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Neutrophils play a complex role during onset of tissue injury and subsequent resolution and healing. To assess neutrophil dynamics upon cardiovascular injury, here we develop a non-invasive, background-free approach for specific mapping of neutrophil dynamics by whole-body magnetic resonance imaging using targeted multimodal fluorine-loaded nanotracers engineered with binding peptides specifically directed against murine or human neutrophils. Intravenous tracer application before injury allowed non-invasive three-dimensional visualization of neutrophils within their different hematopoietic niches over the entire body and subsequent monitoring of their egress into affected tissues. Stimulated murine and human neutrophils exhibited enhanced labeling due to upregulation of their target receptors, which could be exploited as an in vivo readout for their activation state in both sterile and nonsterile cardiovascular inflammation. This non-invasive approach will allow us to identify hidden origins of bacterial or sterile inflammation in patients and also to unravel cardiovascular disease states on the verge of severe aggravation due to enhanced neutrophil infiltration or activation.

Regenerating the injured heart remains one of the most vexing challenges in cardiovascular medicine. Cell therapy has shown potential for treatment of myocardial infarction, but low cell retention so far has limited its success. Here we show that intramyocardial injection of highly apoptosis-resistant unrestricted somatic stem cells (USSC) into infarcted rat hearts resulted in an unprecedented thickening of the left ventricular wall with cTnT

Early macrophage rich vascular inflammation is a key feature in the pathophysiology of restenosis after angioplasty.19F MRI with intravenously applied perfluorooctyl bromide-nanoemulsion (PFOB-NE) could offer ideal features for serial imaging of the inflammatory response after angioplasty. We aimed to non-invasively image monocyte/macrophage infiltration in response to angioplasty in pig carotid arteries using Fluorine-19 magnetic resonance imaging (19F MRI) to assess early inflammatory response to mechanical injury. Early macrophage rich vascular inflammation is a key feature in the pathophysiology of restenosis after angioplasty.19F MRI with intravenously applied perfluorooctyl bromide-nanoemulsion (PFOB-NE) could offer ideal features for serial imaging of the inflammatory response after angioplasty. In eight minipigs, injury of the right carotid artery was induced by either balloon oversize angioplasty only (BA, n=4) or in combination with endothelial denudation (BA + ECDN, n=4). PFOB-NE was administered intravenously three days after injury followed by1H and19F MRI to assess vascular inflammatory burden at day six. Vascular response to mechanical injury was validated using immunohistology. Angioplasty was successfully induced in all eight pigs. Response to injury was characterized by positive remodeling with predominantly adventitial wall thickening and adventitial infiltration of monocytes/macrophages.19F signal could be detectedin vivoin four pigs following BA + ECDN with a robust signal-to-noise ratio (SNR) of 14.7 ± 4.8.Ex vivoanalysis revealed a linear correlation of19F SNR to local monocyte/macrophage cell density. Minimum detection limit of infiltrated monocytes/macrophages was as about 400 cells/mm2. Therefore,19F MRI enables quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity. This might open an avenue to non-invasively monitor inflammatory response to mechanical injury after angioplasty and thus to identify individuals with distinct patterns of vascular inflammation promoting restenosis.One Sentence Summary19F MRI enables radiation-free quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity.

High-grade gliomas, the most common and aggressive primary brain tumors, are characterized by a complex tumor microenvironment (TME). Among the immune cells infiltrating the glioma TME, tumor-associated microglia and macrophages (TAMs) constitute the major compartment. In patients with gliomas, increased TAM abundance is associated with more aggressive disease. Alterations in TAM phenotypes and functions have been reported in preclinical models of multiple cancers during tumor development and after therapeutic interventions, including radiotherapy and molecular targeted therapies. These findings indicate that it is crucial to evaluate TAM abundance and dynamics over time. Current techniques to quantify TAMs in patients rely mainly on histological staining of tumor biopsies. Although informative, these techniques require an invasive procedure to harvest the tissue sample and typically only result in a snapshot of a small region at a single point in time. Fluorine isotope 19 MRI ( 19 F MRI) represents a powerful means to noninvasively and longitudinally monitor myeloid cells in pathological conditions by intravenously injecting perfluorocarbon-containing nanoparticles (PFC-NP). In this study, we demonstrated the feasibility and power of 19 F MRI in preclinical models of gliomagenesis, breast-to-brain metastasis, and breast cancer and showed that the major cellular source of 19 F signal consists of TAMs. Moreover, multispectral 19 F MRI with two different PFC-NP allowed us to identify spatially and temporally distinct TAM niches in radiotherapy-recurrent murine gliomas. Together, we have imaged TAMs noninvasively and longitudinally with integrated cellular, spatial, and temporal resolution, thus revealing important biological insights into the critical functions of TAMs, including in disease recurrence.

Glycosylation is a vital post-translational modification involved in a range of biological processes including protein folding, signaling, and cell-cell interactions. In 2011, a new type of

Increased sodium intake aggravates cardiovascular diseases. Salt substitute containing potassium chlorideimproves cardiovascular outcome in hypertensive patients with high cardiovascular risk, however it isunknown whether it is attributed to potassium (K+) supplementation or sodium reduction. Here, weinvestigate the role of high K+ intake on the development of hypertensive cardiac damage in the presenceor absence of high salt diet. 8-10 weeks old apolipoproteinE-deficient mice (apoE-KO) were fed a normalK+ (0,55%) or high K+ (5%) diet throughout the experiment. Two weeks after diet start,mice were infusedwith angiotensin (Ang)II (500ng/kg/min) for 28 days. Cardiac function was assessed by MRI. High K+ dietincreased serum K+ levels compared to mice fed a normal K+ diet (5.3±1.2 vs. 3.9±0.4mmol/L, p

Carbohydrate-binding antibodies play diverse and critical roles in human health. Endogenous carbohydrate-binding antibodies that recognize bacterial, fungal, and other microbial carbohydrates prevent systemic infections and help maintain microbiome homeostasis. Anti-glycan antibodies can have both beneficial and detrimental effects. For example, alloantibodies to ABO blood group carbohydrates can help reduce the spread of some infectious diseases, but they also impose limitations for blood transfusions. Antibodies that recognize self-glycans can contribute to autoimmune diseases, such as Guillain-Barre syndrome. In addition to endogenous antibodies that arise through natural processes, a variety of vaccines induce anti-glycan antibodies as a primary mechanism of protection. Some examples of approved carbohydrate-based vaccines that have had a major impact on human health are against pneumococcus, Haemophilus influeanza type b, and Neisseria meningitidis. Monoclonal antibodies specifically targeting pathogen associated or tumor associated carbohydrate antigens (TACAs) are used clinically for both diagnostic and therapeutic purposes. This review aims to highlight some of the well-studied and critically important applications of anti-carbohydrate antibodies.

The high mannose patch (HMP) of the HIV envelope protein (Env) is the structure most frequently targeted by broadly neutralizing antibodies; therefore, many researchers have attempted to use mimics of this region as a vaccine immunogen. In our previous efforts, vaccinating rabbits with evolved HMP mimic glycopeptides containing Man9 resulted in an overall antibody response targeting the glycan core and linker rather than the full glycan or Manα1→2Man tips of Man9 glycans. A possible reason could be processing of our immunogen by host serum mannosidases. We sought to test whether more prolonged dosing could increase the antibody response to intact glycans, possibly by increasing the availability of intact Man9 to germinal centers. Here, we describe a study investigating the impact of immunization regimen on antibody response by testing immunogen delivery through bolus, an exponential series of mini doses, or a continuously infusing mini-osmotic pump. Our results indicate that, with our glycopeptide immunogens, standard bolus immunization elicited the strongest HIV Env-binding antibody response, even though higher overall titers to the glycopeptide were elicited by the exponential and pump regimens. Antibody selectivity for intact glycan was, if anything, slightly better in the bolus-immunized animals.

The immune system produces a diverse collection of antiglycan antibodies that are critical for host defense. At present, however, we know very little about the binding properties, origins, and sequences of these antibodies because of a lack of access to a variety of defined individual antibodies. To address this challenge, we used a glycan microarray with over 800 different components to screen a panel of 516 human monoclonal antibodies that had been randomly cloned from different B-cell subsets originating from healthy human subjects. We obtained 26 antiglycan antibodies, most of which bound microbial carbohydrates. The majority of the antiglycan antibodies identified in the screen displayed selective binding for specific glycan motifs on our array and lacked polyreactivity. We found that antiglycan antibodies were about twice as likely than expected to originate from IgG

Germline antibodies, the initial set of antibodies produced by the immune system, are critical for host defense, and information about their binding properties can be useful for designing vaccines, understanding the origins of autoantibodies, and developing monoclonal antibodies. Numerous studies have found that germline antibodies are polyreactive with malleable, flexible binding pockets. While insightful, it remains unclear how broadly this model applies, as there are many families of antibodies that have not yet been studied. In addition, the methods used to obtain germline antibodies typically rely on assumptions and do not work well for many antibodies. Herein, we present a distinct approach for isolating germline antibodies that involves immunizing activation-induced cytidine deaminase (AID) knockout mice. This strategy amplifies antigen-specific B cells, but somatic hypermutation does not occur because AID is absent. Using synthetic haptens, glycoproteins, and whole cells, we obtained germline antibodies to an assortment of clinically important tumor-associated carbohydrate antigens, including Lewis Y, the Tn antigen, sialyl Lewis C, and Lewis X (CD15/SSEA-1). Through glycan microarray profiling and cell binding, we demonstrate that all but one of these germline antibodies had high selectivity for their glycan targets. Using molecular dynamics simulations, we provide insights into the structural basis of glycan recognition. The results have important implications for designing carbohydrate-based vaccines, developing anti-glycan monoclonal antibodies, and understanding antibody evolution within the immune system.

Background Perfluorocarbon (PFC)-nanoemulsions (NE) are a convenient tool for 19F magnetic resonance imaging in cell and animal experiments. Typical preparation methods, like high-pressure homogenization or microfluidization, produce nanoemulsions in mL-scale. However, experiments usually require only miniscule amounts of PFC-NE, several 100 µL. For site-specific imaging tissue-specific ligands, e.g. peptides or antibodies, are covalently bound to the NE surface. This requires the use of expensive functionalized phospholipids containing reactive groups (e.g. maleimide), which often deteriorate quickly in liquid storage, rendering the manufacturing process highly cost-inefficient. A technique to manufacture storage stable NE that maintain their functionality for coupling of various ligands is desired. Methods and results Different PFC-NE formulations and preparation techniques were compared and the most suitable of these was tested in short-, as well as long-term stability tests. Droplet size stability was investigated by dynamic light scattering and cryogenic transmission electron microscopy over 1.5 a. Surface modifiability was assessed by a fluorescence assay. The utility of these NE was proven in an in vitro model. Conclusion The established PFC-NE platform offers a cost-efficient way to produce larger amounts of long-term storable imaging agents, which can be surface-modified on demand for application in targeted 19F MRI.

We have previously studied the generation of immune responses after vaccination with tumor-associated carbohydrate antigen (TACA)-containing glycopeptides from the tandem repeat (TR) sequence of MUC4, an aberrantly expressed mucin in pancreatic adenocarcinomas. A specific lead antigen from that study containing the Thomsen-Friedenreich TACA disaccharide facilitated the pursuit of a monoclonal antibody to this synthetic hapten. Initial evaluation of polyclonal antiserum resulting from immunization with a KLH conjugate of this glycopeptide into rabbits showed high titer antibodies by ELISA assays, and selective immunoreactivity with MUC4+ cells by western blot and flow cytometry techniques. Glycan microarray analysis showed an intriguing binding pattern where the antiserum showed near complete specificity for MUC4 TR glycopeptides and peptides, relative to all components on the array. Tissue staining also showed distinct tumor specificity to pancreatic tumor tissue in relation to normal pancreatic tissue, with a preference for more aggressive tumor foci. Based on this data, we produced a monoclonal antibody whose binding and reactivity profile was similar to that of the polyclonal serum, with the added benefit of being more specific for the N-terminal glycosylated peptide domain. This epitope represents a novel immunogen to potentially develop diagnostic antibodies or immunotherapies against various MUC4-positive cancers.

A research letter describing an innovative method for imaging activated platelets using advanced fluorine‐19 magnetic resonance imaging.

Reduced potassium (k+) intake has been linked to cardiovascular diseases. The underlying mechanisms remain unknown. Here, we investigate the effect of low (0%), normal (0.5%) and high K+ (5%) diet on the development of atherosclerosis and hypertensive cardiac damage. To induce atherosclerosis, apolipoprotein-deficient mice were infused with angiotensin (Ang) II (500ng/kg/min) for 28 days. Potassium treatment was initiated 2 weeks before Ang II infusion. Cardiac function was assessed by MRI. Levels of K+ in the serum and urine were significantly different between groups. The Ang II infused mice from the K+ low group had significantly higher atherosclerotic plaques in the aortic arch (21±3%) compared to K+ high (10±2%) and K+ normal (11±2%) groups. The atherosclerosis development was blood pressure independent since no differences in blood pressure between the groups were observed. Although heart to body-weight ratio did not differ between three groups, K+ low diet was associated with a lower ejection fraction rate and increased mRNA expression levels of cardiac ANP, BNP, collagen and fibronectin compared to the K+ normal and K+ high group. After Ang II infusion, assessment of aldosterone levels in urine showed significant higher aldosterone levels in the high K+ (214±72ng/24h) compared to normal K+ (26±6ng/24h) and low K+ (18±4ng/24h) groups. Aldosterone induced cardiovascular damage is known to be aggravated by sodium. To, evaluate whether high sodium diet unmask aldosterone mediated cardiovascular damage in the high K+ group, mice fed a high or normal K+ diet were additionally treated with high sodium (1% NaCl) in the drinking water. During Ang II treatment, high salt diet accelerated atherosclerosis in the aortic arch of both groups but no differences were observed between the high K+ high/high Na+ (41±7%) and normal K+/high Na+ (49±2%) group. In contrast, high K+/high Na+ group have significantly more severe cardiac hypertrophy compared to normal K+ high Na+ group (8.9±0.7 vs 6.4±0.7mg/g). These results were confirmed by MRI. K+ deficient diet induces atherosclerosis and cardiac damage during Ang II induced hypertension. K+ enriched diet exacerbates cardiac hypertrophy only under high Na+ conditions most likely in an aldosterone-dependent mechanism.

Placental hypoperfusion and hypoxia are key drivers in complications during fetal development such as fetal growth restriction and preeclampsia. In order to study the mechanisms of disease in mouse models, the development of quantitative biomarkers of placental hypoxia is a prerequisite. The goal of this exploratory study was to establish a technique to noninvasively characterize placental partial pressure of oxygen (PO2) in vivo in the Lgals1 (lectin, galactoside-binding, soluble, 1) deficient mouse model of preeclampsia using fluorine magnetic resonance imaging. We hypothesized a decrease in placental oxygenation in knockout mice. Wildtype and knockout animals received fluorescently labeled perfluoro-5-crown-15-ether nanoemulsion i.v. on day E14-15 during pregnancy. Placental PO2 was assessed via calibrated 19F MRI saturation recovery T1 mapping. A gas challenge with varying levels of oxygen in breathing air (30%, 60% and 100% O2) was used to validate that changes in oxygenation can be detected in freely breathing, anesthetized animals. At the end of the experiment, fluorophore-coupled lectin was injected i.v. to label the vasculature for histology. Differences in PO2 between breathing conditions and genotype were statistically analyzed with linear mixed-effects modeling. As expected, a significant increase in PO2 with increasing oxygen in breathing air was found. PO2 in Lgals1 knockout animals was decreased but this effect was only present at 30% oxygen in breathing air, not at 60% and 100%. Histological examinations showed crossing of the perfluorocarbon nanoemulsion to the fetal blood pool but the dominating contribution of 19F MR signal is estimated at > 70% from maternal plasma based on volume fraction measurements of previous studies. These results show for the first time that 19F MRI can characterize oxygenation in mouse models of placental malfunction.

Among the preclinical molecular imaging approaches, lately fluorine (19 F) magnetic resonance imaging (MRI) has garnered significant scientific interest in the biomedical research community, due to the unique properties of fluorinated materials and the 19 F nucleus. Fluorine is an intrinsically sensitive nucleus for MRI-there is negligible endogenous 19 F in the body and, thus, no background signal which allows the detection of fluorinated materials as "hot spots" by combined 1 H/19 F MRI and renders fluorine-containing molecules as ideal tracers with high specificity. In addition, perfluorocarbons are a family of compounds that exhibit a very high fluorine payload and are biochemically as well as physiologically inert. Perfluorocarbon nanoemulsions (PFCs) are well known to be readily taken up by immunocompetent cells, which can be exploited for the unequivocal identification of inflammatory foci by tracking the recruitment of PFC-loaded immune cells to affected tissues using 1 H/19 F MRI. The required 19 F labeling of immune cells can be accomplished either ex vivo by PFC incubation of isolated endogenous immune cells followed by their re-injection or by intravenous application of PFCs for in situ uptake by circulating immune cells. With both approaches, inflamed tissues can unambiguously be detected via background-free 19 F signals due to trafficking of PFC-loaded immune cells to affected organs. To extend 19 F MRI tracking beyond cells with phagocytic properties, the PFC surface can further be equipped with distinct ligands to generate specificity against epitopes and/or types of immune cells independent of phagocytosis. Recent developments also allow for concurrent detection of different PFCs with distinct spectral signatures allowing the simultaneous visualization of several targets, such as various immune cell subtypes labeled with these PFCs. Since ligands and targets can easily be adapted to a variety of problems, this approach provides a general and versatile platform for inflammation imaging which will strongly extend the frontiers of molecular MRI. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Cardiovascular Disease.

Perfluorocarbons hold great promise both as imaging agents, particularly for

Perfluorocarbon nanoemulsions (PFCs) tagged with fluorescence dyes have been intensively used to confirm the in vivo 19F magnetic resonance imaging (MRI) localization of PFCs by post mortem histology or flow cytometry. However, only limited data are available on tagged PFCs and the potential dissociation of fluorescence and 19F label after cellular uptake over time. PFCs were coupled to rhodamine (Rho) or carboxyfluorescein (Cfl) and their fate was analyzed after in vitro uptake by J774, RAW and CHO cells by flow cytometry and 19F MRI. In separate in vivo experiments, the dual-labelled emulsions were intravenously applied into mice and their distribution was monitored in spleen and liver over 24 h. In a final step, time course of fluorescence and 19F signals from injected emulsions were tracked in a local inflammation model making use of a subcutaneous matrigel depot doped with LPS (lipopolysaccharide). Internalization of fluorescence-labelled PFCs was associated with a substantial whitening over 24 h in all macrophage cell lines while the 19F signal remained stable over time. In all experiments, CflPFCs were more susceptible to bleaching than RhoPFCs. After intravenous injection of RhoPFCs, the fluorescence signal in spleen and liver peaked after 30 min and 2 h, respectively, followed by a successive decrease over 24 h, whereas the 19F signal continuously increased during this observation period. Similar results were found in the matrigel/LPS model, where we observed increasing 19F signals in the inflammatory hot spot over time while the fluorescence signal of immune cells isolated from the matrigel depot 24 h after its implantation was only marginally elevated over background levels. This resulted in a massive underestimation of the true PFC deposition in the reticuloendothelial system and at inflammatory hot spots. Cellular uptake of fluorescently tagged PFCs leads to a dissociation of the fluorescence and the 19F label signal over time, which critically impacts on interpretation of long-term experiments validated by histology or flow cytometry.

Carboxylic acid-functionalized Pd and Pt PNNNP pincer complexes were used for the assembly of two porous Zr metal-organic frameworks (MOFs), 2-PdX and 2-PtX. Powder X-ray diffraction analysis shows that the new MOFs adopt cubic framework structures similar to the previously reported Zr6O4(OH)4[(POCOP)PdX]3, [POCOP = 2,6-(OPAr2)2C6H3); Ar = p-C6H4CO2-, X = Cl-, I-] (1-PdX). Elemental analysis and spectroscopic characterization indicate the presence of missing linker defects, and 2-PdX and 2-PtX were formulated as Zr6O4(OH)4(OAc)2.4[M(PNNNP)X]2.4 [M = Pd, Pt; PNNNP = 2,6-(HNPAr2)2C5H3N; Ar = p-C6H4CO2-; X = Cl-, I-]. Postsynthetic halide ligand exchange reactions were carried out by treating 2-PdX with Ag(O3SCF3) or NaI followed by PhI(O2CCF3)2. The latter strategy proved to be more effective at activating the MOF for the catalytic intramolecular hydroamination of an o-substituted alkynyl aniline, underscoring the advantage of using halide exchange reagents that produce soluble byproducts.

Purpose To evaluate key molecular and cellular features of Graves orbitopathy (GO) by simultaneous monitoring of alterations in morphology, inflammatory patterns, and tissue remodeling. Methods To this end, we utilized a murine model of GO induced by immunization with a human thyroid-stimulating hormone receptor A-subunit plasmid. Altogether, 52 mice were used: 27 GOs and 25 controls (Ctrl) immunized with β-galactasidose plasmid. From these, 17 GO and 12 Ctrl mice were subjected to multimodal MRI at 9.4T, whereas 23 mice only underwent histology. Beyond anatomical hydrogen-1 (1 H) MRI, we employed transverse relaxation time (T2 ) mapping for visualization of edema, chemical exchange saturation transfer (CEST) for detection of hyaluronan, and fluorine-19 (19 F) MRI for tracking of in situ-labeled immune cells after intravenous injection of perfluorcarbons (PFCs). Results 1 H/19 F MRI demonstrated substantial infiltration of PFC-loaded immune cells in peri and retro-orbital regions of GO mice, whereas healthy Ctrls showed only minor 19 F signals. In parallel, T2 mapping indicated onset of edema in periorbital tissue and adjacent ocular glands (P = 0.038/0.017), which were associated with enhanced orbital CEST signals in GO mice (P = 0.031). Concomitantly, a moderate expansion of retrobulbar fat (P = 0.029) was apparent; however, no signs for extraocular myopathy were detectable. 19 F MRI-based visualization of orbital inflammation exhibited the highest significance level to discriminate between GO and Ctrl mice (P = 0.006) and showed the best correlation with the clinical score (P = 0.0007). Conclusion The present approach permits the comprehensive characterization of orbital tissue and holds the potential for accurate GO diagnosis in the clinical setting. Magn Reson Med 80:711-718, 2018. © 2018 International Society for Magnetic Resonance in Medicine.

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.

Among the preclinical molecular imaging approaches, lately fluorine (

Chronic high salt (NaCl) intake is a major risk factor for cardiovascular diseases. Recent studies showed that excessive chronic NaCl intake affects immune response and thereby contributes to the pathogenesis of vascular injury. However, it remains unclear whether NaCl intake initially primes immune cell response by promoting memory T cells and sensitize the development of vascular injury in response to a subsequent stimulus. Therefore, we investigate whether 14 days of NaCl treatment (1% NaCl) followed by a 7 day washout period affects the incidence and outcome of abdominal aortic aneurysms (AAA) in apolipoprotein E deficient (ApoE-KO) mice infused chronically with angiotensin II (Ang II) (1000ng/kg/min) via osmotic minipumps for 10 days. Assessment of AAA was done by magnetic resonance imaging (MRI). To visualize vascular inflammation in vivo , perfluorocarbon nano-emulsions (PFC) injected intravenously before and 2, 4, 7 and 10 days after chronic Ang II infusion were detected by combining 1H/19F MRI using a 19 F RARE sequence. After 10 days of Ang II infusion, aortas with perivascular fat were dissected. Aortic inflammation and immune cells infiltration were analyzed by flow cytometery, immunohistology and realtime PCR. The survival rate of ApoE-KO mice pretreated with NaCl was significant lower and the AAA incidence rate was significant higher compared to non-NaCl treated apoE-KO mice. The amount of 19 F signal detected in areas of AAA was significantly increased in apoE-KO mice pretreated with NaCl. Histological analysis confirmed these results. At day 10, proinflammatory cytokines like TNF-alpha, iNOS and TGF-ß as well as numbers of neutrophils, monocytes as well as effector memory CD4 and CD8 T cells were significantly higher in aortas of NaCl pretreated apoE-KO mice compared to non-NaCl treated mice. In conclusion, these results suggest a significant role of NaCl in priming immune cell phenotype and thereby mediating vascular inflammation and damage in Ang II infused apoE-KO mice.

MHC class II molecules play a pivotal role for the induction and maintenance of immune responses against pathogens, but are also implicated in pathological conditions like autoimmune diseases or rejection of transplanted organs. Human antigen-presenting cells express three human leukocyte antigen (HLA) class II isotypes (DR, DP, and DQ), which are, with the exception of DRα, composed of highly polymorphic α and β subunits. The combination of α- and β-chains results in a multitude of MHC-II αβ-heterodimers of the same isotype, but also isotype-mixed MHC class II molecules have been identified. Invariant chain chaperones the assembly of MHC-II molecules within the endoplasmatic reticulum and also facilitates the intracellular transport to MHC class II loading compartments (MIICs). MHC-II molecules are loaded with antigenic peptides and shuttled to the cell surface for inspection by CD4 T-cells. Alternatively, class-II molecules enriched on intraluminal vesicles can be released via exosomes into the extracellular space. Since some of the αβ-combinations may yield mismatched nonfunctional heterodimers, it is not entirely clear which type of HLA class II peptide receptors are transported to MIICs and found on the cell surface of antigen-presenting cells. We present techniques to inspect assembly, intracellular transport, cell surface expression, and exosomal release of MHC class II heterodimers.

INTRODUCTION:We explored the use of a perfluoro-15-crown-5 ether nanoemulsion (PFC) for measuring tissue oxygenation using a mouse model of vascular cognitive impairment.METHODS:Seventeen C57BL/6 mice underwent stereotactic injection of PFC coupled to a fluorophore into the striatum and corpus callosum. Combined 1H/19F magnetic resonance imaging (MRI) to localize the PFC and R1 mapping to assess pO2 were performed. The effect of gas challenges on measured R1 was investigated. All mice then underwent bilateral implantation of microcoils around the common carotid arteries to induce global cerebral hypoperfusion. 19F-MRI and R1 mapping were performed 1 day, 1 week, and 4 weeks after microcoil implantation. In vivo R1 values were converted to pO2 through in vitro calibration. Tissue reaction to the PFC was assessed through ex vivo immunohistochemistry of microglial infiltration.RESULTS:R1 increased with increasing oxygen concentrations both in vitro and in vivo and the strength of the 19F signal remained largely stable over 4 weeks. In the two mice that received all four scans, tissue pO2 decreased after microcoil implantation and recovered 4 weeks later. We observed infiltration of the PFC deposits by microglia.DISCUSSION:Despite remaining technical challenges, intracerebrally injected PFC is suitable for monitoring brain oxygenation in vivo

Herpes simplex virus (HSV)-encoded glycoprotein B (gB) is the most abundant protein in the viral envelope and promotes fusion of the virus with the cellular membrane. In the present study, we found that gB impacts on the major histocompatibility complex (MHC)-II pathway of antigen presentation by fostering homotypic fusion of early endosomes and trapping MHC-II molecules in these altered endosomes. By using an overexpression approach, we demonstrated that transient expression of gB induces giant vesicles of early endosomal origin, which contained Rab5, early endosomal antigen 1 (EEA1), and large amounts of MHC-II molecules [human leukocyte antigen (HLA)-DR, and HLA-DM], but no CD63. In HSV-1-infected and stably transfected cell lines that expressed lower amounts of gB, giant endosomes were not observed, but strongly increased amounts of HLA-DR and HLA-DM were found in EEA1

MHC class II molecules play a pivotal role for the induction and maintenance of immune responses against pathogens, but are also implicated in pathological conditions like autoimmune diseases or rejection of transplanted organs. Human antigen-presenting cells express three human leukocyte antigen (HLA) class II isotypes (DR, DP, and DQ), which are, with the exception of DRα, composed of highly polymorphic α and β subunits. The combination of α- and β-chains results in a multitude of MHC-II αβ-heterodimers of the same isotype, but also isotype-mixed MHC class II molecules have been identified. Invariant chain chaperones the assembly of MHC-II molecules within the endoplasmatic reticulum and also facilitates the intracellular transport to MHC class II loading compartments (MIICs). MHC-II molecules are loaded with antigenic peptides and shuttled to the cell surface for inspection by CD4 T-cells. Alternatively, class-II molecules enriched on intraluminal vesicles can be released via exosomes into the extracellular space. Since some of the αβ-combinations may yield mismatched nonfunctional heterodimers, it is not entirely clear which type of HLA class II peptide receptors are transported to MIICs and found on the cell surface of antigen-presenting cells. We present techniques to inspect assembly, intracellular transport, cell surface expression, and exosomal release of MHC class II heterodimers.

Specific detection of target structures or cells lacking particular surface epitopes still poses a serious problem for all imaging modalities. Here, we demonstrate the capability of synthetic “cargo internalization receptors” (CIRs) for tracking of individual cell populations by 1H/19F magnetic resonance imaging (MRI). To this end, a nanobody for green fluorescent protein (GFP) was used to engineer cell-surface-expressed CIRs which undergo rapid internalization after GFP binding. For 19F MR visibility, the GFP carrier was equipped with “contrast cargo”, in that GFP was coupled to perfluorocarbon nanoemulsions (PFCs). To explore the suitability of different uptake mechanisms for this approach, CIRs were constructed by combination of the GFP nanobody and three different cytoplasmic tails that contained individual internalization motifs for endocytosis of the contrast cargo (CIR1–3). Exposure of CIR+ cells to GFP-PFCs resulted in highly specific binding and internalization as confirmed by fluorescence micros...

Perfluorocarbon nanoemulsions (PFCs) tagged with fluorescence dyes have been intensively used to confirm the in vivoPFCs were coupled to rhodamine (Rho) or carboxyfluorescein (Cfl) and their fate was analyzed after in vitro uptake by J774, RAW and CHO cells by flow cytometry andInternalization of fluorescence-labelled PFCs was associated with a substantial whitening over 24 h in all macrophage cell lines while theCellular uptake of fluorescently tagged PFCs leads to a dissociation of the fluorescence and the