J. Yoon

Bis deficiency results in early lethality with metabolic deterioration and involution of spleen and thymus

Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and pathological conditions. To better define the physiological function of Bis in vivo, we developed bis-deficient mice with a cre-loxP system. Targeted disruption of exon 4 of the bis gene was demonstrated by Southern blotting and PCR, and Western blotting showed that no intact or truncated Bis protein was synthesized in bis(-/-) mice. While heterozygotes were fertile and appeared normal, Bis-deficient mice showed growth retardation and died by 3 wk after birth. The relative weight of the thymus and spleen was reduced and the total numbers of white blood cells, splenocytes, and thymocytes were significantly reduced compared with wild-type littermates. Serum profiles indicated significant hypoglycemia as well as decrease in triglyceride and cholesterol levels. Expression profiles of metabolic genes indicated that gluconeogenesis and beta-oxidation are activated in the liver of bis(-/-) mice. This activation, as well as a decrease in peripheral fat and an induction of fatty liver, appears to be an adaptive response to hypoglycemia. Our study reveals that the absence of Bis has considerable influences on postnatal growth and survival, possibly due to a nutritional impairment.

Bis Is Involved in Glial Differentiation of P19 Cells Induced by Retinoic Acid

Previous observations suggest that Bis, a Bcl-2-binding protein, may play a role the neuronal and glial differentiation in vivo. To examine this further, we investigated Bis expression during the in vitro differentiation of P19 embryonic carcinoma cells induced by retinoic acid (RA). Western blotting and RT-PCR assays showed that Bis expression was temporarily decreased during the free floating stage and then began to increase on day 6 after the induction of differentiation. Double immunostaining indicated that Bis-expressing cells do not express several markers of differentiation, including NeuN, MAP-2 and Tuj-1. However, some of the Bis-expressing cells also were stained with GFAP-antibodies, indicating that Bis is involved glial differentiation. Using an shRNA strategy, we developed bis-knock down P19 cells and compared them with control P19 cells for the expression of NeuroD, Mash-1 and GFAP during RA-induced differentiation. Among these, only GFAP induction was significantly attenuated in P19-dnbis cells and the population showing GFAP immunoreactivity was also decreased. It is noteworthy that distribution of mature neurons and migrating neurons was disorganized, and the close association of migrating neuroblasts with astrocytes was not observed in P19-dnbis cells. These results suggest that Bis is involved in the migration-inducing activity of glial cells.

Deletion of the bis gene results in a marked increase in the production of corticosterone that is associated with thymic atrophy in mice.

Bis (Bag3) is known to be involved in cell survival, migration, the regulating of chaperones, and protein quality control. We reported recently on the production of bis gene-deleted mice, which show early lethality within 3 wk after birth with a phenotype showing severe malnutrition and shrinkage of the thymus. In this report, we provide evidence to show that an intrinsic problem of adrenal gland is the the primary cause for the severe atrophy of the thymus in bis(-/-) mice. The bis(-/-) mice show significantly higher levels of corticosterone, but CRH and ACTH levels were considerably lower than those of wild littermates. The transcription of steroidogenic enzymes was increased in the adrenal glands of bis(-/-) mice, accompanied by an increase in the thickness of the zona reticularis. An analysis of thymus tissue from bis(-/-) mice revealed that the severe atrophy of the thymus is due to the specific loss of immature double-positive (CD4(+)CD8(+)) cortical thymocytes by apoptosis, as evidenced by immunohistochemical examination and flow cytometric analysis, which were restored by injection of an inhibitor of glucocorticoid synthesis. In vitro cultures of thymocytes with increasing doses of dexamethasone exhibited a similar degree of apoptosis between wild and bis(-/-) thymocytes. The corticosterone levels from fasted wild littermates were one-half those of bis(-/-) mice, although serum glucose levels were similar. Thus, the deletion of the bis gene resulted in the intrinsic defect in the adrenal gland, leading to a marked increase in glucocorticoid levels, probably upon starvation stress, which accounts for the massive apoptosis of the thymus.

Expression of Bis in the mouse gastrointestinal system.

The Bcl-2 interacting death suppressor (Bis) protein is known to be involved in a variety of pathophysiological conditions. We recently generated bis-deficient mice, which exhibited early lethality with typical nutritional deprivation status. To further investigate the molecular basis for the malnutrition phenotype of bis deficient mice, we explored Bis expression in the digestive system of normal mice. Western blot analysis and quantitative real time reverse transcription polymerase chain reaction analysis indicated that Bis expression is highest in the esophagus, followed by the stomach, colon, jejunum and ileum. Immunohistochemical data indicated that Bis expression is restricted to the stratified squamous epitheliums in the esophagus and forestomach, and was not notable in the columnar epitheliums in the stomach, small intestine and colon. In addition, strong Bis immunoreactivity was detected in the striated muscles surrounding the esophagus and smooth muscles at a lesser intensity throughout the gastrointestinal (GI) tract. Ganglionated plexuses, located in submucous layers, as well as intermuscular layers, were specifically immunoreactive for Bis. Immunofluorescence studies revealed that Bis is co-localized in glial fibrillary acidic protein-expressing enteric glial cells. Immunostaining with neuron specific esterase antibodies indicate that Bis is also present in the cell bodies of ganglions in the enteric nervous system (ENS). Our findings indicate that Bis plays a role in regulating GI functions, such as motility and absorption, through modulating signal transmission between the ENS and smooth muscles or the intestinal epitheliums.

SU‐FF‐J‐30: Experimental Investigation of Moving Average Algorithm for Tracking Organ Motion

Purpose: To investigate the geometric and dosimetric properties of the moving average tracking algorithm compared with real time tracking and static beam. Method and Materials: Patient motion traces from the Cyberknife Synchrony™ were categorized into 3 types of motion: a regular motion with/without baseline drift and irregular motion. Each motion trace was reproduced with the help of a 4D motion platform. Time averaged mean position of the phantom was used to control dMLC position in the perpendicular direction to the leaf travel. Geometric accuracy, delivery efficiency and dose distribution were evaluated for static, IMRT step‐and‐shoot and sliding window plan using simulated and patient motion traces. The geometric accuracy was calculated from the displacement between the target center and the beam center using a real‐time beams‐eye‐view videos with a circular aperture. For dosimetric measurements, PTW ion chamber array (model 729) was sandwiched between 2 cm thickness of solid water phantoms and irradiated for one field. Dosimetric impact was quantified via gamma index with a 3% dose‐3mm criterion. Results: The difference of RMS geometric error (mm) between the real‐time tracking and moving average tracking was about 3 mm which was similar to errors caused by random motion. The percentages of failing points in the gamma analysis for moving average tracking were reduced by above half of the percent difference between no compensation and real‐time tracking. Delivery efficiencies for moving average tracking were above 97% comparable to static delivery case. Conclusions: Mean position tracking was useful for compensating a baseline drift to improve a geometric and delivery accuracy with high delivery efficiency.

SU‐FF‐J‐39: The Impact of Improving Techniques for Phase Reassignment in 4D CT Reconstruction

Purpose: To develop the respiratory signal analysis program for a phase‐based retrospective 4D CT using RPM, spirometer, stereo‐camera and removed the irregular peaks of respiratory signals for accurate phase assignment in 4D CT.Methods: In this study, an in‐house respiratory signal analysis program was developed for the phase reassignment and the analysis of the irregular respiratory signals. Various irregular respiratory patterns were obtained from clinical experimental volunteers. After then, the in‐house program analyzed the factors affecting to phase assignment which is directly related to irradiate sector. The algorithms for separating individual respiratory signals are presented, which made use of baseline signals of input signal to identify the nodes between baseline signals and input signal. Therefore, we could achieve irregular peak reduction and noise elimination in phase assignment. Subsequently, accuracy of phase assignment was improved with removal of irregular signals by self‐developed algorithm. Furthermore, this program also has a function to detect period and amplitude automatically. Result: The standard deviation (SD) of breathing period representing the baseline method was shown stable compare with gradient method, but the difference was not statistically significant. Gradient method shows inaccurate phase assignment when all irregular peaks were detected and assigned. On the other hand, baseline method which was self‐developed algorithm confirmed accurate phase assignment with removal of error peak detection due to tussis. It was also verified superior peak detection with the suggested baseline method at respiratory signals which contained irregular period changes and drifts of amplitude. Conclusion: This study is considered to be useful for not only image reconstruction and elevation of irradiating accuracy through phase assignment of RPM system but also analysis of respiratory signals. Moreover, this respiratory analysis program can be applied to the 4D CT reconstruction using various respiratory sensors for the reduction of motion artifact.

SU‐GG‐J‐69: Development of a Programmable Moving Phantom for the Verification of 4D Dose Delivery

Purpose: To develop a programmable moving phantom to simulate internal organ motion and investigate the impact of the respiratory motion on dose delivery by comparison with phantom measurements. Method and Materials: A moving phantom system was constructed to simulate internal organ motion. The system consists of a motion system and an acrylic phantom. A mechanical cam is responsible for the motion in the anterior‐posterior direction. A worm gear allows 1‐D linear motion in the superior‐inferior direction. The acrylic phantom has lung and prostate inserts including a film cassette and an ion chamber. The motion control system was composed of two AC servo motors, a controller and a PC‐based motion program. The motion control program was implemented to move the phantom insert with a non‐linear (sinusoidal or irregular) motion according to a user‐defined motion profile. The user‐defined motion profile can be generated in a spreadsheet either by calculation using an analytic model or by acquisition of real patient data. An external stereo camera was used to acquire the motion data for verification. Results: User‐defined motion trajectory can be produced using the programmable moving phantom with an accuracy of 0.5 mm. Although a period for motion update is 10ms, camera measurement result indicates that the resulting motion profile was smooth enough to simulate internal organ motion with the help of an interpolator in the motion controller. Conclusion: This work indicates the prospect for 4D patient specific QA using the programmable phantom with a patient internal organ motion data. Real 4D dose verification could be achieved with independent 3‐axis moving mechanics. Conflict of Interest: This work was supported by a grant of Seoul R&BD Program (10550).

SU-FF-J-77: An Analysis of the Time Delay and Relationship Between External Sensor Signals and Internal Organ Motion for Respiratory Gated Radiotherapy

Purpose: To estimate the delay time and relationship between external signals and internal organ motion for respiratory gated radiotherapyMethods and Materials: In 5 patients, we measured the external respiratory sensor signals, which included respiratory volume, respiratory temperature, and abdominal displacement with three sensors (spirometry, belt‐transducer, and thermistor), and internal organ motion with the fluoroscopy. To evaluate the relationship of the internal organ and external sensor signals, a linear least‐square fit was performed with two signals, and the correlation coefficient (R values) was determined. In order to test the presence of a time‐varying phase relationship, a unique cross‐correlation of the respiratory motion signals and internal organ motion data were performed. Cross‐correlation function (CCF) analysis allows for the identification and estimation of a phase or time delay in two related signals. Results: The correlation coefficient of respiratory signal showed that the internal organ motion to abdominal displacement by piezo respiratory belt‐transducer exhibited high correlation of 0.94 (range 0.98–0.85) with a standard deviation of about 0.06, whereas the respiratory volume and temperature to organ motion was a poor correlation (average 0.70, 0.71). The result of respiratory volume and temperature shows the influence of the phase shift, regarding time delay of 0.2 – 0.4 s. Two sensor signals considered the time delay correction generally correlated well with internal organ motion. Conclusion: This correlation in this study can be used to predict internal organ motion, based on the external sensor signals. If the time delay of external sensor signals was corrected carefully, the use of the respiratory sensor would improve the accuracy for respiratory gated radiotherapy. Thus, it is expected that the respiratory sensors will come into wider use.

SU-FF-J-100: A PC-Based Multiple Respiratory Sensor System for the Correlation Study with An Organ Motion

Purpose: To acquire various respiratory signals simultaneously from a spirometer, a belt transducer and a CCDcamera for the correlation study with organ motion Method and Materials: A PC‐based sensor monitoring system was developed to acquire signals from the multiple respiratory sensors. The system incorporated a data acquisition board featuring a 16‐bit A/D converter. Respiratory signals generated by a spirometer and a belt transducer were fed into the board. Patient surface markers were imaged by two IEEE‐1394 cameras for obtaining 3‐D positions of surface markers. The respiratory signals were displayed in real time after correction for zero‐drift and time integration for tidal volume. Fluoroscopic imaging was performed to assess the correlation of each respiratory signal with a variety of organ motion. Results: A multiple respiratory sensor system was implemented with the DAQ board and PC‐based program. A phase delay of around 0.2 second was observed between the spirometer and the belt transducer signal. Software triggered digital output was provided for gating a LINAC. Conclusion: A multiple respiratory sensor system is feasible using a PC‐based data acquisition technology. The system is flexible in implementing a post‐processing algorithm for a stabilized respiratory signal and can evaluate the correlation of a different kind of respiratory signal with a specific organ motion.

SU‐FF‐I‐50: A Mechanical Alignment Method for Removing Moiré Pattern in Digital Radiography with a Carbon‐Interspaced Grid

Purpose: To remove moire patterns of a carbon‐interspaced X‐ray anti‐scatter grid in use with a digital radiographicdetector by matching a grid line frequency with a DR sampling frequency. Method and Materials: A carbon interspaced grid with a line frequency a little higher than the sampling frequency of DR was processed by a sawing machine which was controlled as micro unit. An amorphous selenium DR panel was used in imaging. A motion‐control jig was developed to change the disposition of the grid with respect to the detector pixels. An alignment process was divided into horizontal and vertical directions. The detector underneath of the X‐ray grid was translated and rotated with the resolution of 2 microns and 0.01 degrees respectively so that the pattern lines were oriented perpendicular to a horizontal axis in an image plane. A height of the grid from the detector was varied by 4 micrometers to magnify the shades of the grid lines at the detector and, hence, to exactly match with the sampling frequency of the detector.Results: A moire frequency was proportional to a difference between the grid shade frequency and the DR sampling frequency. An angular displacement of the detector caused a frequency difference to indicate a higher frequency of moire. The horizontal translation did not change the moire frequency but only phases. An impact of the phase shift on image became larger at lower frequency of moire patterns. Conclusion: A Moire pattern in the use of a fixed type of grid can be removed by matching a grid line frequency with a DR sampling frequency. High straightness and uniformity of grid lines of carbon‐interspaced grids and micro‐controlled alignment methods enable frequency matching to remove moire patterns without a software filtering and a moving grid.