Nanoparticle-based autoantigen delivery to Treg-inducing liver sinusoidal endothelial cells enables control of autoimmunity in mice.

BACKGROUND & AIMS: It is well-known that the liver can induce immune tolerance, yet this knowledge could, thus far, not be translated into effective treatments for autoimmune diseases. We have previously shown that liver sinusoidal endothelial cells (LSECs) could substantially contribute to hepatic tolerance through their ability to induce CD4+ Foxp3+ regulatory T cells (Tregs). Here, we explored whether the Treg-inducing potential of LSECs could be harnessed for the treatment of autoimmune disease. METHODS: We engineered a polymeric nanoparticle (NP) carrier for the selective delivery of autoantigen peptides to LSECs in vivo. In the well-characterized autoimmune disease model of experimental autoimmune encephalomyelitis (EAE), we investigated whether administration of LSEC-targeting autoantigen peptide-loaded NPs could protect mice from autoimmune disease. RESULTS: We demonstrate that NP-based autoantigen delivery to LSECs could completely and permanently prevent the onset of clinical EAE. More importantly, in a therapeutic approach, mice with already established EAE improved rapidly and substantially following administration of a single dose of autoantigen peptide-loaded NPs, whereas the control group deteriorated. Treatment efficacy seemed to depend on Tregs. The Treg frequencies in the spleens of mice treated with autoantigen peptide-loaded NPs were significantly higher than those in vehicle-treated mice. Moreover, NP-mediated disease control was abrogated after Treg depletion by repeated administration of Treg-depleting antibody. CONCLUSION: Our findings provide proof of principle that the selective delivery of autoantigen peptides to LSECs by NPs can induce antigen-specific Tregs and enable effective treatment of autoimmune disease. These findings highlight the importance of Treg induction by LSECs for immune tolerance.

TGF-ß-dependent induction of CD4⁺CD25⁺Foxp3⁺ Tregs by liver sinusoidal endothelial cells.

BACKGROUND & AIMS: CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) have a profound ability to control immune responses. We have previously shown that the liver is a major source of peripherally induced Tregs. Here, we investigate the liver cell types and molecular mechanisms responsible for hepatic Treg induction. METHODS: To assess the Treg-inducing potential of liver resident antigen-presenting cell types, we studied the conversion of Foxp3(-) non-Tregs into Foxp3(+) Tregs induced by liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), or Kupffer cells (KCs). The dependency of Treg induction on TGF-ß was tested in Treg conversion assays using T cells with reduced TGF-ß sensitivity. The suppressive potential of liver cell-induced Tregs was assessed by an in vitro suppression assay and in vivo, in the model of experimental autoimmune encephalomyelitis (EAE). RESULTS: All tested liver cell types were capable of inducing Foxp3(+) Tregs; however, LSECs were most efficient in inducing Tregs. Treg-induction was antigen-specific and depended on TGF-ß. LSECs featured membrane-bound LAP/TGF-ß and the anchor molecule GARP, which is required for tethering LAP/TGF-ß to the cell membrane. LSEC-induced Tregs suppressed proliferation and cytokine secretion of effector T cells in vitro. LSEC-induced Tregs were also functional suppressors in vivo, as neuroantigen-specific Tregs induced by LSECs were able to suppress EAE. CONCLUSIONS: We demonstrate that LSECs are the major liver cell type responsible for TGF-ß dependent hepatic Treg induction. The extraordinary capacity of LSECs to induce Tregs was associated with their unique ability to tether TGF-ß to their membrane.

Bioavailability of chromium(III)-supplements in rats and humans.

Chromium(III) is long regarded as essential trace element but the biochemical function and even basic transport ways in the body are still unclear. For a more rational discussion on beneficial as well as toxic effects of Cr(III), we re-investigated the bioavailability of the most important oral Cr supplements by using radiolabeled compounds and whole-body-counting in rats and in the first time also in humans. The apparent absorption of (51)Cr(III) from Cr-picolinate, Cr-nicotinate, Cr-phenylalaninate, Cr-proprionate, or Cr-chloride was generally low (0.04-0.24 %) in rats with slightly higher values for Cr-chloride and -phenylalaninate. Taking a fast urine excretion into account, the true absorption of (51)Cr was clearly higher for CrPic(3) (0.99 %), probably indicating a different uptake mechanism of this rather stable organic Cr complex. The bioavailability of CrPic(3) and Cr(D: -Phen)(3), the leading compounds in actual investigations, was analysed also in human volunteer by intraindividual comparison. The apparent absorption (=Cr bioavailability) of (51)Cr from both compounds was substantially higher in humans (0.8-1 %) than in rats. Again, most of freshly absorbed CrPic(3) was excreted into the urine resulting in the same low whole-body retention after 7 days for both compounds. In summary, the bioavailability of Cr from pharmaceutical Cr compound is lower than hitherto assumed. Importantly, humans absorb Cr(III) clearly better than rats. The absorption mechanism of CrPic(3) seems to be different from ionic Cr(III) but, as only the same low amount of Cr is retained from this compound, it is also not more bioavailable than other Cr compounds.

Error specific restrictions for older drivers: promoting continued independence and public safety.

OBJECTIVES: To describe a population of older drivers with driving restrictions, their most common restrictions, and to compare restricted drivers to their safe and unsafe counterparts. Safe drivers are those who do not commit hazardous errors or traffic violations. Unsafe drivers are those who commit hazardous errors and/or traffic violations that place them in hazardous situations. Restricted drivers are those who have committed traffic or rule violations only under certain driving conditions. DESIGN: A retrospective, cross-sectional study with mixed methodology. SETTING: A clinical driving evaluation program within an academic geriatrics department. PARTICIPANTS: Drivers age 60+ (N=108) referred for clinical driving evaluation and who consented to allow their data to be used for research purposes. INTERVENTION: Drivers performing at an intermediate level driving fitness were issued error specific driving restrictions. MEASUREMENT: Driving evaluation included clock drawing test (CDT), mini-mental status exam (MMSE), Trailmaking, geriatric depression scale (GDS), and simulated driving. RESULTS: The three most common restrictions were limited driving distance (N=8), limited driving time (N=8), and daytime only driving (N=8). Safe, restricted, and unsafe drivers significantly differed on MMSE (F[2,104]=10.75, p<0.001), Trailmaking Part B (F[2,76]=9.96, p<0.001), CDT (F[2,98]=29.88, p<0.001), and total number of hazardous errors (F[2,97]=39.06, p<0.001). Tukey's test indicated safe and restricted drivers scored significantly better than unsafe drivers on MMSE (safe: p<0.001; restricted: p=0.008), CDT (p<0.001), and hazardous errors (p<0.001). Restricted and unsafe drivers required significantly more time to complete Trailmaking B than safe drivers (p=0.004). CONCLUSION: Preliminary data indicate restricted drivers perform more like safe than unsafe drivers. Driving simulation is instrumental in discerning error specific limitations and categorizing patients as conditionally safe. This clinical evaluation pilots an effective alternative to premature driving cessation.

In my car the brake is on the right: pedal errors among older drivers.

PURPOSE: To assess to what extent specific cognitive functions contribute to pedal errors among older drivers. METHODS: 180 subjects aged 65 and older completed a 30 min driving evaluation on a simulator as well as three cognitive tests, the Mini-Mental State Exam (MMSE), the Clock Drawing Test, and Trailmaking Part A and B. Analyses based on logistic regressions were performed using age, gender, MMSE, Trailmaking Part A and B, and Clock Drawing Test as independent variables. RESULTS: Results indicate that Clock Drawing is the best predictor of pedal errors (odds ratio=10.04, p<.0001, 95% CI: 3.80, 26.63) followed by age > or =84 (odds ratio 6.10, p<.05, 95% CI: 1.77, 21.03). In contrast, Trailmaking Part A and B, gender, and the MMSE were not significantly related to pedal errors. CONCLUSION: Executive dysfunction may be an important contributor to pedal errors and thus unsafe driving. Practitioners may wish to consider measures of executive function when evaluating patients for driving safety.

Continued driving and time to transition to nondriver status through error-specific driving restrictions.

We developed driving restrictions that are linked to specific driving errors, allowing cognitively impaired individuals to continue to independently meet mobility needs while minimizing risk to themselves and others. The purpose of this project was to evaluate the efficacy and duration expectancy of these restrictions in promoting safe continued driving. We followed 47 drivers age 60 years and older for 18 months, evaluating driving performance at 6-month intervals. Results demonstrated restricted drivers had safety profiles similar to safe drivers and gained additional driving time to transition to nondrivers.

Quantitative Activity Measurements of Brown Adipose Tissue at 7 T Magnetic Resonance Imaging After Application of Triglyceride-Rich Lipoprotein 59Fe-Superparamagnetic Iron Oxide Nanoparticle: Intravenous Versus Intraperitoneal Approach.

OBJECTIVES: The aim of this study was to determine metabolic activity of brown adipose tissue (BAT) with in vivo magnetic resonance imaging (MRI) after intravenous (IV) and intraperitoneal (IP) injection of radioactively labeled superparamagnetic iron oxide nanoparticles (SPIOs) embedded into a lipoprotein layer. MATERIALS AND METHODS: Fe-labeled SPIOs were either polymer-coated or embedded into the lipid core of triglyceride-rich lipoproteins (TRL-Fe-SPIOs). First biodistribution and blood half time analysis in thermoneutral mice after IP injection of either TRL-Fe-SPIOs or polymer-coated Fe-SPIOs (n = 3) were performed. In the next step, cold-exposed (24 hours), BAT-activated mice (n = 10), and control thermoneutral mice (n = 10) were starved for 4 hours before IP (n = 10) or IV (n = 10) injection of TRL-Fe-SPIOs. In vivo MRI was performed before and 24 hours after the application of the particles at a 7 T small animal MRI scanner using a T2*-weighted multiecho gradient echo sequence. R2* and ΔR2* were estimated in the liver, BAT, and muscle. The biodistribution of polymer-coated Fe-SPIOs and TRL-Fe-SPIOs was analyzed ex vivo using a sensitive, large-volume Hamburg whole-body radioactive counter. The amount of Fe-SPIOs in the liver, BAT, and muscle was correlated with the MRI measurements using the Pearson correlation coefficient. Tissue uptake of Fe-SPIOs was confirmed by histological and transmission electron microscopy analyses. RESULTS: Triglyceride-rich lipoprotein Fe-SPIOs exhibited a higher blood concentration after IP injection (10.1% ± 0.91% after 24 hours) and a greater [INCREMENT]R2* in the liver (103 ± 5.0 s), while polymer-coated SPIOs did not increase substantially in the blood stream (0.19% ± 0.01% after 24 hours; P < 0.001) and the liver (57 ± 4.08 s; P < 0.001). In BAT activity studies, significantly higher uptake of TRL-Fe-SPIOs was detected in the BAT of cold-exposed mice, with [INCREMENT]R2* of 107 ± 5.5 s after IV application (control mice: [INCREMENT]R2* of 22 ± 5.8 s; P < 0.001) and 45 ± 5.5 s after IP application (control mice: [INCREMENT]R2* of 11 ± 2.9 s; P < 0.01). Fe radioactivity measurements and [INCREMENT]R2* values correlated strongly in BAT (r > 0.85; P < 0.001) and liver tissue (r > 0.85; P < 0.001). Histological and transmission electron microscopy analyses confirmed the uptake of TRL-Fe-SPIOs within the liver and BAT for both application approaches. CONCLUSIONS: Triglyceride-rich lipoprotein-embedded SPIOs were able to escape the abdominal cavity barrier, whereas polymer-coated SPIOs did not increase substantially in the blood stream. Brown adipose tissue activity can be determined via MRI using TRL-Fe-SPIOs. The quantification of [INCREMENT]R2* using TRL-Fe-SPIOs is feasible and may serve as a noninvasive tool for the quantitative estimation of BAT activity.

Drawing clocks and driving cars.

OBJECTIVE: The purpose of the study was to determine whether a new method of scoring the Clock Drawing Test (CDT) is a reliable and valid method for identifying older adults with declining driving competence. DESIGN: Prospective cohort study. SETTING: An outpatient driving evaluation clinic. PARTICIPANTS: One hundred nineteen community-dwelling, active drivers with a valid driver's license, aged 60 and older referred for driving evaluation. MAIN OUTCOME MEASURES: The CDT and a driving test using a STISIM Drive simulator. RESULTS: The CDT showed a high level of accuracy in predicting driving simulation outcome (area under the receiver-operator curve, 0.90; 95% confidence interval, 0.82 to 0.95). CDT scoring scales were comparable and all correlations between CDT scores and driving performance were negative, implying that as the CDT score decreases, the number of errors increases. Interrater reliability of CDT scores was 0.95. Subjects scoring less than 5 out of 7 points on the CDT made significantly more driving errors, hazardous and in total (P<.001). CONCLUSIONS: The CDT can help establish problems with executive function and indicate the need for a formal driving evaluation. Our CDT scoring scale is a reliable, valid, and time-effective screening tool for identifying elderly drivers in need of further evaluation.

Self-rated driving performance among elderly drivers referred for driving evaluation.

PURPOSE: To explore whether elderly drivers of varying driving skill levels (1) differ in their perception of their driving evaluation performance and (2) determine if self-rated driving evaluation performance is related to cognitive ability. METHODS: One hundred and fifty-two drivers aged 65 years or older and referred for a driving evaluation were enrolled into the study. Subjects were asked the question, "how well do you think you will perform today on your driving evaluation compared to others your own age?" Subjects also completed the Mini-Mental State Exam and a 30-min drive on a STISIM Drivetrade mark simulation (Systems Technology, Inc., Hawthorne, CA). Only 47 subjects completed both the simulated drive and self-rated item. RESULTS: Sixty-five percent of drivers rated themselves as performing better on a driving test than others of their age. Another 31.9% felt they would perform the same as others of their age on a driving test. A 50.0% of those considering themselves "a little better" and 52.9% of those considering themselves "a lot better" had an unsafe driving performance. As self-rated driving evaluation performance increased, there was a significantly increased risk of unsafe driving (p=0.02) in the study population. Drivers who considered themselves at least a little better than others of their age were over four times more likely to be unsafe drivers compared to others who believed they were comparable to or worse than other drivers of their age (RR=4.13, 95% CI=1.08-15.78). There was no significant difference in MMSE between self-rating groups (p=0.76). CONCLUSION: Older drivers assign high ratings to their driving performance, even in the presence of suspected skill decline. Cognitive ability was not related to self-rated driving evaluation performance.

The distribution and degradation of radiolabeled superparamagnetic iron oxide nanoparticles and quantum dots in mice.

(51)Cr-labeled, superparamagnetic, iron oxide nanoparticles ((51)Cr-SPIOs) and (65)Zn-labeled CdSe/CdS/ZnS-quantum dots ((65)Zn-Qdots) were prepared using an easy, on demand, exchange-labeling technique and their particokinetic parameters were studied in mice after intravenous injection. The results indicate that the application of these heterologous isotopes can be used to successfully mark the nanoparticles during initial distribution and organ uptake, although the (65)Zn-label appeared not to be fully stable. As the degradation of the nanoparticles takes place, the individual transport mechanisms for the different isotopes must be carefully taken into account. Although this variation in transport paths can bring new insights with regard to the respective trace element homeostasis, it can also limit the relevance of such trace material-based approaches in nanobioscience. By monitoring (51)Cr-SPIOs after oral gavage, the gastrointestinal non-absorption of intact SPIOs in a hydrophilic or lipophilic surrounding was measured in mice with such high sensitivity for the first time. After intravenous injection, polymer-coated, (65)Zn-Qdots were mainly taken up by the liver and spleen, which was different from that of ionic (65)ZnCl2. Following the label for 4 weeks, an indication of substantial degradation of the nanoparticles and the release of the label into the Zn pool was observed. Confocal microscopy of rat liver cryosections (prepared 2 h after intravenous injection of polymer-coated Qdots) revealed a colocalization with markers for Kupffer cells and liver sinusoidal endothelial cells (LSEC), but not with hepatocytes. In J774 macrophages, fluorescent Qdots were found colocalized with lysosomal markers. After 24 h, no signs of degradation could be detected. However, after 12 weeks, no fluorescent nanoparticles could be detected in the liver cryosections, which would confirm our (65)Zn data showing a substantial degradation of the polymer-coated CdSe/CdS/ZnS-Qdots in the liver.

Determination of liver-specific r2 * of a highly monodisperse USPIO by (59) Fe iron core-labeling in mice at 3 T MRI.

Accurate determination of tissue concentration of ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) using T2 * MR relaxometry is still challenging. We present a reliable quantification method for local USPIO amount with the estimation of the liver specific relaxivity r2 * using monodisperse (59) Fe-core-labeled USPIO ((59) FeUSPIO). Dynamic and relaxometric in vivo characteristics of unlabeled monodisperse USPIO were determined in MRI at 3 T. The in vivo MR studies were performed for liver tissue with (59) FeUSPIO using iron dosages of 9 (n = 3), 18 (n = 2) and 27 (n = 3) µmol Fe kg(-1) body weight. The R2 * of the liver before and after USPIO injection (∆R2 *) was measured and correlated with (59) Fe activity measurements of excised organs by a whole body radioactivity counter (HAMCO) to define the dependency of ∆R2 * and (59) FeUSPIO liver concentration and calculate the r2 * of (59) FeUSPIO for the liver. Ultrastructural analysis of liver uptake was performed by histology and transmission electron microscopy. ∆R2 * of the liver revealed a dosage-dependent accumulation of (59) FeUSPIO with a percentage uptake of 70-88% of the injection dose. Hepatic ∆R2 * showed a dose-dependent linear correlation to (59) FeUSPIO activity measurements (r = 0.92) and an r2 * in the liver of 481 ± 74.9 mm(-1) s(-1) in comparison to an in vitro r2 * of 60.5 ± 3.3 mm(-1) s(-1) . Our results indicate that core-labeled (59) FeUSPIO can be used to quantify the local amount of USPIO and to estimate the liver-specific relaxivity r2 *.

Effects of cognition on driving involvement among the oldest old: variations by gender and alternative transportation opportunities.

PURPOSE: This study explored the impact of cognition and the availability of other drivers on driving restriction and cessation among older adults. DESIGN AND METHODS: Survey data from the first wave of the Asset and Health Dynamics Among the Oldest Old data were analyzed, using multinomial logistic regressions. RESULTS: Cognitive impairment is associated with driving restriction and cessation, although a noteworthy minority of mildly and severely cognitively impaired individuals continue to drive. Partner's driving and involvement and presence of other drivers in the household moderated the effect of cognition on driving restriction and cessation. IMPLICATIONS: The decision processes surrounding an individual's restricting or stopping driving are complex and may include consideration not only of competence, but also of sense of self-worth and relationship with a partner.

Problems in transition following bone marrow transplantation: psychosocial aspects.

The number of patients achieving long-term survival following bone marrow transplantation is increasing. Psychosocial issues that may arise for the pediatric patient and for the family following discharge from the hospital are described. Implications for other situations in which there is a lengthy hospitalization for an organ transplant or life-threatening condition are highlighted.

The cell-type specific uptake of polymer-coated or micelle-embedded QDs and SPIOs does not provoke an acute pro-inflammatory response in the liver.

Semiconductor quantum dots (QD) and superparamagnetic iron oxide nanocrystals (SPIO) have exceptional physical properties that are well suited for biomedical applications in vitro and in vivo. For future applications, the direct injection of nanocrystals for imaging and therapy represents an important entry route into the human body. Therefore, it is crucial to investigate biological responses of the body to nanocrystals to avoid harmful side effects. In recent years, we established a system to embed nanocrystals with a hydrophobic oleic acid shell either by lipid micelles or by the amphiphilic polymer poly(maleic anhydride-alt-1-octadecene) (PMAOD). The goal of the current study is to investigate the uptake processes as well as pro-inflammatory responses in the liver after the injection of these encapsulated nanocrystals. By immunofluorescence and electron microscopy studies using wild type mice, we show that 30 min after injection polymer-coated nanocrystals are primarily taken up by liver sinusoidal endothelial cells. In contrast, by using wild type, Ldlr (-/-) as well as Apoe (-/-) mice we show that nanocrystals embedded within lipid micelles are internalized by Kupffer cells and, in a process that is dependent on the LDL receptor and apolipoprotein E, by hepatocytes. Gene expression analysis of pro-inflammatory markers such as tumor necrosis factor alpha (TNFα) or chemokine (C-X-C motif) ligand 10 (Cxcl10) indicated that 48 h after injection internalized nanocrystals did not provoke pro-inflammatory pathways. In conclusion, internalized nanocrystals at least in mouse liver cells, namely endothelial cells, Kupffer cells and hepatocytes are at least not acutely associated with potential adverse side effects, underlining their potential for biomedical applications.

Intraperitoneal injection improves the uptake of nanoparticle-labeled high-density lipoprotein to atherosclerotic plaques compared with intravenous injection: a multimodal imaging study in ApoE knockout mice.

BACKGROUND: The aim of this study was to assess whether high-density lipoprotein (HDL) labeled with superparamagnetic iron oxide nanoparticles (SPIOs) and quantum dots was able to detect atherosclerotic lesions in mice after intravenous and intraperitoneal injection by multimodal imaging. METHODS AND RESULTS: Nanoparticle-labeled HDLs (NP-HDLs) were characterized in vitro by dynamic light scattering and size exclusion chromatography with subsequent cholesterol and fluorescence measurements. For biodistribution and blood clearance studies, NP-HDL(SPIOs) radiolabeled with (59)Fe (NP-HDL(59Fe-SPIOs)) were injected intravenously or intraperitoneally into ApoE knockout mice (n=6), and radioactivity was measured using a gamma counter. NP-HDL accumulation within atherosclerotic plaques in vivo and ex vivo was estimated by MRI at 7 Tesla, ex vivo confocal fluorescence microscopy, x-ray fluorescence microscopy, and histological analysis (n=3). Statistical analyses were performed using a 2-tailed Student t-test. In vitro characterization of NP-HDL confirmed properties similar to endogenous HDL. Blood concentration time curves showed a biexponential decrease for the intravenous injection, whereas a slow increase followed by a steady state was noted for intraperitoneal injection. Radioactivity measurements showed predominant accumulation in the liver and spleen after both application approaches. NP-HDL(59Fe-SPIOs) uptake into atherosclerotic plaques increased significantly after intraperitoneal compared with intravenous injection (P<0.01). In vivo MRI showed an increased uptake of NP-HDL into atherosclerotic lesions after intraperitoneal injection, which was confirmed by ex vivo MRI, x-ray fluorescence microscopy, confocal fluorescence microscopy, and histological analysis. CONCLUSIONS: In vivo MRI and ex vivo multimodal imaging of atherosclerotic plaque using NP-HDL is feasible, and intraperitoneal application improves the uptake within vessel wall lesions compared with intravenous injection.

High interstitial fluid pressure is associated with low tumour penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes.

The human epithelial cell adhesion molecule (EpCAM) is highly expressed in a variety of clinical tumour entities. Although an antibody against EpCAM has successfully been used as an adjuvant therapy in colon cancer, this therapy has never gained wide-spread use. We have therefore investigated the possibilities and limitations for EpCAM as possible molecular imaging target using a panel of preclinical cancer models. Twelve human cancer cell lines representing six tumour entities were tested for their EpCAM expression by qPCR, flow cytometry analysis and immunocytochemistry. In addition, EpCAM expression was analyzed in vivo in xenograft models for tumours derived from these cells. Except for melanoma, all cell lines expressed EpCAM mRNA and protein when grown in vitro. Although they exhibited different mRNA levels, all cell lines showed similar EpCAM protein levels upon detection with monoclonal antibodies. When grown in vivo, the EpCAM expression was unaffected compared to in vitro except for the pancreatic carcinoma cell line 5072 which lost its EpCAM expression in vivo. Intravenously applied radio-labelled anti EpCAM MOC31 antibody was enriched in HT29 primary tumour xenografts indicating that EpCAM binding sites are accessible in vivo. However, bound antibody could only be immunohistochemically detected in the vicinity of perfused blood vessels. Investigation of the fine structure of the HT29 tumour blood vessels showed that they were immature and prone for higher fluid flux into the interstitial space. Consistent with this hypothesis, a higher interstitial fluid pressure of about 12 mbar was measured in the HT29 primary tumour via "wick-in-needle" technique which could explain the limited diffusion of the antibody into the tumour observed by immunohistochemistry.

A simple and widely applicable method to 59Fe-radiolabel monodisperse superparamagnetic iron oxide nanoparticles for in vivo quantification studies.

A simple, fast, efficient, and widely applicable method to radiolabel the cores of monodisperse superparamagnetic iron oxide nanoparticles (SPIOs) with (59)Fe was developed. These cores can be used as precursors for a variety of functionalized nanodevices. A quality control using filtration techniques, size-exclusion chromatography, chemical degradation methods, transmission electron microscopy, and magnetic resonance imaging showed that the nanoparticles were stably labeled with (59)Fe. Furthermore, the particle structure and the magnetic properties of the SPIOs were unchanged. In a second approach, monodisperse SPIOs stabilized with (14)C-oleic acid were synthesized, and the stability of this shell labeling was studied. In proof of principle experiments, the (59)Fe-SPIOs coated with different shells to make them water-soluble were used to evaluate and compare in vivo pharmacokinetic parameters such as blood half-life. It could also be shown that our radiolabeled SPIOs embedded in recombinant lipoproteins can be used to quantify physiological processes in closer detail than hitherto possible. In vitro and in vivo experiments showed that the (59)Fe label is stable enough to be applied in vivo, whereas the (14)C label is rapidly removed from the iron core and is not adequate for in vivo studies. To obtain meaningful results in in vivo experiments, only (59)Fe-labeled SPIOs should be used.

Investigations on the usefulness of CEACAMs as potential imaging targets for molecular imaging purposes.

Members of the carcinoembryonic antigen cell adhesion molecules (CEACAMs) family are the prototype of tumour markers. Classically they are used as serum markers, however, CEACAMs could serve as targets for molecular imaging as well.In order to test the anti CEACAM monoclonal antibody T84.1 for imaging purposes, CEACAM expression was analysed using this antibody. Twelve human cancer cell lines from different entities were screened for their CEACAM expression using qPCR, Western Blot and FACS analysis. In addition, CEACAM expression was analyzed in primary tumour xenografts of these cells. Nine of 12 tumour cell lines expressed CEACAM mRNA and protein when grown in vitro. Pancreatic and colon cancer cell lines showed the highest expression levels with good correlation of mRNA and protein level. However, when grown in vivo, the CEACAM expression was generally downregulated except for the melanoma cell lines. As the CEACAM expression showed pronounced expression in FemX-1 primary tumours, this model system was used for further experiments. As the accessibility of the antibody after i.v. application is critical for its use in molecular imaging, the binding of the T84.1 monoclonal antibody was assessed after i.v. injection into SCID mice harbouring a FemX-1 primary tumour. When applied i.v., the CEACAM specific T84.1 antibody bound to tumour cells in the vicinity of blood vessels. This binding pattern was particularly pronounced in the periphery of the tumour xenograft, however, some antibody binding was also observed in the central areas of the tumour around blood vessels. Still, a general penetration of the tumour by i.v. application of the anti CEACAM antibody could not be achieved despite homogenous CEACAM expression of all melanoma cells when analysed in tissue sections. This lack of penetration is probably due to the increased interstitial fluid pressure in tumours caused by the absence of functional lymphatic vessels.

Brown adipose tissue activity controls triglyceride clearance.

Brown adipose tissue (BAT) burns fatty acids for heat production to defend the body against cold and has recently been shown to be present in humans. Triglyceride-rich lipoproteins (TRLs) transport lipids in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase (LPL). Peripheral organs such as muscle and adipose tissue take up the fatty acids, whereas the remaining cholesterol-rich remnant particles are cleared by the liver. Elevated plasma triglyceride concentrations and prolonged circulation of cholesterol-rich remnants, especially in diabetic dyslipidemia, are risk factors for cardiovascular disease. However, the precise biological role of BAT for TRL clearance remains unclear. Here we show that increased BAT activity induced by short-term cold exposure controls TRL metabolism in mice. Cold exposure drastically accelerated plasma clearance of triglycerides as a result of increased uptake into BAT, a process crucially dependent on local LPL activity and transmembrane receptor CD36. In pathophysiological settings, cold exposure corrected hyperlipidemia and improved deleterious effects of insulin resistance. In conclusion, BAT activity controls vascular lipoprotein homeostasis by inducing a metabolic program that boosts TRL turnover and channels lipids into BAT. Activation of BAT might be a therapeutic approach to reduce elevated triglyceride concentrations and combat obesity in humans.