Download Isolation of an ImmunosuppressivePeptide Fraction from the Serum

[CANCER
RESEARCH
35, 1489-1494,
June 1975]
Isolation of an Immunosuppressive Peptide Fraction from the Serum
of Cancer Patients'
Richard B. Nimberg,2 Arthur H. Glasgow, James 0. Menzoian, Mark B. Constantian,
Sidney R. Cooperband, John A. Mannick, and Karl Schmid
Departments ofBiochemistrv.
02118
Surgery, and Medicine, Boston University School ofMedicine,
SUMMARY
An immunosuppressive peptide fraction was isolated by
means of gel filtration,
membrane
partition,
and ion
exchange chromatography
from the sera of patients hospi
talized for cancer. The resulting peptide fraction, which was
heterogeneous
as judged by high-voltage electrophoresis,
was found to suppress both phytohemagglutinin-induced
proliferation
of lymphocytes
in vitro and the in vivo
induction of splenic plaque-forming
cells in mice. The
specific activity of the peptide fraction, which was isolated
from the sera of cancer patients, was significantly increased
over that of the unfractionated starting material. Moreover,
in control experiments,
when the sera of normals or
non-cancer-bearing
hospitalized individuals were subjected
to the same chromatographic techniques, no active peptide
fraction could be obtained.
INTRODUCTION
For a number of years our laboratory has been involved
in the isolation of an immunosuppressive
peptide fraction
from Cohn Fraction
IV, derived from normal-human
plasma, which we have called immunoregulatory
a-globulin
(17). This peptide fraction has been shown to inhibit a
number of T-cell-dependent immunological reactions in
cluding delayed hypersensitivity responses, the rejection of
transplantable tumors (2), and the stimulation of peripheral
blood lymphocytes by specific antigens and by PHA3 (1).
Since we have recently further demonstrated that a positive
correlation exists between diminished delayed hypersensitiv
ity responses and suppressive activity in the serum of cancer
patients (15), we decided to study the immunosuppressive
Boston University Medical Center, Boston, Massachusetts
possesses imniunosuppressive
activity both in vitro and in
vivo. Furthermore, when the same fractionation procedures
were applied to the sera of normal volunteers or non-cancer
bearing hospitalized patients, no immunosuppressive
pep
tide could be recovered.
MATERIALS
AND METHODS
Collection of Sera. Aliquots of blood (30 ml) were
collected from individual donors by peripheral venipunc
ture. Following clot formation and clot retraction the serum
was separated by centrifugation
at 2000 x g for 30 mm.
Sera were then either fractionated directly or immediately
frozen and stored at —20°
prior to usage.
Blood Donors. Cancer patients, ranging in age from 42 to
87 years, were all hospitalized individuals in whom the
diagnosis of cancer was substantiated by tissue histology. Of
the 3 males and 3 females studied, all had solid tumors and
none were receiving chemotherapy or irradiation.
The normal group consisted of 3 males and 2 females and
a poo1 of 12 individuals (4 males and 8 females) ranging in
age from 23 to 37 years. These individuals were medical
school personnel in good health.
As a further control, 28 patients who were hospitalized
for routine surgical procedures were also studied. This
group consisted of 15 males and 13 females ranging in age
from 40 to 83 years and diagnosed as suffering from a wide
range of chronic and acute noncancerous diseases.
Ion-Exchange Chromatography of the Sera of Cancer
Patients and Normal Individuals.Aliquots of human sera (10
ml), which were adjusted to pH 5.2 with 7% acetic acid
(the immunosuppressive
activity
of human
blood
is not
sera of patients hospitalized with cancer to determine
whether the immunological hyporeactivity of these patients
was due to a peptide fraction similar to immunoregulatory
adversely affected by the addition of this acid or by ly
ophilization in sodium acetate buffers, as judged by in vivo
a-globulin.
on DEAE-cellulose 52 (H. Reeve Angel & Co., Inc.,
Clifton, N. J.) previously equilibrated with F/2 0.005
In this paper we describe the isolation, from small
aliquots of sera of cancer patients, of a peptide fraction that
1 Thrs
study
was
supported
by
NIH
Grants
GM-l0374,
CA-l2209,
AM-10824.
2 Recipient
of
National
Institute
of
Dental
Research
Special
5-F02-DE44743.
3 The
abbreviation
used
is:
PHA,
phytohemagglutinin.
ReceivedNovember 11, 1974;acceptedFebruary 28, 1975.
Fellowship
and
and in vitro analyses),
were subjected
to chromatography
sodium acetate buffer, pH 5.0. The chromatograms
were
developed at a constant pH, first with a linear
@/2gradi
ent from 0.005 to 0.03 and then by stepwise elution with
T/2 0.03, 0. 1, and 0.2 sodium acetate buffers. The result
ing fractions were then concentrated
by lyophilization;
redissolved in a small volume of distilled water; and, in
order to avoid the loss of dialyzable peptides, desalted by
JUNE 1975
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1489
R. B. Nimberg
et a!.
gel filtration
on Sephadex G-25 that was previously
equilibrated against 0.05% (NH4)2CO3 buffer.
Dissociation of the Peptide-Protein Complex. In order to
dissociate the peptide-protein complex, active fractions that
were obtained by ion-exchange chromatography
as de
scribed above, were dissolved in distilled water to give a
final concentration
of 7%, acidified to pH 3.0 with 10%
acetic acid, and fractionated by membrane-partition
chro
matography.
PM-lO Diaflo membranes (Amicon Corp.,
Lexington, Mass.) that retain plasma constituents
with
molecular weights greater than 10,000 were used, and 1.5%
acetic acid was used as the exchange liquid. Those peptides
that were not retained by the membrane were then lyophi
lized and analyzed for suppressive activity.
Electrophoretic Analyses. Cellulose acetate electrophore
sis of the obtained protein fractions was performed using a
Model R-l01 Microzone@ cell (Beckman Instruments, Inc.,
Fullerton, Calif.), at pH 8.6, F/2 0. 1 citrate:diethylbar
biturate buffer. Electrophoresis was carried out at 250 V for
control tubes contained a concentration of reference pooled
normal human serum equal to the greatest concentration
of experimental protein or peptide being tested. The refer
ence serum has been used in all our experiments and,
consistently, the counts in these tubes are equal to the
counts of tubes that have no additional human serum
added. The cultures were then maintained in stationary
racks in a 5% CO2 humidifed environment at 37°for 48
hr. They were then pulsed by the addition of 4 sCi of
[3H]thymidine for 16 to 18 hr. Before killing, viabilities
were ascertained by trypan blue dye exclusion. Viabilities
were equal in control and experimental groups. Following
incubation the tubes were centrifuged, the cell pellets were
washed 4 times with 2-ml aliquots of 0.9% NaCl solution,
and finally they were dissolved in 1 ml of 0.01 N sodium
hydroxide. To this were added 5 ml of a scintillation cock
tail of dioxane, naphthalene, and PPO. Radioactivity was
then counted in a Beckman scintillation counter.
All sera were initially tested for immunosuppressive
activity at a 10% concentration of tissue culture. The extent
30 mm and the electrophoretograms
were stained with
Ponceau Red S. High-voltage electrophoresis
of the iso
lated peptide fractions was performed using a Savant
type horizontal
apparatus
in pyridine:acetic
acid:water
buffer (80:[email protected]), pH 6.5, at 4000 V for 1 hr on Whatman
No. 3MM chromatography
paper. The resulting electro
phoretogram was stained with ninhydrin.
Chemical Analyses. The polypeptide moiety was deter
mined by the biuret procedure of MehI (13) utilizing bovine
serum albumin as a standard. For the determination of the
total neutral hexoses and sialic acid, the orcinol method of
Those sera that possessed
SØrensen and Haugaard (18) and the Warren technique
70% or greater suppression of PHA-induced DNA synthe
(20), respectively, were used. Lysozyme and a,-acid glyco
protein served as references. The content of DNA and RNA
was determined by the procedure of Dische (4). Cortisol was
measured by the competitive protein-binding
assay (16),
RNase activity was assayed by the method of Kalnitsky et
a!. (10), and prostaglandin E, was determined by radioim
munoassay after conversion to prostaglandin F1a (1 1).
Bioassay. The test for immunosuppressive
activity mea
sured inhibition of PHA-induced
proliferation
of human
peripheral
lymphocytes,
a method described by us in
previous publications (5, 6). Human peripheral lymphocytes
were obtained from the plasma after gravity sedimentation
sis, at the screening dose were chromatographed
as de
scribed above. Furthermore,
the quantity of immunosup
pressive activity for each of the fractions obtained after
ion-exchange chromatography
was expressed as “units―
using the following calculation:
of heparinized blood at room temperature for 2 hr. The
lymphocytes .were then purified on sterile cotton wool
columns and eluted with Eagle's minimal essential medium
(Grand Island Biological Co., Grand Island, N. Y.). After 3
washings the cells were counted and the viability determined
with trypan blue dye in a hemocytometer chamber. The cells
were generally 95% small lymphocytes and were 95% viable.
Tissue cultures were performed with 106 lymphocytes in 1
ml of Eagle's minimal essential medium containing addi
tives. All tubes contained 10% fetal calf serum (from the
same batch for all experiments), penicillin, streptomycin,
and L-glutamine. All tubes except the unstimulated con
trols contained 10 sg of PHA, a dose predetermined
to
of immunosuppressive activity was calculated as follows:
cpm of experimental tubes with
PHA —cpm of control tubes
without PHA
% suppression = I —
‘x100
cpm of control tubes with PHA
—cpm of control tubes without
PHA
significant
activity,
defined
as
Immunosuppressive
units = Total weight of pro
tein fraction/smallest
dose producing 100% sup
pression of PHA stimulation in vitro
In vivo testing for immunosuppressive
activity was per
formed by a modification of the Jerne hemolytic plaque
technique (19). Adult CD-l mice (Charles River Breeding
Laboratory, Wilmington, Mass.) were given i.p. injections
of the protein or peptide to be tested 24 hr prior to
immunization with approximately 4 x 108 sheep erythro
cytes. These animals were sacrificed 4 days after immuniza
tion and the number of plaques per spleen was determined
(9). As a control,normalhumanserumandhumanserum
albumin, in concentrations equal to the greatest concentra
tion of sample being tested, were used in all experiments.
RESULTS
In Vitro Assay for Immunosuppressive
Activity.
In order
Experimental tubes contained
to obtain starting material for this study the sera of patients
hospitalized with solid tissue cancer and those of normal
various concentrations
of protein or peptide samples from
cancer patients or from normal individuals. Additional
volunteers were first tested for immunosuppression
in vitro.
Of the 53 cancer patients screened, 27 (50%) had im
give optimal stimulation.
1490
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ImmunosuppressivePeptidefrom Sera of Cancer Patients
4.0
munosuppressive serum as demonstrated by the ability of
such sera to inhibit by 70% or more the stimulation of
normal peripheral blood lymphocytes by PHA. In contrast
to the 17 normal volunteers and 28 hospitalized non-cancer
bearing patients tested, none had immunosuppressive serum
(Table 1). Because the objective of the present investigation
was to define the immunological
@
hyporeactivity
3.5
.@ 3.0
‘:@
2.5
of the sera
of cancer patients and to distinguish this from the sera of
normal volunteers, only those cancer sera that were im
munosuppressive (70 to 85% suppressive at 6 mg/ml) were
used for the subsequent studies. Furthermore, since none of
the sera of the normal donors and those of the non-cancer
bearing patients were significantly suppressive, as judged by
in vitro and in vivo analyses, the sera obtained from these
individuals were used as controls.
Ion-Exchange Chromatography of Sera. Chromatography
of the sera of normal donors and cancer patients on
DEAE-cellulose
consistently yielded 6 fractions (Chart 1).
In vitro analyses for immunosuppression
indicated that, in
cancer patients, although some individual variability was
noted, the protein moiety capable of suppressing PHA
induced stimulation of normal lymphocytes was present in
all of the 6 serum fractions (Table I). Fraction I from
cancer patients, which contained primarily albumins and
fl-globulins (Chart 2), possessed the greatest amount of
immunosuppressive
activity with a mean of 239 ±61.47
(S.E.) immunosuppressive units. For Fractions II, III, IV,
V, and VI, mean immunosuppressive units were found to be
2.0
tb
.s
,@
.0
0.5
0
200
400
600
bearingpatients
disease500103Mitral artery
400
600
800
2000
were then eluted by the stepwiseadditions of 0.03, 0.1, and 0.2 M sodium
acetate buffers, pH 5.0 (arrows). Appropriate
were pooled.
fractions (Roman numerals)
‘1@@
II
•@
Ill
IL
R
®
lv
V
(%)21Atherosclerotic
DonorDiagnosisAge@sion
disease50095Coronary
artery
200
pH 5.0, to 250 ml of 0.3 M sodium acetate, pH 5.0. The retained proteins
Suppres
disease762322Atherosclerotic
vascular
disease78823Atherosclerotic
vascular
disease621024Hernia505825Hernia786334Coronary
vascular
000
was used and 5-ml fractions were collected. A linear salt gradient Was
establishedby the appropriatemixing of 250ml of 0.005Msodiumacetate,
Table 1
immunosuppressive activity in vitro ofthe sera ofhospitalized, non-cancer
800
EFFLUENT VOLUME (ml)
Chart 1. Ion-exchange chromatography of the sera obtained from
normal and cancer patients on DEAE-cellulose. A flow rate of 10 ml/hr
@.
vl.&.
NORMAL
CANCER
Chart 2. Cellulose-acetate electrophoresis of the 6 fractions (I, II, III,
IV, and VI) isolated from the sera of normal and cancer patients after
chromatography on DEAE-cellulose (Chart I). Normal human serum
(R) served as reference.
stenosis400I
failure8301
14Renal
18Coronary
disease520126Coronary
artery
disease502133Coronary
artery
disease5445138Coronary
artery
disease540150Aortic artery
disease6129154Coronary
and mitral valvular
disease6319158Mitralartery
stenosis4613162Actinomycosis568165Pulmonary
abscess519216Urethral
diverticulosis4015220Benign
hypertrophy7036223Gastric
prostatic
obstruction5540230Atherosclerotic
outlet
disease6524238Atherosclerotic
vascular
disease4037243Benign
vascular
hypertrophy5017245Benign
prostatic
hypertrophy6846250Chronic
prostatic
infection5633263Chronic
urinary tract
urinary tract infection6716
a Average
JUNE
age, 58 years.
51.41 ±19.81, 39.50 ±15.88, 26.66 ±11.22, 35.00 ±10.53,
and 42.66 ±19.11, respectively. The mean total immuno
suppressive units of the cancer
was 452.50 ±77.88.
serum used in this study
In markedcontrast,in vitro analysisof the fractions
obtained from the control groups demonstrated that there
was no immunosuppressive
activity in Fraction I (Table 2).
Furthermore,
while the immunosuppressive
activity of the
control groups was also found in Peaks II, III, IV, and VI,
the activity of these fractions was extremely low and the
mean total immunosuppressive units of the normal donors
and patients hospitalized for routine elective procedures
were found to be only 8.2 ±1.50; (p < 0.001) and 10.0 ±
1.0; (p < 0.001), respectively.
Isolation of the Immunosuppressive Peptide Fractions.
Since Peak I, isolated from the sera of cancer patients,
possessed the greatest amount of suppressive material, this
1975
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1491
R. B. Nimberg
et a!.
Table 4
immunosuppressiveactivity in vivo of DEA E-Fraction i derivedfrom the
sera of normal individuals and cancerpatients and the peptidefractions
obtained by ultrafiltration on a PM-JO membrane
fraction was next subjected to ultrafiltration in order to
determine whether its immunosuppressive activity was
contained in a peptide fraction similar to immunoregulatory
a-globulin isolated from normal human serum. Ultrafiltra
tion of the major active fraction of cancer sera resulted in a
significant decrease in the immunosuppressive activity of
the retained high-molecular-weight
%suppres
GroupsPlaques/spleenasionControl
proteins (Table 3). The
protein fractions that were suppressive at doses ranging
between
1 and 6 mg/mI
prior to ultrafiltration,
(no serumor serumfrac
19,200―tions)Normal
serum(6 mg/mouse)102,100
28,4000Normal
serumpeptide(3 mg/mouse)62,400
21,80035Cancer
serum(6 mg/mouse)43,600
17,00054Cancer
serumpeptide(3 mg/mouse)26,500
assayed for inhibition of lymphocyte stimulation in vitro
after ultrafiltration, were found to have little activity at
doses that were initially active. Moreover, the fractions that
passed through
the filter were significantly
95,700±
when
suppressive
±
±
±
±l9,200c72
at
doses between 0. 1 and 0.3 mg/ml. In addition, in vivo
analyses (Table 4) further demonstrated that the obtained
peptide fractions were immunosuppressive in the intact
a Direct
plaque-forming
cell
response
in
adult
CD-I
mice
4 days
after
injection of sheep red blood cells. Duplicate determinations performed in
eachof 5 animals per experimentalgroup.
6 Mean
mouse at a dose of 3.0 mg/mouse.
Cp
normal
In order to demonstrate
that the immunosuppressive
<
± S.D.
0.05,
compared
to
control,
to
pooled
normal
serum,
and
to
pooled
serum peptide.
Table 2
Comparison ofthe immunosuppressive activity in vitro ofthe sera ofnormal
individuals and cancer
patientsfollowing chromatography on DEA E-cellulose
units―6
FractionImmunosuppressive
patientsI239cancer patientsControl6
normal patients3
61.47―0(<0.0l)'@0(<0.Ol)II51.41
±
± 19.811.60
±t03
±15.881.20
±0.37
± 11.222.40±
1.94
(NS)V35.00
± 10.531
.60 ±1.48
<0.02)VI42.66
± 19.111.40
(NS)Total452.50
±1.64
±77.888.20
±1.50
(<0.05)III39.50
(<0.05)IV26.66
a 5@
hospitalized
(<0.05)0.66
±0.33
±0.66
(<0.05)1.33
(NS)―2.33±
(<0.02)3.0
(NS)2.3
(<0.001)10.0
1.15
±I .0
±0.33
(
±1.0
(<0.001)
text.
b Mean
± S.D.
C Numbers
in
d NS,
not
parentheses,
p
values.
significant.
Table 3
immunosuppressive activity in vitro oJDEAE-fraction
I derivedfrom
the sera ofnormal
individuals
and cancerpatients following ultrafiltration on a PM-JO membrane
ultrafiltration
(%)Activity
at 6 mg/mI
NS at 6 mg/mI
2
I mg/mI
7
1.75mg/mI
91 at 0.3 mg/mI
NS at 3 mg/mI
77 at 6 mg/mI76at0.l75mg/ml95 at 0. 1 mg/mI6
98 at 3 mg/ml
28l0Oat 100at 3 mg/mINSat
a Activity
fraction by
wt
peptide fraction
(%)Peptide
at 0.6 mg/mI
NS at 0.6 mg/ml
NSatO.6mg/ml8
at 6 mg/mI
NS at 6 mg/mI
NSat6mg/mlNS
7Cancer22
3NSC NSat6mg/mlNS
23
of
after
prior to
ultrafiltration
(%)°NormalI (%)“Activity
SubjectActivity
is expressed as percentage
of suppression
of PHA-stimulated
7
6
lymphocytes
at the given
concentrationof the analyzedfractions per ml of nutrient media. Values,meansof at least 3 tissue
culture experiments.
nonsuppressive.
6 Recovery
C NS,
1492
of
each
Those values that produced less than 70% suppression were considered
fraction
as
a
percentage
of
the
starting
material
(DEAE-Fraction
I).
nonsuppressive.
CANCER
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Immunosuppressive
Peptide from Sera of Cancer Patients
peptide fraction obtained by ultrafiltration
was not an
artifact of preparation, Peak I from the normal group was
subjected to the same chromatographic
technique. The
results (Tables 3 and 4) indicate that, although a peptide
fraction could be isolated from this protein fraction, no
detectable immunosuppressive
activity could be identified.
Chemical and Physicochemical Analyses. Chemical analy
sis by the biuret procedure on the active low-molecular
weight fraction, isolated from cancer patients, indicated
that the total weight of this fraction could be accounted for
by polypeptide. This finding was confiriped by the absence
major amount of immunosuppressive
activity of the sera of
cancer patients was found in a fraction that appeared to be
devoid of a-globulins. This protein fraction (DEAE-Peak I)
was never active if isolated from the sera of non-cancer
bearing individuals and, when isolated from cancer sera,
possessed about 30 times niore activity than did the com
bined active fractions of normal serum. In contrast, the
suppressive activity contained in the sera of normal mdi
viduals or those ill from nonmalignant
diseases is ex
tremely small in amounts and cannot be detected by in
vitro assays prior to chromatographic
procedures. How
of
ever, after ion-exchange chromatography
neutral
hexoses,
sialic
acid,
cortisone,
DNA,
RNA,
and
prostaglandin E1. Furthermore, analyses for RNase activity
demonstrated
that this fraction possessed a negligible
quantity of this enzyme.
On high-voltage electrophoresis
the isolated active pep
tide fraction was found to be heterogeneous,
revealing
ninhydrin-positive
compounds
with acidic, neutral, and
basic electrophoretic characteristics (Chart 3). This fraction
appeared to be similar electrophoretically
to that isolated
from pooled normal human plasma immunoregulatory
a-globulin and the inactive peptide fraction isolated from a
normal donor.
KR
0
D
I
One might suggest that the immunosuppressive substance
present in blood is initially carried by an a-globulin(s);
however,
as the suppressive
compound
increases
in
amount, as it seems to in some cancer patients, carrier
sites become saturated and the active factor is then picked
up by proteins that chromatograph
in non-a-globulin
containing fractions.
More important,
I
of the sera of
control subjects, the activity was then found to reside in the
a-globulin-rich
fractions of their blood as judged by cellu
lose acetate electrophoresis.
These protein fractions ac
counted for all of the immunosuppressive
activity found
in control donors and represented less than 2% of the pro
teins of their serum on a weight basis. The a-globulin-con
taming peaks isolated from cancer sera were also immuno
suppressive, although more so than in normal donors.
however, was the finding that the
immunosuppressive
activity of the cancer sera used in this
study could be removed from the high-molecular-weight
+
protein fractions by ultrafiltration
and that this activity is
c.s-p
then contained in a low-molecular-weight
peptide fraction.
This peptide fraction was found to be readily dissociable
1@
•
IRA-P
from its protein carrier and would have been easily lost had
conventional dialysis procedures been used in this study.
I
0
Also, the active protein fractions isolated from normal
KR
donors or patients without cancer could not be subjected to
techniques because the amount and
Chart 3. High-voltageelectrophoresisofthe immunosuppressivepeptide the same ultrafiltration
activity
of
material
isolated
from normal sera was too small
fractions obtained from DEAE-Fraction
I by ultrafiltration
(CaS-P),
to allow further investigation.
from normal-human serum (NHS-P), and from Cohn Fraction IV
Furthermore,
the peptide fraction isolated from the sera
(IRA-P). The latter 2 fractions appear to be similar electrophoretically
to
NHS-P
the active peptide fraction isolated from the sera of cancer patients.
of patients with cancer closely resembles, both chemically
Arginine (R), lysine (K), glycine (G), and aspartic acid (D) served as
references.
and physicochemically,
immunoregulatory
a-globulin, an
immunosuppressive
peptide fraction that we have previously
isolated in trace amounts from large pools of normal human
blood (17). Immunoregulatory a-globulin, like the peptide
DISCUSSION
Although it is well known that there is a generalized
depression
of cellular immunity in some individuals afflicted
with cancer, little information is available to explain this
important immunological phenomenon. In order better to
understand these findings and because we have recently
shown that 66% of the sera of cancer patients
who lacked
delayed hypersensitivity responsiveness are capable of inhib
iting PHA
stimulation
of normal
lymphocytes
decided to study the immunosuppressive
patients in order to determine
(15), we
sera of cancer
what might account for these
potentially significant observations.
The results
JUNE
of this investigation
demonstrate
that the
fraction isolated from cancer patients, has been shown to
cross species barriers and is a peptide fraction that inhibits a
wide variety of T-cell-mediated
immune responses without
affecting B-cell responses (2, 3, 12, 14). We believe that
immunoregulatory a-globulin may act biologically as a
feedback regulator of cell-mediated immunity. If this is so,
and if as has been shown in this study, immunoregulatory
a-globulin or an immunoregulatory
a-globulin-like peptide
is synthesized in extremely large amounts in some cancer
patients, then a high level of this circulating, naturally
occurring immunosuppressive agent might be responsible
for the failure of some cancer patients to mount an effective
immune response against their tumors (7).
1975
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1493
R. B. Nimberg
et a!.
ACKNOWLEDGMENTS
9. Jerne, M. K., Nordin, A. A., and Henry, C. in: B. Amos and H.
Koprowski (eds.), Cell-Bound Antibodies, pp. 109-132. Philadelphia:
We gratefully acknowledge the generous help of Dr. L. Levine of
Brandeis University for prostaglandin analyses and we wish to express our
thanks to Inna Saporoschetzand Kathy Wirthwein for their excellent
technical assistance.
REFERENCES
The Wistar Institute Press,1963.
10. Kalnitsky, G., Hummel, J. P., and Diertes, C. Some Factors Which
Affect the Enzymatic Digestion of Ribonucleic Acid. J. Biol. Chem.,
234: 1512-1516,1959.
11. Levine, L. Antibodies to Pharmacologically Active Molecules: Speci
ficities and Some Applications of Antiprostaglandins.
Pharmacol.
Rev., 25: 293-307, 1973.
12. Mannick, J. A., and Schmid, K. Prolongation ofAllograft
1. Cooperband, S. R., Badger, A. M., and Davis, R. C. The Effect of
Immunoregulatory
Alpha Globulin (IRA) upon Lymphocytes in
Vitro. J. Immunol., 109: 154—163,1972.
2. Cooperband,S. R., Bodevik, H. E., and Schmid, K. Transformation
of Human Lymphocytes: Inhibition by Homologous Alpha Globulin.
Science, 159: 1243- 1244, 1968.
3. Davis, R. C., Cooperband, S. R., and Mannick, J. A. The Effect of
Immunoregulatory Alpha Globulin on Antigen Mediated Macrophage
Immobilization In Vitro. J. Immunol., 106: 755—760,1971.
4. Dische, Z. Color Reactions of Nucleic Acid Components. in: E.
Chargaff and J. N. Davidson (eds.), The Nucleic Acids. Vol. 1,
pp. 285—287.New York: Academic Press Inc., 1955.
5. Glasgow, A. H., Cooperband, S. R., Occhino, J., Schmid, K., and
Mannick, J. A. Inhibition ofSecondary Immune Responsein Vivo by
Immunoregulatory Alpha Globulin. Proc. Soc. Exptl. Biol. Med., 138:
753-757, 1971.
6. Glasgow, A. H., Cooperband, S. R., Schmid, K., Parlar, J. T.,
Occhino, J., and Mannick, J. A. Inhibition of Secondary Immune
Responses by Immunoregulatory Alphaglobulin. Transplant. Proc., 3:
835—837,1971.
7. Glasgow, A. H., Menzoian, J. 0., Mimberg, R. B., Saporoschetz, I.,
Cooperband,S. R., Schmid, K., and Mannick, J. A. Association of
Anergy with an ImmunosuppressivePeptide in the Serum of Cancer
Patients. New EngI. J. Med., 291: 1263-1267,1974.
8. Glasgow, A. H., Schmid, K., and Mannick, J. A. Immunoregulatory
Alpha Globulin (IRA) and Tumor Immunity. Surg. Forum, 23:
120-122, 1972.
1494
Survival by
Alpha Globulin Isolated from Normal Blood. Transplantation. 5:
1231—1245,
1967.
13. Mehl, J. The Biuret Reaction of Proteinsin the Presenceof Ethylene
Glycol. J. Biol. Chem., 157: 173-180, 1945.
14. Menzoian,J. 0., Glasgow,A. H., Nimberg, R. B., Cooperband,S. R.,
Schmid. K., Saporoschetz,I., and Mannick, J. A. Regulation of “T―
lymphocyte
Function by Immunoregulatory
Alpha Globulin. J. Im
munol., 113:266-273, 1974.
15. Menzoian, J. 0., Scheinman, L., Glasgow, A. H., Saporoschetz, I.,
and Mannick, J. A. Skin Test Reactivity and in Vitro Immunosup
pressive Activity of Serum of Cancer Patients. Surg. Forum, 24:
309—311,
1973.
16. Murphy, B. E. P. SomeStudiesofthe Protein-Binding ofSteroids and
Their Application to the Routine Micro and Ultramicro
Measurement
of Various Steroids in Body Fluids by Competitive Protein-Binding
Radioassay. J. Clin. Endocrinol. Metab., 27: 973-990, 1967.
17. Occhino, J., Glasgow, A. H., Cooperband, S. R., Mannick, J. A., and
Schmid, K. Isolation of an Immunosuppressive Peptide Fraction from
Human Plasma.J. Immunol., 110:685-694, 1973.
18. S#rensen, M., and Haug@ard,G. Uber die Anwendbarkeit der
Oreinreaktion zur Bestimmung der Art und Menge von Kohlenhy
dratgruppen in Eiweisstoffen. Biochem. Z., 260: 247-277, 1933.
19. Tannenberg, W. J. K., and Melariya, A. N. The Life Cycle of
Antibody-Forming
Cells. J. Exptl. Med., 128: 895-926, 1967.
20. Warren, L. The Thiobarbituric Acid Assay of Sialic Acids. J. Biol.
Chem., 234: 1971-1975, 1959.
CANCER RESEARCH
VOL. 35
Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1975 American Association for Cancer Research.
Isolation of an Immunosuppressive Peptide Fraction from the
Serum of Cancer Patients
Richard B. Nimberg, Arthur H. Glasgow, James O. Menzoian, et al.
Cancer Res 1975;35:1489-1494.
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