Citations

ABSTRACT The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.

Abstract Purpose: Reovirus is a naturally occurring oncolytic virus in clinical trials. Although tumor infection by reovirus can generate adaptive antitumor immunity, its therapeutic importance versus direct viral oncolysis is undefined. This study addresses the requirement for viral oncolysis and replication, and the relative importance of antitumor immunity and direct oncolysis in therapy. Experimental Design: Nonantigen specific T cells loaded with reovirus were delivered i.v. to C57BL/6 and severe combined immunodeficient mice bearing lymph node and splenic metastases from the murine melanoma, B16ova, with assessment of viral replication, metastatic clearance by tumor colony outgrowth, and immune priming. Human cytotoxic lymphocyte priming assays were done with dendritic cells loaded with Mel888 cells before the addition of reovirus. Results: B16ova was resistant to direct oncolysis in vitro, and failed to support reovirus replication in vitro or in vivo. Nevertheless, reovirus purged lymph node and splenic metastases in C57BL/6 mice and generated antitumor immunity. In contrast, reovirus failed to reduce tumor burden in severe combined immunodeficient mice bearing either B16ova or reovirus-sensitive B16tk metastases. In the human system, reovirus acted solely as an adjuvant when added to dendritic cells already loaded with Mel888, supporting priming of specific antitumor cytotoxic lymphocyte, in the absence of significant direct tumor oncolysis; UV-treated nonreplicating reovirus was similarly immunogenic. Conclusion: The immune response is critical in mediating the efficacy of reovirus, and does not depend upon direct viral oncolysis or replication. The findings are of direct relevance to fulfilling the potential of this novel anticancer agent.

Abstract The invasion of malignant glioma cells into the surrounding normal brain precludes effective clinical treatment. In this report, we investigated the role of the NH2-terminal FERM domain in the regulation of the promigratory function of Pyk2. We report that the substitution of residues that constitute a small cleft on the surface of the F3 module of the FERM domain do not significantly alter Pyk2 expression but result in the loss of Pyk2 phosphorylation. A monoclonal antibody, designated 12A10, specifically targeting the Pyk2 FERM domain was generated and recognizes an epitope located on the β5C-α1C surface of the F3 module of the FERM domain. Amino acid substitutions in the F3 module that resulted in the loss of Pyk2 phosphorylation also inhibited the binding of 12A10, suggesting that the 12A10 epitope overlaps a site that plays a role in Pyk2 activity. Conjugation of 12A10 to a membrane transport peptide led to intracellular accumulation and inhibition of glioma cell migration in a concentration-dependent manner. A single chain Fv fragment of 12A10 was stable when expressed in the intracellular environment, interacted directly with Pyk2, reduced Pyk2 phosphorylation, and inhibited glioma cell migration in vitro. Stable intracellular expression of the 12A10 scFv significantly extended survival in a glioma xenograft model. Together, these data substantiate a central role for the FERM domain in regulation of Pyk2 activity and identify the F3 module as a novel target to inhibit Pyk2 activity and inhibit glioma progression. [Mol Cancer Ther 2009;8(6):1505–14]

Abstract Using biopsy specimens from patients with B-cell non-Hodgkin's lymphoma, we observed a significantly low frequency of TH17 cells, including several samples with no detectable amount of interleukin (IL)-17–producing cells present in the tumor microenvironment. We found that, in the absence of lymphoma B cells, treatment with IL-1β/IL-6 or lipopolysaccharide (LPS) enhanced IL-17 expression in CD4+ T cells and this enhancement was attenuated when CD4+ T cells were cocultured with lymphoma B cells. Blockade of CD27-CD70 or CD28-CD80/86 interactions by anti-CD70 or anti-CD80/86 antibodies restored LPS-mediated induction of IL-17 expression in CD4+ T cells cocultured with lymphoma B cells. Because a subset of lymphoma B cells express IL-2 and given that IL-2 signaling is critically important in the development of regulatory T (Treg) cells, we tested the role of IL-2 signaling in TH17 cell development. We found that treatment with anti-IL-2 antibody to interrupt IL-2 signaling significantly inhibited Foxp3 expression in CD4+ T cells. In contrast, interruption of IL-2 signaling up-regulated IL-17 expression in CD4+ T cells and restored lymphoma-mediated down-regulation of IL-17–producing cells. Furthermore, the reversal of Treg cell activity by LPS or CpG-A resulted in an enhancement of IL-17–producing cells. Taken together, our study indicated that lymphoma B cells play an important role in skewing the balance between Treg and TH17 cells resulting in the establishment of a profoundly inhibitory tumor microenvironment. [Cancer Res 2009;69(13):5522–30]

ABSTRACT We established a murine model of Candida albicans central nervous system (CNS) infection and evaluated the efficacy of anidulafungin. Ten milligrams/kg/day anidulafungin, amphotericin B, or voriconazole significantly reduced mortality and fungal burden in brain tissue, although amphotericin B and 10 mg/kg/day anidulafungin reduced fungal burden in brain tissue to a greater extent than did voriconazole. This suggests a potential role for anidulafungin in the treatment of candidal CNS infection.