Effect of a smoke-free policy on staff attitudes and behaviours within an Australian metropolitan health service: a 3 year cross-sectional study.

Objective In 2010, Peninsula Health (Vic., Australia), became smoke free as part of the locally developed smoking prevention and cessation strategy. The aim of the present study was to determine the effect of a smoke-free policy on smoking status and employee attitudes over a 3-year period. Methods Data were collected by three surveys 6 months before and 6 months and 3 years after policy introduction. Demographic data, smoking status and attitudes to the introduction of the smoke-free policy were collected for analysis. Results There were 3224 individual responses collected over three time points with similar demographics at each time. There were fewer employees smoking at 6 months (P=0.010) and 3 years (P<0.001) after implementation of the policy. There were more employees who felt positive towards the policy 3 years after its introduction (P=0.028). There were greater odds of an employee not identifying as a smoker after the policy was in place than before the policy was implemented. Conclusions The introduction of a smoke-free policy within a health service was an upstream health intervention that was well accepted by staff and appeared to have a positive effect on smoking behaviours. What is known about the topic? There are an increasing number of environmental changes that seek to decrease smoking behaviours. Bans within workplaces have a direct effect on employee smoking behaviour. What does this paper add? Some employee groups demonstrated the greater odds of smoking when a smoke-free policy was in place. Employees felt positive towards this policy. What are the implications for practitioners? This policy change supports environmental changes affecting individual health-related behaviours.

Oncolytic vesicular stomatitis virus in an immunocompetent model of MUC1-positive or MUC1-null pancreatic ductal adenocarcinoma.

Vesicular stomatitis virus (VSV) is a promising oncolytic agent against various malignancies. Here, for the first time, we tested VSV in vitro and in vivo in a clinically relevant, immunocompetent mouse model of pancreatic ductal adenocarcinoma (PDA). Our system allows the study of virotherapy against PDA in the context of overexpression (80% of PDA patients) or no expression of human mucin 1 (MUC1), a major marker for poor prognosis in patients. In vitro, we tested three VSV recombinants, wild-type VSV, VSV-green fluorescent protein (VSV-GFP), and a safe oncolytic VSV-ΔM51-GFP, against five mouse PDA cell lines that either expressed human MUC1 or were MUC1 null. All viruses demonstrated significant oncolytic abilities independent of MUC1 expression, although VSV-ΔM51-GFP was somewhat less effective in two PDA cell lines. In vivo administration of VSV-ΔM51-GFP resulted in significant reduction of tumor growth for tested mouse PDA xenografts (+MUC1 or MUC1 null), and antitumor efficacy was further improved when the virus was combined with the chemotherapeutic drug gemcitabine. The antitumor effect was transient in all tested groups. The developed system can be used to study therapies involving various oncolytic viruses and chemotherapeutics, with the goal of inducing tumor-specific immunity while preventing premature virus clearance.

The use of a novel MUC1 antibody to identify cancer stem cells and circulating MUC1 in mice and patients with pancreatic cancer.

BACKGROUND AND OBJECTIVES: MUC1 is over-expressed and aberrantly glycosylated in >60% of human pancreatic cancer (PC). Development of novel approaches for detection and/or targeting of MUC1 are critically needed and should be able to detect MUC1 on PC cells (including cancer stem cells) and in serum. METHODS: The sensitivity and specificity of the anti-MUC1 antibody, TAB 004, was determined. CSCs were assessed for MUC1 expression using TAB 004-FITC on in vitro PC cell lines, and on lineage(-) cells from in vivo tumors and human samples. Serum was assessed for shed MUC1 via the TAB 004 EIA. RESULTS: In vitro and in vivo, TAB 004 detected MUC1 on >95% of CSCs. Approximately, 80% of CSCs in patients displayed MUC1 expression as detected by TAB 004. Shed MUC1 was detected serum in mice with HPAF-II (MUC1(high) ) but not BxPC3 tumors (MUC1(low)). The TAB 004 EIA was able to accurately detect stage progression in PC patients. CONCLUSIONS: The TAB 004 antibody may be explored as a therapeutic targeting agent for CSCs in PC. The TAB 004 EIA detected circulating MUC1 in a stage-dependent manner in patients with PC and thus may be explored as a PC stage diagnostic biomarker.

Resistance of pancreatic cancer cells to oncolytic vesicular stomatitis virus: role of type I interferon signaling.

Oncolytic virus (OV) therapy takes advantage of common cancer characteristics, such as defective type I interferon (IFN) signaling, to preferentially infect and kill cancer cells with viruses. Our recent study (Murphy et al., 2012. J. Virol. 86, 3073-87) found human pancreatic ductal adenocarcinoma (PDA) cells were highly heterogeneous in their permissiveness to vesicular stomatitis virus (VSV) and suggested at least some resistant cell lines retained functional type I IFN responses. Here we examine cellular responses to infection by the oncolytic VSV recombinant VSV-ΔM51-GFP by analyzing a panel of 11 human PDA cell lines for expression of 33 genes associated with type I IFN pathways. Although all cell lines sensed infection by VSV-ΔM51-GFP and most activated IFN-α and ß expression, only resistant cell lines displayed constitutive high-level expression of the IFN-stimulated antiviral genes MxA and OAS. Inhibition of JAK/STAT signaling decreased levels of MxA and OAS and increased VSV infection, replication and oncolysis, further implicating IFN responses in resistance. Unlike VSV, vaccinia and herpes simplex virus infectivity and killing of PDA cells was independent of the type I IFN signaling profile, possibly because these two viruses are better equipped to evade type I IFN responses. Our study demonstrates heterogeneity in the type I IFN signaling status of PDA cells and suggests MxA and OAS as potential biomarkers for PDA resistance to VSV and other OVs sensitive to type I IFN responses.

Current status of gene therapy for brain tumors.

Glioblastoma (GBM) is the most common and deadliest primary brain tumor in adults, with current treatments having limited impact on disease progression. Therefore the development of alternative treatment options is greatly needed. Gene therapy is a treatment strategy that relies on the delivery of genetic material, usually transgenes or viruses, into cells for therapeutic purposes, and has been applied to GBM with increasing promise. We have included selectively replication-competent oncolytic viruses within this strategy, although the virus acts directly as a complex biologic anti-tumor agent rather than as a classic gene delivery vehicle. GBM is a good candidate for gene therapy because tumors remain locally within the brain and only rarely metastasize to other tissues; the majority of cells in the brain are post-mitotic, which allows for specific targeting of dividing tumor cells; and tumors can often be accessed neurosurgically for administration of therapy. Delivery vehicles used for brain tumors include nonreplicating viral vectors, normal adult stem/progenitor cells, and oncolytic viruses. The therapeutic transgenes or viruses are typically cytotoxic or express prodrug activating suicide genes to kill glioma cells, immunostimulatory to induce or amplify anti-tumor immune responses, and/or modify the tumor microenvironment such as blocking angiogenesis. This review describes current preclinical and clinical gene therapy strategies for the treatment of glioma.

Vesicular stomatitis virus as an oncolytic agent against pancreatic ductal adenocarcinoma.

Vesicular stomatitis virus (VSV) is a promising oncolytic agent against a variety of cancers. However, it has never been tested in any pancreatic cancer model. Pancreatic ductal adenocarcinoma (PDA) is the most common and aggressive form of pancreatic cancer. In this study, the oncolytic potentials of several VSV variants were analyzed in a panel of 13 clinically relevant human PDA cell lines and compared to conditionally replicative adenoviruses (CRAds), Sendai virus and respiratory syncytial virus. VSV variants showed oncolytic abilities superior to those of other viruses, and some cell lines that exhibited resistance to other viruses were successfully killed by VSV. However, PDA cells were highly heterogeneous in their susceptibility to virus-induced oncolysis, and several cell lines were resistant to all tested viruses. Resistant cells showed low levels of very early VSV RNA synthesis, indicating possible defects at initial stages of infection. In addition, unlike permissive PDA cell lines, most of the resistant cell lines were able to both produce and respond to interferon, suggesting that intact type I interferon responses contributed to their resistance phenotype. Four cell lines that varied in their permissiveness to VSV-ΔM51 and CRAd dl1520 were tested in mice, and the in vivo results closely mimicked those in vitro. While our results demonstrate that VSV is a promising oncolytic agent against PDA, further studies are needed to better understand the molecular mechanisms of resistance of some PDAs to oncolytic virotherapy.

Sequence-function analysis of the Sendai virus L protein domain VI.

The large (about 2200 amino acids) L polymerase protein of nonsegmented negative-strand RNA viruses (order Mononegavirales) has six conserved sequence regions ("domains") postulated to constitute the specific enzymatic activities involved in viral mRNA synthesis, 5'-end capping, cap methylation, 3' polyadenylation, and genomic RNA replication. Previous studies with vesicular stomatitis virus identified amino acid residues within the L protein domain VI required for mRNA cap methylation. In our recent study we analyzed four amino acid residues within domain VI of the Sendai virus L protein and our data indicated that there could be differences in L protein sequence requirements for cap methylation in two different families of Mononegavirales - rhabdoviruses and paramyxoviruses. In this study, we conducted a more comprehensive mutational analysis by targeting the entire SeV L protein domain VI, creating twenty-four L mutants, and testing these mutations for their effects on viral mRNA synthesis, cap methylation, viral genome replication and virus growth kinetics. Our analysis identified several residues required for successful cap methylation and virus replication and clearly showed the importance of the K-D-K-E tetrad and glycine-rich motif in the SeV cap methylation. This study is the first extensive sequence analysis of the L protein domain VI in the family Paramyxoviridae, and it confirms structural and functional similarity of this domain across different families of the order Mononegavirales.

Identification of sendai virus L protein amino acid residues affecting viral mRNA cap methylation.

Viruses of the order Mononegavirales all encode a large (L) polymerase protein responsible for the replication and transcription of the viral genome as well as all posttranscriptional modifications of viral mRNAs. The L protein is conserved among all members of the Mononegavirales and has six conserved regions ("domains"). Using vesicular stomatitis virus (VSV) (family Rhabdoviridae) experimental system, we and others recently identified several conserved amino acid residues within L protein domain VI which are required for viral mRNA cap methylation. To verify that these critical amino acid residues have a similar function in other members of the Mononegavirales, we examined the Sendai virus (SeV) (family Paramyxoviridae) L protein by targeting homologous amino acid residues important for cap methylation in VSV which are highly conserved among all members of the Mononegavirales and are believed to constitute the L protein catalytic and S-adenosylmethionine-binding sites. In addition, an SeV L protein mutant with a deletion of the entire domain VI was generated. First, L mutants were tested for their abilities to synthesize viral mRNAs. While the domain VI deletion completely inactivated L, most of the amino acid substitutions had minor effects on mRNA synthesis. Using a reverse genetics approach, these mutations were introduced into the SeV genome, and recombinant infectious SeV mutants with single alanine substitutions at L positions 1782, 1804, 1805, and 1806 or a double substitution at positions 1804 and 1806 were generated. The mutant SeV virions were purified, detergent activated, and analyzed for their abilities to synthesize viral mRNAs methylated at their cap structures. In addition, further studies were done to examine these SeV mutants for a possible host range phenotype, which was previously shown for VSV cap methylation-defective mutants. In agreement with a predicted role of the SeV L protein invariant lysine 1782 as a catalytic residue, the recombinant virus with a single K1782A substitution was completely defective in cap methylation and showed a host range phenotype. In addition, the E1805A mutation within the putative S-adenosylmethionine-binding site of L resulted in a 60% reduction in cap methylation. In contrast to the homologous VSV mutants, other recombinant SeV mutants with amino acid substitutions at this site were neither defective in cap methylation nor host range restricted. The results of this initial study using an SeV experimental system demonstrate similarities as well as differences between the L protein cap methylation domains in different members of the Mononegavirales.