RNAseq reveals extensive metabolic disruptions in the sensitive SF-295 cell line treated with schweinfurthins.

The schweinfurthin family of natural compounds exhibit a unique and potent differential cytotoxicity against a number of cancer cell lines and may reduce tumor growth in vivo. In some cell lines, such as SF-295 glioma cells, schweinfurthins elicit cytotoxicity at nanomolar concentrations. However, other cell lines, like A549 lung cancer cells, are resistant to schweinfurthin treatment up to micromolar concentrations. At this time, the precise mechanism of action and target for these compounds is unknown. Here, we employ RNA sequencing of cells treated with 50 nM schweinfurthin analog TTI-3066 for 6 and 24 h to elucidate potential mechanisms and pathways which may contribute to schweinfurthin sensitivity and resistance. The data was analyzed via an interaction model to observe differential behaviors between sensitive SF-295 and resistant A549 cell lines. We show that metabolic and stress-response pathways were differentially regulated in the sensitive SF-295 cell line as compared with the resistant A549 cell line. In contrast, A549 cell had significant alterations in response genes involved in translation and protein metabolism. Overall, there was a significant interaction effect for translational proteins, RNA metabolism, protein metabolism, and metabolic genes. Members of the Hedgehog pathway were differentially regulated in the resistant A549 cell line at both early and late time points, suggesting a potential mechanism of resistance. Indeed, when cotreated with the Smoothened inhibitor cyclopamine, A549 cells became more sensitive to schweinfurthin treatment. This study therefore identifies a key interplay with the Hedgehog pathway that modulates sensitivity to the schweinfurthin class of compounds.

Experiment assessment of mass effects in the rat: implications for small animal PET imaging.

In vivo imaging using positron emission tomography (PET) is important in the development of new radiopharmaceuticals in rodent animal models for use as biochemical probes, diagnostic agents, or in drug development. We have shown mathematically that, if small animal imaging studies in rodents are to have the same "quality" as human PET studies, the same number of coincidence events must be detected from a typical rodent imaging "voxel" as from the human imaging voxel. To achieve this using the same specific activity preparation, we show that roughly the same total amount of radiopharmaceutical must be given to a rodent as to a human subject. At high specific activities, the mass associated with human doses, when administered to a rodent, may not decrease the uptake of radioactivity at non saturable sites or sites where an enzyme has a high capacity for a substrate. However, in the case of binding sites of low density such as receptors, the increased mass injected could saturate the receptor and lead to physiologic effects and non-linear kinetics. Because of the importance of the mass injected for small animal PET imaging, we experimentally compared high and low mass preparations using ex vivo biodistribution and phosphorimaging of three compounds: 2-fluoro-2-deoxyglucose (FDG), 6-fluoro-L-metatyrosine (FMT) and one receptor-directed compound, the serotonin 5HT1A receptor ligand, trans-4-fluoro-N-[2-[4-(2-methoxylphenyl) piperazino]ethyl]-N-(2-pyridyl) cyclohexane- carboxamide (FCWAY). Changes in the mass injected per rat did not affect the distribution of FDG, FMT, and FCWAY in the range of 0.6-1.9 nmol per rat. Changes in the target to nontarget ratio were observed for injected masses of FCWAY in the range of approximately 5-50 nmol per rat. If the specific activity of such compounds and/or the sensitivity of small animal scanners are not increased relative to human studies, small animal PET imaging will not correctly portray the "true" tracer distribution. These difficulties will only be exacerbated in animals smaller than the rat, e.g., mice.

A novel approach using transcomplementing adenoviral vectors for gene therapy of adrenocortical cancer.

Current therapies for adrenocortical carcinomas do not improve the life expectancy of patients. In this study, we tested whether a gene-transfer therapy based upon a suicide gene/prodrug system would be effective in an animal model of the disease. We employed E4- and E1A/B-depleted, herpes simplex virus-thymidine kinase-expressing adenoviral mutants that transcomplement each other within tumor cells, hereby improving transgene delivery and efficacy by viral replication in situ. Transcomplementation of vectors increased the fraction of transduced of tumor cells. This increase was accompanied by greater tumor volume reduction compared to non-transcomplementing approaches. Survival time improved with non-replicating vectors plus GCV compared to controls. However, transcomplementation/replication of vectors led to a further significant increment in anti-tumor activity and survival time (p < 0.02). In treated animals, we observed a high number of apoptotic nuclei both adjacent to and distant from injection sites and sites of viral oncolysis. Ultrastructural analyses exhibited nuclear inclusion bodies characteristic of virus production in situ, and provided further evidence that this therapy induced apoptotic cell death within tumor cells. We conclude that the efficacy of suicide gene therapy is significantly amplified by viral replication and, in combination with GCV, significantly reduces tumor burden and increases survival time.

Monitoring the correction of glycogen storage disease type 1a in a mouse model using [(18)F]FDG and a dedicated animal scanner.

Monitoring gene therapy of glycogen storage disease type 1a in a mouse model was achieved using [(18)F]FDG and a dedicated animal scanner. The G6Pase knockout (KO) mice were compared to the same mice after infusion with a recombinant adenovirus containing the murine G6Pase gene (Ad-mG6Pase). Serial images of the same mouse before and after therapy were obtained and compared with wild-type (WT) mice of the same strain to determine the uptake and retention of [(18)F]FDG in the liver. Image data were acquired from heart, blood pool and liver for twenty minutes after injection of [(18)F]FDG. The retention of [(18)F]FDG was lower for the WT mice compared to the KO mice. The mice treated with adenovirus-mediated gene therapy had retention similar to that found in age-matched WT mice. These studies show that FDG can be used to monitor the G6Pase concentration in liver of WT mice as compared to G6Pase KO mice. In these mice, gene therapy returned the liver function to that found in age matched WT controls as measured by the FDG kinetics in the liver compared to that found in age matched wild type controls.

High resolution PET, SPECT and projection imaging in small animals.

Positron emission tomography, single photon emission computed tomography and planar projection imaging of radioactive tracers have long been in use for detecting and diagnosing disease in human subjects. More recently, advanced versions of these same technologies have begun to be used across the breadth of modern biomedical research to study non-invasively small laboratory animals in a myriad of experimental settings. In this report, we describe some of the new instruments and techniques that make these measurements possible and illustrate, with a few examples, the potential power of these methods in modern biomedical research.

Performance characteristics of the 3-D OSEM algorithm in the reconstruction of small animal PET images. Ordered-subsets expectation-maximixation.

Rat brain images acquired with a small animal positron emission tomography (PET) camera and reconstructed with the three-dimensional (3-D) ordered-subsets expectation-maximization (OSEM) algorithm with resolution recovery have better quality when the brain is imaged by itself than when inside the head with surrounding background activity. The purpose of this study was to characterize the dependence of this effect on the level of background activity, attenuation, and scatter. Monte Carlo simulations of the imaging system were performed. The coefficient of variation from replicate images, full-width at half-maximum (FWHM) from point sources and image profile fitting, and image contrast and uniformity were used to evaluate algorithm performance. A rat head with the typical levels of five and ten times the brain activity in the surrounding background requires additional iterations to achieve the same resolution as the brain-only case at a cost of 24% and 64% additional noise, respectively. For the same phantoms, object scatter reduced contrast by 3%-5%. However, attenuation degraded resolution by 0.2 mm and was responsible for up to 12% nonuniformity in the brain images suggesting that attenuation correction is useful. Given the effects of emission and attenuation distribution on both resolution and noise, simulations or phantom studies should be used for each imaging situation to select the appropriate number of OSEM iterations to achieve the desired resolution-noise levels.

Evaluation of 99mTc-mercaptoacetyltriglycine-biocytin as a new hepatobiliary imaging agent in mice coinjected with bilirubin.

We evaluated 99mTc-labeled mercaptoacetyltriglycine (99mTc-MAG3)-biocytin as a hepatobiliary imaging agent in the absence and presence of bilirubin in mice. We then compared its pharmacokinetic parameters; peak liver/heart activity ratio (rmax) and half clearance time (HCT) with those of 99mTc-labeled diisopropyl-iminodiacetic acid (99mTc-disofenin). Balb/c mice were injected intravenously with hepatobiliary agent (99mTc-MAG3-biocytin or 99mTc-disofenin) alone or in combination with bilirubin at two doses (7 and 14 mg/kg) dissolved in 5% human serum albumin. Images were acquired every 15 s for 30 min with a gamma-camera equipped with a pinhole collimator. Dynamic images showed rapid hepatic uptake of 99mTc-MAG3-biocytin, with rapid clearance from the blood and rapid excretion via the biliary system. Its hepatic uptake was not affected by bilirubin coinjection, whereas 99mTc-disofenin coinjected with bilirubin showed a higher blood background than 99mTc-disofenin alone. These qualitative findings were reflected in pharmacokinetic parameters, rmax and HCT. The rmax was obtained from plots of time versus liver/heart activity ratios obtained in equal-area regions of interest over the heart and liver. The HCT was calculated from the hepatic clearance curve from plots of time versus liver activity. 99mTc-MAG3-biocytin without bilirubin coinjection showed an rmax of 8.9+/-1.3 and an HCT of 399+/-36 s. These values did not change even when 14 mg/kg of bilirubin were coinjected. By contrast, the parameters for 99mTc-disofenin with bilirubin were significantly (p < 0.01) affected by 14 mg/kg of bilirubin coinjection: rmax was decreased from 7.9+/-2.5 to 1.4+/-0.2 and HCT was increased from 292+/-32 s to 782+/-133 s. 99mTc-MAG3-biocytin hepatobiliary scintigraphy in mice is not affected by bilirubin coinjection, and this hepatobiliary agent appears to offer promise for estimating hepatic function in patients with high bilirubin levels.

Use of 99mTc-mercaptoacetyltriglycine (MAG3)-biocytin hepatobiliary scintigraphy to study the protective effect of a synthetic enzyme inhibitor on acute hepatotoxicity in mice.

Recent data suggest that inhibitors of ethanol-inducible cytochrome P450 (CYP2E1) can protect the liver from injury caused by various substrates of CYP2E1. In this study, we measured the protective effect of isopropyl-2-(1,3-dithioetane-2-ylidene)-2[N-(4-methylthiazol -2-yl)-carbamoyl]acetate (YH439), a transcriptional inhibitor of CYP2E1, against carbon tetrachloride (CCl4)-induced hepatotoxicity by using various conventional methods and dynamic scintigraphy with 99mTc-mercaptoacetyltriglycine (MAG3)-biocytin, a recently developed scintigraphic agent. Balb/c mice were pretreated with two doses of YH439 (50 or 150 mg/kg per day) at 48 h and 24 h and one dose of CCl4 (0.25 mL/kg) at 18 h before scintigraphy. The results were compared with those of two other groups, one that received CCl4 but not YH439, and the other that received neither (control). Scintigraphic images were acquired continuously at 15-sec intervals for 30 min. Pharmacokinetic parameters, such as peak liver/heart ratio (r(max)), peak liver uptake time (t(max)), and hepatic half-clearance time (HCT), were obtained from time-activity curves derived from regions-of-interest (ROI) over the liver and the heart. Acute administration of CCl4 alone caused centrilobular necrosis and serum transaminase levels to rise more than 5 times higher than those of the control group. Pharmacokinetic parameters also changed significantly from those of the control group. Administration of YH439 prevented centrilobular necrosis and significantly improved pharmacokinetic parameters. This study demonstrates for the first time that hepatobiliary scintigraphy can be used to study in vivo biochemistry of the CYP2E1 inhibitor (YH439) against liver toxicity.

Performance characteristics of a compact position-sensitive LSO detector module.

We assembled a compact detector module comprised of an array of small, individual crystals of lutetium oxyorthosilicate:Ce (LSO) coupled directly to a miniature, metal-can, position-sensitive photomultiplier tube (PSPMT). We exposed this module to sources of 511-keV annihilation radiation and beams of 30- and 140-keV photons and measured spatial linearity; spatial variations in module gain, energy resolution, and event positioning; coincidence timing; the accuracy and sensitivity of identifying the crystal-of-first-interaction at 511 keV; and the effects of intercrystal scatter and LSO background radioactivity. The results suggest that this scintillator/phototube combination should be highly effective in the coincidence mode and can be used, with some limitations, to image relatively low-energy single photon emitters. Photons that are completely absorbed on their first interaction at 511 keV are positioned by the module at the center of a crystal. Intercrystal scatter events, even those that lead to total absorption of the incident photon, are placed by the module in a regular "connect-the-dot" pattern that joins crystal centers. As a result, the accuracy of event positioning can be made to exceed 90%, though at significantly reduced sensitivity, by retaining only events that occur within small regions-of-interest around each crystal center and rejecting events that occur outside these regions in the connect-the-dot pattern.

The case of Mr. Martinez: ethical conflicts in home care.

Mr. Martinez recently was accepted into the Valley Hospice Program with a diagnosis of end-stage lung cancer. He currently lives with his daughter, Amelia, after moving from Puerto Rico 2 years ago after his wife died. His two sons and another daughter live within 20 minutes of Amelia's home. They are a close-knit family, and the children are committed to honoring Martinez' wishes to die in his room in Amelia's home.

A head motion measurement system suitable for emission computed tomography.

Subject motion during brain imaging studies can adversely affect the images through loss of resolution and other artifacts related to movement. We have developed and tested a device to measure head motion externally in real-time during emission computed tomographic (ECT) brain imaging studies, to be used eventually to correct ECT data for that motion. The system is based on optical triangulation of three miniature lights affixed to the patient's head and viewed by two position-sensitive detectors. The computer-controlled device converts the three sets of lamp positions into rotational and translational coordinates every 0.7 seconds. When compared against a mechanical test fixture, the optical system was found to be linear and accurate with minimal crosstalk between the coordinates. In a study of two subjects, comparing the angular motions measured by the optical device and a commercially available electromagnetic motion detector, the two systems agreed well, with an root mean square (rms) difference of less than 0.6 degree for all rotations.

Dynamic circular buffering: a technique for equilibrium gated blood pool imaging.

We have devised a software technique called "dynamic circular buffering" (DCB) with which we create a gated blood pool image sequence of the heart in real time using the best features of LIST and FRAME mode methods of acquisition/processing. The routine is based on the concept of independent "agents" acting on the timing and position data continuously written into the DCB. This approach allows efficient asynchronous operation on PC-type machines and enhanced capability on systems capable of true multiprocessing and multithreading.

Head movement in normal subjects during simulated PET brain imaging with and without head restraint.

UNLABELLED: Head movement during brain imaging is recognized as a source of image degradation in PET and most other forms of medical brain imaging. However, little quantitative information is available on the kind and amount of head movement that actually occurs during these studies. We sought to obtain this information by measuring head movement in normal volunteers. METHODS: Head position data were acquired for 40 min in each of 13 supine subjects with and without head restraint. These data were then used to drive a mathematically simulated head through exactly the same set of movements. The positions of point sources embedded in this head were computed at each location and these data summarized as movement at FWHM in each of the three coordinate directions. RESULTS: Head movement increased with the length of the sampling interval for studies of either type (with or without head restraint), but the amount and rate of increase with restraint was much smaller. In contrast, head movement during consecutive, short sampling intervals was small and did not increase with time. Spatial gradients in head movement were detected within each study type, and significant spatial differences in head movement were found between study types. CONCLUSIONS: Head movements in normal, supine subjects, though small, can cause the effective resolution of a brain imaging study to appear to vary in space and time. These effects can be reduced significantly with head restraint and may also be reduced by dividing the acquisition of a single image into a sequence of short images (instead of a single long image), aligning these images spatially and summing the result.

Assessment of coronary artery disease by radionuclide cineangiography. History, current applications, and new directions.

In recent years, the utility of radionuclide cineangiography in prognostication has made it a mainstay of management decision making. With evolution of therapeutic modalities and concomitant alteration in management strategies, however, the technique and its application are undergoing parallel evolution to optimize response to current needs. Thus, in addition to standard planar rest and exercise assessment of left ventricular function, newer approaches are involving combined perfusion and function assessment, application of pharmacological stress to permit technically advantageous variations in imaging protocols, and application of SPECT technology in collecting blood pool data for tomographic display and analysis.

Performance characteristics of a 511-keV collimator for imaging positron emitters with a standard gamma-camera.

Line-source experiments were conducted to assess the performance of a gamma-camera equipped with a specially designed 511-keV collimator for the planar imaging of positron emitters. The results were compared with the camera performance with routinely used collimators and radionuclides (thallium-201, technetium-99m and gallium-67). With positron emitters, scatter contributed less to the widening of the line spread function than with radionuclides emitting lower photon energies. These observations can be explained by the relative deterioration in the discrimination power of the gamma-camera to reject scattered radiation at low energies. Planar 511-keV imaging may provide relevant clinical information, as we showed by fluorodeoxyglucose studies in a patient with a myocardial infarction and in a patient with a malignant lymphoma. It is concluded that positron emitters can be effectively applied for planar imaging with the generally available gamma-cameras. This study implies that radiotracers developed for positron emission tomography may find a place in the practice of conventional nuclear medicine.

A theoretical comparison of first-pass and gated equilibrium methods in the measurement of systolic left ventricular function.

First-pass and gated equilibrium radionuclide studies of left ventricular function have proven extremely useful in the detection and management of patients with heart disease. Despite this practical experience, however, comparison of these methods generally has been confined to procedural differences that do not reflect the intrinsic properties of the methods. Here, we describe the results of a simple theoretical calculation from first principles that compares the methods based on their relative statistical precision. This analysis assumes that each procedure is carried out with the same tracer dose in the same hypothetical patient under identical conditions and with the same ideal imaging equipment. Results obtained with this model suggest that the imaging time required for a gated equilibrium study to achieve the same statistical precision as a first-pass study is typically less than 2 min in resting subjects and less than 1 min during stress. The analysis also indicates that gated equilibrium studies will tend to possess the greater statistical precision when cardiac output is elevated, such as when the heart is imaged during exercise. On the other hand, this analysis indicates that the first-pass method will tend to possess the greater precision when cardiac output is low and when imaging time is highly constrained.

Coronary vasoconstriction induced by vasopressin. Production of myocardial ischemia in dogs by constriction of nondiseased small vessels.

BACKGROUND: We studied the effect of intracoronary administration of arginine-8-vasopressin on blood flow in nondiseased coronary arteries and determined whether this vasoconstriction was severe enough to produce ischemia in 30 dogs. METHODS AND RESULTS: In group 1 (n = 6), after vasopressin administration coronary blood flow was decreased by 41% (p less than 0.002) without changes in heart rate or aortic pressure, and left ventricular ejection fraction measured by radionuclide angiocardiography was decreased by 18% (p less than 0.0005). In group 2 (n = 6), ischemia was confirmed by measurement of transmural pH changes. Administration of vasopressin decreased subendocardial pH of the infused zone from 7.40 +/- 0.03 to 7.31 +/- 0.07 (p less than 0.01). The subendocardial pH of the zone not infused with vasopressin did not change. To overcome the intrinsic regulation of blood flow, operating primarily in small coronary arteries, we hypothesized that vasopressin must increase resistance primarily in large rather than small coronary arteries. After intracoronary infusion in group 3 (n = 6), however, most (94%) of the increase in resistance during vasopressin administration was explained by an increase of resistance in small coronary arteries. In group 4 (n = 9), vasopressin decreased coronary blood flow by 50% and decreased local shortening by 90% at a time when systemic hemodynamics were unchanged. Coronary constriction induced by vasopressin, or the recovery from it, also was not altered by cyclooxygenase blockade. CONCLUSIONS: Thus, vasopressin produces myocardial ischemia by constricting small, nondiseased coronary arteries severely enough to overcome the competition from normal coronary regulation, and this ischemic event is not mediated by prostaglandin products.

Comparison of fixed and variable temporal resolution methods for creating gated cardiac blood-pool image sequences.

In each of 50 resting subjects, two gated blood-pool image sequences were created from the same LIST mode data set. One sequence was created using a sorting method that spans each individual cardiac cycle with the same number of images (the "variable temporal" or VT method), while the other (the "fixed temporal" or FT method) spans the average cardiac cycle with images of fixed temporal duration. Left ventricular time-activity curves were extracted from each sequence using identical regions-of-interest and analyzed with identical methods to obtain estimates of ejection fraction, peak ejection rate, peak filling rate, and the times of occurrence of these peak rates. Differences among certain of these parameters in kind and amount support the hypothesis that estimates of resting cardiac function are more accurately portrayed by the FT method. The magnitudes of these differences are small for systolic parameters but large for early diastolic parameters. Thus, although both methods might be used for measuring systolic function, the FT method will yield a more accurate estimate of peak filling rate in resting subjects.

Diagnosis of coronary artery disease by radionuclide angiography: effect of combining indices of left ventricular function.

The aim of this study was to determine whether the diagnostic capability of radionuclide angiography (RNA) in detecting coronary artery disease (CAD) might be improved by using several indices of left ventricular (LV) function in concert. Three different models (rest data, exercise data, and rest plus exercise data) were derived by stepwise multivariate discriminant analysis of RNA data in 65 normal volunteers and 111 patients with CAD and normal ejection fraction (EF) at rest. The model with only resting indices yielded a diagnostic capability comparable to the simple measure of EF response to exercise (area under receiver operating characteristic curve = 89% and 91%, respectively). Both the exercise and rest plus exercise models gave better results (area = 94% and 97%, respectively), but only the rest plus the exercise model was better than the EF response alone (p less than 0.001). Thus (a) if resting studies alone are performed, the diagnostic potential of RNA may be improved by combining several indices of resting function; and (b) combined rest and exercise data may improve the sensitivity of RNA in detecting CAD over what could be obtained with the EF response to exercise alone.

Influence of temporal smoothing on quantitation of left ventricular function by gated blood pool scintigraphy.

Frequently, indexes of systolic and diastolic left ventricular (LV) function obtained from radionuclide angiography are computed after the LV time-activity curve has been temporally smoothed. This smoothing process may introduce important systematic errors into the analysis. To assess this potential effect, high temporal resolution time-activity curves (20 ms/point) were obtained in 113 normal subjects, 175 patients with coronary artery disease and 171 patients with hypertrophic cardiomyopathy. The curves were then subjected to 0-, 3-, 5-, 7- and 9-point temporal smoothing. In each group, increased smoothing progressively and consistently underestimated ejection fraction by up to 5% (p less than 0.001) and peak ejection rate by up to 14% (p less than 0.001). A greater effect on peak filling rate was observed: 5-point and 9-point smoothing reduced peak filling rate by 10% and 23% in normal subjects, 3% and 10% in patients with coronary artery disease and 7% and 15%, respectively, in patients with hypertrophic cardiomyopathy (all p less than 0.001). These errors were compounded further when the same data obtained at lower temporal resolution (40 ms/point) were analyzed: 5-point and 9-point smoothing resulted in underestimation of peak filling rate by 20% and 46% in normal subjects, 13% and 43% in coronary artery disease and 16% and 34% in hypertrophic cardiomyopathy. The underestimation was not uniform, and its magnitude varied considerably among individuals in each of the 3 groups. Thus, smoothing of LV time-activity curves may result in significant systematic errors in computation of indexes of LV systolic and diastolic function, especially in data with poor temporal resolution. These concepts apply to other imaging methods, such as magnetic resonance imaging and cine-computed tomography, that assess LV function from the LV volume curve. Although ejection fraction is affected only mildly by these errors, both peak filling rate and peak ejection rate may be severely underestimated.