Development and applications of oncolytic Maraba virus vaccines.

Oncolytic activity of the MG1 strain of the Maraba vesiculovirus has proven efficacy in numerous preclinical cancer models, and relied not only on a direct cytotoxicity but also on the induction of both innate and adaptive antitumor immunity. To further expand tumor-specific T-cell effector and long-lasting memory compartments, we introduced the MG1 virus in a prime-boost cancer vaccine strategy. To this aim, a replication-incompetent adenoviral [Ad] vector together with the oncolytic MG1 have each been armed with a transgene expressing a same tumor antigen. Immune priming with the Ad vaccine subsequently boosted with the MG1 vaccine mounted tumor-specific responses of remarkable magnitude, which significantly prolonged survival in various murine cancer models. Based on these promising results, we validated the safety profile of the Ad:MG1 oncolytic vaccination strategy in nonhuman primates and initiated clinical investigations in cancer patients. Two clinical trials are currently under way (NCT02285816; NCT02879760). The present review will recapitulate the discoveries that led to the development of MG1 oncolytic vaccines from bench to bedside.

Maraba virus-vectored cancer vaccines represent a safe and novel therapeutic option for cats.

Direct killing of malignant cells combined with induction of tumour-specific immune responses makes oncolytic vaccines attractive for cancer therapy. We previously developed a heterologous cancer immunization strategy that utilized a replication-defective adenovirus-vectored primary vaccine encoding a tumour antigen followed by boosting with a replication-competent Maraba virus expressing the same antigen. To assess the safety of oncolytic Maraba virus-based booster vaccines and inform the design of clinical trials, we conducted translational studies in cats, which have immune systems that are similar to people and spontaneously develop cancers of comparable types and etiologies. A dose of Maraba virus up to 2.5 × 1011 pfu per cat was well-tolerated, with adverse effects limited to mild, transient pyrexia, weight loss, neutropenia, lymphopenia and thrombocytopenia. Maraba viral genomes were present in some urine, stool and most plasma samples up to one week post-infection, but no infectious viruses were recovered. Post-mortem analysis showed one heart, one lung and all spleen samples contained Maraba virus genomes. No replication-competent viruses were recovered from any tissues. Post-mortem histopathological analyses revealed hyperplasia of lymphoid tissues, but no abnormal lesions were attributed to vaccination. This study demonstrated that Maraba virus-vectored cancer vaccines were well-tolerated and supports their use in treating cats.

A Population Health Management Approach to Oral Health.

Clinical outcomes have been shown to be better, and total costs lower, when patients with chronic illness such as diabetes are managed using a population health strategy in a primary care setting that includes structured coordination of care with specialty services. This "population health management approach" offers a promising new vision for addressing oral disease as a chronic illness through a collaborative partnership between primary care teams and dental professionals.

Comparative functional evaluation of immunocompetent mouse breast cancer models established from PyMT-tumors using small animal PET with [(18)F]FDG and [(18)F]FLT.

Positron emission tomography (PET) allows detection of functional changes in malignant tissue. Establishment of an immortalized immunocompetent breast cancer mouse model would provide a useful platform for the analysis of novel cancer treatment strategies. This study describes a comparative functional evaluation of murine breast cancer models established from polyoma virus middle T antigen (PyMT)-derived tumors using small animal PET imaging with [(18)F]FDG and [(18)F]FLT. Primary PyMT tumor-derived cells and a cell line derived from these tumors (MTHJ) were injected subcutaneously into immunocompetent FVB mice to generate breast cancer xenografts. Tumor growth rates were comparable in both models and tumors were analyzed after 4-5 weeks post-injection. [(18)F]FDG uptake in vitro followed a comparable trend in both models but reached higher uptake levels in primary PyMT cells vs. MTHJ cells after 120 min. At all time points, [(18)F]FLT uptake was significantly higher in MTHJ compared to primary PyMT cells. Dynamic small animal PET imaging with [(18)F]FDG revealed standardized uptake values (SUVs) of 2.5±0.1 (n=8) in tumors from primary cells and 2.8±0.4 (n=6) in MTHJ tumors after 60 min p.i.. The corresponding tumor-muscle-ratios were 9.3±1.5 and 10.4±0.9, respectively. Uptake of [(18)F]FLT resulted in slightly higher SUV(60min) in MTHJ tumors (1.1±0.1, n=6) compared to tumors from primary cells (SUV(60min)=0.9±0.05, n=8, p=0.07). The tumor-muscle-ratio was comparable in both tumors (2.1±0.2 and 1.8±0.1, respectively). The PET imaging data demonstrates that the functional profile of immunocompetent murine breast tumor model MTHJ remains the same as in primary-derived PyMT tumors in vivo. Metabolic and proliferative rates as assessed with [(18)F]FDG and [(18)F]FLT are comparable in both tumor models. The observed high SUV(60min) of 2.8±0.4 with [(18)F]FDG in MTHJ tumors allows one to monitor efficacy of therapeutic interventions connected with changes in metabolic response of the tumor by means of small animal PET.

Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells.

A flow cytometry-based cytotoxicity (FCC) assay was developed using a single fluorophore, calcein-acetoxymethyl diacetylester (calcein-AM), to measure NK cell-mediated cytotoxicity. Non-adherent human K562 and U937 target cells were individually labelled with calcein-AM and co-incubated with effector NK cells to measure calcein loss, and therefore calculate target cell cytotoxicity. This FCC assay also provided a measure of sample viability. Notably, cell viability measured by traditional calcein/7-amino-actinomycin D (7-AAD) double labelling and Trypan Blue methods were comparable to the viability calculated using calcein-loss FCC. This FCC assay may also be used with various effector and target cell types and as a multi-parameter tool to measure viability and immunophenotype cells for tissue engineering purposes.

Retroviral expression of MIR2 decreases both surface MHC class I and the alloimmune CTL response.

The immune response to allogeneic cells in tissue-engineered constructs is a major barrier to their successful application in the treatment of many human diseases. Specifically, the T cell-mediated immune response, initiated through the recognition of cell surface MHCI molecules, is the primary cause of acute cellular allograft rejection. In this study, we altered expression of MHCI through viral immunomodulatory mechanisms to examine whether allogeneic cells could be made to 'mimic' viral evasion of a host CTL response. We demonstrate the successful application of a retroviral vector in vitro to overexpress the Kaposi's sarcoma-associated herpesvirus immunomodulatory protein, MIR2, in human monocyte-like myeloid progenitor cells. This approach led to differential downregulation of cell surface MHCI, ICAM-1 and B7-2 molecules. We also demonstrate that downregulation of immunoactive molecules has the functional effect of significantly reducing T cell-mediated cytotoxicity without altering NK-mediated cytotoxicity in vitro. These results provide proof-of-concept that viral immune evasion strategies allow cell-based tissue-engineered constructs to delay or even prevent acute cellular immune rejection in vivo. Importantly, this methodology could facilitate the development of universal donor cells for tissue engineering applications.

The role of ICP0-Null HSV-1 and interferon signaling defects in the effective treatment of breast adenocarcinoma.

Oncolytic viruses that selectively replicate in cancer cells have been described for over 50 years. Despite the observation by several groups that multimutated herpes simplex type 1 vectors are oncolytic in a variety of murine tumor models, the oncolytic potential of ICP0 null mutants has not been described. This study characterizes a novel second-generation oncolytic herpes simplex type 1 vector null for the ICP0 gene. We tested three mutant viruses and found that all were selectively cytotoxic in a variety of human and murine tumor cells in vitro. Furthermore, we provide evidence of a mechanistic link between ICP0's function in interferon signaling pathways and the observed oncolytic capacity of ICP0 mutants. Using an immunocompetent murine model of breast adenocarcinoma we demonstrate that the ICP0 mutant KM100 completely eradicates tumors in approximately 80% of treated animals and significantly increases survival. Our data suggest that active viral replication is necessary for effective tumor regression. In addition, we characterized the potential of KM100 as an anti-tumor vaccine since cured mice were found to elicit a robust anti-tumor immune response and were refractory to subsequent tumor growth upon rechallenge.

Caring for poorly controlled diabetes mellitus: a randomized pharmacist intervention.

BACKGROUND: There is limited information from randomized controlled studies about the influence of pharmacist interventions on diabetes control. OBJECTIVE: To evaluate the effect of a pharmacist intervention on improving diabetes control; secondary endpoints were medication appropriateness and self-reported adherence. METHODS: A randomized, controlled, multi-clinic trial was conducted in the University of Washington Medicine Neighborhood Clinics. Seventy-seven subjects, > or =18 years old with a hemoglobin (Hb) A(1c) > or =9% at baseline and taking at least one oral diabetes medication, were randomized to receive a pharmacist intervention (n = 43) or usual care (n = 34) for 6 months followed by a 6-month usual-care observation period for both groups. Subjects met with a clinical pharmacist to establish and initiate a diabetes care plan followed by weekly visits or telephone calls to facilitate diabetes management and adherence. HbA(1c), medication appropriateness, and self-reported adherence were assessed at baseline, 6 months, and 12 months. RESULTS: The mean HbA(1c) did not differ between groups over the 12-month period (p = 0.61). A reduction in HbA(1c) was noted for both groups over time compared with baseline (p = 0.001); however, control subjects relied more heavily on provider visits. Medication appropriateness was not improved for diabetes medications (p = 0.65). Self-reported adherence was not significantly improved by the intervention. CONCLUSIONS: This pharmacist intervention did not significantly improve diabetes control, but did allow for similar HbA(1c) control with fewer physician visits. Medication appropriateness and self-reported adherence compared with usual care in individuals with poorly controlled diabetes were not changed.

The Retinoic acid receptor alpha (RARalpha) chimeric proteins PML-, PLZF-, NPM-, and NuMA-RARalpha have distinct intracellular localization patterns.

Retinoic acid receptor alpha (RARalpha) gene rearrangement by reciprocal chromosome translocation is the molecular signature of acute promyelocytic leukemia (APL). Disruption of RARalpha function appears to be the likely cause of aberrant myelopoiesis observed in APL, because PML-RARalpha expression has been shown to deregulate the transcription of genes that control myelopoiesis. To target RARalpha chimeric proteins, we engineered epitope-tagged versions of PML-RARalpha, PLZF-RARalpha, NPM-RARalpha, and NuMA-RARalpha (X-RARalphaV5) and generated a panel of stable COS cell lines expressing X-RARalphaV5. Protein fractionation and Western analysis of these COS lines reveal that X-RARalpha proteins localize to both the cytoplasm and nucleus. NPM-RARalpha is predominantly nuclear whereas NuMA-RARalpha is predominantly cytoplasmic. Confocal immunofluorescent microscopy reveals that PML-RARalpha and PLZF-RARalpha share a primarily diffuse nuclear pattern that excludes the nucleolus. NPM-RARalpha is also diffuse in the nucleus but, in contrast to PML-RARalpha and PLZF-RARalpha, is strongly associated with the nucleolus. Strikingly, NuMA-RARalpha predominantly localizes throughout the cytoplasm in a microspeckled pattern. We further demonstrate that NPM and NuMA interact with NPM-RARalpha and NuMA-RARalpha, respectively. The distinct intracellular localization patterns and the shared ability of X-RARalpha to interact with their respective RARalpha partner proteins (X) further support the hypothesis that deregulation of these partners may play a role in APL pathogenesis.