In Vivo Imaging of Cannabinoid Type 2 Receptors, Functional and Structural Alterations in Mouse Model of Cerebral Ischemia by PET and MRI

Abstract Background and purposeBrain ischemia is one of the most important pathologies of the central nervous system. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and follow alterations of receptor expression and metabolism in ischemic stroke. The aim of this study was to assess the cannabinoid type 2 receptors (CB2R) levels in transient middle cerebral artery occlusion (tMCAO) mouse models at subacute stage using positron emission tomography (PET) with our novel tracer [18F]RoSMA-18-d6, and structural imaging by magnetic resonance imaging (MRI). MethodsOur recently developed CB2R PET tracer [18F]RoSMA-18-d6 was used for imaging the neuroinflammation at 24 h after reperfusion in tMCAO mice. The RNA expression levels of CB2R and other inflammatory markers were analyzed by quantitative real-time polymerase chain reaction using brain tissues from tMCAO (1 h occlusion) and sham-operated mice. [18F]fluorodeoxyglucose (FDG) was included for evaluation of the cerebral metabolic rate of glucose (CMRglc). In addition, diffusion-weighted imaging and T2-weighted imaging were performed for anatomical reference and delineating the lesion in tMCAO mice. ResultsmRNA expressions of inflammatory markers TNF-a, Iba1, MMP9 and GFAP, CNR2 were increased at 24 h after reperfusion in the ipsilateral compared to contralateral hemisphere of tMCAO mice, while mRNA expression of the neuronal marker MAP-2 was markedly reduced. Reduced [18F]FDG uptake was observed in the ischemic striatum of tMCAO mouse brain at 24 h after reperfusion. Although higher activity of [18F]RoSMA-18-d6 in ex-vivo biodistribution studies and higher standard uptake value ratio (SUVR) were detected in the ischemic ipsilateral compared to contralateral striatum in tMCAO mice, the in-vivo specificity of [18F]RoSMA-18-d6 was confirmed only in the CB2R-rich spleen. ConclusionsThis study revealed an increased [18F]RoSMA-18-d6 measure of CB2R and a reduced [18F]FDG measure of CMRglc in ischemic striatum of tMCAO mice at subacute stage. [18F]RoSMA-18-d6 might be a promising PET tracer for detecting CB2R alterations in animal models of neuroinflammation without neuronal loss.

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In vivo Imaging of Cannabinoid Type 2 Receptors: Functional and Structural Alterations in Mouse Model of Cerebral Ischemia by PET and MRI

Molecular Imaging and Biology ◽

10.1007/s11307-021-01655-4 ◽

2021 ◽

Author(s):

Ruiqing Ni ◽

Adrienne Müller Herde ◽

Ahmed Haider ◽

Claudia Keller ◽

Georgios Louloudis ◽

...

Keyword(s):

Inflammatory Markers ◽

Temporal Dynamics ◽

Ex Vivo ◽

Receptor Expression ◽

Neuronal Marker ◽

Subacute Stage ◽

Pet Tracer ◽

Biodistribution Studies

Abstract Purpose Stroke is one of the most prevalent vascular diseases. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and follow alterations of receptor expression and metabolism in ischemic stroke. The aim of this study was to assess the cannabinoid type 2 receptor (CB2R) levels in transient middle cerebral artery occlusion (tMCAO) mouse models at subacute stage using positron emission tomography (PET) with our novel tracer [18F]RoSMA-18-d6 and structural imaging by magnetic resonance imaging (MRI). Procedures Our recently developed CB2R PET tracer [18F]RoSMA-18-d6 was used for imaging neuroinflammation at 24 h after reperfusion in tMCAO mice. The RNA expression levels of CB2R and other inflammatory markers were analyzed by quantitative real-time polymerase chain reaction using brain tissues from tMCAO (1 h occlusion) and sham-operated mice. [18F]fluorodeoxyglucose (FDG) was included for evaluation of the cerebral metabolic rate of glucose (CMRglc). In addition, diffusion-weighted imaging and T2-weighted imaging were performed for anatomical reference and delineating the lesion in tMCAO mice. Results mRNA expressions of inflammatory markers TNF-α, Iba1, MMP9 and GFAP, CNR2 were increased to 1.3–2.5 fold at 24 h after reperfusion in the ipsilateral compared to contralateral hemisphere of tMCAO mice, while mRNA expression of the neuronal marker MAP-2 was markedly reduced to ca. 50 %. Reduced [18F]FDG uptake was observed in the ischemic striatum of tMCAO mouse brain at 24 h after reperfusion. Although higher activity of [18F]RoSMA-18-d6 in ex vivo biodistribution studies and higher standard uptake value ratio (SUVR) were detected in the ischemic ipsilateral compared to contralateral striatum in tMCAO mice, the in vivo specificity of [18F]RoSMA-18-d6 was confirmed only in the CB2R-rich spleen. Conclusions This study revealed an increased [18F]RoSMA-18-d6 measure of CB2R and a reduced [18F]FDG measure of CMRglc in the ischemic striatum of tMCAO mice at subacute stage. [18F]RoSMA-18-d6 might be a promising PET tracer for detecting CB2R alterations in animal models of neuroinflammation without neuronal loss.

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In vivo imaging of cannabinoid type 2 receptors, functional and structural alterations in mouse model of cerebral ischemia by PET and MRI

10.1101/2021.05.08.441033 ◽

2021 ◽

Author(s):

Ruiqing Ni ◽

Adrienne Muller Herde ◽

Ahmed Haider ◽

Claudia Keller ◽

Georgios Louloudis ◽

...

Keyword(s):

Inflammatory Markers ◽

Temporal Dynamics ◽

Receptor Expression ◽

Neuronal Marker ◽

Subacute Stage ◽

Pet Tracer ◽

Biodistribution Studies ◽

18F Fdg

Background and purpose: Brain ischemia is one of the most important pathologies of the central nervous system. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and follow alterations of receptor expression and metabolism in ischemic stroke. The aim of this study was to assess the cannabinoid type 2 receptors (CB2R) levels in transient middle cerebral artery occlusion (tMCAO) mouse models at subacute stage using positron emission tomography (PET) with our novel tracer [18F]RoSMA-18-d6, and structural imaging by magnetic resonance imaging (MRI). Methods: Our recently developed CB2R PET tracer [18F]RoSMA-18-d6 was used for imaging the neuroinflammation at 24 h after reperfusion in tMCAO mice. The RNA expression levels of CB2R and other inflammatory markers were analyzed by quantitative real-time polymerase chain reaction using brain tissues from tMCAO (1 h occlusion) and sham-operated mice. [18F]fluorodeoxyglucose (FDG) was included for evaluation of the cerebral metabolic rate of glucose (CMRglc). In addition, diffusion-weighted imaging an T2- weighted imaging were performed for anatomical reference and for delineating the lesion in tMCAO mice. Results: mRNA expressions of inflammatory markers TNF-a, Iba1, MMP9 and GFAP, CNR2 were increased at 24 h after reperfusion in the ipsilateral compared to contralateral hemisphere of tMCAO mice, while mRNA expression of the neuronal marker MAP-2 was markedly reduced. Reduced [18F]FDG uptake was observed in the ischemic striatum of tMCAO mouse brain at 24 h after reperfusion. Although higher activity of [18F]RoSMA-18-d6 in ex-vivo biodistribution studies and higher standard uptake value ratio (SUVR) were detected in the ischemic ipsilateral compared to contralateral striatum in tMCAO mice, the in-vivo specificity of [18F]RoSMA-18-d6 was confirmed only in the CB2R-rich spleen. Conclusions: This study revealed an increased [18F]RoSMA-18-d6 measure of CB2R and a reduced [18F]FDG measure of CMRglc in ischemic striatum of tMCAO mice at subacute stage. [18F]RoSMA-18-d6 might be a promising PET tracer for detecting CB2R alterations in animal models of neuroinflammation without neuronal loss.

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Effect of ethanol and cocaine on [11C]MPC-6827 uptake in SH-SY5Y cells

Molecular Biology Reports ◽

10.1007/s11033-021-06336-7 ◽

2021 ◽

Author(s):

Naresh Damuka ◽

Miranda Orr ◽

Paul W. Czoty ◽

Jeffrey L. Weiner ◽

Thomas J. Martin ◽

...

Keyword(s):

Mechanism Of Action ◽

Ex Vivo ◽

Neuroblastoma Cells ◽

Proper Function ◽

Brain Functions ◽

Biodistribution Studies ◽

Positron Emission ◽

Vitro Binding

AbstractMicrotubules (MTs) are structural units in the cytoskeleton. In brain cells they are responsible for axonal transport, information processing, and signaling mechanisms. Proper function of these processes is critical for healthy brain functions. Alcohol and substance use disorders (AUD/SUDs) affects the function and organization of MTs in the brain, making them a potential neuroimaging marker to study the resulting impairment of overall neurobehavioral and cognitive processes. Our lab reported the first brain-penetrant MT-tracking Positron Emission Tomography (PET) ligand [11C]MPC-6827 and demonstrated its in vivo utility in rodents and non-human primates. To further explore the in vivo imaging potential of [11C]MPC-6827, we need to investigate its mechanism of action. Here, we report preliminary in vitro binding results in SH-SY5Y neuroblastoma cells exposed to ethanol (EtOH) or cocaine in combination with multiple agents that alter MT stability. EtOH and cocaine treatments increased MT stability and decreased free tubulin monomers. Our initial cell-binding assay demonstrated that [11C]MPC-6827 may have high affinity to free/unbound tubulin units. Consistent with this mechanism of action, we observed lower [11C]MPC-6827 uptake in SH-SY5Y cells after EtOH and cocaine treatments (e.g., fewer free tubulin units). We are currently performing in vivo PET imaging and ex vivo biodistribution studies in rodent and nonhuman primate models of AUD and SUDs and Alzheimer's disease.

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Treatment of type 2 diabetic db/db mice with a novel PPARγ agonist improves cardiac metabolism but not contractile function

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00329.2003 ◽

2004 ◽

Vol 286 (3) ◽

pp. E449-E455 ◽

Cited By ~ 70

Author(s):

Andrew N. Carley ◽

Lisa M. Semeniuk ◽

Yakhin Shimoni ◽

Ellen Aasum ◽

Terje S. Larsen ◽

...

Keyword(s):

Contractile Function ◽

Ex Vivo ◽

Metabolic Changes ◽

Pparγ Agonist ◽

Perfused Hearts ◽

Type 2 Diabetic ◽

Contractile Performance ◽

Exogenous Substrates

Hearts from insulin-resistant type 2 diabetic db/db mice exhibit features of a diabetic cardiomyopathy with altered metabolism of exogenous substrates and reduced contractile performance. Therefore, the effect of chronic oral administration of 2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid (COOH), a novel ligand for peroxisome proliferator-activated receptor-γ that produces insulin sensitization, to db/db mice (30 mg/kg for 6 wk) on cardiac function was assessed. COOH treatment reduced blood glucose from 27 mM in untreated db/db mice to a normal level of 10 mM. Insulin-stimulated glucose uptake was enhanced in cardiomyocytes from COOH-treated db/db hearts. Working perfused hearts from COOH-treated db/db mice demonstrated metabolic changes with enhanced glucose oxidation and decreased palmitate oxidation. However, COOH treatment did not improve contractile performance assessed with ex vivo perfused hearts and in vivo by echocardiography. The reduced outward K+ currents in diabetic cardiomyocytes were still attenuated after COOH. Metabolic changes in COOH-treated db/db hearts are most likely indirect, secondary to changes in supply of exogenous substrates in vivo and insulin sensitization.

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Visualisation of interstitial lung disease by molecular imaging of integrin αvβ3 and somatostatin receptor 2

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2018-214322 ◽

2018 ◽

Vol 78 (2) ◽

pp. 218-227 ◽

Cited By ~ 10

Author(s):

Janine Schniering ◽

Martina Benešová ◽

Matthias Brunner ◽

Stephanie Haller ◽

Susan Cohrs ◽

...

Keyword(s):

Interstitial Lung Disease ◽

Lung Disease ◽

Single Photon ◽

Ex Vivo ◽

Somatostatin Receptor ◽

Photon Emission ◽

Nuclear Imaging ◽

Integrin Αvβ3 ◽

Biodistribution Studies

ObjectiveTo evaluate integrin αvβ3 (alpha-v-beta-3)-targeted and somatostatin receptor 2 (SSTR2)-targeted nuclear imaging for the visualisation of interstitial lung disease (ILD).MethodsThe pulmonary expression of integrin αvβ3 and SSTR2 was analysed in patients with different forms of ILD as well as in bleomycin (BLM)-treated mice and respective controls using immunohistochemistry. Single photon emission CT/CT (SPECT/CT) was performed on days 3, 7 and 14 after BLM instillation using the integrin αvβ3-targeting 177Lu-DOTA-RGD and the SSTR2-targeting 177Lu-DOTA-NOC radiotracer. The specific pulmonary accumulation of the radiotracers over time was assessed by in vivo and ex vivo SPECT/CT scans and by biodistribution studies.ResultsExpression of integrin αvβ3 and SSTR2 was substantially increased in human ILD regardless of the subtype. Similarly, in lungs of BLM-challenged mice, but not of controls, both imaging targets were stage-specifically overexpressed. While integrin αvβ3 was most abundantly upregulated on day 7, the inflammatory stage of BLM-induced lung fibrosis, SSTR2 expression peaked on day 14, the established fibrotic stage. In agreement with the findings on tissue level, targeted nuclear imaging using SPECT/CT specifically detected both imaging targets ex vivo and in vivo, and thus visualised different stages of experimental ILD.ConclusionOur preclinical proof-of-concept study suggests that specific visualisation of molecular processes in ILD by targeted nuclear imaging is feasible. If transferred into clinics, where imaging is considered an integral part of patients’ management, the additional information derived from specific imaging tools could represent a first step towards precision medicine in ILD.

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Multinucleated giant cells in adipose tissue are specialized in adipocyte degradation

10.2337/figshare.13168862 ◽

2020 ◽

Author(s):

Ada Admin ◽

Julia Braune ◽

Andreas Lindhorst ◽

Janine Fröba ◽

Constance Hobusch ◽

...

Keyword(s):

Adipose Tissue ◽

High Fat Diet ◽

Ex Vivo ◽

Giant Cells ◽

Low Grade ◽

Adipose Tissue Macrophages ◽

Visceral Adipose ◽

Low Grade Inflammation

Obesity is associated with a chronic low-grade inflammation in visceral adipose tissue (AT) characterized by an increasing number of adipose tissue macrophages (ATMs) and linked to type 2 diabetes. AT inflammation is histologically indicated by the formation of so-called crown-like structures (CLS), as accumulation of ATMs around dying adipocytes, and the occurrence of multi-nucleated giant cells (MGCs). However to date, the function of MGCs in obesity is unknown. Hence, the aim of this study was to characterize MGCs in AT and unravel the function of these cells. We demonstrate that MGCs occur in obese patients and after 24 weeks of high fat diet (HFD) in mice, accompanying signs of AT inflammation and then represent ~3% of ATMs in mice. Mechanistically, we found evidence that adipocyte death triggers MGC formation. Most importantly, MGCs in obese AT have a higher capacity to phagocytose oversized particles, such as adipocytes, as shown by live-imaging of AT, 45 µm bead uptake ex vivo and a higher lipid content in vivo. Finally, we show that IL-4 treatment is sufficient to increase the number of MGCs in AT, whereas other factors maybe more important for endogenous MGC formation in vivo.

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Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.274.5.l714 ◽

1998 ◽

Vol 274 (5) ◽

pp. L714-L720 ◽

Cited By ~ 34

Author(s):

Sue Buckley ◽

Lora Barsky ◽

Barbara Driscoll ◽

Kenneth Weinberg ◽

Kathryn D. Anderson ◽

...

Keyword(s):

Dna Damage ◽

Epithelial Cells ◽

Cellular Response ◽

Ex Vivo ◽

Keratinocyte Growth Factor ◽

Alveolar Epithelial Cells ◽

Alveolar Epithelial ◽

Dna Laddering

Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.

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Dynamic Pattern of Adhesion Receptor Expression during the Maturation of Ex-Vivo Generated Human Adult and Neonatal Erythroid Cells.

Blood ◽

10.1182/blood.v112.11.997.997 ◽

2008 ◽

Vol 112 (11) ◽

pp. 997-997 ◽

Cited By ~ 1

Author(s):

Giovanni Migliaccio ◽

Massimo Sanchez ◽

Francesca Masiello ◽

Valentina Tirelli ◽

Katija Iliecic ◽

...

Keyword(s):

Cord Blood ◽

Ex Vivo ◽

Receptor Expression ◽

Cellular Interactions ◽

Erythroid Cells ◽

Hematopoietic Growth Factors ◽

Dynamic Pattern ◽

Adhesion Receptor ◽

Class 1

Abstract The maturation of erythroid cells occurs in specialized areas of the marrow in close proximity to macrophages. The mature cell, the reticulocyte, loses its association with the macrophages and egresses into the blood stream. This dynamic pattern of cellular interactions is mediated by specific adhesion receptors, such as CXCR4 (CD184), VLA- 4 (α4 integrin, CD49d) and P-selectin glycoprotein ligand-1 (PSGL-1 or CD162). Culture conditions capable to generate ex vivo human erythroblasts in numbers sufficient for transfusion have been recently established by several investigators. The aim of this study was to evaluate whether these ex vivo-generated erythroblasts would express the adhesion receptor profile required for establishing, once injected in vivo, the cellular interactions necessary to complete their maturation. For this purpose, the pattern of CD184, CD49d and CD162 expression during the maturation of human erythroblasts generated ex vivo from adult and cord blood was investigated. Erythroblasts were divided into 4 classes of maturation by cytofluorimetrical criteria based on the levels of CD36 and CD235a (glycophorin A) expression: class 1, CD36highCD235aneg (CFU-E); class 2, CD36highCD235alow (pro-erythroblasts); class 3, CD36highCD235ahigh (basophilic-polychromatic erythroblasts) and class 4, CD36lowCD235ahigh (orthochromatic erythroblasts). The transition of the different cell populations through the maturation process was tracked by cell cycle analyses and CFSE staining. Large numbers (>5 x 107) of erythroblasts were generated from as little as 10 mL of either cord- or adult blood after 10–11 days of culture in the presence of hematopoietic growth factors, dexamethasone and estradiol (Migliaccio et al, BCMD28: 169, 2002). Cord blood-derived cells remained significantly more immature than the adult blood-derived ones (e.g. 60% vs 10% in class 1). Class 1–2 cells were mostly in G1 (G1=74%, S=21% and G2=3–5%) while a large proportion of the class 3 cells were in S (G1=34–56, S=43–56% and G2=10–11%). Changes in the levels of CSFE staining indicated that class 3 cells completed one division within 24 hrs and did not divide further. On the other hand, class 1–2 cells completed one division in 24 hr and their progeny was composed both by class 1–2 and class 3 cells (in a 50% ratio). The class 1–2 progeny divided at least one more time in the following 24 hrs while the class 3 progeny did not divide progressing directly toward the mature class 4. The majority of class 1–2 cells expressed low level of CD184 (80–85% CD184dim and 15–20% CD184high) and high levels of CD49d and CD162. When these cells were induced to mature by exposure to EPO alone, they rapidly (within 24 hrs) expressed high levels of CD184 and CD49d while the expression of CD162 was reduced. By the end of 4 days of the maturation culture in the presence of EPO, most of the cells had progressed to the mature class 3–4 phenotype. These mature class 4 cells were CD184dim, CD49dlow and CD162low. Therefore, in vitro maturation of ex vivo-generated cord and adult blood erythroblasts was associated with a dynamic pattern of adhesion receptor expression. Although, the changes observed with cord and adult blood-derived erythroblasts were similar, they occurred more rapidly and with a higher magnitude in cord blood-derived cells. In conclusion, the pattern of CD184, CD49d and CD162 expressed by ex vivo-derived human erythroblasts suggests that these cells might be capable to establish proper cellular interactions and to progress in their maturation following in vivo infusion.

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PET imaging of cannabinoid type 2 receptors with [11C]A-836339 did not evidence changes following neuroinflammation in rats

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x16685105 ◽

2017 ◽

Vol 37 (3) ◽

pp. 1163-1178 ◽

Cited By ~ 14

Author(s):

Geraldine Pottier ◽

Vanessa Gómez-Vallejo ◽

Daniel Padro ◽

Raphaël Boisgard ◽

Frédéric Dollé ◽

...

Keyword(s):

Cerebral Ischemia ◽

Pet Imaging ◽

Receptor Expression ◽

Brain Inflammation ◽

Cb2 Receptor ◽

Future Studies ◽

Positron Emission

Cannabinoid type 2 receptors (CB2R) have emerged as promising targets for the diagnosis and therapy of brain pathologies. However, no suitable radiotracers for accurate CB2R mapping have been found to date, limiting the investigation of the CB2 receptor expression using positron emission tomography (PET) imaging. In this work, we report the evaluation of the in vivo expression of CB2R with [11C]A-836339 PET after cerebral ischemia and in two rat models of neuroinflammation, first by intrastriatal LPS and then by AMPA injection. PET images and in vitro autoradiography showed a lack of specific [11C]A-836339 uptake in these animal models demonstrating the limitation of this radiotracer to image CB2 receptor under neuroinflammatory conditions. Further, using immunohistochemistry, the CB2 receptor displayed a modest expression increase after cerebral ischemia, LPS and AMPA models. Finally, [18F]DPA-714-PET and immunohistochemistry demonstrated decreased neuroinflammation by a selective CB2R agonist, JWH133. Taken together, these findings suggest that [11C]A-836339 is not a suitable radiotracer to monitor in vivo CB2R expression by using PET imaging. Future studies will have to investigate alternative radiotracers that could provide an accurate binding to CB2 receptors following brain inflammation.

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