The Calcium Versus Hemorrhage Trial: Developing Diagnostic Criteria for Chronic Intracranial Susceptibility Lesions Using Single-Energy Computed Tomography, Dual-Energy Computed Tomography, and Quantitative Susceptibility Mapping.

PURPOSE: Chronic susceptibility lesions in the brain can be either hemorrhagic (potentially dangerous) or calcific (usually not dangerous) but are difficult to discriminate on routine imaging. We proposed to develop quantitative diagnostic criteria for single-energy computed tomography (SECT), dual-energy computed tomography (DECT), and quantitative susceptibility mapping (QSM) to distinguish hemorrhage from calcium. MATERIALS AND METHODS: Patients with positive susceptibility lesions on routine T2*-weighted magnetic resonance of the brain were recruited into this prospective imaging clinical trial, under institutional review board approval and with informed consent. The SECT, DECT, and QSM images were obtained, the lesions were identified, and the regions of interest were defined, with the mean values recorded. Criteria for quantitative interpretation were developed on the first 50 patients, and then applied to the next 45 patients. Contingency tables, scatter plots, and McNemar test were applied to compare classifiers. RESULTS: There were 95 evaluable patients, divided into a training set of 50 patients (328 lesions) and a validation set of 45 patients (281 lesions). We found the following classifiers to best differentiate hemorrhagic from calcific lesions: less than 68 Hounsfield units for SECT, calcium level of less than 15 mg/mL (material decomposition value) for DECT, and greater than 38 ppb for QSM. There was general mutual agreement among the proposed criteria. The proposed criteria outperformed the current published criteria. CONCLUSIONS: We provide the updated criteria for the classification of chronic positive susceptibility brain lesions as hemorrhagic versus calcific for each major clinically available imaging modality. These proposed criteria have greater internal consistency than the current criteria and should likely replace it as gold standard.

Axonal Transport as an In Vivo Biomarker for Retinal Neuropathy.

We illuminate a possible explanatory pathophysiologic mechanism for retinal cellular neuropathy by means of a novel diagnostic method using ophthalmoscopic imaging and a molecular imaging agent targeted to fast axonal transport. The retinal neuropathies are a group of diseases with damage to retinal neural elements. Retinopathies lead to blindness but are typically diagnosed late, when substantial neuronal loss and vision loss have already occurred. We devised a fluorescent imaging agent based on the non-toxic C fragment of tetanus toxin (TTc), which is taken up and transported in neurons using the highly conserved fast axonal transport mechanism. TTc serves as an imaging biomarker for normal axonal transport and demonstrates impairment of axonal transport early in the course of an N-methyl-D-aspartic acid (NMDA)-induced excitotoxic retinopathy model in rats. Transport-related imaging findings were dramatically different between normal and retinopathic eyes prior to presumed neuronal cell death. This proof-of-concept study provides justification for future clinical translation.

Hyperpolarized Pyruvate MR Spectroscopy Depicts Glycolytic Inhibition in a Mouse Model of Glioma.

BackgroundA generation of therapies targeting tumor metabolism is becoming available for treating glioma. Hyperpolarized MRI is uniquely suited to directly measure the metabolic effects of these emerging treatments.PurposeTo explore the feasibility of the use of hyperpolarized [1-carbon 13 {13C}]-pyruvate for real-time measurement of metabolism and response to treatment with a glycolytic inhibitor in an orthotopic mouse model of glioma.Materials and MethodsIn this animal study, anatomic MRI and dynamic 13C MR spectroscopy were performed at 7 T during intravenous injection of hyperpolarized [1-13C]-pyruvate on mice with orthotopic U87MG glioma and healthy control mice. Anatomic MRI and dynamic 13C MR spectroscopy were repeated after administration of the glycolytic inhibitor WP1122, a prodrug of 2-deoxy-d-glucose. All experiments were conducted in athymic nude mice between October 2016 and March 2017. Hyperpolarized lactate production was quantified as an apparent reaction rate, or kPL, and normalized lactate ratio (nLac). The Wilcoxon signed-rank test was used to assess changes in paired measures of lactate production before and after treatment.ResultsThirteen 12-16-week-old female mice and five healthy female mice underwent anatomic MRI and hyperpolarized [1-13C]-pyruvate spectroscopy. Large contrast agent-enhanced tumors were shown in mice with glioma at T2-weighted and T1-weighted postcontrast MRI by postimplantation day 40. After treatment with WP1122, a decrease in lactate was observed in mice with glioma (baseline and treatment mean kPL, 0.027 and 0.018 sec-1, respectively, P = .01; baseline and posttreatment mean nLac, 0.28 and 0.22, respectively, P = .01) whereas no significant decrease was observed in healthy control mice (baseline and posttreatment mean kPL, 0.011 and 0.017 sec-1, respectively, P = .91; baseline and posttreatment mean nLac, 0.16 and 0.21, respectively, P = .84).ConclusionHyperpolarized carbon 13 measurements of pyruvate metabolism can provide rapid feedback for monitoring treatment response in glioma.© RSNA, 2019.

Differentiation of low-attenuation intracranial hemorrhage and calcification using dual-energy computed tomography in a phantom system.

OBJECTIVES: Calcific and hemorrhagic intracranial lesions with attenuation levels of less than 100 Hounsfield units (HUs) cannot currently be reliably differentiated by single-energy computed tomography (SECT). The proper differentiation of these lesion types would have a multitude of clinical applications. A phantom model was used to test the ability of dual-energy CT (DECT) to differentiate such lesions. MATERIALS AND METHODS: Agar gel-bound ferric oxide and hydroxyapatite were used to model hemorrhage and calcification, respectively. Gel models were scanned using SECT and DECT and organized into SECT attenuation-matched pairs at 16 attenuation levels between 0 and 100 HU. Dual-energy CT data were analyzed using 3-dimensional (3D) Gaussian mixture models (GMMs), as well as a simplified threshold plane metric derived from the 3D GMM, to assign voxels to hemorrhagic or calcific categories. Accuracy was calculated by comparing predicted voxel assignments with actual voxel identities. RESULTS: We measured 6032 voxels from each gel model, for a total of 193,024 data points (16 matched model pairs). Both the 3D GMM and its more clinically implementable threshold plane derivative yielded similar results, with higher than 90% accuracy at matched SECT attenuation levels of 50 HU and greater. CONCLUSIONS: Hemorrhagic and calcific lesions with attenuation levels between 50 and 100 HU were differentiable using DECT in a clinically relevant phantom system with higher than 90% accuracy. This method warrants further testing for potential clinical applications.

Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells.

Phage display screening allows the study of functional protein-protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development.

Development of a targeted gene vector platform based on simian adenovirus serotype 24.

Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations--widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery--justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.

Fluorescence imaging of fast retrograde axonal transport in living animals.

Our purpose was to enable an in vivo imaging technology that can assess the anatomy and function of peripheral nerve tissue (neurography). To do this, we designed and tested a fluorescently labeled molecular probe based on the nontoxic C fragment of tetanus toxin (TTc). TTc was purified, labeled, and subjected to immunoassays and cell uptake assays. The compound was then injected into C57BL/6 mice (N = 60) for in vivo imaging and histologic studies. Image analysis and immunohistochemistry were performed. We found that TTc could be labeled with fluorescent moieties without loss of immunoreactivity or biologic potency in cell uptake assays. In vivo fluorescent imaging experiments demonstrated uptake and retrograde transport of the compound along the course of the sciatic nerve and in the spinal cord. Ex vivo imaging and immunohistochemical studies confirmed the presence of TTc in the sciatic nerve and spinal cord, whereas control animals injected with human serum albumin did not exhibit these features. We have demonstrated neurography with a fluorescently labeled molecular imaging contrast agent based on the TTc.

Optimizing imaging of three-dimensional multicellular tumor spheroids with fluorescent reporter proteins using confocal microscopy.

Tumor spheroids more faithfully mimic tumor biology than monolayer cultures and require three-dimensional microscopy. Our goal in this study was to overcome the limitations of signal to noise ratio that have traditionally limited three-dimensional imaging to depths of 100 microm or less. We studied the expression of hypoxia-inducible factor 1alpha (HIF-1alpha), the main regulator of cellular hypoxic response in C6 glioma spheroids. In our spheroids, red fluorescent protein is expressed constitutively and green fluorescent protein is expressed conditionally under control of a HIF-1alpha promoter. In this article, we show a series of optimizations that allowed us to obtain excellent quality confocal microscopy images at imaging depths of up to 320 microm. The combined use of special objectives, glass-bottomed culture dishes, and depth-dependent laser output modulation extended our depth range beyond previously accepted limits. This allowed us to image up to the equator of spheroids of 650 microm diameter, allowing interrogation of HIF-1alpha expression from the spheroid periphery to its hypoxic center.

Nuclear entry of high-risk human papillomavirus type 16 E6 oncoprotein occurs via several pathways.

The E6 oncoprotein of high-risk human papillomavirus type 16 (HPV16) interacts with several nuclear transcription factors and coactivators in addition to cytoplasmic proteins, suggesting that nuclear import of HPV16 E6 plays a role in the cellular transformation process. In this study we have investigated the nuclear import pathways of HPV16 E6 in digitonin-permeabilized HeLa cells. We found that HPV16 E6 interacted with the karyopherin (Kap) alpha2 adapter and could enter the nucleus via a classical Kap alpha2beta1-mediated pathway. Interestingly, HPV16 E6 also interacted, via its basic nuclear localization signal (NLS) located at the C terminus, with both Kap beta1 and Kap beta2 import receptors. Binding of RanGTP to these Kap betas inhibited their interaction with HPV16 E6 NLS. In agreement with these binding data, nuclear import of the HPV16 E6 oncoprotein in digitonin-permeabilized HeLa cells could be mediated by either Kap beta1 or Kap beta2. Nuclear import via these pathways required RanGDP and was independent of GTP hydrolysis by Ran. Significantly, an E6(R124G) mutant had reduced nuclear import activity, and the E6 deletion mutant lacking (121)KKQR(124) was not imported into the nucleus. The data reveal that the HPV16 E6 oncoprotein interacts via its C-terminal NLS with several karyopherins and exploits these interactions to enter the nucleus of host cells via multiple pathways.