Molecular imaging of chemokine-like receptor 1 (CMKLR1) in experimental acute lung injury.

The lack of techniques for noninvasive imaging of inflammation has challenged precision medicine management of acute respiratory distress syndrome (ARDS). Here, we determined the potential of positron emission tomography (PET) of chemokine-like receptor-1 (CMKLR1) to monitor lung inflammation in a murine model of lipopolysaccharide-induced injury. Lung uptake of a CMKLR1-targeting radiotracer, [64Cu]NODAGA-CG34, was significantly increased in lipopolysaccharide-induced injury, correlated with the expression of multiple inflammatory markers, and reduced by dexamethasone treatment. Monocyte-derived macrophages, followed by interstitial macrophages and monocytes were the major CMKLR1-expressing leukocytes contributing to the increased tracer uptake throughout the first week of lipopolysaccharide-induced injury. The clinical relevance of CMKLR1 as a biomarker of lung inflammation in ARDS was confirmed using single-nuclei RNA-sequencing datasets which showed significant increases in CMKLR1 expression among transcriptionally distinct subsets of lung monocytes and macrophages in COVID-19 patients vs. controls. CMKLR1-targeted PET is a promising strategy to monitor the dynamics of lung inflammation and response to anti-inflammatory treatment in ARDS.

The Clinical Utility of Molecular Imaging in COVID-19: An Update.

The novel pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in Wuhan, China in late 2019 with Coronavirus disease 2019 (COVID-19) declared a global pandemic in March 2020. Primarily involving the lungs, conventional imaging with chest radiography and CT can play a complementary role to RT-PCR in the initial diagnosis, and also in follow up of select patients. As a broader understanding of the multi-systemic nature of COVID-19 has evolved, a potential role for molecular imaging has developed, that may detect functional changes in advance of standard cross-sectional imaging. In this review, we highlight the evolving role of molecular imaging such as fluorine-18 (18F) fluorodeoxyglucose (FDG) with PET/CT and PET/MRI in the evaluation of both pulmonary and extra-pulmonary COVID-19, ventilation and perfusion scan with SPECT/CT for thromboembolic disease, long term follow-up of COVID-19 infection, and COVID-19 vaccine-related complications.

Present status and future trends in molecular imaging of lymphocytes.

Immune system is emerging as a crucial protagonist in a huge variety of oncologic and non-oncologic conditions including response to vaccines and viral infections (such as SARS-CoV-2). The increasing knowledge of molecular biology underlying these diseases allowed the identification of specific targets and the possibility to use tailored therapies against them. Immunotherapies and vaccines are, indeed, more and more used nowadays for treating infections, cancer and autoimmune diseases and, therefore, there is the need to identify, quantify and monitor immune cell trafficking before and after treatment. This approach will provide crucial information for therapy decision-making. Imaging of B and T-lymphocytes trafficking by using tailored radiopharmaceuticals proved to be a successful nuclear medicine tool. In this review, we will provide an overview of the state of art and future trends for "in vivo" imaging of lymphocyte trafficking and homing by mean of specific receptor-tailored radiopharmaceuticals.

Molecular imaging on ACE2-dependent transocular infection of coronavirus.

A transocular infection has been proved as one of the main approaches that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the body, and angiotensin-converting enzyme 2 (ACE2) plays a key role in this procedure. Dynamic and quantitative details on virus distribution are lacking for virus prevention and drug design. In this study, a radiotraceable pseudovirus packed with an enhanced green fluorescent protein (EGFP) gene, 125 I-CoV, was prepared and inoculated in the unilateral eye of humanized ACE2 (hACE2) mice or ACE2-knockout (ACE2-KO) mice. Single-photon emission computed tomography/computed tomography images were acquired at multiple time points to exhibit ACE2-dependent procedures from invasion to clearance. Positron emission tomography (PET) and western blot were performed to quantify ACE2 expression and verify the factors affecting transocular infection. For the transocular infection of coronavirus (CoV), the renin-angiotensin-aldosterone system (RAAS), lungs, intestines, and genital glands were the main targeted organs. Due to the specific anchor to ACE2-expressed host cells, virus concentrations in genital glands, liver, and lungs ranked the top three most and stabilized at 3.75 ± 0.55, 3.30 ± 0.25, and 2.10 ± 0.55% inoculated dose (ID)/mL at 48 h post treatment. Meanwhile, ACE2-KO mice had already completed the in vivo clearance. In consideration of organ volumes, lungs (14.50 ± 3.75%ID) and liver (10.94 ± 0.71%ID) were the main in-store reservoirs of CoV. However, the inoculated eye (5.52 ± 1.85%ID for hACE2, 5.24 ± 1.45%ID for ACE2-KO, p > 0.05) and the adjacent brain exhibited ACE2-independent virus infection at the end of 72 h observation, and absolute amount of virus played a key role in host cell infection. These observations on CoV infection were further manifested by infection-driven intracellular EGFP expression. ACE2 PET revealed an infection-related systematic upregulation of ACE2 expression in the organs involved in RAAS (e.g., brain, lung, heart, liver, and kidney) and the organ that was of own local renin-angiotensin system (e.g., eye). Transocular infection of CoV is ACE2-dependent and constitutes the cause of disturbed ACE2 expression in the host. The brain, genital glands, and intestines were of the highest unit uptake, potentially accounting for the sequelae. Lungs and liver were of the highest absolute amount, closely related to the respiratory diffusion and in vivo duplication. ACE2 expression was upregulated in the short term after infection with CoV. These visual and quantitative results are helpful to fully understanding the transocular path of SARS-CoV-2 and other CoVs.

Molecular Imaging Findings on Acute and Long-Term Effects of COVID-19 on the Brain: A Systematic Review.

Molecular imaging techniques such as PET and SPECT have been used to shed light on how coronavirus disease 2019 (COVID-19) affects the human brain. We provide a systematic review that summarizes the current literature according to 5 predominant topics. First, a few case reports have suggested reversible cortical and subcortical metabolic alterations in rare cases with concomitant para- or postinfectious encephalitis. Second, imaging findings in single patients with the first manifestations of parkinsonism in the context of COVID-19 resemble those in neurodegenerative parkinsonism (loss of nigrostriatal integrity), but scarceness of data and a lack of follow-up preclude further etiologic conclusions (e.g., unmasking/hastening of neurodegeneration vs. infectious or parainfectious parkinsonism). Third, several case reports and a few systematic studies have addressed focal symptoms and lesions, most notably hyposmia. The results have been variable, although some studies found regional hypometabolism of regions related to olfaction (e.g., orbitofrontal and mesiotemporal). Fourth, a case series and systematic studies in inpatients with COVID-19-related encephalopathy (acute to subacute stage) consistently found a frontoparietal-dominant neocortical dysfunction (on imaging and clinically) that proved to be grossly reversible in most cases until 6 mo. Fifth, studies on post-COVID-19 syndrome have provided controversial results. In patients with a high level of self-reported complaints (e.g., fatigue, memory impairment, hyposmia, and dyspnea), some authors found extensive areas of limbic and subcortical hypometabolism, whereas others found no metabolic alterations on PET and only minor cognitive impairments (if any) on neuropsychologic assessment. Furthermore, we provide a critical appraisal of studies with regard to frequent methodologic issues and current pathophysiologic concepts. Finally, we devised possible applications of PET and SPECT in the clinical work-up of diagnostic questions related to COVID-19.

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment.

Recombinant antibodies such as nanobodies are progressively demonstrating to be a valid alternative to conventional monoclonal antibodies also for clinical applications. Furthermore, they do not solely represent a substitute for monoclonal antibodies but their unique features allow expanding the applications of biotherapeutics and changes the pattern of disease treatment. Nanobodies possess the double advantage of being small and simple to engineer. This combination has promoted extremely diversified approaches to design nanobody-based constructs suitable for particular applications. Both the format geometry possibilities and the functionalization strategies have been widely explored to provide macromolecules with better efficacy with respect to single nanobodies or their combination. Nanobody multimers and nanobody-derived reagents were developed to image and contrast several cancer diseases and have shown their effectiveness in animal models. Their capacity to block more independent signaling pathways simultaneously is considered a critical advantage to avoid tumor resistance, whereas the mass of these multimeric compounds still remains significantly smaller than that of an IgG, enabling deeper penetration in solid tumors. When applied to CAR-T cell therapy, nanobodies can effectively improve the specificity by targeting multiple epitopes and consequently reduce the side effects. This represents a great potential in treating malignant lymphomas, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma and solid tumors. Apart from cancer treatment, multispecific drugs and imaging reagents built with nanobody blocks have demonstrated their value also for detecting and tackling neurodegenerative, autoimmune, metabolic, and infectious diseases and as antidotes for toxins. In particular, multi-paratopic nanobody-based constructs have been developed recently as drugs for passive immunization against SARS-CoV-2 with the goal of impairing variant survival due to resistance to antibodies targeting single epitopes. Given the enormous research activity in the field, it can be expected that more and more multimeric nanobody molecules will undergo late clinical trials in the next future. Systematic Review Registration.

Stealth Fluorescence Labeling for Live Microscopy Imaging of mRNA Delivery.

Methods for tracking RNA inside living cells without perturbing their natural interactions and functions are critical within biology and, in particular, to facilitate studies of therapeutic RNA delivery. We present a stealth labeling approach that can efficiently, and with high fidelity, generate RNA transcripts, through enzymatic incorporation of the triphosphate of tCO, a fluorescent tricyclic cytosine analogue. We demonstrate this by incorporation of tCO in up to 100% of the natural cytosine positions of a 1.2 kb mRNA encoding for the histone H2B fused to GFP (H2B:GFP). Spectroscopic characterization of this mRNA shows that the incorporation rate of tCO is similar to cytosine, which allows for efficient labeling and controlled tuning of labeling ratios for different applications. Using live cell confocal microscopy and flow cytometry, we show that the tCO-labeled mRNA is efficiently translated into H2B:GFP inside human cells. Hence, we not only develop the use of fluorescent base analogue labeling of nucleic acids in live-cell microscopy but also, importantly, show that the resulting transcript is translated into the correct protein. Moreover, the spectral properties of our transcripts and their translation product allow for their straightforward, simultaneous visualization in live cells. Finally, we find that chemically transfected tCO-labeled RNA, unlike a state-of-the-art fluorescently labeled RNA, gives rise to expression of a similar amount of protein as its natural counterpart, hence representing a methodology for studying natural, unperturbed processing of mRNA used in RNA therapeutics and in vaccines, like the ones developed against SARS-CoV-2.

Discovery of potential imaging and therapeutic targets for severe inflammation in COVID-19 patients.

The Coronavirus disease 2019 (COVID-19) has been spreading worldwide with rapidly increased number of deaths. Hyperinflammation mediated by dysregulated monocyte/macrophage function is considered to be the key factor that triggers severe illness in COVID-19. However, no specific targeting molecule has been identified for detecting or treating hyperinflammation related to dysregulated macrophages in severe COVID-19. In this study, previously published single-cell RNA-sequencing data of bronchoalveolar lavage fluid cells from thirteen COVID-19 patients were analyzed with publicly available databases for surface and imageable targets. Immune cell composition according to the severity was estimated with the clustering of gene expression data. Expression levels of imaging target molecules for inflammation were evaluated in macrophage clusters from single-cell RNA-sequencing data. In addition, candidate targetable molecules enriched in severe COVID-19 associated with hyperinflammation were filtered. We found that expression of SLC2A3, which can be imaged by [18F]fluorodeoxyglucose, was higher in macrophages from severe COVID-19 patients. Furthermore, by integrating the surface target and drug-target binding databases with RNA-sequencing data of severe COVID-19, we identified candidate surface and druggable targets including CCR1 and FPR1 for drug delivery as well as molecular imaging. Our results provide a resource in the development of specific imaging and therapy for COVID-19-related hyperinflammation.

Identify. Quantify. Predict. Why Immunologists Should Widely Use Molecular Imaging for Coronavirus Disease 2019.

Molecular imaging using PET/CT or PET/MRI has evolved from an experimental imaging modality at its inception in 1972 to an integral component of diagnostic procedures in oncology, and, to lesser extent, in cardiology and neurology, by successfully offering in-vivo imaging and quantitation of key pathophysiological targets or molecular signatures, such as glucose metabolism in cancerous disease. Apart from metabolism probes, novel radiolabeled peptide and antibody PET tracers, including radiolabeled monoclonal antibodies (mAbs) have entered the clinical arena, providing the in-vivo capability to collect target-specific quantitative in-vivo data on cellular and molecular pathomechanisms on a whole-body scale, and eventually, extract imaging biomarkers possibly serving as prognostic indicators. The success of molecular imaging in mapping disease severity on a whole-body scale, and directing targeted therapies in oncology possibly could translate to the management of Coronavirus Disease 2019 (COVID-19), by identifying, localizing, and quantifying involvement of different immune mediated responses to the infection with SARS-COV2 during the course of acute infection and possible, chronic courses with long-term effects on specific organs. The authors summarize current knowledge for medical imaging in COVID-19 in general with a focus on molecular imaging technology and provide a perspective for immunologists interested in molecular imaging research using validated and immediately available molecular probes, as well as possible future targets, highlighting key targets for tailored treatment approaches as brought up by key opinion leaders.

Guidelines on Setting Up Stations for Remote Viewing of Nuclear Medicine and Molecular Imaging Studies During COVID-19.

The current pandemic has created a situation where nuclear medicine practitioners and medical physicists read or process nuclear medicine images remotely from their home office. This article presents recommendations on the components and specifications when setting up a remote viewing station for nuclear medicine imaging.

Non-invasive bioluminescence imaging of HCoV-OC43 infection and therapy in the central nervous system of live mice.

Human coronaviruses (HCoVs) are important pathogens that cause upper respiratory tract infections and have neuroinvasive abilities; however, little is known about the dynamic infection process of CoVs in vivo, and there are currently no specific antiviral drugs to prevent or treat HCoV infection. Here, we verified the replication ability and pathogenicity of a reporter HCoV-OC43 strain expressing Renilla luciferase (Rluc; rOC43-ns2DelRluc) in mice with different genetic backgrounds (C57BL/6 and BALB/c). Additionally, we monitored the spatial and temporal progression of HCoV-OC43 through the central nervous system (CNS) of live BALB/c mice after intranasal or intracerebral inoculation with rOC43-ns2DelRluc. We found that rOC43-ns2DelRluc was fatal to suckling mice after intranasal inoculation, and that viral titers and Rluc expression were detected in the brains and spinal cords of mice infected with rOC43-ns2DelRluc. Moreover, viral replication was initially observed in the brain by non-invasive bioluminescence imaging before the infection spread to the spinal cord of BALB/c mice, consistent with its tropism in the CNS. Furthermore, the Rluc readout correlated with the HCoV replication ability and protein expression, which allowed quantification of antiviral activity in live mice. Additionally, we validated that chloroquine strongly inhibited rOC43-ns2DelRluc replication in vivo. These results provide new insights into the temporal and spatial dissemination of HCoV-OC43 in the CNS, and our methods provide an extremely sensitive platform for evaluating the efficacy of antiviral therapies to treat neuroinvasive HCoVs in live mice.

Clinical PARP inhibitors do not abrogate PARP1 exchange at DNA damage sites in vivo.

DNA breaks recruit and activate PARP1/2, which deposit poly-ADP-ribose (PAR) to recruit XRCC1-Ligase3 and other repair factors to promote DNA repair. Clinical PARP inhibitors (PARPi) extend the lifetime of damage-induced PARP1/2 foci, referred to as 'trapping'. To understand the molecular nature of 'trapping' in cells, we employed quantitative live-cell imaging and fluorescence recovery after photo-bleaching. Unexpectedly, we found that PARP1 exchanges rapidly at DNA damage sites even in the presence of clinical PARPi, suggesting the persistent foci are not caused by physical stalling. Loss of Xrcc1, a major downstream effector of PAR, also caused persistent PARP1 foci without affecting PARP1 exchange. Thus, we propose that the persistent PARP1 foci are formed by different PARP1 molecules that are continuously recruited to and exchanging at DNA lesions due to attenuated XRCC1-LIG3 recruitment and delayed DNA repair. Moreover, mutation analyses of the NAD+ interacting residues of PARP1 showed that PARP1 can be physically trapped at DNA damage sites, and identified H862 as a potential regulator for PARP1 exchange. PARP1-H862D, but not PARylation-deficient PARP1-E988K, formed stable PARP1 foci upon activation. Together, these findings uncovered the nature of persistent PARP1 foci and identified NAD+ interacting residues involved in the PARP1 exchange.

Post-COVID-19 New Normal for Molecular Imaging Departments: A United Kingdom Perspective.

Coronavirus disease 2019 has changed the way the world is navigated and has had a massive impact on health care. Depending on where you are in the world, the guidance on dealing with potential infected patients is varied. With the high risk of a second wave, it is important to learn from initial responses to plan for the future. With proper preparation, it is possible to minimize exposure and risk of contamination to individuals visiting molecular imaging departments. Such precautions will help departments operate at full capacity. From the widespread nature of this pandemic, a global perspective can be useful; what follows is the United Kingdom's perspective.

Nuclear Medicine Clinical Practice in the United States During the COVID-19 Era and Beyond.

Traditionally, practice in nuclear medicine has involved strong emphasis on radiation safety principles. Nuclear medicine technologists (NMTs) focus on practices that keep patients, the public, and the technologist safe from potentially harmful effects of unnecessary radiation exposure using concepts of time, distance, and shielding as well as ALARA (As low as reasonably achievable) principles. The current COVID-19 pandemic has brought to light the need to apply focus on infection prevention in practice and update knowledge and procedures on such measures. In this article, the authors outline the need for NMTs to develop practices and values focused on infection prevention measures.