Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.     

Cutting edge: Protective effect of CX3CR1+ dendritic cells in a vaccinia virus pulmonary infection model

The protective host immune response to viral infections requires both effective innate and adaptive immune responses. Cross-talk between the two responses is coordinated by the chemokine network and professional APCs such as dendritic cells (DCs). In mice, subpopulations of myeloid DCs in peripheral tissues such as lungs and in blood express CX3CR1 depending on the inflammation state. We thus examined the host response of mice deficient in the chemokine receptor CX3CR1 to an intranasal vaccinia virus infection. CX3CR1-deficient mice displayed significantly more severe morbidity and mortality compared with control wild-type mice within 10 d following vaccinia virus infection. CX3CR1(-/-) mice had increased viral loads and a reduced T cell response compared with wild-type mice. Finally, an adoptive transfer of CX3CR1(+/+) DCs completely protected CX3CR1(-/-) mice to a previously lethal infection. This study therefore opens up the possibility of novel antiviral therapeutics targeting lung DC recruitment.     

Structure of an Fab-protease complex reveals a highly specific non-canonical mechanism of inhibition

The vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition but, due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be aided greatly by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 Å resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.     

Cumulative funnel plots for the early detection of interoperator variation: retrospective database analysis of observed versus predicted results of percutaneous coronary intervention

Objective To use funnel plots and cumulative funnel plots to compare in-hospital outcome data for operators undertaking percutaneous coronary interventions with predicted results derived from a validated risk score to allow for early detection of variation in performance.Design Analysis of prospectively collected data.Setting Tertiary centre NHS hospital in the north east of England.Participants Five cardiologists carrying out percutaneous coronary interventions between January 2003 and December 2006.Main outcome measures In-hospital major adverse cardiovascular and cerebrovascular events (in-hospital death, Q wave myocardial infarction, emergency coronary artery bypass graft surgery, and cerebrovascular accident) analysed against the logistic north west quality improvement programme predicted risk, for each operator. Results are displayed as funnel plots summarising overall performance for each operator and cumulative funnel plots for an individual operator’s performance on a case series basis.Results The funnel plots for 5198 patients undergoing percutaneous coronary interventions showed an average observed rate for major adverse cardiovascular and cerebrovascular events of 1.96% overall. This was below the predicted risk of 2.06% by the logistic north west quality improvement programme risk score. Rates of in-hospital major adverse cardiovascular and cerebrovascular events for all operators were within the 3σ upper control limit of 2.75% and 2σ upper warning limit of 2.49%.Conclusion The overall in-hospital major adverse cardiovascular and cerebrovascular events rates were under the predicted event rate. In-hospital rates after percutaneous coronary intervention procedure can be monitored successfully using funnel and cumulative funnel plots with 3σ control limits to display and publish each operator’s outcomes. The upper warning limit (2σ control limit) could be used for internal monitoring. The main advantage of these charts is their transparency, as they show observed and predicted events separately. By this approach individual operators can monitor their own performance, using the predicted risk for their patients but in a way that is compatible with benchmarking to colleagues, encapsulated by the funnel plot. This methodology is applicable regardless of variations in individual operator case volume and case mix.     

Analytical validation of a prognostic prostate cancer gene expression assay using formalin fixed paraffin embedded tissue

Background

There is a clear need for assays that can predict the risk of metastatic prostate cancer following curative procedures. Importantly these assays must be analytically robust in order to provide quality data for important clinical decisions. DNA microarray based gene expression assays measure several analytes simultaneously and can present specific challenges to analytical validation. This study describes the analytical validation of one such assay designed to predict metastatic recurrence in prostate cancer using primary formalin fixed paraffin embedded tumour material.

Methods

Accuracy was evaluated with a method comparison study between the assay development platform (Prostate Disease Specific Array) and an alternative platform (Xcel? microarray) using 50 formalin-fixed, paraffin-embedded prostate cancer patient samples. An additional 70 samples were used to establish the assay reportable range. Determination of assay precision and sensitivity was performed on multiple technical replicates of three prostate cancer samples across multiple variables (operators, days, runs, reagent lots, and equipment) and RNA/cDNA inputs respectively using the appropriate linear mixed model.

Results

The overall agreement between the development and alternative platform was 94.7% (95% confidence interval, 86.9–98.5%). The reportable range was determined to be 0.150 to 1.107 for core needle biopsy samples and???0.214 to 0.844 for radical prostatectomy samples. From the precision study, the standard deviations for assay repeatability and reproducibility were 0.032 and 0.040 respectively. The sensitivity study demonstrated that a total RNA input and cDNA input of 50?ng and 3.5?μg respectively was conservative.

Conclusion

The Metastatic Assay was found to be highly reproducible and precise. In conclusion the studies demonstrated an acceptable analytical performance for the assay and support its potential use in the clinic.

     

Spruce budworm feeding and oviposition are stimulated by monoterpenes in white spruce epicuticular waxes

Monoterpenes, source of the distinctive odor of conifers, are generally considered plant defensive compounds. However, they are also known to act as long‐range insect attractants, as they are volatile and permeate forest airspaces. Moreover, they are lipid soluble and can be absorbed into plant epicuticular waxes. We test their role in short‐range host plant choice by both adult females and larvae of a folivorous forest pest (Choristoneura fumiferana). We conducted laboratory assays testing the responses of Eastern spruce budworm to an artificial monoterpene mix (α‐pinene, β‐pinene, limonene, myrcene) and to white spruce (Picea glauca) epicuticular waxes in closed arenas. Ovipositing females preferred filter paper discs treated with P. glauca waxes to controls, and preferred the waxes + monoterpenes treatment to waxes alone. However, females showed no preference between the monoterpene‐treated disc and the control when presented without waxes. Feeding larvae prefered wax discs to control discs. They also consumed discs treated with realistic monoterpene concentrations and wax preferentially over wax‐only discs, but showed no preference between extremely high monoterpene concentrations and wax‐only controls. In an insect‐free assay, P. glauca epicuticular wax decreased monoterpene volatilization. These results suggest that P. glauca waxes and realistic concentrations of monoterpenes are stimulatory to both egg‐laying females and feeding larvae, and that their effects are synergistic.     

Synthesis of serum proteins by cultured aggregates from endodermal cells of the area opaca of the primitive streak chick embryo

Summary The pattern of serum protein synthesis and secretion in aggregates of extraembryonic endoderm cells (EEC) from the area opaca of primitive streak chick embryos was studied. EEC aggregates were cultured for various time intervals and serum proteins were detected using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Serum proteins were identified based on their comigration with reference proteins from 4 day chick embryo serum and with reference proteins from egg white albumen and chicken serum. A number of serum proteins were detected in EEC aggregates including: two variants of immunoglobulin (IgG), four variants of transferrin, a protein with a molecular weight of 66 500 which may correspond to globulins, prealbumin, and a protein with a molecular weight of 38 600 (serum protein 11) which remains unidentified. These proteins were also detected in the culture medium. The banding profiles of EEC extracts and culture medium were compared over various time intervals of culture (6, 18 and 30 h). The IgGs, transforms and serum protein 11 decreased in concentration in EEC extracts over the culture interval. These proteins as well as prealbumin, were detected in the culture medium. A number of proteins were synthesized by EEC, as determined by radiolabelled amino acid incorporation. All of the labelled serum proteins were detected in the culture medium, not in EEC extracts. These results suggest that serum proteins are synthesized by EEC then rapidly released into the medium. Labelled serum proteins detected in the culture medium include prealbumin and an unidentified serum protein (serum protein 14) which migrates with the tracking dye, both synthesized early in culture (6 h), and transferrin which was synthesized later (18 h) during culture.