18?F-Fluoride positron emission tomography/computed tomography for noninvasive in vivo quantification of pathophysiological bone metabolism in experimental murine arthritis

Introduction

Evaluation of disease severity in experimental models of rheumatoid arthritis is inevitably associated with assessment of structural bone damage. A noninvasive imaging technology allowing objective quantification of pathophysiological alterations of bone structure in rodents could substantially extend the methods used to date in preclinical arthritis research for staging of autoimmune disease severity or efficacy of therapeutical intervention. Sodium ¹⁸ F-fluoride (¹⁸ F-NaF) is a bone-seeking tracer well-suited for molecular imaging. Therefore, we systematically examined the use of ¹⁸ F-NaF positron emission tomography/computed tomography (PET/CT) in mice with glucose-6-phosphate isomerase (G6PI)–induced arthritis for quantification of pathological bone metabolism.

Methods

F-fluoride was injected into mice before disease onset and at various time points of progressing experimental arthritis. Radioisotope accumulation in joints in the fore- and hindpaws was analyzed by PET measurements. For validation of bone metabolism quantified by ¹⁸ F-fluoride PET, bone surface parameters of high-resolution μCT measurements were used.

Results

Before clinical arthritis onset, no distinct accumulation of ¹⁸ F-fluoride was detectable in the fore- and hindlimbs of mice immunized with G6PI. In the course of experimental autoimmune disease, ¹⁸ F-fluoride bone uptake was increased at sites of enhanced bone metabolism caused by pathophysiological processes of autoimmune disease. Moreover, ¹⁸ F-fluoride signaling at different stages of G6PI-induced arthritis was significantly correlated with the degree of bone destruction. CT enabled identification of exact localization of ¹⁸ F-fluoride signaling in bone and soft tissue.

Conclusions

The results of this study suggest that small-animal PET/CT using ¹⁸ F-fluoride as a tracer is a feasible method for quantitative assessment of pathophysiological bone metabolism in experimental arthritis. Furthermore, the possibility to perform repeated noninvasive measurements in vivo allows longitudinal study of therapeutical intervention monitoring.     

Synthesis and in vivo evaluation of ¹⁸F-fluoroethyl GF120918 and XR9576 as positron emission tomography probes for assessing the function of drug efflux transporters

The purpose of this study was to synthesize two new positron emission tomography (PET) probes, N-(4-(2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-[1?F]fluoroethoxy-9-oxo-4-acridine carboxamide ([1?F]3) and quinoline-3-carboxylic acid [2-(4-{2-[7-(2-[1?F]fluoroethoxy)-6-methoxy-3,4-dihydro-1H-isoquinolin-2-yl]ethyl}phenylcarbamoyl)-4,5-dimethoxyphenyl]amide ([1?F]4), and to evaluate the potential of these PET probes for assessing the function of two major drug efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). [1?F]3 and [1?F]4 were synthesized by 1?F-alkylation of each O-desmethyl precursor with [1?F]2-fluoroethyl bromide for injection as PET probes. In vitro accumulation assay showed that treatment with P-gp/BCRP inhibitors (1 and 2) enhanced the intracellular accumulation capacity of P-gp- and BCRP-overexpressing MES-SA/Dx5 cells. In PET studies, the uptake (AUC(brain[0-)?? (min])) of [1?F]3 and [1?F]4 in wild-type mice co-injected with 1 were approximately sevenfold higher than that in wild-type mice, and the uptake of [1?F]3 and [1?F]4 in P-gp/Bcrp knockout mice were eight- to ninefold higher than that in wild-type mice. The increased uptake of [1?F]3 and [1?F]4 was similar to that of parent compounds ([11C]1 and [11C]2) previously described, indicating that radioactivity levels in the brain after injection of [1?F]3 and [1?F]4 are related to the function of drug efflux transporters. Also, these results suggest that the structural difference between parent compounds ([11C]1 and [11C]2) and fluoroethyl analogs ([1?F]3 and [1?F]4) do not obviously affect the potency against drug efflux transporters. In metabolite analysis of mice, the unchanged form in the brain and plasma at 60 min after co-injection of [1?F]4 plus 1 were higher (95% for brain; 81% for plasma) than that after co-injection of [1?F]3 plus 1. [1?F]4 is a promising PET probe to assess the function of drug efflux transporters.     

Electron tomography of RabA4b- and PI-4Kβ1-labeled trans Golgi network compartments in Arabidopsis

The trans Golgi network (TGN) of plant cells sorts and packages Golgi products into secretory (SV) and clathrin-coated (CCV) vesicles. We have analyzed of TGN cisternae in Arabidopsis root meristem cells by cell fractionation and electron microscopy/tomography to establish reliable criteria for identifying TGN cisternae in plant cells, and to define their functional attributes. Transformation of a trans Golgi cisterna into a Golgi-associated TGN cisterna begins with cisternal peeling, the formation of SV buds outside the plane of the cisterna and a 30-35% reduction in cisternal membrane area. Free TGN compartments are defined as cisternae that have detached from the Golgi to become independent organelles. Golgi-associated and free TGN compartments, but not trans Golgi cisternae, bind anti-RabA4b and anti-phosphatidylinositol-4 kinase (PI-4K) antibodies. RabA4b and PI-4Kβ1 localize to budding SVs in the TGN and to SVs en route to the cell surface. SV and CCV release occurs simultaneously via cisternal fragmentation, which typically yields ～30 vesicles and one to four residual cisternal fragments. Early endosomal markers, VHA-a1-green fluorescent protein (GFP) and SYP61-cyan fluorescent protein (CFP), colocalized with RabA4b in TGN cisternae, suggesting that the secretory and endocytic pathways converge at the TGN. pi4k1/pi4k2 knockout mutant plants produce SVs with highly variable sizes indicating that PI-4Kβ1/2 regulates SV size.     

A comparative study in Alzheimer’s and normal brains of trace element distribution using PIXE and INA analyses and glucose metabolism by positron emission tomography

The onset of Alzheimer’s disease has been shown to affect trace element concentrations in the brain when compared to “normal” subjects in ex vivo samples. The techniques used to determine trace element concentrations were proton-induced X-ray emission and instrumental neutron activation analysis. With these methods, significant differences are seen between lobes within a hemisphere and between the same lobes of opposing hemispheres for “normal” brains. The change observed in trace element concentrations may indicate a possible alteration in the function of the blood-brain barrier, the effect of which can be investigated in vivo using the imaging technique of positron emission tomography (PET). A PET study was performed on nine female and nine male subjects to determine whether the regional metabolic rate of glucose (rCMRGlu) varied between hemispheres and sex in the Alzheimer diseased brain as was seen in the trace element study. Glucose metabolism was measured using [F-18]-fluorodeoxyglucose (¹⁸F-FDG). Hemispherical differences were observed for the frontal, occipital, parietal lobes, and the temporolateral region in both males and females for rCMRGlu. Variation was also seen between sexes, where the frontal lobe had a lower rCMRGlu in females compared to that of males.     

Dual localization of β-glucuronidase and acid phosphatase in lysosomes and in microsomes

Summary The dual localization of certain hydrolases in lysosomes and in endoplasmic reticulum as studied in enzyme staining reactions is now supported by cytobiochemical studies on mouse liver and kidney -glucuronidase and acid phosphatase. Use was made of the renal -glucuronidase response to endogenous androgen for both studies. Accordingly, sucrose homogenates were prepared of liver and kidney of male BALB/C mice previously injected with gonadotrophin along with control animals receiving saline instead. The homogenates were subjected to differential ultracentrifugation yielding six fractions. These were characterized as to their organelle composition by measurements of marker enzymes and by observations with the electron microscope. In all subcellular fractions, -glucuronidase was uniformly increased 5 to 8 times over the corresponding control value and, in fractions rich in lysosomes, this enzyme was easily released by alternate freezing and thawing. On the other hand, the microsomal -glucuronidase and acid phosphatase enzymes were not liberated by freezing and thawing nor were they after treatment with 0.1 % Triton X-100 and by employing other reagents and conditions which are known to release lysosomal enzymes. In contrast to microsomal acid phosphatase, microsomal -glucuronidase activity could be liberated by treatment with hyaluronidase. This soluble -glucuronidase showed the same optimum pH, Michaelis Constant and heat inactivation behavior as the lysosomal -glucuronidase prepared by freezing and thawing treatment. These observations define two populations of microsomal vesicles each identifiable by an individual membrane-associated acid hydrolase. One of these -glucuronidase, increases in specific activity in the animal on androgens and is released by hyaluronidase and the other, acid phosphatase, does not respond to androgen and is not released by hyaluronidase. There would appear to be a variety of mechanisms by which hydrolases enter into association with the membranes of the endoplasmic reticulum and from there, a variety of routes to the lysosomes. A comment is made concerning the question of acid phosphatases and -glucuronidase as enzyme markers for lysosomes in mouse kidney.Aided in part by Research Grant, P-106, of the American Cancer Society, Inc., New York, and by U.S.P.H.S. Grant CA-07538 and by a Research Career Award, CA-K6-18453 to William H. Fishman.     

Effects of freeze–thaw and micro-computed tomography irradiation on structure–property relations of porcine trabecular bone

We study the effects of freeze–thaw and irradiation on structure–property relations of trabecular bone. We measure the porosity, apparent density, mineral content, trabecular orientation, trabecular thickness, fractal dimension, surface area, and connectivity of trabecular bone using micro-computed tomography (micro-CT) and relate them to Young?s modulus and ultimate strength measured by uniaxial compression testing. The analysis is done on six-month porcine trabecular bone from femoral heads. The effects of freeze–thaw are studied by using bones from three different groups: fresh bone and bones frozen for one and five years. We find that the porosity and apparent density have most dominant influence on the elastic modulus and strength of fresh bone. Also, five years of freezing lowers both Young?s modulus and ultimate strength of trabecular bone. Additionally, the effects of radiation are investigated by comparing Young?s modulus before and after micro-CT exposure. We find that the micro-CT irradiation has a negligible effect on the Young?s modulus of trabecular bone. These findings provide insights on the effects of tissue preservation and imaging on properties of trabecular bone.     

Yields and accumulations of N and P in farmer-managed intercrops of maize–pigeonpea in semi-arid Africa

Maize (Zea mays L.) is a major staple food in Sub-Saharan Africa but low soil fertility, limited resources and droughts keep yields low. Cultivation of maize intercropped with pigeonpea (Cajanus cajan L. Millsp.) is common in some areas of eastern and southern Africa. The objectives of this study were (1) to investigate dry matter, nitrogen (N) and phosphorus (P) accumulation in different plant components of maize–pigeonpea intercropping systems and (2) to report the effects of the intercrops on soil fertility. Maize–pigeonpea intercrops were compared to sole maize grown using farmersȁ9 practices. Intercropping maize and pigeonpea increased (P < 0.05) total system yield compared to sole maize in terms of biomass, N and P accumulation. Pigeonpea planted in maize did not reduce (P < 0.05) the accumulation of dry matter, N nor P in the maize grain. The harvest indices of maize, calculated on basis dry matter, N or P did not differ either (P < 0.05). Total soil C and N contents and inorganic N content, nitrate and ammonium, were not affected by two seasons of maize–pigeonpea intercropping compared to sole maize (P > 0.11). Nitrate and ammonium levels in soil were still not affected by the treatments after the soils were incubated in anaerobic conditions for 8 days at 37°C (P > 0.11). However, pigeonpea added up to 60 kg of N ha⁻¹ to the system and accumulated up to 6 kg of P ha⁻¹ and only 25% of this N and P were exported in the grain. In conclusion, beside the added grain yield of pigeonpea in the intercropped systems, pigeonpea increased the recirculation of dry matter, N and P, which may have a long-term effect on soil fertility. Furthermore, the stems from pigeonpea contributed to household fuel wood consumption. The intercropped system thus had multiple benefits that gave significant increase in combined yield per unit area without additional labour requirements. The main requirement in order to up-scale the maize–pigeonpea intercropping approach is sufficient supply of high-quality pigeonpea seeds.     

Gender differences in parameters of lipid metabolism and of level of antioxidants in groups of juveniles—the Even and the Europeans

There was performed a comparative evaluation of the mean values of lipid metabolism parameters in youths and girls of the indigenous population (the Even), of the coming population of the north of Irkutsk province, and of the province center (the Europeoids). Gender differences in HDLP have been revealed in adolescents of the coming population, in the glutathione status and the content of liposoluble vitamins in the Even and coming adolescents and in retinol concentration in the province center adolescents. Adaptive changes in the ROS system were found in the Even girls (the higher α-tocopherol and GSH concentrations and the GSH/GSSG ratio as well as a decrease of GSSG relative to the Even youths and the coming population girls, an increase of α-tocopherol relatively to the coming population Europeoids) and youth of the coming population (the higher GSH content and the GSH/GSSG parameter relatively to the Even youths).     

Rate of utilization of glucose and `compartmentation'' of α-oxoglutarate and glutamate in rat brain

1. The rate of incorporation of ¹⁴C into pyruvate, α-oxoglutarate, lactate and glucose of rat tissues was measured after the subcutaneous injection of uniformly labelled glucose. 2. In rat brain the specific radioactivities of lactate and glucose were similar to that of alanine. In liver the specific radioactivity of glucose was considerably higher than that of lactate or alanine. 3. The specific radioactivities of α-oxo acids of rat brain were lower than those of corresponding amino acids, alanine and glutamate. These findings have been explained in relation to metabolic compartments in vivo. 4. The approximate estimated rate of glucose utilization in rat brain in vivo is 0·96μmole/g. of brain/min.     

Correlation of Tc-99 m ethyl cysteinate dimer single-photon emission computed tomography and clinical presentations in patients with low cobalamin status

Background

Cobalamin (Cbl) deficiency has been associated with various neuropsychiatric symptoms of different severities. While some studies dedicated in structural neuroimaging credibly address negative impact of low Cbl status, functional imaging reports are limited. We herein retrospectively review the correlation of Tc-99 m ethyl cysteinate dimer single-photon emission computed tomography (Tc-99 m-ECD SPECT) and clinical presentations among patients with low serum cobalamin (Cbl) status (<250 pg/ml).

Methods

Twelve symptomatic patients with low serum Cbl status were enrolled. Clinical presentations, Tc-99 m-ECD SPECT, and neuropsychological tests were reviewed.

Results

Dysexecutive syndrome (67 %), forgetfulness (50 %), attention deficits (42 %), and sleep disorders (33 %) constituted the major clinical presentations. All patients (100 %) had temporal hypoperfusion on the Tc-99 m-ECD SPECT. Five patients (42 %) had hypoperfusion restricted within temporal regions and deep nuclei; seven patients (58 %) had additional frontal hypoperfusion. In patients with hypoperfusion restricted within temporal regions and deep nuclei, psychiatric symptoms with spared cognition were their main presentations. Among patients with additional frontal hypoperfusion, six of seven patients (86 %) showed impaired cognitive performances (two of them were diagnosed as having dementia). Among ten patients who finished neuropsychological tests, abstract thinking (70 %) was the most commonly affected, followed by verbal fluency (60 %), short-term memory (50 %), and attention (50 %). Anxiety and sleep problems were the major clinically remarkable psychiatric features (33 % both). Four Tc-99 m-ECD SPECT follow-up studies were available; the degree and extent of signal reversal correlated with cognitive changes after Cbl replacement therapy.

Conclusions

Our TC-99 m-ECD SPECT observations provide pivotal information of neurobiological changes within basal ganglia and fronto-temporal regions in conjunction with disease severity among patients with Cbl deficiency. Hypoperfusion within thalamus/basal ganglia and temporal regions may be seen in the earlier state of Cbl deficiency, when psychiatric symptoms predominate. Hypoperfusion beyond thalamus/basal ganglia and involving frontal regions appears when cognitive problems, mostly dysexecutive syndrome, are manifested. Symmetric hypofrontality of SPECT in the context of dysexcutive syndrome serves as a distinguishing feature of non-amnestic mild cognitive impairment attributed to Cbl deficiency. Concordant with TC-99 m-ECD SPECT findings, the psychiatric symptoms and dysexcutive syndrome undergird impaired limbic and dorsolateral prefrontal circuits originating from basal ganglia respectively.

     

Antioxidant mechanisms of Quercetin and Myricetin in the gas phase and in solution – a comparison and validation of semi-empirical methods

Flavonoids have long been recognized for their general health-promoting properties, of which their antioxidant activity may play an important role. In this work we have studied the properties of two flavonols, quercetin and myricetin, using semi-empirical methods in order to validate the application of the recent Parametric Model 6 and to understand the fundamental difference between the two molecules. Their geometries have been optimized and important molecular properties have been calculated. The energetic of the possible antioxidant mechanisms have also been analyzed. The two studied flavonols do not differ significantly in their molecular properties, but the antioxidant mechanisms by which they may act in solution can be rather different. Moreover, we also show that the Parametric Model 6 can produce reliable information for this type of compounds.     

Interaction between Oligomers of Stefin B and Amyloid-�� in Vitro and in Cells

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-β-(1–40) peptide (Aβ). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Aβ is oligomer specific. The dimers and tetramers of stefin B, which bind Aβ, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Aβ fibril formation. When expressed in cultured cells, stefin B co-localizes with Aβ intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Aβ epitope. Thus, stefin B is another APP/Aβ-binding protein in vitro and likely in cells.     

Regulation of calcium in pancreatic α- and β-cells in health and disease

The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca(2+)-influx via voltage-gated Ca(2+)-channels, elevation of [Ca(2+)](i) and initiation of Ca(2+)-dependent exocytosis. Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca(2+)-channel present in α- and β-cells and the modulation of hormone secretion by Ca(2+)-dependent and -independent processes. We also consider how subtle changes in Ca(2+)-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes.     

Assessment of tetralogy of Fallot–associated congenital extracardiac vascular anomalies in pediatric patients using low-dose dual-source computed tomography

Background

To investigate the diagnostic value of dual-source computed tomography (DSCT) in the evaluation of tetralogy of Fallot (TOF)-associated extracardiac vascular abnormalities in pediatric patients compared with transthoracic echocardiography (TTE).

Methods

One hundred and twenty-three pediatric patients diagnosed with TOF were included in this retrospective study. All patients underwent DSCT and TTE preoperatively. All associated extracardiac vascular abnormalities and their percentages were recorded. The diagnostic performances of DSCT and TTE were compared based on the surgical results. The image quality of DSCT was rated, and the effective radiation dose (ED) was calculated.

Results

A total of 159 associated extracardiac vascular deformities were confirmed by surgery. Patent ductus arteriosus (36, 22.64%), right-sided aortic arch (29, 18.24%), and pulmonary valve stenosis (23, 14.47%) were the most common associated extracardiac vascular abnormalities. DSCT was superior to TTE in demonstrating associated extracardiac anomalies (diagnostic accuracy: 99.13% vs. 97.39%; sensitivity: 92.45% vs. 77.07%; specificity: 99.81% vs. 99.42%). The agreement on grading the image quality of DSCT was excellent (κ?=?0.80), and the mean score of the image quality was 3.39?±?0.50. The mean ED of DSCT was 0.86?±?0.47 mSv.

Conclusions

Compared to TTE, low-dose DSCT has high diagnostic accuracy in the depiction of associated extracardiac vascular anomalies in pediatric patients with TOF, and could provide more morphological details for surgeons.

     

Comparison of α and β keratin in reptiles

Summary The different patterns of keratin formation that have evolved in the class Reptilia are all variations of a common process. In Squamata (snakes and lizards), a sequence of layers composed of or keratin is formed periodically, after which the old epidermal generation is shed. In Chelonia (turtles and tortoises), the epidermis of the shell is composed of only keratin, whereas the skin of the neck and leg is composed exclusively of keratin. Molting in toto does not occur and shedding is a continuous process comparable to that in avian and mammalian epidermis. In Crocodilia (crocodiles, caimans, alligators) there is only a single layer of cornified cells, but the composition of the layer varies in different parts of the scale. The hinge regions have many of the morphological characteristics of and keratin whereas the center resembles keratin. The living cells beneath contain accumulations of keratohyalin.There are four ultrastructural characteristics of a keratinized layer: 1) cellular outlines remain distinct, 2) a thickened plasma membrane forms during keratinization, 3) 80 Å filaments embedded in an amorphous matrix can be seen, and 4) PAS-positive material accumulates in extracellular spaces between the desmosomes.The layer exhibits none of these features. Instead the cells more or less (depending on species) coalesce into a compact layer which becomes attenuated in the hinge regions. A 30 Å filament pattern can be seen.The mesos layer of squamates resembles the hinge region of crocodilians, exhibiting a combination of the characteristics of both and keratin.This study constitutes publication No. 464 from the Oregon Regional Primate Research Center, supported in part by NIH Grant No. FR-00163.