Reduce, reuse and recycle: a green solution to Canada's medical isotope shortage.

Due to the unforeseen maintenance issues at the National Research Universal (NRU) reactor at Chalk River and coincidental shutdowns of other international reactors, a global shortage of medical isotopes (in particular technetium-99m, Tc-99m) occurred in 2009. The operation of these research reactors is expensive, their age creates concerns about their continued maintenance and the process results in a large amount of long-lived nuclear waste, whose storage cost has been subsidized by governments. While the NRU has since revived its operations, it is scheduled to cease isotope production in 2016. The Canadian government created the Non-reactor based medical Isotope Supply Program (NISP) to promote research into alternative methods for producing medical isotopes. The NRC was a member of a collaboration looking into the use of electron linear accelerators (LINAC) to produce molybdenum-99 (Mo-99), the parent isotope of Tc-99m. This paper outlines NRC's involvement in every step of this process, from the production, chemical processing, recycling and preliminary animal studies to demonstrate the equivalence of LINAC Tc-99m with the existing supply. This process stems from reusing an old idea, reduces the nuclear waste to virtually zero and recycles material to create a green solution to Canada's medical isotope shortage.

A comparison of rat SPECT images obtained using (99m)Tc derived from 99Mo produced by an electron accelerator with that from a reactor.

Recent shortages of molybdenum-99 ((99)Mo) have led to an examination of alternate production methods that could contribute to a more robust supply. An electron accelerator and the photoneutron reaction were used to produce (99)Mo from which technetium-99m ((99m)Tc) is extracted. SPECT images of rat anatomy obtained using the accelerator-produced (99m)Tc with those obtained using (99m)Tc from a commercial generator were compared. Disks of (100)Mo were irradiated with x-rays produced by a 35 MeV electron beam to generate about 1110 MBq (30 mCi) of (99)Mo per disk. After target dissolution, a NorthStar ARSII unit was used to separate the (99m)Tc, which was subsequently used to tag pharmaceuticals suitable for cardiac and bone imaging. SPECT images were acquired for three rats and compared to images for the same three rats obtained using (99m)Tc from a standard reactor (99)Mo generator. The efficiency of (99)Mo-(99m)Tc separation was typically greater than 90%. This study demonstrated the delivery of (99m)Tc from the end of beam to the end user of approximately 30 h. Images obtained using the heart and bone scanning agents using reactor and linac-produced (99m)Tc were comparable. High-power electron accelerators are an attractive option for producing (99)Mo on a national scale.

Sci-Fri AM: Imaging - 09: Serial estimation of cross-talk for correction in dual-isotope imaging with dynamic tracers.

The recent radioisotope shortage has led to interest in non-Tc99m-based tracers. We have developed a novel I-123-labelled myocardial perfusion imaging tracer. We compare the I123-tracer to the clinical standard of Tc99m tetrofosmin in vivo in a rat model using a small-animal SPECT/CT camera. SPECT distinguishes different isotopes based on the different energies of the emitted gamma rays and thus allows simultaneous comparison of two tracer distributions in the same animal. Dual-isotope imaging is complicated by cross-talk between the energy windows of the isotopes. Standard energy-window-based correction methods are difficult to employ because of the proximity in energy of Tc99m (140keV) and I123 (159keV). Imaging the second tracer's energy window prior to its injection provides an estimate of the cross-talk. However, this estimate is only accurate if the tracer distribution is static. We use serial imaging prior to the introduction of the second tracer to estimate the dynamics of the first tracer and interpolate the cross-talk images to provide a more accurate correction. We used rat models of myocardial disease (n=3). I123 tracer was injected and imaged for one hour at 20min intervals. The Tc99m tetrofosmin was then injected and 30min later, a dual-isotope image was obtained. The impact of this approach is assessed by comparing the differences in the Tc99m-tetrofosmin image using this method with correction by simple correction for physical decay. The interpolative approach improves the accuracy of the correction by 2%-5% and thereby enhances the comparison of the two tracers.

A simple indentation device for measuring micrometer-scale tissue stiffness.

Mechanical properties of cells and extracellular matrices are critical determinants of function in contexts including oncogenic transformation, neuronal synapse formation, hepatic fibrosis and stem cell differentiation. The size and heterogeneity of biological specimens and the importance of measuring their mechanical properties under conditions that resemble their environments in vivo present a challenge for quantitative measurement. Centimeter-scale tissue samples can be measured by commercial instruments, whereas properties at the subcellular (nm) scale are accessible by atomic force microscopy, optical trapping, or magnetic bead microrheometry; however many tissues are heterogeneous on a length scale between micrometers and millimeters which is not accessible to most current instrumentation. The device described here combines two commercially available technologies, a micronewton resolution force probe and a micromanipulator for probing soft biological samples at sub-millimeter spatial resolution. Several applications of the device are described. These include the first measurement of the stiffness of an intact, isolated mouse glomerulus, quantification of the inner wall stiffness of healthy and diseased mouse aortas, and evaluation of the lateral heterogeneity in the stiffness of mouse mammary glands and rat livers with correlation of this heterogeneity with malignant or fibrotic pathology as evaluated by histology.

Sci-Sat AM(1): Imaging-04: Respiratory errors in cardiac PET/CT with manual alignment of the CT image.

Respiratory motion can produce misregistration errors between CT and PET images in cardiac PET/CT imaging. The objective of this study was to determine if manual registration of a single-phase end-expiration CT scan to the PET image would eliminate respiratory-induced artifacts. Listmode data from 71 cardiac PET patient scans were rebinned into a 8-frame respiratory-gated image series based on a respiratory trigger signal obtained with an optical tracking system. CT-based attenuation correction (AC) was performed after registering the CT image with the mean position of the PET images. The 8 phases of the gated PET study were coregistered and the breathing motion was measured. Images from end-inspiration and end-expiration were compared to assess the effect of motion. Studies in which the motion was >8mm were reconstructed again, with the CT scan aligned to end-expiration or end-inspiration, to determine if phase-specific registration could reduce the residual errors. The motion was found to be greatest in the axial direction (mean 4.1mm +\- 1.8mm) and 4 Rb stress studies (17%) had motion >8mm. The maximum displacement during breathing was greater for Rb-stress imaging (<15mm) than for resting (<7.5mm) or NH3-stress (<5.4mm) imaging. No significant differences were noted between the respiratory phases of the rest studies. Errors in myocardial radiotracer uptake of up to 35% were noted between end-inspiration and end-expiration for studies with >8mm of motion. Phase-specific registration of the CT reduced the extent of the errors but did not fully resolve them, suggesting that more sophisticated AC is required.

Increased ADAMTS-13 proteolytic activity in rat hepatic stellate cells upon activation in vitro and in vivo.

INTRODUCTION: ADAMTS-13 is a member of A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS) family, primarily synthesized in hepatic stellate cells (HSCs), one of the major cell types transdifferentiating into myofibroblasts during liver fibrosis. However, the association between ADAMTS-13 expression and HSC activation or liver fibrosis is not known. METHODS: In this study, we determined the ADAMTS-13 mRNA, protein, and activity in isolated primary HSCs upon activation on a plastic dish and in liver after administration of carbon tetrachloride (CCl(4)) in rats. RESULTS: We showed that ADAMTS-13 antigen and proteolytic activity in the activated rat HSCs were dramatically increased, whereas ADAMTS-13 mRNA in these cells was only minimally altered. Similarly, the ADAMTS-13 antigen and proteolytic activity in rat liver after CCl(4) injury were also significantly increased, whereas the ADAMTS-13 mRNAs in these liver tissues were only slightly increased compared with normal. Surprisingly, despite the dramatic up-regulation of ADAMTS-13 protein synthesis in the activated HSCs after CCl(4) administration, the plasma levels of ADAMTS-13 protease in rats did not increase concordantly. CONCLUSION: We conclude that the up-regulation of ADAMTS-13 protein expression in rat HSCs during activation in vitro and in vivo suggests the possibility of ADAMTS-13 proteolysis, an important part of function of the activated HSCs, perhaps through modulation of liver regeneration or formation of liver fibrosis after various injuries. The data also suggest the minimal contribution of the activated HSCs in regulation of plasma levels of ADAMTS-13 protease.

Novel inactivating mutations of transforming growth factor-beta type I receptor gene in head-and-neck cancer metastases.

Carcinoma cell lines are frequently refractory to transforming growth factor-beta (TGF beta)-mediated cell cycle arrest. Whether and how TGF beta signaling is disrupted in the majority of human tumors, however, remains unclear. To investigate whether TGF beta signaling might be disrupted by inactivation of the key signaling molecule, the TGF beta type I (T beta R-I) receptor, and whether or not T beta R-I inactivation is associated with late stage disease, we conducted a comprehensive structural analysis of the T beta R-I gene in fine-needle aspirates of 23 head-&-neck cancer metastases. We encountered 4 different mutations of T beta R-I, 3 of which have not been previously identified. In 1 case, we found a somatic intragenic 4-bp deletion predicting for a truncation of the receptor protein. This is the first example of a true loss-of-function mutation of T beta R-I in a human epithelial neoplasm. In 2 other cases, we identified missense mutations located between the juxtamembrane- and serine-threonine kinase domains. One of these resulted in an alanine-to-threonine substitution (A230T), which disrupts receptor signaling activity by causing rapid protein degradation within the endoplasmatic reticulum. This represents a novel mechanism of inactivation of a TGF beta signaling intermediate. Finally, we identified a serine-to-tyrosine substitution at codon 387 (S387Y) in a metastasis but not in the corresponding primary tumor. We had previously shown this S387Y mutant to be predominantly associated with breast cancer metastases and to have a diminished ability to mediate TGF beta-dependent signaling. In aggregate, these findings provide further support for the hypothesis that inactivation of the TGF beta signaling pathway occurs in a significant subset of human cancers.

Fibrogenesis. V. TGF-beta signaling pathways.

Transforming growth factor (TGF)-beta is a multifunctional peptide growth factor with a wide range of potential effects on growth, differentiation, extracellular matrix deposition, and the immune response. General TGF-beta signaling pathways have been described in detail over the last several years, but factors that determine the nature of the TGF-beta response are poorly understood. In particular, signaling pathways that specifically mediate the matrix effects of TGF-beta have received little attention, although they will be important therapeutic targets in the treatment of pathological fibrosis. This themes article focuses on TGF-beta signaling and highlights potential points for generating matrix-specific responses.

LROC analysis of detector-response compensation in SPECT.

Localization ROC (LROC) observer studies examined whether detector response compensation (DRC) in ordered-subset, expectation-maximization (OSEM) reconstructions helps in the detection and localization of hot tumors. Simulated gallium (Ga-67) images of the thoracic region were used in the study. The projection data modeled the acquisition of attenuated 93- and 185-keV photons with a medium-energy parallel-hole collimator, but scatter was not modeled. Images were reconstructed with five strategies: 1) OSEM with no DRC; 2) OSEM preceded by restoration filtering; 3) OSEM with iterative DRC; 4) OSEM with an ideal DRC; and 5) filtered backprojection (FBP) with no DRC. All strategies included attenuation correction. There were four LROC studies conducted. In a study using a single tumor activity, the ideal DRC offered the best performance, followed by iterative DRC, restoration filtering, OSEM with no DRC, and FBP. Statistical significance at the 5% level was found between all pairs of strategies except for restoration filtering and OSEM with no DRC. A similar ranking was found for a more realistic study using multiple tumor activities. Additional studies considered the effects of OSEM iteration number and tumor activity on the detection improvement that iterative DRC offered with respect to OSEM with no DRC.

Comparing filtered backprojection and ordered-subsets expectation maximization for small-lesion detection and localization in 67Ga SPECT.

UNLABELLED: Iterative reconstruction of SPECT images has recently become clinically available as an alternative to filtered backprojection (FBP). However, there is conflicting evidence on whether iterative reconstruction, such as with the ordered-subsets expectation maximization (OSEM) algorithm, improves diagnostic performance over FBP. The study objective was to determine if the detection and localization of small lesions in simulated thoracic gallium SPECT images are better with OSEM reconstruction than with FBP, both with and without attenuation correction (AC). METHODS: Images were simulated using an analytic projector acting on the mathematic cardiac torso computer phantom. Perfect scatter rejection was assumed. Lesion detection accuracy was assessed using localization receiver operating characteristic methodology. The images were read by 5 nuclear medicine physicians. For each reconstruction strategy and for each observer, data were collected in 2 viewing sessions of 100 images. Two-way ANOVA and, when indicated, the Scheffé multiple comparisons test were applied to check for significant differences. RESULTS: Little difference in the accuracy of detection or localization was seen between FBP with and without AC. OSEM with AC extended the contrast range for accurate lesion detection and localization over that of the other methods investigated. Without AC, no significant difference between OSEM and FBP reconstruction was detected. CONCLUSION: OSEM with AC may improve the detection and localization of thoracic gallium-labeled lesions over FBP reconstruction.

The effect of changing the excreta moisture of caged laying hens on the excreta and microbial contamination of their egg shells.

1. An experiment that included 1440 caged laying hens in 24 experimental units was conducted to determine the effect of differences in excreta moisture on the proportion of dirty eggs and the microbial contamination of eggs that were ostensibly uncontaminated by excreta. Excreta moisture contents were changed by giving the hens diets that contained 4 different concentrations of sodium. 2. Diets containing 1.6, 5, 10 or 15 g/kg dietary sodium were fed ad libitum to 1140 laying hens for a 12-week feeding period. A sample of excreta was collected from each experimental unit each week and its moisture content determined. All eggs produced were classified as clean or dirty according to the European Community Egg Marketing Regulations. A sample of eggs were collected from each experimental unit on 4 separate occasions in the last 4 weeks of the feeding period and the total bacterial numbers on ostensibly clean egg shells were determined. 3. Increasing dietary sodium concentration gave linear (P<0.01) increases in excreta moisture. Each 100 g/kg increase in excreta moisture increased (P<0.001) dirty egg numbers by 0.52% of the total eggs produced. Increasing excreta moisture gave a linear increase (P<0.001) in the (log-transformed) numbers of microorganisms that contaminated ostensibly clean egg shells.

Effect of excess dietary sodium, potassium, calcium and phosphorus on excreta moisture of laying hens.

1. Four experiments were conducted to investigate the effects of dietary concentrations of sodium, potassium, calcium or phosphate on the water intake and excreta moisture of laying hens. A fifth experiment examined the effect on these variables of increasing amounts of 2 different sodium salts (chloride or bicarbonate) and the interactions with 2 levels of dietary phosphorus. 2. All experiments involved individually caged laying hens fed on diets varying in 1 or 2 minerals in replacement for washed sand. The experimental diets contained mineral concentrations that either met or exceeded the expected requirement of the hens. The diets were given for a 7 or 8 d feeding period and food and water intakes were measured and excreta were collected for the last 48 h of each feeding period. These data were corrected for evaporative water loss to the environment during the collection period. 3. Increasing dietary concentrations of sodium, potassium or phosphorus gave linear increases (P<0.001) in the water intake of the laying hens and linear increases (P<0.01) in the moisture content of their excreta. Each 1 g/kg increase in dietary mineral increased the moisture content of the excreta by 9.04 (+/- 1.57), 11.95 (+/- 2.02) and 5.59 (+/- 0.31) g/kg (+/- standard error) for sodium, potassium and phosphorus, respectively. Increasing concentrations of dietary calcium did not significantly affect the water intakes or excreta moisture levels of the laying hens. 4. The fifth experiment showed that, although there was a sodium x phosphorus interaction (P<0.05), the effects of the 2 mineral additions were approximately additive. There were no significant differences (P>0.05) in water intakes or excreta moisture contents due to the 2 different sodium salts (chloride or bicarbonate).

Effect of filtering on the detection and localization of small Ga-67 lesions in thoracic single photon emission computed tomography images.

Tumor detection can be significantly affected by filtering so determining an optimal filter is an important aspect of establishing a clinical reconstruction protocol. The purpose of this study was to identify the cut-off frequency of a Butterworth filter used in a filtered backprojection (FBP) reconstruction that maximized the detection and localization accuracy of 1 cm spherical lesions in Ga-67 citrate, thoracic SPECT images. Image quality was evaluated by means of a localization receiver operating characteristic (LROC) study using computer simulated images. Projection data were generated using the mathematical cardiac-torso digital phantom with a clinically realistic background source distribution. The images were reconstructed using FBP with multiplicative Chang attenuation correction and fifth-order Butterworth filtering. The cut-off frequencies considered were 0.25, 0.32, 0.47, and 0.79 cm(-1) for the case of three-dimensional (3D) post-filtering and 0.25, 0.32, and 0.47 cm(-1) for two-dimensional (2D) post-filtering. The images were read by three research scientists and one board certified nuclear medicine clinician. The area under the LROC curve and the localization accuracy for all test conditions were compared using Scheffé's multiple comparisons test. It was found that 3D post-filtering using filters with cut-off frequencies of 0.32 and 0.47 cm(-1) resulted in the highest lesion detectability and localization accuracy. These two test conditions did not differ significantly from each other but were significantly better (p<0.05) than all of the 2D, and the 3D 0.79 cm(-1) cut-off frequency cases.

Transforming growth factor-beta induces formation of a dithiothreitol-resistant type I/Type II receptor complex in live cells.

Transforming growth factor-beta (TGF-beta) binds to and signals via two serine-threonine kinase receptors, the type I (TbetaRI) and type II (TbetaRII) receptors. We have used different and complementary techniques to study the physical nature and ligand dependence of the complex formed by TbetaRI and TbetaRII. Velocity centrifugation of endogenous receptors suggests that ligand-bound TbetaRI and TbetaRII form a heteromeric complex that is most likely a heterotetramer. Antibody-mediated immunofluorescence co-patching of epitope-tagged receptors provides the first evidence in live cells that TbetaRI. TbetaRII complex formation occurs at a low but measurable degree in the absence of ligand, increasing significantly after TGF-beta binding. In addition, we demonstrate that pretreatment of cells with dithiothreitol, which inhibits the binding of TGF-beta to TbetaRI, does not prevent formation of the TbetaRI.TbetaRII complex, but increases its sensitivity to detergent and prevents TGF-beta-activated TbetaRI from phosphorylating Smad3 in vitro. This indicates that either a specific conformation of the TbetaRI. TbetaRII complex, disrupted by dithiothreitol, or direct binding of TGF-beta to TbetaRI is required for signaling.

Oligomeric structure of type I and type II transforming growth factor beta receptors: homodimers form in the ER and persist at the plasma membrane.

Transforming growth factor beta (TGF-beta) signaling involves interactions of at least two different receptors, types I (TbetaRI) and II (TbetaRII), which form ligand-mediated heteromeric complexes. Although we have shown in the past that TbetaRII in the absence of ligand is a homodimer on the cell surface, TbetaRI has not been similarly investigated, and the site of complex formation is not known for either receptor. Several studies have indicated that homomeric interactions are involved in TGF-beta signaling and regulation, emphasizing the importance of a detailed understanding of the homooligomerization of TbetaRI or TbetaRII. Here we have combined complementary approaches to study these homomeric interactions in both naturally expressing cell lines and cells cotransfected with various combinations of epitope-tagged type I or type II receptors. We used sedimentation velocity of metabolically labeled receptors on sucrose gradients to show that both TbetaRI and TbetaRII form homodimer-sized complexes in the endoplasmic reticulum, and we used coimmunoprecipitation studies to demonstrate the existence of type I homooligomers. Using a technique based on antibody-mediated immunofluorescence copatching of receptors carrying different epitope tags, we have demonstrated ligand-independent homodimers of TbetaRI on the surface of live cells. Soluble forms of both receptors are secreted as monomers, indicating that the ectodomains are not sufficient to mediate homodimerization, although TGF-beta1 is able to promote dimerization of the type II receptor ectodomain. These findings may have important implications for the regulation of TGF-beta signaling.

Biosynthesis of the type I and type II TGF-beta receptors. Implications for complex formation.

The TGF-beta type I and type II receptors (TbetaRI and TbetaRII) are signaling receptors that form heteromeric cell surface complexes with the TGF-betas as one of the earliest events in the cellular response to these multifunctional growth factors. Using TGF-beta-responsive mink lung epithelial cells (Mv1Lu), we have determined the half-lives of the endoplasmic reticulum (ER) and mature forms of these receptors. In metabolically labeled cells, approximately 90% of newly synthesized type II receptor undergoes modification of N-linked sugars in the Golgi, with a half-life of 30-35 min; the Golgi-processed form of the receptor has a relatively short metabolic half-life of 2.5 h. In contrast, only 50% of pulse-labeled type I receptor is converted to the Golgi-processed and therefore endoglycosidase H-resistant form, and the endoglycosidase H-sensitive ER form has a half-life of 2.8-3 h. Addition of 100 pM TGF-beta1 causes the Golgi-processed type II receptor to become less stable, with a half-life of 1.7 h, and also destabilizes the Golgi-processed type I receptor. TGF-beta1 binding and cross-linking experiments on cells treated with tunicamycin for various times confirm different ER to cell surface processing times for TbetaRI and TbetaRII. Our results, which suggest that stable complexes between type I and II TGF-beta receptors do not form until the proteins reach a post-ER compartment (presumably the cell surface), have important implications for our understanding of complex formation and receptor regulation.

Wire stent for tracheomalacia in a five-year-old girl.

A wire stent was used successfully to treat life-threatening tracheomalacia in a 5-year-old girl. Wire stents placed bronchoscopically are nonobstructing and have the potential for balloon expansion to accommodate growth.