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Purpose Isoflurane (ISO) is the most commonly used preclinical inhalation anesthetic. This is a problem in 19F MRI of fluorine contrast agents, as ISO signals cause artifacts that interfere with unambiguous image interpretation and quantification; the two most attractive properties of heteronuclear MRI. We aimed to avoid these artifacts using MRI strategies that can be applied by any pre-clinical researcher. Procedures Three strategies to avoid ISO chemical shift displacement artifacts (CSDA) in 19F MRI are described and demonstrated with measurements of 19F-containing agents in phantoms and in vivo (n = 3 for all strategies). The success of these strategies is compared to a standard Rapid Acquisition with Relaxation Enhancement (RARE) sequence, with phantom and in vivo validation. ISO artifacts can successfully be avoided by (1) shifting them outside the region of interest using a narrow signal acquisition bandwidth, (2) suppression of ISO by planning a frequency-selective suppression pulse before signal acquisition or by (3) preventing ISO excitation with a 3D sequence with a narrow excitation bandwidth. Results All three strategies result in complete ISO signal avoidance (p 1-inhomogeneities are present. Preventing ISO excitation with a narrow excitation pulse in a 3D sequence yields the most robust results (relative SNR 151 ± 28% compared to 2D multislice methods, p = 0.006). Conclusion We optimized three easily implementable methods to avoid ISO signal artifacts and validated their performance in phantoms and in vivo. We make recommendation on the parameters that pre-clinical studies should report in their method section to make the used approach insightful.

BackgroundChronic Q fever is a zoonosis caused by the bacterium Coxiella burnetii which can manifest as infection of an abdominal aortic aneurysm (AAA). Antibiotic therapy often fails, resulting in severe morbidity and high mortality. Whereas previous studies have focused on inflammatory processes in blood, the aim of this study was to investigate local inflammation in aortic tissue.MethodsMultiplex immunohistochemistry was used to investigate local inflammation in Q fever AAAs compared to atherosclerotic AAAs in aorta tissue specimen. Two six-plex panels were used to study both the innate and adaptive immune system.ResultsQ fever AAAs and atherosclerotic AAAs contained similar numbers of CD68+ macrophages and CD3+ T cells. However, in Q fever AAAs the number of CD68+CD206+ M2 macrophages was increased, while expression of GM-CSF was decreased compared to atherosclerotic AAAs. Furthermore, Q fever AAAs showed an increase in both the number of CD8+ cytotoxic T cells and CD3+FoxP3+ regulatory T cells. Lastly, Q fever AAAs did not contain any well-defined granulomas.ConclusionsThese findings demonstrate that despite the presence of pro-inflammatory effector cells, there is an immune suppressive micro environment in Q fever AAA resulting in persistent local infection with C. burnetii.

19F magnetic resonance imaging (19F MRI) is an emerging technique for quantitative imaging of novel therapies, such as cellular therapies and theranostic nanocarriers. A modification of perfluorocarbon (PFC)-loaded, nanocarrier-based 19F MRI probes with paramagnetic chelates can enhance probe’s functionality. Liquid PFC-loaded nanocarriers typically have a core-shell structure with PFC in the core due to the poor miscibility of PFC. However, paramagnetic relaxation enhancement acts only at a distance of a few angstroms. Thus, efficient modulation of 19F signal is possible only with fluorophilic PFC-soluble chelates. Such chelates, however, cannot interact with the surroundings of nanocarriers. Conversely, chelates on the surface typically affect only the aqueous environment but not the 19F signal. We show that the confinement of PFC in biodegradable polymeric nanoparticles with fractal structure enables modulation of longitudinal and transverse 19F relaxation, as well as proton signal, using non-fluorophilic paramagnetic chelates. We compared nanoparticles with fractal multicore versus conventional core-shell structure, where the PFC is encapsulated in the core(s) and the chelate in the surrounding polymeric matrix. Importantly, paramagnetic chelates affected both longitudinal and transverse 19F relaxation in fractal multicore nanoparticles, but not in core-shell nanocapsules. Both relaxation rates of 19F nucleus increased with an increasing concentration of the paramagnetic chelate. Moreover, as the polymeric matrix remained water-permeable, proton enhancement additionally was observed in MRI. In the future, the effects of fractal confinement could be combined with more effective paramagnetic chelates to develop multifunctional imaging probes, for example, for high-sensitivity 19F MRI combined with sensing.

Spraying of agrochemicals (pesticides, fertilizers) causes environmental pollution on a million‐ton scale. A sustainable alternative is target‐specific, on‐demand drug delivery by polymeric nanocarriers. Trunk injections of aqueous nanocarrier dispersions can overcome the biological size barriers of roots and leaves and allow distributing the nanocarriers through the plant. To date, the fate of polymeric nanocarriers inside a plant is widely unknown. Here, the in planta conditions in grapevine plants are simulated and the colloidal stability of a systematic series of nanocarriers composed of polystyrene (well‐defined model) and biodegradable lignin and polylactic‐co‐glycolic acid by a combination of different techniques is studied. Despite the adsorption of carbohydrates and other biomolecules onto the nanocarriers’ surface, they remain colloidally stable after incubation in biological fluids (wood sap), suggesting a potential transport via the xylem. The transport is tracked by fluorine‐ and ruthenium‐labeled nanocarriers inside of grapevines by 19F‐magnetic resonance imaging or induced coupled plasma – optical emission spectroscopy. Both methods show that the nanocarriers are transported inside of the plant and proved to be powerful tools to localize nanomaterials in plants. This study provides essential information to design nanocarriers for agrochemical delivery in plants to sustainable crop protection., Nanocarrier‐mediated release of agrochemicals in planta is a promising approach to treat plant diseases and to reduce spraying of chemicals. This paper investigates the transport of a diverse library of polymeric nanocarriers in vitro and in planta rendering drug delivery in plants with nanocarriers a promising tool for future agriculture.

Background/Introduction Bicuspid aortic valve (BAV) is associated with ascending aorta aneurysms and dissections. Presently, genetic factors and pathological flow patterns are considered responsible for aneurysm formation in BAV. Despite, indication for preventive surgery is presently only defined by vessel diameter which is poor marker for dissection, as it does not take other processes responsible for the vulnerability of the aorta into account. Purpose Inflammation is not considered a player in BAV aortopathy. We introduce a quantitative immunohistochemistry (IHC) approach to sensitively look at the potential role of both the innate and adaptive immune system in BAV aortopathy. Methods Dilated (n=8), non-dilated (n=14) and dissected (n=4) BAV ascending aortas were collected during surgery or from post-mortem donors. Median time from symptoms to surgery for dissections was just over 4 hours. Tissue was stained with a novel 8-colour IHC technique allowing for simultaneous visualization of 6 markers per slide, completed with DAPI nuclear counter-stain and elastin fiber autofluorescence. One panel focused on the adaptive immune system (identifying B cells and classic dendritic cells type 2 (cDC2s) and phenotyping T cells), and the other on the innate immune system (assessing macrophage polarization and neutrophil extravasation). All cells were identified and comprehensively phenotyped using automated quantitative analysis. Results Aneurysm formation was associated with an organized and consistent increase of lymphocytes in the adventitia. B cell follicles and helper T cell expansion were identified, suggestive of a targeted adaptive immune response (Fig. 1a). Only dissected aortas showed a statistically significant increase of helper T (p=0.3) and cDC2s (p=0.3) in the media, when compared to non-dilated and dilated samples (Fig. 1b). The short time between dissection symptoms and surgery suggests these cells were present before the dissection occurred. Furthermore, aneurysms and dissections are associated with a shift in macrophage phenotype to the more aggressive M1-like subset. In summary, we found that a progression of aggressive immune cells in the adventitia and media was correlated to a progression in disease state; from normal to dilated to dissected. Conclusions Aorta dilatation in patients with BAV is associated with an expansion of B and helper T cells in the adventitial compartment without changes in the media. This result might indicate an antigen-driven adaptive immune response. Only dissections show an increase in helper T cells and cDC2s in the media, together with polarization of macrophages to a more M1-like phenotype. We hypothesize that antigen-specific helper T cells expand in the adventitia, migrate to the media, and then potentiate macrophages which can eventually lead to tissue degeneration. These associations could shine light on the final step in the deterioration of the aorta towards a dissection. Figure 1. Microscopy results Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): TTW-NWO open technology grant (STW-14716), ERC-2014-StG-336454-CoNQUeST

Background Chronic Q fever (QCF) is the persistence of C. Burnettii after an acute infection. Atheromas in the abdominal aorta are a preferential location of this bacterial persistence and may result in accelerated abdominal aorta aneurysm (AAA) formation or even rupture. CQF is characterized by a poor response to antibiotic therapy with high mortality. The localization of the bacteria in the acidic lysosomes is thought to be the main cause of poor antibiotic efficacy. Purpose A potent immune system is mandatory for successful antibiotic therapy. PBMC simulation assays have shown potent immune activation on stimulation with C. Burnettii. Here we assess whether this holds true in situ by combining 3 six-marker panels with phenotypical markers describing the adaptive and innate immune system and looking at key check-points in immune regulation. Methods (Tissue) PCR-positive CQF infected AAAs (n=10) are compared to atherosclerotic AAAs (n=13) and normal abdominal aortas from kidney transplant donors (n=3). We combine DAPI and elastin autofluorescence with 3 distinct panels of 6 markers in an 8-color IHC technique (Vectra 3, Perkin Elmer, MA) to describe the local immune landscape. The panels focus on 1) adaptive immune response, 2) innate immune response and 3) checkpoint inhibitors. Cells were phenotyped and quantified using an automated quantitative analysis method. Results In contrast to normal abdominal aortas, both atherosclerotic AAAs and CQF AAAs show impressive lymphocyte recruitment and proliferation with the formulation of very large tertiary lymphoid structures. CQF AAAs show a 2-fold increase in regulatory T cell numbers compared to atherosclerotic AAAs. These regulatory cells are both surrounding germinal centers and are seen throughout the tissue. Additionally, CQF AAAs show more positive cells and a higher expression of PD-L1, which is a key immunosuppressive mediator. Conclusion The immune landscape within the aortic wall shows far greater immune suppression than previously found in in vitro and peripheral blood studies. We show that CQF infections are associated with severely increased numbers of regulatory T cells, even compared to the low-grade and chronic inflammation present in atherosclerosis. Subsequently, a look at the immune checkpoint PD-L1 shows an increased expression in CQF aortas, elucidating a possible underlying mechanism and treatment target. Figure 1. Representative microscopy images of normal (left) atherosclerotic AAA (middle) and CQF AAA (right), full thickness overview (top row) and magnified region off interest (bottom row). Vast increase in regulatory T cells (FoxP3+), recognisable by the yellow nucleus, surrounding germinal centres in only the CQF aorta. Furthermore, DAPI in blue, B cells in green, helper T cells in red, cytotoxic T cells in cyan, classic DC type 2 in magenta. Funding Acknowledgement Type of funding source: Public grant(s) – EU funding. Main funding source(s): ERC starting grant (ERC-2014-StG-336454-CoNQUeST); TTW-NWO open technology grant (STW-14716)

Contains fulltext : 229134.pdf (Publisher’s version ) (Open Access) Perfluorocarbon-loaded nanoparticles are powerful theranostic agents, which are used in the therapy of cancer and stroke and as imaging agents for ultrasound and (19)F magnetic resonance imaging (MRI). Scaling up the production of perfluorocarbon-loaded nanoparticles is essential for clinical translation. However, it represents a major challenge as perfluorocarbons are hydrophobic and lipophobic. We developed a method for continuous-flow production of perfluorocarbon-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles using a modular microfluidic system, with sufficient yields for clinical use. We combined two slit interdigital micromixers with a sonication flow cell to achieve efficient mixing of three phases: liquid perfluorocarbon, PLGA in organic solvent, and aqueous surfactant solution. The production rate was at least 30 times higher than with the conventional formulation. The characteristics of nanoparticles can be adjusted by changing the flow rates and type of solvent, resulting in a high PFC loading of 20-60 wt % and radii below 200 nm. The nanoparticles are nontoxic, suitable for (19)F MRI and ultrasound imaging, and can dissolve oxygen. In vivo (19)F MRI with perfluoro-15-crown-5 ether-loaded nanoparticles showed similar biodistribution as nanoparticles made with the conventional method and a fast clearance from the organs. Overall, we developed a continuous, modular method for scaled-up production of perfluorocarbon-loaded nanoparticles that can be potentially adapted for the production of other multiphase systems. Thus, it will facilitate the clinical translation of theranostic agents in the future.

Perfluorocarbons hold great promise both as imaging agents, particularly for

Poly(lactic-co-glycolic acid) (PLGA) particles are very widely used, particularly for drug delivery, including commercial clinical formulations. Adding perfluorocarbon (PFC) enables in vivo imaging and quantification of the PLGA particles through 19F NMR, MRS or MRI. PFCs are both hydrophobic and lipophobic at the same time. This property makes their encapsulation in particles challenging, as it requires the addition of a third immiscible phase during the emulsification process. Here we explore how different parameters affect the miniemulsion formation of particles loaded with perfluoro-15-crown-5-ether (PFCE). By changing the concentration of surfactant and type of solvent, we were able to control the radius of synthesized particles, between 85-200 nm. We assessed stability and release from the particles at different pH values, showing that hydrophobic agents are released from the particles by diffusion rather than degradation. With cell experiments, we show that primary human dendritic cells take up the particles without any apparent effect, including on cell migration. In summary, the control of synthesis conditions leads to particles with sufficient PFCE encapsulation, which are suitable for drug loading and cell labeling, and do not affect cell viability or functionality. Finally, these nanoparticles can be produced at GMP-grade for clinical use.

Contains fulltext : 229164.pdf (Publisher’s version ) (Open Access) Perfluorocarbons hold great promise both as imaging agents, particularly for (19)F MRI, and in therapy, such as oxygen delivery. (19)F MRI is unique in its ability to unambiguously track and quantify a tracer while maintaining anatomic context, and without the use of ionizing radiation. This is particularly well-suited for inflammation imaging and quantitative cell tracking. However, perfluorocarbons, which are best suited for imaging - like perfluoro-15-crown-5 ether (PFCE) - tend to have extremely long biological retention. Here, we showed that the use of a multi-core PLGA nanoparticle entrapping PFCE allows for a 15-fold reduction of half-life in vivo compared to what is reported in literature. This unexpected rapid decrease in (19)F signal was observed in liver, spleen and within the infarcted region after myocardial infarction and was confirmed by whole body NMR spectroscopy. We demonstrate that the fast clearance is due to disassembly of the ~200 nm nanoparticle into ~30 nm domains that remain soluble and are cleared quickly. We show here that the nanoparticle ultrastructure has a direct impact on in vivo clearance of its cargo i.e. allowing fast release of PFCE, and therefore also bringing the possibility of multifunctional nanoparticle-based imaging to translational imaging, therapy and diagnostics.

Bicuspid aortic valve (BAV) is associated with ascending aorta aneurysms and dissections. Presently, genetic factors and pathological flow patterns are considered responsible for aneurysm formation in BAV. Here we investigate whether the immune response is involved in aneurysm formation. Dilated (n

Background/Aims: Endometriosis has a long diagnostic delay that is influenced by varying socio-economic and healthcare factors. In the Dutch situation, these factors are not yet identified. The aim of this study is to determine the length of the diagnostic delay of endometriosis in the Netherlands and to identify which variables affect this delay. Methods: A retrospective study among 139 patients diagnosed with endometriosis in a secondary care hospital with a specialized multidisciplinary endometriosis team. The diagnostic process was evaluated using a questionnaire-guided telephonic interview. Results: The median time interval from the onset of symptoms to diagnosis was 89 months or 7.4 years, divided in 7 months patient delay, 35 months general practitioner (GP) delay and 5 months gynecologist delay. Determinants for a longer diagnostic delay were young age at onset of symptoms, use of oral contraceptives or analgesics prescribed by GP, alternative diagnoses considered by the GP, and cyclic symptoms. Subfertility as presenting symptom resulted in faster diagnosis. Conclusion: This study shows that the time interval to the diagnosis of endometriosis is long and mainly consists of the period of time the woman consults her first line medical professional.

Contains fulltext : 193511.pdf (Publisher’s version ) (Open Access) Photoacoustic imaging (PAI) is an emerging biomedical imaging technique that is now coming to the clinic. It has a penetration depth of a few centimeters and generates useful endogenous contrast, particularly from melanin and oxy-/deoxyhemoglobin. Indocyanine green (ICG) is a Food and Drug Administration-approved contrast agents for human applications, which can be also used in PAI. It is a small molecule dye with limited applications due to its fast clearance, rapid protein binding, and bleaching effect. Methods: Here, we entrap ICG in a poly(lactic-co-glycolic acid) nanoparticles together with a perfluorocarbon (PFC) using single emulsion method. These nanoparticles and nanoparticle-loaded dendritic cells were imaged with PA, (19)F MR, and fluorescence imaging in vitro and in vivo. Results: We formulated particles with an average diameter of 200 nm. The encapsulation of ICG within nanoparticles decreased its photobleaching and increased the retention of the signal within cells, making it available for applications such as cell imaging. As little as 0.1x10(6) cells could be detected in vivo with PAI using automated spectral unmixing. Furthermore, we observed the accumulation of ICG signal in the lymph node after subcutaneous injection of nanoparticles. Conclusion: We show that we can label primary human dendritic cells with the nanoparticles and image them in vitro and in vivo, in a multimodal manner. This work demonstrates the potential of combining PAI and (19)F MRI for cell imaging and lymph node detection using nanoparticles that are currently produced at GMP-grade for clinical use.

Poly(lactic

Objective: Marfan syndrome (MFS) is a connective tissue disorder associated with severe cardiovascular morbidity and mortality. It is unknown if aorta complications in MFS are associated with progressive pulmonary artery (PA) dilatation. Methods: We measured the PA diameter on routine magnetic resonance imaging in a population of MFS patients seen in our specialised centre with follow up of diameters as well as the outcome. Results: PA dilatation was defined as an increase in diameter of 2 mm or more, and 71 patients (44%) of our total cohort (n = 162) met this criterion, mean follow up between two scans was 8.6 years (standard deviation (SD) &plusmn, 2.7 years). Furthermore, 28 patients suffered from dissections, of which 14 had a type A dissection, 10 had a type B dissection, and 4 patients suffered from both. Of those who suffered from dissection, 64% (18 out of 28) had a dilatation of the PA, versus 39% (53 out of 134) in the patient group without a dissection (p &lt, 0.05). There was a significant association between type B dissection and descending aorta diameter (OR 1.14, 95% CI 1.05&ndash, 1.24 p &lt, 0.01) and PA dilatation (OR 1.69, 95% CI 1.03&ndash, 2.77 p = 0.04). In the multivariable analysis the final model for type B dissection, only systolic blood pressure (OR 1.06, 95% CI 1.01&ndash, 1.11 p = 0.02) and PA dilatation were statistically significant (OR 1.85, 95% CI 1.10&ndash, 3.12 p = 0.02) while descending aorta diameter was not. Conclusions: We report an association between progressive PA dilatation and type B dissection. Our findings encourage a renewed interest in PA dimensions in MFS.