State Of Immunological Tolerance Research Articles

Aspectos inmunológicos de preeclampsia

Preeclampsia is a pathology with high morbidity and mortality worldwide. In this disease, the placenta is an organ of shock where inflammation and the immune response generate the damage that results in the characteristic clinical scenario. The classic triad in preeclampsia is made up of hypertension, edema, and proteinuria, so it is thought that the endothelium must be affected by inflammatory-immunological activity. The immune system acts in the development of pregnancy and does so at different times and regulating physiologically. Both, cellular and humoral components of the innate and acquired response have been studied in patients with preeclampsia and it has been determined that their participation is decisive in the pathophysiology of this disease. The involvement of the immune system in the pathophysiology of preeclampsia reaches a high level of complexity since it interacts with other systems (coagulation, renal, cardiovascular and endocrinological among others) thus favoring the disease. For this reason, treatment must be comprehensive, with a holistic vision of the condition and requires a multidisciplinary team that acts harmoniously to achieve the greatest therapeutic success with the least frequency of sequelae for the mother-fetus or mother-newborn dyads. During pregnancy, the so-called "immunological tolerance of pregnancy" develops, in this state of immunological tolerance the B and T cells can recognize specific antigens (for example, the paternal ones) and later activate and generate the immune response, which is why preeclampsia could being considered an autoimmune pathology, where the loss of immunological tolerance would be the cornerstone of pathophysiology, knowing how to limit or regulate this abnormal cell activation could help to propose new therapeutic approaches and thus control this disease.

Are Aspects of Integrative Concepts Helpful to Improve Pancreatic Cancer Therapy?

Numerous clinical studies have been conducted to improve the outcomes of patients suffering from pancreatic cancer. Different approaches using targeted therapeutic strategies and precision medicine methods have been investigated, and synergies and further therapeutic advances may be achieved through combinations with integrative methods. For pancreatic tumors, a particular challenge is the presence of a microenvironment and a dense stroma, which is both a physical barrier to drug penetration and a complex entity being controlled by the immune system. Therefore, the state of immunological tolerance in the tumor microenvironment must be overcome, which is a considerable challenge. Integrative approaches, such as hyperthermia, percutaneous irreversible electroporation, intra-tumoral injections, phytotherapeutics, or vitamins, in combination with standard-oncological therapies, may potentially contribute to the control of pancreatic cancer. The combined application of standard-oncological and integrative methods is currently being studied in ongoing clinical trials. An actual overview is given here.

LPS enhances CTB-INSULIN induction of IDO1 and IL-10 synthesis in human dendritic cells

Autoantigen-specific immunotherapy promises effective treatment for devastating tissue specific autoimmune diseases like multiple sclerosis (MS) and type 1 diabetes (T1D). Because activated dendritic cells (DCs) stimulate the differentiation of autoreactive T cells involved in the initiation of autoimmunity, blocking the activation of DCs may be an effective strategy for inhibiting tissue specific autoimmunity. Following this approach, immature DCs were shown to remain inactive after treatment with chimeric fusion proteins composed of the cholera toxin B subunit adjuvant linked to autoantigens like proinsulin (CTB-INS). Mass spectrometer analysis of human DCs treated with CTB-INS suggest that upregulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) is responsible for inhibiting DC activation thereby resulting in a state of immunological tolerance within the DC. Here we show that the fusion protein CTB-INS inhibits human monocyte derived DC (moDC) activation through stimulation of IDO1 biosynthesis and that the resultant state of DC tolerance can be further enhanced by the presence of residual E. coli lipopolysaccharide (LPS) present in partially purified CTB-INS preparations. Additional experiments showed that LPS enhancement of DC tolerance was dependent upon stimulation of IDO1 biosynthesis. LPS stimulation of increased levels of IDO1 in the DC resulted in increased secretion of kynurenines, tryptophan degradation products known to suppress DC mediated pro-inflammatory T cell differentiation and to stimulate the proliferation of regulatory T cells (Tregs). Further, the presence of LPS in CTB-INS treated DCs stimulated the biosynthesis of costimulatory factors CD80 and CD86 but failed to upregulate maturation factor CD83, suggesting CTB-INS treated DCs may be maintained in a state of semi-activation. While treatment of moDCs with increasing amounts of LPS free CTB-INS was shown to increase DC secretion of the anti-inflammatory cytokine IL-10, the presence of residual LPS in partially purified CTB-INS preparations dramatically increased IL-10 secretion, suggesting that CTB-INS may enhance DC mediated immunological tolerance by stimulating the proliferation of anti-inflammatory T cells. While the extraction of LPS from bacterial generated CTB-INS may remove additional unknown factors that may contribute to the regulation of IDO1 levels, together, our experimental data suggest that LPS stimulates the ability of CTB-INS to induce IDO1 and IL-10 important factors required for establishment of a state of functional immunological tolerance in human DCs. Regulation of the ratio of LPS to CTB-INS may prove to be an effective method for optimization of readily available “off the shelf” CTB-INS mediated immune-therapy for tissue specific autoimmune diseases including type 1 diabetes.

Targeting tumor tolerance: A new hope for pancreatic cancer therapy?

With a 5-year survival rate of just 8%, pancreatic cancer (PC) is projected to be the second leading cause of cancer deaths by 2030. Most PC patients are not eligible for surgery with curative intent upon diagnosis, emphasizing a need for more effective therapies. However, PC is notoriously resistant to chemoradiation regimens. As an alternative, immune modulating strategies have recently achieved success in melanoma, prompting their application to other solid tumors. For such therapeutic approaches to succeed, a state of immunologic tolerance must be reversed in the tumor microenvironment and that has been especially challenging in PC. Nonetheless, knowledge of the PC immune microenvironment has advanced considerably over the past decade, yielding new insights and perspectives to guide multimodal therapies. In this review, we catalog the historical groundwork and discuss the evolution of the cancer immunology field to its present state with a specific focus on PC. Strategies currently employing immune modulation in PC are reviewed, specifically highlighting 66 clinical trials across the United States and Europe.

Forbearance of the Immune System during pregnancy: Fetal parasite takes an estrogenic way!

Understanding the mission of managing the genetically diverging fetus as “transitory self” by the mother during pregnancy is vital in detailing the state of immunological tolerance. Hormones dictate the whole event and helps in enrolling, differentiating and altering the functions of immune cells. Estrogen the decisive hormone during embryo implantation as it activates embryo for adhesion and determines the window of uterine receptivity, along with progesterone exerts control over differentiating endometrial and immune cells. This review focuses on how estrogen modulates various immune cells to develop a uterine milieu conducive for the growth of the fetus.

Salmonella enterica Serovar Enteritidis Enterocolitis during Late Stages of Gestation Induces an Adverse Pregnancy Outcome in the Murine Model

Foodborne diseases caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) are a significant health problem. Pregnancy, state of immunological tolerance, is a predisposing condition for the development of infections with intracellular pathogens. Salmonella species can cause pregnancy complications such as chorioamnionitis, transplacental fetal infection, pre term labor, abortions, neonatal and maternal septicemia. However, the specific mechanisms by which Salmonella infections trigger these alterations are not clear. In the present work, using a self-limiting enterocolitis murine model, we show that the ingestion of a low dose of S. Enteritidis at late stages of pregnancy (day 15 of gestation) is sufficient to induce massive maternal infection. We found that Salmonella infection leads to 40% of pre term delivery, 33% of abortion and fetal growth restriction. Placental dysfunction during S. Enteritidis enterocolitis was confirmed through cellular infiltration and hypoxia markers (MPO activity and COX-1 and COX-2 expression, respectively). Apoptosis in placental tissue due to Salmonella infection was also evident at day 18 of gestation when investigated by morphometric procedure, DNA fragmentation and Fas/FasL expression. Also, the expression of IFN-γ, TNF-α, IL-17 and IL-10 was up regulated in response to Salmonella not only in placenta, but also in amniotic fluid and maternal serum. Altogether, our results demonstrate that S. Enteritidis enterocolitis during late stages of gestation causes detrimental effect on pregnancy outcome.

Costimulation Blockade Induces Foxp3+ Regulatory T Cells to Human Embryonic Stem Cells

Transplantation of human embryonic stem cells (hESCs), like other allogeneic cellular transplants, require immunomodulation or immunosuppression in order to be maintained in the recipient. Costimulation blockade applied at the time of transplantation inhibits costimulatory signals in the immunological synapse leading to a state of anergy in the donor reactive T-cell population and a state of immunological tolerance in the host. In models of solid organ transplantation, tolerance is maintained by the infiltration of Foxp3+ regulatory T cells into the graft. In order to study if regulatory T cells could be generated to hESC transplants, costimulation blockade (CTLA4Ig, anti-CD40L, anti-LFA-1) was administered for the first week after transplantation of two different hESC lines implanted under the kidney capsule of wild-type mice. hESC transplants were maintained indefinitely, and when harvested at long-term follow-up, Foxp3+ T-cells were found surrounding the graft, implying the maintenance of tolerance through the induction of regulatory T cells. These results imply that costimulation blockade could be a useful treatment strategy for the induction of tolerance to hESC transplants and may down-modulate immune responses locally around the graft.

Adenovirus mediated CTLA4Ig transgene therapy alleviates abortion by inhibiting spleen lymphocyte proliferation and regulating apoptosis in the feto-placental unit

Pregnancy is thought to be a state of immunological tolerance. The mechanisms underlying this phenomenon are still poorly understood. In our previous study, adenovirus mediated CTLA4Ig transgene (Ad-CTLA4Ig) therapy was demonstrated to improve pregnancy outcome in an abortion-prone mouse model by skewing the Th2/Th1 cytokine balance, expanding peripheral CD4+ CD25+ regulatory T cell populations and inducing indoleamine 2,3 dioxygenase (IDO) mRNA expression at the maternal–fetal interface. However, it is still not clear whether other mechanisms are involved in the protective effect of CTLA-4 on pregnancy outcome in abortion-prone matings. In this study, we focused on the effect of CTLA4Ig on spleen lymphocyte proliferation and apoptosis at the maternal–fetal interface. We demonstrated that Ad-CTLA4Ig therapy inhibited the proliferation of CBA/J splenocytes and IL-2 secretion in response to DBA/2 stimulator cells in the abortion-prone mice model. Ad-CTLA4Ig therapy also skewed cytokine production toward a Th2 bias and regulated the expression of anti-apoptosis factor Bcl-2 and pro-apoptosis factor Bax at the maternal–fetal interface. However, it did not influence the apoptosis and cell cycles of splenocytes in pregnant CBA/J mice. On the whole, these findings indicated that Ad-CTLA4Ig therapy could ameliorate the outcome of spontaneous abortion by inhibiting proliferation of maternal spleen lymphocytes and regulating apoptosis in the feto-placental unit.

ORIGINAL ARTICLE: The Persistence of Paternal Antigens in the Maternal Body is Involved in Regulatory T‐Cell Expansion and Fetal‐Maternal Tolerance in Murine Pregnancy

Citation Zenclussen ML, Thuere C, Ahmad N, Wafula PO, Fest S, Teles A, Leber A, Casalis PA, Bechmann I, Priller J, Volk H‐D, Zenclussen AC. The persistence of paternal antigens in the maternal body is involved in regulatory T‐cell expansion and fetal‐maternal tolerance in murine pregnancy. Am J Reprod Immunol 2010; 63: 200–208Problem Mammalian pregnancy is a state of immunological tolerance and CD4+ CD25+ regulatory T cells (Treg) contribute to its maintenance. Knowing that Treg act in an antigen‐specific way during pregnancy, we hypothesized that they are generated after maternal immune cells encounter paternal antigens.Method of study We mated wild type females with transgenic green fluorescent protein (GFP) males in an allogenic setting and killed them on different days of pregnancy.Results Presence of paternal and maternal MHC class II+ cells in vaginal lavage on day 0.5 of pregnancy was confirmed. Thus, antigen presentation may take place early during pregnancy in the periphery either by the direct or indirect pathways. Foxp3+ cells known to have regulatory activity could be detected on day 2 of pregnancy in lymph nodes and shortly after implantation at the fetal‐maternal interface.Conclusion Our data suggest that paternal antigens are processed early during pregnancy, which leads to the generation of Treg. The continuous release of placental antigens into the maternal circulation allows the maintenance of a Treg population which is specific for paternal antigens and mediates tolerance toward the semi‐allogeneic fetus until the time point of birth.

Mechanisms of Immunization Against Cancer Using Chimeric Antigens

Successful approaches to tumor immunotherapy must overcome the physiological state of tolerance of the immune system to self-tumor antigens. Immunization with appropriate variants of syngeneic antigens can achieve this. However, improvements in vaccine design are needed for efficient cancer immunotherapy. Here we explore nine different chimeric vaccine designs, in which the antigen of interest is expressed as an in-frame fusion with polypeptides that impact antigen processing or presentation. In DNA immunization experiments in mice, three of nine fusions elevated relevant CD8(+) T-cell responses and tumor protection relative to an unfused melanoma antigen. These fusions were: Escherichia coli outer membrane protein A (OmpA), Pseudomonas aeruginosa exotoxin A, and VP22 protein of herpes simplex virus-1. The gains of immunogenicity conferred by the latter two are independent of epitope presentation by major histocompatibility complex class II (MHC II). This finding has positive implications for immunotherapy in individuals with CD4(+) T-cell deficiencies. We present evidence that antigen instability is not a sine qua non condition for immunogenicity. Experiments using two additional melanoma antigens identified different optimal fusion partners, thereby indicating that the benefits of fusion vectors remain antigen specific. Therefore large fusion vector panels such as those presented here can provide information to promote the successful advancement of gene-based vaccines.

T-cell-based immunotherapy in multiple sclerosis: induction of regulatory immune networks by T-cell vaccination

Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS with presumed autoimmune origin. Pathogenic autoimmune responses in MS are thought to be the result of a breakdown of self tolerance. Several mechanisms account for the natural state of immunological tolerance to self antigens, including clonal deletion of self-reactive T cells in the thymus. However, autoimmune T cells are also part of the normal T-cell repertoire, supporting the existence of peripheral regulatory mechanisms that keep these potentially pathogenic T cells under control. One such mechanism involves active suppression by regulatory T cells. It has been indicated that regulatory T cells do not function properly in autoimmune disease. Immunization with attenuated autoreactive T cells, T-cell vaccination, may enhance or restore the regulatory immune networks to specifically suppress autoreactive T cells, as shown in experimental autoimmune encephalomyelitis, an animal model for MS. In the past decade, T-cell vaccination has been tested for MS in several clinical trials. This review summarizes these clinical trials and updates our current knowledge on the induction of regulatory immune networks by T cell vaccination.

CD4 +CD25 + T regulatory cells in murine pregnancy

Mammalian pregnancy is thought to be a state of immunological tolerance and immunological pregnancy complications may result from incomplete allo-tolerance. We reported recently a higher frequency of Th1 cytokine-producing T cells specific against paternal antigens in abortion-prone mice compared to normal pregnant mice. Since Th2 cells were shown to be not essential for normal pregnancy; alloreactive Th1 cells must be differently regulated. In this context, T regulatory cells (Treg) were proposed to play an essential role. Normal pregnant mice show an expansion of CD4 +CD25 + and IL-10 + Treg cells at the periphery compared to non-pregnant animals. Further, we reported significantly lower frequencies of Treg in abortion-prone mice. Interestingly, CD4 +CD25 + Treg cells from normal pregnant mice were able to prevent fetal rejection. Accordingly, down-regulated levels of Treg were also reported during human miscarriage. The putative mechanisms involved in Treg-induced tolerance in mice and humans are discussed in this review.

Abnormal T-Cell Reactivity against Paternal Antigens in Spontaneous Abortion: Adoptive Transfer of Pregnancy-Induced CD4 +CD25 + T Regulatory Cells Prevents Fetal Rejection in a Murine Abortion Model

Mammalian pregnancy is thought to be a state of immunological tolerance. The mechanisms underlying this phenomenon are still poorly understood. Here, we determined whether an inappropriate function of T regulatory (Treg) cells is involved in the pathogenesis of spontaneous abortion. We evaluated spleen and decidual lymphocytes from CBA/J mice undergoing immunological abortion (DBA/2J-mated) or having normal pregnancy (BALB/c-mated) on day 14 of gestation for ex vivo cytokine production after PMA or paternal antigen (alloantigen) stimulation. Treg activity was characterized by quantifying CD4(+)CD25(+) cells, foxp3 expression, and interleukin-10 secretion. Decidual lymphocytes from abortion CBA/J mice contained a significantly higher frequency of interferon-gamma-producing T cells specific for paternal antigens compared to those from normal pregnancy (7.8% versus 2.7%, P < 0.05). Compared to virgin CBA/J females, normal pregnant mice showed strongly elevated numbers of CD4(+)CD25(+) and interleukin-10(+) Treg cells in the thymus whereas significantly lower frequencies of Treg cells were observed in abortion mice. Very interestingly, CD4(+)CD25(+) Treg cells from normal pregnant and nonpregnant CBA/J mice could inhibit both proliferation and interferon-gamma secretion of lymphocytes from abortion mice in vitro whereas in vivo prevention of fetal rejection could only be achieved after adoptive transfer of Treg cells from normal pregnant mice. Our data suggest that pregnancy-induced Treg cells play a vital role in maternal tolerance to the allogeneic fetus.

Transplantation Immunology from the Historical Perspective

Transplantation would be the only way to cure the end-stage organ failure involving heart, lung, liver, kidney and pancreas. The replacement of the parts of the body damaged to lose its function or lost to trauma must be a dream of human-being. Human history is replete with chimeras, from sphinxes to mermaids, making one wonder if the ancients might actually have dreamed of what now is called 'xenotransplantation'. In the 20th century, the transplantation of organs and tissues to cure disease has become a clinical reality. The development in the fields of surgical techniques, physiology and immunology attributed to the successful transplantation in human. In the center of the successful transplantation lies the progress in understanding the cellular and molecular biology of immune system which led to the development of immunosuppressive drugs and the invention of the concept of immunological tolerance. The mandatory side effects of immunosuppressive drugs including infection and cancer forced us to search alternative approaches along with the development of new immunosuppressive agents. Among the alternative approaches, the induction of a state of immunologic tolerance would be the most promising and the most generic applicability as a future therapy. Recent reports documenting long-term graft survival without immunosuppression suggest that tolerance-based therapies may become a clinical reality. Last year, we saw the epoch making success of overcoming hyperacute rejection in porcine to primate xenotransplantation which will lead porcine to human xenotransplantation to clinical reality. In this review, I dare to summarize the development of transplantation immunology from the perspective of history.

Differential localization and function of ADP-ribosylation factor-6 in anergic human T cells: a potential marker for their identification.

Anergy is a state of immunologic tolerance in which T cells are viable but incapable of responding to antigenic stimulation. Recent data indicate that anergic cells have a distinct gene expression program that determines their unique function. In this study we show that anergic human T cells selectively express the small GTPase ADP-ribosylation factor-6 (ARF6), which is involved in membrane traffic and regulation of the cortical actin cytoskeleton. ARF6 was expressed in the GTP-bound form that localizes at the plasma membrane, resulting in a distinct morphologic appearance of anergic cells. Forced expression of ARF6-GTP in Jurkat T cells prevented TCR-mediated reorganization of cortical actin, extracellular signal-regulated kinase1/2 activation, and IL-2 transcription. Forced expression of ARF6-GTP in primary human T cells inhibited extracellular signal-regulated kinase1/2 activation and proliferative responses. Importantly, T cells with the distribution pattern of ARF6-GTP were detected in peripheral blood, suggesting that anergic T cells may constitutively exist in vivo.

Fetal immunization of baboons induces a fetal-specific antibody response.

Neonates face a high risk of infection because of the immaturity of their immune systems. Although the transplacental transfer of maternal antibodies to the fetus may convey improved postnatal immunity, this transfer occurs late in gestation and may fail to prevent in utero infection. Both fetal immunization and in utero exposure to antigen can result in a state of immunologic tolerance in the neonate. Tolerance induction of fetal and premature infant lymphocytes has become a paradigm for neonatal responsiveness. However, fetal IgM responses have been demonstrated to maternal immunization with tetanus toxoid and to congenital infections such as rubella, toxoplasma, cytomegalovirus and human immunodeficiency virus. Moreover, 1-week-old infants can respond to standard pediatric vaccination, and neonates immunized with polysaccharide antigens do not develop immunologic tolerance. Here, direct immunization of the baboon fetus with recombinant hepatitis B surface antigen produced a specific fetal IgG antibody response. No specific maternal antibody response was detected, eliminating the possibility of vertical antibody transmission to the fetus. Some infants also responded to later vaccinations with hepatitis B surface antigen, indicating that no immunological tolerance was induced by prior fetal immunization. These results characterize the ability of the fetal immune system to respond to in utero vaccination. We demonstrate that active fetal immunization can serve as a safe and efficient vaccination strategy for the fetus and neonate.

Induction of murine acquired immunodeficiency syndrome (MAIDS) in allophenic mice generated from strains susceptible and resistant to disease.

To examine whether a retroviral disease can be controlled in animals in which cells from a resistant strain coexist in a state of immunological tolerance with cells from a susceptible strain, allophenic mice were constructed and infected with LP-BM5 murine leukemia viruses which induce a fatal disorder, termed murine acquired immunodeficiency syndrome (MAIDS), characterized by lymphoproliferation and immunodeficiency in susceptible inbred strains of mice. We found that in two different strain combinations, resistance to MAIDS was contingent on the presence in individual animals of >50% of lymphocytes of resistant strain origin and correlated with reduction or elimination of retrovirus. In contrast, animals harboring substantial, but less than predominant, numbers of genetically resistant lymphocytes developed disease and died within the same time frame as susceptible control mice with uncontained proliferation of retrovirus.

Does corticosteroid pretreatment enhance the effect of alfa interferon treatment in chronic hepatitis B?

In recent years alfa interferon (IFN) treatment has become accepted therapy for chronic hepatitis B. Several studies have documented that treatment with IFN is accompanied or followed by a loss of hepatitis B virus (HBV) DNA, hepatitis B e antigen (HBeAg) to antibody to hepatitis B e antigen (anti-HBe) seroconversion and a decrease in inflammatory disease activity in approximately 35% of patients with replicative, and inflammatory active chronic hepatitis B (1,2). It is a common observation that the highest response rates occur in patients with more active inflammatory disease activity and in patients with lower levels of HBV DNA (2,3). It is also clear that the poorest response rates are achieved in immuno-compromised patients, in disease acquired in childhood and in ethnic groups where vertical transmission of hepatitis B is predominant (4-6). Whether the varying response rates observed simply reflect the difference in the underlying tendency to spontaneous HBeAg to anti-HBe seroconversion or whether there actually is a difference in the relative efficacy of IFN treatment is a matter of debate (7). Furthermore, despite treatment of several thousand patients with IFN, there are still substantial uncertainties about the optimal dose of IFN, duration of treatment, significance of development of IFN antibodies and whether there are therapeutic differences between the IFN types. Clinically the effect of IFN is unsatisfactory. The lowest response rates are obtained in patients who in the long run may have the greatest risk of developing significant liver disease, including hepatocellular carcinoma; and even in immunologically intact Caucasians with inflammatory active disease acquired as adults, a beneficial response may be expected in fewer than half of the treated patients. Moreover, it is still not known whether IFN therapy actually diminishes morbidity and mortality in chronic hepatitis B. These facts must be weighed against the inconveniences, side effects, risks and cost of IFN therapy. Increases in overall response rates and in the response in children and patients with vertically transmitted disease are therefore some of the issues that should be given priority. One of the suggested ways to improve the results of IFN therapy is combination with immune modulating therapy. In this issue of the Journal o f Hepatology, three randomised controlled trials focus on corticosteroid treatment followed by IFN in chronic hepatitis B. Dealing with treatment in children (Utili et al. (8)), Chinese adults (Liaw et al. (9)) and improvement of response in adult Caucasians (Reichen et al. (10)), these studies provide valuable information on some of the key issues in the treatment of chronic hepatitis B. Long-term treatment of chronic hepatitis B with corticosteroids was abandoned in the early eighties when it became evident that this treatment regimen was harmful (11). So what is the rationale for short-term treatment with corticosteroids? Early clinical observations showed that patients with chronic hepatitis B often cleared markers of viral replication following tapering or discontinuation of corticosteroid treatment (12-14). The corticosteroid discontinuation was generally followed by a marked flare in disease activity, which may also be observed in spontaneous seroconversion or following superinfection with other hepatotropic viruses. These findings generated the hypothesis that a state of immunological tolerance induced by corticosteroids was followed by an immunological rebound phenomenon upon discontinu-

Protective action of polyinosinic-polycytidylic acid in viral and transplanted leukemia in mice

The inhibitory effects of polynucleotides on leukemia development and transplantability have been studied. RFM/Un mice, injected neonatally with leukemogenic Graffi virus, received 0.02 mg polyinosinic-polycytidylic acid (Poly I:C) intraperitoneally, twice a week, for two months starting 24 hrs after virus injection. Leukemia incidence in treated mice was significantly reduced, i.e., 56% with a mean latent period of 19 weeks as compared to 87% and 15 weeks latency in the control animals. RFM/Un adult mice, injected intravenously with different cell doses of two transplantable syngeneic leukemias, received four doses of 0.1 mg Poly I:C on alternate days starting 24 hrs after cell transplant. The evaluation of neoplastic nodules on the spleen surface according to the method of Bruce and Van Der Gaag (2) was the parameter of leukemic growth. Mice receiving Poly I:C showed a marked reduction in spleen nodule counts for all cell doses of both leukemia lines and histological examination of the spleen confirmed actual decrease in leukemic foci and disclosed enlargement of germinal centers and periarteriolar areas of lymphoid follicles. To investigate whether Poly I:C is capable of abolishing the state of immunological tolerance (operationally speaking) to cellular virus-induced antigens (CBA x C57BL)F1 adult mice, injected neonatally with Graffi virus, received 0.20 mg Poly I:C i.p. together with 104 or 105 cells from a syngeneic transplantable leukemia originally induced by Graffi virus. No differences in leukemic deaths were observed between Poly I:C treated virus-injected animals and controls, even though normal mice receiving Poly I:C and 10* cells showed 50% survival compared to 0% in control groups. Leukemic cells incubated in vitro in HE-MEM containing 0.1, 1, 10 or 100 μg Poly I:C/ml for 2, 4, 8 and 24 hrs (37 °C, 5% CO2) did not exhibit significant changes in viability as evaluated by phase contrast microscopy. On the basis of present and previous results (6), the possible mechanisms of antineoplastic action exerted by polynucleotides are discussed. It is concluded that while interferon production by synthetic RNAs may represent a major inhibiting factor in virus-induced tumors, non specific stimulation of immune reactivity and direct effect on cell metabolism and replication may play a significant role in other neoplastic conditions.

Immunological Tolerance in Amphibians

SYNOPSIS. A state of immunological tolerance, or specific unresponsiveness to foreign antigen, can be induced in amphibians by joining two nonsibling embryos in parabiotic union. The induced tolerance is immunologically specific; the co-partners are tolerant of each other' santigens but do respond to unrelated antigens from third parties. The parabionts are blood-cell chimeras; each contains blood elements of the type proper to the other. The degree of chimerism wanes in the later life of the parabionts, which may be the consequence of the appearance of a new clone of uninhibited reactive cells derived from the bone marrow. Tolerance to a particular antigen is interpreted as arising by the deletion of the population of competent lymphocytes capable of responding to that antigen. The breakdown of tolerance occurs through the emergence of a new population of antigen-reactive cells. In essence, a tolerant cell as a discrete entity does not exist.