Noninvasive Quantification of Radiation-Induced Lung Injury Using a Targeted Molecular Imaging Probe.

PURPOSE: Radiation-induced lung injury (RILI) is a progressive inflammatory process seen after irradiation for lung cancer. The disease can be insidious, often characterized by acute pneumonitis followed by chronic fibrosis with significant associated morbidity. No therapies are approved for RILI, and accurate disease quantification is a major barrier to improved management. Here, we sought to noninvasively quantify RILI using a molecular imaging probe that specifically targets type 1 collagen in mouse models and patients with confirmed RILI. METHODS AND MATERIALS: Using a murine model of lung radiation, mice were imaged with EP-3533, a type 1 collagen probe, to characterize the development of RILI and to assess disease mitigation after losartan treatment. The human analog probe 68Ga-CBP8, targeting type 1 collagen, was tested on excised human lung tissue containing RILI and was quantified via autoradiography. 68Ga-CBP8 positron emission tomography was used to assess RILI in vivo in 6 human subjects. RESULTS: Murine models demonstrated that probe signal correlated with progressive RILI severity over 6 months. The probe was sensitive to mitigation of RILI by losartan. Excised human lung tissue with RILI had increased binding versus unirradiated control tissue, and 68Ga-CBP8 uptake correlated with collagen proportional area. Human imaging revealed significant 68Ga-CBP8 uptake in areas of RILI and minimal background uptake. CONCLUSIONS: These findings support the ability of a molecular imaging probe targeted at type 1 collagen to detect RILI in preclinical models and human disease, suggesting a role for targeted molecular imaging of collagen in the assessment of RILI.

Detection of Pulmonary Fibrosis with a Collagen-Mimetic Peptide.

Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology that is characterized by excessive deposition and abnormal remodeling of collagen. IPF has a mean survival time of only 2-5 years from diagnosis, creating a need to detect IPF at an earlier stage when treatments might be more effective. We sought to develop a minimally invasive probe that could detect molecular changes in IPF-associated collagen. Here, we describe the design, synthesis, and performance of [68Ga]Ga·DOTA-CMP, which comprises a positron-emitting radioisotope linked to a collagen-mimetic peptide (CMP). This peptide mimics the natural structure of collagen and detects irregular collagen matrices by annealing to damaged collagen triple helices. We assessed the ability of the peptide to detect aberrant lung collagen selectively in a bleomycin-induced mouse model of pulmonary fibrosis using positron emission tomography (PET). [68Ga]Ga·DOTA-CMP PET demonstrated higher and selective uptake in a fibrotic mouse lung compared to controls, minimal background signal in adjacent organs, and rapid clearance via the renal system. These studies suggest that [68Ga]Ga·DOTA-CMP identifies fibrotic lungs and could be useful in the early diagnosis of IPF.

Noninvasive Quantification of Radiation-Induced Lung Injury using a Targeted Molecular Imaging Probe.

Rationale: Radiation-induced lung injury (RILI) is a progressive inflammatory process commonly seen following irradiation for lung cancer. The disease can be insidious, often characterized by acute pneumonitis followed by chronic fibrosis with significant associated morbidity. No therapies are approved for RILI, and accurate disease quantification is a major barrier to improved management. Objective: To noninvasively quantify RILI, utilizing a molecular imaging probe that specifically targets type 1 collagen in mouse models and patients with confirmed RILI. Methods: Using a murine model of lung radiation, mice were imaged with EP-3533, a type 1 collagen probe to characterize the development of RILI and to assess disease mitigation following losartan treatment. The human analog probe targeted against type 1 collagen, 68Ga-CBP8, was tested on excised human lung tissue containing RILI and quantified via autoradiography. Finally, 68Ga-CBP8 PET was used to assess RILI in vivo in six human subjects. Results: Murine models demonstrated that probe signal correlated with progressive RILI severity over six-months. The probe was sensitive to mitigation of RILI by losartan. Excised human lung tissue with RILI had increased binding vs unirradiated control tissue and 68Ga-CBP8 uptake correlated with collagen proportional area. Human imaging revealed significant 68Ga-CBP8 uptake in areas of RILI and minimal background uptake. Conclusions: These findings support the ability of a molecular imaging probe targeted at type 1 collagen to detect RILI in preclinical models and human disease, suggesting a role for targeted molecular imaging of collagen in the assessment of RILI.Clinical trial registered with www.clinicaltrials.gov (NCT04485286, NCT03535545).

Optimization of an Allysine-Targeted PET Probe for Quantifying Fibrogenesis in a Mouse Model of Pulmonary Fibrosis.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a destructive lung disease with a poor prognosis, an unpredictable clinical course, and inadequate therapies. There are currently no measures of disease activity to guide clinicians making treatment decisions. The aim of this study was to develop a PET probe to identify lung fibrogenesis using a pre-clinical model of pulmonary fibrosis, with potential for translation into clinical use to predict disease progression and inform treatment decisions. METHODS: Eight novel allysine-targeting chelators, PIF-1, PIF-2, …, PIF-8, with different aldehyde-reactive moieties were designed, synthesized, and radiolabeled with gallium-68 or copper-64. PET probe performance was assessed in C57BL/6J male mice 2 weeks after intratracheal bleomycin challenge and in naïve mice by dynamic PET/MR imaging and with biodistribution at 90 min post injection. Lung hydroxyproline and allysine were quantified ex vivo and histological staining for fibrosis and aldehyde was performed. RESULTS: In vivo screening of probes identified 68GaPIF-3 and 68GaPIF-7 as probes with high uptake in injured lung, high uptake in injured lung versus normal lung, and high uptake in injured lung versus adjacent liver and heart tissue. A crossover, intra-animal PET/MR imaging study of 68GaPIF-3 and 68GaPIF-7 confirmed 68GaPIF-7 as the superior probe. Specificity for fibrogenesis was confirmed in a crossover, intra-animal PET/MR imaging study with 68GaPIF-7 and a non-binding control compound, 68GaPIF-Ctrl. Substituting copper-64 for gallium-68 did not affect lung uptake or specificity indicating that either isotope could be used. CONCLUSION: A series of allysine-reactive PET probes with variations in the aldehyde-reactive moiety were evaluated in a pre-clinical model of lung fibrosis. The hydrazine-bearing probe, 68GaPIF-7, exhibited the highest uptake in fibrogenic lung, low uptake in surrounding liver or heart tissue, and low lung uptake in healthy mice and should be considered for further clinical translation.

Biodistribution, Dosimetry, and Pharmacokinetics of 68Ga-CBP8: A Type I Collagen-Targeted PET Probe.

The 68Ga-Collagen Binding Probe #8, 68Ga-CBP8, is a peptide-based, type I collagen-targeted probe developed for imaging of tissue fibrosis. The aim of this study was to determine the biodistribution, dosimetry, and pharmacokinetics of 68Ga-CBP8 in healthy human subjects. Methods: Nine healthy volunteers (5 male and 4 female) underwent whole-body 68Ga-CBP8 PET/MRI using a Biograph mMR scanner. The subjects were imaged continuously for up to 2 h after injection of 68Ga-CBP8. A subset of subjects underwent an additional imaging session 2-3 h after probe injection. OLINDA/EXM software was used to calculate absorbed organ and effective dose estimates based on up to 17 regions of interest (16 for men) defined on T2-weighted MR images and copied to the PET images, assuming a uniform distribution of probe concentration in each region. Serial blood sampling up to 90 min after probe injection was performed to assess blood clearance and metabolic stability. Results: The mean injected activity (±SD) of 68Ga-CBP8 was 220 ± 100 MBq (range, 113-434 MBq). No adverse effects related to probe administration were detected. 68Ga-CBP8 demonstrated an extracellular distribution with predominantly rapid renal clearance. Doses on the urinary bladder were 0.15 versus 0.19 mGy/MBq for men versus women. The highest absorbed doses for the rest of the organs were measured in the kidneys (0.078 vs. 0.088 mGy/MBq) and the liver (0.032 vs. 0.041 mGy/MBq). The mean effective dose was 0.018 ± 0.0026 mSv/MBq using a 1-h voiding model. The 68Ga-CBP8 signal in the blood demonstrated biexponential pharmacokinetics with an initial distribution half-life of 4.9 min (95% CI, 2.4-9.4 min) and a 72-min elimination half-life (95% CI, 47-130 min). The only metabolite observed had a long blood plasma half-life, suggesting protein-bound 68Ga. Conclusion: 68Ga-CBP8 displays favorable in-human characteristics and dosimetry similar to that of other gallium-based probes. 68Ga-CBP8 could therefore be used for noninvasive collagen imaging across a range of human fibrotic diseases.

Spatiotemporal 3D image registration for mesoscale studies of brain development.

Comparison of brain samples representing different developmental stages often necessitates registering the samples to common coordinates. Although the available software tools are successful in registering 3D images of adult brains, registration of perinatal brains remains challenging due to rapid growth-dependent morphological changes and variations in developmental pace between animals. To address these challenges, we introduce CORGI (Customizable Object Registration for Groups of Images), an algorithm for the registration of perinatal brains. First, we optimized image preprocessing to increase the algorithm's sensitivity to mismatches in registered images. Second, we developed an attention-gated simulated annealing procedure capable of focusing on the differences between perinatal brains. Third, we applied classical multidimensional scaling (CMDS) to align ("synchronize") brain samples in time, accounting for individual development paces. We tested CORGI on 28 samples of whole-mounted perinatal mouse brains (P0-P9) and compared its accuracy with other registration algorithms. Our algorithm offers a runtime of several minutes per brain on a laptop and automates such brain registration tasks as mapping brain data to atlases, comparing experimental groups, and monitoring brain development dynamics.

Molecular MR Contrast Agents.

Molecular magnetic resonance (MR) imaging utilizes molecular probes to provide added biochemical or cellular information to what can already be achieved with anatomical and functional MR imaging. This review provides an overview of molecular MR and focuses specifically on molecular MR contrast agents that provide contrast by shortening the T1 time. We describe the requirements for a successful molecular MR contrast agent and the challenges for clinical translation. The review highlights work from the last 5 years and places an emphasis on new contrast agents that have been validated in multiple preclinical models. Applications of molecular MR include imaging of inflammation, fibrosis, fibrogenesis, thromboembolic disease, and cancers. Molecular MR is positioned to move beyond detection of disease to the quantitative staging of disease and measurement of treatment response.

Revisiting dithiadiaza macrocyclic chelators for copper-64 PET imaging.

Synthesis and characterisation of a dithiadiaza chelator NSNS2A, as well as copper complexes thereof are reported in this paper. Solution structures of copper(i/ii) complexes were calculated using density functional theory (DFT) and validated by both NMR and EPR spectroscopy. DFT calculations revealed a switch in the orientation of tetragonal distortion upon protonation, which might be responsible for poor stability of the Cu(II)NSNS2A complex in aqueous media, whilst the same switch in tetragonal distortion was experimentally observed by changing the solvent. The chelator was radiolabeled with 64Cu and evaluated using PET/MRI in rats. Despite a favorable redox potential to stabilize the cuprous state in vivo, the 64Cu(II)NSNS2A complex showed suboptimal stability compared to its tetraazamacrocyclic analogue, 64Cu(TE2A), with a significant 64Cu uptake in the liver.

Addressing prosthetic joint infections via gentamicin-eluting UHMWPE spacer.

AIMS: We propose a state-of-the-art temporary spacer, consisting of a cobalt-chrome (CoCr) femoral component and a gentamicin-eluting ultra-high molecular weight polyethylene (UHMWPE) tibial insert, which can provide therapeutic delivery of gentamicin, while retaining excellent mechanical properties. The proposed implant is designed to replace conventional spacers made from bone cement. METHODS: Gentamicin-loaded UHMWPE was prepared using phase-separated compression moulding, and its drug elution kinetics, antibacterial, mechanical, and wear properties were compared with those of conventional gentamicin-loaded bone cement. RESULTS: Gentamicin-loaded UHMWPE tibial components not only eradicated planktonic Staphylococcus aureus, but also prevented colonization of both femoral and tibial components. The proposed spacer possesses far superior mechanical and wear properties when compared with conventional bone cement spacers. CONCLUSION: The proposed gentamicin-eluting UHMWPE spacer can provide antibacterial efficacy comparable with currently used bone cement spacers, while overcoming their drawbacks. The novel spacer proposed here has the potential to drastically reduce complications associated with currently used bone cement spacers and substantially improve patients' quality of life during the treatment. Cite this article: Bone Joint J 2020;102-B(6 Supple A):151-157.

Neural Networks With Motivation.

Animals rely on internal motivational states to make decisions. The role of motivational salience in decision making is in early stages of mathematical understanding. Here, we propose a reinforcement learning framework that relies on neural networks to learn optimal ongoing behavior for dynamically changing motivation values. First, we show that neural networks implementing Q-learning with motivational salience can navigate in environment with dynamic rewards without adjustments in synaptic strengths when the needs of an agent shift. In this setting, our networks may display elements of addictive behaviors. Second, we use a similar framework in hierarchical manager-agent system to implement a reinforcement learning algorithm with motivation that both infers motivational states and behaves. Finally, we show that, when trained in the Pavlovian conditioning setting, the responses of the neurons in our model resemble previously published neuronal recordings in the ventral pallidum, a basal ganglia structure involved in motivated behaviors. We conclude that motivation allows Q-learning networks to quickly adapt their behavior to conditions when expected reward is modulated by agent's dynamic needs. Our approach addresses the algorithmic rationale of motivation and makes a step toward better interpretability of behavioral data via inference of motivational dynamics in the brain.

Click histochemistry for whole-mount staining of brain structures.

Labeling of the replicating DNA with synthetic thymidine analogs is commonly used for marking the dividing cells. However, until now this method has only been applied to histological sections. A growing number of current approaches for three-dimensional visualization of large tissue samples requires detection of dividing cells within whole organs. Here we describe a method for labeling dividing cells with 5-ethynyl-2'-deoxyuridine (EdU) and their further detection in whole brain structures (for example, hippocampus) using the Cu (I) -catalyzed [3 + 2] cycloaddition reaction (so-called click-reaction). The presented method can be used for brain neurogenesis studies as well as for whole-mount staining of any preparations in which the terminal ethynyl group has been introduced. •New click histochemistry method based on Cu (I) -catalyzed [3 + 2] cycloaddition reaction allows whole-mount staining of brain structures and other tissues.•Our whole-mount click histochemistry method allows to visualize dividing cells in 3D and can be used in neurogenesis studies, i.e. for birthdating dividing early progenitors and further tracking of proliferation, survival, migration, differentiation, and fate of their progeny.•Our whole-mount click histochemistry staining demonstrates high staining specificity, high signal intensity, and low background levels in young and adult mouse brain tissue.

Peroxidase Sensitive Amplifiable Probe for Molecular Magnetic Resonance Imaging of Pulmonary Inflammation.

An amplifiable magnetic resonance imaging (MRI) probe that combines the stability of the macrocyclic Gd-DOTAGA core with a peroxidase-reactive 5-hydroxytryptamide (5-HT) moiety is reported. The incubation of the complex under enzymatic oxidative conditions led to a 1.7-fold increase in r1 at 1.4 T that was attributed to an oligomerization of the probe upon oxidation. This probe, Gd-5-HT-DOTAGA, provided specific detection of lung inflammation by MRI in bleomycin-injured mice.

Suppressed neurogenesis without cognitive deficits: effects of fast neutron irradiation in mice.

This study assessed the effects of combined low-dose neutron and γ-ray irradiation on hippocampal neurogenesis and hippocampal-dependent memory. Neural progenitor cell division and survival were evaluated in brain sections and whole hippocampal preparations following head irradiation at a dose of 0.34 Gy for neutron radiation and 0.36 Gy for γ-ray radiation. Hippocampal-dependent memory formation was tested in a contextual fear conditioning task following irradiation at doses of 0.4 Gy for neutron radiation and 0.42 Gy for γ-ray radiation. Cell division was suppressed consistently along the entire dorsoventral axis of the hippocampus 24 h after the irradiation, but quiescent stem cells remained unaffected. The control and irradiated mice showed no differences in terms of exploratory behavior or anxiety 6 weeks after the irradiation. The ability to form hippocampus-dependent memory was also unaffected. The data may be indicative of a negligible effect of the low-dose of fast neutron irradiation and the neurogenesis suppression on animal behavior at 6 weeks after irradiation.

Network cloning using DNA barcodes.

The connections between neurons determine the computations performed by both artificial and biological neural networks. Recently, we have proposed SYNSeq, a method for converting the connectivity of a biological network into a form that can exploit the tremendous efficiencies of high-throughput DNA sequencing. In SYNSeq, each neuron is tagged with a random sequence of DNA-a "barcode"-and synapses are represented as barcode pairs. SYNSeq addresses the analysis problem, reducing a network into a suspension of barcode pairs. Here, we formulate a complementary synthesis problem: How can the suspension of barcode pairs be used to "clone" or copy the network back into an uninitialized tabula rasa network? Although this synthesis problem might be expected to be computationally intractable, we find that, surprisingly, this problem can be solved efficiently, using only neuron-local information. We present the "one-barcode-one-cell" (OBOC) algorithm, which forces all barcodes of a given sequence to coalesce into the same neuron, and show that it converges in a number of steps that is a power law of the network size. Rapid and reliable network cloning with single-synapse precision is thus theoretically possible.

A near-IR luminescent ratiometric ytterbium pH probe.

The hypersensitive 2F5/2 to 2F7/2 transition of Yb3+ can be used to monitor perturbations of the coordination sphere in ytterbium(iii) complexes. An envelope of Stark components gives rise to a relatively broad and asymmetric emission band, whilst changes in their relative intensity and energy enable a ratiometric response. We report a new ytterbium complex with a sulphonamide arm that binds reversibly to Yb3+ as a function of pH, giving rise to significant pH dependent changes in the Yb emission spectrum.

Selective signalling of glyphosate in water using europium luminescence.

A series of four emissive europium complexes has been evaluated for the binding of glyphosate in various aqueous media, including river water and grain extracts. Binding selectivity toward inorganic phosphate and bicarbonate was enhanced by measuring samples at pH 5.9, above the pKa of glyphosate itself. The highest affinity was shown with [Eu·L1], which creates an exocyclic tripicolylamine moiety when one pyridine group dissociates from Eu. Glyphosate was bound selectively over dihydrogenphosphate, glycinate, aminomethylphosphonate and the related herbicide glufosinate. The complex was used to measure glyphosate over the range 5 to 50 µM, in river water and grain extracts.

Chiral probes for α1-AGP reporting by species-specific induced circularly polarised luminescence.

Luminescence spectroscopy has been used to monitor the selective and reversible binding of pH sensitive, macrocyclic lanthanide complexes, [LnL1], to the serum protein α1-AGP, whose concentration can vary significantly in response to inflammatory processes. On binding α1-AGP, a very strong induced circularly-polarised europium luminescence signal was observed that was of opposite sign for human and bovine variants of α1-AGP - reflecting the differences in the chiral environment of their drug-binding pockets. A mixture of [EuL1] and [TbL1] complexes allowed the ratiometric monitoring of α1-AGP levels in serum. Moreover, competitive displacement of [EuL1] from the protein by certain prescription drugs could be monitored, allowing the determination of drug binding constants. Reversible binding of the sulphonamide arm as a function of pH, led to a change of the coordination environment around the lanthanide ion, from twisted square antiprism (TSAP) to a square antiprismatic geometry (SAP), signalled by emission spectral changes and verified by detailed computations and the fitting of NMR pseudocontact shift data in the sulphonamide bound TSAP structure for the Dy and Eu examples. Such analyses allowed a full definition of the magnetic susceptibility tensor for [DyL1].

Monitoring of the ADP/ATP Ratio by Induced Circularly Polarised Europium Luminescence.

A series of three europium complexes bearing picolyl amine moieties was found to possess differing binding affinities towards Zn2+ and three nucleotides: AMP, ADP, and ATP. A large increase in the total emission intensity was observed upon binding Zn2+ , followed by signal amplification upon the addition of nucleotides. The resulting adducts possessed strong induced circularly polarised emission, with ADP and ATP signals of opposite sign. Model DFT geometries of the adducts suggest the Δ diastereoisomer is preferred for ATP and the Λ isomer for ADP/AMP. This change in sign allows the ADP/ATP (or AMP/ATP) ratio to be assessed by monitoring changes in the emission dissymmetry factor, gem .

Selectively switching on europium emission in drug site one of human serum albumin.

A luminescent europium probe has been discovered that binds selectively to drug-site I in human serum albumin, signalled by a 'switching on' of europium emission, and accompanied by strong induced circularly polarised luminescence.

Responsive, Water-Soluble Europium(III) Luminescent Probes.

The design principles, mechanism of action and performance of europium(III) complexes that serve as strongly emissive and responsive molecular probes in water are critically discussed. Examples of systems designed to assess pH, selected metal ions and anions, including chiral species, as well as selected small molecules and biopolymers are considered, and prospects evaluated for improved performance in more complex biological media such as in bio-fluids and within living cells. Modulation of the emission spectral form, lifetime and degree of circular polarisation can be used to quantify the spectral response and permit calibration.