SU-E-J-102: The Impact of the Number of Subjects for Atlas-Based Automatic Segmentation.

PURPOSE: To determine the impact of atlas size on the performance of atlas-based automatic segmentation (ABAS) in delineation of organs at risk for adaptive radiation therapy. METHODS: A total of 25 patients who had undergone intensity modulated radiation therapy for various head and neck cancers were retrospectively selected for inclusion in a library to be used for ABAS with the MIM VISTA software package (MIM Software, Cleveland OH). Treatment planning computed tomography (CT) scans and subsequent organ at risk (OAR) contours generated as part of the treatment planning process for these patients were added to the library. This library of 25 patients was then successively pruned to generate 5 atlases with 25, 20, 15, 10, and 5 patient subjects respectively. Atlas based segmentation was performed on 10 retrospectively selected treatment planning CT scans to automatically generate right and left parotid glands and brainstem contours. These planning CT scans belonged to a unique set of 10 patient subjects different from the ones used for generating the atlases. One physician (JW), who was blinded to the ABAS results, manually delineated gold-standard contours for the right and left parotid glands and brainstem. Dice similarity coefficients were calculated and analyzed as a function of atlas subject size. RESULTS: For the sites selected in this study, the performance of ABAS was relatively insensitive to atlas size. Furthermore, some patient subjects were repeatedly selected implying that the adoption of a single standard patient for ABAS may be of benefit. CONCLUSIONS: Our preliminary results indicate that the performance of the atlas based segmentation module in MIM VISTA Version 5.2 for the organs studied here may be relatively insensitive to the atlas size.

Imaging of nanoparticles with dual-energy computed tomography.

A simulation study was performed to determine the feasibility and performance of imaging nanoparticles as contrast agents in dual-energy computed tomography. An analytical simulation model was used to model the relevant signal-to-noise ratio (SNR) in dual-energy imaging for the specific case of a three-material patient phantom consisting of water, calcium hydroxyapatite and contrast agent. Elemental gold and iodine were both considered as contrast agents. Simulations were performed for a range of monoenergetic (20-150 keV) and polyenergetic (20-150 kVp) beam spectra. A reference configuration was defined with beam energies of 80 and 140 kVp to match current clinical practice. The effect of adding a silver filter to the high-energy beam was also studied. A figure of merit (FOM), which normalized the dual-energy SNR to the square root of the patient integral dose, was calculated for all cases. The units of the FOM were keV(-1/2). A simple Rose model of detectability was used to estimate the minimum concentration of either elements needed to be detected (SNR > 5). For monoenergetic beams, the peak FOM of gold was 6.4 × 10(-6) keV(-1/2), while the peak FOM of iodine was 3.1 × 10(-6) keV(-1/2), a factor of approximately 2 greater for gold. For polyenergetic spectra, at the reference energies of 80 and 140 kVp, the FOM for gold and iodine was 1.65 × 10(-6) and 5.0 × 10(-7) keV(-1/2), respectively, a factor of approximately 3.3 greater. Also at these energies, the minimum detectable concentration of gold was estimated to be 58.5 mg mL(-1), while iodine was estimated to be 117.5 mg mL(-1). The results suggest that the imaging of a gold nanoparticle contrast agent is well suited to current conditions used in clinical imaging. The addition of a silver filter of 800 µm further increased the image quality of the gold signal by approximately 50% for the same absorbed dose to the patient.

Scatter correction in digital mammography based on image deconvolution.

X-ray scatter is a major cause of nonlinearity in densitometry measurements using digital mammography. Previous scatter correction techniques have primarily used a single scatter point spread function to estimate x-ray scatter. In this study, a new algorithm to correct x-ray scatter based on image convolution was implemented using a spatially variant scatter point spread function which is energy and thickness dependent. The scatter kernel was characterized in terms of its scattering fraction (SF) and scatter radial extent (k) on uniform Lucite phantoms with thickness of 0.8-8.0 cm. The algorithm operates on a pixel-by-pixel basis by grouping pixels of similar thicknesses into a series of mask images that are individually deconvolved using Fourier image analysis with a distinct kernel for each image. The algorithm was evaluated with three Lucite step phantoms and one anthropomorphic breast phantom using a full-field digital mammography system at energies of 24, 28, 31 and 49 kVp. The true primary signal was measured with a multi-hole collimator. The effect on image quality was also evaluated. For all 16 studies, the average mean percentage error in estimating the true primary signal was found to be -2.13% and the average rms percentage error was 2.60%. The image quality was seen to improve at every energy up to 25% at 49 kVp. The results indicate that a technique based on a spatially variant scatter point spread function can accurately estimate x-ray scatter.

Computed tomography of necrotizing meningoencephalitis in 3 Yorkshire Terriers.

A necrotizing meningoencephalitis of Yorkshire terriers has recently been reported in 6 dogs in Switzerland, 1 dog in Japan and 1 dog in the United States. The purpose of this report is to describe the computed tomographic (CT) findings in 3 dogs with this disease, and to correlate the CT abnormalities with the clinical and pathologic findings in each case. Three Yorkshire Terriers between 2 and 10 years old were evaluated. Physical and neurologic examinations, complete blood count (CBC), serum biochemistry profile, cerebrospinal fluid analysis, and CT scan were performed on all 3 dogs. Brainstem auditory evoked responses (BAER) were evaluated for 2 dogs. Two dogs were euthanized at the owners' request and necropsies were performed. Neurologic examination findings were consistent with a multifocal/diffuse encephalitis involving the cerebrum and brainstem in all 3 dogs. Complete blood count and biochemistry profiles were normal. Elevated protein concentration and a mononuclear pleocytosis were demonstrated in 2 of 3 dogs on cerebrospinal fluid evaluation. Multifocal, extensive areas of decreased opacity throughout the cerebral hemispheres, asymmetric ventriculomegaly, and lack of contrast enhancement were appreciated on CT images of all three dogs. No mass effect was seen. These findings correlated well with pathologic findings at necropsy, which included multiple malacic cavitations within the brain, representing areas of locally extensive necrosis. CT abnormalities in combination with signalment, clinical findings and cerebrospinal fluid analysis should facilitate a presumptive diagnosis of Yorkshire Terrier necrotizing meningoencephalitis.

Azathioprine therapy for acquired myasthenia gravis in five dogs.

Five dogs with acquired myasthenia gravis (MG), verified via positive serum acetylcholine (ACh) receptor antibody concentrations, were treated with a drug protocol including azathioprine (AZA). Four of the five dogs were concurrently treated with pyridostigmine. Azathioprine was used as the sole immunosuppressive agent in four dogs. One dog was temporarily treated with a combination of an immunosuppressive dose of prednisone and AZA, then maintained on AZA as the sole immunosuppressive drug. Three patients experienced complete remission of clinical signs within three months of therapy. In the four dogs for which follow-up serum ACh receptor antibody concentrations were available, initial versus final concentrations decreased substantially (81%), coincident with clinical improvement. One dog died suddenly due to a suspected myasthenic crisis before attaining the target dose of AZA. Two of the four surviving dogs were euthanized approximately one and seven years after diagnosis. One of these two dogs was euthanized because of a rib osteosarcoma, and the other dog was euthanized because of paraparesis of undetermined cause. The remaining two dogs were alive and doing well at the time of final follow-up evaluation, approximately six months and one year after diagnosis. The use of AZA as a therapeutic agent for acquired canine MG has not been investigated. The cases presented in this report suggest a potentially important role for AZA in the treatment of acquired MG in dogs.

Bisindolylmaleimide protein-kinase-C inhibitors delay the decline in DNA synthesis in mouse hair follicle organ cultures.

We have used a series of bisindolylmaleimide selective protein-kinase C (PKC) inhibitors to investigate the role of this enzyme in the regulation of cell proliferation in mouse hair follicle organ cultures. Mouse whisker follicles were isolated by microdissection, and rates of DNA synthesis during culture were determined from 3H-thymidine incorporation. The bisindolylmaleimides Ro 31-7549, Ro 31-8161, Ro 31-8425 and Ro 31-8830 inhibit isolated brain PKC with IC50 values of 8-80 nM, are > 60-fold less potent against protein kinase A, and inhibit PKC-mediated protein phosphorylation in platelets with IC50 values in the range 0.25-4.4 microM. These PKC inhibitors were found to increase levels of mouse hair follicle DNA synthesis, with EC50 values in the range 1-4 microM and maximal levels in the range 151-197% of control. Ro 31-7549 had an IC50 value 50-fold lower than that of minoxidil, while the maximal level of DNA synthesis for the PKC inhibitor was 86% higher. Incubation of mouse hair follicles with Ro 31-7549 resulted in a delay of approximately 24 h in the onset of decline in follicular DNA synthesis rates. Ro 31-6045 and Ro 31-7208, bisindolylmaleimides without activity in the platelet PKC assay, did not affect mouse hair follicle DNA synthesis rates. Taken together, these findings show that PKC mediates, at least in part, the rapid loss of proliferative activity that occurs in mouse whisker follicles in culture, and provide further evidence that PKC plays a role as a negative proliferative signal in hair follicles.

Thapsigargin induces rapid, transient growth inhibition and c-fos expression followed by sustained growth stimulation in mouse keratinocyte cultures.

Although the sesquiterpene lactone thapsigargin has been shown to possess hyperplastic and tumor-promoting activities when applied topically to mouse skin in vivo, the cellular mechanism(s) which underlie these effects are unclear. We show here that thapsigargin treatment of Primary mouse epidermal keratinocytes increased intracellular free Ca2+ concentration (Cai) in a concentration-dependent manner. Thapsigargin induced a rapid, transient elevation in keratinocyte Cai, in part due to the release of Ca2+ from intracellular stores. This response was followed by a sustained elevation in Ca2+, resulting entirely from calcium influx. Thapsigargin elicited a biphasic effect on keratinocyte DNA synthesis: a rapid inhibitory effect (50-60% inhibition at 4-8 h), followed by a very marked and sustained elevation. Prolonged treatment of keratinocytes with thapsigargin at relatively high concentrations resulted in cytotoxicity (inhibition of neutral red uptake). The rapid antiproliferative effect of thapsigargin was not associated with cytotoxicity, as determined by either neutral red uptake or by trypan blue exclusion, and was not blocked by pretreatment with Ro 31-7349, a selective inhibitor of protein kinase C. The rapid antiproliferative effect of thapsigargin was associated with rapid, transient activation of keratinocyte c-fos expression and rapid inhibition of total protein synthesis. Taken together, these findings raise the possibility that the hyperplastic and tumor-promoting activities of thapsigargin on epidermis in vivo result from direct keratinocyte growth stimulation as a consequence of a prolonged elevation in levels of Cai.

Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has been proposed as a physiologic regulator of keratinocyte growth and differentiation. Utilizing a proliferative serum-free culture system, we have found that a physiologic (picomolar) concentrations this hormone stimulated proliferation of primary mouse epidermal keratinocytes; at higher (nanomolar to micromolar) doses, growth was inhibited by 1,25(OH)2D3. We investigated the nature of the signal transduction mechanism underlying the response to 1,25(OH)2D3 and observed little or no effect of either low or high concentrations of the hormone on cytosolic calcium levels or Fos expression. Furthermore, the protein kinase C inhibitor, Ro 31-7549, had very little effect on the growth inhibition induced by a high dose (1 microM) of 1,25(OH)2D3. This lack of rapid signal transduction events was consistent with the inability of a short (4-hour) exposure to 1,25(OH)2D3 to initiate a complete growth-inhibitory response as measured using [3H]thymidine incorporation. Our results indicate that physiologic concentrations of 1,25(OH)2D3 are required for optimal keratinocyte growth. Furthermore, we found no evidence of rapid effects of 1,25(OH)2D3 and suggest that in mouse epidermal keratinocytes, the response to this hormone is mediated by a slow transduction pathway, such as that activated by the intracellular 1,25(OH)2D3 receptor (VDR).

Regulation of fos-lacZ fusion gene expression in primary mouse epidermal keratinocytes isolated from transgenic mice.

The expression of a fos-lacZ fusion gene was studied in primary mouse epidermal keratinocytes obtained from transgenic mice. This gene construct contains the entire upstream regulatory sequence of c-fos, and expression of the endogenous and fusion gene was shown by Northern analysis to correlate upon induction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Using a chromogenic substrate of beta-galactosidase, we also demonstrated that expression of the fusion gene product, like that of Fos, was localized to the cell nucleus. In addition, we showed that epidermal keratinocytes responded to dialysed fetal bovine serum (FBS), TPA and high-calcium medium with enhanced Fos-lacZ expression and an inhibition of proliferation. The time course of induction of Fos-lacZ expression was similar for dialysed FBS and TPA, with a peak approximately 2 h after exposure. Exposure for approximately 24 h to an elevated extracellular calcium concentration was required to elicit an increase in Fos-lacZ expression. The lack of an immediate effect of raising medium calcium levels on Fos-lacZ expression contrasted with the rapidity of its effect on DNA synthesis, which was significantly inhibited within 6-8 h. In addition, we found that the protein kinase C inhibitor Ro 31-7549 blocked Fos-lacZ expression induced by TPA but had little or no effect on that elicited by high calcium levels. Thus, although our results indicate that the fos gene product may be involved in mediating epidermal keratinocyte growth arrest in response to differentiative agents such as FBS, TPA and high medium calcium levels, the exact role of this gene product remains unclear.

Effects of the selective protein kinase C inhibitor, Ro 31-7549, on the proliferation of cultured mouse epidermal keratinocytes.

We have investigated the effects of Ro 31-7549, a selective protein kinase C (PKC) inhibitor, on DNA synthesis and proliferation in two primary mouse epidermal keratinocyte culture systems. In differentiating keratinocytes incubated in medium containing 10% serum and high calcium (approximately 0.5 mM), Ro 31-7549 blocked the inhibitory effect of the phorbol ester 12-0-tetradecanoyl-13-acetate (TPA) (a PKC activator) on keratinocyte DNA synthesis at 24 h [50% maximal response concentration (EC50) = 1 microM], consistent with inhibition of PKC-mediated differentiation. Continuous treatment of the differentiative culture system with the PKC inhibitor resulted in a marked (fourfold) stimulation of [3H]thymidine incorporation at day 7 of exposure, with an EC50 of 0.25 microM. The potencies of these effects of Ro 31-7549 are comparable to that reported for inhibition of TPA-induced platelet 47-kD protein phosphorylation [50% inhibitory concentration (IC50) = 4.4 microM]. The time course of [3H]thymidine incorporation indicated that Ro 31-7549 did not directly stimulate DNA synthesis but instead prevented the loss of proliferative capacity associated with continued culture in this medium. Maximal stimulation (2.6 times) of DNA synthesis was observed on day 4, whereas DNA synthesis at day 1 was unaffected. In a highly proliferative culture system using serum-free medium containing 25 microM calcium, TPA dose-dependently inhibited proliferation with an IC50 of approximately 0.3 mM. This antiproliferative effect of TPA was largely reversed by 0.1 microM Ro 31-7549. In the proliferative culture system, 0.75 microM Ro 31-7549 also essentially reversed the inhibition of proliferation caused by switching to high (1.0 mM) calcium. These results suggest that the loss of proliferative capacity in differentiating epidermal keratinocyte cultures may be mediated, at least in part, by PKC.

Potassium channel openers stimulate DNA synthesis in mouse epidermal keratinocyte and whole hair follicle cultures.

We have conducted studies using primary mouse epidermal keratinocyte and whole hair follicle cultures to investigate the mechanism of the hypertrichotic activity of potassium channel openers. In a time course study, the extent of stimulation of epidermal keratinocyte DNA synthesis by minoxidil increased as the rate of DNA synthesis in control cultures declined. Minoxidil stimulation of DNA synthesis in 7-day cultures required prolonged (> 1 day) exposure to the agent. Pinacidil and diazoxide also stimulated DNA synthesis in mouse epidermal keratinocyte cultures. In addition, minoxidil, pinacidil, diazoxide, and cromakalim stimulated DNA synthesis in whole-organ cultures of mouse hair follicles. These results suggest that potassium channel openers retard the loss of proliferative activity of differentiating keratinocytes and support the hypothesis that these agents stimulate hair growth through a direct effect on hair follicles.

Methyl mercury stimulates chain elongation by purified HeLa RNA polymerase II.

Methyl mercury (MeHg) inhibited the overall RNA synthetic reaction of HeLa RNA polymerase II. However, when RNA chain initiation was allowed to occur in its absence, MeHg stimulated the rate of the subsequent elongation stage of the reaction. Chain elongation with both double-stranded and single-stranded DNA templates was stimulated. This stimulatory effect was specific for MeHg; both p-hydroxymercuribenzoate and HgCl2 inhibited chain elongation (to about the same degree as they inhibited the overall reaction). The stimulatory effect was also specific for the HeLa polymerase; with Escherichia coli RNA polymerase, MeHg inhibited elongation (to the same degree as it inhibited the overall reaction).

A cell line with decreased sensitivity to the methyl mercury-induced stimulation of alpha-amanitin sensitive RNA synthesis in isolated nuclei.

1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on alpha-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275-6279). 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.

The enhanced rate of transcription of methyl mercury-exposed DNA by RNA polymerase is not sufficient to explain the stimulatory effect of methyl mercury on RNA synthesis in isolated nuclei.

Previous work demonstrated two stimulatory effects of methyl mercury on nucleic acid synthesis: (1) in isolated nuclei, methyl mercury stimulates RNA synthesis which is catalyzed by RNA polymerase II [Frenkel and Randles, J. Biol. Chem. 257, 6275-6279 (1982)]. (2) Brief exposure of purified DNA to methyl mercury increases the rate of its transcription by purified RNA polymerase II [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)]. The latter effect was considered as a possible mechanism of the former. Two lines of evidence are presented here which demonstrate that the latter effect is not a sufficient explanation for the former. (1) Mercuric perchlorate has been found to increase the rate of DNA transcription by purified polymerase and the template properties of the mercuric perchlorate-exposed DNA have been found to resemble those of methyl mercury-exposed DNA. Nevertheless, mercuric perchlorate has been shown not to stimulate RNA synthesis in isolated HeLa nuclei. (2) In isolated nuclei of the B50 rat neuroblastoma cell line, RNA synthesis has been found to be stimulated only minimally by methyl mercury. Nevertheless, RNA polymerase II purified from the B50 cells has been found to transcribe methyl mercury-exposed DNA at a higher rate than unexposed control DNA.

Effects of exposure of DNA to methyl mercury on its activity as a template-primer for DNA polymerases.

A previous publication [Frenkel, Cain, and Chao, Biochem. Biophys. Res. Commun. 127, 849-856 (1985)] described the observation that double-stranded DNA which was briefly exposed to methyl mercury (MeHg) and purified to remove free methyl mercury was transcribed at a higher rate by RNA polymerase II from wheat germ. The specificity of this phenomenon has now been investigated by examining the activity of this MeHg-exposed DNA as a template-primer for DNA polymerases. DNA synthesis by the bacteriophage T4-induced DNA polymerase was higher with the MeHg-exposed DNA as a template-primer than with control DNA. In contrast, the rate of DNA synthesis by E. coli DNA polymerase I was lower with the MeHg-exposed DNA as template-primer. With both enzymes (as well as with RNA polymerase II), after denaturation of the MeHg-exposed and control DNAs the differences in template activity were either eliminated or markedly reduced. The enzymes are thus able to detect a MeHg-induced alteration in DNA. In contrast, circular dichroism, a physical method that is sensitive to conformational changes in DNA, did not detect any difference between the MeHg-exposed and control DNAs.