Lobar selective internal radiation therapy (SIRT) is widely used to treat liver tumors inducing atrophy of the treated lobe and contralateral hypertrophy. The lack of animal model has precluded further investigations to improve this treatment. We developed an animal model of liver damage and atrophy-hypertrophy complex after SIRT. Three groups of 5-8 rabbits received transportal SIRT with Yttrium 90 resin microspheres of the cranial lobes with different activities (0.3, 0.6 and 1.2 GBq), corresponding to predicted absorbed radiation dose of 200, 400 and 800 Gy, respectively. Another group received non-loaded microspheres (sham group). Cranial and caudal lobes volumes were assessed using CT volumetry before, 15 and 30 days after SIRT. Liver biochemistry, histopathology and gene expression were evaluated. Four untreated rabbits were used as controls for gene expression studies. All animals receiving 1.2 GBq were euthanized due to clinical deterioration. Cranial SIRT with 0.6 GBq induced caudal lobe hypertrophy after 15 days (median increase 34% -ns-) but produced significant toxicity. Cranial SIRT with 0.3 GBq induced caudal lobe hypertrophy after 30 days (median increase 82%, p = 0.04). No volumetric changes were detected in sham group. Transient increase in serum transaminases was detected in all treated groups returning to normal values at 15 days. There was dose-dependent liver dysfunction with bilirubin elevation and albumin decrease. Histologically, 1.2 GBq group developed permanent severe liver damage with massive necrosis, 0.6 and 0.3 GBq groups developed moderate damage with inflammation and portal fibrosis at 15 days, partially recovering at 30 days. There was no difference in the expression of hepatocyte function and differentiation genes between 0.3 GBq and control groups. Cranial SIRT with 0.3 GBq of Y-90 resin microspheres in rabbits is a reliable animal model to analyse the atrophy-hypertrophy complex and liver damage without toxicity.

Dopaminergic denervation in patients with Parkinson's disease is associated with changes in brain metabolism. Cerebral in-vivo mapping of glucose metabolism has been studied in severe stable parkinsonian monkeys, but data on brain metabolic changes in early stages of dopaminergic depletion of this model is lacking. Here, we report cerebral metabolic changes associated with progressive nigrostriatal lesion in the pre-symptomatic and symptomatic stages of the progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model of Parkinson's Disease. Monkeys (Macaca fascicularis) received MPTP injections biweekly to induce progressive grades of dopamine depletion. Monkeys were sorted according to motor scale assessments in control, asymptomatic, recovered, mild, and severe parkinsonian groups. Dopaminergic depletion in the striatum and cerebral metabolic patterns across groups were studied in vivo by positron emission tomography (PET) using monoaminergic ([11C]-dihydrotetrabenazine; 11C-DTBZ) and metabolic (2-[18F]-fluoro-2-deoxy-d-glucose; 18F-FDG) radiotracers. 11C-DTBZ-PET analysis showed progressive decrease of binding potential values in the striatum of monkeys throughout MPTP administration and the development of parkinsonian signs. 18F-FDG analysis in asymptomatic and recovered animals showed significant hypometabolism in temporal and parietal areas of the cerebral cortex in association with moderate dopaminergic nigrostriatal depletion. Cortical hypometabolism extended to involve a larger area in mild parkinsonian monkeys, which also exhibited hypermetabolism in the globus pallidum pars interna and cerebellum. In severe parkinsonian monkeys, cortical hypometabolism extended further to lateral-frontal cortices and hypermetabolism also ensued in the thalamus and cerebellum. Unbiased histological quantification of neurons in Brodmann's area 7 in the parietal cortex did not reveal neuron loss in parkinsonian monkeys versus controls. Early dopaminergic nigrostriatal depletion is associated with cortical, mainly temporo-parietal hypometabolism unrelated to neuron loss. These findings, together with recent evidence from Parkinson's Disease patients, suggest that early cortical hypometabolism may be associated and driven by subcortical changes that need to be evaluated appropriately. Altogether, these findings could be relevant when potential disease modifying therapies become available.

Wilson's disease (WD) is an inherited disorder of copper metabolism associated with mutations in ATP7B gene. We have shown that the administration of an adeno-associated vector (AAV) encoding a mini version of human ATP7B (VTX-801) provides long-term correction of copper metabolism in a murine WD model. In preparation of a future clinical trial, we have evaluated by positron emission tomography (PET) the value of 64Cu biodistribution, excretion pattern, and blood kinetics as pharmacodynamic biomarkers of VTX-801 effects. Six-week-old WD mice were injected intravenously with increasing doses of VTX-801 and 3 weeks or 3 months later with [64Cu]CuCl2. Untreated WD and wild-type (WT) mice were included as controls. Control WD mice showed increased hepatic 64Cu retention, reduced fecal excretion of the radiotracer, and altered 64Cu blood kinetics (BK) compared with WT mice. VTX-801 treatment in WD mice resulted in a significant reduction of hepatic 64Cu accumulation, the restoration of fecal 64Cu excretion, and the correction of 64Cu BK. This study showed that VTX-801 restores physiological copper metabolism in WD mice, confirming the mechanism of action of VTX-801, and demonstrated the translational potential of [64Cu]CuCl2-PET to explore VTX-801 pharmacodynamics in a minimally invasive and sensitive manner in WD patients.

Enterotoxigenic Escherichia coli (ETEC) represents a major cause of morbidity and mortality in the human population. In particular, ETEC infections affect children under the age of five from low-middle income countries. However, there is no licensed vaccine against this pathogen. ETEC vaccine development is challenging since this pathotype expresses a wide variety of antigenically diverse virulence factors whose genes can be modified due to ETEC genetic plasticity. To overcome this challenge, we propose the use of outer membrane vesicles (OMVs) isolated from two ETEC clinical strains. In these OMVs, proteomic studies revealed the presence of important immunogens, such as heat-labile toxin, colonization factors, adhesins and mucinases. Furthermore, these vesicles proved to be immunogenic after subcutaneous administration in BALB/c mice. Since ETEC is an enteropathogen, it is necessary to induce both systemic and mucosal immunity. For this purpose, the vesicles, free or encapsulated in zein nanoparticles coated with a Gantrez(R)-mannosamine conjugate, were administered orally. Biodistribution studies showed that the encapsulation of OMVs delayed the transit through the gut. These results were confirmed by in vivo study, in which OMV encapsulation resulted in higher levels of specific antibodies IgG2a. Further studies are needed to evaluate the protection efficacy of this vaccine approach.

The aim was to study the performance of the U-SPECT6/CT E-class system for preclinical imaging, to later demonstrate the viability of simultaneous multi-animal and multi-isotope imaging with reliable quantitative accuracy. The performance of the SPECT was evaluated for two collimators dedicated for mouse (UHS-M) and rat imaging (UHR-RM) in terms of sensitivity, energy resolution, uniformity and spatial resolution. Point sources, hot-rod and uniform phantoms were scanned, and additional tests were carried out to evaluate singular settings such as simultaneous multi-isotope acquisition and imaging with a multi-bed system. For in-vivo evaluation, simultaneous triple-isotope and multi-animal studies were performed on mice. Sensitivity for Tc-99m was 2370 cps/MBq for the UHS-M collimator and 493 cps/MBq for the UHR-RM. Rods of 0.6 mm and 0.9 mm were discernible with the UHS-M and UHR-RM collimators respectively, with optimized reconstruction. Uniformity in low counting conditions has proven to be poor (> 75%). Multi-isotope and multi-bed phantom acquisitions demonstrated accurate quantification. In mice, simultaneous multi-isotope imaging provided the separate distribution of 3 tracers and image quality of the multi-mouse bone scan was adequate. The U-SPECT6/CT E-class has shown good sensitivity and spatial resolution. This system provides quantitative images with suitable image quality for multi-mouse and multi-isotope acquisitions.

Negatively charged microspheres (NCMs) are postulated as a new form of treatment for chronic wounds. Despite the efficacy shown at clinical level, more studies are required to demonstrate their safety and local effect. The objective of the work was to confirm the lack of NCM systemic absorption performing a biodistribution study of the NCMs in an open wound rat animal model. To this end, radiolabeling of NCMs with technetium-99m was optimized and biodistribution studies were performed by in vivo SPEC/CT imaging and ex vivo counting during 24 h after topical administration. The studies were performed on animals treated with a single or repeated dose to study the effect of macrophages during a prolonged treatment. NCM radiolabeling was achieved in a simple, efficient and stable manner with high yield. SPECT/CT images showed that almost all NCMs (about 85 %) remained on the wound for 24 h either after single or multiple administrations. Ex vivo biodistribution studies confirmed that there was no accumulation of NCMs in any organ or tissue except in the wound area, suggesting a lack of absorption. In conclusion, NCMs can be considered safe as local wound treatment since they remain at the administration area.

Brown adipose tissue (BAT) dysfunction in aging and obesity has been related to chronic unresolved inflammation, which could be mediated by an impaired production of specialized proresolving lipid mediators (SPMs), such as Lipoxins-LXs, Resolvins-Rvs, Protectins-PDs, and Maresins-MaRs. Our aim was to characterize the changes in BAT SPMs signatures and their association with BAT dysfunction during aging, especially under obesogenic conditions, and their modulation by a docosahexaenoic acid (DHA)-rich diet. Lipidomic, functional, and molecular studies were performed in BAT of 2- and 18-month-old lean (CT) female mice and in 18-month-old diet-induced obese (DIO) mice fed with a high-fat diet (HFD), or a DHA-enriched HFD. Aging downregulated Prdm16 and UCP1 levels, especially in DIO mice, while DHA partially restored them. Arachidonic acid (AA)-derived LXs and DHA-derived MaRs and PDs were the most abundant SPMs in BAT of young CT mice. Interestingly, the sum of LXs and of PDs were significantly lower in aged DIO mice compared to young CT mice. Some of the SPMs most significantly reduced in obese-aged mice included LXB4, MaR2, 4S,14S-diHDHA, 10S,17S-diHDHA (a.k.a. PDX), and RvD6. In contrast, DHA increased DHA-derived SPMs, without modifying LXs. However, MicroPET studies showed that DHA was not able to counteract the impaired cold exposure response in BAT of obese-aged mice. Our data suggest that a defective SPMs production could underlie the decrease of BAT activity observed in obese-aged mice, and highlight the relevance to further characterize the physiological role and therapeutic potential of specific SPMs on BAT development and function.

Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome-reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm-associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome-reduced bacterium that can fight against clinically relevant biofilm-associated bacterial infections.

Mutations in the GBA1 gene coding for glucocerebrosidase (GCase) are the main genetic risk factor for Parkinson's disease (PD). Indeed, identifying reduced GCase activity as a common feature underlying the typical neuropathological signatures of PD-even when considering idiopathic forms of PD-has recently paved the way for designing novel strategies focused on enhancing GCase activity to reduce alpha-synuclein burden and preventing dopaminergic cell death. Here we have performed bilateral injections of a viral vector coding for the mutated form of alpha-synuclein (rAAV9-SynA53T) for disease modeling purposes, both in mice as well as in nonhuman primates (NHPs), further inducing a progressive neuronal death in the substantia nigra pars compacta (SNpc). Next, another vector coding for the GBA1 gene (rAAV9-GBA1) was unilaterally delivered in the SNpc of mice and NHPs one month after the initial insult, together with the contralateral delivery of an empty/null rAAV9 for control purposes. Obtained results showed that GCase enhancement reduced alpha-synuclein burden, leading to improved survival of dopaminergic neurons. Data reported here support using GCase gene therapy as a disease-modifying treatment for PD and related synucleinopathies, including idiopathic forms of these disorders.

The aim of this work was to study the biodistribution of bevacizumab-loaded HSA nanoparticles (NP-Ab) crosslinked with PEG35000 by SPECT/CT in vivo imaging. For this purpose, NP-Abs were prepared by a desolvation process, coated with PEG35000 and radiolabeled with technetium-99 m using a pre-tinning method ([99mTc]TcNP-Ab). The Ab was labeled using [99mTc][Tc(CO)3(H2O)3]+ and used to prepare nanoparticles (NP-[99mTc]TcAb). Particle size was similar in both formulations. Chemical and radiochemical purity of the two nanosystems were >95%. Bevacizumab-labeling conditions were tested by in vitro stability studies. More than 87% of the radiolabeled antibody remained intact for 24 h after incubation with plasma. SPECT/CT imaging of the two nanoparticles was performed in healthy female Wistar rats. Ex vivo gamma counting of selected organs was also carried out in all animals. The results showed different clearance rates of the nanoparticle shell and the antibody, providing valuable information by the use of molecular imaging in the evaluation of drug delivery nanosystems.

Variegate porphyria (VP) results from haploinsufficiency of pro-toporphyrinogen oxidase (PPDX), the seventh enzyme in the heme synthesis pathway. There is no VP model that recapitulates the clinical manifestations of acute attacks. Combined administrations of 2-allyl-2-isopropylacetamide and rifampicin in rabbits halved hepatic PP OX activity, resulting in increased accumulation of a potentially neurotoxic heme precursor, lipid peroxidation, inflammation, and hepatocyte cytoplasmic stress. Rabbits also showed hypertension, motor impairment, reduced activity of critical mitochondrial hemoprotein functions, and altered glucose homeostasis. Hemin treatment only resulted in a slight drop in heme precursor accumulation but further increased hepatic heme catabolism, inflammation, and cytoplasmic stress. Hemin replenishment did protect against hypertension, but it failed to restore action potentials in the sciatic nerve or glucose homeostasis. Systemic porphobilinogen deaminase (PBGD) mRNA administration increased hepatic PBGD activity, the third enzyme of the pathway, and rapidly normalized serum and urine porphyrin precursor levels. All features studied were improved, including those related to critical hemoprotein functions. In conclusion, the VP model recapitulates the biochemical characteristics and some clinical manifestations associated with severe acute attacks in humans.

Bevacizumab (as other monoclonal antibodies) has now become a mainstay in the treatment of several cancers in spite of some limitations, including poor tumour penetration and the development of resistance mechanisms. Its nanoencapsulation may be an adequate strategy to minimize these problems. The aim of this work was to evaluate the efficacy of bevacizumab-loaded nanoparticles (B-NP-PEG) on a xenograft model of human colorectal cancer. For this purpose, human serum albumin nanoparticles were prepared by coacervation, then coated with poly(ethylene glycol) and freeze-dried. B-NP-PEG displayed a mean size of about 300 nm and a bevacizumab loading of approximately 145 ¿g/mg. An in vivo study was conducted in the HT-29 xenograft model of colorectal cancer. Both, free and nanoencapsulated bevacizumab, induced a similar reduction in the tumour growth rate of about 50%, when compared to controls. By microPET imaging analysis, B-NP-PEG was found to be a more effective treatment in decreasing the glycolysis and metabolic tumour volume than free bevacizumab, suggesting higher efficacy. These results correlated well with the capability of B-NP-PEG to increase about fourfold the levels of intratumour bevacizumab, compared with the conventional formulation. In parallel, B-NP-PEG displayed six-times lower amounts of bevacizumab in blood than the aqueous formulation of the antibody, suggesting a lower incidence of potential undesirable side effects. In summary, albumin-based nanoparticles may be adequate carriers to promote the delivery of monoclonal antibodies (i.e. bevacizumab) to tumour tissues.

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G(2)-M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in down-regulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD. Significance: These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.

Re-activation of the healing process is a major challenge in the field of chronic wound treatment. For that purpose, lipid-nanoparticles, especially nanostructured lipid carriers (NLC), possess extremely useful characteristics such as biodegradability, biocompatibility and long-term stability, besides being suitable for drug delivery. Moreover, they maintain wound moisture due to their occlusive properties, which have been associated with increased healing rates. In the light of above, NLC have been extensively used topically for wound healing; but to date, there are no safety-preclinical studies concerning such type of application. Thus, in this work, biodistribution studies were performed in rats with the NLC previously developed by our research group, using technetium-99 m (99mTc-NLC) as radiomarker, topically administered on a wound. 99mTc-NLC remained on the wound for 24 h and systemic absorption was not observed after administration. In addition, toxicological studies were performed to assess NLC safety after topical administration. The results obtained demonstrated that NLC were non-cytotoxic, non-sensitizing and non-irritant/corrosive. Overall, it might be concluded that developed NLC remained at the administration area, potentially exerting a local effect, and were safe after topical administration on wounds.

BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children and young adults that produces aberrant osteoid. The aim of this study was to assess the utility of 2-deoxy-2-[18F-] fluoro-D-glucose ([18F] FDG) and sodium [18F] Fluoride (Na [18F] F) PET scans in orthotopic murine models of osteosarcoma to describe the metabolic pattern of the tumors, to detect and diagnose tumors and to evaluate the efficacy of a new treatment based in oncolytic adenoviruses. METHODS: Orthotopic osteosarcoma murine models were created by the injection of 143B and 531MII cell lines. [18F]FDG and Na [18F] F PET scans were performed 30 days (143B) and 90 days (531MII) post-injection. The antitumor effect of two doses (107 and 108 pfu) of the oncolytic adenovirus VCN-01 was evaluated in 531 MII model by [18F] FDG PET studies. [18F] FDG uptake was quantified by SUVmax and Total Lesion Glycolysis (TLG) indexes. For Na [18F] F, the ratio tumor SUVmax/hip SUVmax was calculated. PET findings were confirmed by histopathological techniques. RESULTS: The metabolic pattern of tumors was different between both orthotopic models. All tumors showed [18F] FDG uptake, with a sensitivity and specificity of 100%. The [18F] FDG uptake was significantly higher for the 143B model (p < 0.001). Sensitivity for Na [18F] F was around 70% in both models, with a specificity of 100%. 531MII tumors showed a heterogeneous Na [18F] F uptake, significantly higher than 143B tumors (p < 0.01).

Background: The feasibility of beta cell mass (BCM) imaging and quantification with positron emission tomography (PET) in the pancreas is controversial. In an effort to shed some light on this topic, we have used a xenograft model of rat insulinoma (RIN) in mice, mimicking an intramuscular islet transplantation situation. Methods: A total of 105 RIN cells were subcutaneously implanted in nude mice (N.=8). Tumor size and glycaemia levels were determined daily. Rat C-peptide was measured to demonstrate rat insulin production. PET imaging with 11C-(+)-¿-dihydrotetrabenazine (11C-DTBZ) was done at 3 and 4 weeks and compared with 18F-FDG and 18F-DOPA studies in the same mice. Ex-vivo autoradiography with 11C-DTBZ was carried out in frozen sections of tumors. VMAT2 expression was measured by Western-blot and immunohistochemistry in tumors and RIN cells. Results: Functional rat insulin production in mice was demonstrated by substantial decrease in glycaemia (<50 mg/dL by week 4) and rat C-peptide levels (7.2±2.6 ng/mL) similar to those measured in control rats. PET studies showed that tumor imaging with 11C-DTBZ at four (N.=8) and five (N.=5) weeks was negative; only bigger tumors could be seen with 18F-DOPA. In explanted tumors 11C-DTBZ autoradiography was negative, albeit VMAT2 expression measured by Western-blot and immunohistochemistry was lower than in cultured RIN cells. Conclusions: Although insulinomas are fully functional it does not seem feasible to use 11C-DTBZ for in-vivo measuring of BCM. This might either be due to inherent technical limitations of PET, decrease in VMAT2 expression in the tumors due to unknown reasons, or other biological limiting facts.

PET/CT imaging of 18F-FDG-labeled CSC allows quantifying biodistribution and acute retention of implanted cells in a clinically relevant pig model of chronic myocardial infarction. Similar levels of acute retention are achieved when cells are IM or IC administered. However, acute cell retention does not correlate with cell engraftment, which is improved by IM injection.

Id1 promotes carcinogenesis and metastasis, and predicts prognosis of non-small cell lung cancer (NSCLC)-adenocarcionoma patients. We hypothesized that Id1 may play a critical role in lung cancer colonization of the liver by affecting both tumor cells and the microenvironment. Depleted levels of Id1 in LLC (Lewis lung carcinoma cells, LLC shId1) significantly reduced cell proliferation and migration in vitro. Genetic loss of Id1 in the host tissue (Id1(-/-) mice) impaired liver colonization and increased survival of Id1 animals. Histologically, the presence of Idl in tumor cells of liver metastasis was responsible for liver colonization. Microarray analysis comparing liver tumor nodules from Id1(+/+) mice and Idl(-/-) mice injected with LLC control cells revealed that Id1 loss reduces the levels of EMT-related proteins, such as vimentin. In tissue microarrays containing 532 NSCLC patients' samples, we found that Idl significantly correlated with vimentin and other EMT-related proteins. Idl loss decreased the levels of vimentin, integrin beta 1, TGF beta 1 and snail, both in vitro and in vivo. Therefore, Id1 enables both LLC and the host microenvironment for an effective liver colonization, and may represent a novel therapeutic target to avoid NSCLC liver metastasis. (C) 2017 Elsevier B.V. All rights reserved.

Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria, AIP) is characterized by neurovisceral attacks when hepatic heme synthesis is activated by endogenous or environmental factors including fasting. While the molecular mechanisms underlying the nutritional regulation of hepatic heme synthesis have been described, glucose homeostasis during fasting is poorly understood in porphyria. Our study aimed to analyse glucose homeostasis and hepatic carbohydrate metabolism during fasting in PBGD-deficient mice. To determine the contribution of hepatic PBGD deficiency to carbohydrate metabolism, AIP mice injected with a PBGD-liver gene delivery vector were included. After a 14 h fasting period, serum and liver metabolomics analyses showed that wild-type mice stimulated hepatic glycogen degradation to maintain glucose homeostasis while AIP livers activated gluconeogenesis and ketogenesis due to their inability to use stored glycogen. The serum of fasted AIP mice showed increased concentrations of insulin and reduced glucagon levels. Specific over-expression of the PBGD protein in the liver tended to normalize circulating insulin and glucagon levels, stimulated hepatic glycogen catabolism and blocked ketone body production. Reduced glucose uptake was observed in the primary somatosensorial brain cortex of fasted AIP mice, which could be reversed by PBGD-liver gene delivery. In conclusion, AIP mice showed a different response to fasting as measured by altered carbohydrate metabolism in the liver and modified glucose consumption in the brain cortex. Glucose homeostasis in fasted AIP mice was efficiently normalized after restoration of PBGD gene expression in the liver.

BACKGROUND: Index lesion characterization is important in the evaluation of primary prostate carcinoma (PPC). The aim of this study was to analyze the contribution of (11) C-Choline PET/CT and the Apparent Diffusion Coefficient maps (ADC) in detecting the Index Lesion and clinically significant tumors in PPC. METHODS: Twenty-one untreated patients with biopsy-proven PPC and candidates for radical prostatectomy (RP) were prospectively evaluated by means of Ultra-High Definition PET/CT and 3T MRI, which included T2-weighted imaging (T2WI) and ADC maps obtained from diffusion weighted imaging (DWI). Independent experts analyzed all the images separately and were unaware of the pathological data. In each case, the Index lesion was defined as the largest tumor measured on histopathology (Index H). In addition, the largest lesion observed on MRI (Index MRI) and the highest avid (11) C-Choline uptake lesion (Index PET) were obtained. The Gleason scores (GS) of the tumors were determined. PET/CT and ADC map quantitative parameters were also calculated. Measures of correlation among imaging parameters as well as the sensitivity (S), specificity (Sp), negative and positive predictive values (NPV and PPV) for tumor detection were analyzed. All data was validated with the pathological study. RESULTS: In the morphological study, 139 foci of carcinoma were identified, 47 of which corresponded to clinically significant tumors (>0.5¿cm(3) ). The remaining foci presented a maximum diameter (dmax ) of 0.1¿cm¿±¿SD 0.75 and were not classified as clinically significant. Thirty-two tumors presented a GS (3¿+¿3), nine GS (3¿+¿4), and six GS (4¿+¿3). A total of 21 Index H (dmax ¿=¿1.37¿cm SD¿±¿0.61) were identified. The S, Sp, NPV, and PPV for tumor detection with PET were 100%, 70%, 83%, 100%, and for MRI were 46%, 100%, 100%, 54%, respectively. Both Index PET and Index MRI were complementary and identified 95% of the Index H when quantitative criteria were used. CONCLUSION: In spite of the fact that PET imaging has higher tumor sensitivity than MRI, (11) C-Choline PET and ADC maps have complementary roles in the evaluation of Index Lesion in PPC. Index PET and Index MRI could be complementary targets in the therapeutic planning of PPC.

We studied the role of matrix metalloproteinase-10 (MMP-10) during skeletal muscle repair after ischemia using a model of femoral artery excision in wild-type (WT) and MMP-10 deficient (Mmp10(-/-)) mice. Functional changes were analyzed by small animal positron emission tomography and tissue morphology by immunohistochemistry. Gene expression and protein analysis were used to study the molecular mechanisms governed by MMP-10 in hypoxia. Early after ischemia, MMP-10 deficiency resulted in delayed tissue reperfusion (10%, P < 0.01) and in increased necrosis (2-fold, P < 0.01), neutrophil (4-fold, P < 0.01), and macrophage (1.5-fold, P < 0.01) infiltration. These differences at early time points resulted in delayed myotube regeneration in Mmp10(-/-) soleus at later stages (regenerating myofibers: 30 ± 9% WT vs. 68 ± 10% Mmp10(-/-), P < 0.01). The injection of MMP-10 into Mmp10(-/-) mice rescued the observed phenotype. A molecular analysis revealed higher levels of Cxcl1 mRNA (10-fold, P < 0.05) and protein (30%) in the ischemic Mmp10(-/-) muscle resulting from a lack of transcriptional inhibition by MMP-10. This was further confirmed using siRNA against MMP-10 in vivo. Our results demonstrate an important role of MMP-10 for proper muscle repair after ischemia, and suggest that chemokine regulation such as Cxcl1 by MMP-10 is involved in muscle regeneration

PRGF is a platelet concentrate within a plasma suspension that forms an in situ-generated fibrin-matrix delivery system, releasing multiple growth factors and other bioactive molecules that play key roles in tissue regeneration. This study was aimed at exploring the angiogenic and myogenic effects of PRGF on in vitro endothelial cells (HUVEC) and skeletal myoblasts (hSkMb) as well as on in vivo mouse subcutaneously implanted matrigel and on limb muscles after a severe ischemia. Human PRGF was prepared and characterized. Both proliferative and anti-apoptotic responses to PRGF were assessed in vitro in HUVEC and hSkMb. In vivo murine matrigel plug assay was conducted to determine the angiogenic capacity of PRGF, whereas in vivo ischemic hind limb model was carried out to demonstrate PRGF-driven vascular and myogenic regeneration. Primary HUVEC and hSkMb incubated with PRGF showed a dose dependent proliferative and anti-apoptotic effect and the PRGF matrigel plugs triggered an early and significant sustained angiogenesis compared with the control group. Moreover, mice treated with PRGF intramuscular infiltrations displayed a substantial reperfusion enhancement at day 28 associated with a fibrotic tissue reduction. These findings suggest that PRGF-induced angiogenesis is functionally effective at expanding the perfusion capacity of the new vasculature and attenuating the endogenous tissue fibrosis after a severe-induced skeletal muscle ischemia. (C) 2015 Elsevier B.V. All rights reserved.

Background-Microvascular endothelium in different organs is specialized to fulfill the particular needs of parenchymal cells. However, specific information about heart capillary endothelial cells (ECs) is lacking. Methods and Results-Using microarray profiling on freshly isolated ECs from heart, brain, and liver, we revealed a genetic signature for microvascular heart ECs and identified Meox2/Tcf15 heterodimers as novel transcriptional determinants. This signature was largely shared with skeletal muscle and adipose tissue endothelium and was enriched in genes encoding fatty acid (FA) transport-related proteins. Using gain-and loss-of-function approaches, we showed that Meox2/Tcf15 mediate FA uptake in heart ECs, in part, by driving endothelial CD36 and lipoprotein lipase expression and facilitate FA transport across heart ECs. Combined Meox2 and Tcf15 haplodeficiency impaired FA uptake in heart ECs and reduced FA transfer to cardiomyocytes. In the long term, this combined haplodeficiency resulted in impaired cardiac contractility. Conclusions-Our findings highlight a regulatory role for ECs in FA transfer to the heart parenchyma and unveil 2 of its intrinsic regulators. Our insights could be used to develop new strategies based on endothelial Meox2/Tcf15 targeting to modulate FA transfer to the heart and remedy cardiac dysfunction resulting from altered energy substrate usage.

Carbon-11 labeled dihydrotetrabenazine (11C-DTBZ) binds to the vesicular monoamine transporter 2 and has been used to assess nigro-striatal integrity in animal models and patients with Parkinson's disease. Here, we applied 11C-DTBZ positron emission tomography (PET) to obtain longitudinally in-vivo assessment of striatal dopaminergic loss in the classic unilateral and in a novel bilateral 6-hydroxydopamine (6-OHDA) lesion rat model. Forty-four Sprague-Dawley rats were divided into 3 sub-groups: 1. 6-OHDA-induced unilateral lesion in the medial forebrain bundle, 2. bilateral lesion by injection of 6-OHDA in the third ventricle, and 3. vehicle injection in either site. 11C-DTBZ PET studies were investigated in the same animals successively at baseline, 1, 3 and 6weeks after lesion using an anatomically standardized volumes-of-interest approach. Additionally, 12 rats had PET and Magnetic Resonance Imaging to construct a new 11C-DTBZ PET template. Behavior was characterized by rotational, catalepsy and limb-use asymmetry tests and dopaminergic striatal denervation was validated post-mortem by immunostaining of the dopamine transporter (DAT). 11C-DTBZ PET showed a significant decrease of striatal binding (SB) values one week after the unilateral lesion. At this point, there was a 60% reduction in SB in the affected hemisphere compared with baseline values in 6-OHDA unilaterally lesioned animals. A 46% symmetric reduction over baseline SB values was found in bilaterally lesioned rats at the first week after lesion. SB values remained constant in unilaterally lesioned rats whereas animals with bilateral lesions showed a modest (22%) increase in binding values at the 3rd and 6th weeks post-lesion. The degree of striatal dopaminergic denervation was corroborated histologically by DAT immunostaining. Statistical analysis revealed a high correlation between 11C-DTBZ PET SB and striatal DAT immunostaining values (r=0.95, p<0.001). The data presented here indicate that 11C-DTBZ PET may be used to ascertain changes occurring in-vivo throughout the evolution of nigro-striatal dopaminergic neurodegeneration, mainly in the unilateral 6-OHDA lesion rat.

Background: [F-18]-tetrafluoroborate is a PET radiotracer taken up by the sodium/iodide symporter (NIS). Albeit the in vivo behavior in rodents is similar to the (99)mTc-pertechnetate, no studies exist in primates or in humans. The aims of this study were to evaluate the biodistribution of [F-18]-tetrafluoroborate in non-human primates with PET and to estimate the absorbed dose in organs. Methods: Whole-body PET imaging was done in a Siemens ECAT HR+ scanner in two male Macaca fascicularis monkeys. After an i.v. injection of 24.93 +/- 0.05 MBq/kg of [F-18]-tetrafluoroborate, prepared by isotopic exchange of sodium tetrafluoroborate with [F-18]-fluoride under acidic conditions, eight sequential images from the head to the thigh (five beds) were collected for a total duration of 132 min. The whole-body emission scan was reconstructed applying attenuation and scatter corrections. After image reconstruction, three-dimensional volumes of interest (VOIs) were hand-drawn on the PET transaxial or coronal slices of the frame where the organ was most conspicuous. Time-activity curves for each VOI were obtained, and the organ residence times were calculated by integration of the time-activity curves. Human absorbed doses were estimated using the OLINDA/EXM software and the standard human model. Results: [F-18]-tetrafluoroborate was able to discriminate clearly the thyroid gland with an excellent signal-to-noise ratio. Most of the radiotracers (residence time) are localised in the orga

A mouse model was developed for in vivo monitoring of infection and the effect of antimicrobial treatment against Staphylococcus aureus biofilms, using the [(18)F]fluoro-deoxyglucose-MicroPET ([(18)F]FDG-MicroPET) image technique. In the model, sealed Vialon catheters were briefly precolonized with S. aureus strains ATCC 15981 or V329, which differ in cytotoxic properties and biofilm matrix composition. After subcutaneous implantation of catheters in mice, the S. aureus strain differences found in bacterial counts and the inflammatory reaction triggered were detected by the regular bacteriological and histological procedures and also by [(18)F]FDG-MicroPET image signal intensity determinations in the infection area and regional lymph node. Moreover, [(18)F]FDG-MicroPET imaging allowed the monitoring of the rifampin treatment effect, identifying the periods of controlled infection and those of reactivated infection due to the appearance of bacteria naturally resistant to rifampin. Overall, the mouse model developed may be useful for noninvasive in vivo determinations in studies on S. aureus biofilm infections and assessment of new therapeutic approaches.

Much controversy exists concerning the effect of levodopa on striatal dopaminergic markers in Parkinson's disease (PD) and its influence on functional neuroimaging. To deal with this issue we studied the impact of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and chronic levodopa administration on striatal (18)F-DOPA uptake (Ki) in an animal model of PD. The levels of several striatal dopaminergic markers and the number of surviving dopaminergic neurons in the substantia nigra (SN) were also assessed. Eleven Macaca fascicularis were included in the study. Eight animals received weekly intravenous injections of MPTP for 7weeks and 3 intact animals served as controls. MPTP-monkeys were divided in two groups. Group I was treated with placebo while Group II received levodopa. Both treatments were maintained for 11months and then followed by a washout period of 6months. (18)F-DOPA positron emission tomography (PET) scans were performed at baseline, after MPTP intoxication, following 11months of treatment, and after a washout period of 1, 3 and 6months. Monkeys were sacrificed 6months after concluding either placebo or levodopa treatment and immediately after the last (18)F-DOPA PET study. Striatal dopamine transporter (DAT) content, tyrosine hydroxylase (TH) content and aromatic l-amino acid decarboxylase (AADC) content were assessed. In Group II (18)F-DOPA PET studies performed at 3 and 6months after interrupting levodopa showed a significantly increased Ki in the anterior putamen as compared to Group I. Levodopa and placebo treated animals exhibited a similar number of surviving dopaminergic cells in the SN. Striatal DAT content was equally reduced in both groups of animals. Animals in Group I exhibited a significant decrease in TH protein content in all the striatal regions assessed. However, in Group II, TH levels were significantly reduced only in the anterior and posterior putamen. Surprisingly, in the levodopa-treated animals the TH levels in the posterior putamen were significantly lower than those in the placebo group. AADC levels in MPTP groups were similar to those of control animals in all striatal areas analyzed. This study shows that chronic levodopa administration to monkeys with partial nigrostriatal degeneration followed by a washout period induces modifications in the functional activity of the dopaminergic nigrostriatal pathway.

La imagen preclínica engloba distintas técnicas de imagen molecular que utilizan radiaciones ionizantes, destacando la tomografía por emisión de fotón único (SPECT), la tomografía de emisión de positrones (PET) y la imagen autorradiográfica. Cada una de ellas utiliza distintas sondas que permiten obtener imágenes de una gran variedad de procesos metabólicos. En ocasiones se emplean junto a equipos de rayos X que permiten obtener imágenes anatómicas. En consecuencia, la investigación en las instalaciones de imagen molecular preclínica deberá realizarse en un contexto en el que se aplique la protección radiológica. De entre los riesgos radiológicos del personal que opera este tipo de instalaciones destaca la irradiación de las manos producida tanto por la administración de los radiotrazadores a los animales como por la manipulación de los mismos, siendo importante el entrenamiento y el uso de los blindajes. El diseño de la instalación radiactiva estará determinado por la actividad que en ella se realice. En particular dependerá tanto de las distintas técnicas de imagen molecular preclínica que se desarrollen como de la relación funcional que la instalación tenga con el resto del centro en el que se encuentre, en particular con el animalario y la unidad de radiofarmacia.

A precise equilibrium between cellular differentiation and proliferation is fundamental for tissue homeostasis. Maintaining this balance is particularly important for the liver, a highly differentiated organ with systemic metabolic functions that is endowed with unparalleled regenerative potential. Carcinogenesis in the liver develops as the result of hepatocellular de-differentiation and uncontrolled proliferation. Here, we identified SLU7, which encodes a pre-mRNA splicing regulator that is inhibited in hepatocarcinoma, as a pivotal gene for hepatocellular homeostasis. SLU7 knockdown in human liver cells and mouse liver resulted in profound changes in pre-mRNA splicing and gene expression, leading to impaired glucose and lipid metabolism, refractoriness to key metabolic hormones, and reversion to a fetal-like gene expression pattern. Additionally, loss of SLU7 also increased hepatocellular proliferation and induced a switch to a tumor-like glycolytic phenotype. Slu7 governed the splicing and/or expression of multiple genes essential for hepatocellular differentiation, including serine/arginine-rich splicing factor 3 (Srsf3) and hepatocyte nuclear factor 4¿ (Hnf4¿), and was critical for cAMP-regulated gene transcription. Together, out data indicate that SLU7 is central regulator of hepatocyte identity and quiescence.

There is a need for comparative studies to determine which cell types are better candidates to remedy ischemia. Here, we compared human AC133(+) cells and multipotent adult progenitor cells (hMAPC) in a mouse model reminiscent of critical limb ischemia. hMAPC or hAC133(+) cell transplantation induced a significant improvement in tissue perfusion (measured by microPET) 15 days posttransplantation compared to controls. This improvement persisted for 30 days in hMAPC-treated but not in hAC133(+)-injected animals. While transplantation of hAC133(+) cells promoted capillary growth, hMAPC transplantation also induced collateral expansion, decreased muscle necrosis/fibrosis, and improved muscle regeneration. Incorporation of differentiated MC 133(+) or hMAPC progeny into new vessels was limited; however, a paracrine angio/arteriogenic effect was demonstrated in animals treated with hMAPC. Accordingly, hMAPC-conditioned, but not hAC133(+)-conditioned, media stimulated vascular cell proliferation and prevented myoblast, endothelial, and smooth muscle cell apoptosis in vitro. Our study suggests that although hAC133(+) cell and hMAPC transplantation both contribute to vascular regeneration in ischemic limbs, hMAPC exert a more robust effect through trophic mechanisms, which translated into collateral and muscle fiber regeneration. This, in turn, conferred tissue protection and regeneration with longer term functional improvement.

Purpose: Study by molecular imaging the biodistribution of poly(anhydride) nanoparticles after oral administration. Procedures: Poly (anhydride) nanoparticles (NP) and cyclodextrin-tagged nanoparticles (CD-NP) were radiolabelled with Tc-99m. Radiochemical purity was measured with a double-solvent chromatography system and the absence of undesirable components was confirmed by size and polydispersion measurement of the technetium-labelled nanoparticles by photon correlation spectroscopy. Single photon emission computed tomography (SPECT) fused computed tomography (CT) in vivo molecular imaging was used for biodistribution studies in small animals. Results: SPECT-CT images revealed activity only in the gastrointestinal tract. Thirteen percent of the given dose of CD-NP and 3% of the given dose of conventional NP were found in the stomach at 8 h. Conclusion: No evidence of translocation or distribution out of gastrointestinal tract was found. CD-NP moved significantly more slowly inside the gut than conventional NP, probably due to their physico-chemical structure that allows stronger interactions with the gut mucosa.

This study focuses on the occupational doses of technologists working at an Animal Research Unit using PET radiotracers and on the environmental dose rates produced by the animals (mice, rats and monkeys). In particular, whole body and extremity monitoring is reported and related with the workload. The study shows that doses not only depend on the amount of activity injected but also on the type of animals and radiotracers managed. The extremities, with a great variability of the doses received, are the limiting organs as far as regulatory dose limits for workers are concerned. Mean H¿(10) rates in contact and at 20 cm from the animals, when they are handled by the technologist, range from around 1 mSv/h to 20 ¿Sv/h, respectively.

This study demonstrates that any difference detected with SPM analysis of MOSAIC PET images of rat brain is detected also by the gold standard autoradiographic technique, confirming that this methodology provides reliable results, although partial volume effects might make it difficult to detect slight differences in small regions.

BACKGROUND: Leptin and nitric oxide (NO) on their own participate in the control of non-shivering thermogenesis. However, the functional interplay between both factors in this process has not been explored so far. Therefore, the aim of the present study was to analyze the impact of the absence of the inducible NO synthase (iNOS) gene in the regulation of energy balance in ob/ob mice. METHODS AND FINDINGS: Double knockout (DBKO) mice simultaneously lacking the ob and iNOS genes were generated, and the expression of molecules involved in the control of brown fat cell function was analyzed by real-time PCR, western-blot and immunohistochemistry. Twelve week-old DBKO mice exhibited reduced body weight (p<0.05), decreased amounts of total fat pads (p<0.05), lower food efficiency rates (p<0.05) and higher rectal temperature (p<0.05) than ob/ob mice. Ablation of iNOS also improved the carbohydrate and lipid metabolism of ob/ob mice. DBKO showed a marked reduction in the size of brown adipocytes compared to ob/ob mutants. In this sense, in comparison to ob/ob mice, DBKO rodents showed an increase in the expression of PR domain containing 16 (Prdm16), a transcriptional regulator of brown adipogenesis. Moreover, iNOS deletion enhanced the expression of mitochondria-related proteins, such as peroxisome proliferator-activated receptor gamma coactivator-1 alpha (Pgc-1alpha), sirtuin-1 (Sirt-1) and sirtuin-3 (Sirt-3). Accordingly, mitochondrial uncoupling proteins 1 and 3 (Ucp-1 and Ucp-3) were upregulated in brown adipose tissue (BAT) of DBKO mice as compared to ob/ob rodents. CONCLUSION: Ablation of iNOS improved the energy balance of ob/ob mice by decreasing food efficiency through an increase in thermogenesis. These effects may be mediated, in part, through the recovery of the BAT phenotype and brown fat cell function improvement.

The aim of this study is to assess the long-term effect of mesenchymal stem cells (MSC) transplantation in a rat model of chronic myocardial infarction (MI) in comparison with the effect of bone marrow mononuclear cells (BM-MNC) transplant. Five weeks after induction of MI, rats were allocated to receive intramyocardial injection of 10(6) GFP-expressing cells (BM-MNC or MSC) or medium as control. Heart function (echocardiography and (18)F-FDG-microPET) and histological studies were performed 3 months after transplantation and cell fate was analyzed along the experiment (1 and 2 weeks and 1 and 3 months). The main findings of this study were that both BM-derived populations, BM-MNC and MSC, induced a long-lasting (3 months) improvement in LVEF (BM-MNC: 26.61 +/- 2.01% to 46.61 +/- 3.7%, p <0.05; MSC: 27.5 +/- 1.28% to 38.8 +/- 3.2%, p < 0.05) but remarkably, only MSC improved tissue metabolism quantified by (18)F-FDG uptake (71.15 +/- 1.27 to 76.31 +/- 1.11, p<0.01), which was thereby associated with a smaller infarct size and scar collagen content and also with a higher revascularization degree. Altogether, results show that MSC provides a long-term superior benefit than whole BM-MNC transplantation in a rat model of chronic MI.