Tumor endothelial specific expression of Robo4 in adults identifies this plasma membrane protein as an anti-cancer target for immunotherapeutic approaches, such as vaccination. In this report, we describe how vaccination against Robo4 inhibits angiogenesis and tumor growth. To break tolerance to the auto-antigen Robo4, mice were immunised with the extracellular domain of mouse Robo4, fused to the Fc domain of human immunoglobulin within an adjuvant. Vaccinated mice show a strong antibody response to Robo4, with no objectively detectable adverse effects on health. Robo4 vaccinated mice showed impaired fibrovascular invasion and angiogenesis in a rodent sponge implantation assay, as well as a reduced growth of implanted syngeneic Lewis lung carcinoma. The anti-tumor effect of Robo4 vaccination was present in CD8 deficient mice but absent in B cell or IgG1 knockout mice, suggesting antibody dependent cell mediated cytotoxicity as the anti-vascular/anti-tumor mechanism. Finally, we show that an adjuvant free soluble Robo4-carrier conjugate can retard tumor growth in carrier primed mice. These results point to appropriate Robo4 conjugates as potential anti-angiogenic vaccines for cancer patients.

Background: Radiotherapy is an effective treatment of intermediate/high-risk locally advanced prostate cancer, however,> 30% of patients relapse within 5 years. Clinicopathological parameters currently fail to identify patients prone to systemic relapse and those whom treatment intensification may be beneficial. The purpose of this study was to independently validate the performance of a 70-gene Metastatic Assay in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Patients and methods: A bridging cohort of prostate cancer diagnostic biopsy specimens was profiled to enable optimization of the Metastatic Assay threshold before further independent clinical validation in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Multivariable Cox proportional hazard regression analysis was used to assess assay performance in predicting biochemical failure-free survival (BFFS) and metastasisfree survival (MFS). Results: Gene expression analysis was carried out in 248 patients from the independent validation cohort and the Metastatic Assay applied. Ten-year MFS was 72% for Metastatic Assay positive patients and 94% for Metastatic Assay negative patients [HR = 3.21 (1.35-7.67); P = 0.003]. On multivariable analysis the Metastatic Assay remained predictive for development of distant metastases [HR = 2.71 (1.11-6.63); P = 0.030]. The assay retained independent prognostic performance for MFS when assessed with the Cancer of the Prostate Assessment Score (CAPRA) [HR = 3.23 (1.22-8.59); P = 0.019] whilst CAPRA itself was not significant [HR = 1.88, (0.52-6.77); P = 0.332]. A high concordance [100% (61.5-100)] for the assay result was noted between two separate foci taken from 11 tumours, whilst Gleason score had low concordance. Conclusions: The Metastatic Assay demonstrated significant prognostic performance in patients treated with radical radiotherapy both alone and independent of standard clinical and pathological variables. The Metastatic Assay could have clinical utility when deciding upon treatment intensification in high-risk patients. Genomic and clinical data are available as a public resource.

Our aim was to investigate whether molecular classification can be used to refine prognosis in grade 3 endometrial endometrioid carcinomas (EECs). Grade 3 EECs were classified into 4 subgroups: p53 abnormal, based on mutant-like immunostaining (p53abn); MMR deficient, based on loss of mismatch repair protein expression (MMRd); presence of POLE exonuclease domain hotspot mutation (POLE); no specific molecular profile (NSMP), in which none of these aberrations were present. Overall survival (OS) and recurrence-free survival (RFS) rates were compared using the Kaplan-Meier method (Log-rank test) and univariable and multivariable Cox proportional hazard models. In total, 381 patients were included. The median age was 66 years (range, 33 to 96 y). Federation Internationale de Gynecologie et d'Obstetrique stages (2009) were as follows: IA, 171 (44.9%); IB, 120 (31.5%); II, 24 (6.3%); III, 50 (13.1%); IV, 11 (2.9%). There were 49 (12.9%) POLE, 79 (20.7%) p53abn, 115 (30.2%) NSMP, and 138 (36.2%) MMRd tumors. Median follow-up of patients was 6.1 years (range, 0.2 to 17.0 y). Compared to patients with NSMP, patients with POLE mutant grade 3 EEC (OS: hazard ratio [HR], 0.36 [95% confidence interval, 0.18-0.70]; P=0.003; RFS: HR, 0.17 [0.05-0.54]; P=0.003) had a significantly better prognosis; patients with p53abn tumors had a significantly worse RFS (HR, 1.73 [1.09-2.74]; P=0.021); patients with MMRd tumors showed a trend toward better RFS. Estimated 5-year OS rates were as follows: POLE 89%, MMRd 75%, NSMP 69%, p53abn 55% (Log rank P=0.001). Five-year RFS rates were as follows: POLE 96%, MMRd 77%, NSMP 64%, p53abn 47% (P=0.000001), respectively. In a multivariable Cox model that included age and Federation Internationale de Gynecologie et d'Obstetrique stage, POLE and MMRd status remained independent prognostic factors for better RFS; p53 status was an independent prognostic factor for worse RFS. Molecular classification of grade 3 EECs reveals that these tumors are a mixture of molecular subtypes of endometrial carcinoma, rather than a homogeneous group. The addition of molecular markers identifies prognostic subgroups, with potential therapeutic implications.

Background and Aims: From 2004 to 2013, the vineyard area in the United Kingdom (UK) increased 148%. Observed climate change and underlying weather variability were assessed for their influence on the development and viability of UK viticulture.Methods and Results: The perspectives of grapegrowers in the UK on climate change and weather variability were complemented by a quantitative analysis of climate and weather data (1954-2013) for the main UK viticultural regions. The variability of growing season average temperature (GST) was calculated and also mapped using a modelling approach. Since 1993, GST has consistently been above the 13 degrees C cool climate viticulture threshold. Alone, GST does not reliably assure yield predictability but does correlate more closely following the recent increasing UK focus on sparkling wine cultivars. June precipitation demonstrates the strongest relationship with yield.Conclusions: Increasing GST superficially suggests enhanced UK cool climate viticultural opportunities, but critically masks the additional impact of shorter term temperature and precipitation events and a high degree of inter-annual variability that continues to threaten productivity. A recent change in dominant UK vine cultivars appears to have increased viticultural sensitivity to inter-annual weather variability.Significance of the Study: This first quantitative and qualitative analysis of climate vulnerability in UK viticulture identifies threats and opportunities and helps steer studies of the impact of future climate change.

This paper describes a traceability system developed for the Stem cells for Biological Assays of Novel drugs and prediCtive toxiCology consortium. The system combines records and labels that to biological material across geographical locations and scientific processes from sample donation to induced pluripotent stem cell line. The labeling system uses a unique identification number to link every aliquot of sample at every stage of the reprogramming pathway back to the original donor. Only staff at the clinical recruitment site can reconnect the unique identification number to the identifying details of a specific donor. This ensures the system meets ethical and legal requirements for protecting privacy while allowing full traceability of biological material. The system can be adapted to other projects and for use with different primary sample types.

Tumor endothelial specific expression of Robo4 in adults identifies this plasma membrane protein as an anti-cancer target for immunotherapeutic approaches, such as vaccination. In this report, we describe how vaccination against Robo4 inhibits angiogenesis and tumor growth. To break tolerance to the auto-antigen Robo4, mice were immunised with the extracellular domain of mouse Robo4, fused to the Fc domain of human immunoglobulin within an adjuvant. Vaccinated mice show a strong antibody response to Robo4, with no objectively detectable adverse effects on health. Robo4 vaccinated mice showed impaired fibrovascular invasion and angiogenesis in a rodent sponge implantation assay, as well as a reduced growth of implanted syngeneic Lewis lung carcinoma. The anti-tumor effect of Robo4 vaccination was present in CD8 deficient mice but absent in B cell or IgG1 knockout mice, suggesting antibody dependent cell mediated cytotoxicity as the anti-vascular/anti-tumor mechanism. Finally, we show that an adjuvant free soluble Robo4-carrier conjugate can retard tumor growth in carrier primed mice. These results point to appropriate Robo4 conjugates as potential anti-angiogenic vaccines for cancer patients.

Current understanding of the behaviour of sea breezes in the offshore environment is limited but rapidly requires improvement due, not least, to the expansion of the offshore wind energy industry. Here we report on contrasting characteristics of three sea-breeze types on five coastlines around the southern North Sea from an 11 year model-simulated climatology. We present and test an identification method which distinguishes sea-breeze types which can, in principle, be adapted for other coastlines around the world. The coherence of the composite results for each type demonstrates that the method is very effective in resolving and distinguishing characteristics and features. Some features, such as jets and calm zones, are shown to influence offshore wind farm development areas, including the sites of the proposed wind farms up to 200 km offshore. A large variability in sea-breeze frequency between neighbouring coastlines of up to a factor of 3 is revealed. Additionally, there is a strong association between sea-breeze type on one coastline and that which may form coincidentally on another nearby. This association can be as high as 86% between, for example, the North Norfolk and East Norfolk coasts. We show, through associations between sea-breeze events on coastlines with contrasting orientations, that each coastline can be important for influencing the wind climate of another. Furthermore, we highlight that each sea-breeze type needs separate consideration in wind power resource assessment and that future larger turbines will be more sensitive to sea-breeze impacts.

ABSTRACTDeep moist convection (DMC) requires three ingredients: instability, moisture and lift. One measure that incorporates two of these, instability and moisture, is convective available potential energy (CAPE). A 10‐year climatology of CAPE over Great Britain is presented covering the period 1 June 2002–31 May 2012, based on a 9‐km grid spacing implementation of the Weather Research and Forecasting (WRF) model, with two‐way interactive nesting. Appropriate tests are carried out to verify model reliability by comparing simulated and observed CAPE. CAPE is found to be highly variable both spatially and temporally, the highest values being produced during Spanish plume events. A strong relationship is confirmed between surface temperature and CAPE magnitude, the highest CAPE across Great Britain during this period locally exceeding 3000 J kg−1. In an average year, 15 days produce CAPE in excess of 500 J kg−1 somewhere in Great Britain, 4 days > 1000 J kg−1 and 1 day > 1500 J kg−1. Three main CAPE seasons are identified: ‘land dominated CAPE’ between April and September, ‘sea dominated CAPE’ between September and January and ‘low CAPE’ from January to April. The southern North Sea witnesses significant CAPE all year round because of a combination of favourable synoptic situations, including warm air plumes in spring/summer and cold air incursions over warmer seas in winter. CAPE is not a direct predictor of thunderstorm incidence, due in part to the confounding effect of convective inhibition (CIN). However, at the annual scale, when comparing against an existing days of thunder climatology, we observe a close correspondence with >500 J kg−1 CAPE frequency.

Merkel cell carcinoma (MCC) is an aggressive poorly differentiated neuroendocrine cutaneous carcinoma associated with older age, immunodeficiency and Merkel cell polyomavirus (MCPyV) integrated within malignant cells. The presence of intra-tumoural CD8+ lymphocytes reportedly predicts better MCC-specific survival. In this study, the distribution of inflammatory cells and properties of CD8+ T lymphocytes within 20 primary MCC specimens were characterised using immunohistochemistry and multicolour immunofluorescent staining coupled to confocal microscopy. CD8+ cells and CD68+ macrophages were identified in 19/20 primary MCC. CD20+ B cells were present in 5/10, CD4+ cells in 10/10 and FoxP3+ cells in 7/10 specimens. Only two specimens had almost no inflammatory cells. Within specimens, inflammatory cells followed the same patchy distribution, focused at the edge of sheets and nodules and, in some cases, more intense in trabecular areas. CD8+ cells were outside vessels on the edge of tumour. Those few within malignant sheets typically lined up in fine septa not contacting MCC cells expressing MCPyV large T antigen. The homeostatic chemokine CXCL12 was expressed outside malignant nodules whereas its receptor CXCR4 was identified within tumour but not on CD8+ cells. CD8+ cells lacked CXCR3 and granzyme B expression irrespective of location within stroma versus malignant nodules or of the intensity of the intra-tumoural infiltrate. In summary, diverse inflammatory cells were organised around the margin of malignant deposits suggesting response to aberrant signaling, but were unable to penetrate the tumour microenvironment itself to enable an immune response against malignant cells or their polyomavirus.

Abstract. The behaviour and characteristics of the marine component of sea breeze cells have received little attention relative to their onshore counterparts. Yet there is a growing interest and dependence on the offshore wind climate from, for example, a wind energy perspective. Using idealized model experiments, we investigate the sea breeze circulation at scales which approximate to those of the southern North Sea, a region of major ongoing offshore wind farm development. We also contrast the scales and characteristics of the pure and the little known corkscrew and backdoor sea breeze types, where the type is pre-defined by the orientation of the synoptic scale flow relative to the shoreline. We find, crucially, that pure sea breezes, in contrast to corkscrew and backdoor types, can lead to substantial wind speed reductions offshore and that the addition of a second eastern coastline emphasises this effect through generation of offshore "calm zones". The offshore extent of all sea breeze types is found to be sensitive to both the influence of Coriolis acceleration and to the boundary layer scheme selected. These extents range, for example for a pure sea breeze produced in a 2 m s−1 offshore gradient wind, from 0 km to 21 km between the Mellor-Yamada-Nakanishi-Niino and the Yonsei State University schemes respectively. The corkscrew type restricts the development of a backdoor sea breeze on the opposite coast and is also capable of traversing a 100 km offshore domain even under high along-shore gradient wind speed (>15 m s−1) conditions. Realistic variations in sea surface skin temperature and initializing vertical thermodynamic profile do not significantly alter the resulting circulation, though the strengths of the simulated sea breezes are modulated if the effective land-sea thermal contrast is altered. We highlight how sea breeze impacts on circulation need to be considered in order to improve the accuracy of both assessments of the offshore wind energy climate and forecasts of wind energy output.

The Demonstration Test Catchments (DTC) project is a UK Government funded initiative to test the effectiveness of on-farm mitigation measures designed to reduce agricultural pollution without compromising farm productivity. Three distinct catchments in England have been chosen to test the efficacy of mitigation measures on working farms in small tributary sub-catchments equipped with continuous water quality monitoring stations. The Hampshire Avon in the south is a mixed livestock and arable farming catchment, the River Wensum in the east is a lowland catchment with predominantly arable farming and land use in the River Eden catchment in the north-west is predominantly livestock farming. One of the many strengths of the DTC as a national research platform is that it provides the ability to investigate catchment hydrology and biogeochemical response across different landscapes and geoclimatic characteristics, with a range of differing flow behaviours, geochemistries and nutrient chemistries. Although numerous authors present studies of individual catchment responses to storms, no studies exist of multiple catchment responses to the same rainfall event captured with in situ high-resolution nutrient monitoring at a national scale. This paper brings together findings from all three DTC research groups to compare the response of the catchments to a major storm event in April 2012. This was one of the first weather fronts to track across the country following a prolonged drought period affecting much of the UK through 2011–2012, marking an unusual meteorological transition when a rapid shift from drought to flood risk occurred. The effects of the weather front on discharge and water chemistry parameters, including nitrogen species (NO3-N and NH4-N) and phosphorus fractions (total P (TP) and total reactive P (TRP)), measured at a half-hourly time step are examined. When considered in the context of one hydrological year, flow and concentration duration curves reveal that the weather fronts resulted in extreme flow, nitrate and TP concentrations in all three catchments but with distinct differences in both hydrographs and chemographs. Hysteresis loops constructed from high resolution data are used to highlight an array of potential pollutant sources and delivery pathways. In the Hampshire Avon DTC, transport was dominated by sub-surface processes, where phosphorus, largely in the soluble form, was found to be transport-limited. In the Wensum DTC, transport was largely dominated by rapid sub-surface movement due to the presence of under-drainage, which mobilised large quantities of nitrate during the storm. In the Eden DTC, transport was found to be initially dominated by surface runoff, which switched to subsurface delivery on the falling limb of the hydrograph, with the surface delivery transporting large amounts of particulate phosphorus to the river, with a transport-limited response. The lack of exhaustion of nutrient delivery in response to such extreme flow generation indicates the size of the nutrient pools stored in these catchments, and highlights the scale of the challenges faced by environmental managers when designing mitigation measures to reduce the flux of nutrients to UK river systems from diffuse agricultural sources.

This trial tested a dendritic cell (DC) therapeutic cancer vaccine in which antigen is loaded using a novel non-viral transfection method enabling the uptake of plasmid DNA condensed with a cationic peptide. Proof of principle required the demonstration of diverse T lymphocyte responses following vaccination, including multiple reactivities restricted through both major histocompatibility complex (MHC) class I and II. Patients with advanced melanoma were offered four cycles of vaccination with autologous DC expressing melan A and gp100. Disease response was measured using Response Evaluation Criteria in Solid Tumours. Circulating MHC class I- and II-restricted responses were measured against peptide and whole antigen targets using interferon-γ ELIspot and enzyme-linked immunosorbent assay assays, respectively. Responses were analyzed across the trial population and presented descriptively for some individuals. Twenty-five patients received at least one cycle. Vaccination was well tolerated. Three patients had reduction in disease volume. Across the trial population, vaccination resulted in an expansion of effector responses to both antigens, to the human leukocyte antigen A2-restricted modified epitope, melan A ELAGIGILTV, and to a panel of MHC class I- and II-restricted epitopes. Vaccination with mature DC non-virally transfected with DNA encoding antigen had biological effect causing tumour regression and inducing diverse T lymphocyte responses.

Proteasomes represent the major non-lysosomal mechanism responsible for the degradation of proteins. Following interferon γ treatment 3 proteasome subunits are replaced producing immunoproteasomes. Adenovirus E1A interacts with components of the 20S and 26S proteasome and can affect presentation of peptides. In light of these observations we investigated the relationship of AdE1A to the immunoproteasome. AdE1A interacts with the immunoproteasome subunit, MECL1. In contrast, AdE1A binds poorly to the proteasome β2 subunit which is replaced by MECL1 in the conversion of proteasomes to immunoproteasomes. Binding sites on E1A for MECL1 correspond to the N-terminal region and conserved region 3. Furthermore, AdE1A causes down-regulation of MECL1 expression, as well as LMP2 and LMP7, induced by interferon γ treatment during Ad infections or following transient transfection. Consistent with previous reports AdE1A reduced IFNγ-stimulated STAT1 phosphorylation which appeared to be responsible for its ability to reduce expression of immunoproteasome subunits.

This is a phase II clinical trial investigating the safety and efficacy of intravenous vaccination with mature autologous dendritic cells (DCs) pulsed ex vivo with a liver tumor cell line lysate (HepG2) in patients with advanced hepatocellular carcinoma (HCC). HCC is an attractive target for immunotherapy as evidenced by an active recruitment of tumor-infiltrating lymphocytes that are capable of lysing autologous tumor cells in ex vivo studies. DCs are the most potent antigen-presenting cells, with the capacity to take up, process, and present tumor antigens to T cells and stimulate an immune response, thus providing a rational platform for vaccine development. Thirty-five patients with advanced HCC and not suitable for radical or loco-regional therapies received a maximum of six DC vaccinations each at 3-week intervals. In total, 134 DC infusions were administered with no significant toxicity and no evidence of autoimmunity. Twenty-five patients who received at least three vaccine infusions were assessed clinically for response. The radiologically determined disease control rate (combined partial response and stable disease >= 3 months) was 28%. In 17 patients the baseline serum alpha-fetoprotein (AFP) was >= 1,000 ng/ml; in four of these patients, it fell to <30% of baseline following vaccination. In one patient there was a radiological partial response associated with a fall in AFP to <10% of baseline. Immune responses were assessed using an ELIspot assay of interferon-gamma (IFN-gamma) release. In several cases there was induction of T cell responses to the vaccine and/or AFP following vaccination. Conclusion: Autologous DC vaccination in patients with HCC is safe and well tolerated with evidence of antitumor efficacy assessed radiologically and serologically, with generation of antigen-specific immune responses in some cases. (HEPATOLOGY 2009;49:124-132.)

ABSTRACT We have evaluated a neutralizing antibody assay which uses human papillomavirus (HPV) type 16 (HPV-16) and HPV-18 pseudovirions carrying a secretory alkaline phosphatase reporter gene and which can potentially measure functionally relevant HPV type-specific neutralizing antibodies. The reproducibility of the assay was excellent; for HPV-16, the intra- and interassay kappa values were 0.95 and 0.90, respectively; and for HPV-18, the corresponding values were 0.90 and 0.90. This assay was used to describe the kinetics of the neutralizing antibody response in a cohort of 42 young women who were recruited soon after first intercourse and who first tested positive for HPV-16 DNA or HPV-18 DNA, or both, during follow-up. Most women seroconverted following the first detection of type-specific HPV DNA and remained seropositive until the end of follow-up. Our findings are broadly consistent with those of two other cohort studies which have measured the serological response following an incident infection by using the technically simpler virus-like-particle-based enzyme-linked immunosorbent assay.

Solid tumors are composed of the malignant cell itself (most commonly a carcinoma) and supporting cells that comprise the stroma. Significant stromal components include the extracellular matrix, supporting fibroblasts, vessels comprised of endothelium, pericytes and in some cases vascular smooth muscle, lymphatics and usually a major leukocyte infiltration. Indeed, macrophages may constitute up to 50% of the viable cells within the tumor. For many years, researchers have concentrated almost exclusively on the malignant carcinoma and looked for ways to either selectively kill or restrict its growth. In recent years the frustrating lack of advances in cytotoxic cancer therapy provoked a search for more novel strategies and foremost amongst these were anti-angiogenesis and vascular targeting. The purpose of this article is to illustrate how the stroma is now being pursued as an anti-cancer target. The article will briefly touch on antiangiogenics that are now entering the clinic but concentrate on recent studies looking at vascular disrupting agents, stromal tumor fibroblasts and macrophages. Target identification is illustrated by the search for tumor endothelial markers. Finally, we draw attention to efforts to develop a cancer vaccine. The genetic instability and variation found in carcinoma cells made vaccination in the past a near impossibility. In contrast, genetically stable tumor endothelium with its unique accessibility to blood borne agents, together with recent advances in immunotherapy means that the possibility of a cancer vaccine now takes on a reality not previously recognised.

AIM:To determine self-reported use of New Zealand's Guidelines Group (NZGG) and BPAC resources by practice nurses (PNs) in the Greater Auckland area of New Zealand.METHOD:A postal survey of all PNs registered on the University of Auckland's Department of General Practice and Primary Health Care's database.RESULTS:A total of 419 of 917 (46%) PNs working in 280 general practices returned completed questionnaires. The majority of PNs did not use either the NZGG (53%) or BPAC guidelines (57%) and 35% did not use any evidence resources. The main reason these resources were not used was lack of knowledge about them, one-third of PNs had never heard of NZGG guidelines and 42% had never heard of BPAC guidelines. Of those who knew of NZGG guidelines, 74% found them useful, (a 'fair amount' or 'very') and 94% found BPAC guidelines useful (a 'fair amount' or 'very'). When PNs knew of these resources, 74% used NZGG guidelines and 69% used BPAC guidelines for patient care.CONCLUSION:PNs who knew of New Zealand Guidelines and BPAC found them useful in patient management. Practice nurses are not routinely on the mailing list of these two organisations. Strategies to increase PN awareness of these publicly funded evidence-based resources may increase their use and thus contribute to the reduction in health inequalities between ethnic groups in New Zealand.