Synthetic immunoreglators, and methods of use and preparation

To regulate the immune response to an antigen, a subject with the antigen is injected with polyanionic polysaccharide derivative: (1) having a molecular weight of between 1,000 and 600,000; (2) selected to correspond to the antigen; (3) not being cytotoxic at an effective dosage; and (4) stimulating a cell-mediated immune response. For virus, as an example, mycodextran sulfate or pustulan sulfate may be used in sufficient quantities to stimulate cell-mediated immune responses without stimulating synthesis of gamma-E globulin and gamma-G globulins. The polyanionic polysaccharide derivative may be injected alone to stimulate cell-mediated immune responses to antigens in a diseased subject.

BACKGROUND OF THE INVENTION 
This invention relates to immunoregulators and their methods of use and 
preparation. 
It is known to use synthetic adjuvants or immunoregulators. However, the 
selection of such regulators has been experimental and not based on sound 
theoretical principles because several factors necessary for such 
selection were not understood, such as: (1) the exact mechanism for the 
immune response; (2) if T-cells or B-cells or both T-cells and B-cells 
were involved; (3) the manner in which regulators interact with the immune 
system for a specific response; and (4) the manner in which compounds or 
mixtures can be synthesized to enable them to elicit the desired response. 
A prior art synthetic adjuvant, dextran sulfate, has a polysaccharide 
molecule with anionic groups attached. The use of dextran sulfate as an 
adjuvant was disclosed in McCarthy, R. E., Arnold, L. W., and Babcock, G. 
F.: "Dextran Sulfate: An Adjuvant for Cell-Mediated Immune Responses, 
"Immunology, 32:964, 1977. The immune response was based upon trial and 
error and it was not known if it would stimulate a T-cell response without 
a B-cell response. It stimulated both T-cell response and B-cell response. 
Thus, prior art techniques and adjuvants have had several disadvantages, 
such as: (1) they are not predictable except by trial and error; (2) new 
adjuvants cannot be easily discovered or synthesized; (3) it is difficult 
to know the best manner and time of using them; (4) they are not usable to 
elicit only certain responses such as to select one that stimulates a 
T-cell response but does not stimulate antibody synthesis; and (5) many of 
them are cytotoxic in effective dosages. 
SUMMARY OF THE INVENTION 
Accordingly, it is an object of this invention to provide novel synthetic 
immunoregulators. 
It is a further object of the invention to provide a novel technique for 
obtaining, using and preparing synthetic immunoregulators. 
It is a still further object of the invention to provide a novel technique 
for the use of synthetic immunoregulators to study specific immune 
reactions. 
It is a still further object of the invention to provide a novel method for 
immunizing patients or animals. 
It is a still further object of the invention to provide a novel method for 
using synthetic immunoregulators to regulate immune responses. 
It is a still further object of the invention to provide a novel technique 
for enhancing cell-mediated immune responses without stimulating the 
synthesis of gamma-E globulin or gamma-G globulins. 
It is a still further object of the invention to provide a novel technique 
for enhancing cell-mediated immune responses with reduced probability of 
anaphylactic shock reactions. 
It is a still further object of the invention to provide a novel technique 
for specifically enhancing cell-mediated immune responses without 
simultaneously stimulating synthesis of immune globulins G and immune 
globulin E. 
It is a still further object of the invention to provide a novel technique 
for utilizing a regulator that selectively regulates only one of the 
actions of T-cells or B-cells in the immune response. 
It is a still further object of the invention to provide a novel technique 
for utilizing an adjuvant that is incorporated into a viral vaccine so as 
to minimize anaphylactic shock reactions. 
It is a still further object of the invention to provide a novel method of 
synthesizing immunoregulators by substituting chemical anionic groups onto 
a high-molecular-weight backbone of a compound that is not regulatory to 
selectively arrive at a regulator which causes a predetermined stimulating 
response. 
It is a still further object of the invention to provide a novel technique 
for selecting certain derivatives of polysaccharide compounds which affect 
the humoral and cellular immune responses in a predetermined manner. 
It is a still further object of the invention to provide a novel technique 
for controlling the humoral hemagglutinating antibody responses (IgM and 
IgG) of animals and the responses of the classes of immunoglobulin E and 
G.sub.1 which are associated with anaphylactic reactions. 
It is a still further object of the invention to provide a novel technique 
for synthesizing compounds to cause specific immunoregulatory effects. 
It is a still further object of the invention to provide novel 
high-molecular-weight sulfate compounds which have known immunoregulatory 
effects. 
In accordance with the above and further objects of the invention, the 
immune response of a subject to an antigen is regulated by an injection of 
a synthetic immunoregulator in sufficient amount to provide a selected 
type and degree of immune response in the subject. The synthetic 
immunoregulator includes a high-molecular-weight compound, which is not by 
itself regulatory, but following the addition of anionic groups on it, 
interacts with specific lymphocytes. The high-molecular-weight compound 
may be a polysacchaaride and the interacting groups may be anionic groups, 
such as sulfate. If they are sulfates, the polysaccharide should be 
sulfated to provide at least two sulfate groups. 
The synthetic immunoregulators are not cytotoxic at effective doses and 
have high specific binding values. The polysaccharide and ionic groups, 
because of their specific binding sites, may provide selective, specific 
regulatory effects on immune response without excessive toxicity. They may 
be selected to have a time of effectiveness long enough to permit 
intermittent doses, such as every two to four weeks. Because of the time 
span between doses, the synthetic immunoregulator is economical for 
treatment of chronic diseases by stimulating the appropriate immune 
response for a long term with reasonably spaced intervals. The intervals 
should be at least one week long. Moreover, rejection of foreign bodies 
such as prosthetic devices, skin grafts or the like, may be regulated to 
some extent. 
As can be understood from the above summary, the immunoregulators of this 
invention and its methods of use and preparation have several advantages, 
such as: (1) they can be used for T-cell-mediated responses only and thus 
avoid anaphylactic shock; (2) specific synthetic regulators are available 
to operate in a predictable manner; (3) it is possible to use a dosage and 
regulator for a known type of response; and (4) it is possible to 
synthesize regulators for use with particular antigens. 
SPECIFIC DESCRIPTION 
Broadly, a novel regulator is provided which stimulates a specific immune 
response without stimulating other unnecessary immune responses and thus 
reduces undesirable effects that might be caused by the administration of 
the immunizing dose of antigen. 
The regulator (referred to in the art at times as an adjuvant implying only 
enhanacement of immune response) is administered in conjunction with the 
administration of an antigen or by itself to stimulate the immune response 
to an antigen already in the subject such as cancer cells or intracellular 
infecting organisms such as M. liprae or HIV virus (AIDS). It may be 
administrated in any conventional manner such as by inoculation or orally 
but preferably by inoculation. 
It may be used to regulate response to foreign bodies such as prosthetic 
devices or skin grafts so as to reduce rejection problems. While the 
regulators are referred to as stimulating a response, it is believed that 
they may, under some circumstances, suppress an immune response. The 
regulators may also inhibit the suppressive effects on the cell mediated 
immune response exerted by certain agents such as some pathogens and tumor 
cells. 
The synthetic immunoregulators are not cytotoxic at effective doses and 
have specific binding properties. The polysaccharide and ionic groups, 
because of their specific binding sites, may provide selective, specific 
regulatory effects on immune response without excessive toxicity. They may 
be selected to have an effective time long enough to permit intermittent 
doses, such as every two weeks, so as to be economical for treatment of 
chronic diseases by stimulating the appropriate immune response for a long 
term with reasonably spaced intervals. For example, pustulan sulfate may 
be an effective treatment for chronic diseases caused by intracellular 
parasites such as M. liprae because it is not cytotoxic at effective 
doses, stimulates cell-mediated immune response without stimulating 
gamma-immunoglobulin or E immunoglobulin responses, and continues to be 
effective in the subject for two weeks. 
In one embodiment, regulators enhance cell-mediated immune responses 
without stimulating synthesis of gamma-E globulin and gamma-G globulin. 
Since the cell-mediated immune responses are the effective mechanisms 
against vital diseases and the gamma-G globulin and the gamma-E globulin 
classes of immunoglobulin cause anaphylactic shock reactions, the 
regulator increases the effectiveness of immune injections against viruses 
without creating shock effects. Moreover, certain regulators may be 
selected to enhance one cell-mediated immune response and not another such 
as shown by enhancing footpad swelling responses without affecting 
allograft rejection. 
Derivatives of the polysaccharide are used which show specific binding to 
the lymphatic system, as determined by the techniques described in the 
publication by Paul, Marangus and Skulmick, 1981 "The Benzodiazepene. 
GABA--Chloride Ionophase Receptor Complex: Common Site of Minor 
Tranquilizer Action," Biol-Psychi 16(3):213-229, the disclosure of which 
is incorporated herein by reference with appropriate modification as 
described in the examples of this case. Dosages are used which are not 
toxic but effective to enhance a selected immune response. 
To prepare the regulator, certain active groups and high-molecular-weight 
compounds which are not regulatory are selected and the groups attached to 
convert the high-molecular-weight compound from an immunologically inert 
compound to a compound that exerts regulatory effects on the immune 
response system by specific binding. For example, certain polyanionic 
groups on polysaccharides having molecular weights of between 1,000 and 
600,000 have been shown to stimulate T-cells selectively. Sulfation of 
such high-molecular-weight compounds has provided effective regulators. 
The resulting regulator has an effect that reflects both the 
high-molecular-weight backbone and the nature and the amount of the 
attached group and the selection is made to achieve a desired result 
taking all three of these factors into consideration. 
Polysaccharides that are either inert when tested in their unsulfated 
condition or which produce minimal immunological response changes are 
active immunoregulators following sulfation. The degree of sulfation can 
be modulated so that: (1) some compounds have saturation of all available 
sites that accept the sulfate; and (2) some compounds have partial 
sulfation. When modulated, complete sulfation results in the greatest 
immunoregulatory effect while moderate sulfation yields a lesser 
immunoregulatory effect. 
The polysaccharide sulfate derivatives stimulate cell-mediated 
T-cell-dependent immune responses without stimulating antibody-mediated 
immune responses that are B-cell dependent. Certain unmodified 
polysaccharides stimulate only B-cells and other materials are known which 
under certain conditions stimulate B-cell and T-cell responses to 
different degrees. Consequently, the proper approach may be selected and 
used according to the needs of the subject under certain circumstances. 
Cell-mediated immune stimulation increases as the number of sites for each 
unit of backbone molecule that is bound to an anionic group increases 
without an increase in B-cell stimulation. For purposes of this 
discussion: (1) T-cell response is a delayed sensitivity reaction of more 
than ten percent of a control usually 18 to 24 hours after challenge; and 
(2) B-cell response is a sensitivity reaction earlier than or four hours 
after challenge that is more than ten percent of a control. 
Two compounds which provide an adequately large molecular backbone for 
formulation into selective T-cell adjuvants are mycodextran and pustulan. 
Mycodextran consists of a polymer of D-glucosyl residues with alternating 
alpha (1-4) and alpha (1-3) linkages. A method of synthesizing mycodextran 
is disclosed by E. T. Reese and Mandels, Canadian Journal of Microbiology, 
v. 10, p. 103, 1964. Pustulan is a polysaccharide of 20,000 median 
molecular weight consisting of D-glucosyl residues with beta (1-6) 
linkages and 2.2 sulfate groups for each anhydrous glucose unit as made by 
the method described by Schweiger, R. G. 1972 "Polysaccharide sulfate. I. 
Cellulose Sulfate with a High Degree of Substitution. Carb. Res., 
21:219-228, the disclosure of which is incorporated herein. 
More specifically, a polysaccharide having a molecular weight of between 
1,000 and 600,000 is selected. The polysaccharide can be postulan or 
micodextran or substantially any other polysaccharide fitting this 
criteria. The selection is partly by trial and error as will be explained 
hereinafter. 
The polysaccharide is sulfonated or acylated by any conventional means but 
the degree of sulfonation or acylation is controlled. Samples with 
different degrees of sulfonation or acylation have differing 
immunoregulatory effects. Hypersensitivity testing may be done by standard 
delayed foot pad swelling reaction. 
In the alternative, instead of sulfonating the polysaccharide, it may be 
acylated. The degree of acylation or sulfonation is determined by 
preparing samples with different reaction times and testing them for 
immunoregulatory effect until the reaction time required for a suitable 
level of sulfonation or acylation is determined. Generally, the compound 
used is of the structural formula shown in formula 1, where R is a 
monosacchride and it equals a number of individual. monosacchrides of 
between 60 and 3,600. The unsubstituted carboxyl units are indicated by X 
and are equal in number to up to 58 or between 58 and 0 and the number of 
sulfate ions substituted as indicated by Y are between 2 and 3,600. 
##STR1## 
The invention is illustrated by the following examples:

EXAMPLES 
GENERAL 
In tests for selective enhancement of cell-mediated response without 
antibody-mediated response, examples 1-7, two different basic 
polysaccharide backbone molecules were selected for use which varied in 
the degree of rigidity of the molecule. Two different anionic groups for 
binding and four control compounds were selected. The polysaccharides are 
mycodextran and pustulan and the anionic groups were acetyl and sulfate. 
For each polysaccharide backbone either acetyl or sulfate groups were 
attached. The degree of sulfation was modulated in such a way that the 
compounds were tested with: (1) saturation of all available sites that 
accept the sulfate or (2) with partial sulfation. 
Two methods of sulfation of the polysaccharides were used. Both involved 
the DMF-S03 complex. In one method, the complex was prepared first and 
added to the polysaccharide suspended in DMF as described by R. G. 
Schweiger, Carb. Res. 21 (1972) 219-228. In the other method liquid S03 
was added slowly (Sulfan B) to a cooled suspension of the polysaccharide 
in DMF (N, N-Dimethylformamide). Product isolation is also different from 
that described by Schweiger in that a dialysis step was added and the 
product was obtained by lyophilization. A similar method was used for 
acylation. 
Three strains of mice were used for all studies except one done on one 
strain of rats as a control. The strains of mice are: (1) B6D2F1/J 
(Jackson Laboratories, Bar Harbor, Maine); (2) CFW (Charles River, 
Wilmington, Mass.); and (3) ESP (Eppley Cancer Institute, Omaha, Nebr.). 
The strain of rats is COBS COF. On day seven, blood was collected by 
bleeding from the retroorbital venous plexus. 
Candidate immunoregulators were evaluated by the footpad swelling assay. 
The antigen used was sheep red blood cells (SRBC) obtained from Colorado 
Serum Company in Denver, Colo. The SRBC were washed three times in Hank's 
balanced salt solution (HBBS) and resuspended to a concentration of ten 
percent of the volume of SRBC in HBBS v/v (volume-to-volume percentage). 
The immunoregulators were dissolved in HBBS and mixed with an equal volume 
of the SRBC to give a final concentration of five percent SRBC and the 
desired concentration of the immunoregulator. 
The control group of mice were sensitized subcutaneously (s.c.) with five 
percent SRBC and the appropriate concentration of the immunoregulator. All 
the solutions were made just prior to sensitization. In the event that the 
immunoregulators were lytic for SRBC, they were injected uncombined at the 
same site one after the other at the same time, the immunoregulator being 
administered first. 
Blood was collected by retroorbital venous puncture on days seven and 
fifteen post sensitization and the mice were challenged in the footpads 
with 0.05 ml of twenty percent SRBC (in HBSS). The right footpad received 
the SRBC suspension; the left foot20 pad received an equal amount of 
phosphate-buffered saline. The thickness of the footpads was measured at 
4, 24 and 48 hours after challenge. The difference between the left and 
right footpads of each mouse was expressed as a percentage. The average 
percent footpad swelling (APFS) for each group of mice was determined by 
dividing the sum of the individual swellings by the number of mice in the 
group. The average percentage increase (API) was determined by subtracting 
the APFS of the control group from the APFS of the treated group. 
The following steps were followed in this procedure: 
1. Regulatory Compounds 
The polysaccharide-compound regulators are: (1) mycodextran; (2) sulfated 
mycodextran; (3) pustulan; (4) sulfated pustulan; and (5) acylated 
pustulan. 
2. Toxicity Studies and Dose Determination 
Preliminary toxicity studies were performed to determine concentration of 
the regulators which can be tolerated by the mice. Only doses of adjuvants 
which do not exhibit systemic or local toxic effects were used in the 
assays. In examples 1-9 infra, four groups of five mice each were injected 
with a range of doses of candidate regulators and observed for seven days 
for gross toxic effects. Animals were sacrificed, autopsied and examined 
for localized toxic effects. 
More specifically, four groups of five mice each were injected 
subcutaneously with 25 mg, (milligrams) 50 mg, 100 mg, and 200 mg of 
pustulan sulfate per killogram of body weight and observed for seven days. 
Surviving animals were then sacrificed and autopsies performed. The dose 
used in following experiments was one at which no systemic or local toxic 
effects were noted. 
3. Immediate and Delayed Hypersensitivity Reactions 
Mice were immunized with sheep red blood cells (SRBC) with or without 
regulators. Mice were sensitized on day zero subcutaneously in the scruff 
of the neck. Groups of ten mice each were injected with two doses of 
regulator compound and 0.02 milliliter per gram body weight of five 
percent SRBC in a carrier, or in other words, five percent of the total 
volume of SRBC and carrier in volume of SRBC added (v/v). Control mice 
received only the SRBC. Mice of both sexes were used. 
The blood from mice of the same group was pooled and the serum used to 
determine hemagglutinin titers. On day eight, the mice were challenged in 
the right hind footpad with 0.05 milliliter of a twenty percent (v/v) 
suspension of SRBC in phosphate-buffered saline (PBS) and in the left hind 
footpad with 0.05 milliliter of PBS only. The thickness of the footpads 
was measured at 4, 24 and 48 hours with Vernier calipers after challenge. 
Bleeding and challenge were repeated on days fifteen and sixteen, 
respectively. 
The difference in thickness between the left and right footpads of each 
mouse was expressed as a percentage. The average percent footpad swelling 
(APFS) for each group of mice was determined by dividing the sum of the 
individual swellings by the number of mice in the group. Significance was 
determined using the Student's t-test at ninety-five percent confidence 
limits as described by G. W. Snedecor and W. G. Cochran, 1967, Statistical 
Methods, Iowa State University Press, Ames, Iowa. 
Positive footpad swellings occurring at four hours are immediate-type 
hypersensitivity reactions (antibody-mediated). Positive reactions 
occurring at 24 and 48 hours are delayed-type hypersensitivity reactions 
(cell-mediated). 
4. Hemagglutinin Titers 
Antibody responses of the IgM and IgG classes against SRBC in immunized 
mice were determined using the microtiter method. All tests were performed 
in V-bottom microtiter plates (Cooke Engineering Co., Alexandria, Va.). 
Two-fold dilutions of sera (obtained as described above) were made in 
bovine serum albumin saline (BSA) (100 milligrams in 100 milliliters PBS). 
The titer was considered to be the reciprocal of the highest dilution 
showing positive agglutination. Hemagglutinating antibody titers were 
determined using the procedure described by McCarthy et al 1977, "Dextram 
Sulfate: An Adjuvant for Cell Mediated Immune Responses, Immunology, 
32:9637, 1977. 
5. Study of IgG.sub.1 and IgE Responses 
Two types of experiments were conducted to study the responses of 
immunoglobulin associated with anaphylactic shock reactions. The first of 
these is active systemic anaphylaxis, which detects both IgG1 and IgE 
simultaneously and the second one is passive cutaneous anaphylaxis, which 
allows a determination of the presence or absence and relative 
concentrations of IgG1 and IgE in animals that respond. 
6. Active Systemic Anaphylaxis 
Groups of ten mice were immunized subcutaneously on day zero with fifty 
micrograms of ovalbumin with or without the candidate adjuvant. Two doses 
of adjuvant were used and a third group received antigen only. On day 
fourteen, the mice were challenged intravenously with one milligram of 
ovalbumin and observed for four hours. The number of mice dying within 
this time was recorded and the percent of anaphylactic shock determined as 
the number of deaths divided by the number challenged. This protocol and 
dosage of antigen has been shown to result in predominantly IgE responses 
in mice. There is, however, some IgGl produced. 
7. Passive Cutaneous Anaphylaxis (PCA) 
Groups of ten mice were immunized with ovalbumin and the candidate 
adjuvant. The mice were bled from the retroorbital venous plexus fourteen 
days following immunization. The sera were titrated for IgGl and IgE 
levels using PCA reactions at 2 and 48 hours, respectively, following 
intradermal injection of serum. Mice were challenged intravenously on day 
eighteen with one milligram of ovalbumin to assess systemic active 
anaphylaxis. 
EXAMPLE 1 
NON-ACYLATED PUSTULAN 
The dosage of pustulan was determined in toxicity studies as described 
under the heading "2. Toxicity Studies" above and the dosage was selected 
to be one hundred milligrams per kilogram of body weight of the mice. 
Three strains of mice were used, as shown in Table 1, five male and five 
female of each strain. 
The immediate- and delayed-type hypersensitivity reactions were determined 
as described under the heading "3. Immediate and Delayed Hypersensitivity 
Reactions" above. The results are shown in Table 1, in which a "0" (zero) 
indicates that the footpad swelling was less than ten percent over the 
control, a "+" (plus) sign means that there was significant footpad 
swelling greater than ten percent over the control and a "-" (minus) sign 
means there was a significant suppression of footpad swelling greater than 
ten percent over the control. 
The hemagglutinin titers were determined using the procedure described 
under the heading "4. Hemagglutinin Titers". The results are shown in 
Table 1. 
EXAMPLE 2 
ACYLATED PUSTULAN 
The dosage of acylated pustulan was determined in toxicity studies as 
described under the heading "2. Toxicity Studies" above and the dosage was 
selected to be one hundred milligrams per kilogram of body weight of the 
mice. Three strains of mice 
TABLE 1 
______________________________________ 
NON-ACYLATED PUSTULAN 
(used at 100 mg/kg body weight) 
First Challenge 
Second Challenge 
Strain Sex 4 hr 24 hr 48 hr 
4 hr 24 hr 
48 hr 
______________________________________ 
ESP M 0 + 0 0 0 + 
ESP F 0 + + + + + 
CFW M 0 + 0 0 0 0 
CFW F 0 0 0 0 + 0 
B6D2F1 M 0 + + + + 0 
B6D2F1 F Strain Not Available Presently 
______________________________________ 
Hemagglutinin Titers: Significant Increase 
were used, as shown in Table 2, five male and five female of each strain. 
The immediate and delayed hypersensitivity reactions were determined as 
described under the heading "3. Immediate and Delayed Hypersensitivity 
Reactions" above. The results are shown in Table 2, in which a "0" (zero) 
indicates that the footpad swelling was less than ten percent over the 
control, a "+" (plus sign) means that there was significant footpad 
swelling greater than ten percent over the control and a "-" (minus sign) 
means there was a significant suppression of footpad swelling greater than 
ten percent over the control. 
The hemagglutinin titers were determined using the procedure described 
under the heading "4. Hemagglutinin Titers". The results are shown in 
Table 2. 
EXAMPLE 3 
PUSTULAN SULFATE 
The dosage of pustulan sulfate was determined in toxicity studies as 
described under the heading "2. Toxicity Studies" above and the dosage was 
selected to be fifty milligrams per kilogram of body weight of 
TABLE 2 
______________________________________ 
ACYLATED PUSTULAN (used at 100 mg/kg body weight) 
First Challenge 
Second Challenge 
Strain Sex 4 hr 24 hr 48 hr 
4 hr 24 hr 
48 hr 
______________________________________ 
ESP M 0 + + 0 0 0 
ESP F 0 + + 0 + 0 
CFW M 0 + 0 0 + 0 
CFW F + + 0 + + 0 
B6D2F1 M 0 + + + + 0 
B6D2F1 F 0 0 + 0 + 0 
______________________________________ 
Hemagglutinin Titers: Not Significant 
the mice. Three strains of mice were used, as shown in Table 3, five male 
and five female of each strain. 
The immediate and delayed hypersensitivity reactions were determined as 
described under the heading "3. Immediate and Delayed Hypersensitivity 
Reactions" above. The results are shown in Table 3, in which a "0" (zero) 
indicates that the footpad swelling was less than ten percent over the 
control, a "+" (plus sign) means that there was significant footpad 
swelling greater than ten percent over the control and a "-" (minus sign) 
means there was a significant suppression of footpad swelling greater than 
ten percent over the control. 
The hemagglutinin titers were determined using the procedure described 
under the heading "4. Hemagglutinin Titers". The results are shown in 
Table 3. 
Groups of ten mice were immunized subcutaneously with fifty micrograms of 
ovalbumin for each kilogram of body weight, with each group being a 
different of the three species and five of each group being male and five 
female. A control group was given only the ovalbumin at a dose of forty 
micrograms for each kilogram of body weight and the experimental group was 
given pustulan sulfate at a dose of one hundred 
TABLE 3 
______________________________________ 
PUSTULAN SULFATE (used at 50 mg/kg body weight) 
First Challenge 
Second Challenge 
Strain Sex 4 hr 24 hr 48 hr 
4 hr 24 hr 
48 hr 
______________________________________ 
ESP M 0 + + + + + 
ESP F 0 + + 0 + + 
CFW M 0 0 + 0 + + 
CFW F + + + 0 + 0 
B6D2F1 M -- + 0 + + 0 
B6D2F1 F 0 + + 0 + 0 
______________________________________ 
Hemagglutinin Titers: Significant Increase 
micrograms per kilogram of body weight, following procedure 6 above under 
the heading "6. Active Systemic Anaphylaxis". The results are shown in 
Table 4. 
EXAMPLE 4 
MYCODEXTRAN 
The dosage of mycodextran was determined in toxicity studies as described 
under the heading "2. Toxicity Studies" above and the dosage was selected 
to be fifty milligrams per kilogram of body weight of the mice. Three 
strains of mice were used, as shown in Table 5, five male and five female 
of each strain. 
The immediate and delayed hypersensitivity reactions were determined as 
described under the heading "3. Immediate and Delayed Hypersensitivity 
Reactions" above. The results are shown in Table 5, in which a "0" (zero) 
indicates that the footpad swelling was less ten percent over the control, 
a "+" (plus sign) means that there was significant footpad swelling 
greater than ten percent over the control and a "-" (minus sign) means 
there was a significant suppression of footpad swelling greater than ten 
percent over the control. 
TABLE 4 
______________________________________ 
OVALBUMIN-INDUCED ANAPHYLACTIC SHOCK 
Effect of Pustulan Sulfate 
on Anaphylactic Shock in Mice 
Percent Survivors 
ESP CFW B6D2F1 
Male Female Male Female Male Female 
______________________________________ 
Control Group Given Ovalbumin Only 
90 70 0 80 100 100 
(9/10) 
(7/10) (0/10) (8/10) (10/10) 
(10/10) 
Experimental Group Given Ovalbumin 
Plus Pustulan Sulfate 
10 40 30 20 100 100 
(1/10) 
(4/10) (3/10) (2/10) (10/10) 
(10/10) 
______________________________________ 
TABLE 5 
______________________________________ 
MYCODEXTRAN 
First Challenge 
Second Challenge 
Strain Sex 4 hr 24 hr 48 hr 
4 hr 24 hr 
48 hr 
______________________________________ 
ESP M 0 + 0 0 0 0 
ESP F 0 + 0 0 0 0 
CFW M + 0 0 0 0 0 
CFW F 0 0 + 0 + + 
B6D2F1 M + 0 + + + + 
B6D2F1 F -- + 0 0 0 + 
______________________________________ 
Hemagglutinin Titers: Not Significant 
The hemagglutinin titers in Table 5 were determined using the procedure 
described under the heading "4. Hemagglutinin Titers." 
EXAMPLE 5 
MYCODEXTRAN SULFATE 
The dosage of mycodextran sulfate was determined in toxicity studies as 
described under the heading "2. Toxicity Studies" above and the dosage was 
selected to be fifty milligrams per kilogram of body weight of mice. Three 
strains of mice were used, as shown in Table 6, five male and five female 
of each strain. 
The immediate and delayed hypersensitivity reactions were determined as 
described under the heading "3. Immediate and Delayed Hypersensitivity 
Reactions" above. The results are shown in Table 6, in which a "0" (zero) 
indicates that the footpad swelling was less than ten percent over the 
control, a "+" (plus sign) means that there was significant footpad 
swelling greater than ten percent over the control and a "-" (minus sign) 
means there was a significant suppression of footpad swelling greater than 
ten percent over the control. 
TABLE 6 
______________________________________ 
MYCODEXTRAN SULFATE 
First Challenge 
Second Challenge 
Strain Sex 4 hr 24 hr 48 hr 
4 hr 24 hr 
48 hr 
______________________________________ 
ESP M 0 + + 0 + 0 
ESP F 0 + + 0 + 0 
CFW M 0 + 0 + + + 
CFW F 0 + + + + 0 
B6D2F1 M 0 + + 0 + 0 
B6D2F1 F + + + 0 + 0 
______________________________________ 
Hemagglutinin Titers: Not Significant 
The hemagglutinin titers were determined using the procedure described 
under the heading "4. Hemagglutinin Titers". The results are shown in 
Table 6. 
Groups of ten mice were immunized subcutaneously with fifty micrograms of 
ovalbumin. Each group of ten mice is a different strain of the three 
above-mentioned strains of mice, with five of the mice in each group being 
male and five female. A control group was given only the ovalbumin at a 
dose of forty milligrams per kilogram of body weight and the experimental 
group was given mycodextran sulfate at a dose of fifty milligrams per 
kilogram of body weight, following procedure 6 above under the heading "6. 
Active Systemic Anaphylaxis." The results are shown in Table 7. Similar 
groups of ten mice were immunized with ovalbumin and an adjuvant and bled 
from the retroorbital venous plexus, following procedure 7 under the 
heading "7. Passive Cutaneous Anaphylaxis (PCA)" above, to determine 
systemic active anaphylaxis. The results are shown in Table 7. 
EXAMPLE 6 
MYCODEXTRAN SULFATE IN RATS 
The dosage of mycodextran sulfate was determined in toxicity studies as 
described under the heading 
TABLE 7 
______________________________________ 
OVALBUMIN-INDUCED ANAPHYLACTIC SHOCK 
Effect of Mycodextran Sulfate 
on Anaphylaxis in Mice 
Percent Survivors 
ESP CFW B6D2F1 
Male Female Male Female Male Female 
______________________________________ 
Control Group Given Ovalbumin Only 
70 100 100 100 100 100 
(7/10) 
(10/10) (10/10) (10/10) (10/10) 
(10/10) 
Experimental Group Given Ovalbumin Plus Mycodextran 
60 70 0 0 100 100 
(6/10) 
(7/30) (0/10) (0/10) (10/10) 
(10/10) 
______________________________________ 
"2. Toxicity Studies" above and the dosage was selected to be fifty 
milligrams per kilogram of body weight of the rats. The strain of rats 
used is as shown in Table 8, five male and five female rats were used. 
The immediate and delayed hypersensitivity reactions were determined as 
described under the heading "3. Immediate and Delayed Hypersensitivity 
Reactions" above. The results are shown in Table 8, in which a "0" (zero) 
indicates that the footpad swelling was less than ten percent over the 
control, a "+" (plus sign) means that there was significant footpad 
swelling greater than ten percent over the control and a "-" (minus sign) 
means there was a significant suppression of footpad swelling greater than 
ten percent over the control. 
The hemagglutinin titers were determined using the procedure described 
under the heading "4. Hemagglutinin Titers." The results are shown in 
Table 8. 
EXAMPLE 7 
PUSTULAN SULFATE 
The dosage of pustulan sulfate was determined in toxicity studies as 
described under the heading "2. 
TABLE 8 
______________________________________ 
MYCODEXTRAN EXPERIMENTS IN RATS 
MYCODEXTRAN SULFATE 
First Challenge 
Strain Sex 4 hr 24 hr 
48 hr 
______________________________________ 
COBS CDF M 0 + 0 
COBS CDF F 0 + 0 
______________________________________ 
Hemagglutinin Titers: Significant Increase in Male and Female Rats 
Toxicity Studies" above and the dosage was selected to be fifty milligrams 
per kilogram of body weight of the mice. Three strains of mice were used, 
as shown in Tables 9-14, five male and five female of each strain. 
The immediate and delayed hypersensitivity reactions were determined as 
described under the heading "3. Immediate and DHR (Delayed 
Hypersensitivity Reactions) above. The results are shown in Tables 9-14, 
in which a "0" (zero) indicates that the footpad swelling was less than 
ten percent over the control, a "+" (plus sign) means that there was 
significant footpad swelling greater than ten percent over the control and 
a "-" (minus sign) means there was a significant suppression of footpad 
swelling greater than ten percent over the control. 
The hemagglutinin titers were determined using the procedure described 
under the heading "4. Hemagglutinin Titers". The results are shown in 
Table 15. 
EXAMPLE 8 
In tests for selective enhancement of footpad swelling cell-mediated 
response without allograft 
TABLE 9 
______________________________________ 
Hypersensitivity 
Immediate Type 
Adjuvant No Suppress 
Strain Sex Effect Effect 
Effect 
______________________________________ 
ESP M x 
ESP F x 
CFW M x 
CFW F x 
slight 
B6D2F1/J M x 
slight 
B6D2F1/J F x 
______________________________________ 
TABLE 10 
______________________________________ 
Hypersensitivity 
Immediate Type 
Adjuvant No Suppress 
Strain Sex Effect Effect 
Effect 
______________________________________ 
ESP M x 
ESP F x 
CFW M x 
CFW F x 
B6D2F1/J M x x 
B6D2F1/J F x 
______________________________________ 
TABLE 11 
______________________________________ 
Hypersensitivity 
24-Hr Delayed Type 
Adjuvant No Suppress 
Strain Sex Effect Effect 
Effect 
______________________________________ 
ESP M x 
ESP F x 
CFW M x 
CFW F x 
B6D2F1/J M x 
B6D2F1/J F x 
______________________________________ 
TABLE 12 
______________________________________ 
Hypersensitivity 
24-Hr Delayed Type 
Adjuvant No Suppress 
Strain Sex Effect Effect 
Effect 
______________________________________ 
ESP M x 
ESP F x 
CFW M x 
CFW F x 
B6D2F1/J M 
B6D2F1/J F x 
______________________________________ 
TABLE 13 
______________________________________ 
Hypersensitivity 
48-Hr Delayed Type 
Adjuvant No Suppress 
Strain Sex Effect Effect 
Effect 
______________________________________ 
ESP M x 
ESP F x 
CFW M x 
CFW F x 
B6D2F1/J M x 
B6D2F1/J F x 
______________________________________ 
TABLE 14 
______________________________________ 
Hypersensitivity 
48-Hr Delayed Type 
Adjuvant No Suppress 
Strain Sex Effect Effect 
Effect 
______________________________________ 
ESP M x 
ESP F x 
CFW M x 
CFW F x 
B6D2F1/J M x 
B6D2FI/J F x 
______________________________________ 
TABLE 15 
______________________________________ 
PUSTULAN SULFATE IN MICE 
Hemagglutinin Titers 
Pre- Post- 
Strain Dosage Challenge Challenge 
______________________________________ 
ESP 50 mg/kg 2 512 
ESP Control 0 256 
ESP 50 mg/kg 0 512 
ESP Control 2 256 
CFW 50 mg/kg 0 256 
CFW Control 2 32 
CFW 50 mg/kg 4 256 
CFW Control 4 256 
B6D2F1/J 50 mg/kg 2 512 
B6D2F1/J Control 2 128 
B6D2F1/J 50 mg/kg 2 512 
B6D2F1/J Control 2 128 
______________________________________ 
rejection cell-mediated response, four doses of the pustulan sulfate were 
studied: 25 mg/kg, 50 mg/kg, 100 mg/kg and 200 mg/kg of body weight using 
the procedures described with respect to Examples 1-8. Toxicity for the 
three strains of mice tested are shown in Table 16. 
In these tests, groups of five mice were injected subcutaneously in the 
scruff of the neck with the indicated dose, observed for seven days and 
sacrificed for autopsy. At the doses of 100 mg/kg and 200 mg/kg pustulan 
sulfate was shown to be toxic. Only five of the thirty mice tested with 
200 mg/kg survived through day seven. Four of the thirty mice tested with 
a 100 mg/kg dose died before day seven. Two of the 26 surviving animals of 
this group were found to have subcutaneous scarring at the injection site. 
At the lower dose of 25 mg/kg, no deaths occurred and no toxic effects were 
found. At 50 mg/kg, no deaths occurred and only two of the animals tested 
showed subcutaneous scarring. The 50 mg/kg dose was chosen for use in in 
vivo studies. 
Tests for footpad swelling of mice due to SRBC alone and SRBC with pustulan 
sulfate were made with mice sensitized with SRBC and experimental mice 
TABLE 16 
______________________________________ 
Pustulan Sulfate Toxicity Study 
dose at day 7 
mg/kg animals with subcutaneous 
strain 
sex body weight 
deaths 
scars per # of survivors 
______________________________________ 
CFW m 25 0 0/5 
CFW f 25 0 0/5 
CFW m 50 0 0/5 
CFW f 50 0 0/5 
CFW m 100 0 0/5 
CFW f 100 0 0/5 
CFW m 200 4 0/1 
CFW f 200 5 0/0 
ESP m 25 0 0/5 
ESP f 25 0 0/5 
ESP m 50 0 2/5 
ESP f 50 0 0/5 
ESP m 100 1 4/5 
ESP f 100 0 2/5 
ESP m 200 4 0/1 
ESP f 200 3 0/2 
BDF m 25 0 0/5 
BDF f 25 0 0/5 
BDF m 50 0 0/5 
BDF f 50 0 0/5 
BDF m 100 1 0/4 
BDF f 100 2 0/3 
BDF m 200 5 0/0 
BDF f 200 5 0/0 
______________________________________ 
injected with SRBC and pustulan sulfate. P values less than 0.05 were 
considered significant. Values were calculated using Student's t-test for 
the difference between two means. 
All strains of mice tested showed an increased DTH reaction to SRBC when 
pustulan sulfate was administered at the time of sensitization as shown in 
Table 17. All strains showed a significant increase in footpad swelling 
over controls at 24 hours after the first challenge with one exception 
(male CFW mice did not show a statistically significant increase due to 
wide variance between individuals of the group, although pronounced 
swelling was observed). Increases in 48 hour swellings occurred, but 
results varied from strain to strain and from male to female of the same 
strain. Increased DTH responses were also noted after the second challenge 
in all but ESP male m ice. 
Survival times of allogenic skin grafts were not altered from controls as 
shown in Table 18. This result was consistent in the three dosage and 
treatment schedules tested. The free skin grafting technique described in 
the publication of Billingham & Medawar (1951) "The Technique of Free Skin 
Graftings in Mammals" J. Exp. Biol., 28:385-402, the 
TABLE 17 
__________________________________________________________________________ 
Effect of Pustulan Sulfate on Footpad Swelling Responses 
Group Mean % Change 
% Increase over Controls 
P Value 
Strain 
Sex 
Group 
24 hours 
48 hours 
24 hours 
48 hours 
24 hours 
48 hours 
__________________________________________________________________________ 
BDF M con 24.5 10.1 -- -- -- -- 
exp 41.7 16.2 17.2 6.1 0.049 
0.08 
BDF F con 22.7 9.1 -- -- -- -- 
exp 55.9 22.8 33.2 13.7 0.001 
0.005 
ESP M con 13.1 8.2 -- -- -- -- 
exp 44.2 24.1 31.1 15.9 0.001 
0.001 
ESP F con 34.4 17.5 -- -- -- -- 
exp 51.1 29.7 16.7 12.2 0.006 
0.01 
CFW M con 18.4 13.1 -- -- -- -- 
exp 34.4 21.1 6.0 8.0 0.44 0.09 
CFW F con 26.9 9.6 -- -- -- -- 
exp 45.8 23.1 18.9 13.5 0.002 
0.004 
__________________________________________________________________________ 
TABLE 18 
__________________________________________________________________________ 
Effect of Pustulan Sulfate on Allogeneic Skil Graft Rejection 
Dose, mg/kg 
Animals per Mean Days to 
Group # 
Body Weight 
Group Treatment 
Rejection 
S.E. 
__________________________________________________________________________ 
1 -- 11 control, graft 
13.5 0.92 
only 
2 50 10 1 dose with 
14 0.44 
graft 
3 5 11 daily i.p., 5 days 
12.75 0.53 
prior to grafting 
through 
rejection 
4 5 10 daily i.p., day of 
13.29 0.52 
grafting 
through 
rejection 
__________________________________________________________________________ 
disclosure of which is incorporated herein, was used in all experiments. 
Donor skin was taken from C3H/HeJ male mice and grafted to CFW male mice. 
Criteria for rejection have been described in the publication of Babcock, 
et al., (1977) "Suppression of Cell-Mediated Immune responses by Dextran 
Sulphate", Immunology, 33:925-929, the disclosure of which is incorporated 
herein. 
EXAMPLE 9 
LEUKEMIA P388 
GENERAL 
In tests to determine increase in mouse survival time in mice innoculated 
with mouse leukemia P388 with the use of mycodextran sulfate, five doses 
of mycodextran sulfate were studied, which are: 100 mg/kg (milligram for 
each kilogram of body weight using the procedures described with respect 
to examples 1-8); 50 mg/kg; 25 mg/kg; 12.5 mg/kg; and 6.25 mg/kg. These 
doses were applied to each of eight B6d2f1/j male mice, 12 weeks old, and 
having weights within three grams of each other. In the control group, 
thirty mice were used. 
The mice were injected interparietally with tumor cells conventionally 
designated as P-388 having 106 cells for each 0.1 milliliter HBSS injected 
at the start of the test. For a positive control, the same tumor cells 
were injected into eight of the same strain of mice, for a negative 
control it was injected into 30 mice of the same strain and it was 
injected into eight mice who were also injected with a saline solution. 
The materials were prepared in sterile saline and injected interparietally 
within 15 minutes in a solution of 0.02 milliliters per gram of mouse body 
weight for nine days. 
The results are shown in Table 19. The experiment was repeated without 
applying a dosage of 100 mg/kg because of the number of toxic deaths at 
this number and the results are shown in Table 20. The median survival 
rate was calculated by adding the number of days expired until the total 
number of deaths in a group is half of the mice in the group to the 
earliest day when the cumulative number of deaths is one more than half of 
the number of mice in the group divided by two. The test to control ratio 
(t/c) is significant when it is above 125 percent (test group median 
survival time divided by the negative control median survival time 
multiplied by 100). The toxic deaths are the number of mice in each group 
which died before day six. 
TABLE 19 
______________________________________ 
Experimental 
Anti-Tumor Dosage Median T/C Toxic 
Group Agent (mg/kg) Survival 
70 deaths 
______________________________________ 
1 MS 100 3.0 22 7 
2 MS 50 10.5 77 4 
3 MS 25 19.5 144 2 
4 MS 12.5 17.0 126 0 
5 MS 6.25 16.0 119 0 
6 5-FU 20 25.0 185 0 
7 Saline 15.5 115 0 
8 None 13.5 0 
(Neg Cont) 
______________________________________ 
TABLE 20 
______________________________________ 
Experimental 
Anti-Tumor Dosage Median T/C Toxic 
Group Agent (mg/kg) Survival 
70 deaths 
______________________________________ 
1 MS 50 21.5 179 2 
2 MS 25 15.5 129 1 
3 MS 12.5 18.5 154 0 
4 MS 6.25 14.0 117 0 
5 5-FU 20 19.5 163 0 
6 Saline 11.5 95 0 
7 None 12.0 0 
(Neg Cont) 
______________________________________ 
From these tests, it can be seen that doses of mycodextran sulfate at 
non-toxic levels above a threshold, which was 6.25 milligrams per kilogram 
of weight in the above tests have shown anti-tumor activity. 
EXAMPLE 10 
DETERMINATION OF BINDING SITES 
To prepare single cell suspensions: (1) mice were sacrificed by cervical 
dislocation; (2) spleens were dissected out and placed in a 10.times.35 
millimeter plastic petri dish with five milliliter of phosphate buffered 
saline and five percent PBS, at 4 degrees Centigrade and pH 7.1 to 7.2; 
(3) cells were removed from the organ; (4) the removed cells were washed 
by suspending in a medium and decanting; (5) the red cells were lysed and 
the suspension washed; and the spleen cell suspension prepared. 
To remove and wash the cells: (1) a cell suspension was made by squashing 
the organ between the frosted ends of two microscope slides and rinsing 
the slides with the medium in the petri dishes; (2) the cells and medium 
were transferred by Pasteur pipette to 12.times.75 millimeter glass tubes, 
leaving the capsule in the petri dish; (3) debris was allowed to settle 
for 5 minutes; (4) supernatant was transferred by Pasteur pipette to a 
clean 12.times.75 millimeter tube; and (5) the cells were spun down at 300 
gravity for five minutes at 4 degrees Centigrade. 
To separate and prepare the single-cell suspension of spleen cells, the 
supernatant was poured off and red cells were lysed by mixing the pellet 
for 15 seconds with 0.5 milliliters distilled water. Lysing was stopped by 
the addition of 3.5 milliliters of medium and the cells were spun as above 
and washed twice with four milliliters of medium. After the final wash, 
spleen cells were resuspended in one to two milliliters of RPMI 
1640+Hepes, depending on pellet size. An aliquot of the cells was stained, 
counted and viability was assessed by the trypan blue dye exclusion 
method. All preparations used contained ninety percent or more viable 
cells. Suspensions were adjusted to 5.times.10.sup.6 cells/mi. 
Membrane binding studies were performed using techniques described in the 
publication of Paul, Maragnos & Skolnick (1981) cited earlier with the 
following modifications. Total binding of tritiated pustulan sulfate to 
cells was determined by incubating 100 ul (microliters) 500,000 cells of a 
single cell suspension with 50 ul of media and 50 ul of tritiated pustulan 
sulfate in a flat bottom microtiter plate (Flow Laboratories, Inc., 
McLean, Va.). 
The concentrations of tritiated pustulan sulfate examined ranged from ten 
nanograms added/well to 400 ng/well. Non-specific binding to cells was 
determined by incubating 100 microliters of cells with 50 microliters 
tritiated pustulan sulfate (ranging from 10 ng/well to 400 ng/well) and 50 
microliter of parent compound (1 microgram added per well) . 
The reaction was terminated by separating bound and free ligand by 
collecting cells on glass fibre filters (Whatman, Clinton, N.J.) with two 
quick rinses using a cell harvester (Otto Hiller Co., Madison, Wis.). 
Radioactive ligand was detected by placing dried filter discs in seven 
milliliters of scintillation cocktail (Hydrocount, J. L. Baker), mixing 
and then standing for at least 1 hour and counting with a Beckman LS7500 
LSC using an appropriate program for tritium detection. 
Specific binding values may be calculated as the difference between total 
and non-specific binding. The total ligand (tritiated pustulan sulfate) 
bound was determined by incubating cells and radiolabeled compound in 
RPMI-1640 +Hepes for 1 hour at 4 degrees Centigrade. Non-specific binding 
was determined in a similar manner with the addition of a large excess of 
non-labeled compound. The amount of specific ligand bound is computed as 
the difference between total and non-specific ligand bound. P values less 
than 0.05 were considered significant. Values were computed using 
Student's t-test for the difference between two means. The results are 
shown in Table 19. 
As indicated in Table 21, low levels of specific binding were consistently 
detected. Results for one experiment in Table 21 are representative of 
three separate experiments. Specific binding was detected in the three 
experiments in wells with tritiated pustulan sulfate concentration levels 
ranging from 10 to 100 ng/well. Specific binding at 200/well is 
detectable, but is not consistently found. When higher concentrations of 
radioactive pustulan sulfate are added, non-specific binding levels 
increase sharply and no specific binding is detectable. 
For toxicity studies with pustulan at 50, 100, 200 or 400 milligrams per 
kilogram of body weight, some local inflammation at the injection site is 
observed. For pustulan sulfate at 25, 50, 100 or 200 milligrams per 
kilogram of body weight, no toxicity 
TABLE 21 
______________________________________ 
Spleen Cell Membrane Binding of Pustulan Sulfate (PS) 
total PS non-specific PS specific PS 
ng added 
bound* bound p value 
bound 
______________________________________ 
10 859 768 0.013 91 
50 5033 4555 0.024 443 
100 9560 9219 0.038 341 
______________________________________ 
*bound ligand expressed as decay per minute (dpm) 
is observed at 25 or 50 milligrams per kilogram of body weight but toxic 
effects and some deaths are observed in animals receiving 100 or 100 
milligrams per kilogram of body weight. 
In the delayed hypersensitivity response-footpad swelling test, the 24 and 
48 hour delayed hypersensitivity responses with pustulan sulfate adjuvant 
are indicated in Tables 11-14. The results indicate that there was a 
response by five or six groups of animals at 24 and 48 hours after the 
first challenge. Following the second challenge, five of six groups of 
mice indicate a significant delayed hypersensitivity response. 
There are some increases in titers of hemagglutinating antibodies in the 
animals receiving pustulan sulfate. The results are shown in Table 15. 
There were no deaths from anaphylactic shock in the groups of animals 
following the first challenge with antigen. Five of ten male ESP mice died 
following the second challenge. No signs of shock were observed in the 
remaining animals. Pustulan sulfate exerts significant adjuvant action in 
the system employed. The effect is greatest on the delayed 
hypersensitivity reaction. Changes in immunoglobulin synthesis are 
minimal. Pustulan sulfates selectively enhance one delayed immune response 
(footpad swelling) but not another (allograft rejection) and show specific 
binding sites. The amount of specific binding is approximately 5 percent 
which corresponds to the percentage of T4 helper cells available for 
attachment to virus parts such as by the HIV glycoprotein attachment to 
the cellular protein of the helper T4 cells. The suppression of virus in 
the HIV family is discussed in "Dextran Sulfate Suppression of Viruses in 
the HIV Family: Inhibition of Virion Binding to CD4+Cells" by Mitsuya et 
al., Science, v. 240, pp. 646-649. 
Each of the classes of high-molecular-weight polysaccharides is either 
inert in its unsulfated condition or produces minimal immunological 
response changes and is an active immunoregulator following sulfation. The 
degree of sulfation may be modulated in such a way that the compounds have 
all available sites saturated that accept the sulfate or have partial 
sulfation. Complete sulfation results in the greatest immunoregulatory 
effect while moderate sulfation yields a lesser immunoregulataory effect 
in the case of such modulation. 
The results of this study are summarized in Tables 1 through 19 and are 
consistent with the prior results of studies on dextran sulfate as 
reported by R. E. McCarthy, L. W. Arnold and G. F. Babcock in Immunology, 
1977, 32, pp.963-974, entitled "Dextran Sulphate: An Adjuvant for 
Cell-mediated Immune Responses," the disclosure of which is incorporated 
herewith for reference. 
Non-acylated pustulan, (i.e., pustulan molecule with no substitutent 
groups,) has a certain degree of regulatory effect in that the delayed 
hypersensitivity footpad swelling response is increased significantly at 
24 hours following both first and second challenges with antigen. Acylated 
pustulan, the form in which it is commercially available, is seen to have 
a positive effect on the delayed footpad swelling reaction, which in 
general is greater than that observed for the unsubstituted pustulan 
molecule. Pustulan sulfate is observed to have an even greater effect on 
the immune response than either of the other two forms of pustulan. The 
effect of pustulan sulfate on anaphylactic shock reactions in mice is 
reported in Table 4. 
The incidence of fatal anaphylaxis in ESP and CFW mice is greatly increased 
when pustulan sulfate is used as an adjuvant. The B6D2F1 strain of animal 
is extremely resistant to the induction of anaphylaxis under any 
circumstances and it has been our experience to date that no compound acts 
as an anaphylactic shock inducer in this strain. 
The results of experiments with mycodextran are reported in Tables 5 
through 8. Unsubstituted mycodextran has a minimal effect on the immune 
responses as indicated by changes in footpad swelling reaction. Sulfation 
(Tables 6 and 7) converts this material into a highly active 
immunoregulator. Moreover, mycodextran sulfate has a significant effect on 
delayed hypersensitivity responses in rats as well (Table 8). The effect 
of mycodextran sulfate on anaphylaxis in mice is presented in Table 7. 
This compound has its greatest effect in CFW mice, in which there was one 
hundred percent lethality. 
While experimental work has been done with SRBC because it is an accepted 
antigen, antigens other than SRBC may be used. For example, antigens used 
to assess whether there is diminished delayed hypersensitivity or anergy 
in selected patients may be among one of the following: (1) Candidin; (2) 
Mixed respiratory vaccine such as: (a) Staphylococcus auareus, (b) 
Streptococcus, (c) Streptococcus pneumonia, (d) Neisseria catarrhalis, (e) 
Klebsiella pneumoniae or (f) Haemophilus influenzae; (3) Purified protein 
derivative of tuberculin (PPD); (4) Strephtokinase-streptodornase (SK-50); 
(5) Syaphylococcal antigens (Staphage Lystate); (6) Trichophytin; or (7) 
Mumps virus. 
If use of the above antigen produces a negative response, the following 
have been used to sensitize the patient and later test for response: (1) 
Dinitrochlorobenzene (DNCB); or (2) Keyhole limpet hemocyanin. These 
proceedings have been disclosed in Spitlet, Lynne E., 1980, "Delayed 
Hypersensitivity Skin Testing." Manual of Criminal Immunology (M. R. Rose 
and H. Friedman, Esq), American Society for Microbiology, Washington, 
D.C., pp.200-212. 
As can be understood from the above description, the immunoregulators of 
this invention and its methods of use and preparation have several 
advantages, such as: (1) They can be used for T-cell-mediated responses 
only and thus reduce anaphylactic shock; (2) specific synthetic regulators 
are available to operate in a predictable manner; (3) it is possible to 
use a dosage and regulator for a known type of response; and (4) it is 
possible to synthesize regulators for particular antigens. 
Although a preferred embodiment has been described with some particularity, 
many modifications and variations may be made in the preferred embodiment 
without deviating from the invention. Accordingly, it is to be understood 
that, within the scope of the appended claims, the invention may be 
practiced other than as specifically described.