159 research outputs found

    The phorbol derivatives thymeleatoxin and 12-deoxyphorbol-13-O-phenylacetate-10-acetate cause translocation and down-regulation of multiple protein kinase C isozymes

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    AbstractPhorbol esters such as phorbol 12-myristate,13-acetate (PMA) are potent activators of protein kinase C (PKC), and activate all PKC isozymes except ζ, and λ. Recently, 12-deoxyphorbol-13-O-phenylacetate-20-acetate (dPPA) and thymeleatoxin (Tx) were reported to selectively activate PKCβ1 (dPPA) and PKCα, -β, and -γ (Tx), but not PKCδ or PKCϵ in vitro. We examined the ability of these phorbol derivatives to translocate and down-regulate PKC isozymes in intact cells. Our findings demonstrate that dPPA and Tx cause translocation and down-regulation of multiple PKC isozymes, including δ and ϵ

    Kinetic Modelling of [<sup>68</sup>Ga]Ga-DOTA-Siglec-9 in Porcine Osteomyelitis and Soft Tissue Infections

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    Background: [68Ga]Ga-DOTA-Siglec-9 is a positron emission tomography (PET) radioligand for vascular adhesion protein 1 (VAP-1), a protein involved in leukocyte trafficking. The tracer facilitates the imaging of inflammation and infection. Here, we studied the pharmacokinetic modelling of [68Ga]Ga-DOTA-Siglec-9 in osteomyelitis and soft tissue infections in pigs. Methods: Eight pigs with osteomyelitis and soft tissue infections in the right hind limb were dynamically PET scanned for 60 min along with arterial blood sampling. The fraction of radioactivity in the blood accounted for by the parent tracer was evaluated with radio-high-performance liquid chromatography. One- and two-tissue compartment models were used for pharmacokinetic evaluation. Post-mortem soft tissue samples from one pig were analysed with anti-VAP-1 immunofluorescence. In each analysis, the animal&rsquo;s non-infected left hind limb was used as a control. Results: Tracer uptake was elevated in soft tissue infections but remained low in osteomyelitis. The kinetics of [68Ga]Ga-DOTA-Siglec-9 followed a reversible 2-tissue compartment model. The tracer metabolized quickly; however, taking this into account, produced more ambiguous results. Infected soft tissue samples showed endothelial cell surface expression of the Siglec-9 receptor VAP-1. Conclusion: The kinetics of [68Ga]Ga-DOTA-Siglec-9 uptake in porcine soft tissue infections are best described by the 2-tissue compartment model

    Pretargeted PET Imaging of trans-Cyclooctene-Modified Porous Silicon Nanoparticles

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    Pretargeted positron emission tomography (PET) imaging based on bioorthogonal chemical reactions has proven its potential in immunoimaging. It may also have great potential in nanotheranostic applications. Here, we report the first successful pretargeted PET imaging of trans-cyclooctene-modified mesoporous silicon nanoparticles, using F-18-labeled tetrazine as a tracer. The inverse electron-demand Diels-Alder cycloaddition (IEDDA) reaction was fast, resulting in high radioactivity accumulation in the expected organs within 10 min after the administration of the tracer. The highest target-to-background ratio was achieved 120 min after the tracer injection. A clear correlation between the efficiency of the in vivo IEDDA labeling reaction and the injected amount of the tracer was observed. The radioactivity accumulation decreased with the increased amount of the co-injected carrier, indicating saturation in the reaction sites. This finding was supported by the in vitro results. Our study suggests that pretargeted imaging has excellent potential in nanotheranostic PET imaging when using high-specific-activity tracers

    Evaluation of 68Ga-labeled peptide tracer for detection of gelatinase expression after myocardial infarction in rat

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    BACKGROUND: Matrix metalloproteinases 2 and 9 (MMP-2/9) play a role in extracellular matrix remodeling after an ischemic myocardial injury. We evaluated 68Ga-DOTA-peptide targeting MMP-2/9 for the detection of gelatinase expression after myocardial infarction (MI) in rat.METHODS: Rats were injected with 43 ± 7.7 MBq of 68Ga-DOTA-peptide targeting MMP-2/9 at 7 days (n = 7) or 4 weeks (n = 8) after permanent coronary ligation or sham operation (n = 5 at both time points) followed by positron emission tomography (PET). The left ventricle was cut in frozen sections for autoradiography and immunohistochemistry 30 minutes after tracer injection.RESULTS: Immunohistochemical staining showed MMP-2 and MMP-9 expressing cells, CD31-positive endothelial cells, and CD68-positive macrophages in the infarcted myocardium. Autoradiography showed increased tracer uptake in the infarcted area both at 7 days and 4 weeks after MI (MI-to-remote area ratio 2.5 ± 0.46 and 3.1 ± 1.0, respectively). Tracer uptake in damaged tissue correlated with the amount of CD68-positive macrophages at 7 days after MI, and CD31-positive endothelial cells at 7 days and 4 weeks after MI. The tracer was rapidly metabolized, radioactivity in the blood exceeded that of the myocardium, and tracer accumulation in the heart was not detectable by in vivo PET.CONCLUSIONS: 68Ga-DOTA-peptide targeting MMP-2/9 accumulates in the damaged rat myocardium after an ischemic injury, but tracer instability and slow clearance in vivo make it unsuitable for further evaluation.</p

    The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes.

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    addresses: Institute of Biomedical and Clinical Sciences, Peninsula Medical School, Plymouth, UK.The final publication is available at link.springer.com/article/10.1007%2Fs00125-009-1276-0Evidence that the beta cells of human patients with type 1 diabetes can be infected with enterovirus is accumulating, but it remains unclear whether such infections occur at high frequency and are important in the disease process. We have now assessed the prevalence of enteroviral capsid protein vp1 (vp1) staining in a large cohort of autopsy pancreases of recent-onset type 1 diabetic patients and a range of controls

    Mesenchymal Cell-Derived Juxtacrine Wnt1 Signaling Regulates Osteoblast Activity and Osteoclast Differentiation

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    Human genetic evidence demonstrates that WNT1 mutations cause osteogenesis imperfecta (OI) and early‐onset osteoporosis, implicating WNT1 as a major regulator of bone metabolism. However, its main cellular source and mechanisms of action in bone remain elusive. We generated global and limb bud mesenchymal cell–targeted deletion of Wnt1 in mice. Heterozygous deletion of Wnt1 resulted in mild trabecular osteopenia due to decreased osteoblast function. Targeted deletion of Wnt1 in mesenchymal progenitors led to spontaneous fractures due to impaired osteoblast function and increased bone resorption, mimicking the severe OI phenotype in humans with homozygous WNT1 mutations. Importantly, we showed for the first time that Wnt1 signals strictly in a juxtacrine manner to induce osteoblast differentiation and to suppress osteoclastogenesis, in part via canonical Wnt signaling. In conclusion, mesenchymal cell‐derived Wnt1, acting in short range, is an essential regulator of bone homeostasis and an intriguing target for therapeutic interventions for bone diseases.</p

    Type 2 diabetes enhances arterial uptake of choline in atherosclerotic mice: an imaging study with positron emission tomography tracer F-18-fluoromethylcholine

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    Background: Diabetes is a risk factor for atherosclerosis associated with oxidative stress, inflammation and cell proliferation. The purpose of this study was to evaluate arterial choline uptake and its relationship to atherosclerotic inflammation in diabetic and non-diabetic hypercholesterolemic mice.Methods: Low-density lipoprotein-receptor deficient mice expressing only apolipoprotein B100, with or without type 2 diabetes caused by pancreatic overexpression of insulin-like growth factor II (IGF-II/LDLR−/−ApoB100/100 and LDLR−/−ApoB100/100) were studied. Distribution kinetics of choline analogue 18F-fluoromethylcholine (18F-FMCH) was assessed in vivo by positron emission tomography (PET) imaging. Then, aortic uptakes of 18F-FMCH and glucose analogue 18F-fluorodeoxyglucose (18F-FDG), were assessed ex vivo by gamma counting and autoradiography of tissue sections. The 18F-FMCH uptake in atherosclerotic plaques was further compared with macrophage infiltration and the plasma levels of cytokines and metabolic markers. Results: The aortas of all hypercholesterolemic mice showed large, macrophage-rich atherosclerotic plaques. The plaque burden and densities of macrophage subtypes were similar in diabetic and non-diabetic animals. The blood clearance of 18F-FMCH was rapid. Both the absolute 18F-FMCH uptake in the aorta and the aorta-to-blood uptake ratio were higher in diabetic than in non-diabetic mice. In autoradiography, the highest 18F-FMCH uptake co-localized with macrophage-rich atherosclerotic plaques. 18F-FMCH uptake in plaques correlated with levels of total cholesterol, insulin, C-peptide and leptin. In comparison with 18F-FDG, 18F-FMCH provided similar or higher plaque-to-background ratios in diabetic mice. Conclusions: Type 2 diabetes enhances the uptake of choline that reflects inflammation in atherosclerotic plaques in mice. PET tracer 18F-FMCH is a potential tool to study vascular inflammation associated with diabetes.</p

    EEG in fitness to drive evaluations in people with epilepsy - Considerable variations across Europe

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    PURPOSE: Epilepsy patients consider driving issues to be one of their most serious concerns. Ideally, decisions regarding fitness to drive should be based upon thorough evaluations by specialists in epilepsy care. In 2009, an EU directive was published aiming to harmonize evaluation practices within European countries, but, despite these recommendations, whether all epileptologists use the same criteria is unclear. We therefore conducted this study to investigate routine practices on how epileptologists at European epilepsy centers evaluate fitness to drive. METHODS: A questionnaire was sent to 63 contact persons identified through the European Epi-Care and the E-pilepsy network. The questionnaire addressed how fitness-to-drive evaluations were conducted, the involvement of different professionals, the use and interpretation of EEG, and opinions on existing regulations and guidelines. RESULTS: The questionnaire was completed by 35 participants (56 % response rate). Results showed considerable variation regarding test routines and the emphasis placed on the occurrence and extent of epileptiform discharges revealed by EEG. 82 % of the responders agreed that there was a need for more research on how to better evaluate fitness-to-drive in people with epilepsy, and 89 % agreed that regulations on fitness to drive evaluations should be internationally coordinated. CONCLUSION: Our survey showed considerable variations among European epileptologists regarding use of EEG and how findings of EEG pathology should be assessed in fitness-to-drive evaluations. There is a clear need for more research on this issue and international guidelines on how such evaluations should be carried out would be of value

    Imaging of αvβ3 integrin expression in experimental myocardial ischemia with [68Ga]NODAGA-RGD positron emission tomography

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    BACKGROUND: Radiolabeled RGD peptides detect αvβ3 integrin expression associated with angiogenesis and extracellular matrix remodeling after myocardial infarction. We studied whether cardiac positron emission tomography (PET) with [68Ga]NODAGA-RGD detects increased αvβ3 integrin expression after induction of flow-limiting coronary stenosis in pigs, and whether αvβ3 integrin is expressed in viable ischemic or injured myocardium.METHODS: We studied 8 Finnish landrace pigs 13 ± 4 days after percutaneous implantation of a bottleneck stent in the proximal left anterior descending coronary artery. Antithrombotic therapy was used to prevent stent occlusion. Myocardial uptake of [68Ga]NODAGA-RGD (290 ± 31 MBq) was evaluated by a 62 min dynamic PET scan. The ischemic area was defined as the regional perfusion abnormality during adenosine-induced stress by [15O]water PET. Guided by triphenyltetrazolium chloride staining, tissue samples from viable and injured myocardial areas were obtained for autoradiography and histology.RESULTS: Stent implantation resulted in a partly reversible myocardial perfusion abnormality. Compared with remote myocardium, [68Ga]NODAGA-RGD PET showed increased tracer uptake in the ischemic area (ischemic-to-remote ratio 1.3 ± 0.20, p = 0.0034). Tissue samples from the injured areas, but not from the viable ischemic areas, showed higher [68Ga]NODAGA-RGD uptake than the remote non-ischemic myocardium. Uptake of [68Ga]NODAGA-RGD correlated with immunohistochemical detection of αvβ3 integrin that was expressed in the injured myocardial areas.CONCLUSIONS: Cardiac [68Ga]NODAGA-RGD PET demonstrates increased myocardial αvβ3 integrin expression after induction of flow-limiting coronary stenosis in pigs. Localization of [68Ga]NODAGA-RGD uptake indicates that it reflects αvβ3 integrin expression associated with repair of recent myocardial injury.</p
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