In situ positron emission tomography monitoring of endothelial cells embedded in perfused fibrin gels

In tissue engineering, the continuous monitoring of cell and tissue cultures in vitro is crucial to assess their functional status over time. However, these constructs can be large, thick and non-transparent. Medical imaging techniques can allow real-time in situ monitoring of cell and tissue cultures in thick solid scaffolds. Here, human endothelial cells were embedded in fibrin gels that were continuously perfused by a culture medium. Positron emission tomography (PET) imaging was used to assess cell viability non-destructively over periods extending up to a few weeks. PET imaging protocols were adapted and validated to measure culture perfusion and cell metabolism using [¹⁸F]-fluorodeoxyglucose (¹⁸FDG). Cell densities down to 100,000 cells/mL were detectable after 12 h of culture and cell structures were localized within the fibrin gels after 1–2 weeks of culture. PET is a promising tool to investigate a wide range of cellular properties and reveal information on tissue development.     

Synthesis of fluorinated analogues of sphingosine-1-phosphate antagonists as potential radiotracers for molecular imaging using positron emission tomography

Sphingosine-1-phosphate (S1P) receptors play major roles in cardiovascular, immunological and neurological diseases. The recent approval of the sphingolipid drug Fingolimod (Gilenya®), a sphingosine-1-phosphate agonist for relapsing multiple sclerosis, in 2010 exemplifies the potential for targeting sphingolipids for the treatment of human disorders. Moreover, non-invasive in vivo imaging of S1P receptors that are not available till now would contribute to the understanding of their role in specific pathologies and is therefore of preclinical interest. Based on fluorinated analogues of the S1P₁ receptor antagonist W146 showing practically equal in vitro potency as the lead structure, the first S1P receptor antagonist [¹⁸F]-radiotracer has been synthesized and tested for in vivo imaging of the S1P₁ receptor using positron emission tomography (PET). Though the tracer is serum stable, initial in vivo images show fast metabolism and subsequent accumulation of free [¹⁸F]fluoride in the bones.     

Decreased levels of alpha‐synuclein in cerebrospinal fluid of patients with clinically isolated syndrome and multiple sclerosis

Cerebrospinal fluid (CSF) α‐synuclein (ASYN) levels are emerging as a possible biomarker in a number of neurodegenerative conditions; however, there has been little study of such levels in demyelinating conditions with neurodegeneration such as multiple sclerosis (MS). In this study, we aimed to assess CSF ASYN levels in MS spectrum [clinically isolated syndrome (CIS) and MS] patients and compare them to those obtained in control subjects with benign neurological conditions (BNC). We used a recently developed, ultra‐sensitive sandwich enzyme‐linked immunosorbent assay to measure and compare CSF ASYN levels in three categories of subjects: BNC (n = 38), CIS (n = 36) and MS [Relapsing Remitting (RRMS, n = 22) and Primary Progressive (PPMS, n = 15)]. We also performed secondary analyses, including relationship of CSF ASYN levels to aging, gender, presence of CSF oligoclonal bands (OB) and gadolinium (Gd)‐enhancing demyelinating lesions on T1‐weighted MRIs. CSF ASYN levels were found to be significantly lower in the CIS (78.2 ± 7.5 pg/mL), RRMS (76.8 ± 5.1 pg/mL), and PPMS (76.3 ± 6.7 pg/mL) groups compared to the BNC (125.7 ± 13.6 pg/mL) group. Secondary analyses did not reveal additional correlations. Our results suggest that in a cohort of CIS and MS patients, CSF ASYN levels are decreased, thus providing another possible link between MS and neurodegeneration. Future studies will need to be performed to confirm and extend these findings, to lead to a fuller understanding of the possible biological link between ASYN and MS.

     

Performance and limitations of positron emission tomography (PET) scanners for imaging very low activity sources

Emerging applications for positron emission tomography (PET) may require the ability to image very low activity source distributions in the body. The performance of clinical PET scanners in the regime where activity in the field of view is <1 MBq has not previously been explored. In this study, we compared the counting rate performance of two clinical PET/CT scanners, the Siemens Biograph Reveal 16 scanner which is based on lutetium oxyorthosilicate (LSO) detectors and the GE Discovery-ST scanner which is based on bismuth germanate (BGO) detectors using a modified National Electrical Manufacturers Association (NEMA) NU 2-2007 protocol. Across the activity range studied (2–100 kBq/mL in a 5.5 mL line source in the NEMA scatter phantom), the BGO-based scanner significantly outperformed the LSO-based scanner. This was largely due to the effect of background counts emanating from naturally occurring but radioactive ¹⁷⁶Lu within the LSO detector material, which dominates the observed counting rate at the lowest activities. Increasing the lower energy threshold from 350 keV to 425 keV in an attempt to reduce this background did not significantly improve the measured NECR performance. The measured singles rate due to ¹⁷⁶Lu emissions within the scanner energy window was also found to be dependent on temperature, and to be affected by the operation of the CT component, making approaches to correct or compensate for the background more challenging. We conclude that for PET studies in a very low activity range, BGO-based scanners are likely to have better performance because of the lack of significant background.     

Synthesis of 18F-labeled streptozotocin derivatives and an in-vivo kinetics study using positron emission tomography

Glucose transporter 2 (GLUT2) is involved in glucose uptake by hepatocytes, pancreatic beta cells, and absorptive cells in the intestine and proximal tubules in the kidney. Pancreatic GLUT2 also plays an important role in the mechanism of glucose-stimulated insulin secretion. In this study, novel Fluorine-18-labeled streptozotocin (STZ) derivatives were synthesized to serve as glycoside analogs for in-vivo GLUT2 imaging. Fluorine was introduced to hexyl groups at the 3′-positions of the compounds, and we aimed to synthesize compounds that were more stable than STZ. The nitroso derivatives exhibited relatively good stability during purification and purity analysis after radiosynthesis. We then evaluated the compounds in PET imaging and ex-vivo biodistribution studies. We observed high levels of radioactivity in the liver and kidney, which indicated accumulation in these organs within 5 min of administration. In contrast, the denitroso derivatives accumulated only in the kidney and bladder shortly after administration. Compounds with nitroso groups are thus expected to accumulate in GLUT2-expressing organs, and the presence of a nitroso group is essential for in-vivo GLUT2 imaging.     

Synthesis and biodistribution of an 18F-labelled resveratrol derivative for small animal positron emission tomography

Summary. Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is a naturally occurring phytoalexin and polyphenol existing in grapes and various other plants, and one of the best known ‘nutriceuticals’. It shows a multiplicity of beneficial biological effects, particularly, by attenuating atherogenic, inflammatory, and carcinogenic processes. However, despite convincing evidence from experimental and clinical studies, data concerning the role of resveratrol and other members of the large polyphenols family for human health is still a matter of debate. One reason for this is the lack of suitable sensitive and specific methods, which would allow direct assessment of biodistribution, biokinetics, and the metabolic fate of these compounds in vivo. The unique features of positron emission tomography (PET) as a non-invasive in vivo imaging methodology in combination with suitable PET radiotracers have great promise to assess quantitative information on physiological effects of polyphenols in vivo. Herein we describe the radiosynthesis of an ¹⁸F-labelled resveratrol derivative, 3,5-dihydroxy-4′-[¹⁸F]fluoro-trans-stilbene ([¹⁸F]-1), using the Horner-Wadsworth-Emmons reaction as a novel radiolabelling technique in PET radiochemistry for subsequent functional imaging of polyphenol metabolism in vivo. In a typical “three-step/one-pot” reaction, ¹⁸F-labelled resveratrol derivative [¹⁸F]-1 could be synthesized within 120–130 min including HPLC separation at a specific radioactivity of about 90 GBq/μmol. The radiochemical yield was about 9% (decay-corrected) related to [¹⁸F]fluoride and the radiochemical purity exceeded 97%. First radiopharmacological evaluation included measurement of biodistribution ex vivo and positron emission tomography (PET) studies in vivo after intravenous application of [¹⁸F]-1 in male Wistar rats using a dedicated small animal PET camera with very high spatial resolution. Concordantly with data on bioavailability and metabolism of native resveratrol from the literature, these investigations revealed an extensive uptake and metabolism in the liver and kidney, respectively, of [¹⁸F]-1. This study represents the first investigation of polyphenols in vivo by means of PET.     

Benefits and risks of transforming data from dynamic positron emission tomography, with an application to hepatic encephalopathy

Transforming data sets to bring out expected model features can be valuable within limits and misleading outside them. Here we establish such limits for the widely used Gjedde-Patlak representation of dynamic PET data, with an application to hepatic encephalopathy.     

Increasing blood oxygen increases an index of 5-HT synthesis in human brain as measured using alpha-[(11)C]methyl-L-tryptophan and positron emission tomography

The main objective of this investigation was to test the hypothesis that brain serotonin (5-HT) synthesis, as measured by trapping of alpha-[(11)C]methyl-L-tryptophan (alpha-MTrp) using positron emission tomography (PET), can be modulated by changes in blood oxygen. The study involved six healthy participants (three male and three female), who breathed a 15% or 60% oxygen mixture starting 15 min before the injection of tracer and continuing during the entire acquisition period. Participants were injected with up to 12m Ci of alpha-MTrp. Two sets of PET images were acquired while the participants were breathing each of the oxygen mixtures and, after reconstruction, all images were converted into brain functional images illustrating the brain trapping constant K(*) (microL/g/min). The K(*) values were obtained for 12 regions of interest outlined on the magnetic resonance images. The K(*) values obtained at high and low blood oxygen content were compared by paired statistics using Tukey's post hoc correction. As there were no difference in plasma tryptophan concentrations, these K(*) values are directly related to regional 5-HT synthesis. The results showed highly significant increases (50% on average) in brain serotonin synthesis (K(*) values) at high (mean value of 223+/-41 mmHg) relative to low (mean value 77.1+/-7.7 mmHg) blood oxygen levels. This suggests that tryptophan hydroxylase is not saturated with oxygen in the living human brain and that increases in blood oxygen can elevate brain serotonin synthesis.     

Application of positron emission tomography to neuroimaging in sports sciences

To investigate exercise-induced regional metabolic and perfusion changes in the human brain, various methods are available, such as positron emission tomography (PET), functional magnetic resonance imaging (fMRI), near-infrared spectroscopy (NIRS) and electroencephalography (EEG). In this paper, details of methods of metabolic measurement using PET, [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) and [¹⁵O]radio-labelled water ([¹⁵O]H₂O) will be explained.Functional neuroimaging in the field of neuroscience was started in the 1970s using an autoradiography technique on experimental animals. The first human functional neuroimaging exercise study was conducted in 1987 using a rough measurement system known as ¹³³Xe inhalation. Although the data was useful, more detailed and exact functional neuroimaging, especially with respect to spatial resolution, was achieved by positron emission tomography. Early studies measured the cerebral blood flow changes during exercise. Recently, PET was made more applicable to exercise physiology and psychology by the use of the tracer [¹⁸F]FDG. This technique allowed subjects to be scanned after an exercise task is completed but still obtain data from the exercise itself, which is similar to autoradiography studies.In this report, methodological information is provided with respect to the recommended protocol design, the selection of the scanning mode, how to evaluate the cerebral glucose metabolism and how to interpret the regional brain activity using voxel-by-voxel analysis and regions of interest techniques (ROI).Considering the important role of exercise in health promotion, further efforts in this line of research should be encouraged in order to better understand health behavior. Although the number of research papers is still limited, recent work has indicated that the [¹⁸F]FDG-PET technique is a useful tool to understand brain activity during exercise.     

Simultaneous determination of 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid, and homovanillic acid in cerebrospinal fluid with high-performance liquid chromatography using electrochemical detection

An improved high-performance liquid chromatographic method with electrochemical detection (HPLC-EC) for the simultaneous determination of 3-methoxy-4-hydroxyphenylglycol (MHPG), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) in cerebrospinal fluid (CSF) of humans and nonhuman primates is described. Quantitation is based on the use of an internal standard, 5-fluoro-HVA. Sample preparation consists of mixing an aliquot of CSF with a solution of the internal standard followed by ultrafiltration. The precision of the method is high, with within-run and between-run coefficients of variation of 2-6% and less than 10%, respectively, in the concentration ranges of the metabolites encountered in human lumbar CSF. Accuracy was tested by comparing the present HPLC method with specific gas chromatographic-mass spectrometric (GS-MS) assays for MHPG and HVA and a GC-MS-validated HPLC assay for 5-HIAA: the correlations obtained were 0.968 for MHPG, 0.989 for 5-HIAA, and 0.999 for HVA, with no systematic bias between the methods. The use of ascorbate as a preserving agent for monoamine metabolites in CSF was not found to be necessary when proper care was exercised in sample handling and storage. The analysis of samples with up to 2% ascorbic acid was possible as well, but MHPG had to be assayed separately using an extraction procedure and an alternative internal standard, 3-ethoxy-4-hydroxyphenylglycol.     

The use of positron emission tomography for studies of long-distance transport in plants: uptake and transport of 18F

Abstract. Positron emission tomography (PET) has been utilized to obtain dynamic images of long distance nutrient translocation in plants. Positron emitting ¹⁸F, produced by a Van de Graaff accelerator using the reaction ¹⁸O(p,n)¹⁸F, was fed in solution to excised stems of Glycine max positioned vertically in a large-aperture PET detector system. Images of tracer activity were recorded with a time resolution of 0.5 min and a spatial resolution of 4 mm. Maximum tracer activities at stem sites were obtained within 3 min of the pulse feed. A model is presented enabling evaluation of regional values for tracer flow, tracer binding, flow speed and flow volume. Analysis of data for one stem position yielded a flow volume of 2.1mm³ min⁻¹ and a flow speed of 36cm min⁻¹. Comparison with the distribution of ¹⁴C-inulin, which was simultaneously fed to the cut stems, indicates the ¹⁸F is suitable for use as an apoplastic tracer; 92% of the tracer activity accumulated in the leaves. The fraction of ¹⁸F that remained bound was most concentrated at stem nodal regions, an observation consistent with the existence of transfer cells at these sites. Advantages and limitations of PET applied to plant physiological investigations are discussed.     

Assessment of the partitioning capacity of high abundant proteins in human cerebrospinal fluid using affinity and immunoaffinity subtraction spin columns

The performance of three different affinity and immunoaffinity subtraction spin columns was investigated for the removal of the most abundant proteins in human cerebrospinal fluid (CSF). A pool of human CSF was processed with the spin columns and both the bound and flow through fractions were compared with each other and with intact CSF using 1D gel electrophoresis and nanoLC–MALDI-TOF/TOF-MS analysis. MASCOT MS/MS ionscores were compared before and after processing with the columns. The non-specific co-removal of proteins bound to the high abundant proteins, so called “sponge effect” was also examined for each spin column. The reproducibility of one of the spin columns, ProteomeLab IgY-12 proteome partitioning spin column, was further investigated by isobaric tags for relative and absolute quantification (iTRAQ) labeling and MS/MS analysis. Overall, 173 unique proteins were identified on a 95% MudPIT confidence scoring level. For all three spin columns, the number of proteins identified and their MASCOT scores were increased up to 10 times. The largest degree of non-specific protein removal was observed for a purely affinity based albumin removal column, where 28 other proteins also were present. The ProteomeLab IgY-12 proteome partitioning spin column showed very high reproducibility when combined with iTRAQ labeling and MS/MS analysis. The combined relative standard deviation (R.S.D.) for the high abundant protein removal, iTRAQ labeling and nanoLC–MALDI-TOF/TOF-MS analysis was less than 17.5%.     

The genetic diversity of SMLS (Sitobion miscanthi L type symbiont) and its effect on the fitness,mitochondrial DNA diversity and Buchnera aphidicola dynamic of wheat aphid,Sitobion miscanthi (Hemiptera: Aphididae)

SMLS (Sitobion miscanthi L type symbiont) is a recently discovered aphid secondary symbiont. Using evidence extracted from 16S rRNA sequences, previous studies indicate that SMLS is the most widely distributed and most recently transferred secondary symbiont in Chinese Sitobion miscanthi populations. Here, we further investigated genetic diversity among SMLS geographic strains with multiloci data. Furthermore, the influence of SMLS on S. miscanthi was uncovered with ecological and evolutionary evidence. The results indicated that there was limited influence of infection with SMLS on variation and evolutionary patterns of S. miscanthi mitochondrial DNA. By hemolymph injection, the SMLS‐infected and SMLS‐uninfected S. miscanthi clones with the identical genetic background were built in this study. Although similar Buchnera aphidicola dynamics were observed between SMLS‐infected and SMLS‐uninfected S. miscanthi population, B. aphidicola density of SMLS‐infected S. miscanthi population was always significantly higher than SMLS‐uninfected ones. The results of fitness measurements indicated that under laboratory rearing conditions, transfection of SMLS could confer modest advantages to some fitness components of S. miscanthi, that is, total number of offspring, longevity, age of first reproduction and weight of adult. However, as SMLS is not strictly associated with S. miscanthi, further investigations are needed to uncover the mechanisms responsible for this inconceivable association.     

Effects of Light Quality on the Interaction between Cucumber Mosaic Virus and Nicotiana tabacum

Light is an important environment factor controlling plant growth, development, and nutritional quality and is also one of the most important factors inducing plant defence. In this study, we assayed the potential effects of light quality on the interaction between Nicotiana tabacum and cucumber mosaic virus (CMV). Our results indicated that white light‐treated N. tabacum plants displayed obvious symptoms at early stage postinoculation, while the symptoms were significantly inhibited under red light and blue light. Western blotting and quantitative real‐time PCR (qRT‐PCR) analyses showed that blue light and red light can effectively delay the replication of CMV compared with white light. The activities of various reactive oxygen species (ROS)‐scavenging enzymes and reducing substances [reduced glutathione (GSH) and ascorbic acid (ASA)] were increased under blue light and red light. In addition, hormone measurements and qRT‐PCR analyses revealed that salicylic acid (SA)‐mediated signalling pathway plays positive role in the related regulation, and cytokinin (CTK) may also participate in them. Furthermore, we found that the formation of dark green islands (DGIs) was significantly suppressed in plants under red light and blue light at 30 days postinoculation (Dpi). However, the accumulation of virus in plants under different light conditions had no notable differences at later stage of postinoculation. Taken together, these results indicated that red light and blue light could effectively delay symptom expression and replication of CMV on N. tabacum at the relatively earlier stage postinoculation.