Mesenchymal stem cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model

AbstractLung cancer is a major public health problem in the western world, and gene therapy strategies to tackle this disease systemically are often impaired by inefficient delivery of the vector to the tumour tissue. Some of the main factors inhibiting systemic delivery are found in the blood stream in the form of red and white blood cells (WBCs) and serum components. Mesenchymal stem cells (MSCs) have been shown to home to tumour sites and could potentially act as a shield and vehicle for a tumouricidal gene therapy vector. Here, we describe the ability of an adenoviral vector expressing TRAIL (Ad.TR) to transduce MSCs and show the apoptosis‐inducing activity of these TRAIL‐carrying MSCs on A549 lung carcinoma cells. Intriguingly, using MSCs transduced with Ad.enhanced‐green‐fluorescent‐protein (EGFP) we could show transfer of viral DNA to cocultured A549 cells resulting in transgenic protein production in these cells, which was not inhibited by exposure of MSCs to human serum containing high levels of adenovirus neutralizing antibodies. Furthermore, Ad.TR‐transduced MSCs were shown not to induce T‐cell proliferation, which may have resulted in cytotoxic T‐cell‐mediated apoptosis induction in the Ad.TR‐transduced MSCs. Apoptosis was also induced in A549 cells by Ad.TR‐transduced MSCs in the presence of physiological concentrations of WBC, erythrocytes and sera from human donors that inhibit or neutralize adenovirus alone. Moreover, we could show tumour growth reduction with TRAIL‐loaded MSCs in an A549 xenograft mouse model. This is the first study that demonstrates the potential therapeutic utility of Ad.TR‐transduced MSCs in cancer cells and the stability of this vector in the context of the blood environment.</jats:p

Advances in mesenchymal stem cell-mediated gene therapy for cancer

Mesenchymal stem cells have a natural tropism for tumours and their metastases, and are also considered immunoprivileged. This remarkable combination of properties has formed the basis for many studies investigating their potential as tumour-specific delivery vehicles for suicide genes, oncolytic viruses and secreted therapeutic proteins. The aim of the present review is to discuss the range of approaches that have been used to exploit the tumour-homing capacity of mesenchymal stem cells for gene delivery, and to highlight advances required to realize the full potential of this promising approach

Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

Introduction: A combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia. Methods: Transduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-alpha-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated. Results: The second generation lentiviral vector rHIV-pWPT-EF1-alpha-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression. Conclusions: Collectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies

Clinical decision support systems for opioid prescribing for chronic non-cancer pain in primary care : a scoping review

Background and Objectives: Clinical decision support systems (CDSSs) may help clinicians prescribe opioids for chronic noncancer pain (CNCP) more appropriately. This scoping review determined the extent and range of the current evidence on CDSSs for opioid prescribing for CNCP in primary care, and whether investigators followed best evidence and current guidance in designing, implementing and evaluating these complex interventions. Methods: We searched 9 electronic databases and other data sources for studies from January 1, 2008 to October 11, 2019. Two reviewers independently screened the citations. One reviewer extracted data and a second verified for accuracy. INCLUSION CRITERIA: study of a CDSS for opioid prescribing for CNCP in a primary care clinical setting. We reported quantitative results in tables and qualitative results in narrative form. Results: Our search yielded 5068 records, of which 14 studies met our inclusion criteria. All studies were conducted in the United States. Six studies examined local (eg, health center) CDSSs and 8 examined prescription drug monitoring program CDSSs. Three CDSSs incorporated evidence-based components. Study aims were heterogeneous and study designs included both quantitative and qualitative methodologies. No studies assessed patient health outcomes. Few studies appeared to be following guidance for evaluating complex interventions.  Conclusions: Few studies have rigorously assessed the use of CDSSs for opioid prescribing for CNCP in primary care settings. Going forward, investigators should include evidence-based components into the design of CDSSs and follow guidance for the development and evaluation of complex interventions.PostprintPeer reviewe

Construction of a highly enriched marsupial Y chromosome-specific BAC sub-library using isolated Y chromosomes

The Y chromosome is perhaps the most interesting element of the mammalian genome but comparative analysis of the Y chromosome has been impeded by the difficulty of assembling a shotgun sequence of the Y. B AC-based sequencing has been successful for the human and chimpanzee Y but is difficult to do efficiently for an atypical mammalian model species (Skaletsky et al. 2003, Kuroki et al. 2006). We show how Y-specific sub-libraries can be efficiently constructed using DNA amplified from microdissected or flow-sorted Y chromosomes. A Bacterial Artificial Chromosome (BAC) library was constructed from the model marsupial, the tammar wallaby (Macropus eugenii). We screened this library for Y chromosome-derived BAC clones using DNA from both a microdissected Y chromosome and a flow-sorted Y chromosome in order to create a Y chromosome-specific sub-library. We expected that the tammar wallaby Y chromosome should detect ∼100 clones from the 2.2 times redundant library. The microdissected Y DNA detected 85 clones, 82% of which mapped to the Y chromosome and the flow-sorted Y DNA detected 71 clones, 48% of which mapped to the Y chromosome. Overall, this represented a ∼330-fold enrichment for Y chromosome clones. This presents an ideal method for the creation of highly enriched chromosome-specific sub-libraries suitable for BAC-based sequencing of the Y chromosome of any mammalian species

Derivation of an analytical expression for the power coupling coefficient for offset launch into multimode fiber

The demand for higher bandwidth in local area networks (LANs) has fuelled considerable research in techniques for mitigating modal dispersion in multimode fiber (MMF).These techniques include selective mode excitation, offset launching, angular multiplexing and electronic dispersion compensation, all of which strive to optimize the channel impulse response of a MMF.To obtain the optimal bandwidth-enhancement results from these techniques, knowledge of the distribution of power coupling coefficients given an arbitrary offset launch in a MMF is important.In this paper, an analytical expression for the power coupling coefficient for an incident Gaussian beam launched with a radial offset into a MMF having an infinite parabolic refractive index profile is derived.This expression is useful in understanding the parameters which may affect the power coupling coefficient and how they may enhance the MMF bandwidth. The power coupling coefficients obtained from the derived analytical expression are compared with numerical results and are in excellent agreement.The analytical expression may be extended to manufactured MMF

PIPc study: development of indicators of potentially inappropriate prescribing in children (PIPc) in primary care using a modified Delphi technique

OBJECTIVE: There is limited evidence regarding the quality of prescribing for children in primary care. Several prescribing criteria (indicators) have been developed to assess the appropriateness of prescribing in older and middle-aged adults but few are relevant to children. The objective of this study was to develop a set of prescribing indicators that can be applied to prescribing or dispensing data sets to determine the prevalence of potentially inappropriate prescribing in children (PIPc) in primary care settings. DESIGN: Two-round modified Delphi consensus method. SETTING: Irish and UK general practice. PARTICIPANTS: A project steering group consisting of academic and clinical general practitioners (GPs) and pharmacists was formed to develop a list of indicators from literature review and clinical expertise. 15 experts consisting of GPs, pharmacists and paediatricians from the Republic of Ireland and the UK formed the Delphi panel. RESULTS: 47 indicators were reviewed by the project steering group and 16 were presented to the Delphi panel. In the first round of this exercise, consensus was achieved on nine of these indicators. Of the remaining seven indicators, two were removed following review of expert panel comments and discussion of the project steering group. The second round of the Delphi process focused on the remaining five indicators, which were amended based on first round feedback. Three indicators were accepted following the second round of the Delphi process and the remaining two indicators were removed. The final list consisted of 12 indicators categorised by respiratory system (n=6), gastrointestinal system (n=2), neurological system (n=2) and dermatological system (n=2). CONCLUSIONS: The PIPc indicators are a set of prescribing criteria developed for use in children in primary care in the absence of clinical information. The utility of these criteria will be tested in further studies using prescribing databases

Cytokinin response factors regulate PIN-FORMED auxin transporters

Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development