Syngeneic Serum Research Articles

Cure of Mice with Advanced Ovarian Adenocarcinoma CaO1 by the Serum Blood Proteins.

After removal of the primary tumor node, tumor-specific activity appears in the serum that blocks tumor growth in mice. This activity is observed at the time interval when activity of the tumor growth-stimulating factor is not determined. Administration of blood serum (0.1 ml) from mice with removed tumor to mice with CaO1 adenocarcinoma for 14 days led first to a stop of its growth, and then to tumor regression. The animals cured of adenocarcinoma lived for at least one year without signs of relapse. The cured animals did not develop resistance to repeated tumor transplantation. Repeated transplantation led to the growth of the new tumor. No cellular immune response was observed on histological slides of the regressing tumor. It was concluded that a serum factor is required for the growth of a tumor in the body and the state of the serum with blocked activity of this tumor-stimulating factor can be used for tumor treatment in oncology patients. This is the first result in the syngeneic system, when the tumor was cured by syngeneic serum proteins.

Излечение мышей с распространённой аденокарциномой яичников СаО1 с помощью белков сыворотки крови

After removal of the primary tumor node, tumor-specific activity appears in the serum that blocks tumor growth in mice. This activity is observed at the time interval when activity of the tumor growth-stimulating factor is not determined. Administration of blood serum (0.1 ml) from mice with removed tumor to mice with CaO1 adenocarcinoma for 14 days led first to a stop of its growth, and then to tumor regression. The animals cured of adenocarcinoma lived for at least one year without signs of relapse. The cured animals did not develop resistance to repeated tumor transplantation. Repeated transplantation led to the growth of the new tumor. No cellular immune response was observed on histological slides of the regressing tumor. It was concluded that a serum factor is required for the growth of a tumor in the body and the state of the serum with blocked activity of this tumor-stimulating factor can be used for tumor treatment in oncology patients. This is the first result in the syngeneic system, when the tumor was cured by syngeneic serum proteins.

Blood transfusion and recurrence of colorectal cancer: The role of platelet derived growth factors

Efforts to explain the possible effects of blood transfusion on the recurrence of colorectal cancer have been based entirely on the immunosuppressive effects of blood transfusion. However, the relationship between solid tumour development and the immune system is inconclusive. We have investigated an alternative mechanism involving the potential role of growth factors in this phenomenon. Using a human fibroblast: [125I]deoxyuridine uptake mitogenesis assay, the relative amounts of growth factor in the plasma of stored blood were measured. There was a progressive increase in mitogenesis from day 0 (n = 6) to day 28 (n = 6; P less than 0.001, Mann-Whitney U test). The effect of growth factors on the development of liver and intraperitoneal metastases was studied in Hooded Lister rats. Following an intraportal injection of 10(5) MC28 tumour cells, the experimental group (n = 25) received 2 ml of syngeneic serum intravenously for 4 days. Likewise, colonic anastomoses were performed on omentectomized rats and the peritoneal cavity seeded with 10(3) cells. The experimental groups (n = 20) received either 2 ml serum intravenously repeatedly or 3 ml serum intraperitoneally (n = 19). There was no significant increase in liver metastases or peritoneal disease following intravenous infusion of serum but serum delivered intraperitoneally resulted in a significant increase in tumour from 22 per cent in the controls to 89 per cent in the study group (P less than 0.01). Growth factors released from platelets following blood loss into the peritoneal cavity may be important in enhancing local recurrence of colorectal cancer.

Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells.

Presentation of MHC class I-restricted peptides by dendritic cells (DCs) can elicit vigorous antigen-specific CTL responses in vivo. It is well established, however, that T cell help can augment CTL function, raising the question of how best to present tumor-associated MHC class I epitopes to induce effective tumor immunity. To this end, we have examined the role of MHC class II peptide-complexes present on the immunizing DCs in a murine melanoma model. To present MHC class I- and II-restricted Ags reliably on the same cell, we retrovirally transduced bone marrow-derived DCs with the model Ag OVA encoding well-defined class I- and II-restricted epitopes. The importance of CD4+ T cells activated by the immunizing DCs in this model is demonstrated by the following findings: 1) transduced DCs presenting class I and class II epitopes are more efficient than class I peptide-pulsed DCs; 2) MHC class II-deficient DCs fail to induce tumor protection; 3) CD4+ T cell depletion abolishes induction of tumor protection; and 4) DCs presenting bovine serum Ags are more effective in establishing tumor immunity than DCs cultured in syngeneic serum. When MHC class II-deficient DCs were directly activated via their CD40 receptor, we indeed observed a moderate elevation of OVA-specific CTL activity. However, this increase in CTL activity was not sufficient to induce in vivo tumor rejection. Thus, our results demonstrate the potency of genetically modified DCs that express both MHC class I and II epitopes, but caution against the use of DCs presenting only the former.

Reduced tumorigenicity of threshold syngeneic tumor inocula in XID‐bearing mice treated with natural antibodies

Several correlative experimental approaches have provided evidence that polyclonal serum natural antibodies (NAb) participate in the defense against small syngeneic tumor foci in vivo. The threshold subcutaneous tumor inoculum model of incipient neoplasia has consistently revealed an inverse relationship between tumorigenicity in vivo and the NAb binding capacity of the tumor injected or the anti-tumor NAb levels of the recipient animals, including B cell-deficient mice bearing the xid mutation of the CBA/N mouse strain. Now passive i.v. administration of whole normal syngeneic serum NAb in bolus injections, given I each day beginning on day -2, -1 or 0 prior to the threshold s.c. challenge of xid-bearing CBA/N or male (CBA/N x CBA/J)F1 mice with syngeneic RI-28 lymphoma cells, consistently and significantly reduced their tumorigenicity assayed as tumor appearance and latency. Three similar injections of an ammonium sulfate-precipitated fraction of whole serum NAb also reduced tumor frequency and latency, while a combination of purified IgG and IgM NAb reduced the appearance of tumors slightly and increased the survival of recipients. The reconstitution of the xid-defect in anti-tumor NAb provides more direct evidence to substantiate a role for NAb in the defense against early tumor development in vivo and establishes a model for the dissection of the in vivo mechanisms of the NAb anti-tumor activity.

Simultaneous intraperitoneal administration of OK-432 and serum enhances superoxide generation from migrated polymorphonuclear leukocytes, with special emphasis on the role of complements.

Superoxide and its derived active oxygen species are responsible for the polymorphonuclear leukocyte (PMN)-mediated tumoricidal activity which is typically shown in the intraperitoneal administration of OK-432, a biological response modifier, for cancer ascites. We examined the effects of intraperitoneal administration of OK-432 with or without syngeneic serum on superoxide generation from PMNs which migrated in the peritoneal cavity using the new method of Cypridina luciferin analog-dependent chemiluminescence for the detection of superoxide. PMNs harvested from rat peritoneal cavity 6 h after the intraperitoneal administration of OK-432 (0.25KE/kg, or 2.5KE/kg) generated larger amounts of superoxide than those harvested after intraperitoneal injection of oyster glycogen (75mg/kg) when stimulated by opsonized zymosan or phorbol myristate acetate. Simultaneous intraperitoneal administration of OK-432 and syngeneic serum induced a greater increase in PMN superoxide generation than OK-432 alone, which was reversed by a complement activation inhibitor (MX-1). Simultaneous injection of OK-432 and heat-inactivated syngeneic serum did not exhibit a significant increase in PMN superoxide generation as compared with OK-432 alone. These results provide pharmacological evidence to the satisfactory therapeutic effects of the intraperitoneal administration of OK-432 with or without serum for patients with cancer ascites, and indicate that complements, in particular C5a, are involved in this enhanced PMN-derived superoxide generation induced by the simultaneous injection of OK-432 and serum.

Interactions between Oligodendrocytes and Complement

A unique relationship between oligodendrocytes and complement has emerged from studies of cultured glial cells and syngeneic serum. Rat oligodendrocytes bear a surface component that may activate the first component of the classical pathway of complement, C1; they also lack a key complement regulatory protein, protectin (CD59), rendering them unduly sensitive to complement attack. This may have implications for developmental neurobiology and for clinical disorders affecting oligodendrocytes. Furthermore, in cell culture techniques, negative selection strategies for the purification of glia may include the sole addition of fresh syngeneic serum as a means of removing rat oligodendrocytes. Alternatively, methods for removing oligodendrocytes, bipotential progenitor cells, or microglia using cell-specific antibodies are also available.

Interaction between thymocytes and thymus-derived macrophages: II. Engulfment of thymocytes by macrophages

A high percentage (80–90%) of immature thymocytes were engulfed by syngeneic thymus-derived macrophages (TDMφ) following cocultivation for 3 days. Elimination occurred via internalization of thymocytes by the macrophages. We unequivocally demonstrated the presence of many live thymocytes inside the TDMφ by means of specific staining. Mature PNA − thymocytes were phagocytized to a lower degree than immature thymocytes, and T splenocytes were not eliminated at all. Bone marrow-derived macrophages internalized immature thymocytes to a degree similar to TDMφ. Since thymocyte survival was not at all affected by Mφ culture supernatants alone, we conclude that cell to cell contact is necessary for thymocyte elimination. To identify the surface molecules which participate in internalization of thymocytes by the macrophages, both cell types were pretreated with a variety of agents. Treatment of thymocytes with tunicamycin (N-glycosylation inhibitor) and anti-Lyt-2 mAb decreased their elimination by Mφ. Similarly, treatment of Mφ with neuraminidase, trypsin, and anti-Ia mAb markedly suppressed their capacity to engulf thymocytes. On the other hand, thymocyte elimination was unaffected by (1) cell cultivation in syngeneic serum rather than heterologous serum; (2) use of allogeneic rather than syngeneic thymocytes; and (3) use of X-irradiated Mφ and LPS-activated Mφ rather than nontreated Mφ.

Serotherapy of cancer: cellular changes in primary rat mammary carcinomas after infusion of syngeneic sera absorbed with protein A-Sepharose.

Serotherapy and plasma therapy have proved to be effective in the treatment of diverse neoplasms. The mechanisms of the tumoricidal or growth-inhibitory effects are unknown. We previously reported that activation of the alternative pathway of complement in absorbed sera correlated with the presence of anti-tumor activity. Complement components generated during absorption may serve as the initial mediators of cytotoxicity; for example, C5a may function in its role as a chemo-attractant. To further investigate the anti-tumor mechanisms, we undertook a series of sequential histological studies of in vivo changes in tumors following i.v. serotherapy. We found diffuse inflammatory cellular infiltrates in the interstitial compartments of primary mammary carcinomas of rats within 3-4 hr of administration of protein A-Sepharose absorbed syngeneic serum. The number of inflammatory cells was significantly higher in tumors from treated rats: total infiltrating cells (p = 0.002), eosinophils (p = 0.001), neutrophils (p = 0.001), macrophages (p = 0.001), lymphocytes (p = 0.004) and plasma cells (p = 0.001). Also, the mitotic index of tumor cells was significantly lower 4 hr after serotherapy when compared with that of untreated rat tumor cells. C3 in tumor tissue was decreased at 4 hr following serotherapy. Fibrosis was present in tumor nodules with retarded growth 5 weeks after the start of serotherapy. Localization of the infiltrating cells to tumor interstitial compartments prevents direct contact between inflammatory cells and neoplastic cells, making it unlikely that direct cell-cell killing occurs. Indirect cell killing within the tumor bed apparently occurs through several mechanisms involving interactions between serotherapy-initiated humoral mediators and inflammatory cells. The resulting anti-tumor effects include microvascular injury leading to localized ischemia, tumor infarction, and fibroblastic reactions obstructing tumor invasion and growth.

Mouse T-cell-dependent proliferative response to syngeneic serum modified with various agents used in root canal therapy

Syngeneic sera modified with seven agents were used as the immunogens to induce lymph-node cell proliferation in C57BL/6 mice. Immunogenicity could be conferred upon tolerated syngeneic serum by modification with diluted tincture of iodine (J) or formocresol (FC). However, the synergistic action of formalin and cresol appeared to be an indispensable condition in acquiring immunogenicity of FC-modified syngeneic serum (FC-MS). Moreover, immunological tolerance to native serum was not terminated after injection of J-MS or FC-MS. These proliferative functions were dependent on Lyt-1 cells. The cells directed against J- or FC-modified syngeneic serum were specific for the immunizing hapten J or FC. However, they did not recognize the hapten per se, but rather, the conjugate-specific determinants, i.e. these contributed by both the hapten and the self-carrier.

Autoimmune orchitis can be induced by thymic lymphocytes autosensitized against syngeneic sertoli cells in vitro.

Thymic lymphocytes from normal inbred Lewis/Wistar rats were cocultured with syngeneic Sertoli cell-peritubular cell preparations in the presence of heterologous or allogeneic serum. Thymic cells cultured in this manner bound to Sertoli cells, became autosensitized , and markedly altered syngeneic Sertoli cell surface properties and remodeling functions in vitro. In contrast, control thymic cells incubated with Sertoli cells in autologous or syngeneic serum did not become sensitized. Coculture of autosensitized thymic cells with syngeneic seminiferous tubule segments, or local transfer of such lymphocytes into syngeneic rat testes, resulted in intratubular infiltration by "light cells." Intratesticular injection of autosensitized thymic cells was followed by derangement of the seminiferous epithelium, and by morphologic changes characteristic of experimental autoimmune orchitis. Thymic cells incubated with Sertoli cells in autologous or syngeneic serum did not elicit these changes. Thymic cells incubated with peritubular cells in heterologous or autologous serum behaved like control thymocytes, and were not sensitized. Data presented indicate that thymic cells are potentially capable of recognizing syngeneic Sertoli cell self-antigens. We speculate that factors normally present in serum may inhibit the recognition by thymic lymphocytes of antigenic determinants present on Sertoli cells. We discuss the possibility that the modulation of interactions between immature thymic lymphocytes and Sertoli cells is implicated in the prevention of autoimmune reactions against the testis.

Spontaneous proliferation in unfractionated spleen cell cultures: Autologous mixed-lymphocyte reactions (AMLR) which can be differentially regulated by prostaglandins and lymphokines

The present studies were undertaken to define the contribution of the autologous or syngeneic mixed-leukocyte reactions (AMLR/SMLR) to the cellular proliferation observed in unfractionated spleen cell cultures. Proliferation was studied in whole, untreated 6-day murine spleen cell cultures supplemented with syngeneic serum. These cultures exhibited relatively low but significant levels of cellular proliferation as measured by uptake of radioactive thymidine ([ 3H]TdR). Treatment of spleen cells with monoclonal anti-Thy 1.2 antibody and complement before culture, the addition of specific anti- I- A monoclonal antibodies to the cultures or removal of Ia + adherent cells before initiation of culture all inhibited the proliferative response significantly. Thus, the autologous proliferation of untreated and unfractionated spleen cells manifests the main characteristics of the AMLR/SMLR, namely, its dependence on T (responder) and Ia + (stimulator) cells and specific inhibition by anti- I- A antibodies. A marked augmentation in cellular proliferation was observed in unfractionated spleen cell cultures treated for the initial 24 hr of culture with 5 × 10 −6 M indomethacin, an inhibitor of prostaglandin synthesis. Conversely, the addition of 7 × 10 −9 M prostaglandin E 1 (PGE 1) to these cultures depressed cellular proliferation. This suppression of autologous splenic cell proliferation induced by PGE 1 could be partially reversed by the addition of concanavalin A-induced lymphokine (LK) preparations early in the culture. These findings indicate that (a) the proliferation of unfractionated spleen cell cultures occurring in the absence of exogenous stimulatory signals is due largely to an ongoing AMLR, and (b) biologically active mediators with opposing influences, namely, prostaglandins and immunostimulatory LK, participate in the regulation of the AMLR.

Chromosome Fragility in New Zealand Black Mice: Effect of Syngeneic, Allogeneic, and Xenogeneic Sera on Fetal Fibroblasts In Vitro<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The sensitivity of fetal fibroblasts from New Zealand Black (NZB) and BALB/c mice to syngeneic, allogenic, and xenogeneic sera was tested. The NZB fetal fibroblasts manifested significantly more chromosome abnormalities when grown in the presence of syngeneic sera or in one of three tested allogeneic sera but no in xenogeneic sera. In contrast, no chromosome fragility was observed in BALB/c fetal fibroblasts tested under similar conditions. These findings suggested that NZB fetal fibroblasts are not generally unstable but that the instability is expressed only in response to specific factors(s) present in syngeneic sera from young and old mice.

MURINE AUTOGENEIC MIXED LYMPHOCYTE CULTURE ROLE OF CULTURE CONDITIONS

In an attempt to determine whether culture conditions significantly influence autoreactivity, we tested the effects of fetal calf, syngeneic, and autogeneic serum with and without 2-mercaptoethanol on the murine self-reactive mixed lymphocyte culture. We used both unfractionated and T-enriched lymph node cells as responders and unfractionated and B-enriched irradiated spleen cells as stimulators in a primary mixed lymphocyte culture (MLC). Using unfractionated cells as responders to various stimulator cell populations, we found excellent allogeneic reactions in all media tested but minimal or no self-reactivity in syngeneic of autogeneic serum. When using T-enriched lymph node cells as responders, allogeneic reactivity was excellent but self-reactivity was present only in the cultures supplemented with fetal calf serum and 2-mercaptoethanol. The possibility that the substances in fetal calf serum and/or 2-mercaptoethanol may be needed to enhance minimal, but biologically relevant, self-reactivity is discussed, as well as the possibility that the culture supplements may be inducing "nonspecific" self-reactivity.

Augmentation by serum-induced helper cells of T cell-mediated cytotoxic response against tumor associated antigens and alloantigens.

In vitro T cell-mediated cytotoxic responses to tumor associated antigens or alloantigens can be augmented by the addition of small amounts (0.1 to 1%) of syngeneic (mouse) or xenogeneic (rabbit) serum in the standard lymphocyte culture medium. Further studies showed that the augmentation is mediated by helper cells, which are induced by culturing the spleen cells or lymph node cells in the presence of these sera. In the syngeneic system performed with mixed lymphocyte tumor cell cultures (MLTC), the serum-induced helper cells are found to be resistant to the lysis of anti-Thy 1.2 antibody and are radioresistant; thus they have the characteristics of macrophages. In the allogeneic system performed with mixed lymphocyte culture (MLC), the serum-induced helper cells are also found to be resistant to the lysis of anti-Thy 1.2 antibody but are radiosensitive. In the latter case, however, removal of T cells abolishes the helper cell generation and only the T cell-enriched fraction provides for the generation of helper cells, indicating that the helper cells for MLC are probably derived from T cells but lose their susceptibility to anti-Thy 1.2 antibody lysis upon culturing in vitro. A study of the mode of action of the helper cells for MLC showed that they are probably needed at a later stage of cytotoxic response for the amplification of the killing efficiency of the T effector cells whereas the helper cells for MLTC are needed in the early induction phase of the immune response. These results indicate that although serum can augment the cytotoxic responses both in the syngeneic and in the allogeneic systems, the mechanism for the augmentation differs: macrophagelike helper cells are responsible for the augmentation of cytotoxic response to tumor associated antigens, whereas augmentation of cytotoxic response to alloantigens appears to be mediated by a subpopulation of T helper cells.

Incorporation of labeled cortisol into different types of connective tissue

The properties of early (BFUen) and late (CFUen) erythropoietin-independent progenitors were studied in bone marrow plasma clot cultures of mice. Syngeneic serum was used as a stimulant of colony formation. It was revealed that serum from polycytemic mice did not alter the efficacy of cloning the erythroid progenitors. Varying effects produced on erythropoiesis in vivo did not significantly change the number of CFUen and BFUen, with the exception of posttransfusion polycytemia where the number of CFUen showed a statistically significant rise. Assessment of radiosensitivity and proliferative activity of the erythroid progenitors showed that for CFUen D0=206 rad and n=1.3; for BFUen D0=118 rad and n=1.4; the proportion of proliferating CFUen was 42% +/- 12.5 and that of BFUen 46% +/- 4.5. The data obtained confirm the hypothesis of the erythropoietin-independent nature of the erythroid progenitors expressed under the culture conditions used.

Effect of prolonged immunization of mice with bovine serum albumin on the incidence of chemically induced tumors.

The effect of prolonged immunization of mice with bovine serum albumin (BSA) on tumors elicited by injection of 3-methylcholanthrene was studied. The incidence of tumors and rate of tumor growth were higher in mice immunized with BSA than in control mice injected with syngeneic serum. The possibility of an immunosuppressive effect of the prolonged immunization with BSA is discussed.

A sensitive assay for the detection of cytotoxic antibodies to mammalian cell surface antigens

A microcytotoxicity assay for the detection of complement-dependent serum toxicity against adherent cells has been modified to include post-labeling of the target cells with [ 3H]leucine or [ 3H]thymidine. The adaptation represents a significant improvement over the visual counting of residual cells. The technique is more objective and less tedious than the original procedure, while retaining the features of economy of cells and sera, reproducibility, and sensitivity. Examples are given which demonstrate the suitability of the modified assay for the analysis of human and mouse tumor antigens using autologous, allogeneic, and syngeneic sera.

Modification of the immunogenicity and antigenicity of rat hepatoma cells. I. Cell-surface stabilization with glutaraldehyde

gamma-Irradiated rat hepatoma cells are immunogenic in syngeneic WAB/Not rats, so that immunized animals are protected against tumour-cell challenge and circulating tumour-specific antibody is produced. Treatment of the immunizing cells with glutaraldehyde at concentrations of 0.001% or greater for 30 min rendered these cells non-protective in tumour-rejection tests and no longer able to induce significant formation of specific antibody. However, tumour-specific antigens were shown to be expressed upon treated cells; they specifically bound tumour-specific antibody from syngeneic immune sera assessed in indirect membrane-immunofluorescence tests. Also, these cells specifically absorbed antibody from immune or tumour-bearer sera, as demonstrated in the indirect membrane-immunofluorescence test or a complement-dependent 51Cr-release test. Alloantigen expression was not influenced by glutaraldehyde treatment, although glutaraldehyde-treated hepatoma cells failed to induce alloantibody formation in KX/Not rats. Polyacrylamide-gel electrophoresis of treated cells, surface-labelled with 125I, indicated that extensive cross-linking of the surface protein occurred as a result of glutaraldehyde treatment. The present findings establish that although the expression of a tumour-specific antigen is necessary for the induction of immuno-protection against tumour-cell challenge, this alone is not a sufficient condition for eliciting tumour immunity.

Murine natural anti‐tumor antibodies. I. Rapid in vivo binding of natural antibody by tumor cells in syngeneic mice

A competitive radioimmunoassay (RIA) for the detection of cell-bound antibody was used to study the in vivo acquisition of immunoglobulin (Ig) by tumor cells. Several tumor lines acquired Ig rapidly between 3 and 18 h after intraperitoneal implantation into normal syngeneic mice and this Ig was recovered by elution with basic or acid buffers. The Ig eluted from the L5178Y lymphoma showed higher binding to the L5178Y than to thymocytes, bone-marrow cells, 1509a sarcoma and P-815-X2 mastocytoma. In addition, binding of the eluates to the L5178Y was specifically inhibited by L5178Y cells or by solubilized membrane antigens of the L5178Y. The in vivo acquisition of Ig by the L5178Y could also be blocked by the IV and IP injections of tumor antigen although both L5178Y and 1509a solubilized membrane antigens were effective. Some of the Ig acquired by the tumor cells was found to be complement-fixing antibody since normal rabbit complement lysed 80% of L5178Y cells obtained from the peritoneal cavity of syngeneic mice 18 h after implantation, but did not lyse in vitro L5178Y cells. The in vivo binding of the complement-fixing antibodies was also inhibited by tumor antigens in the same way as the acquisition of Ig detected by RIA. It was shown that the acquisition of Ig during the first 18h of IP growth was a T-independent phenomenon because tumor cells acquire as much Ig in AT X BM mice as in sham-thymectomized controls. In a study with 11 different clones derived from the L5178Y lymphoma, a high correlation (r = 0.75, p less than 0.005) was found between the amount of Ig acquired after in vivo implantation and the amount of Ig bound to the cells after in vitro incubation with normal syngeneic serum. It is suggested that the rapid in vivo acquisition of Ig was due to the in vivo binding of natural antibodies to tumor cells.