Immunosuppressive Substance Research Articles

Determination of an immunosuppressive substance in serum by latex particle electrophoresis

The level of immunosuppressive substance (IS), which increases in the serum of patients with cancer, was determined by an assay based on particle electrophoresis. Polystyrene latex particles (PLP) were coated with IS, which was extracted from the ascitic fluid of patients with cancer. The IS was a glycoprotein with a molecular weight of about 52 000, and an isoelectric point in the range pH 2.7–3.3. When the IS on the surface of the PLP reacted with the anti-IS antibody, the mean electrophoretic mobility of the PLP changed from −3.16 to + 0.21 μm·s −1·V −1·cm in the medium of pH 7.2 and ionic strength I = 0.0154. After preincubation of anti-IS antiserum and tested serum, the PLP coated with IS were added to this solution. It was incubated again and then the surface charge of the PLP was measured by an automatic cell-electrophoretic instrument. This method was used to determine the IS concentration in the serum of cancer patients and pregnant women. When compared to healthy controls, the serum IS level was significantly higher in patients with cancer, and lower in pregnant women. The assay based on latex-particle electrophoresis proved to be a sensitive and rapid method for determining the IS level in serum.

Natural killer activity of peripheral blood lymphocytes and its relation to histopathological factors of lung cancer.

This investigation was intended to determine whether the natural killer (NK) activity of peripheral blood lymphocytes (PBL) correlated with the histopathological factor, which is thought to be a result of a balance between tumor aggression and host resistance. The NK activity of PBL from 60 patients with lung cancer was measured by the lysis of 51Cr-labelled K562 target cells. The activity was significantly decreased with advancing stages of the disease, and inversely correlated with increased immunosuppressive substance levels of the serum. Histopathological factors, such as low grade pleural invasion of the tumor and abundant lymphoid cell infiltration around the tumor, were significantly associated with the high NK activity of PBL. These results show that a decrease in NK activity may play a role in identifying those individuals with a greater risk of cancer development.

Purification and characterization of immunosuppressive (IS) substance obtained from ascitic fluids of patients with gastrointestinal cancer

An immunosuppressive substance was isolated from ascitic fluids of patients with advanced colon cancer by means of ammonium sulphate precipitation and preparative isoelectric focusing. This was a glycoprotein with an isoelectric point of pH 2.7-3.3, a molecular weight of about 52,000, and a sedimentation coefficient of 4.0S. The substance showed a single band on ordinary disc electrophoresis, but it was separated into several bands by gel isoelectric focusing, due to the structural variety of the sugar moiety. The results of physicochemical analysis indicate that the amino acid composition of this glycoprotein was indistinguishable from that of alpha 1-acid glycoprotein (alpha 1-AG), but its molecular weight, its carbohydrate content, and composition were distinctly different. Furthermore, this glycoprotein was found to have higher immunosuppressive activity than that of alpha 1-AG in both the in vitro and in vivo assays. This glycoprotein, which we called "IS substance," is a cancer-related substance synthesized in cancer patients.

Characterization of immunosuppressive substances in the basic protein fraction of uterine secretions from pregnant ewes.

The basic protein fraction of ovine uterine secretions collected late in pregnancy (Days 125-140) contains a substance capable of inhibiting in vitro blastogenic responses of lymphocytes to phytohemagglutinin (PHA) or mixed lymphocyte reactions. In this study, the immunosuppressive substance in the basic protein fraction of uterine secretions was further defined by gel filtration. The immunosuppressive activity resided in a group of high molecular weight proteins eluting at the void volume of Sephacryl S-200 and Sepharose CL-6B columns. For example, incorporation of thymidine by PHA-stimulated lymphocytes incubated with 20, 40, 80, and 120 micrograms/ml of protein from the void volume of Sepharose CL-6B was 65, 28, 2, and 0 percent of control lymphocytes, respectively. Based on polyacrylamide-gel electrophoresis in the presence of sodium dodeylsulfate (SDS), the immunosuppressive fraction from Sepharose CL-6B chromatography contained aggregates of uterine milk proteins (UTM-proteins) and a pair of proteins running at the top of a 5% (w/v) polyacrylamide gel. Other protein peaks resolved by Sephacryl S-200 and Sepharose CL-6B contained aggregates of UTM-proteins but were not immunosuppressive. The substance inhibiting in vitro lymphocyte function was not of conceptus origin, because it was found in fluid from the ligated uterine horn of unilaterally pregnant ewes and from the uterus of an ovariectomized ewe treated for 60 days with progesterone and estrone.

Detection of Early Pregnancy Factor in Human Sera

The rosette inhibition titers (RIT) for sera from 94 women at various stages of gestation were detected with a standardized rosette inhibition test. The results showed that an immunosuppressive substance, named early pregnancy factor (EPF), did exist in the pregnancy sera. We confirmed that the EPF activity was very high in the early pregnant stage (the mean RIT = 6.30), gradually decreased with the continuance of gestation, and disappeared at 8 weeks before delivery when the mean RIT for sera (less than 4) had fallen in the RIT nonpregnant range. In addition, observations for the EPF dynamic changes before and at varying stages after the induced abortion in 21 pregnant women showed that the mean RIT was 5.9 +/- 0.25 (SE) before the abortion and dropped below 4 (EPF activity could not be detected) at 3-5 days after the abortion.

Immunosuppressive Acidic Protein and Immunosuppressive Substance in Middle Ear Effusion (second report)

Immunosuppressive Acidic Protein and Immunosuppressive Substance in Middle Ear Effusion (second report)

STUDY ON STRAIN DIFFERENCE AND IMMUNOLOGICAL BACKGROUND OF 1, 2-DIMETHYLHYDRAZINE-INDUCED COLON TUMORS IN THE RAT

Rats of three strains, ACI, Buffalo and Wistar, were treated with 1, 2-dimethylhydrazine (DMH) 20 mg/kg/week, serially for nine weeks and sacrificed at the 6th week after the last injection. In control rats, saline was administered ad libitum. In Wistar rats, the incidence of colon tumors (32/ 50) was significantly higher than that in ACI rats (10/44) and Buffalo rats (8/50) (P <0.005).Both the saline-treated and DMH-treated groups of ACI rats showed a higher peripheral lymphocyte count than those in Buffalo and Wistar rats. There was no strain difference in natural killer cell activity among the saline-treated groups. On the other hand, Wistar rats showed significantly higher natural killer cell activity among the three rat strains after DMH administration. The serum immunosuppressive substance level was high in the tumor-free and colonic tumor-bearing groups of Wistar rats. Thus immunological parameters selected in this study seemed to be insufficient to explain the strain difference in susceptibility to DMH.

Decrease of mononuclear phagocyte cell functions and prolongation of graft survival in experimental transplantation by (±)-15-deoxyspergualin

The new anti-tumor substance 15-deoxyspergualin was shown to have immunosuppressive properties. Immunosuppression seemed to be predominantly directed against mononuclear phagocytes and to monocyte dependent lymphocyte effector functions. The substance was shown to be effective in the prevention of the rejection of allogeneic transplants. This was shown in a rat tail skin transplantation model (LEW → Fischer). Prolongation of graft survival time was increased about two fold after either i.p. or p.o. administration of 15-deoxyspergualin. Suppression of monocyte activity would thus be a novel mode of action for an immunosuppressive substance being effective in organ transplantation.

Serum immunosuppressive substance in patients with gynecologic malignancies and in pregnant women.

The presence of serum immunosuppressive substance (IS) was determined in 134 patients with malignant gynecologic tumors (105 cervical cancers, 15 endometrial cancers, and 14 ovarian cancers), 45 patients with benign gynecologic tumors (33 uterine myomas and 12 ovarian tumors), 10 patients with severe inflammatory diseases, 326 pregnant women, and 48 healthy controls. The mean levels and percentages of positive levels (greater than 750 micrograms/ml) in both the groups of cancer patients and the group of patients with severe inflammatory diseases were significantly higher than those in the groups of patients with benign tumors or the control group. In pregnant women, however, the majority of serum levels were within a normal range, showing relatively elevated levels in the first trimester. In the patients with malignancies (cervical, endometrial, and ovarian cancers), extremely elevated levels (greater than 1000 micrograms/ml) suggested an active or a progressive state of the malignancies. It became clear that serial IS determinations are valuable for monitoring the disease state or judging the effect of therapy.

Immunosuppressive Acidic Protein and Immunosuppressive Substance in Middle Ear Effusion

Immunosuppressive Acidic Protein and Immunosuppressive Substance in Middle Ear Effusion

A novel method of screening for immunomodulating substances, establishment of an assay system and its application to culture broths of microorganisms.

A novel method of screening for immunomodulating substances is developed employing lymphocytes and three mitogens. Concanavalin A (Con A) and phytohemagglutinin (PHA) are applied as T cell specific stimulants and lipopolysaccharide (LPS) as a B cell specific stimulant respectively. The lymphocytes obtained from mouse spleen are cultured with an antibiotic or a sample extract in the presence or absence of mitogen for three days and pulsed with [3H]thymidine for five hours before harvest. Differential effects of a sample compound on [3H]thymidine incorporation by the activated and quiescent lymphocytes are scored. In this procedure most of the tested antibiotics or chemical compounds with different mode of actions show non-specific effects. Cyclosporin A, a potent immunosuppressive substance, suppresses both Con A and PHA responses more extensively than LPS response and quiescent cell growth, and two cytochrome bc1 complex inhibitors, funiculosin and antimycin A3, are less suppressive to PHA response than to the others. The present system was also applied to the methanol extracts of the culture broths prepared from the type strains of Actinomycetes and Penicillium.

Immunosuppressive Activity Associated with Early Pregnancy in the Bovine1

Immunosuppressive activity was assessed in uterine flushings (UF) and uterine vein serum and plasma from nonpregnant and early-pregnant cows, and in media from the short-term culture of Day 18 bovine embryos. The preparations were tested for their ability to inhibit [3H] thymidine (3H-TdR) incorporation into phytohemagglutinin-stimulated bovine lymphocytes. On Days 2-3 (called Day 3), Days 9-10 (called Day 10), and Days 17-19 (called Day 18) of the estrous cycle (estrus = Day 0) and pregnancy, untreated and superovulated cows were anesthesized and jugular vein and uterine vein blood was collected. The uteri were removed and flushed to obtain UF and embryos. Uterine flushings were concentrated and tested for immunosuppressive activity at 400 micrograms uterine protein/ml culture fluid. Uterine flushings from both Day 18 pregnant and Day 18 nonpregnant cows were immunosuppressive (8/8), whereas Day 10 UF were usually not immunosuppressive (7/10). Day 3 UF were usually stimulatory or only marginally suppressive (8/8). Uterine vein serum and plasma from Day 18 cows were not suppressive when compared to jugular vein serum or plasma from the same cow; neither were Day 18 uterine vein serum or plasma suppressive when compared to those same samples taken from Day 3 cows. Embryo culture media obtained from the 48-h culture of Day 18 embryos was consistently suppressive. The activity was lost after dialysis in 1000-Mr cut-off tubing, removed by charcoal, and reduced by protease digestion. These results suggest two mechanisms whereby the embryo could escape immune rejection: 1) the progesterone-induced secretion of a uterine immunosuppressive substance(s) and 2) the production by the embryo of a low molecular weight immunosuppressive substance(s).

Combined therapeutic effects of an immunomodulator, PSK, and chemotherapy with carboquone on rat bladder carcinoma.

Responses of bladder cancer in ACI rats to combination therapy with an immunomodulator, PSK, and an alkylating agent, carboquone, are reported. PSK is a protein-bound polysaccharide isolated from Basidiomycetes, and carboquone is the alkylating agent 2,5-bis(1-aziridinyl)-3-(2-hydroxy-1-methanoxyethyl)-6-methyl-p- benzoquinone carbonate, molecular weight 3,214. The immunomodulator, PSK, was shown to enhance the effectiveness of the chemotherapeutic agent, carboquone. The therapeutic effect of combination treatment was monitored by measuring growth rates of tumors transplanted SC and by measuring decreases in metastatic spread to lungs in tumor-bearing animals. Effects of PSK on host immunity were monitored by measuring serum levels of immunosuppressive substance.

Effect of a protein-bound polysaccharide preparation, PS-K, on dimethyl hydrazine induction of intestinal tumors in rats.

The effect of a protein-bound polysaccharide preparation, PS-K, on the induction of intestinal tumors by dimethyl hydrazine (DMH) was assessed in Wistar rats. One hundred and fifty-one rats were randomly divided into two groups. Seventy-two rats were treated with DMH alone and 79 rats were treated with DMH and PS-K. All animals were subjected to a sequential autopsy and all lesions within the gastro-intestinal tract were examined macroscopically and histologically. Tumor incidence in the DMH plus PS-K-treated group was significantly lower than that in the group treated with DMH alone. The most interesting histological finding was marked lymphoid infiltration in and around the tumors of the rats in the PS-K-treated group. The number of circulating lymphocytes dropped below the control range in the 25th and 35th weeks for the group treated with DMH alone, but the drop in the PS-K-treated group was smaller. Delayed-type hypersensitivity reaction to purified protein derivative was well maintained in the PS-K-treated rats. The most interesting findings in these experiments were differences in the serum blocking activities and serum immunosuppressive substance in these two groups; they were markedly reduced in the PS-K-treated rats. The present results may be explained in terms of competitive action of PS-K against the immunosuppressive factor produced by a tumor-bearing host.

Tissue ferritin concentration in carcinoma of the breast

Ferritin concentration was measured in cytosol extracts of 44 mammary carcinomas and 14 benign breast tissues. A six-fold difference was observed (mean, 364.6 +/- 223.3 ng/mcp in malignant tissue versus mean, 60.2 +/- 42.1 ng/mcp in benign tissue P less than 0.001). Thirty-five malignant tissue specimens were reviewed independently by a pathologist without knowledge of their ferritin contents. Higher concentrations of ferritin were present in malignancies with greater degrees of epithelial proliferation and plemorphism suggesting the malignant epithelium as the major site of the increased ferritin. There was no correlation between desmoplastic reaction within the tumors or inflammation within or adjacent to the tumors and ferritin concentration. Ferritin in breast tissue may be important as a marker of neoplasia, a source of elevated serum ferritin, an indicator of clinical prognosis or an immunosuppressive substance.

Mechanisms by which human gliomas may escape cellular immune attack.

Whereas substantial evidence indicates that the majority of glioma patients make humoral immune responses to their own tumours, the evidence that glioma patients make significant cellular immune responses is more tenuous and controversial. In order to study those properties of human gliomas that might contribute to their ability to escape cell-mediated immune attack, we have examined the ability of cultured human glioma cells to elicit allogeneic cytolytic lymphocyte responses in vitro. Five of ten glioma lines were unable to elicit allogeneic cytolytic lymphocyte responses in mixed lymphocyte-tumour cultures, despite the presence of serologically detectable alloantigens on the surface of the glioma cells. Analysis of the reasons why certain glioma lines failed to stimulate cytolytic lymphocyte responses revealed three distinct mechanisms by which human gliomas may escape cellular immune attack: 1. a defect in immunogenicity which can be overcome by "help" from an allogeneic mixed lymphocyte reaction, 2. the secretion of a protective mucopolysaccharide coat, and 3. the production of macromolecular immunosuppressive substance(s). The implications of these findings for the immunotherapy of human gliomas are discussed.

Effect of PSK against immunosuppressive substance in tumor bearing hosts

Effect of PSK against immunosuppressive substance in tumor bearing hosts

Immunosuppression in murine malaria: a soluble immunosuppressive factor derived from Plasmodium berghei-infected blood.

Mice infected with Plasmodium berghei have a depressed immune response to a variety of antigens. We report the extraction, purification, and characterization of a soluble immunosuppressive substance derived from P. berghei-infected mouse blood. A crude extract, prepared by solubilization of infected erythrocytes in a Parr cell disruption bomb, reduced the anti-DNP PFC response of mice injected with the extract 1 day before immunization. Purification of the immunosuppressant was accomplished by precipitation with 50% saturated ammonium sulfate followed by chromatography on Sephadex G-150 in the presence of 6 M guanidine hydrochloride. The immunosuppressive activity was recovered in the last fraction eluted from the Sephadex G-150 column that contained low m.w. components. The activity was abrogated by trypsin digestion, but not by periodate oxidation. Volume for volume, the purified immunosuppressant had a 100-fold greater activity than the crude extract from which it was derived. It suppressed the response to the T-dependent antigens DNP-KLH and SRBC, but not to the T-independent antigen DNP-Ficoll.

Immunosuppressive effect of ovine uterine secretory protein upon lymphocytes in vitro.

The immunosuppressive effect of ovine uterine protein upon phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes was investigated in vitro. Uterine secretory protein was collected and concentrated in sheep uterine flushings (SUF) obtained from nonpregnant and pregnant Dorset ewes on Days 4, 9, and 14 of the cycle. Varying concentrations of protein (2.5–1600μg/ml) were added to cultures each containing 1 × 106 ovine lymphocytes and 0.6 μg of PHA. Lymphocyte response to PHA was suppressed in cultures containing SUF protein from Day 4, 9, and 14 nonpregnant and pregnant ewes. The regression of SUF protein concentration with lymphocyte suppression (as percent of control) for each day and reproductive stage was significant (P<0.001). Almost complete suppression occurred with much less SUF protein (100 μg/ml) obtained on Day 14 of pregnancy than was obtained at most other times (1600 μg/ml). Ovine serum and bovine serum albumin had no significant immunosuppressive effect. The immunosuppressive effect of SUF protein was not mediated by cytotoxicity. It is unknown whether lymphocyte suppression observed in vitro occurs in vivo. If a parallelism exists, data from this investigation strongly suggest that the ovine uterus may possess a continual immunosuppressive substance(s) during pregnancy and the estrous cycle and that the activity of this substance increases considerably by Day 14 of pregnancy.

Fractionation and characterization of the immunosuppressive substance in crude extracellular products released by Streptococcus intermedius.

The noncytotoxic immunosuppressive substance detected in crude extracellular products of Streptococcus intermedius (CEP-SI) was fractionated by two steps of preparative isoelectric focusing in sucrose gradients using ampholytes of pH range from 3.5 to 6 and 4 to 5, respectively. The in vitro and in vivo suppressor effects of the most highly purified fraction of CEP-Si, designated fraction 3' (F3'EP-Si), corresponded well with those of the original CEP-Si. F3'EP-Si was sensitive to the effects of alpha, gamma, and delta chymotrypsin, trypsin, and heating. It contained approximately 1% of the total amount of protein found in the original CEP-Si, corresponding to a single band on analytical isoelectric focusing, stainable by Coomassie Blue and of isoelectric point of 4.25. The absorption spectrum of F3'EP-Si had a maximum at 260 nm but its biological activity was resistant to deoxyribonuclease and ribonuclease A and it did not contain material stainable by methylene blue. It was also resistant to neuraminidase and did not contain material stainable by periodic acid schiff. We conclude that the substance responsible for the suppressor activity of CEP-Si is a protein of molecular weight approximately 90,000, which adheres to Sephadex of cellulose acetate and forms complexes with other, nonactive constituents of CEP-Si.