharmacol. 14: 173-183 (1992)) by both particulate and soluble forms 
of .beta.-glucans. In contrast, soluble .beta.(1-3)-glucan does not 
directly activate leukocyte functions such as oxidative burst activity 
(Mackin et al. FASEB J. 8:A216 (1994)), cytokine secretion (Putsiaka et 
al. Blood 82: 3695-3700 (1993)) or proliferation (Wakshull et al. J. Cell. 
Biochem. suppl. 18A: 22 (1994)). Instead, soluble .beta.(1-3)-glucan 
primes cells for activation by secondary stimuli (Mackin et al. (1994); 
Brunke-Reese and Mackin, FASEB J. 8: A488 (1994); and Wakshull et al. 
(1994)). 
The biological activity of .beta.-glucans is mediated through specific 
receptors on target cells. Several investigators have described receptors 
which bind particulate .beta.-glucan preparations. For example, receptors 
for particulate .beta.-glucan (e.g., zymosan-like particles) have been 
described by Czop and colleagues (Czop and Kay, J. Exp. Med. 173: 
1511-1520 (1991); Szabo et al., J. Biol. Chem. 270: 2145-2151 (1995)) and 
Goldman (Immunology 63 319-324 (1988); Exp. Cell Res. 174: 481-490 
(1988)). The leukocyte complement receptor 3 (CR3, also known as MAC 1 or 
CD11b/CD18) has been shown to have the capacity to bind particulate and 
some soluble .beta.-glucans, as well as other polysaccharides (Thornton et 
al., J. Immunol. 156: 1235-1246 (1996)). A soluble aminated .beta.-glucan 
preparation has been shown to bind to murine peritoneal macrophages 
(Konopski et al., Biochim Biophys. Acta 1221: 61-65 (1994)), and a 
phosphorylated .beta.-glucan derivative has been reported to bind to 
monocyte cell lines (Muller et al., J. Immunol. 156: 3418-3425 (1996)). 
The ability of salmon macrophages (Engstad and Robertson, Dev. Comp. 
Immunol. 18: 397-408 (1994)) and brain microglial cells (Muller et al., 
Res. Immunol. 145: 267-275 (1994)) to phagocytose .beta.-glucan particles, 
presumably through a receptor-mediated process, has also been described. 
Each of the foregoing studies utilized .beta.-glucan preparations varying 
widely in source, method of preparation, purity and degree of 
characterization. Because of this, little information is available 
regarding the relationship between .beta.-glucan structure/conformation 
and biological activity. There is, thus, a need for an improved 
understanding of .beta.-glucan structure/activity relationships to aid in 
the development of novel .beta.-glucan compositions with improved 
biological activity. 
SUMMARY OF THE INVENTION 
The present invention relates to soluble, non-gelling, .beta.-glucan 
compositions. In one embodiment, the invention provides a soluble 
.beta.-glucan composition which is substantially in a triple helix 
conformation under physiological conditions, such as physiological 
temperature and pH. Such a composition is characterized by an aggregate 
number greater than about 6 (i.e., an aggregate of more than two triple 
helices composed of six .beta.-glucan chains). 
The present invention also provides a method of producing a soluble 
.beta.-glucan composition having a greater aggregate number than a 
starting soluble .beta.-glucan composition, comprising separating a high 
molecular weight portion from the starting soluble .beta.-glucan, said 
high molecular weight portion having a greater aggregate number than the 
starting composition. In one embodiment, the high molecular weight portion 
is separated from the remainder of the starting composition by gel 
permeation chromatography. 
In another embodiment, the present invention also provides a method of 
preparing a soluble .beta.-glucan composition having an aggregate number 
lower than that of a starting soluble .beta.-glucan composition. The 
method comprises separating a low molecular weight portion from a starting 
soluble .beta.-glucan composition. The low molecular weight portion is 
enriched in the single triple helix and/or single helix conformation 
compared to the starting composition. 
In a further embodiment, the invention includes a method of forming a 
.beta.-glucan composition wherein at least a portion of the .beta.-glucan 
polymer chains adopt a triple helix aggregate conformation under 
physiological conditions. The method comprises the steps of (1) reacting a 
highly branched .beta.-glucan under conditions sufficient to remove a 
portion of the branches, thereby forming a partially debranched 
.beta.-glucan and (2) maintaining the partially debranched .beta.-glucan 
under conditions sufficient for formation of a triple helix aggregate 
conformation. 
In yet another embodiment, the invention provides a method of determining 
the aggregate number distribution across the entire molecular weight range 
of a polysaccharide composition. The method comprises the steps of (1) 
directing a first aliquot of a .beta.-glucan solution through a gel 
permeation chromatography column to obtain a .beta.-glucan fraction; (2) 
determining the molecular weight of the .beta.-glucan fraction; (3) 
directing a second aliquot of the .beta.-glucan solution through a gel 
permeation chromatography column to obtain a second .beta.-glucan fraction 
corresponding to the .beta.-glucan fraction of step (1); (4) contacting 
the second .beta.-glucan fraction with alkali to denature the second 
.beta.-glucan fraction into single polymer chains; (5) determining the 
molecular weight of the .beta.-glucan fraction as single polymer chains; 
and (6) dividing the molecular weight determined in step (2) by the 
molecular weight determined in step (5) to obtain the aggregate number of 
the .beta.-glucan fraction. This method enables the determination of the 
aggregate number distribution across the entire range of .beta.-glucans. 
The present invention offers several advantages. For example, the soluble 
.beta.-glucan compositions of the invention are enriched in one or more 
triple helix aggregate conformations compared to prior art .beta.-glucans. 
The invention also provides novel methods of preparing and characterizing 
such .beta.-glucans.

DETAILED DESCRIPTION OF THE INVENTION 
PGG-glucan is a polysaccharide composed of glucopyranose units linked in 
chains via .beta.(1,3)-glycosidic bonds, with branches intermittently 
linked to the main chain via .beta.(1,6)-glycosidic bonds. Single chains 
can be isolated, i.e., not substantially interacting with another chain. 
Three single helix chains can also combine to form a triple helix 
structure which is held together by interchain hydrogen bonding. Two or 
more .beta.-glucan triple helices can join together to form a triple helix 
aggregate. A .beta.-glucan polysaccharide can exist in at least four 
distinct conformations: single disordered chains, single helix, single 
triple helix and triple helix aggregates. Preparations of the 
.beta.-glucan can comprise one or more of these forms, depending upon such 
conditions as pH and temperature. 
The term "single triple helix", as used herein, refers to a .beta.-glucan 
conformation wherein three single chains are joined together to form a 
triple helix structure. In this conformation, there is no higher ordering 
of these triple helices, that is, there is no substantial aggregation of 
triple helices. 
The term "triple helix aggregate", as used herein, refers to a 
.beta.-glucan conformation in which two or more triple helices are joined 
together via non-covalent interactions. 
The "molecular weight" of a .beta.-glucan composition, as the term is used 
herein, is the mass average molar mass of the collection of polymer 
molecules within the composition. The characterization of a collection of 
polymer molecules in terms of polymer mass average molar mass is well 
known in the art of polymer science. 
The "aggregate number" of a .beta.-glucan conformation is the number of 
single chains which are joined together in that conformation. The 
aggregate number of a single helix is 1, the aggregate number of a single 
triple helix is 3, and the aggregate number of a triple helix aggregate is 
greater than 3. For example, a triple helix aggregate consisting of two 
triple helices joined together has an aggregate number of 6. 
The aggregate number of a .beta.-glucan sample under a specified set of 
conditions can be determined by determining the average molecular weight 
of the polymer under those conditions. The .beta.-glucan is then 
denatured, that is, subjected to conditions which separate any aggregates 
into their component single polymer chains. The average molecular weight 
of the denatured polymer is then determined. The ratio of the molecular 
weights of the aggregated and denatured forms of the polymer is the 
aggregate number. A typical .beta.-glucan composition includes molecules 
having a range of chain lengths, conformations and molecular weights. 
Thus, the measured aggregate number of a .beta.-glucan composition is the 
mass average aggregate number across the entire range of .beta.-glucan 
molecules within the composition. It is to be understood that any 
reference herein to the aggregate number of a .beta.-glucan composition 
refers to the mass average aggregate number of the composition under the 
specified conditions. The aggregate number of a composition indicates 
which conformation is predominant within the composition. For example, a 
measured aggregate number of about 6 or more is characteristic of a 
composition in which the .beta.-glucan is substantially in the triple 
helix aggregate conformation. 
The present invention is predicated upon the discovery that the 
conformation of a PGG-glucan preparation is temperature dependent. For 
example, as described in Example 1, an aqueous PGG-glucan solution 
prepared according to the method disclosed in U.S. Pat. No. 5,622,939, 
incorporated herein by reference, elutes from a gel permeation 
chromatography column (GPC, also referred to as size exclusion 
chromatography) at 25.degree. C. as a single symmetric peak. When the 
elution is conducted at 37.degree. C. however, two distinct peaks are 
observed, denoted Fraction A, which elutes first, and Fraction C, which 
elutes last. The elution profiles of the PGG-glucan preparation at 
25.degree. C. and 37.degree. C. are shown in FIG. 1. 
The molecular weights of fractions A and C were determined at 25.degree. C. 
at both pH 7 and pH 13, and at 37.degree. C. at pH 7. At pH 13, PGG-glucan 
is in an unaggregated or single chain conformation. Thus, at a given 
temperature the ratio of the molecular weights determined at pH 7 and pH 
13 is the aggregate number at pH 7 at that temperature. 
At pH 7 and 25.degree. C., Fraction A had a molecular weight of 238,000 and 
an aggregate number of 15.0. Upon increasing the temperature to 37.degree. 
C., the molecular weight of Fraction A decreased to 164,000 and the 
aggregate number decreased to 10.3. At 75.degree. C. the molecular weight 
of this fraction was 52,600 with an aggregate number of 3.3. The 
temperature dependence of molecular weight and aggregate number was more 
pronounced for Fraction C. At pH 7.0 and 25.degree. C., Fraction C had a 
molecular weight of 71,500 and an aggregate number of 6.0. At 37.degree. 
C., the molecular weight of Fraction C was 32,000 and the aggregate number 
was 2.7. At 75.degree. C., the molecular weight of this fraction was 
17,200 and the aggregate number was 1.4. 
The results of this study indicate that at 25.degree. C. and pH 7, both 
Fraction A and Fraction C exist predominantly in a triple helix aggregate 
conformation. When the temperature is increased to 37.degree. C., Fraction 
A remains predominantly in a triple helix aggregate conformation, while 
Fraction C is primarily in a single triple helix conformation. At 
75.degree. C., Fraction A remains predominantly in a single triple helix 
conformation, while Fraction C is primarily in a single chain random coil 
conformation. 
In another series of experiments, described in Example 3, the original 
PGG-glucan preparation described above was subjected to preparative scale 
GPC at 25.degree. C., resulting in a single broad elution band. Portions 
from the leading and trailing edges and the center of this band were 
collected to provide, in order of elution, Fractions 1, 2 and 3. An 
overlay of the analytical GPC elution profile of each of these fractions 
is shown in FIG. 7. The average molecular weight of each fraction was 
determined at both pH 7 and pH 13. The results showed that both molecular 
weight and aggregate number decreased with increasing elution time. The 
molecular weights determined at 25.degree. C. ranged from 244,100 for 
Fraction 1, 156,600 for Fraction 2, and 104,300 for Fraction 3. The 
aggregate numbers determined at 25.degree. C. were 11.3 for Fraction 1, 
8.6 for Fraction 2 and 7.7 for Fraction 3. 
The average molecular weight and aggregate number of each fraction were 
temperature dependent. For each fraction, both average molecular weight 
and aggregate number decreased upon warming from 25.degree. C. to 
37.degree. C. The molecular weights (aggregate numbers) determined at 
37.degree. C. were 164,100 (7.6) for Fraction 1, 109,100 (6.0) for 
Fraction 2, and 51,760 (3.8) for Fraction 3. 
These results indicate that in each fraction the PGG-glucan is 
predominantly in a triple helix aggregate conformation at 25.degree. C. At 
37.degree. C., however, Fractions 1 and 2 remain predominantly in a triple 
helix aggregate conformation, while Fraction 3, however, is primarily in a 
single triple helix conformation. 
The aggregation state of another .beta.-glucan, known as scleroglucan, was 
also examined. Scleroglucan is a .beta.-glucan polymer which is 
substantially more branched than PGG-glucan. Based upon the molecular 
weights of a scleroglucan sample at 25.degree. C. at pH 7 and pH 13 and at 
37.degree. C. and pH 7, the aggregate number of this sample was determined 
to be about 3 at both temperatures. Thus while PGG-glucan exists in a 
triple helix aggregate conformation at 25.degree. C. and pH 7, under these 
conditions scleroglucan exists primarily in a single triple helix 
conformation. 
The differences in the conformations of scleroglucan and PGG-glucan can be 
ascribed to structural differences between the two .beta.-glucans. As the 
primary structural difference is the extent of branching, this suggests 
that scleroglucan is too highly branched to form triple helix aggregates 
under these conditions. This indicates that a .beta.-glucan which forms 
triple helix aggregates at physiological temperature and pH can be formed 
by debranching a highly branched .beta.-glucan such as scleroglucan. 
The present invention provides a soluble .beta.-glucan composition which is 
substantially in a triple helix aggregate conformation under physiological 
conditions. A "soluble .beta.-glucan composition", as the term is used 
herein, is an underivatized .beta.-glucan composition which dissolves in 
an aqueous medium at room temperature (about 20-25.degree. C.) and neutral 
pH (from about pH 5.5 to about 7.5) to form a visually clear solution at a 
concentration up to about 100 mg/mL. An "aqueous medium", as the term is 
used herein, refers to water or a water-rich phase, particularly 
physiologically acceptable aqueous phases, including phosphate-buffered 
saline, saline and dextrose solutions. 
The term "physiological conditions", as used herein, refers to 
physiological pH, about pH 7, and physiological temperature, about 
37.degree. C. In a preferred embodiment, under physiological conditions 
the .beta.-glucan composition consists essentially of .beta.-glucan chains 
in one or more triple helix aggregate conformations. 
As used herein, a soluble .beta.-glucan composition is "substantially in a 
triple helix conformation" if greater that about 50% by weight of the 
composition is in a triple helix aggregate conformation under 
physiological conditions. Preferably, greater than about 60%, and more 
preferably, greater than about 70% by weight of the composition is in a 
triple helix aggregate conformation under physiological conditions. In one 
embodiment, the soluble .beta.-glucan composition of the invention is 
characterized by an aggregate number under physiological conditions of 
greater than about 6. Preferably, the aggregate number of the 
.beta.-glucan composition under physiological conditions is at least about 
7, and, more preferably, at least about 8. In the most preferred 
embodiment, the aggregate number of the .beta.-glucan composition under 
physiological conditions is at least about 9. 
The soluble .beta.-glucan composition can be prepared from insoluble glucan 
particles, preferably derived from yeasts, as described in U.S. Pat. No. 
5,622,939. A general procedure for the preparation of insoluble yeast 
glucans is provided by Manners et al., Biol. J. 135: 19-30 (1973). Glucan 
particles which are particularly useful as starting materials in the 
present invention are whole glucan particles as described by Jamas et al. 
in U.S. Pat. Nos. 4,810,646, 4,992,540, 5,082,936, 5,028,703 and 
5,622,939, the teachings of each of which are incorporated herein by 
reference in their entirety. The source of the whole glucan particles can 
be any fungal organism which contains .beta.-glucans in its cell walls. 
Particularly useful are whole glucan particles obtained from the strains 
Saccharomyces cerevisiae R4 (NRRL Y-15903) and R4 Ad (ATCC No. 74181). 
Other strains of yeast which are suitable sources of whole glucan 
particles include Saccharomyces delbruekii, Saccharomyces rosei, 
Saccharomyces microellipsodes, Saccharomyces carlsbergensis, 
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, 
Kluyveromyces polysporus, Candida albicans, Candida cloacae, Candida 
tropicalis, Candida utilis, Hansenula wingei, Hansenula arni, Hansenula 
henricii and Hansenula americana. 
In another embodiment, the present invention provides a method of preparing 
a soluble .beta.-glucan composition having an aggregate number greater 
than that of a starting soluble .beta.-glucan composition. The method 
comprises separating a high molecular weight portion from a starting 
soluble .beta.-glucan composition. The high molecular weight portion is 
enriched in the triple helix aggregate conformation compared to the 
starting composition. The starting composition can be, for example, a 
.beta.-glucan composition having an aggregate number less than about 6 
under specified conditions. In one embodiment, the high molecular weight 
fraction which is separated from the starting composition is substantially 
in a triple helix aggregate conformation under physiological conditions. 
The high molecular weight portion can be any portion of the starting 
composition, as long as it has a greater average molecular weight than 
that of the starting composition. In one embodiment, the isolated portion 
represents about 60% or less, by weight, of the starting composition. The 
fraction of the starting composition isolated will depend upon the 
dispersion of molecular weights within the starting composition and the 
aggregate number desired and can be readily determined by one of skill in 
the art. 
The high molecular weight portion can be separated from the starting 
composition using a variety of techniques. In a preferred embodiment, the 
high molecular weight portion is separated from the remainder of the 
starting composition using gel permeation chromatography (GPC). In this 
embodiment, the high molecular weight portion is separated from the 
starting composition by a method comprising the steps of (1) directing a 
.beta.-glucan composition through a gel permeation chromatography column, 
and (2) collecting a high molecular weight fraction or a high molecular 
weight portion of a fraction of the starting composition. 
In one embodiment, the starting .beta.-glucan composition is separated into 
two or more fractions by GPC. In this case, the faster eluting fraction is 
a high molecular weight portion of the starting composition and all or a 
part of this fraction can be collected. In another embodiment, the 
starting .beta.-glucan composition elutes as a single fraction or two or 
more overlapping fractions. In this case, the leading edge of the fraction 
or overlapping fractions can be collected. 
The "leading edge" of a fraction eluting from a chromatography column is 
the portion of the fraction which elutes first. For example, if the 
fraction elutes in a given volume of eluent, the first 10 to 50% by volume 
of the fraction can be collected. The amount of the .beta.-glucan fraction 
to be collected depends upon the nature of the original .beta.-glucan 
composition, for example, the distribution of molecular weights and 
conformations, and the chromatography conditions, such as the type of GPC 
column employed, the eluent and the flow rate. Optimization of these 
parameters is within the ordinary level of skill in the art. .beta.-Glucan 
molecules having higher aggregate numbers are expected to elute first. 
Therefore, if the portion collected has an aggregate number under 
physiological conditions which is lower than desired, the original 
.beta.-glucan composition can be fractionated again, and a smaller leading 
edge portion can be collected to obtain a .beta.-glucan composition having 
a larger aggregate number under physiological conditions. Preferably, the 
parameters are optimized using an analytical scale GPC column. 
A suitable .beta.-glucan composition having an aggregate number at 
physiological temperature of less than about 6 is a PGG-glucan composition 
previously described in U.S. Pat. No. 5,622,939. Preparative scale GPC can 
be performed to fractionate such a composition as described in Example 3. 
For example, if the .beta.-glucan composition elutes from the GPC column 
as a single band, the earlier-eluting, or leading edge, portion of the 
elution band can be collected to yield a PGG-glucan composition having an 
aggregate number greater than about 6. Such a .beta.-glucan composition 
will have an increased triple helix aggregate conformer population at 
physiological temperature and pH compared to the original preparation. 
The present invention also provides a method of preparing a soluble 
.beta.-glucan composition having an aggregate number lower than that of a 
starting soluble .beta.-glucan composition. The method comprises 
separating a low molecular weight portion from a starting soluble 
.beta.-glucan composition. The low molecular weight portion is enriched in 
a single triple helix and/or single helix conformation compared to the 
starting composition. In one embodiment, the low molecular weight portion 
which is separated from the starting composition is substantially in a 
single triple helix conformation under physiological conditions. The low 
molecular weight portion can be any portion of the starting composition, 
as long as it has a lower average molecular weight than that of the 
starting composition. In one embodiment, the isolated portion represents 
about 60% or less, by weight, of the starting composition. The fraction of 
the starting composition separated will depend upon the dispersion of 
molecular weights within the starting composition and the aggregate number 
desired and can be readily determined by one of skill in the art. 
The low molecular weight portion can be separated from the starting 
composition using a variety of techniques. In a preferred embodiment, the 
low molecular weight portion is separated from the remainder of the 
starting composition using gel permeation chromatography. In this 
embodiment, the high molecular weight portion is separated from the 
starting composition by a method comprising the steps of (1) directing a 
.beta.-glucan composition through a gel permeation chromatography column, 
and (2) collecting a low molecular weight fraction or a low molecular 
weight portion of a fraction of the starting composition. 
In one embodiment, the starting .beta.-glucan composition is separated into 
two or more fractions by GPC. In this case, the more slowly eluting 
fraction is a low molecular weight portion of the starting composition and 
all or a part of this fraction can be collected. In another embodiment, 
the starting .beta.-glucan composition elutes as a single fraction or two 
or more overlapping fractions. In this case, the trailing edge of the 
fraction or overlapping fractions can be collected. 
The "trailing edge" of a fraction eluted from a chromatography column is 
that portion of the fraction which elutes last. For example, if the 
fraction elutes in a given volume of eluent, the last 10 to 50% of the 
fraction can be collected. The amount of the .beta.-glucan fraction to be 
collected depends upon the nature of the original .beta.-glucan 
composition, for example, the distribution of molecular weights and 
conformations, and the chromatography conditions, such as the type of gel 
permeation chromatography column employed, the eluent and the flow rate. 
Optimization of these parameters is within the ordinary level of skill in 
the art. .beta.-Glucan molecules which adopt a single triple helix 
conformation under physiological conditions are expected to elute last. 
Therefore, if the portion collected has an aggregate number under 
physiological conditions which is greater than desired, the original 
.beta.-glucan composition can be fractionated again, and a smaller 
trailing edge portion can be collected to obtain a .beta.-glucan 
composition having a smaller aggregate number under physiological 
conditions. Preferably, the parameters are optimized using an analytical 
scale GPC column. 
The present invention also includes a method for determining the aggregate 
number distribution of a .beta.-glucan composition across the entire 
molecular weight range of the composition. The method comprises the steps 
of (1) directing a first aliquot of a .beta.-glucan solution through a gel 
permeation chromatography column to obtain a .beta.-glucan fraction; (2) 
determining the molecular weight of the .beta.-glucan fraction; (3) 
directing a second aliquot of the .beta.-glucan solution through a gel 
permeation chromatography column to obtain a .beta.-glucan fraction 
corresponding to the .beta.-glucan fraction of step (1); (4) denaturing 
the .beta.-glucan fraction into single .beta.-glucan chains; (5) 
determining the molecular weight of the fraction as single polymer chains; 
and (6) dividing the molecular weight determined in step (2) by the 
molecular weight determined in step (5) to obtain the aggregate number of 
the .beta.-glucan fraction. In this way, the aggregate number distribution 
of a .beta.-glucan composition across the entire molecular weight range of 
the composition is obtained. 
The gel permeation chromatography column can be a gravity column or a high 
performance liquid chromatography (HPLC) column. Optionally, the 
.beta.-glucan solution can be fractionated by passage through two or more 
gel permeation chromatography columns. 
The .beta.-glucan fraction can be denatured, for example, by treatment with 
aqueous base or dimethyl sulfoxide. The aqueous base can be any alkaline 
solution having a sufficiently high pH to denature the .beta.-glucan into 
single helices or disordered polymer chains. Suitable bases include 
aqueous KOH, NaOH and LiOH. For example, the aqueous solution can have a 
pH of about pH 12 or greater, preferably about pH 13 or greater. 
In one embodiment, the method of determining the aggregate number of a 
.beta.-glucan fraction described above is carried out using an apparatus 
comprising a gel permeation chromatography column, a post-column reaction 
system and a device for determining the molecular weight of the resulting 
fractions. The post-column reaction system enables the delivery of the 
denaturing reagent, such as an aqueous base, to the effluent from the GPC 
column, such that each fraction eluted from the GPC column can be 
denatured prior to determining the molecular weight of the fraction. 
The molecular weight of the .beta.-glucan fraction can be determined using 
a variety of methods. For example, suitable methods include light 
scattering techniques, such as multi-angle laser light scattering (MALLS), 
on-line viscometry, ultracentrifugation, osmometry, and other methods 
known in the art of polymer science. 
An example of an apparatus which is of use in the method of the invention 
is depicted schematically in FIG. 3. The apparatus includes a standard 
HPLC system comprising first solvent reservoir 1, pump 2, an injector 3, 
and one or more gel permeation chromatography columns 4. Second solvent 
reservoir 5 is linked via line 6 to second pump 7, which is in turn linked 
via tee 8 to effluent line 9. Effluent line 9 is connected to heating 
reactor 10 which is connected by line 11 to multi-angle laser light 
scattering detector (MALLS) 12. The MALLS is connected by line 13 to 
refractive index (RI) detector 14 which is connected to waste outlet 15. 
Reservoirs 1 and 5 can be, for example, Kontes 5 liter Ultra-Ware Conical 
Bottom reservoirs (Kontes). Suitable pump for use in this system (pumps 2 
and 7) include Hitachi L-6000 (Hitachi). Injector 3 can be, for example, 
an Hitachi AS-4000 injector. The system can include one or more gel 
permeation chromatography columns, for example, two Shodex KB-804 columns 
and one Shodex KB-803 columns. Tee 8 can be an Upchurch static mixing tee. 
A suitable heating reactor is an Eppendorf CH-460 heater with temperature 
controller. Multi-angle laser light scattering detector 12 can be, for 
example, a Wyatt Technology MINIDAWN.TM.. Suitable refractive index 
detectors include the Knauer Model 98 detector. 
In a further embodiment, the present invention provides a method of forming 
a .beta.-glucan composition comprising .beta.-glucan chains which are in a 
triple helix aggregate conformation. The method comprises the steps of (1) 
reacting a highly branched .beta.-glucan under conditions sufficient to 
remove at least a portion of the branches to form a debranched 
.beta.-glucan and (2) maintaining the debranched .beta.-glucan under 
conditions sufficient for formation of a triple helix aggregate form. 
The highly branched .beta.-glucan is a .beta.-glucan which is substantially 
more branched than PGG-glucan, for example, a .beta.-glucan which is too 
highly branched to form triple helix aggregates. For example, the highly 
branched .beta.-glucan can be at least about 25% branched. In a preferred 
embodiment, the branches are joined to the main chain via 
.beta.(1,6)-glycosidic bonds. Suitable examples of highly branched 
.beta.-glucans of this type include scleroglucan, which is about 30-33% 
branched, schizophyllan, lentinan, cinerean, grifolan and pestalotan. 
The highly branched .beta.-glucan can be debranched by cleaving a portion 
of the bonds joining the branches to the main polymer chain. For example, 
when the branches are joined to the main polymer chain by 
.beta.(1,6)-glycosidic bonds, the .beta.(1,6)-glycosidic bonds can be 
hydrolyzed under conditions which leave the main polymer chain 
substantially intact. For example, hydrolysis of the 
.beta.(1,6)-glycodsidic bonds can be catalyzed by an enzyme which 
preferentially cleaves .beta.(1,6)-glycosidic bonds over 
.beta.(1,3)-glycosidic bonds. Such enzymes of this type include hydrolases 
which are specific for or preferentially cleave .beta.(1,6)-glycosidic 
bonds, for example, endoglycosidases, such as .beta.(1,6)-glycosidases 
(Sasaki et al., Carbohydrate Res. 47: 99-104 (1976)). 
The highly branched .beta.-glucan can also be debranched using chemical 
methods. A preferred chemical debranching method is the Smith degradation 
(Whistler et al., Methods Carbohydrate Chem. 1: 47-50 (1962)). In this 
method the .beta.-glucan is treated for about 3 days in the dark with a 
limiting amount of NaIO.sub.4, based on the extent of debranching desired. 
The reaction is next quenched with ethylene glycol and dialyzed. The 
reaction mixture is then treated with excess NaBH.sub.4, then quenched 
with acetic acid and dialyzed. The reaction mixture is then heated for 
about 3 hours at 80.degree. C. with 0.2 M trifluoroacetic acid. The 
reaction mixture is then dialyzed and concentrated. 
The debranching reaction is performed under conditions suitable for forming 
a .beta.-glucan composition which is sufficiently debranched to permit 
triple helix aggregate formation. For example, in one embodiment, the 
extent of branching of the debranched .beta.-glucan is less than about 
10%. In a preferred embodiment, the debranched .beta.-glucan is branched 
to substantially the same extent as PGG-glucan (about 7%). 
The soluble .beta.-glucan compositions of the present invention have 
utility as safe, effective, therapeutic and/or prophylactic agents, either 
alone or as adjuvants, to enhance the immune response in humans and 
animals. Soluble .beta.-glucans produced by the present method preferably 
selectively activate only those components that are responsible 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 is generally 
defined as a person who exhibits an attenuated or reduced ability to mount 
a normal cellular or humoral defense to challenge by infectious agents, 
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 deleteriously affects 
the immune system. The method is useful to treat patients who have a 
disease or disorder which causes 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 post-operative 
complications because of a disease, disorder or treatment resulting in a 
reduced ability to mobilize the body's normal metabolic responses to 
infection. Treatment with the soluble glucans has been shown to be 
particularly effective in mobilizing the host's normal immune defenses, 
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 soluble glucan can be oral, enteral, parenteral, 
intravenous, subcutaneous, intraperitoneal, intramuscular, topical or 
intranasal. The form in which the composition will be administered (e.g., 
powder, tablet, capsule, solution, emulsion) will depend on 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 least 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 compositions of the present invention can be administered 
to an individual periodically as necessary 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 on 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 defenses which the body mobilizes against infections. 
Soluble .beta.-glucan compositions can be used for the prevention and 
treatment of infections caused by a broad spectrum of bacterial, fungal, 
viral and protozoan pathogens. The prophylactic administration of soluble 
.beta.-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 
classified as contaminated or potentially contaminated (e.g., 
gastrointestinal surgery, hysterectomy, cesarean section, transurethral 
prostatectomy) and in patients in whom infection at the operative site 
would present a serious risk (e.g., prosthetic arthroplasty, 
cardiovascular surgery), concurrent initial therapy with an appropriate 
antibacterial agent and the present soluble glucan preparation will reduce 
the incidence and severity of infectious complications. 
In patients who are immunosuppressed, not only by disease (e.g., cancer, 
AIDS) but by courses of chemotherapy and/or radiotherapy, the prophylactic 
administration of the soluble glucan will reduce the incidence of 
infections caused by a broad spectrum of opportunistic pathogens including 
many unusual bacteria, fungi and viruses. Therapy using soluble 
.beta.-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 soluble 
.beta.-glucan may be started empirically before catheterization, use of 
respirators, drainage tubes, intensive care units, prolonged 
hospitalizations, etc. to help prevent the infections that are commonly 
associated with these procedures. Concurrent therapy with antimicrobial 
agents and the soluble .beta.-glucan is indicated for the treatment of 
chronic, severe, refractory, complex and difficult to treat infections. 
The compositions administered in the method of the present invention can 
optionally include other components, in addition to the soluble 
.beta.-glucan. The other components that can be included in a particular 
composition are determined primarily by the manner in which the 
composition is to be administered. For example, a composition to be 
administered orally in tablet form can include, in addition to soluble 
.beta.-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 soluble 
.beta.-glucan and, optionally, an emulsifying agent, a flavoring agent 
and/or a coloring agent. A composition for parenteral administration can 
be mixed, dissolved or emulsified in water, sterile saline, PBS, dextrose 
or other biologically acceptable carrier. A composition 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. 
The soluble glucan composition of the invention 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 disease, among others, can be treated according to the methods 
of this invention to accelerate the healing process. Alternatively, the 
soluble .beta.-glucan can be injected into the wound or afflicted area. In 
addition to wound repair, the composition can be used to treat infection 
associated therewith or the causative agents that result in the wound. A 
composition 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. 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 use of the compositions of this invention is for the 
treatment of myelodysplastic 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 resulting in death typically accompany MDS. Thus, in 
order to reduce the severity of the disease 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 this invention, in order to specifically increase the infection 
fighting activity of the patient's white blood cells. Other bone marrow 
disorders, such as aplastic anemia (a condition of quantitatively reduced 
and defective hematopoiesis) can be treated to reduce infection and 
hemorrhage that are associated with this disease state. 
The soluble .beta.-glucan compositions of the present invention enhance the 
non-specific defenses of mammalian mononuclear cells and significantly 
increases their ability to respond to an infectious challenge. The unique 
property of soluble glucan macrophage activation is 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 soluble glucan may lie in the natural profile of responses it 
mediates in white blood cells. 
The soluble .beta.-glucan compositions of the invention are also of use in 
methods of inducing or enhancing mobilization of peripheral blood 
precursor cells, elevating circulating levels of peripheral blood 
precursor cells and enhancing or facilitating hematopoietic reconstitution 
or engraftment in mammals, including humans. Peripheral blood precursor 
cells include stem cells and early progenitor cells which, although more 
differentiated than stem cells, have a greater potential for proliferation 
than stem cells. These methods comprise administering to the mammal an 
effective amount of a .beta.-glucan composition of the present invention. 
Such methods are of use, for example, in the treatment of patients 
undergoing cytoreductive therapy, such as chemotherapy or radiation 
therapy. 
The invention is further illustrated by the following Examples. 
EXAMPLES 
Example 1 
Temperature Dependence of PGG-glucan Conformation 
Fractionation of PGG-glucan at 25.degree. C. and 37.degree. C. 
A PGG-glucan composition prepared as described in U.S. Pat. No. 5,622,939 
was concentrated to approximately 20 mg/mL. An aliquot of the concentrated 
sample was fractionated at 25.degree. C. on a preparative scale gel 
permeation chromatography column (5 cm TSK HW55F resin column) using 0.15 
M sodium chloride as the mobile phase. As shown in FIG. 1, the composition 
eluted as a single symmetrical band. 
A second aliquot of the concentrated sample was fractionated on a 
preparative GPC column maintained in a hot room at 37.degree. C. The 
PGG-glucan eluted from the column in two distinct fractions, as shown in 
FIG. 1. These fractions were collected and designated Fraction A and 
Fraction C. 
Characterization of Fractions A and C 
The molecular weights of each fraction at pH 7 and 25.degree. C., 
37.degree. C., and 75.degree. C., and at pH 13 and 25.degree. C. are 
presented in Table 1. For each fraction, molecular weight decreases with 
increasing temperature. The aggregate numbers of each fraction at pH 7 and 
25.degree. C., 37.degree. C., and 75.degree. C. are shown in Table 2. At 
25.degree. C., each fraction has an aggregate number greater than 6, 
indicating that each fraction is predominantly in a triple helix aggregate 
conformation at this temperature. At 37.degree. C., Fraction A remains 
predominantly in a triple helix aggregate conformation, while Fraction C 
is predominantly in a single triple helix conformation. At 5.degree. C., 
however, Fraction A remains predominantly in a single triple helix 
conformation, while Fraction C is predominantly in a single chain 
random-coil conformation. 
The aggregate number distribution of each fraction at pH 7 and 25.degree. 
C. was studied using the gel permeation chromatography/MALLS/post-column 
reaction system previously described and shown in FIG. 3. FIG. 4 shows the 
aggregate number distribution for Fractions A and C. Fraction A includes 
aggregate numbers from about 30 to about 9, while Fraction C has aggregate 
numbers from about 7 to 1. At 25.degree. C., Fraction A is predominantly 
in a triple helix aggregate conformation (aggregates of about 3 to 10 
triple helices), while Fraction 4 includes both triple helix aggregates 
and single triple helices. 
The temperature dependence of the PGG-Glucan conformation was also 
characterized by dynamic light scattering methods. The hydrodynamic size 
of each fractions at pH 7 and both 25.degree. C. and 40.degree. C. were 
measured using a Nicomp 370 submicron Particle Sizer with an argonion 
laser (NICOMP Particle Sizing Systems). Table 3 presents the hydrodynamic 
size of Fraction A and Fraction C at both 25.degree. C. and 40.degree. C. 
The hydrodynamic size of Fraction A does not change with increasing 
temperature from 25.degree. C. to 40.degree. C., while the hydrodynamic 
size of Fraction C is significantly reduced. 
The temperature dependence of the conformation of Fractions A and C was 
studied using a Nano Differential Scanning Calorimeter (Nano-DSC, 
Calorimetry Sciences Corp.). The calorimeter was run from 0.degree. C. to 
125.degree. C. at a scan rate of 1.degree. C./min. FIG. 5 displays two 
thermal transitions from about 30 to 70.degree. C. and 75 to 120.degree. 
C. for Fraction A and from about 30 to 40.degree. C. and 50 to 90.degree. 
C. for Fraction C. Based on the molecular weight and aggregate number data 
(see Tables 1 and 2), the first transition is a helix-helix transition 
caused by disaggregation of triple helix aggregates to single triple 
helices, while the second transition represents a helix-coil transition, 
such as denaturation of a single triple helix to a single chain 
random-coil conformation. Fraction A completely denatures at 120.degree. 
C., while Fraction C completely denatures at 90.degree. C. For 
shizophyllan, a triple helix to random-coil transition occurs at about 
130.degree. C. (Stokke et al., Biopolymers, 33: 193-198 (1993)). 
Polarimetry techniques have also been used to study the conformational 
transition of polysaccharides (Ogawa et al., Carbohydrate Research, 23: 
399-405 (1972)). Temperature dependence of Fraction A and Fraction C 
conformational changes were measured using an Auto-Poll V (Rudolph, Inc.) 
polarimeter. FIG. 6 presents specific optical rotation changes of 
Fractions A and C with increasing temperature. These results confirm that 
conformation of Fraction A is substantially triple helix aggregate or 
single triple helix within the temperature range from 25.degree. C. to 
90.degree. C. However, while Fraction C is substantially in a triple helix 
aggregate or single triple helix conformation from 25.degree. C. to 
70.degree. C., above 80.degree. C. the conformation is predominantly a 
denatured random coil. 
TABLE 1 
______________________________________ 
Average Molecular Weight Values 
of Fractions A and C at 25.degree. C. and 37.degree. C. 
Molecular Weight 
pH 13, pH 7, pH 7, pH 7, 
Fraction 25.degree. C. 25.degree. C. 37.degree. C. 75.degree. 
______________________________________ 
C. 
Fraction A 
16,000 238,000 164,00 
52,600 
Fraction C 12,000 71,500 32,000 17,200 
______________________________________ 
TABLE 2 
______________________________________ 
Aggregate Number of Fractions A and C at 25.degree. C. and 37.degree. C. 
Aggregate Number 
pH 7, pH 7, pH 7, 
Fraction 25.degree. C. 37.degree. C 75.degree. C. 
______________________________________ 
Fraction A 
15.0 10.3 3.3 
Fraction C 6.0 2.7 1.4 
______________________________________ 
TABLE 3 
______________________________________ 
Hydrodynamic size of Fractions A and 
C measured by dynamic light scattering 
20.degree. C. (mass 
40.degree. C. (mass 
Fraction distribution) distribution) 
______________________________________ 
Fraction A 12.5 nm (100%) 
11.4 nm (63%) 
3.5 nm (37%) 
Fraction C 10.2 nm (100%) 9.2 nm (23%) 
3.5 nm (73%) 
______________________________________ 
Example 2 
Fractionation of PGG-glucan and Characterization of Fractions 
A PGG-glucan composition prepared according to the method described in U.S. 
Pat. No. 5,622,939 was concentrated from 3.1 mg/mL to about 20 mg/mL. The 
concentrated sample was fractionated by preparative scale GPC on a 5 cm 
TSK HW55F resin column (Toso Hass) at 25.degree. C. Three samples were 
collected from the single symmetrical elution band. Samples from the 
leading edge, the trailing edge and the center of the band were collected 
to provide Fractions 1, 2 and 3 in order of elution. 
FIG. 7 is an overlay of the elution profiles of Fractions 1, 2 and 3. 
Characterization of Fractions 1, 2 and 3 
The conformations of Fractions 1, 2 and 3 were further characterized by a 
variety of techniques, including GPC and multi-angle laser light 
scattering (MALLS). 
The molecular weights of each fraction at pH 7 and both 25.degree. C. and 
37.degree. C. are presented in Table 4, along with the polydispersity of 
each value. For each fraction, molecular weight decreases with increasing 
temperature. Table 5 present the molecular weight for each fraction at 
25.degree. C. and pH 13. Table 6 presents the aggregate number of each 
fraction at pH 7 and both 25.degree. C. and 37.degree. C. At 25.degree. 
C., each of the fractions has an aggregate number greater than 6, 
indicating that each fraction is predominantly in a triple helix aggregate 
conformation at this temperature. At 37.degree. C., however, Fractions 1 
and 2 remain predominantly in a triple helix aggregate conformation while 
Fraction 3 is predominantly in a single triple helix conformation. 
TABLE 4 
______________________________________ 
Absolute Mw measured by HR-GPC/MALLS at pH 7 
25.degree. C. 37.degree. C. 
SAMPLES Mw Pd Mw Pd 
______________________________________ 
Fraction 1 244,100 1.22 164,100 
1.70 
Fraction 2 156,600 1.09 109,100 1.30 
Fraction 3 104,300 1.14 51,760 1.14 
______________________________________ 
TABLE 5 
______________________________________ 
Absolute Mw measured by GPC/MALLS at pH 13 and 25.degree. C. 
SAMPLES Mw Pd 
______________________________________ 
Fraction 1 21,500 1.1 
Fraction 2 18,280 1.1 
Fraction 3 13,620 1.1 
______________________________________ 
TABLE 6 
______________________________________ 
Aggregate Numbers at 25.degree. C. and 37.degree. C. 
Aggregate Number 
SAMPLES 25.degree. C. 
37.degree. C. 
______________________________________ 
Fraction 1 11.3 7.6 
Fraction 2 8.6 6.0 
Fracticn 3 7.7 3.8 
______________________________________ 
EQUIVALENTS 
Those skilled in the art will recognize or be able to ascertain, using no 
more than routine experimentation, many equivalents to the specific 
materials and components described herein. Such equivalents are intended 
to be encompassed in the scope of the following claims: