Immune modulation in advanced radiotherapies: Targeting out-of-field effects

By virtue of being a localized treatment modality, radiotherapy is unable to deliver a tumoricidal radiation dose to tissues outside of the irradiated field. Nevertheless, ionizing radiation may result in radiation damage mediated by a bystander like effect away from the irradiated field, but this response is likely to be modest when radiotherapy is the sole treatment modality. Over the last decade there has been a re-emergence of immune modulating therapies as anti-cancer treatment modalities. Clinical trials on vaccines have on the whole been largely disappointing, but greater response rates have been observed from the immune checkpoint modulators. A clinical benefit of using such agents has been shown in disease sites such as melanoma and non-small cell lung cancer. There is growing pre-clinical data and a number of case reports which suggest the presence of abscopal effects when radiotherapy is co-administered with immune checkpoint inhibitors, suggesting that this combination may lead to an enhanced tumour response outside of the primary treatment field. In this review, the mechanisms of such an enhanced out-of-field tumour response, the potential clinical utilities, the optimal radiotherapy delivery and considerations for clinical follow-up following treatment are discussed.

Rationale and Means to Target Pro-Inflammatory Interleukin-8 (CXCL8) Signaling in Cancer

It is well established that chronic inflammation underpins the development of a number of human cancers, with pro-inflammatory signaling within the tumor microenvironment contributing to tumor progression and metastasis. CXCL8 is an ELR+ pro-inflammatory CXC-chemokine which mediates its effects via signaling through two G protein-coupled receptors, CXCR1 and CXCR2. Elevated CXCL8-CXCR1/2 signaling within the tumor microenvironment of numerous cancers is known to enhance tumor progression via activation of signaling pathways promoting proliferation, angiogenesis, migration, invasion and cell survival. This review provides an overview of established roles of CXCL8-CXCR1/2 signaling in cancer and subsequently, discusses the possible strategies of targeting CXCL8-CXCR1/2 signaling in cancer, covering indirect strategies (e.g., anti-inflammatories, NFκB inhibitors) and direct CXCL8 or CXCR1/2 inhibition (e.g., neutralizing antibodies, small molecule receptor antagonists, pepducin inhibitors and siRNA strategies). Reports of pre-clinical cancer studies and clinical trials using CXCL8-CXCR1/2-targeting strategies for the treatment of inflammatory diseases will be discussed. The future translational opportunities for use of such agents in oncology will be discussed, with emphasis on exploitation in stratified populations.

Patience, Persistence and Pragmatism: Experiences and Lessons Learnt from the Implementation of Clinically Integrated Teaching and Learning of Evidence-Based Health Care - A Qualitative Study

Background

Clinically integrated teaching and learning are regarded as the best options for improving evidence-based healthcare (EBHC) knowledge, skills and attitudes. To inform implementation of such strategies, we assessed experiences and opinions on lessons learnt of those involved in such programmes.

Methods and Findings

We conducted semi-structured interviews with 24 EBHC programme coordinators from around the world, selected through purposive sampling. Following data transcription, a multidisciplinary group of investigators carried out analysis and data interpretation, using thematic content analysis. Successful implementation of clinically integrated teaching and learning of EBHC takes much time. Student learning needs to start in pre-clinical years with consolidation, application and assessment following in clinical years. Learning is supported through partnerships between various types of staff including the core EBHC team, clinical lecturers and clinicians working in the clinical setting. While full integration of EBHC learning into all clinical rotations is considered necessary, this was not always achieved. Critical success factors were pragmatism and readiness to use opportunities for engagement and including EBHC learning in the curriculum; patience; and a critical mass of the right teachers who have EBHC knowledge and skills and are confident in facilitating learning. Role modelling of EBHC within the clinical setting emerged as an important facilitator. The institutional context exerts an important influence; with faculty buy-in, endorsement by institutional leaders, and an EBHC-friendly culture, together with a supportive community of practice, all acting as key enablers. The most common challenges identified were lack of teaching time within the clinical curriculum, misconceptions about EBHC, resistance of staff, lack of confidence of tutors, lack of time, and negative role modelling.

Conclusions

Implementing clinically integrated EBHC curricula requires institutional support, a critical mass of the right teachers and role models in the clinical setting combined with patience, persistence and pragmatism on the part of teachers.

Microphysiological modeling of the reproductive tract: A fertile endeavor

Preclinical toxicity testing in animal models is a cornerstone of the drug development process, yet it is often unable to predict adverse effects and tolerability issues in human subjects. Species-specific responses to investigational drugs have led researchers to utilize human tissues and cells to better estimate human toxicity. Unfortunately, human cell-derived models are imperfect because toxicity is assessed in isolation, removed from the normal physiologic microenvironment. Microphysiological modeling often referred to as 'organ-on-a-chip' or 'human-on-a-chip' places human tissue into a microfluidic system that mimics the complexity of human in vivo physiology, thereby allowing for toxicity testing on several cell types, tissues, and organs within a more biologically relevant environment. Here we describe important concepts when developing a repro-on-a-chip model. The development of female and male reproductive microfluidic systems is critical to sex-based in vitro toxicity and drug testing. This review addresses the biological and physiological aspects of the male and female reproductive systems in vivo and what should be considered when designing a microphysiological human-on-a-chip model. Additionally, interactions between the reproductive tract and other systems are explored, focusing on the impact of factors and hormones produced by the reproductive tract and disease pathophysiology.

Genomics and the impact of new technologies on the management of colorectal cancer

High-throughput genomic technologies have the potential to have a major impact on preclinical and clinical drug development and the selection and stratification of patients in clinical trials. These technologies, which are at varying stages of commercialization, include array-based comparative genomic hybridization, single-nucleotide polymorphism arrays, and (the most mature example) expression-based arrays. One of the rate-limiting steps in the routine clinical application of expression array-based technology is the need for suitable clinical samples. One of the major challenges moving forward, therefore, relates to the ability to use formalin-fixed, paraffin-embedded--derived tissue in expression profiling-based approaches.

Pro-neural transcription factors as cancer markers

BACKGROUND: The aberrant transcription in cancer of genes normally associated with embryonic tissue differentiation at various organ sites may be a hallmark of tumour progression. For example, neuroendocrine differentiation is found more commonly in cancers destined to progress, including prostate and lung. We sought to identify proteins which are involved in neuroendocrine differentiation and differentially expressed in aggressive/metastatic tumours.

RESULTS: Expression arrays were used to identify up-regulated transcripts in a neuroendocrine (NE) transgenic mouse model of prostate cancer. Amongst these were several genes normally expressed in neural tissues, including the pro-neural transcription factors Ascl1 and Hes6. Using quantitative RT-PCR and immuno-histochemistry we showed that these same genes were highly expressed in castrate resistant, metastatic LNCaP cell-lines. Finally we performed a meta-analysis on expression array datasets from human clinical material. The expression of these pro-neural transcripts effectively segregates metastatic from localised prostate cancer and benign tissue as well as sub-clustering a variety of other human cancers.

CONCLUSION: By focussing on transcription factors known to drive normal tissue development and comparing expression signatures for normal and malignant mouse tissues we have identified two transcription factors, Ascl1 and Hes6, which appear effective markers for an aggressive phenotype in all prostate models and tissues examined. We suggest that the aberrant initiation of differentiation programs may confer a selective advantage on cells in all contexts and this approach to identify biomarkers therefore has the potential to uncover proteins equally applicable to pre-clinical and clinical cancer biology.

An in vitro model of nociceptors in human trigeminal nerves

Background: The oro-facial region is densely innervated by the trigeminal nerve, which when stimulated can induce noxious pain sensation and contribute to neurogenic inflammation in local tissues. Recent research on the expression of specialised ion channels on the trigeminal nerve has highlighted the need to undertake more extensive studies on ion channel expression/functionality with the aim of elucidating their role in pain sensations. A major family of such ion channels is the transient receptor potential (TRP) channels which are activated by a wide variety of thermal, mechanical or chemical stimuli and merit investigation as possible druggable targets for future analgesics.
Objective: Study of TRP channel expression and regulation in oro-facial tissues is hindered by the fact that the cell bodies of neurons innervating these tissues are located in the trigeminal ganglion. Using dental pulp stem cells differentiated towards peripheral neuronal equivalents (PNEs), we sought to determine TRP channel expression, functionality and potential modulation by cytokines in this novel model.
Method: Dental pulp stem cells (DPSCs) were grown on substrate-coated tissue culture plates and differentiated towards a neuronal phenotype using neuronal induction media. Quantitative polymerase chain reaction (qPCR) was performed on PNEs +/-cytokine treatment. Ion channel functionality was investigated using whole cell patch clamping.
Result: qPCR analysis showed that PNEs expressed the TRP channels TRPA1, TRPV1, TRPV4 and TRPM8. TRPA1 was the most abundantly expressed TRP channel studied whereas TRPM8 was lowly expressed. TRP channel expression was shown to be regulated by treatment with inflammatory cytokines. Patch clamp studies using specific agonists and antagonists for TRPA1 and TRPV1 showed these channels were functional.
Conclusion: PNEs differentiated from DPSCs provide a suitable model for TRP channel expression, regulation, and sensistisation in oro-facial tissues. This human neuronal model has potential for use in pre-clinical studies of novel analgesics.

Mitochondrial Transfer via Tunneling Nanotubes (TNT) is an Important Mechanism by which Mesenchymal Stem Cells Enhance Macrophage Phagocytosis in the in vitro and in vivo Models of ARDS

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in pre-clinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the anti-microbial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC anti-microbial effect in the in vivo model of E.coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct co-culture of MSC with monocyte-derived macrophages (MDMs) enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through TNT-like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the anti-microbial effect of MSC in vivo.

Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the anti-microbial effect of MSC in ARDS.

A Design & Feasibility Study of the Spaced Learning Programme

Objectives There is evidence from neuroscience, cognitive psychology and educational research that the delivery of a stimulus in a spaced format (over time) rather than a massed format (all at once) leads to more effective learning. This project aimed to pilot spaced learning materials using various spacing lengths for GCSE science by exploring the feasibility of introducing spaced leaning into regular classrooms and by evaluating teacher fidelity to the materials. The spaced learning methods will then be compared with traditional science revision techniques and a programme manual will be produced. Design A feasibility study. Methods A pilot study (4 schools) was carried out to examine the feasibility and teacher fidelity to the materials, using pupil workshops and teacher interviews. A subsequent random assignment experimental study (12 schools) will involve pre and post testing of students on a science attainment measure and a post-test implementation questionnaire. Results The literature review found that longer spacing intervals between repetitions of material (>24 hours) may be optimal for long term memory formation than shorter intervals. A logic model was developed to inform the design of various programme variants for the pilot and experimental study. This paper will report qualitative data from the initial pilot study. Conclusions The paper uses this research project as an example to explain the importance of conducting pilot work and small scale experimental studies to explore the feasibility and inform the design of educational interventions, rather than prematurely moving to RCT type studies.

Prior knowledge transfer across transcriptional data sets and technologies using compositional statistics yields new mislabelled ovarian cell line

Here, we describe gene expression compositional assignment (GECA), a powerful, yet simple method based on compositional statistics that can validate the transfer of prior knowledge, such as gene lists, into independent data sets, platforms and technologies. Transcriptional profiling has been used to derive gene lists that stratify patients into prognostic molecular subgroups and assess biomarker performance in the pre-clinical setting. Archived public data sets are an invaluable resource for subsequent in silico validation, though their use can lead to data integration issues. We show that GECA can be used without the need for normalising expression levels between data sets and can outperform rank-based correlation methods. To validate GECA, we demonstrate its success in the cross-platform transfer of gene lists in different domains including: bladder cancer staging, tumour site of origin and mislabelled cell lines. We also show its effectiveness in transferring an epithelial ovarian cancer prognostic gene signature across technologies, from a microarray to a next-generation sequencing setting. In a final case study, we predict the tumour site of origin and histopathology of epithelial ovarian cancer cell lines. In particular, we identify and validate the commonly-used cell line OVCAR-5 as non-ovarian, being gastrointestinal in origin. GECA is available as an open-source R package.

Psychosocial impact of breast/ovarian (BRCA 1/2) cancer-predictive genetic testing in a UK multi-centre clinical cohort

This multi-centre UK study assesses the impact of predictive testing for breast and ovarian cancer predisposition genes (BRCA 1/2) in the clinical context. In the year following predictive testing, 261 adults (59 male) from nine UK genetics centres participated; 9 I gene mutation carriers and 170 noncarriers. Self-report questionnaires were completed at baseline (pre-genetic testing) and 1, 4 and 12 months following the genetic test result. Men were assessed for general mental health (by general health questionnaire (GHQ)) and women for general mental health, cancer-related worry, intrusive and avoidant thoughts, perception of risk and risk management behaviour. Main comparisons were between female carriers and noncarriers on all measures and men and women for general mental health. Female noncarriers benefited psychologically, with significant reductions in cancer-related worry following testing (P

RAS testing of colorectal carcinoma—a guidance document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Group

Analysis of colorectal carcinoma (CRC) tissue for KRAS codon 12 or 13 mutations to guide use of anti-epidermal growth factor receptor (EGFR) therapy is now considered mandatory in the UK. The scope of this practice has been recently extended because of data indicating that NRAS mutations and additional KRAS mutations also predict for poor response to anti-EGFR therapy. The following document provides guidance on RAS (i.e., KRAS and NRAS) testing of CRC tissue in the setting of personalised medicine within the UK and particularly within the NHS. This guidance covers issues related to case selection, preanalytical aspects, analysis and interpretation of such RAS testing.

Biomarker Testing and Pre-emptive Therapy in Preventing Heart Failure

In an attempt to reduce the heart failure epidemic,screening and prevention will become an increasing focus ofmanagement in the wider at-risk population. Refining riskprediction through the use of biomarkers in isolation or incombination is emerging as a critical step in this process.The utility of biomarkers to identify disease manifestationsbefore the onset of symptoms and detrimental myocardialdamage is proving to be valuable. In addition, biomarkers thatpredict the likelihood and rate of disease progression over timewill help streamline and focus clinical efforts and therapeuticstrategies. Importantly, several recent early intervention studiesusing biomarker strategies are promising and indicate thatnot only can new-onset heart failure be reduced but also thedevelopment of other cardiovascular conditions.

Drug Delivery of Aminolevulinic Acid from Topical Formulations Intended for Photodynamic Therapy

Topical photodynamic therapy is used for a variety of malignant and pre-malignant skin disorders, including Bowen's Disease and Superficial Basal Cell Carcinoma. A haem precursor, typically 5-aminolevulinic acid (ALA), acting as a prodrug, is absorbed and converted by the haem biosynthetic pathway to photoactive protoprophyrin IX (PpIX), which accumulates preferentially in rapidly dividing
cells. Cell destruction occurs when PpIx is activated by an intense light source of appropriate wavelength. Topical delivery of ALA avoids the prolonged photosensitivity reactions associated with systemic administration of photosensitisers but its clinical utility is influenced by the tissue penetration characteristics of the drug, its ease of application and the stability of the active agent in the applied dose. This review, therefore, focuses on drug delivery applications for topical, ALA-based PDT. Issues considered in detail include physical and chemical enhancement strategies for tissue penetration of ALA and subsequent intracellular accumulation of PpIX, together with formulation strategies and drug delivery design solutions appropriate to various clinical applications. The fundamental aspects of drug diffusion in
relation to the physicochemical properties of ALA are reviewed and specific consideration is given to the degradation pathways of ALA in formulated systems that, in turn, influence the design of stable topical formulations.