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The human commensal yeast Candida is the fourth most common cause of hospital-acquired bloodstream infections, with Candida albicans accounting for the majority of the >400,000 life-threatening infections annually. Diagnosis of invasive candidiasis (IC), a disease encompassing candidemia (blood-borne yeast infection) and deep-seated organ infections, is a major challenge since clinical manifestations of the disease are indistinguishable from viral, bacterial and other fungal diseases, and diagnostic tests for biomarkers in the bloodstream such as PCR, ELISA, and pan-fungal β-D-glucan lack either standardization, sensitivity, or specificity. Blood culture remains the gold standard for diagnosis, but test sensitivity is poor and turn-around time slow. Furthermore, cultures can only be obtained when the yeast resides in the bloodstream, with samples recovered from hematogenous infections often yielding negative results. Consequently, there is a pressing need for a diagnostic test that allows the identification of metastatic foci in deep-seated Candida infections, without the need for invasive biopsy. Here, we report the development of a highly specific mouse IgG3 monoclonal antibody (MC3) that binds to a putative β-1,2-mannan epitope present in high molecular weight mannoproteins and phospholipomannans on the surface of yeast and hyphal morphotypes of C. albicans , and its use as a [ 64 Cu]NODAGA-labeled tracer for whole-body pre-clinical imaging of deep-seated C. albicans infections using antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI). When used in a mouse intravenous (i.v.) challenge model that faithfully mimics disseminated C. albicans infections in humans, the [ 64 Cu]NODAGA-MC3 tracer accurately detects infections of the kidney, the principal site of blood-borne candidiasis in this model. Using a strain of the emerging human pathogen Candida auris that reacts with MC3 in vitro , but which is non-infective in i.v. challenged mice, we demonstrate the accuracy of the tracer in diagnosing invasive infections in vivo . This pre-clinical study demonstrates the principle of using antibody-guided molecular imaging for detection of deep organ infections in IC, without the need for invasive tissue biopsy.

The goal of this study was to validate the use of an MR-compatible blood sampler (BS) with a detector system based on a lutetium oxyorthosilicate scintillator and avalanche photodiodes for small-animal PET. Methods: Five rats underwent a 60-min 18 F-FDG study. For each animal, the arterial input function (AIF) was derived from the BS recording, from manual sampling (MS), and from the PET image. These AIFs were applied for kinetic modeling of the striatum using the irreversible 2-tissue-compartment model. The MS-based technique with a dispersion correction served as a reference approach, and the kinetic parameters that were estimated with the BS- and the image-derived AIFs were compared with the reference values. Additionally, the effect of applying a population-based activity ratio for plasma to whole blood ( p / wb ) and the dispersion correction was assessed. Results: The K 1 , k 2 , and k 3 values estimated with the reference approach were 0.174 ± 0.037 mL/min/cm 3 , 0.342 ± 0.080 1/min, and 0.048 ± 0.009 1/min, respectively. The corresponding parameters obtained with the BS- and image-derived AIFs deviated from these values by 0.6%-18.8% and 16.7%-47.9%, respectively. To compensate for the error in the BS-based technique, data from one MS collected at the end of the experiment were combined with the data from the first 10 min of the BS recording. This approach reduced the deviation in the kinetic parameters to 1.8%-6.3%. Using p / wb led to a 1.7%-8.3% difference from the reference parameters. The sensitivity of the BS was 23%, the energy resolution for the 511-keV photopeak was 19%, and the timing resolution was 11.2 ns. Conclusion: Online recording of the blood activity level with the BS allows precise measurement of AIF, without loss of blood volume. Combining the BS data with one MS is the most accurate approach for the data analysis. The high sensitivity of the device may allow application of lower radioactivity doses., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

Over the last decade, the combination of PET and MRI in one system has proven to be highly successful in basic preclinical research, as well as in clinical research. Nowadays, PET/MRI systems are well established in preclinical imaging and are progressing into clinical applications to provide further insights into specific diseases, therapeutic assessments, and biological pathways. Certain challenges in terms of hardware had to be resolved concurrently with the development of new techniques to be able to reach the full potential of both combined techniques. This review provides an overview of these challenges and describes the opportunities that simultaneous PET/MRI systems can exploit in comparison with stand-alone or other combined hybrid systems. New approaches were developed for simultaneous PET/MRI systems to correct for attenuation of 511 keV photons because MRI does not provide direct information on gamma photon attenuation properties. Furthermore, new algorithms to correct for motion were developed, because MRI can accurately detect motion with high temporal resolution. The additional information gained by the MRI can be employed to correct for partial volume effects as well. The development of new detector designs in combination with fast-decaying scintillator crystal materials enabled time-of-flight detection and incorporation in the reconstruction algorithms. Furthermore, this review lists the currently commercially available systems both for preclinical and clinical imaging and provides an overview of applications in both fields. In this regard, special emphasis has been placed on data analysis and the potential for both modalities to evolve with advanced image analysis tools, such as cluster analysis and machine learning., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The hemolytic uremic syndrome (HUS) is a life-threatening disease of the kidney that is induced by shiga toxin-producing E.coli. Major changes in the monocytic compartment and in CCR2-binding chemokines have been observed. However, the specific contribution of CCR2-dependent Gr1 high monocytes is unknown. To investigate the impact of these monocytes during HUS, we injected a combination of LPS and shiga toxin into mice. We observed an impaired kidney function and elevated levels of the CCR2-binding chemokine CCL2 after shiga toxin/LPS- injection, thus suggesting Gr1 high monocyte infiltration into the kidney. Indeed, the number of Gr1 high monocytes was strongly increased one day after HUS induction. Moreover, these cells expressed high levels of CD11b suggesting activation after tissue entry. Non-invasive PET-MR imaging revealed kidney injury mainly in the kidney cortex and this damage coincided with the detection of Gr1 high monocytes. Lack of Gr1 high monocytes in Ccr2-deficient animals reduced neutrophil gelatinase-associated lipocalin and blood urea nitrogen levels. Moreover, the survival of Ccr2-deficient animals was significantly improved. Conclusively, this study demonstrates that CCR2-dependent Gr1 high monocytes contribute to the kidney injury during HUS and targeting these cells is beneficial during this disease., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that are expanded in cancer and act as potent suppressors of the anti-tumor immune response. MDSCs consist of two major subsets, namely monocytic (M-) MDSCs and granulocytic (G-) MDSCs that differ with respect to their phenotype, morphology and mechanisms of suppression. Here, we cultured bone marrow cells with IL-6 and GM-CSF in vitro to generate a population of bone marrow MDSCs (BM-MDSCs) similar to G-MDSCs from tumor-bearing mice in regards to phenotype, morphology and suppressive-function. Through fluorescent labeling of these BM-MDSCs and optical imaging, we could visualize the recruitment and localization of BM-MDSCs in breast tumor-bearing mice in vivo. Furthermore, we were able to demonstrate that BM-MDSCs home to primary and metastatic breast tumors, but have no significant effect on tumor growth or progression. Ex vivo flow cytometry characterization of BM-MDSCs after adoptive transfer demonstrated both organ-and tumor-specific effects on their phenotype and differentiation, demonstrating the importance of the local microenvironment on MDSC fate and function. In this study, we have developed a method to generate, visualize and detect BM-MDSCs in vivo and ex vivo through optical imaging and flow cytometry, in order to understand the organ-specific changes rendered to MDSCs in breast cancer.

Phenotypic heterogeneity is commonly observed in diseased tissue, specifically in tumors. Multimodal imaging technologies can reveal tissue heterogeneity noninvasively in vivo, enabling imaging-based profiling of receptors, metabolism, morphology, or function on a macroscopic scale. In contrast, in vitro multiomics, immunohistochemistry, or histology techniques accurately characterize these heterogeneities in the cellular and subcellular scales in a more comprehensive but ex vivo manner. The complementary in vivo and ex vivo information would provide an enormous potential to better characterize a disease. However, this requires spatially accurate coregistration of these data by image-driven sampling as well as fast sample-preparation methods. Here, a unique image-guided milling machine and workflow for precise extraction of tissue samples from small laboratory animals or excised organs has been developed and evaluated. The samples can be delineated on tomographic images as volumes of interest and can be extracted with a spatial accuracy better than 0.25 mm. The samples remain cooled throughout the procedure to ensure metabolic stability, a precondition for accurate in vitro analysis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

This study depicts the evaluation of a SiPM detector with depth of interaction (DOI) capability via a dual-sided readout that is suitable for high-resolution positron emission tomography and magnetic resonance (PET/MR) imaging. Two different 12  ×  12 pixelated LSO scintillator arrays with a crystal pitch of 1.60 mm are examined. One array is 20 mm-long with a crystal separation by the specular reflector Vikuiti enhanced specular reflector (ESR), and the other one is 18 mm-long and separated by the diffuse reflector Lumirror E60 (E60). An improvement in energy resolution from 22.6% to 15.5% for the scintillator array with the E60 reflector is achieved by taking a nonlinear light collection correction into account. The results are FWHM energy resolutions of 14.0% and 15.5%, average FWHM DOI resolutions of 2.96 mm and 1.83 mm, and FWHM coincidence resolving times of 1.09 ns and 1.48 ns for the scintillator array with the ESR and that with the E60 reflector, respectively. The measured DOI signal ratios need to be assigned to an interaction depth inside the scintillator crystal. A linear and a nonlinear method, using the intrinsic scintillator radiation from lutetium, are implemented for an easy to apply calibration and are compared to the conventional method, which exploits a setup with an externally collimated radiation beam. The deviation between the DOI functions of the linear or nonlinear method and the conventional method is determined. The resulting average of differences in DOI positions is 0.67 mm and 0.45 mm for the nonlinear calibration method for the scintillator array with the ESR and with the E60 reflector, respectively; Whereas the linear calibration method results in 0.51 mm and 0.32 mm for the scintillator array with the ESR and the E60 reflector, respectively; and is, due to its simplicity, also applicable in assembled detector systems.

Aim: To determine the accuracy of locating the apical constriction using apex locators., Methodology: Extracted teeth were micro-CT scanned preoperatively to localize the apical constriction. Electronic length measurements of 91 root canals were made using nine electronic apex locators (EAL) connected to a mounting model. Distances from the major foramen were recorded at each scale bar of the EALs, and a file was fixed in the canal at a position indicated by each EAL to be the apical constriction. A second micro-CT scan was conducted, and distances from the file tip to constriction and major foramen were calculated for each EAL. The accuracy of EALs was determined with a tolerance of 0.1, 0.25, 0.5 and 1 mm, and the 95% confidence interval was used to compare the EALs. A rank analysis was performed in which measurements beyond the major foramen were considered as inaccurate., Results: Regardless of the type of teeth, there was no significant difference in the accuracy of determining the apical constriction and major foramen between the nine EALs within a tolerance of ±0.5 mm and 1 mm, but there was a significant difference for the tolerances of ±0.1 and 0.25 mm. The highest ranks close to the constriction (98% and 94%) and to the major foramen (86% and 73%) were observed in Dentaport ZX and Elements Diagnostic Unit, respectively. Overestimation of working length beyond the major foramen was observed in all EALs (5% to 71%) when the scale for the major foramen, as recommended by the manufacturers, was used. However, when the scale for the constriction was used, only 3% of the measurements were beyond the major foramen., Conclusion: Electronic apex locators were able to determine the apical constriction. Using EALs to determine the major foramen led to overestimation of the working length. Therefore, it may be recommended to use the EAL scale of the constriction., (© 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Noninvasive imaging technologies are increasingly used in preclinical drug research for the pharmacokinetic analysis of therapeutic compounds in living animals over time. The different preclinical imaging modalities available differ intrinsically in their detection principle and thus might exhibit limitations for a specific application. Here, we systematically investigated the performance of advanced fluorescence-mediated tomography (FMT)/CT in comparison to PET/MRI for quantitative analysis of the biodistribution of different antibody formats and dependence on the required imaging label in squamous cell carcinoma xenografts. Methods: Different formats of an antibody (monoclonal antibody and the antigen binding fragments F(ab') 2 and Fab) targeting epidermal growth factor receptor were labeled with Alexa750 or 64 Cu-NODAGA and injected intravenously into separate cohorts of nude mice bearing subcutaneous A-431 tumors. Two and 24 h after injection, the mice were measured by FMT/CT and PET/MRI. Probe accumulation was quantitatively assessed in organs and tumors. In vivo data were compared between modalities and correlated with ex vivo fluorescence, γ-counting, and electrochemiluminescence immunoassay. Results: Both imaging methods faithfully monitored the biodistribution and elimination routes of the compounds, and organ accumulation measured by FMT/CT and PET/MRI correlated significantly with ex vivo measurements. In addition, the accumulation in kidney, muscle, and tumor tissue correlated between FMT/CT and PET/MRI. However, the pharmacokinetics of the Alexa750-labeled antibody formats showed shorter blood half-times and higher liver uptake than the radiolabeled counterparts. Conclusion: FMT/CT imaging allows quantifying the biodistribution of antibodies in nude mice and provides an alternative to PET analysis in preclinical drug research. However, even for large molecules, such as monoclonal antibodies, Alexa750 labeling can change pharmacokinetics and trigger liver uptake., (© 2018 by the Society of Nuclear Medicine and Molecular Imaging.)

Purpose: Non-invasive in vivo positron emission tomography (PET) provides high detection sensitivity in the nano- to picomolar range and in addition to other advantages, the possibility to absolutely quantify the acquired data. The present study focuses on the comparison of transmission data acquired with an X-ray computed tomography (CT) scanner or a Co-57 source for the Inveon small animal PET scanner (Siemens Healthcare, Knoxville, TN, USA), as well as determines their influences on the quantification accuracy and partial volume effect (PVE). A special focus included the impact of the performed calibration on the quantification accuracy., Procedures: Phantom measurements were carried out to determine the quantification accuracy, the influence of the object size on the quantification, and the PVE for different sphere sizes, along the field of view and for different contrast ratios., Results: An influence of the emission activity on the Co-57 transmission measurements was discovered (deviations up to 24.06 % measured to true activity), whereas no influence of the emission activity on the CT attenuation correction was identified (deviations <3 % for measured to true activity). The quantification accuracy was substantially influenced by the applied calibration factor and by the object size. The PVE demonstrated a dependency on the sphere size, the position within the field of view, the reconstruction and correction algorithms and the count statistics. Depending on the reconstruction algorithm, only ∼30-40 % of the true activity within a small sphere could be resolved. The iterative 3D reconstruction algorithms uncovered substantially increased recovery values compared to the analytical and 2D iterative reconstruction algorithms (up to 70.46 % and 80.82 % recovery for the smallest and largest sphere using iterative 3D reconstruction algorithms). The transmission measurement (CT or Co-57 source) to correct for attenuation did not severely influence the PVE., Conclusions: The analysis of the quantification accuracy and the PVE revealed an influence of the object size, the reconstruction algorithm and the applied corrections. Particularly, the influence of the emission activity during the transmission measurement performed with a Co-57 source must be considered. To receive comparable results, also among different scanner configurations, standardization of the acquisition (imaging parameters, as well as applied reconstruction and correction protocols) is necessary.

Purpose: Non-invasive techniques allowing quantitative determination of the functional liver mass are of great interest for patient management in a variety of clinical settings. Recently, we presented [ 68 Ga]DTPA-GSA to target the hepatic asialoglycoprotein receptor for this purpose. Here, we introduce [ 68 Ga]NOTA-GSA to improve metabolic stability of the radiopharmaceutical and compare the imaging properties with [ 68 Ga]DTPA-GSA., Procedures: Labeling of the compounds was carried out at room temperature using 1.9 M sodium acetate as buffer. For quality control, thin-layer, high-performance liquid, and size exclusion chromatographies were used. Metabolic stability was studied in rat and human serums. For in vivo evaluation, Fischer rats were scanned by positron emission tomography and magnetic resonance imaging and subsequently sacrificed for biodistribution studies. Time activity curves (TACs) for heart and liver were generated and corresponding parameters (T 50 , T 90 , LHL15, HH15) were calculated., Results: [ 68 Ga]NOTA-GSA can be produced in high radiochemical yield and purity (>95 %) within 15 min. Stability studies revealed almost no metabolite formation over the 2-h observation period. Analysis of the TACs showed comparable results for most of the investigated parameters. The only significant difference was found in the T 90 value, where [ 68 Ga]NOTA-GSA showed slower uptake in comparison with 68 Ga-DTPA-GSA (123 ± 10 vs. 89 ± 3 s, p < 0.01)., Conclusions: [ 68 Ga]NOTA-GSA showed a significant increase of the metabolic stability and in most organs lower background activity. However, comparison of LHL15 and HH15 indicates that the increased stability did not further improve the diagnostic value. Thus, [ 68 Ga]NOTA-GSA and [ 68 Ga]DTPA-GSA can be used equivalent for imaging hepatic function with positron emission tomography.

Many pathophysiological processes are associated with proliferation, migration or death of distinct cell populations. Monitoring specific cell types and their progeny in a non-invasive, longitudinal and quantitative manner is still challenging. Here we show a novel cell-tracking system that combines Cre/lox-assisted cell fate mapping with a thymidine kinase (sr39tk) reporter gene for cell detection by positron emission tomography (PET). We generate Rosa26-mT/sr39tk PET reporter mice and induce sr39tk expression in platelets, T lymphocytes or cardiomyocytes. As proof of concept, we demonstrate that our mouse model permits longitudinal PET imaging and quantification of T-cell homing during inflammation and cardiomyocyte viability after myocardial infarction. Moreover, Rosa26-mT/sr39tk mice are useful for whole-body characterization of transgenic Cre mice and to detect previously unknown Cre activity. We anticipate that the Cre-switchable PET reporter mice will be broadly applicable for non-invasive long-term tracking of selected cell populations in vivo.Non-invasive cell tracking is a powerful method to visualize cells in vivo under physiological and pathophysiological conditions. Here Thunemann et al. generate a mouse model for in vivo tracking and quantification of specific cell types by combining a PET reporter gene with Cre-dependent activation that can be exploited for any cell population for which a Cre mouse line is available.

Preclinical imaging benefits from simultaneous acquisition of high-resolution anatomical and molecular data. Additionally, PET/MRI systems can provide functional PET and functional MRI data. To optimize PET sensitivity, we propose a system design that fully integrates the MRI coil into the PET system. This allows positioning the scintillators near the object but requires an optimized design of the MRI coil and PET detector. It further requires a new approach in realizing the radiofrequency (RF) shielding. Thus, we propose the use of an optically transparent RF shielding material between the PET scintillator and the light sensor, suppressing the interference between both systems. We evaluated two conductive foils (ITO, 9900) and a wire mesh. The PET performance was tested on a dual-layer scintillator consisting of 12  ×  12 LSO matrices, shifted by half a pitch. The pixel size was 0.9  ×  0.9 mm 2 ; the lengths were 10.0 mm and 5.0 mm, respectively. For a light sensor, we used a 4  ×  4 SiPM array. The RF attenuation was measured from 320 kHz to 420 MHz using two pick-up coils. MRI-compatibility and shielding effect of the materials were evaluated with an MRI system. The average FWHM energy resolution at 511 keV of all 144 crystals of the layer next to the SiPM was deteriorated from 15.73  ±  0.24% to 16.32  ±  0.13%, 16.60  ±  0.25%, and 19.16  ±  0.21% by the ITO foil, 9900 foil, mesh material, respectively. The average peak-to-valley ratio of the PET detector changed from 5.77  ±  0.29 to 4.50  ±  0.39, 4.78  ±  0.48, 3.62  ±  0.16, respectively. The ITO, 9900, mesh attenuated the scintillation light by 11.3  ±  1.6%, 11.0  ±  1.8%, 54.3  ±  0.4%, respectively. To attenuate the RF from 20 MHz to 200 MHz, mesh performed better than copper. The results show that an RF shielding material that is sufficiently transparent for scintillation light and is MRI compatible can be obtained. This result enables the development of a fully integrated PET detector and MRI coil assembly.

Manganese in its divalent state (Mn 2+ ) has features that make it a unique tool for tracing neuronal pathways. It is taken up and transported by neurons in an activity-dependent manner and it can cross synapses. It also acts as a contrast agent for magnetic resonance imaging (MRI) enabling visualization of neuronal tracts. However, due to the limited sensitivity of MRI systems relatively high Mn 2+ doses are required. This is undesirable, especially in long-term studies, because of the known toxicity of the metal. In order to overcome this limitation, we propose 52 Mn as a positron emission tomography (PET) neuronal tract tracer. We used 52 Mn for imaging dopaminergic pathways after a unilateral injection into the ventral tegmental area (VTA), as well as the striatonigral pathway after an injection into the dorsal striatum (STR) in rats. Furthermore, we tested potentially noxious effects of the radioactivity dose with a behavioral test and histological staining. 24 h after 52 Mn administration, the neuronal tracts were clearly visible in PET images and statistical analysis confirmed the observed distribution of the tracer. We noticed a behavioral impairment in some animals treated with 170 kBq of 52 Mn, most likely caused by dysfunction of dopaminergic cells. Moreover, there was a substantial DNA damage in the brain tissue after applying 150 kBq of the tracer. However, all those effects were completely eliminated by reducing the 52 Mn dose to 20-30 kBq. Crucially, the reduced dose was still sufficient for PET imaging., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of hematological malignancy or bone marrow transplant patients caused by the ubiquitous environmental fungus Aspergillus fumigatus . Current diagnostic tests for the disease lack sensitivity as well as specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. In a previous study, we reported the development of a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI) using a [ 64 Cu]DOTA-labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus . To enable translation of the tracer to the clinical setting, we report here the development of a humanised version of the antibody (hJF5), and pre-clinical imaging of lung infection using a [ 64 Cu]NODAGA-hJF5 tracer. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to its radiolabeled murine counterpart [ 64 Cu]NODAGA-mJF5. Using reverse genetics of the pathogen, we show that the antibody binds to the antigenic determinant β1,5-galactofuranose (Gal f ) present in a diagnostic mannoprotein antigen released by the pathogen during invasive growth in the lung. The absence of the epitope Gal f in mammalian carbohydrates, coupled with the enhanced imaging capabilities of the hJF5 antibody, means that the [ 64 Cu]NODAGA-hJF5 tracer developed here represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting., Competing Interests: Competing Interests: B.J.P. receives grant/research support from: Bayer Healthcare, Boehringer-Ingelheim, and Siemens; however, none of the grants are directly related to this work. C.R.T. is director of ISCA Diagnostics Ltd.

The clinical use of Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET) has proven to be a strong diagnostic tool in the field of neurology. The reliability of these methods to confirm clinical diagnoses has guided preclinical research to utilize these techniques for the characterization of animal disease models. Previously, we demonstrated that an endothelial cell-specific ablation of the murine Serum Response Factor (Srf iECKO ) results in blood brain barrier (BBB) breakdown and hemorrhagic stroke. Taking advantage of this mouse model we here perform a comprehensive longitudinal, multiparametric and in vivo imaging approach to reveal pathophysiological processes occurring before and during the appearance of cerebral microbleeds using combined PET and MRI. We complement our imaging results with data regarding animal behavior and immunohistochemistry. Our results demonstrate diffusion abnormalities in the cortical brain tissue prior to the onset of cerebral microbleeds. Diffusion reductions were accompanied by significant increments of [ 18 F]FAZA uptake before the onset of the lesions in T2WI. The Open Field behavioral tests revealed reduced activity of Srf iECKO animals, whereas histology confirmed the presence of hemorrhages in cortical regions of the mouse brain and iron deposition at lesion sites with increased hypoxia inducible factor 1α, CD31 and glial fibrillary acidic protein expression. For the first time, we performed a thorough evaluation of the prodromal period before the occurrence of spontaneous cerebral microbleeds. Using in vivo PET and MRI, we show the pathological tissue changes that occur previous to gross blood brain barrier (BBB) disruption and breakage. In addition, our results show that apparent diffusion coefficient (ADC) reduction may be an early biomarker of BBB disruption proposing an alternate clinical interpretation. Furthermore, our findings remark the usefulness of this novel Srf iECKO mouse model to study underlying mechanisms of hemorrhagic stroke., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aim: To validate the use of longitudinal sections against cross sections using micro-CT for disclosing the topography and location of the apical constriction., Methodology: Seventy extracted human teeth with 117 completely developed roots were micro-CT scanned and reconstructed at a voxel size of 27 μm. The 3DSlicer program was used to navigate the longitudinal sections parallel to the long axis of the canal and also to rotate and tilt the views. Each root canal was evaluated in both mesio-distal and bucco-lingual planes. Constriction topographies were identified as described in the literature. In each canal, the number of different topographies detected was recorded. Further, serial cross-sectional analysis of the apical portion of the canal was performed. Reconstructed plots of canal areas were assessed to locate the constriction and determine its form. A descriptive analysis of both longitudinal and cross section methods was conducted. In each canal, the frequency of constriction forms was calculated in the mesio-distal or bucco-lingual aspects and the 99% confidence interval was computed., Results: When both aspects of the longitudinal sections were pooled, all root canals had two or more topographies and consequently different locations of the apical constriction. In contrast, cross-sectional analysis constantly yielded one constriction form per canal., Conclusion: Compared to cross-sectional analysis, longitudinal sections of the root canal conveyed inconsistent results regarding the topography and the location of the apical constriction., (© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Purpose: We aimed to precisely estimate intra-tumoral heterogeneity using spatially regularized spectral clustering (SRSC) on multiparametric MRI data and compare the efficacy of SRSC with the previously reported segmentation techniques in MRI studies., Procedures: Six NMRI nu/nu mice bearing subcutaneous human glioblastoma U87 MG tumors were scanned using a dedicated small animal 7T magnetic resonance imaging (MRI) scanner. The data consisted of T2 weighted images, apparent diffusion coefficient maps, and pre- and post-contrast T2 and T2* maps. Following each scan, the tumors were excised into 2-3-mm thin slices parallel to the axial field of view and processed for histological staining. The MRI data were segmented using SRSC, K-means, fuzzy C-means, and Gaussian mixture modeling to estimate the fractional population of necrotic, peri-necrotic, and viable regions and validated with the fractional population obtained from histology., Results: While the aforementioned methods overestimated peri-necrotic and underestimated viable fractions, SRSC accurately predicted the fractional population of all three tumor tissue types and exhibited strong correlations (r necrotic  = 0.92, r peri-necrotic  = 0.82 and r viable  = 0.98) with the histology., Conclusions: The precise identification of necrotic, peri-necrotic and viable areas using SRSC may greatly assist in cancer treatment planning and add a new dimension to MRI-guided tumor biopsy procedures.

Hypoxia is essential for the development of autoimmune diseases such as rheumatoid arthritis (RA) and is associated with the expression of reactive oxygen species (ROS), because of the enhanced infiltration of immune cells. The aim of this study was to demonstrate the feasibility of measuring hypoxia noninvasively in vivo in arthritic ankles with PET/MRI using the hypoxia tracers 18 F-fluoromisonidazole ( 18 F-FMISO) and 18 F-fluoroazomycinarabinoside ( 18 F-FAZA). Additionally, we quantified the temporal dynamics of hypoxia and ROS stress using L-012, an ROS-sensitive chemiluminescence optical imaging probe, and analyzed the expression of hypoxia-inducible factors (HIFs). Methods: Mice underwent noninvasive in vivo PET/MRI to measure hypoxia or optical imaging to analyze ROS expression. Additionally, we performed ex vivo pimonidazole-/HIF-1α immunohistochemistry and HIF-1α/2α Western blot/messenger RNA analysis of inflamed and healthy ankles to confirm our in vivo results. Results: Mice diseased from experimental RA exhibited a 3-fold enhancement in hypoxia tracer uptake, even in the early disease stages, and a 45-fold elevation in ROS expression in inflamed ankles compared with the ankles of healthy controls. We further found strong correlations of our noninvasive in vivo hypoxia PET data with pimonidazole and expression of HIF-1α in arthritic ankles. The strongest hypoxia tracer uptake was observed as soon as day 3, whereas the most pronounced ROS stress was evident on day 6 after the onset of experimental RA, indicating that tissue hypoxia can precede ROS stress in RA. Conclusion: Collectively, for the first time to our knowledge, we have demonstrated that the noninvasive measurement of hypoxia in inflammation using 18 F-FAZA and 18 F-FMISO PET imaging represents a promising new tool for uncovering and monitoring rheumatic inflammation in vivo. Further, because hypoxic inflamed tissues are associated with the overexpression of HIFs, specific inhibition of HIFs might represent a new powerful treatment strategy., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

This protocol illustrates the production of 64 Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64 Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described. In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64 Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4 + interferon (IFN)-γ-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64 Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-γ enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented. The 64 Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g. 64 Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) ( 64 Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

In this study, we described and validated an unsupervised segmentation algorithm for the assessment of tumor heterogeneity using dynamic 18 F-FDG PET. The aim of our study was to objectively evaluate the proposed method and make comparisons with compartmental modeling parametric maps and SUV segmentations using simulations of clinically relevant tumor tissue types. Methods: An irreversible 2-tissue-compartmental model was implemented to simulate clinical and preclinical 18 F-FDG PET time-activity curves using population-based arterial input functions (80 clinical and 12 preclinical) and the kinetic parameter values of 3 tumor tissue types. The simulated time-activity curves were corrupted with different levels of noise and used to calculate the tissue-type misclassification errors of spectral clustering (SC), parametric maps, and SUV segmentation. The utility of the inverse noise variance- and Laplacian score-derived frame weighting schemes before SC was also investigated. Finally, the SC scheme with the best results was tested on a dynamic 18 F-FDG measurement of a mouse bearing subcutaneous colon cancer and validated using histology. Results: In the preclinical setup, the inverse noise variance-weighted SC exhibited the lowest misclassification errors (8.09%-28.53%) at all noise levels in contrast to the Laplacian score-weighted SC (16.12%-31.23%), unweighted SC (25.73%-40.03%), parametric maps (28.02%-61.45%), and SUV (45.49%-45.63%) segmentation. The classification efficacy of both weighted SC schemes in the clinical case was comparable to the unweighted SC. When applied to the dynamic 18 F-FDG measurement of colon cancer, the proposed algorithm accurately identified densely vascularized regions from the rest of the tumor. In addition, the segmented regions and clusterwise average time-activity curves showed excellent correlation with the tumor histology. Conclusion: The promising results of SC mark its position as a robust tool for quantification of tumor heterogeneity using dynamic PET studies. Because SC tumor segmentation is based on the intrinsic structure of the underlying data, it can be easily applied to other cancer types as well., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Introduction: Radiolabeled 2-nitroimidazoles (azomycins) are a prominent class of biomarkers for PET imaging of hypoxia. [ 18 F]Fluoro-azomycin-α-arabinoside ([ 18 F]FAZA) - already in clinical use - may be seen as α-configuration nucleoside, but enters cells only via diffusion and is not transported by cellular nucleoside transporters. To enhance image contrast in comparison to [ 18 F]FAZA our objective was to 18 F-radiolabel an azomycin-2´-deoxyriboside with β-configuration ([ 18 F]FAZDR, [ 18 F]-β-8) to mimic nucleosides more closely and comparatively evaluate it versus [ 18 F]FAZA., Methods: Precursor and cold standards for [ 18 F]FAZDR were synthesized from methyl 2-deoxy-d-ribofuranosides α- and β-1 in 6 steps yielding precursors α- and β-5. β-5 was radiolabeled in a GE TRACERlab FX F-N synthesizer in DMSO and deprotected with NH 4 OH to give [ 18 F]FAZDR ([ 18 F]-β-8). [ 18 F]FAZA or [ 18 F]FAZDR was injected in BALB/c mice bearing CT26 colon carcinoma xenografts, PET scans (10min) were performed after 1, 2 and 3h post injection (p.i.). On a subset of mice injected with [ 18 F]FAZDR, we analyzed biodistribution., Results: [ 18 F]FAZDR was obtained in non-corrected yields of 10.9±2.4% (9.1±2.2GBq, n=4) 60min EOB, with radiochemical purity >98% and specific activity >50GBq/μmol. Small animal PET imaging showed a decrease in uptake over time for both [ 18 F]FAZDR (1h p.i.: 0.56±0.22% ID/cc, 3h: 0.17±0.08% ID/cc, n=9) and [ 18 F]FAZA (1h: 1.95±0.59% ID/cc, 3h: 0.87±0.55% ID/cc), whereas T/M ratios were significantly higher for [ 18 F]FAZDR at 1h (2.76) compared to [ 18 F]FAZA (1.69, P<0.001), 3h p.i. ratios showed no significant difference. Moreover, [ 18 F]FAZDR showed an inverse correlation between tracer uptake in carcinomas and oxygen breathing, while muscle tissue uptake was not affected by switching from air to oxygen., Conclusions: First PET imaging results with [ 18 F]FAZDR showed advantages over [ 18 F]FAZA regarding higher tumor contrast at earlier time points p.i. Availability of precursor and cold fluoro standard together with high output radiosynthesis will allow for a more detailed quantitative evaluation of [ 18 F]FAZDR, especially with regard to mechanistic studies whether active transport processes are involved, compared to passive diffusion as observed for [ 18 F]FAZA., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Great advances have recently been made in treating patients with metastatic melanoma. However, existing therapies are less effective on cerebral than extracerebral metastases. This highlights the potential role of the brain environment on tumor progression and drug resistance and underlines the need for "brain-specific" therapies. We previously showed that the PI3K-AKT survival pathway is hyperactivated in brain but not extracerebral melanoma metastases and that astrocyte-conditioned medium activates AKT in melanoma cells in vitro We therefore tested the PI3K inhibitor buparlisib as an antitumor agent for melanoma brain metastases., Experimental Design and Results: Buparlisib inhibited AKT activity, decreased proliferation, and induced apoptosis in metastatic melanoma cell lines and short-term brain melanoma cells, irrespective of their BRAF and NRAS mutation status. In addition, buparlisib inhibited hyperactivated AKT and induced apoptosis in melanoma cells that were stimulated with astrocyte-conditioned medium. The growth of tumors induced by injecting human BRAF- and NRAS-mutant metastatic melanoma cells into the brain of mice was significantly inhibited by buparlisib., Conclusions: These results emphasize the value of targeting the PI3K pathway as a strategy to develop drugs for melanoma brain metastases. Clin Cancer Res; 22(23); 5818-28. ©2016 AACR., (©2016 American Association for Cancer Research.)

Antibody-based therapies gain momentum in clinical therapy, thus the need for accurate imaging modalities with respect to target identification and therapy monitoring are of increasing relevance. Cerenkov luminescence imaging (CLI) are a novel method detecting charged particles emitted during radioactive decay with optical imaging. Here, we compare Position Emission Tomography (PET) with CLI in a multimodal imaging study aiming at the fast and efficient screening of monoclonal antibodies (mAb) designated for targeting of the neuroblastoma-characteristic epitope disialoganglioside GD2. Neuroblastoma-bearing SHO mice were injected with a 64Cu-labeled GD2-specific mAb. The tumor uptake was imaged 3 h, 24 h and 48 h after tracer injection with both, PET and CLI, and was compared to the accumulation in GD2-negative control tumors (human embryonic kidney, HEK-293). In addition to an in vivo PET/CLI-correlation over time, we also demonstrate linear correlations of CLI- and γ-counter-based biodistribution analysis. CLI with its comparably short acquisition time can thus be used as an attractive one-stop-shop modality for the longitudinal monitoring of antibody-based tumor targeting and ex vivo biodistribution.These findings suggest CLI as a reliable alternative for PET and biodistribution studies with respect to fast and high-throughput screenings in subcutaneous tumors traced with radiolabeled antibodies. However, in contrast to PET, CLI is not limited to positron-emitting isotopes and can therefore also be used for the visualization of mAb labeled with therapeutic isotopes like electron emitters.

Most frequently, gram-negative bacterial infections in humans are caused by Enterobacteriaceae and remain a major challenge in medical diagnostics. We non-invasively imaged moderate and severe systemic Yersinia enterocolitica infections in mice using the positron emission tomography (PET) tracer 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), which is a marker of proliferation, and compared the in vivo results to the ex vivo biodistributions, bacterial loads, and histologies of the corresponding organs. Y. enterocolitica infection is detectable with histology using H&E staining and immunohistochemistry for Ki 67. [18F]FLT revealed only background uptake in the spleen, which is the main manifestation site of systemic Y. enterocolitica-infected mice. The uptake was independent of the infection dose. Antibody-based thymidine kinase 1 (Tk-1) staining confirmed the negative [18F]FLT-PET data. Histological alterations of spleen tissue, observed via Ki 67-antibody-based staining, can not be detected by [18F]FLT-PET in this model. Thus, the proliferation marker [18F]FLT is not a suitable tracer for the diagnosis of systemic Y. enterocolitica infection in the C57BL/6 animal model of yersiniosis., Competing Interests: BJP received grant/research support from AstraZeneca, Bayer Healthcare, Boehringer-Ingelheim, Bruker, Oncodesign, Merck and Siemens. This did not alter our adherence to PLOS ONE policies on sharing data and materials.

Differential diagnosis and therapy of heterogeneous breast tumors poses a major clinical challenge. To address the need for a comprehensive, noninvasive strategy to define the molecular and functional profiles of tumors in vivo, we investigated a novel combination of metabolic PET and diffusion-weighted (DW)-MRI in the polyoma virus middle T antigen transgenic mouse model of breast cancer. The implementation of a voxelwise analysis for the clustering of intra- and intertumoral heterogeneity in this model resulted in a multiparametric profile based on [(18)F]Fluorodeoxyglucose ([(18)F]FDG)-PET and DW-MRI, which identified three distinct tumor phenotypes in vivo, including solid acinar, and solid nodular malignancies as well as cystic hyperplasia. To evaluate the feasibility of this approach for clinical use, we examined estrogen receptor-positive and progesterone receptor-positive breast tumors from five patient cases using DW-MRI and [(18)F]FDG-PET in a simultaneous PET/MRI system. The postsurgical in vivo PET/MRI data were correlated to whole-slide histology using the latter traditional diagnostic standard to define phenotype. By this approach, we showed how molecular, structural (microscopic, anatomic), and functional information could be simultaneously obtained noninvasively to identify precancerous and malignant subtypes within heterogeneous tumors. Combined with an automatized analysis, our results suggest that multiparametric molecular and functional imaging may be capable of providing comprehensive tumor profiling for noninvasive cancer diagnostics. Cancer Res; 76(18); 5512-22. ©2016 AACR., (©2016 American Association for Cancer Research.)

Purpose: NHS-IL12 is an immunocytokine targeting necrotic tumour areas. IL12 shows anti-tumour activity. As local irradiation might induce additional necrosis in solid tumours, we aimed to evaluate the increase in intratumoural accumulation of NHS-IL12 after irradiation and correlate the findings with diffusion-weighted MRI studies in two xenograft models., Methods: Human rhabdomyosarcoma (A204) and prostate cancer (PC3) cells were studied in vitro and as subcutaneous xenografts. Radiation sensitivity of the cell lines was assessed in vitro by colony formation assays. In vivo tumour necrosis was assessed based on apparent diffusion coefficients (ADC). Biodistribution of NHS-IL12 was evaluated with and without tumour irradiation using in vivo small-animal PET and ex vivo biodistribution., Results: A204 and PC3 differed in their intrinsic radiation sensitivity. Accordingly, radiation-induced tumour necrosis was found only in A204 xenografts. In comparison with control, ADC was significantly increased after irradiation of A204 tumours with 1 × 8.0 Gy and 5 × 2.0 Gy, whereas no change in ADC was observed in PC3 xenografts in all irradiation regimes. ADC correlated with histology. An enhanced uptake of radiolabelled NHS-IL12 in A204 tumours was detected by PET and ex vivo biodistribution after tumour irradiation. In PC3 tumours, no increase in NHS-IL12 uptake was observed., Conclusions: In dependence of the tumour model, tumour irradiation enhanced tumour necrosis measured in MRI and histology. In vivo PET and ex vivo biodistribution showed enhanced binding of NHS-IL12 in rhabdomyosarcoma xenografts. Thus, enhanced binding of necrosis-targeting immunocytokines might be a novel mechanism of additive effects in combination with irradiation.

Non-invasive in vivo small animal computed tomography (CT) imaging provides high resolution bone scans but cannot differentiate between soft tissues. For most applications injections of contrast agents (CAs) are necessary. Aim of this study was to uncover the advantages and disadvantages of commercially available CT CAs (ExiTron nano 12 000 and 6000, eXIA 160 and 160XL, Fenestra VC and LC) regarding their pharmacokinetics, toxicological side-effects and the influence of anesthesia on the biodistribution, based on an injection volume of 100 μL/25 g body weight. The pharmacokinetics of the CAs were determined for up to five days. The CA-induced toxicological/physiological side-effects were evaluated by determining blood counts, liver enzymes, thyroxine and total protein values, pro-inflammatory mediators (messenger ribonucleic acid (mRNA)), histology and immunohistochemistry. ExiTron nano 12 000 and 6000 yielded a long-term contrast enhancement (CE) in the liver and spleen for up to five days. Some of the evaluated CAs did not show any CE at all. Anesthesia did not impair the CAs' biodistribution. The CAs differentially affected the body weight, blood counts, liver enzymes, thyroxine and total protein values. ExiTron nano 12 000 and 6000 induced histiocytes in the liver and spleen. Moreover, ExiTron nano 12 000 and eXIA 160 enhanced tumor necrosis factor (TNF) mRNA expression levels in the kidneys. Thus, we recommend ExiTron nano 12 000 and 6000 when multiple injections should be avoided. We recommend careful selection of the employed CA in order to achieve an acceptable CE in the organs of interest and to avoid influences on the animal physiology. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Metabolic syndrome is characterized by insulin resistance, obesity, and dyslipidemia. It is the consequence of an imbalance between caloric intake and energy consumption. Adiponectin protects against metabolic syndrome. Insulin-induced signaling includes activation of PI3 kinase and protein kinase B (PKB)/Akt. PKB/Akt in turn inactivates glycogen synthase kinase (GSK) 3, a major regulator of metabolism. Here, we studied the significance of PI3K-dependent GSK3 inactivation for adiponectin formation in diet-induced metabolic syndrome. Mice expressing PI3K-insensitive GSK3 (gsk3(KI)) and wild-type mice (gsk3(WT)) were fed a high-fat diet. Compared with gsk3(WT) mice, gsk3(KI) mice were protected against the development of metabolic syndrome as evident from a markedly lower weight gain, lower total body and liver fat accumulation, better glucose tolerance, stronger hepatic insulin-dependent PKB/Akt phosphorylation, lower serum insulin, cholesterol, and triglyceride levels, as well as higher energy expenditure. Serum adiponectin concentration and the activity of transcription factor C/EBPα controlling the expression of adiponectin in adipose tissue was significantly higher in gsk3(KI) mice than in gsk3(WT) mice. Treatment with GSK3 inhibitor lithium significantly decreased the serum adiponectin concentration of gsk3(KI) mice and abrogated the difference in C/EBPα activity between the genotypes. Taken together, our data demonstrate that the expression of PI3K-insensitive GSK3 stimulates the production of adiponectin and protects from diet-induced metabolic syndrome.

Unlabelled: The natural phytoestrogen genistein is known as protein kinase inhibitor and tumor suppressor in various types of cancers. We studied its antitumor effect in two different xenograft models using positron emission tomography (PET) in vivo combined with ex vivo histology and nuclear magnetic resonance (NMR) metabolic fingerprinting., Procedures: A431 and Colo205 tumor-bearing mice were treated with vehicle or genistein (500 mg/kg/d) over a period of 12 days. Imaging was performed with 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and 3'-deoxy-3'-[18F]fluorothymidine ([18F] FLT). In a second study A431 tumor-bearing mice were treated with vehicle, genistein (500 mg/kg/d), cetuximab (1 mg/3d) or a combination of the compounds and imaged using [18F]FDG, [18F]FLT and [64Cu]NODAGA-cetuximab. Data were compared to histology and principal components analysis (PCA) of NMR fingerprinting data., Results: Genistein reduced tumor growth significantly in both xenografts. [18F] FLT uptake was consistent in both models and corresponded to histological findings and also PCA whereas [18F]FDG and [64Cu]NODAGA-cetuximab were not suitable for therapy monitoring., Conclusions: As mono-therapy the natural isoflavone genistein has a powerful therapeutic effect in vivo on A431 and Colo205 tumors. [18F]FLT has superior consistency compared to the other tested tracers in therapy monitoring, while the treatment effect could be shown on the molecular level by histology and metabolic fingerprinting., Competing Interests: Bernd Pichler: grant/research support: Siemens, AstraZeneca, Bayer Healthcare, Boehringer-Ingelheim, Oncodesign, Merck, Bruker. Other authors: no conflicts of interest.

Purpose: Positron emission tomography (PET) and diffusion-weighted MRI (DW-MRI) were used to characterize the treatment effects of the MEK1/2 inhibitor selumetinib (AZD6244), docetaxel, and their combination in HCT116 tumor-bearing mice on the molecular level., Procedures: Mice were treated with vehicle, selumetinib (25 mg/kg), docetaxel (15 mg/kg), or a combination of both drugs for 7 days and imaged at four time points with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) or 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) followed by DW-MRI to calculate the apparent diffusion coefficient (ADC). Data was cross-validated using the Pearson correlation coefficient (PCC) and compared to histology (IHC)., Results: Each drug led to tumor growth inhibition but their combination resulted in regression. Separate analysis of PET or ADC could not provide significant differences between groups. Only PCC combined with IHC analysis revealed the highest therapeutic impact for combination therapy., Conclusion: Combination treatment of selumetinib/docetaxel was superior to the respective mono-therapies shown by PCC of PET and ADC in conjunction with histology.

The specific and rapid detection of Enterobacteriaceae, the most frequent cause of gram-negative bacterial infections in humans, remains a major challenge. We developed a non-invasive method to rapidly detect systemic Yersinia enterocolitica infections using immunoPET (antibody-targeted positron emission tomography) with [64Cu]NODAGA-labeled Yersinia-specific polyclonal antibodies targeting the outer membrane protein YadA. In contrast to the tracer [18F]FDG, [64Cu]NODAGA-YadA uptake co-localized in a dose dependent manner with bacterial lesions of Yersinia-infected mice, as detected by magnetic resonance (MR) imaging. This was accompanied by elevated uptake of [64Cu]NODAGA-YadA in infected tissues, in ex vivo biodistribution studies, whereas reduced uptake was observed following blocking with unlabeled anti-YadA antibody. We show, for the first time, a bacteria-specific, antibody-based, in vivo imaging method for the diagnosis of a Gram-negative enterobacterial infection as a proof of concept, which may provide new insights into pathogen-host interactions.

Unlabelled: The aim of our study was to create a novel Gaussian mixture modeling (GMM) pipeline to model the complementary information derived from(18)F-FDG PET and diffusion-weighted MRI (DW-MRI) to separate the tumor microenvironment into relevant tissue compartments and follow the development of these compartments longitudinally., Methods: Serial (18)F-FDG PET and apparent diffusion coefficient (ADC) maps derived from DW-MR images of NCI-H460 xenograft tumors were coregistered, and a population-based GMM was implemented on the complementary imaging data. The tumor microenvironment was segmented into 3 distinct regions and correlated with histology. ANCOVA was applied to gauge how well the total tumor volume was a predictor for the ADC and (18)F-FDG, or if ADC was a good predictor of (18)F-FDG for average values in the whole tumor or average necrotic and viable tissues., Results: The coregistered PET/MR images were in excellent agreement with histology, both visually and quantitatively, and allowed for validation of the last-time-point measurements. Strong correlations were found for the necrotic (r = 0.88) and viable fractions (r = 0.87) between histology and clustering. The GMM provided probabilities for each compartment with uncertainties expressed as a mixture of tissues in which the resolution of scans was inadequate to accurately separate tissues. The ANCOVA suggested that both ADC and (18)F-FDG in the whole tumor (P = 0.0009, P = 0.02) as well as necrotic (P = 0.008, P = 0.02) and viable (P = 0.003, P = 0.01) tissues were a positive, linear function of total tumor volume. ADC proved to be a positive predictor of (18)F-FDG in the whole tumor (P = 0.001) and necrotic (P = 0.02) and viable (P = 0.0001) tissues., Conclusion: The complementary information of (18)F-FDG and ADC longitudinal measurements in xenograft tumors allows for segmentation into distinct tissues when using the novel GMM pipeline. Leveraging the power of multiparametric PET/MRI in this manner has the potential to take the assessment of disease outcome beyond RECIST and could provide an important impact to the field of precision medicine., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [(64)Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [(64)Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [(18)F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.

Unlabelled: Serotonin, or 5-hydroxytryptamine (5-HT), plays a key role in the central nervous system and is involved in many essential neurologic processes such as mood, social behavior, and sleep. The serotonin transporter ligand (11)C-3-amino-4(2-dimethylaminomethyl-phenylsufanyl)-benzonitrile ((11)C-DASB) has been used to determine nondisplaceable binding potential (BPND), which is defined as the quotient of the available receptor density (Bavail) and the apparent equilibrium dissociation rate constant (1/appKD) under in vivo conditions. Because of the increasing number of animal models of human diseases, there is a pressing need to evaluate the applicability of (11)C-DASB to rats and mice. Here, we assessed the feasibility of using (11)C-DASB for quantification of serotonin transporter (SERT) density and affinity in vivo in rats and mice., Methods: Rats and mice underwent 4 PET scans with increasing doses of the unlabeled ligand to calculate Bavail and appKD using the multiple-ligand concentration transporter assay. An additional PET scan was performed to calculate test-retest reproducibility and reliability. BPND was calculated using the simplified reference tissue model, and the results for different reference regions were compared., Results: Displaceable binding of (11)C-DASB was found in all brain regions of both rats and mice, with the highest binding being in the thalamus and the lowest in the cerebellum. In rats, displaceable binding was largely reduced in the cerebellar cortex, which in mice was spatially indistinguishable from cerebellar white matter. Use of the cerebellum with fully saturated binding sites as the reference region did not lead to reliable results. Test-retest reproducibility in the thalamus was more than 90% in both mice and rats. In rats, Bavail, appKD, and ED50 were 3.9 ± 0.4 pmol/mL, 2.2 ± 0.4 nM, and 12.0 ± 2.6 nmol/kg, respectively, whereas analysis of the mouse measurements resulted in inaccurate fits due to the high injected tracer mass., Conclusion: Our data showed that in rats, (11)C-DASB can be used to quantify SERT density with good reproducibility. BPND agreed with the distribution of SERT in the rat brain. It remains difficult to estimate quantitative parameters accurately from mouse measurements because of the high injected tracer mass and underestimation of the binding parameters due to high displaceable binding in the reference region., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Unlabelled: Klotho, a protein expressed mainly in the kidney, is required for the inhibitory effect of FGF23 on renal 1,25(OH)2D3 formation. Klotho counteracts vascular calcification and diverse age-related disorders. Klotho-hypomorphic mice (kl/kl) suffer from severe vascular calcification and rapid aging. The calcification is at least in part caused by excessive 1,25(OH)2D3, Ca(2+), and phosphate concentrations in blood, which trigger osteogenic signaling including upregulation of alkaline phosphatase (Alpl). As precipitation of calcium and phosphate is fostered by alkaline pH, extracellular acidosis could counteract tissue calcification. In order to induce acidosis, acetazolamide was added to drinking water (0.8 g/l) of kl/kl and wild-type mice. As a result, acetazolamide treatment of kl/kl mice partially reversed the growth deficit, tripled the life span, almost completely reversed the calcifications in trachea, lung, kidney, stomach, intestine, and vascular tissues, the excessive aortic alkaline phosphatase mRNA levels and the plasma concentrations of osteoprotegerin, osteopontin as well as fetuin-A, without significantly decreasing FGF23, 1,25(OH)2D3, Ca(2+), and phosphate plasma concentrations. In primary human aortic smooth muscle cells, acidotic environment prevented phosphate-induced alkaline phosphatase mRNA expression. The present study reveals a completely novel effect of acetazolamide, i.e., interference with osteoinductive signaling and tissue calcification in kl/kl mice., Key Messages: Klotho deficient (kl/kl) mice suffer from hyperphosphatemia with dramatic tissue calcification. Acetazolamide (ACM) treatment partially reversed the growth deficit of kl/kl mice. In kl/kl mice, ACM reversed tissue calcification despite continued hyperphosphatemia. ACM tripled the life span of kl/kl mice. In human aortic smooth muscle cells, low extracellular pH prevented osteogenic signaling.

Huntington's disease is a fatal human neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene, which translates into a mutant huntingtin protein. A key event in the molecular pathogenesis of Huntington's disease is the proteolytic cleavage of mutant huntingtin, leading to the accumulation of toxic protein fragments. Mutant huntingtin cleavage has been linked to the overactivation of proteases due to mitochondrial dysfunction and calcium derangements. Here, we investigated the therapeutic potential of olesoxime, a mitochondria-targeting, neuroprotective compound, in the BACHD rat model of Huntington's disease. BACHD rats were treated with olesoxime via the food for 12 months. In vivo analysis covered motor impairments, cognitive deficits, mood disturbances and brain atrophy. Ex vivo analyses addressed olesoxime's effect on mutant huntingtin aggregation and cleavage, as well as brain mitochondria function. Olesoxime improved cognitive and psychiatric phenotypes, and ameliorated cortical thinning in the BACHD rat. The treatment reduced cerebral mutant huntingtin aggregates and nuclear accumulation. Further analysis revealed a cortex-specific overactivation of calpain in untreated BACHD rats. Treated BACHD rats instead showed significantly reduced levels of mutant huntingtin fragments due to the suppression of calpain-mediated cleavage. In addition, olesoxime reduced the amount of mutant huntingtin fragments associated with mitochondria, restored a respiration deficit, and enhanced the expression of fusion and outer-membrane transport proteins. In conclusion, we discovered the calpain proteolytic system, a key player in Huntington's disease and other neurodegenerative disorders, as a target of olesoxime. Our findings suggest that olesoxime exerts its beneficial effects by improving mitochondrial function, which results in reduced calpain activation. The observed alleviation of behavioural and neuropathological phenotypes encourages further investigations on the use of olesoxime as a therapeutic for Huntington's disease., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This paper summarises key themes and discussions from the 4th international workshop dedicated to the advancement of the technical, scientific and clinical applications of combined positron emission tomography (PET)/magnetic resonance imaging (MRI) systems that was held in Tübingen, Germany, from February 23 to 27, 2015. Specifically, we summarise the three days of invited presentations from active researchers in this and associated fields augmented by round table discussions and dialogue boards with specific topics. These include the use of PET/MRI in cardiovascular disease, paediatrics, oncology, neurology and multi-parametric imaging, the latter of which was suggested as a key promoting factor for the wider adoption of integrated PET/MRI. Discussions throughout the workshop and a poll taken on the final day demonstrated that attendees felt more strongly that PET/MRI has further advanced in both technical versatility and acceptance by clinical and research-driven users from the status quo of last year. Still, with only minimal evidence of progress made in exploiting the true complementary nature of the PET and MRI-based information, PET/MRI is still yet to achieve its potential. In that regard, the conclusion of last year's meeting "the real work has just started" still holds true.

Unlabelled: We present a combined PET/7 T MR imaging and 16.4 T microscopic MR imaging dual-modality imaging approach enabling quantification of the amyloid load at high sensitivity and high resolution, and of regional cerebral blood flow (rCBF) in the brain of transgenic APP23 mice. Moreover, we demonstrate a novel, voxel-based correlative data analysis method for in-depth evaluation of amyloid PET and rCBF data., Methods: We injected 11C-Pittsburgh compound B (PIB) intravenously in transgenic and control APP23 mice and performed dynamic PET measurements. rCBF data were recorded with a flow-sensitive alternating inversion recovery approach at 7 T. Subsequently, the animals were sacrificed and their brains harvested for ex vivo microscopic MR imaging at 16.4 T with a T2*-weighted gradient-echo sequence at 30-μm spatial resolution. Additionally, correlative amyloid histology was performed. The 11C-PIB PET data were quantified to nondisplaceable binding potentials (BPND) using the Logan graphical analysis; flow-sensitive alternating inversion recovery data were quantified with a simplified version of the Bloch equation., Results: Amyloid load assessed by both 11C-PIB PET and amyloid histology was highest in the frontal cortex of transgenic mice (11C-PIB BPND: 0.93±0.08; amyloid histology: 15.1%±1.5%), followed by the temporoparietal cortex (11C-PIB BPND: 0.75±0.08; amyloid histology: 13.9%±0.7%) and the hippocampus (11C-PIB BPND: 0.71±0.09; amyloid histology: 9.2%±0.9%), and was lowest in the thalamus (11C-PIB BPND: 0.40±0.07; amyloid histology: 6.6%±0.6%). However, 11C-PIB BPND and amyloid histology linearly correlated (R2=0.82, P<0.05) and were significantly higher in transgenic animals (P<0.01). Similarly, microscopic MR imaging allowed quantifying the amyloid load, in addition to the detection of substructures within single amyloid plaques correlating with amyloid deposition density and the measurement of hippocampal atrophy. Finally, we found an inverse relationship between 11C-PIB BPND and rCBF MR imaging in the voxel-based analysis that was absent in control mice (slopetg: -0.11±0.03; slopeco: 0.004±0.005; P=0.014)., Conclusion: Our dual-modality imaging approach using 11C-PIB PET/7 T MR imaging and 16.4 T microscopic MR imaging allowed amyloid-load quantification with high sensitivity and high resolution, the identification of substructures within single amyloid plaques, and the quantification of rCBF., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Checkpoint kinase 2 (Chk2) is the main effector kinase of ataxia telangiectasia mutated (ATM) and responsible for cell cycle regulation. ATM signaling has been shown to upregulate interferon-regulating factor-1 (IRF-1), a transcription factor also expressed in the kidney. Calcitriol (1,25 (OH)2D3), a major regulator of mineral metabolism, is generated by 25-hydroxyvitamin D 1α-hydroxylase in the kidney. Since 25-hydroxyvitamin D 1α-hydroxylase expression is enhanced by IRF-1, the present study explored the role of Chk2 for calcitriol formation and mineral metabolism. Chk2-deficient mice (chk2 (-/-)) were compared to wild-type mice (chk2 (+/+)). Transcript levels of renal 25-hydroxyvitamin D 1α-hydroxylase, Chk2, and IRF-1 were determined by RT-PCR; Klotho expression by Western blotting; bone density by μCT analysis; serum or plasma 1,25 (OH)2D3, PTH, and C-terminal FGF23 concentrations by immunoassays; and serum, fecal, and urinary calcium and phosphate concentrations by photometry. The renal expression of IRF-1 and 25-hydroxyvitamin D 1α-hydroxylase as well as serum 1,25 (OH)2D3 and FGF23 levels were significantly lower in chk2 (-/-) mice compared to chk2 (+/+) mice. Plasma PTH was not different between the genotypes. Renal calcium and phosphate excretion were significantly higher in chk2 (-/-) mice than in chk2 (+/+) mice despite hypophosphatemia and normocalcemia. Bone density was not different between the genotypes. We conclude that Chk2 regulates renal 25-hydroxyvitamin D 1α-hydroxylase expression thereby impacting on calcium and phosphate metabolism.

Intracerebral hemorrhagic stroke and vascular dementia are age- and hypertension-associated manifestations of human cerebral small vessel disease (SVD). Cerebral microvessels are formed by endothelial cells (ECs), which are connected through tight junctions, adherens junctions, and stabilizing basement membrane structures. These endothelial connections ensure both vessel stability and blood-brain barrier (BBB) functions, the latter enabling selective exchange of ions, bioactive molecules, and cells between the bloodstream and brain tissue. Srf(iECKO) mice, permitting conditional EC-specific depletion of the transcription factor Serum Response Factor (SRF), suffer from loss of BBB integrity and intracerebral hemorrhaging. Cerebral microbleeds and larger hemorrhages developed upon postnatal and adult depletion of either SRF or its cofactors Myocardin Related Transcription Factor (MRTF-A/-B), revealing essential requirements of ongoing SRF/MRTF activity for maintenance of cerebral small vessel integrity. In vivo magnetic resonance imaging allowed detection, localization, and time-resolved quantification of BBB permeability and hemorrhage formation in Srf(iECKO) brains. At the molecular level, direct and indirect SRF/MRTF target genes, encoding structural components of tight junctions (Claudins and ZO proteins), adherens junctions (VE-cadherin, α-Actinin), and the basement membrane (Collagen IV), were down-regulated upon SRF depletion. These results identify SRF and its MRTF cofactors as major transcriptional regulators of EC junctional stability, guaranteeing physiological functions of the cerebral microvasculature. We hypothesize that impairments in SRF/MRTF activity contribute to human SVD pathology.

Purpose: 2-Deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) has been used as a standard clinical positron emission tomography (PET) tracer for the follow-up of the rare but life-threatening parasitic disease alveolar echinococcosis (AE). Given that the disease is endemic in many countries in the northern hemisphere and the diagnosis is still challenging, the aim of our study was to evaluate further clinically relevant PET tracers as possible diagnostic tools for AE in vitro and in vivo., Procedures: Various clinically used PET tracers were evaluated in vitro and assessed in an in vivo AE animal model based on PET/magnetic resonance (MR) measurements., Results: In vitro binding assays displayed high uptake of [(18)F]FDG in a cell suspension of E. multilocularis tissue, whereas 3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) and [(11)C]choline were found to be taken up strongly by E. multilocularis vesicles. [(18)F]FDG and [(18)F]FLT displayed an elevated uptake in vivo, which appeared as several foci throughout the parasite tissue as opposed to [(18)F]fluoro-azomycinarabinofuranoside ([(18)F]FAZA) and [(11)C]choline., Conclusions: Our data clearly demonstrate that the clinically applied PET tracer [(18)F]FDG is useful for the diagnosis and disease staging of AE but also has drawbacks in the assessment of currently inactive or metabolically weak parasitic lesions. The different tested PET tracers do not show the potential for the replacement or supplementation of current diagnostic strategies. Hence, there is still the need for novel diagnostic tools.

Unlabelled: Pediatric imaging is regarded as a key application for combined PET/MR imaging systems. Because existing MR-based attenuation-correction methods were not designed specifically for pediatric patients, we assessed the impact of 2 potentially influential factors: inter- and intrapatient variability of attenuation coefficients and anatomic variability. Furthermore, we evaluated the quantification accuracy of 3 methods for MR-based attenuation correction without (SEGbase) and with bone prediction using an adult and a pediatric atlas (SEGwBONEad and SEGwBONEpe, respectively) on PET data of pediatric patients., Methods: The variability of attenuation coefficients between and within pediatric (5-17 y, n = 17) and adult (27-66 y, n = 16) patient collectives was assessed on volumes of interest (VOIs) in CT datasets for different tissue types. Anatomic variability was assessed on SEGwBONEad/pe attenuation maps by computing mean differences to CT-based attenuation maps for regions of bone tissue, lungs, and soft tissue. PET quantification was evaluated on VOIs with physiologic uptake and on 80% isocontour VOIs with elevated uptake in the thorax and abdomen/pelvis. Inter- and intrapatient variability of the bias was assessed for each VOI group and method., Results: Statistically significant differences in mean VOI Hounsfield unit values and linear attenuation coefficients between adult and pediatric collectives were found in the lungs and femur. The prediction of attenuation maps using the pediatric atlas showed a reduced error in bone tissue and better delineation of bone structure. Evaluation of PET quantification accuracy showed statistically significant mean errors in mean standardized uptake values of -14% ± 5% and -23% ± 6% in bone marrow and femur-adjacent VOIs with physiologic uptake for SEGbase, which could be reduced to 0% ± 4% and -1% ± 5% using SEGwBONEpe attenuation maps. Bias in soft-tissue VOIs was less than 5% for all methods. Lung VOIs showed high SDs in the range of 15% for all methods. For VOIs with elevated uptake, mean and SD were less than 5% except in the thorax., Conclusion: The use of a dedicated atlas for the pediatric patient collective resulted in improved attenuation map prediction in osseous regions and reduced interpatient bias variation in femur-adjacent VOIs. For the lungs, in which intrapatient variation was higher for the pediatric collective, a patient- or group-specific attenuation coefficient might improve attenuation map accuracy. Mean errors of -14% and -23% in bone marrow and femur-adjacent VOIs can affect PET quantification in these regions when bone tissue is ignored., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Unlabelled: Simultaneous PET/MR imaging is an emerging hybrid modality for clinical and preclinical imaging. The static magnetic field of the MR imaging device affects the trajectory of the positrons emitted by the PET radioisotopes. This effect translates into an improvement of the spatial resolution in transaxial images. However, because of the elongation of the positron range distribution along the magnetic field, the axial resolution worsens and shine-through artifacts may appear. These artifacts can lead to misinterpretation and overstaging. The aim of this work was to study the relevance of this effect., Methods: Measurements were performed in a 3-tesla PET/MR scanner. A 1-cm(2) piece of paper, soaked with a radioisotope and placed in air, was scanned, and the magnitude of the shine-through was quantified from the PET images for various radioisotopes. Additionally, PET/MR and PET/CT images of the lungs and the larynx with trachea of a deceased swine were obtained after injecting a mixture of NiSO4 and (68)Ga to simulate hot tumor lesions., Results: For the radioactive paper, shine-through artifacts appeared in the location of the acrylic glass backplane, located 3 cm from the source in the axial direction. The ratio between the activity of the shine-through and the activity reconstructed in the original location ranged from 0.9 ((18)F) to 5.7 ((68)Ga). For the larynx-with-trachea images, the magnitude of the artifacts depended on the organ orientation with respect to the magnetic field. The shine-through activity could reach 46% of the reconstructed activity (larynx lesion). The lesion within the trachea produced 2 artifacts, symmetrically aligned with the magnetic field and characterized by artifact-to-lesion volume-of-interest ratios ranging from 21% to 30%., Conclusion: In simultaneous PET/MR imaging, the effect of the magnetic field on positrons may cause severe artifacts in the PET image when the lesions are close to air cavities and high-energy radioisotopes are used. For accurate staging and interpretation, this effect needs to be recognized and adequate compensation techniques should be developed., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Especially for neuroscience and the development of new biomarkers, a direct correlation between in vivo imaging and histology is essential. However, this comparison is hampered by deformation and shrinkage of tissue samples caused by fixation, dehydration and paraffin embedding. We used magnetic resonance (MR) imaging and computed tomography (CT) imaging to analyze the degree of shrinkage on murine brains for various fixatives. After in vivo imaging using 7 T MRI, animals were sacrificed and the brains were dissected and immediately placed in different fixatives, respectively: zinc-based fixative, neutral buffered formalin (NBF), paraformaldehyde (PFA), Bouin-Holland fixative and paraformaldehyde-lysine-periodate (PLP). The degree of shrinkage based on mouse brain volumes, radiodensity in Hounsfield units (HU), as well as non-linear deformations were obtained. The highest degree of shrinkage was observed for PLP (68.1%, P < 0.001), followed by PFA (60.2%, P<0.001) and NBF (58.6%, P<0.001). The zinc-based fixative revealed a low shrinkage with only 33.5% (P<0.001). Compared to NBF, the zinc-based fixative shows a slightly higher degree of deformations, but is still more homogenous than PFA. Tissue shrinkage can be monitored non-invasively with CT and MR. Zinc-based fixative causes the smallest degree of brain shrinkage and only small deformations and is therefore recommended for in vivo ex vivo comparison studies.

Methods: CT26 colon carcinoma-bearing mice were anesthetized with isoflurane (IF) or ketamine/xylazine (KX) while breathing air or oxygen (O2). We performed 10 min static PET scans 1 h, 2 h and 3 h after [18F]FAZA injection and calculated the [18F]FAZA-uptake and tumor-to-muscle ratios (T/M). In another experimental group, we placed a pO2 probe in the tumor as well as in the gastrocnemius muscle to measure the pO2 and perfusion., Results: Ketamine/xylazine-anesthetized mice yielded up to 3.5-fold higher T/M-ratios compared to their isoflurane-anesthetized littermates 1 h, 2 h and 3 h after [18F]FAZA injection regardless of whether the mice breathed air or oxygen (3 h, KX-air: 7.1 vs. IF-air: 1.8, p = 0.0001, KX-O2: 4.4 vs. IF-O2: 1.4, p < 0.0001). The enhanced T/M-ratios in ketamine/xylazine-anesthetized mice were mainly caused by an increased [18F]FAZA uptake in the carcinomas. Invasive pO2 probe measurements yielded enhanced intra-tumoral pO2 values in air- and oxygen-breathing ketamine/xylazine-anesthetized mice compared to isoflurane-anesthetized mice (KX-air: 1.01 mmHg, IF-air: 0.45 mmHg; KX-O2 9.73 mmHg, IF-O2: 6.25 mmHg). Muscle oxygenation was significantly higher in air-breathing isoflurane-anesthetized (56.9 mmHg) than in ketamine/xylazine-anesthetized mice (33.8 mmHg, p = 0.0003)., Conclusion: [18F]FAZA tumor uptake was highest in ketamine/xylazine-anesthetized mice regardless of whether the mice breathed air or oxygen. The generally lower [18F]FAZA whole-body uptake in isoflurane-anesthetized mice could be due to the higher muscle pO2-values in these mice compared to ketamine/xylazine-anesthetized mice. When performing preclinical in vivo hypoxia PET studies, oxygen should be avoided, and ketamine/xylazine-anesthesia might alleviate the identification of tumor hypoxia areals.

This study aims at evaluating the combination of the tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL)-receptor 2 (TRAIL-R2)-specific antibody Drozitumab and the Smac mimetic BV6 in preclinical glioblastoma models. To this end, the effect of BV6 and/or Drozitumab on apoptosis induction and signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures and glioblastoma stem-like cells. Here, we report that BV6 and Drozitumab synergistically induce apoptosis and reduce colony formation in several glioblastoma cell lines (combination index<0.1). Also, BV6 profoundly enhances Drozitumab-induced apoptosis in primary glioblastoma cultures and glioblastoma stem-like cells. Importantly, BV6 cooperates with Drozitumab to suppress tumor growth in two glioblastoma in vivo models including an orthotopic, intracranial mouse model, underlining the clinical relevance of these findings. Mechanistic studies reveal that BV6 and Drozitumab act in concert to trigger the formation of a cytosolic receptor-interacting protein (RIP) 1/Fas-associated via death domain (FADD)/caspase-8-containing complex and subsequent activation of caspase-8 and -3. BV6- and Drozitumab-induced apoptosis is blocked by the caspase inhibitor zVAD.fmk, pointing to caspase-dependent apoptosis. RNA interference-mediated silencing of RIP1 almost completely abolishes the BV6-conferred sensitization to Drozitumab-induced apoptosis, indicating that the synergism critically depends on RIP1 expression. In contrast, both necrostatin-1, a RIP1 kinase inhibitor, and Enbrel, a TNFα-blocking antibody, do not interfere with BV6/Drozitumab-induced apoptosis, demonstrating that apoptosis occurs independently of RIP1 kinase activity or an autocrine TNFα loop. In conclusion, the rational combination of BV6 and Drozitumab presents a promising approach to trigger apoptosis in glioblastoma, which warrants further investigation.