Epidemiological and molecular characterization of a novel adenovirus of squirrel monkeys after fatal infection during immunosuppression.

Adenoviruses are a frequent cause of acute upper respiratory tract infections that can also cause disseminated disease in immunosuppressed patients. We identified a novel adenovirus, squirrel monkey adenovirus 1 (SqMAdV-1), as the cause of fatal infection in an immunocompromised squirrel monkey (Saimiri boliviensis) at the Keeling Center for Comparative Medicine and Research (KCCMR). Sequencing of SqMAdV-1 revealed that it is most closely related (80.4 % pairwise nucleotide identity) to the titi monkey (Plecturocebus cupreus) adenovirus (TMAdV). Although identified in the titi monkey, TMAdV is highly lethal in these monkeys, and they are not thought to be the natural host. While SqMAdV-1 is similar to other primate adenoviruses in size and genomic characteristics, a nucleotide polymorphism at the expected stop codon of the DNA polymerase gene results in a 126 amino acid extension at the carboxy terminus, a feature not previously observed among other primate adenoviruses. PCR testing and partial sequencing of 95 archived faecal samples from other squirrel monkeys (Saimiri boliviensis and Saimiri sciureus) housed at the KCCMR revealed the presence of three distinct, and apparently endemic species of adenoviruses. A grouping of ten squirrel monkey adenovirus variants has high similarity to SqMAdV-1. A single adenovirus variant (designated SqMAdV-3), detected in five monkeys, has similarity to tufted capuchin (Sapajus apella) adenoviruses. The largest group of adenovirus variants detected (designated SqMAdV-2.0-2.16) has very high similarity (93-99 %) to the TMAdV, suggesting that squirrel monkeys may be the natural host of the TMAdV.

Supporting a role for the GTPase Rab7 in prostate cancer progression.

Invasion and subsequent metastasis is the major cause of death from most cancers including prostate cancer. Herein we report on the potential tumor suppressive properties of Rab7, a GTPase that regulates trafficking of lysosomes. The movement of lysosomes to the cell surface in response to environmental cues increases the secretion of proteinases and cell invasion. We determined that Troglitazone and other members of the Thiazolidinedione family inhibit cell-surface directed lysosome trafficking and cathepsin B secretion through a Rab7-dependent mechanism. Moreover, Rab7 shRNA expressing cells were found to be more invasive in vitro and in vivo. Increased invasiveness was accompanied by elevated expression of the c-Met receptor and prolonged downstream signaling, thereby supporting a role for Rab7 as a mediator of signaling down-regulation. Taken together, these results suggested that Rab7 acts as a negative regulator of prostate tumor growth and invasion, providing further evidence for its potential as a tumor suppressor.

Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector.

Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

Rapamycin inhibits lymphatic endothelial cell tube formation by downregulating vascular endothelial growth factor receptor 3 protein expression.

Mammalian target of rapamycin (mTOR) controls lymphangiogenesis. However, the underlying mechanism is not clear. Here we show that rapamycin suppressed insulin-like growth factor 1 (IGF-1)- or fetal bovine serum (FBS)-stimulated lymphatic endothelial cell (LEC) tube formation, an in vitro model of lymphangiogenesis. Expression of a rapamycin-resistant and kinase-active mTOR (S2035T, mTOR-T), but not a rapamycin-resistant and kinase-dead mTOR (S2035T/D2357E, mTOR-TE), conferred resistance to rapamycin inhibition of LEC tube formation, suggesting that rapamycin inhibition of LEC tube formation is mTOR kinase activity dependent. Also, rapamycin inhibited proliferation and motility in the LECs. Furthermore, we found that rapamycin inhibited protein expression of VEGF receptor 3 (VEGFR-3) by inhibiting protein synthesis and promoting protein degradation of VEGFR-3 in the cells. Down-regulation of VEGFR-3 mimicked the effect of rapamycin, inhibiting IGF-1- or FBS-stimulated tube formation, whereas over-expression of VEGFR-3 conferred high resistance to rapamycin inhibition of LEC tube formation. The results indicate that rapamycin inhibits LEC tube formation at least in part by downregulating VEGFR-3 protein expression.

Association of core-binding factor ß with the malignant phenotype of prostate and ovarian cancer cells.

Core binding factor (CBF) is a transcription factor complex that plays roles in development, stem-cell homeostasis, and human disease. CBF is a heterodimer composed of one of three DNA-binding RUNX proteins plus the non-DNA-binding protein, CBFß. Recent studies have showed that the RUNX factors exhibit complex expression patterns in prostate, breast, and ovarian cancers, and CBF has been implicated in the control of cancer-related genes. However, the biologic roles of CBF in solid tumors have not been fully elucidated. To test whether CBF is required for the malignant phenotype of various epithelial cancers, we used lentiviral delivery of CBFß-specific shRNA to significantly decrease CBFß expression in two prostate cancer cell lines (PPC1 and PC-3) and the SKOV-3 ovarian cancer cell line. We found that knockdown of CBFß significantly inhibited anchorage independent growth of each cell line. Further, CBFß knockdown in PPC1 cells suppressed xenograft tumor growth compared to controls. Mice injected with SKOV-3 ovarian cancer cells knocked-down for CBFß exhibited a survival time similar to control mice. However, human cells recovered from the ascites fluid of these mice showed CBFß expression levels similar to those from mice injected with control SKOV-3 cells, suggesting that CBFß knockdown is incompatible with tumor cell growth. Gene expression profiling of CBFß knockdown cells revealed significant changes in expression in genes involved in various developmental and cell signaling pathways. These data collectively suggest that CBFß is required for malignancy in some human cancers.

Design, synthesis and evaluation of novel 2-thiophen-5-yl-3H-quinazolin-4-one analogues as inhibitors of transcription factors NF-kappaB and AP-1 mediated transcriptional activation: Their possible utilization as anti-inflammatory and anti-cancer agents.

In an attempt to discover novel inhibitors of NF-kappaB and AP-1 mediated transcriptional activation utilizing the concept of chemical lead based medicinal chemistry and bioisosterism a series of 2-(2,3-disubstituted-thiophen-5-yl)-3H-quinazolin-4-one analogs was designed. A facile and simple route for the synthesis of the designed molecules was developed. Synthesized molecules were evaluated for their activity as inhibitors towards NF-kappaB and AP-1 mediated transcriptional activation in a cell line report-based assay. This series provides us with a substantial number of compounds inhibiting the activity of NF-kappaB and/or AP-1 mediated transcriptional activation. These compounds also exhibit anti-inflammatory and anti-cancer activity in in vivo models of inflammation and cancer. The 4-pyridyl group is found to be the most important pharmacophore on the third position of thiophene ring for inhibiting NF-kappaB and AP-1 mediated transcriptional activation. The relationships between the activities shown by these compounds in the in vivo and in vitro models have been established by using FVB transgenic mice model. These results suggest the suitability of the designed molecular framework as a potential scaffold for the design of molecules with inhibitory activity towards NF-kappaB and AP-1 mediated transcriptional activation, which may also exhibit anti-inflammatory and anti-cancer activity. This series of molecules warrants further study to explore their potential as therapies for use in chronic inflammatory conditions and cancer. Development of the synthetic protocol for the synthesis of this series of molecules, biological activities and a structure-activity relationship (SAR) have been discussed herein.

Bovine alveolar macrophage neurokinin-1 and response to substance P.

In this study bovine alveolar macrophage neurokinin-1 (NK-1) and the in vitro response to substance P (SP) exposure were investigated. Bovine alveolar macrophage membrane extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted using anti-NK-1 antiserum demonstrated the presence of an approximately 60kDa band. Phagocytosis of fluorescent bioparticles by SP-exposed macrophages was 39% greater than that of non-exposed macrophages (P=0.0089). Likewise, there was 28% greater TNF production by macrophages following SP exposure compared to non-exposed controls (P=0.116). These results suggest that bovine alveolar macrophages respond to SP at least in part by enhancing phagocytosis and TNF production.