Development of Allogeneic Stem Cell-Based Platform for Delivery and Potentiation of Oncolytic Virotherapy.

We describe the repurposing and optimization of the TK-positive (thymidine kinase) vaccinia virus strain ACAM1000/ACAM2000™ as an oncolytic virus. This virus strain has been widely used as a smallpox vaccine and was also used safely in our recent clinical trial in patients with advanced solid tumors and Acute Myeloid Leukemia (AML). The vaccinia virus was amplified in CV1 cells and named CAL1. CAL1 induced remarkable oncolysis in various human and mouse cancer cells and preferentially amplified in cancer cells, supporting the use of this strain as an oncolytic virus. However, the therapeutic potential of CAL1, as demonstrated with other oncolytic viruses, is severely restricted by the patients' immune system. Thus, to develop a clinically relevant oncolytic virotherapy agent, we generated a new off-the-shelf therapeutic called Supernova1 (SNV1) by loading CAL1 virus into allogeneic adipose-derived mesenchymal stem cells (AD-MSC). Culturing the CAL1-infected stem cells allows the expression of virally encoded proteins and viral amplification prior to cryopreservation. We found that the CAL1 virus loaded into AD-MSC was resistant to humoral inactivation. Importantly, the virus-loaded stem cells (SNV1) released larger number of infectious viral particles and virally encoded proteins, leading to augmented therapeutic efficacy in vitro and in animal tumor models.

Sharing on platforms: Reducing perceived risk for peer-to-peer platform consumers through trust-building and regulation.

Sharing a flat with strangers is no longer hypothetical but well accepted by many consumers who participate in peer-to-peer (P2P) services. Online P2P sharing platforms act as intermediaries between providers and consumers who do not know each other personally. Sharing via platforms entails a certain amount of risk for consumers. Thus, in order to attract consumers, platforms need to apply mechanisms to reduce the perceived risk of potential consumers. In a prestudy and two experimental online surveys, the current research investigates whether trust-building measures and regulation mechanisms presented on a website can reduce the potential consumers' level of perceived risk and increase their willingness to participate in a platform's sharing offer. First, an analysis of existing P2P accommodation platforms showed a lack of regulation mechanisms. Second, the manipulation of information on P2P accommodation platforms' websites in two online experiments revealed that regulation mechanisms led to lower perceived risk and a higher intention to engage in sharing. However, commonly used trust-building measures on P2P accommodation platforms show no significant effect on risk perception and the intention to engage in sharing in the present study. We point out relevant managerial possibilities to minimise the perceived risk of potential consumers of P2P platforms.

The Influence of Regulation on Trust and Risk Preference in Sharing Communities.

Sharing within communities has gained popularity in recent years. However, taking part in a community also comes with a certain amount of risk. This perceived amount of risk can be contained by regulations within a community as well as by potential participants' trust in the community and the other members. We argue for a relation between regulation and the willingness to take the risk of joining a sharing community with trust as a mediator. Thereby, we distinguish between two kinds of regulation (soft and harsh regulation) and two kinds of trust (implicit and reason-based trust) on two different levels (vertical and horizontal trust). In one laboratory and one online experiment with 432 participants overall, we found that the compound of high soft and low harsh regulation increases participants' willingness to take the risk of participation and that the effect of soft regulation is mediated mainly by vertical and horizontal reason-based trust. Based on our results, we encourage sharing communities to count on soft regulation in order to increase potential members' trust in the community and therefore take the risk to participate.

The impact of powerful authorities and trustful taxpayers: evidence for the extended slippery slope framework from Austria, Finland, and Hungary.

Tax authorities utilize a wide range of instruments to motivate honest taxpaying ranging from strict audits to fair procedures or personalized support, differing from country to country. However, little is known about how these different instruments and taxpayers' trust influence the generation of interaction climates between tax authorities and taxpayers, motivations to comply, and particularly, tax compliance. The present research examines the extended slippery slope framework (eSSF), which distinguishes tax authorities' instruments into different qualities of power of authority (coercive and legitimate) and trust in authorities (reason-based and implicit), to shed light on the effect of differences between power and trust. We test eSSF assumptions with survey data from taxpayers from three culturally different countries (N = 700) who also vary concerning their perceptions of power, trust, interaction climates, and tax motivations. Results support assumptions of the eSSF. Across all countries, the relation of coercive power and tax compliance was mediated by implicit trust. The connection from legitimate power to tax compliance is partially mediated by reason-based trust. The relationship between implicit trust and tax compliance is mediated by a confidence climate and committed cooperation. Theoretical and practical implications are discussed.

Take me on a ride: The role of environmentalist identity for carpooling.

Sharing does not need to involve corporate providers but can also happen on a peer-to-peer (P2P) basis. P2P sharing platforms who match private providers and users are thus dealing with two different customer segments. An example of this is carpooling, the sharing of a car journey. Recent years have seen considerable research on why people use sharing services. In contrast, there is little knowledge of why people may offer a good for sharing purposes. Drawing on identity theory, this paper suggests that users and providers of carpooling need to be addressed differently. A pilot study and two studies, including both actual car owners and nonowners confirm that the extent to which one identifies as an environmentalist predicts car owners' willingness to offer carpooling, but does not affect nonowners' willingness to use carpooling services. These findings remain robust when controlling for various potential confounds. Furthermore, Study 2 suggests that an environmentalist identity plays an important role for car owners' actual decision to offer a ride via an online platform. These results suggest that marketers of P2P platforms need to pursue different strategies when addressing potential users and providers on the same platform.

The Relationship Between Austrian Tax Auditors and Self-Employed Taxpayers: Evidence From a Qualitative Study.

A constructive, highly professional relationship between tax authorities and taxpayers is essential for tax compliance. The aim of the present paper was to explore systematically the determinants of this relationship and related tax compliance behaviors based on the extended slippery slope framework. We used in-depth qualitative interviews with 33 self-employed taxpayers and 30 tax auditors. Interviewees described the relationship along the extended slippery slope framework concepts of power and trust. However, also novel sub-categories of power (e.g., setting deadlines) and trust (e.g., personal assistance) were mentioned. Furthermore, also little-studied categories of tax behavior emerged, such as accepting tax behavior, e.g., being available to the tax authorities, or stalling tax behavior, e.g., the intentional creation of complexity. The results comprehensively summarize the determinants of the tax relationship and tax compliance behaviors. Additionally, results highlight future research topics and provide insights for policy strategies.

A Retrospective Assessment of Four Antigen Assays for the Detection of Invasive Candidiasis Among High-Risk Hospitalized Patients.

Because of their high mortality rates and non-specific symptoms, invasive Candida infections pose a huge diagnostic and therapeutic challenge. In this study, we evaluated the three mannan antigen assays Platelia, Platelia Plus and Serion, and the (1-3)-ß-D-glucan assay Fungitell in a group of high-risk (hematological and surgical) patients. Test results of 305 patients hospitalized at the Vienna General Hospital and the University Hospital of Innsbruck were retrospectively analyzed. We assessed the test accuracy by means of descriptive statistics. Nine (2.95%) patients were affected by invasive candidiasis (IC), and 25 (8.2%) patients had a probable/possible infection. The majority of patients (271; 88.9%) showed no signs of infection. The Platelia and Serion mannan assays had a low sensitivity (65% and 52%, respectively), but high specificity (98% for both tests). The newer version of the Platelia assay, the Platelia Plus, had a higher sensitivity (85%) but a lower specificity (89%). The sensitivity of the Fungitell assay was high (100%), while its specificity was low (58%). The positive predictive values were 0.48 for the Platelia and 0.41 for the Serion assay, 0.26 for the Platelia Plus and 0.09 for the Fungitell assay. Our limited, retrospective study suggests the efficacy of mannan assays as screening (Platelia Plus) and confirmatory (Serion) tests, while the Fungitell assay can be used to exclude invasive Candida infections.

Authorities' Coercive and Legitimate Power: The Impact on Cognitions Underlying Cooperation.

The execution of coercive and legitimate power by an authority assures cooperation and prohibits free-riding. While coercive power can be comprised of severe punishment and strict monitoring, legitimate power covers expert, and informative procedures. The perception of these powers wielded by authorities stimulates specific cognitions: trust, relational climates, and motives. With four experiments, the single and combined impact of coercive and legitimate power on these processes and on intended cooperation of n1 = 120, n2 = 130, n3 = 368, and n4 = 102 student participants is investigated within two exemplary contexts (tax contributions, insurance claims). Findings reveal that coercive power increases an antagonistic climate and enforced compliance, whereas legitimate power increases reason-based trust, a service climate, and voluntary cooperation. Unexpectedly, legitimate power is additionally having a negative effect on an antagonistic climate and a positive effect on enforced compliance; these findings lead to a modification of theoretical assumptions. However, solely reason-based trust, but not climate perceptions and motives, mediates the relationship between power and intended cooperation. Implications for theory and practice are discussed.

Does the sole description of a tax authority affect tax evasion?--the impact of described coercive and legitimate power.

Following the classic economic model of tax evasion, taxpayers base their tax decisions on economic determinants, like fine rate and audit probability. Empirical findings on the relationship between economic key determinants and tax evasion are inconsistent and suggest that taxpayers may rather rely on their beliefs about tax authority's power. Descriptions of the tax authority's power may affect taxpayers' beliefs and as such tax evasion. Experiment 1 investigates the impact of fines and beliefs regarding tax authority's power on tax evasion. Experiments 2-4 are conducted to examine the effect of varying descriptions about a tax authority's power on participants' beliefs and respective tax evasion. It is investigated whether tax evasion is influenced by the description of an authority wielding coercive power (Experiment 2), legitimate power (Experiment 3), and coercive and legitimate power combined (Experiment 4). Further, it is examined whether a contrast of the description of power (low to high power; high to low power) impacts tax evasion (Experiments 2-4). Results show that the amount of fine does not impact tax payments, whereas participants' beliefs regarding tax authority's power significantly shape compliance decisions. Descriptions of high coercive power as well as high legitimate power affect beliefs about tax authority's power and positively impact tax honesty. This effect still holds if both qualities of power are applied simultaneously. The contrast of descriptions has little impact on tax evasion. The current study indicates that descriptions of the tax authority, e.g., in information brochures and media reports, have more influence on beliefs and tax payments than information on fine rates. Methodically, these considerations become particularly important when descriptions or vignettes are used besides objective information.

Evaluation of a new recombinant oncolytic vaccinia virus strain GLV-5b451 for feline mammary carcinoma therapy.

Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis. In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model.

Inducible gene expression in tumors colonized by modified oncolytic vaccinia virus strains.

UNLABELLED: Exogenous gene induction of therapeutic, diagnostic, and safety mechanisms could be a considerable improvement in oncolytic virotherapy. Here, we introduced a doxycycline-inducible promoter system (comprised of a tetracycline repressor, several promoter constructs, and a tet operator sequence) into oncolytic recombinant vaccinia viruses (rVACV), which were further characterized in detail. Experiments in cell cultures as well as in tumor-bearing mice were analyzed to determine the role of the inducible-system components. To accomplish this, we took advantage of the optical reporter construct, which resulted in the production of click-beetle luciferase as well as a red fluorescent protein. The results indicated that each of the system components could be used to optimize the induction rates and had an influence on the background expression levels. Depending on the given gene to be induced in rVACV-colonized tumors of patients, we discuss the doxycycline-inducible promoter system adjustment and further optimization. IMPORTANCE: Oncolytic virotherapy of cancer can greatly benefit from the expression of heterologous genes. It is reasonable that some of those heterologous gene products could have detrimental effects either on the cancer patient or on the oncolytic virus itself if they are expressed at the wrong time or if the expression levels are too high. Therefore, exogenous control of gene expression levels by administration of a nontoxic inducer will have positive effects on the safety as well as the therapeutic outcome of oncolytic virotherapy. In addition, it paves the way for the introduction of new therapeutic genes into the genome of oncolytic viruses that could not have been tested otherwise.

Characterization of metastasis formation and virotherapy in the human C33A cervical cancer model.

More than 90% of cancer mortalities are due to cancer that has metastasized. Therefore, it is crucial to intensify research on metastasis formation and therapy. Here, we describe for the first time the metastasizing ability of the human cervical cancer cell line C33A in athymic nude mice after subcutaneous implantation of tumor cells. In this model, we demonstrated a steady progression of lumbar and renal lymph node metastases during tumor development. Besides predominantly occurring lymphatic metastases, we visualized the formation of hematogenous metastases utilizing red fluorescent protein (RFP) expressing C33A-RFP cells. RFP positive cancer cells were found migrating in blood vessels and forming micrometastases in lungs of tumor-bearing mice. Next, we set out to analyze the influence of oncolytic virotherapy in the C33A-RFP model and demonstrated an efficient virus-mediated reduction of tumor size and metastatic burden. These results suggest the C33A-RFP cervical cancer model as a new platform to analyze cancer metastases as well as to test novel treatment options to combat metastases.

A ubiquitin-specific protease possesses a decisive role for adenovirus replication and oncogene-mediated transformation.

Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies.

Preferential colonization of metastases by oncolytic vaccinia virus strain GLV-1h68 in a human PC-3 prostate cancer model in nude mice.

Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a significant therapeutic potential in treating lymph node metastases of human PC-3 prostate carcinoma in tumor xenografts. In this study, underlying mechanisms of the virus-mediated metastases reduction were analyzed. Immunohistochemistry demonstrated that virus-treatment resulted in a drastically decrease of blood and lymph vessels, representing essential routes for PC-3 cell migration, in both tumors and metastases. Thus, GLV-1h68 drastically reduced essential routes for the metastatic spread of PC-3 cells. Furthermore, analysis of viral distribution in GLV-1h68-injected tumor-bearing mice by plaque assays, revealed significantly higher virus titers in metastases compared to solid tumors. To elucidate conditions potentially mediating the preferential viral colonization and eradication of metastases, microenvironmental components of uninfected tumors and metastases were compared by microscopic studies. These analyses revealed that PC-3 lymph node metastases showed increased vascular permeability, higher proliferation status of tumor cells as determined by BrdU- and Ki-67 assays and lesser necrosis of PC-3 cells than solid tumors. Moreover, an increased number of immune cells (MHCII(+)/CD68(+) macrophages, MHCII(+)/CD19(+) B lymphocytes) combined with an up-regulated expression of pro-inflammatory cytokines was observed in metastases in comparison to primary PC-3 tumors. We propose that these microenvironmental components mediated the metastatic tropism of GLV-1h68. Therefore, vaccinia virus-based oncolytic virotherapy might offer a novel treatment of metastatic prostate carcinomas in humans.

Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies.

BACKGROUND: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. METHODS: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. RESULTS: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) renal excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. CONCLUSION: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells.

Intranuclear targeting and nuclear export of the adenovirus E1B-55K protein are regulated by SUMO1 conjugation.

We have investigated the requirements for CRM1-mediated nuclear export and SUMO1 conjugation of the adenovirus E1B-55K protein during productive infection. Our data show that CRM1 is the major export receptor for E1B-55K in infected cells. Functional inactivation of the E1B-55K CRM1-dependent nuclear export signal (NES) or leptomycin B treatment causes an almost complete redistribution of the viral protein from the cytoplasm to the nucleus and its accumulation at the periphery of the viral replication centers. Interestingly, however, this nuclear restriction imposed on the wild type and the NES mutant protein is fully compensated by concurrent inactivation of the adjacent SUMO1 conjugation site. Moreover, the same mutation fully reverses defects of the NES mutant in the nucleocytoplasmic transport of Mre11 and proteasomal degradation of p53. These results show that nuclear export of E1B-55K in infected cells occurs via CRM1-dependent and -independent pathways and suggest that SUMO1 conjugation and deconjugation provide a molecular switch that commits E1B-55K to a CRM1-independent export pathway.

Blockage of CRM1-dependent nuclear export of the adenovirus type 5 early region 1B 55-kDa protein augments oncogenic transformation of primary rat cells.

The 55-kDa gene product from subgroup C adenovirus type 5 (Ad5) early region 1 (E1B-55kDa) plays a central role in the oncogenic transformation of primary rodent cells primarily by inactivating transcriptional and presumably other functional properties of the tumor suppressor protein p53. We have previously shown that Ad5 E1B-55kDa possesses a leucine-rich nuclear export signal (NES), which confers rapid nucleocytoplasmic shuttling via the CRM1-dependent export pathway. In this study we report that an export-deficient mutant of the viral protein (E1B-NES) substantially enhances focus formation of primary baby rat kidney cells in combination with Ad E1A. Transformed rat cells stably expressing the E1B-NES protein exhibited increased tumorigenicity and accelerated tumor growth in nude mice compared to transformants containing the wild-type E1B product. This 'gain of function' correlated with enhanced inhibition of p53 transactivation in transient reporter assays and the accumulation of the mutant protein and p53 in several dot-like subnuclear aggregates. Interestingly, these structures also contained a large fraction of cellular promyelocytic leukemia protein (PML), a known regulator of p53. These data indicate that E1B-NES promotes oncogenic transformation by combinatorial mechanisms that involve modulation of p53 in the context of PML nuclear bodies. In sum, these results extend our previous observation that inhibition of PML activities by E1B-55kDa is required for efficient focus formation and provide further support for the view that blocking p53 transcriptional functions is the principal mechanism by which the Ad protein contributes to complete cell transformation in conjunction with Ad E1A.