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19 F magnetic resonance imaging ( 19 F MRI) is an emerging technique for quantitative imaging in novel therapies, such as cellular therapies and theranostic nanocarriers. Nanocarriers loaded with liquid perfluorocarbon (PFC) typically have a (single) core-shell structure with PFC in the core due to the poor miscibility of PFC with organic and inorganic solvents. Paramagnetic relaxation enhancement acts only at a distance of a few angstroms. Thus, efficient modulation of the 19 F signal is possible only with fluorophilic PFC-soluble chelates. However, these chelates cannot interact with the surrounding environment and they might result in image artifacts. Conversely, chelates bound to the nanoparticle shell typically have a minimal effect on the 19 F signal and a strong impact on the aqueous environment. We show that the confinement of PFC in biodegradable polymeric nanoparticles (NPs) with a multicore structure enables the modulation of longitudinal ( T 1 ) and transverse ( T 2 ) 19 F relaxation, as well as proton ( 1 H) signals, using non-fluorophilic paramagnetic chelates. We compared multicore NPs versus a conventional single core structure, where the PFC is encapsulated in the core(s) and the chelate in the surrounding polymeric matrix. This modulated relaxation also makes multicore NPs sensitive to various acidic pH environments, while preserving their stability. This effect was not observed with single core nanocapsules (NCs). Importantly, paramagnetic chelates affected both T 1 and T 2 19 F relaxation in multicore NPs, but not in single core NCs. Both relaxation times of the 19 F nucleus were enhanced with an increasing concentration of the paramagnetic chelate. Moreover, as the polymeric matrix remained water permeable, proton enhancement additionally was observed in MRI.

Background: 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 while the exact role of inflammatory processes remains unknown., Methods: In order to objectify inflammation, we employ a highly sensitive, quantitative immunohistochemistry approach. Whole slides of dissected, dilated and non-dilated ascending aortas from BAV patients were quantitatively analyzed., Results: Dilated aortas show a 4-fold increase of lymphocytes and a 25-fold increase in B lymphocytes in the adventitia compared to non-dilated aortas. Tertiary lymphoid structures with B cell follicles and helper T cell expansion were identified in dilated and dissected aortas. Dilated aortas were associated with an increase in M1-like macrophages in the aorta media, in contrast the number of M2-like macrophages did not change significantly., Conclusion: This study finds unexpected large numbers of immune cells in dilating aortas of BAV patients. These findings raise the question whether immune cells in BAV aortopathy are innocent bystanders or contribute to the deterioration of the aortic wall., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Staal, Cortenbach, Gorris, van der Woude, Srinivas, Heijmen, Geuzebroek, Grewal, Hebeda, de Vries, DeRuiter and van Kimmenade.)

Background: Aim of this study was to investigate immune cells and subsets in different stages of human coronary artery disease with a novel multiplex immunohistochemistry (mIHC) technique., Methods: Human left anterior descending coronary artery specimens were analyzed: eccentric intimal thickening (N = 11), pathological intimal thickening (N = 10), fibroatheroma (N = 9), and fibrous plaque (N = 9). Eccentric intimal thickening was considered normal, and pathological intimal thickening, fibroatheroma, and fibrous plaque were considered diseased coronary arteries. Two mIHC panels, consisting of six and five primary antibodies, autofluoresence, and DAPI, were used to detect adaptive and innate immune cells. Via semi-automated analysis, (sub)types of immune cells in whole plaques and specific plaque regions were quantified., Results: Increased numbers of CD3 + T cells (P < 0.001), CD20 + B cells (P = 0.013), CD68 + macrophages (P = 0.003), CD15 + neutrophils (P = 0.017), and CD31 + endothelial cells (P = 0.024) were identified in intimas of diseased coronary arteries compared to normal. Subset analyses of T cells and macrophages showed that diseased coronary arteries contained an abundance of CD3 + CD8 - non-cytotoxic T cells and CD68 + CD206 - non-M2-like macrophages. Proportions of CD3 + CD45RO + memory T cells were similar to normal coronary arteries. Among pathological intimal thickening, fibroatheroma, and fibrous plaque, all immune cell numbers and subsets were similar., Conclusions: The type of immune response does not differ substantially between different stages of plaque development and may provide context for mechanistic research into immune cell function in atherosclerosis. We provide the first comprehensive map of immune cell subtypes across plaque types in coronary arteries demonstrating the potential of mIHC for vascular research., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Background: Chronic 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., Methods: Multiplex 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 systems., Results: Q 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 + CD8 - FoxP3 + regulatory T cells. Finally, Q fever AAAs did not contain any well-defined granulomas., Conclusions: These findings demonstrate that despite the presence of pro-inflammatory effector cells, persistent local infection with C. burnetii is associated with an immune-suppressed microenvironment., Funding: This work was supported by SCAN consortium: European Research Area - CardioVascualar Diseases (ERA-CVD) grant [JTC2017-044] and TTW-NWO open technology grant [STW-14716]., Competing Interests: KC, AS, TS, MG, LV, AJ, PP, RV, PW, CB, MS, KH, Mv, JD, RV No competing interests declared, JV Senior editor, eLife, (© 2022, Cortenbach et al.)

Purpose: Isoflurane (ISO) is the most commonly used preclinical inhalation anesthetic. This is a problem in 19 F 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 19 F MRI are described and demonstrated with measurements of 19 F-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 < 0.0001 for all methods). Using a narrow acquisition bandwidth can result in loss of signal to noise ratio and distortion of the image, and a frequency-selective suppression pulse can be incomplete when B 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., (© 2021. The Author(s).)

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 19 F-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., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

The use of polymeric nanoparticles (NPs) as therapeutics has been steadily increasing over past decades. In vivo imaging of NPs is necessary to advance the therapeutic performance. 19 F Magnetic Resonance Imaging ( 19 F MRI) offers multiple advantages for in vivo imaging. However, design of a probe for both biodistribution and degradation has not been realized yet. We developed polymeric NPs loaded with two fluorocarbons as promising imaging tools to monitor NP biodistribution and degradation by 19 F MRI. These 200 nm NPs consist of poly(lactic-co-glycolic acid) (PLGA) loaded with perfluoro-15-crown-5 ether (PFCE) and PERFECTA. PERFECTA/PFCE-PLGA NPs have a fractal sphere structure, in which both fluorocarbons are distributed in the polymeric matrix of the fractal building blocks, which differs from PFCE-PLGA NPs and is unique for fluorocarbon-loaded colloids. This structure leads to changes of magnetic resonance properties of both fluorocarbons after hydrolysis of NPs. PERFECTA/PFCE-PLGA NPs are colloidally stable in serum and biocompatible. Both fluorocarbons show a single resonance in 19 F MRI that can be imaged separately using different excitation pulses. In the future, these findings may be used for biodistribution and degradation studies of NPs by 19 F MRI in vivo using "two color" labeling leading to improvement of drug delivery agents., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutrophils kill ingested pathogens by the so-called oxidative burst, where reactive oxygen species (ROS) are produced in the lumen of phagosomes at very high rates (mM/s), although these rates can only be maintained for a short period (minutes). In contrast, dendritic cells produce ROS at much lower rates, but they can sustain production for much longer after pathogen uptake (hours). It is becoming increasingly clear that this slow but prolonged ROS production is essential for antigen cross-presentation to activate cytolytic T cells, and for shaping the repertoire of antigen fragments for presentation to helper T cells. However, despite this importance of ROS production by dendritic cells for activation of the adaptive immune system, their actual ROS production rates have never been quantified. Here, we quantified ROS production in human monocyte-derived dendritic cells by measuring the oxygen consumption rate during phagocytosis. Although a large variation in oxygen consumption and phagocytic capacity was present among individuals and cells, we estimate a ROS production rate of on average ~0.5 mM/s per phagosome. Quantitative microscopy approaches showed that ROS is produced within minutes after pathogen encounter at the nascent phagocytic cup. H 2 DCFDA measurements revealed that ROS production is sustained for at least ~10 h after uptake. While ROS are produced by dendritic cells at an about 10-fold lower rate than by neutrophils, the net total ROS production is approximately similar. These are the first quantitative estimates of ROS production by a cell capable of antigen cross-presentation. Our findings provide a quantitative insight in how ROS affect dendritic cell function.

Tyrosine kinase inhibitors (TKIs) have changed the landscape of treatment for patients with chronic myeloid leukemia (CML) leading to a life expectancy comparable to the general population. Side effects commonly encountered during TKI treatment are pleural effusion due to use of dasatinib and vascular side effects due to nilotinib and ponatinib. Coronary artery spasm (CAS), although encountered during treatment with other chemotherapeutic drugs, have to our knowledge never been reported during TKI treatment. Here, we describe two cases of coronary artery spasms which are likely due to TKIs.

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., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

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 19 F 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., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)