Decoding Functional High-Density Lipoprotein Particle Surfaceome Interactions.

High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDL's journey throughout the body, its functions are mediated through interactions with cell surface receptors on different cell types. To characterize and better understand the functional interplay between HDL particles and tissue, we analyzed the surfaceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (auto-CSC) and HATRIC-based ligand-receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL receptor scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors representing potential HDL interaction candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the auto-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL.

Reproducible Determination of High-Density Lipoprotein Proteotypes.

High-density lipoprotein (HDL) is a heterogeneous mixture of blood-circulating multimolecular particles containing many different proteins, lipids, and RNAs. Recent advancements in mass spectrometry-based proteotype analysis show promise for the analysis of proteoforms across large patient cohorts. In order to create the required spectral libraries enabling these data-independent acquisition (DIA) strategies, HDL was isolated from the plasma of more than 300 patients with a multiplicity of physiological HDL states. HDL proteome spectral libraries consisting of 296 protein groups and more than 786 peptidoforms were established, and the performance of the DIA strategy was benchmarked for the detection of HDL proteotype differences between healthy individuals and a cohort of patients suffering from diabetes mellitus type 2 and/or coronary heart disease. Bioinformatic interrogation of the data using the generated spectral libraries showed that the DIA approach enabled robust HDL proteotype determination. HDL peptidoform analysis enabled by using spectral libraries allowed for the identification of post-translational modifications, such as in APOA1, which could affect HDL functionality. From a technical point of view, data analysis further shows that protein and peptide quantities are currently more discriminative between different HDL proteotypes than peptidoforms without further enrichment. Together, DIA-based HDL proteotyping enables the robust digitization of HDL proteotypes as a basis for the analysis of larger clinical cohorts.

'Break the Chains 2015' community-based HIV prevention campaign for men who have sex with men in Switzerland: non-randomised evaluation and cost analysis.

OBJECTIVES: To study the implementation, effects and costs of Break the Chains, a community-based HIV prevention campaign for men who have sex with men (MSM) in Switzerland, from March to May 2015, which aimed to reduce early HIV transmission by promoting the campaign message to adopt short-term risk reduction followed by HIV testing. DESIGN: Non-randomised evaluation and cost analysis. SETTING: Gay venues in 11 of 26 cantons in Switzerland and national online media campaign. PARTICIPANTS: MSM in online surveys (precampaign n=834, postcampaign n=688) or attending HIV testing centres (n=885); campaign managers (n=9); and campaign staff (n=38) or further intermediaries (n=80) in an online survey. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome measure was the proportion of MSM at risk of HIV acquisition or transmission who adhered to the campaign message. Secondary outcomes were postcampaign test uptake, knowledge about HIV primary infection and sense of belonging to the gay community. RESULTS: Campaign staff estimated that they contacted 17 145 MSM in 11 cantons. Among 688 respondents to the postcampaign survey, 311 (45.2%) were categorised as MSM at risk. Of 402/688 (58.5%) MSM who had heard about Break the Chains 2015, MSM categorised as being at risk were less likely to report adherence to the campaign message than MSM not at risk (adjusted OR 0.24; 95% CI 0.14 to 0.42). Twenty per cent of MSM with a defined risk of HIV acquisition or transmission who adopted risk reduction declared having done so because of the campaign. Costs for one MSM at risk to adhere to the campaign message were estimated at USD purchasing power parity 36-55. The number of HIV tests in the month after the campaign was twice the monthly average. CONCLUSION: Break the Chains increased HIV testing, implying that community-based campaigns are useful HIV prevention strategies for MSM. Additional interventions are needed to reach MSM at the highest risk of infection more effectively.

Immediate versus modified release hydrocortisone in mitotane-treated patients with adrenocortical cancer.

OBJECTIVE: Mitotane induces hepatic CYP3A4 activity, resulting in accelerated cortisol inactivation, and also increases cortisol binding globulin (CBG). Therefore, higher hydrocortisone doses are required in patients with adrenocortical cancer (ACC) on mitotane treatment. Modified release hydrocortisone has not been used in mitotane-treated ACC patients yet. AIM: Case series to compare serum cortisol, calculated free serum cortisol and ACTH levels in ACC patients on mitotane treatment with immediate and modified release hydrocortisone. DESIGN: Pharmacokinetics of immediate and modified release hydrocortisone, each administered at a dose of 40-20-0 mg, in nine patients with ACC and adjuvant mitotane treatment. For comparison, ten patients with secondary adrenal insufficiency (SAI) on three different hydrocortisone regimens and ten healthy males were included. METHODS: Serum cortisol and plasma ACTH were measured by chemiluminescent enzyme immunoassay, and CBG by RIA, followed by calculation of free cortisol. RESULTS: Calculated free serum cortisol levels after 40 mg immediate release hydrocortisone in ACC patients (46 ± 14 nmol/l) were similar to those after 10 mg immediate release hydrocortisone intake in men with SAI (64 ± 16 nmol/l) or to the physiological morning free cortisol levels in healthy subjects (31 ± 5 nmol/l). Compared to immediate release hydrocortisone, free cortisol levels after 40 mg modified release hydrocortisone in ACC patients were significantly lower (12 ± 3 nmol/l; P = 0·03) resulting in a generally lower AUC (98 ± 21 vs 149 ± 37 nmol h/l; P = 0·02). CONCLUSIONS: 40-20-0 mg immediate release, but not modified release hydrocortisone, resulted in sufficient glucocorticoid coverage in patients with ACC receiving mitotane treatment. The use of equivalent doses of modified release hydrocortisone preparation should be avoided in patients on mitotane treatment.

Variable RBE in proton therapy: comparison of different model predictions and their influence on clinical-like scenarios.

BACKGROUND: In proton radiation therapy a constant relative biological effectiveness (RBE) of 1.1 is usually assumed. However, biological experiments have evidenced RBE dependencies on dose level, proton linear energy transfer (LET) and tissue type. This work compares the predictions of three of the main radio-biological models proposed in the literature by Carabe-Fernandez, Wedenberg, Scholz and coworkers. METHODS: Using the chosen models, a spread-out Bragg peak (SOBP) as well as two exemplary clinical cases (single field and two fields) for cranial proton irradiation, all delivered with state-of-the-art pencil-beam scanning, have been analyzed in terms of absorbed dose, dose-averaged LET (LET D ), RBE-weighted dose (D RBE) and biological range shift distributions. RESULTS: In the systematic comparison of RBE predictions by the three models we could show different levels of agreement depending on (α/ß) x and LET values. The SOBP study emphasizes the variation of LET D and RBE not only as a function of depth but also of lateral distance from the central beam axis. Application to clinical-like scenario shows consistent discrepancies from the values obtained for a constant RBE of 1.1, when using a variable RBE scheme for proton irradiation in tissues with low (α/ß) x , regardless of the model. Biological range shifts of 0.6- 2.4 mm (for high (α/ß) x ) and 3.0 - 5.4 mm (for low (α/ß) x ) were found from the fall-off analysis of individual profiles of RBE-weighted fraction dose along the beam penetration depth. CONCLUSIONS: Although more experimental evidence is needed to validate the accuracy of the investigated models and their input parameters, their consistent trend suggests that their main RBE dependencies (dose, LET and (α/ß) x ) should be included in treatment planning systems. In particular, our results suggest that simpler models based on the linear-quadratic formalism and LETD might already be sufficient to reproduce important RBE dependencies for re-evaluation of plans optimized with the current RBE = 1.1 approximation. This approach would be a first step forward to consider RBE variations in proton therapy, thus enabling a more robust choice of biological dose delivery. The latter could in turn impact clinical outcome, especially in terms of reduced toxicities for tumors adjacent to organs at risk.

Clinical implementation and range evaluation of in vivo PET dosimetry for particle irradiation in patients with primary glioma.

PURPOSE: The physical and biological properties of ion-beams offer various advantages in comparison to conventional radiotherapy, though uncertainties concerning quality assurance are still left. Due to the inverted depth dose profile, range accuracy is of paramount importance. We investigated the range deviations between planning simulation and post-fractional PET/CT measurement from particle therapy in primary glioblastoma. METHODS AND MATERIALS: 20 patients with glioblastoma undergoing particle therapy at our institution were selected. 10 received a proton-boost, 10 a carbon-ion-boost in addition to standard treatment. After two fractions, we performed a PET/CT-scan of the brain. We compared the resulting range deviation based on the Most-likely-shift method between the two measurements, and the measurements with corresponding expectations, calculated with the Monte-Carlo code FLUKA. RESULTS: A patient's two measurements deviated by 0.7mm (±0.7mm). Overall comparison between measurements and simulation resulted in a mean range deviation of 3.3mm (±2.2mm) with significant lower deviations in the (12)C-arm. CONCLUSION: The used planning concepts display the actual dose distributions adequately. The carbon ion group's results are below the used PTV safety margins (3mm). Further adjustments to the simulation are required for proton irradiations. Some anatomical situations require particular attention to ensure highest accuracy and safety.