Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension

BACKGROUND: Inflammation is a feature of pulmonary arterial hypertension (PAH), and increased circulating levels of cytokines are reported in patients with PAH. However, to date, no information exists on the significance of elevated cytokines or their potential as biomarkers. We sought to determine the levels of a range of cytokines in PAH and to examine their impact on survival and relationship to hemodynamic indexes. METHODS AND RESULTS: We measured levels of serum cytokines (tumor necrosis factor-alpha, interferon-gamma and interleukin-1beta, -2, -4, -5, -6, -8, -10, -12p70, and -13) using ELISAs in idiopathic and heritable PAH patients (n=60). Concurrent clinical data included hemodynamics, 6-minute walk distance, and survival time from sampling to death or transplantation. Healthy volunteers served as control subjects (n=21). PAH patients had significantly higher levels of interleukin-1beta, -2, -4, -6, -8, -10, and -12p70 and tumor necrosis factor-alpha compared with healthy control subjects. Kaplan-Meier analysis showed that levels of interleukin-6, 8, 10, and 12p70 predicted survival in patients. For example, 5-year survival with interleukin-6 levels of >9 pg/mL was 30% compared with 63% for patients with levels < or = 9 pg/mL (P=0.008). In this PAH cohort, cytokine levels were superior to traditional markers of prognosis such as 6-minute walk distance and hemodynamics. CONCLUSIONS: This study illustrates dysregulation of a broad range of inflammatory mediators in idiopathic and familial PAH and demonstrates that cytokine levels have a previously unrecognized impact on patient survival. They may prove to be useful biomarkers and provide insight into the contribution of inflammation in PAH

New insights into the intracellular distribution pattern of cationic amphiphilic drugs

Cationic amphiphilic drugs (CADs) comprise a wide variety of different substance classes such as antidepressants, antipsychotics, and antiarrhythmics. It is well recognized that CADs accumulate in certain intracellular compartments leading to specific morphological changes of cells. So far, no adequate technique exists allowing for ultrastructural analysis of CAD in intact cells. Azidobupramine, a recently described multifunctional antidepressant analogue, allows for the first time to perform high-resolution studies of CADs on distribution pattern and morphological changes in intact cells. We showed here that the intracellular distribution pattern of azidobupramine strongly depends on drug concentration and exposure time. The mitochondrial compartment (mDsRed) and the late endolysosomal compartment (CD63-GFP) were the preferred localization sites at low to intermediate concentrations (i.e. 1 mu M, 5 mu M). In contrast, the autophagosomal compartment (LC3-GFP) can only be reached at high concentrations (10 mu M) and long exposure times (72 hrs). At the morphological level, LC3-clustering became only prominent at high concentrations (10 mu M), while changes in CD63 pattern already occurred at intermediate concentrations (5 mu M). To our knowledge, this is the first study that establishes a link between intracellular CAD distribution pattern and morphological changes. Therewith, our results allow for gaining deeper understanding of intracellular effects of CADs

Prediction of second neurological attack in patients with clinically isolated syndrome using support vector machines

The aim of this study is to predict the conversion from clinically isolated syndrome to clinically definite multiple sclerosis using support vector machines. The two groups of converters and non-converters are classified using features that were calculated from baseline data of 73 patients. The data consists of standard magnetic resonance images, binary lesion masks, and clinical and demographic information. 15 features were calculated and all combinations of them were iteratively tested for their predictive capacity using polynomial kernels and radial basis functions with leave-one-out cross-validation. The accuracy of this prediction is up to 86.4% with a sensitivity and specificity in the same range indicating that this is a feasible approach for the prediction of a second clinical attack in patients with clinically isolated syndromes, and that the chosen features are appropriate. The two features gender and location of onset lesions have been used in all feature combinations leading to a high accuracy suggesting that they are highly predictive. However, it is necessary to add supporting features to maximise the accuracy. © 2013 IEEE

Quantum dot synthesis towards integrin receptor detection

Säugetierzellen exprimieren bestimmte Rezeptoren, welche in den komplexen Vorgängen der Zelladhäsion, Proliferation und Angiogenese relevant sind. Allerdings spielt ein Rezeptor, die Integrine, auch eine entscheidende Rolle bei pathogenen Prozessen. Beispielhaft für diese Spezies ist das Integrin v3. Ziel dieser Arbeit beschreibt die Entwicklung einer neuartigen Assaystrategie, um das räumlich-zeitliche Verhalten von Integrinen während der Ligandenbindung zu verfolgen. Integrin-bezogene Prozesse werden typischerweise auf der Oberfläche von lebenden Zellen untersucht. Jedoch ist auf Grund der zellulären Integrinvielfalt und -redundanz, die Charakterisierung spezifischer Integrinwechselwirkungen auf zellulären Oberflächen oft sehr stark eingeschränkt, wodurch diese Untersuchungen schwer zu realisieren sind. Wir waren in der Lage zum ersten Mal die in vitro membranunterstützte Proteinsynthese (iMAPS) eines voneinander abhängigen, heterodimeren Membranrezeptors in eine künstliche Membranarchitektur zu demonstrieren. Diese membranstabilisierten Integrine, in sogenannten Proteopolymersomen, mussten jedoch vor einer weiteren Charakterisierung und Verwendung gereinigt werden. Um dies zu erreichen haben wir hierzu eine spezifische Immunpräzipitationstechnik entwickelt. Immunoanfärbungen litten oft unter schwachen Signalen mit einem reduzierten Signal-Rausch-Verhältnis, welches auf nicht-spezifische Antikörperbindung zurückzuführen ist. Um die integrinspezifische Signalintensität zu erhöhen, entschieden wir uns einen neuen Antikörper-Tracers zu synthetisieren, welcher auf Quantum Dot Nanokristallen (QDs) basierte. Wir entschieden uns daher QDs zu synthetisieren, welche eine proteinresistente Oberfläche aufwiesen. Um dies zu erreichen haben wir ein neues Syntheseverfahren entwickelt, bei der native Monothiolliganden teilweise mit Dithiol-PEG substituiert wurden. Die Adsorptionsbedingungen wurden so abgestimmt, dass ein Gleichgewicht zwischen effizienter Oberflächenpassivierung und Lumineszenz beibehalten wurde und gleichzeitig eine ausreichend dichte PEG-Hülle für eine starke kolloidale Stabilität und Proteinresistenz sorgt.Mammalian cells synthesize certain receptors on their surface relevant in the complex process of cell adhesion, proliferation and angiogenesis. However, such receptor family, the integrins, plays as well a crucial role in pathogenic processes, such as metastasis. An example for a relevant receptor species is the integrin v3. The allover aim of this thesis describes the development of an assay strategy in order to trace the spatio-temporal behavior of integrins upon ligand binding. Integrin-related processes are typically studied on the surface of living cells. However, due to cellular integrin diversity and redundancy, the characterization of specific interactions on cellular surfaces is impeded, making the study difficult. Set-ups based on isolated and surface-immobilized integrin receptors are also difficult to develop, as the isolation of integrin molecules from cells is notoriously difficult. To address this problem, we demonstrated for the first time the in vitro membrane-assisted protein synthesis (iMAPS) of an interdependent heterodimeric membrane receptor into a stable polymeric architecture. These membrane-stabilized integrins, so-called proteopolymersomes, had to be purified from the crude reaction mixture prior to characterization and use. To do this, we developed a specific immunoprecipitation technique in order to purify the proteopolymersomes. Immuno-staining however, often suffered in weak signals stemming from reduced signal-to-noise ratios due to non-specific antibody binding. To enhance the specific signal intensity we chose to address this need by developing a novel tracer strategy, based on Quantum Dot-nanocrystals (QDs). We therefore chose to synthesize QDs, and functionalize them with a protein-resistant, water soluble surface. This required us to establish a novel synthesis method for the partial exchange of native ligands for dithiol-PEG on QDs. The adsorption conditions where fine-tuned until a balance between efficient surface passivation and a sufficiently dense PEG-grafting achieving strong colloidal stability and protein resistance. The QDs will serve as a precursor for further functionalization with an integrin specific target. Furthermore, the samples could also be used directly for transmission electron microscopy (TEM), as electron density and elemental composition of the QDs permit this extension.eingereicht von Dipl.-Chem. Christoph ZabaZusammenfassung in deutscher SpracheUniversität für Bodenkultur Wien, Dissertation, 2016OeBB(VLID)193161

Fluorescent Magnetopolymersomes: A Theranostic Platform to Track Intracellular Delivery

We present a potential theranostic delivery platform based on the amphiphilic diblock copolymer polybutadiene-block-poly (ethylene oxide) combining covalent fluorescent labeling and membrane incorporation of superparamagnetic iron oxide nanoparticles for multimodal imaging. A simple self-assembly and labeling approach to create the fluorescent and magnetic vesicles is described. Cell uptake of the densely PEGylated polymer vesicles could be altered by surface modifications that vary surface charge and accessibility of the membrane active species. Cell uptake and cytotoxicity were evaluated by confocal microscopy, transmission electron microscopy, iron content and metabolic assays, utilizing multimodal tracking of membrane fluorophores and nanoparticles. Cationic functionalization of vesicles promoted endocytotic uptake. In particular, incorporation of cationic lipids in the polymersome membrane yielded tremendously increased uptake of polymersomes and magnetopolymersomes without increase in cytotoxicity. Ultrastructure investigations showed that cationic magnetopolymersomes disintegrated upon hydrolysis, including the dissolution of incorporated iron oxide nanoparticles. The presented platform could find future use in theranostic multimodal imaging in vivo and magnetically triggered delivery by incorporation of thermorepsonsive amphiphiles that can break the membrane integrity upon magnetic heating via the embedded superparamagnetic nanoparticles

Doping Method Determines Para- or Superparamagnetic Properties of Photostable and Surface-Modifiable Quantum Dots for Multimodal Bioimaging

Semiconductor quantum dots (QDs) are widely used for optical applications and bioimaging. In comparison to organic dyes used for fluorescent labeling, QDs exhibit very high photostability and can be further surface modified. Equipping QDs with magnetic properties (mQDs) makes it possible to combine fluorescence and magnetic resonance imaging analyses. For this purpose, we have prepared water-dispersible and magnetic CdTe/ZnS mQDs, whereby ferrous ions are selectively incorporated in either their cores or their shells. This study aims at understanding the differences in optical, structural, and magnetic properties between these core- and shell-doped mQDs. Field-dependent isothermal magnetic susceptibility measurements show that shell-doped mQDs exhibit paramagnetic and their core-doped equivalents super­paramagnetic behavior near room temperature. Shell doping results in about 1.7 times higher photoluminescence quantum yields and 1.4 times higher doping efficiency than core doping. X-ray diffraction patterns reveal that core doping leads to defects in the lattice and hence to a severe decrease in crystallinity, whereas shell doping has no significant impact on the crystal structure and consequently fewer disadvantages regarding the mQD’s quantum yield. These selective doping approaches, particularly shell doping, allow for the tailored design of paramagnetic QDs having modifiable and biocompatible particle surfaces. The organic ligandsin this study <i>N</i>-acetyl-l-cysteinesufficiently prevent leakage of toxic metal ions, as shown by cytotoxicity assays with HepG2 cells. Confocal laser scanning microscopy shows that mQDs are internalized by these cells and accumulated near their nuclei. This study shows that biocompatible, fluorescent, and paramagnetic QDs are promising photostable labels for multimodal bioimaging

Current strategies for managing chronic thromboembolic pulmonary hypertension: results of the worldwide prospective CTEPH Registry

Pulmonary endarterectomy (PEA), pulmonary arterial hypertension (PAH) therapy and balloon pulmonary angioplasty (BPA) are currently accepted therapies for chronic thromboembolic pulmonary hypertension (CTEPH). This international CTEPH Registry identifies clinical characteristics of patients, diagnostic algorithms and treatment decisions in a global context. 1010 newly diagnosed consecutive patients were included in the registry between February 2015 and September 2016. Diagnosis was confirmed by right heart catheterisation, ventilation-perfusion lung scan, computerised pulmonary angiography and/or invasive pulmonary angiography after at least 3 months on anticoagulation. Overall, 649 patients (64.3%) were considered for PEA, 193 (19.1%) for BPA, 20 (2.0%) for both PEA and BPA, and 148 (14.7%) for PAH therapy only. Reasons for PEA inoperability were technical inaccessibility (n=235), comorbidities (n=63) and patient refusal (n=44). In Europe and America and other countries (AAO), 72% of patients were deemed suitable for PEA, whereas in Japan, 70% of patients were offered BPA as first choice. Sex was evenly balanced, except in Japan where 75% of patients were female. A history of acute pulmonary embolism was reported for 65.6% of patients. At least one PAH therapy was initiated in 35.8% of patients (26.2% of PEA candidates, 54.5% of BPA candidates and 54.1% of those not eligible for either PEA or BPA). At the time of analysis, 39 patients (3.9%) had died of pulmonary hypertension-related causes (3.5% after PEA and 1.8% after BPA). The registry revealed noticeable differences in patient characteristics (rates of pulmonary embolism and sex) and therapeutic approaches in Japan compared with Europe and AAO