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[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 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1975 American Association for Cancer Research. 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 CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1975 American Association for Cancer Research. 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 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1975 American Association for Cancer Research. 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 RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1975 American Association for Cancer Research. 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 Downloaded from cancerres.aacrjournals.org on June 12, 2017. © 1975 American Association for Cancer Research. 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. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/35/6/1489 Sign up to receive free email-alerts related to this article or journal. 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