Barbara A. Torres

Antiviral activity of the pregnancy recognition hormone ovine trophoblast protein-1

Ovine trophoblast protein-1 (oTP-1) is an antiluteolytic protein which plays an important role in maternal recognition of pregnancy. It exhibits a 45-70% amino acid sequence homology with alpha interferons (IFN-alpha s) from various species. We show here that purified oTP-1 has high specific antiviral activity (2-3 X 10(8) units/mg protein) and is thus as potent as any known IFN. oTP-1 is antigenically distinct but does show antigenic relation to both ovine and bovine IFN-alpha s. The antiviral activity of oTP-1 could be demonstrated in Day 12 - Day 16 conceptus culture medium and in allantoic fluid from Day 60 of pregnancy. Our functional and antigenic characterization suggests that oTP-1 is a type of IFN-alpha which performs classic roles associated with IFN-alpha, as well as being the primary conceptus secretory product responsible for maternal recognition of pregnancy.

IFNγ inhibition of cell growth in glioblastomas correlates with increased levels of the cyclin dependent kinase inhibitor p21 WAF1/CIP1

Glioblastoma is a highly aggressive form of brain cancer characterized by uncontrolled cell growth resulting from a loss of cell cycle regulation. In this study we determined the antiproliferative effects of interferon gamma (IFNγ) on the glioblastoma cell lines T98G, SNB-19 and U-373, focusing on the ability of IFNγ to increase levels of p21WAF1/CIP1, an important negative regulator of cell cycle events. IFNγ was found to inhibit the growth of all cell lines, with inhibition ranging from 82.2% to 45.4%. Flow cytometry analysis showed that IFNγ treatment caused a cell cycle delay in the G1 or S phases. The strength of this delay varied, correlating with the degree by which IFNγ inhibited proliferation of each cell line. IFNγ treatment increased the production of the cyclin dependent kinase inhibitor (CKI) p21WAF1/CIP1 in all cell lines, the level and kinetics of production of which correlated with the degree and stage of inhibition of cellular proliferation. Further, immunoprecipitation of p21WAF1/CIP1 in complexes of p21WAF1/CIP1/cyclin-dependent kinase 2 (cdk2)/cyclin showed that the amount of p21WAF1/CIP1 in the complexes and the inhibition of cdk2-cyclin kinase activity correlated with the level of p21WAF1/CIP1 produced in the cells by IFNγ. These results show that IFNγ has significant antiproliferative effects on the glioblastoma cell lines and suggest that p21WAF1/CIP1 plays a role in mediating these effects.

Superantigens: The Good, the Bad, and the Ugly:

Increasing evidence suggests that superantigens play a role in Immune-mediated diseases. Superantigens are potent activators of CD4* T cells, causing rapid and massive proliferation of cells and cytokine production. This characteristic of superantigens can be exploited in diseases where strong immunologic responses are required, such as in the B16F10 animal model of melanoma. Superantigen administration is able to significantly enhance Ineffective anti-tumor Immune responses, resulting in potent and long-lived protective anti-tumor immunity. However, superantigens are more well-known for the role they play in diseases. Studies using an animal model for neurologic demy-elinatlng diseases such as multiple sclerosis show that superantigens can induce severe relapses and activate auto-reactive T cells not involved in the Initial bout of disease. This may also involve epitope spreading of disease. Superantigens have also been implicated in acute diseases such as food poisoning and TSS, and in chronic diseases such as psoriasis and rheumatoid arthritis. Viral superantigens are also involved in the disease process, including superantigens derived from human Immunodeficiency virus and mouse mammary tumor virus. Finally, immunotherapies that ameliorate the role played by superantigens in disease are discussed.

Enhancement of the in vitro antibody response by thyrotropin.

The pituitary hormone thyrotropin (TSH) has been shown to enhance in a dose dependent manner the in vitro antibody response. Highly purified preparations of bovine and human TSH enhanced up to 375% the number of cells producing antibody to sheep erythrocytes. TSH had to be present prior to 24-48h of the initiation of culture for enhancement of the antibody response. An analogy is discussed between TSH and B lymphocyte growth and differentiation factors.

Superantigens: Structure and Relevance to Human Disease∗:

Abstract Superantigens are a class of immunostimulatory molecules produced by bacterla and viruses. Their potent immune effects are due to their unique ability to bind to the major histocompatibility complex (MHC) outside the antigen-binding cleft and to stimulate T cells in a T-cell receptor (TCR) Vβ-specific manner. Structural studies have revealed the binding sites involved in the MHC/superantigen/TCR complex. The bacterial superantigens are responsible for a number of syndromes, including food poisoning and toxic shock syndrome, but their effects may be not only acute but also chronic and complex. Recent evidence suggests that superantigens may be relevant to the pathogenesis of autoimmune and immunodeficiency disorders. To illustrate the detrimental effects of superantigens on disease outcome, evidence demonstrating the modulation of experimental allergic encephalomyelitis, an animal model for multiple sclerosis, by superantigen, as well as the potential role of superantigens in HIV pathogenesis of AIDS, will be presented. The information presented may provide valuable insight into the role of superantigens in autoimmunity and HIV infection.

IFN-gamma Induction of p21WAF1 Is Required for Cell Cycle Inhibition and Suppression of Apoptosis

Interferons (IFN) inhibit the growth of tumor cells by blocking the progression of their cell cycle. Recently, we showed that this cell cycle inhibition correlates with the ability of IFN to upregulate the cyclin-dependent kinase inhibitor p21(WAF1). This, however, is not proof of a causal relationship. Using p21(WAF1)-deficient cells derived from the HCT116 colon adenocarcinoma cell line, we now show that p21(WAF1) is indeed responsible for the antiproliferative effects of the type II IFN, IFN-gamma. IFN-gamma upregulated p21(WAF1) expression in a p53-independent manner, decreased cyclin-dependent kinase 2 activity, and inhibited entry into the S phase of the cell cycle in p21+/+ but not in p21-/- HCT116 cells. We additionally found that the lack of p21(WAF1) expression resulted in an increase in the ability of IFN-gamma to induce apoptosis, as reflected by an earlier induction of DNA fragmentation and caspase 3 activity in p21-/- cell. Our results indicate that p21(WAF1) expression is necessary for IFN-gamma-mediated cell cycle inhibition and suppression of IFN-gamma-induced apoptosis.

Nuclear Translocation of IFN-γ Is an Intrinsic Requirement for Its Biologic Activity and Can Be Driven by a Heterologous Nuclear Localization Sequence

We have previously identified a nuclear localization sequence (NLS) in interferon-gamma (IFN-gamma). This NLS functions intracellularly by forming a complex with its transcription factor Stat1alpha and the nuclear importer of Stat1alpha, the importin-alpha analog NPI-1. The stability of this complex and the subsequent nuclear translocation of the complexed Stat1alpha are dependent on the integrity of this NLS, showing that Stat1alpha nuclear import is mediated by the IFN-gamma NLS. In this study, to directly evaluate the intrinsic requirement of nuclear IFN-gamma toward its biologic activities, we engineered a chimeric in which the IFN-gamma NLS has been substituted by a heterologous NLS, namely, the prototypical NLS of the SV40 large T antigen, which would drive nuclear translocation of IFN-gamma in a sequence-nonspecific manner. The chimeric, IFN-gamma-SV, was equally active in antiviral and antiproliferative assays as the wild-type IFN-gamma. Interestingly, IFN-gamma-SV was also translocated to the nucleus and was also recovered intracellularly as a complex with the Stat1alpha importer NPI-1, like wild-type IFN-gamma. Comparison with an NLS deletion mutant showed that deletion or changes within the NLS motif of IFN-gamma were inconsequential to the high-affinity extracellular binding to the IFN-gamma receptor complex, yet the presence of an NLS was critical to the expression of the biologic activities of IFN-gamma and its NPI-1 complexation ability. Our data conclusively demonstrate that nuclear translocation of IFN-gamma is an intrinsic requirement for the full expression of the biologic activities of IFN-gamma and strengthen the conclusion that nuclear chaperoning of Stat1alpha is the primary role of IFN-gamma nuclear translocation. This type of ligand imprinting by sequestering of activated Stat may contribute to the specificity of Stat nuclear transcription.

Inhibition of the glioblastoma cell cycle by type I IFNs occurs at both the G1 and S phases and correlates with the upregulation of p21WAF1/CIP1

The antiproliferative effect of IFNα was tested on the human glioblastoma cell lines, U-373MG and T98G. IFNα significantly inhibited the growth of both cell lines, but was more effective in retarding the growth of U-373MG cells. Flow cytometry analysis indicated that synchronized IFNα-treated U-373MG cells showed a strong block in the progression of cells out of the S phase of the cell cycle. T98G cells, on the other hand, showed a moderate delay in the transition of cells from G1 to S phase and only a slight delay in the S phase, consistent with the decreased antiproliferative effect of IFNα on this cell line. IFNα-treated cells were then tested for the induction of the tumor suppressor gene product, p21WAF1/CIP1. Higher levels of p21WAF1/CIP1 were detected in lysates from IFNα-treated U-373MG cells as compared to media controls for as long as 18 h. In IFNα-treated T98G cells, p21WAF1/CIP1 levels were slightly elevated at 4 and 6 h, but decreased to levels similar to controls thereafter, correlating with the antiproliferative effects of IFNα on each cell line. Immunoprecipitation studies on lysates from IFNα-treated U-373MG and T98G cells indicated that increased amounts of p21WAF1/CIP1 were complexed to both cyclin D1 and cyclin E. Further, reduced cyclin-dependent kinase 2 (cdk2) activity was found in both IFNα-treated U-373MG and T98G cells, suggesting a mechanism by which p21WAF1/CIP1 exerted its antiproliferative effects. Lastly, we analyzed the time-dependent production of the cyclins Dl, E, and A. No differences in cyclin D1 levels were found between IFNα-treated and media-treated U-373MG and T98G cells. However, both IFNα-treated U-373MG and T98G cells showed a prolonged elevation in cyclin E, correlating with the G1 to S phase delays observed in these cell lines. Further, the duration of cyclin E production corresponded with the magnitude of the cell cycle delays seen in IFNα-treated U-373MG and T98G cells. Prolonged elevation of cyclin A was also seen in both IFNα-treated U-373MG and T98G cells, the magnitude of which correlated with the S phase delay observed in these cell lines. Thus, the data indicate that IFNα has significant antiproliferative activity against glioblastoma cells that is mediated, at least in part, by the tumor suppressor gene product, p21WAF1/CIP1.

Modulation of disease by superantigens

Increasing evidence suggests that bacterial and viral superantigens are involved in immune-mediated disease. Studies using an animal model for multiple sclerosis show that superantigens can induce relapses and bring into play autoreactive T cells with restricted usage of T cell receptor V beta families that may be indirectly involved in the initial episode of disease. This may also involve epitope spreading. Superantigens have also been implicated in other autoimmune diseases such as rheumatoid arthritis and psoriasis. Superantigens encoded by viruses such as mouse mammary tumor virus play an important role in disease progression.

Superantigen Enhancement of Specific Immunity: Antibody Production and Signaling Pathways

Superantigens are microbial proteins that induce massive activation, proliferation, and cytokine production by CD4+ T cells via specific Vβ elements on the TCR. In this study we examine superantigen enhancement of Ag-specific CD4+ T cell activity for humoral B cell responses to T-dependent Ags BSA and HIV gp120 envelope, type I T-independent Ag LPS, and type II T-independent Ag pneumococcal polysaccharides. Injection of BSA followed by a combination of superantigens staphylococcal enterotoxin A and staphylococcal enterotoxin B (SEB) 7 days later enhanced the anti-BSA Ab response in mice ∼4-fold as compared with mice given BSA alone. The anti-gp120 response was enhanced ∼3-fold by superantigens. The type II T-independent Ag pneumococcal polysaccharide response was enhanced ∼2.3-fold by superantigens, whereas no effect was observed on the response to the type I T-independent Ag LPS. The superantigen effect was completely blocked by the CD4+ T cell inhibitory cytokine IL-10. SEB-stimulated human CD4+ T cells were examined to determine the role of the mitogen-activated protein (MAP) kinase signal transduction pathway in superantigen activation of T cells. Inhibitors of the mitogen pathway of MAP kinase blocked SEB-induced proliferation and IFN-γ production, while an inhibitor of the p38 stress pathway had no effect. Consistent with this, SEB activated extracellular signal-regulated kinase/MAP kinase as well as MAP kinase-interacting kinase, a kinase that phosphorylates eIF4E, which is an important component of the eukaryotic protein synthesis initiation complex. Both kinases were inhibited by IL-10. Thus, superantigens enhance humoral immunity via Ag-specific CD4+ T cells involving the stress-independent pathway of MAP kinase.