Identification of the sensory nerve fiber responsible for lysophosphatidic acid-induced allodynia in mice.

Lysophosphatidic acid (LPA) has been considered one of the molecular culprits for neuropathic pain. Understanding how LPA changes the function of primary afferent fibers might be an essential step for clarifying the pathogenesis of neuropathic pain. The present study was designed to identify the primary afferent fibers (Aß, Aδ, or C) participating in LPA-induced allodynia in ddY mice. Mechanical allodynia and thermal hyperalgesia were evaluated by the von Frey filament test and thermal paw withdrawal test, respectively. Sensory nerve fiber responsiveness was measured using a Neurometer. Daily repeated intrathecal treatment with LPA led to a decrease in the mechanical, but not thermal nociceptive threshold, and a reduction in the threshold for paw withdrawal induced by 2000-Hz (Aß fiber) and 250-Hz (Aδ fiber), but not 5-Hz (C fiber) sine-wave electrical stimulation. When the transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor agonist resiniferatoxin (RTX) was administered subcutaneously before the start of LPA treatment, LPA-induced mechanical allodynia and Aß and Aδ fiber hypersensitivity demonstrated by neurometry were not affected, indicating that TRPV1-expressing nerve fibers (possibly C fibers) might not be essential for LPA-induced allodynia. LPA-induced allodynia was reversed by treatment with RTX at 7 days after the start of LPA treatment. Expression of TRPV1 on myelinated nerve fibers after repeated intrathecal LPA treatment was observed in the dorsal root ganglion. These results suggest that sensitization of Aß and Aδ fibers, but not C fibers, contributes to the development of intrathecally administered LPA-induced mechanical allodynia. Moreover, increased or newly expressed TRPV1 receptors in Aß and Aδ fibers are considered to be involved in the maintenance of LPA-induced allodynia.

SU-E-T-106: Evaluation of Sensitivity and Uniformity of New Radiochromic Film with Two Commercial Scanners.

PURPOSE: A newly introduced radiochromic film, the GAFCHROMIC EBT3, has been expected as much useful device for the IMRT dosimetry. The purpose of this study was to investigate the sensitivity and the uniformity of the films between an Epson ES-10000G flatbed scanner and a Vidar DosimetryPRO Advantage (Red) scanner. METHODS: Doses ranging from 1 cGy to 1600 cGy with 15-MV photon beam was irradiated to the film in a solid water phantom, respectively. All of the films were then digitized after irradiation using both two scanners. Sensitivities, local fluctuations of the film with two scanners were evaluated. Local fluctuations were defined as the relative (percent) standard deviation of the film response in ROIs (3 cmx3 cm). RESULTS: As to the Vidar scanner, the sensitivity of the film was higher for low dose range (below <400 cGy). While, as to the Epson scanner, the sensitivity using the red color channel was higher than others for low dose range. At high dose range (above >400 cGy), the green color channel had higher sensitivity than others. The Vidar scanner exhibited the lower local fluctuations than the Epson scanner for all dose ranges. For the Epson scanner, the red color channel had the lower local fluctuations than the green and blue color channel for all dose ranges. CONCLUSIONS: This study shows the characteristics of the new EBT3 films, in conjunction with the Epson ES-10000G flatbed scanner and the Vidar DosimetryPRO Advantage (Red) scanner.

SU-E-T-525: Dosimetric Validation of the Algorithm Based on Linear Boltzmann Transport Equations for Photon 4MV Dose Calculation.

PURPOSE: To evaluate a dosimetric accuracy of AcurosXB dose calculation algorithm for 4 MV photon beam. METHODS: Four MV beam (Clinac-6EX) and AAA and AcurosXB algorithms (pre-release version 11.0.03.) were used in this study. The differences of the calculation with AAA (EAAA) and AcurosXB (EAXB) to the measurement were evaluated in the depth doses to 25 cm depth and dose profiles within the water and slab phantoms (water, lung and bone equivalent). In addition, the clinical cases, including three whole breast plans and three head and neck IMRT plans, were evaluated. First the AAA plans were calculated, then AcurosXB plans were recalculated with dose-to-medium with identical beam setup and monitor units as in the AAA plan. RESULTS: In the water phantom study, the EAAA and EAXB were up to 2.2% and 1.5% in the depth doses for the open field (field size = 4 - 40cm square), respectively. Under the heterogeneity conditions, the EAAA and EAXB were less than 4.4% and 2.2% in lung region, and less than 12.5% and 6.3% in bone region, respectively. In the re-buildup region after passing through the lung phantom, the AAA overestimated the doses about 10%; however AcurosXB had good agreement with measurement within 3%. Dose profiles with AcurosXB were better agreement with measurement than AAA. In the clinical cases, the dose of the skin surface region with AcurosXB were higher than AAA by at least 10%, and the dose differences over 5% appeared in heterogeneous region. However, DVH shapes of each organ were similar between AAA and AcurosXB within 2%. CONCLUSIONS: In phantom study, AcurosXB had better agreement to measurement than AAA, especially in heterogeneous region and re-buildup region. In the clinical cases, there were large differences between AcurosXB and AAA in the surface region. Evaluation Agreement of non-clinical versions of Acuros XB with　Varian Medical Systems.

SU-E-J-142: Gafchromic Film Dosimetry in Fluoroscopy for Dynamic Tumor Tracking Irradiation of the Lung Using XR-SP2 Model - A Phantom Study.

PURPOSE: We have recently developed a dynamic tumor tracking irradiation system using Vero4DRT (MHI-Tm2 000). It is needed to create a 4D correlation model between a fiducial marker implanted near a tumor and an external surrogate as a function of time by continuously acquiring both fluoroscopy images and external surrogate signals. The purpose of this study was to propose a new dosimetry method using Gafchromic XR-SP2 films to measure surface dose by fluoroscopy imaging. METHODS: First, half-value layers (HVLs) were measured using aluminum (Al) thicknesses (15 mm) at 40125 kVp. Subsequently, several films were irradiated using various milliampere second values on a solid water phantom. The surface air kerma were also measured using the chamber to calculate the surface doses under the same condition. Then, the calibration curve of dose vs. pixel values was calculated. Finally, surface dose by fluoroscopy imaging was measured using several pieces of film taped on the chest phantom. Orthogonal X-ray fluoroscopy imaging was simultaneously performed until completion of data acquisition for creating a 4D correlation model. Those films were scanned after irradiation using a flat-bed scanner and converted to dose by calibration curve. RESULTS: The HVLs for tube voltage within 40125 kVp ranged from 2.35 to 5.98 mm Al. The calibration curve between surface dose and pixel values was reasonably smooth. The differences between the measured and the calibrated doses were less than 3%. The hot spots with the maximum dose of 37.12 mGy were observed around the area overlapped by both fluoroscopic fields. CONCLUSIONS: We have proposed a new dosimetry method using Gafchromic XR-SP2 films to measure surface dose by fluoroscopy imaging. This phantom study has demonstrated that it may be feasible to assess surface dose to patients during dynamic tumor tracking irradiation in clinic with ease after further investigation. This research was supported by the Japan Society for the Promotion of Science (JSPS) through its Funding Program for World-Leading Innovation R&D on Science and Technology (FIRST Program). Research sponsored in part by Mitsubishi Heavy Industries, Ltd.

WE-G-213CD-09: Quality Assurance of the Surrogate Signal-Based Dynamic Tumor-Tracking Irradiation with Vero4DRT (MHI-TM2000).

PURPOSE: To perform the quality assurance for the dynamic tumor-tracking (DTT) irradiation with Vero4DRT (MHI-Tm2 000). METHODS: Vero4DRT swings its gimbaled 6-MV C-band x-ray head along the pan and tilt direction to track a moving tumor. Surrogate signal-based DTT system implemented in Vero4DRT was used. Before DTT irradiation, the correlation model (4D-model) between motion of the IR markers on the abdominal wall and the tumor position was created with synchronously monitoring by the IR camera and orthogonal kV x-ray imaging subsystem. During beam delivery, the 4D-model predicted the future tumor position from the displacement of the IR markers in real-time, and then contentiously transferred the corresponding tracking orientation to the gimbaled x-ray head.Water-equivalent phantoms were set on a 1D motor-driven base with IR markers. A film placed at a depth of 10 cm in the phantom was irradiated under the following conditions: stationary state, and tracking and non- tracking state for sinusoidal patterns. In addition, the geometric accuracy was evaluated using a 3D moving phantom and Polaris Spectra for the previously-acquired patient's respiratory pattern. RESULTS: Compared to the stationary conditions, reductions in lateral distance between 95% doses of the dose profile were 1.2 mm for tracking and 29.6 mm for non-tracking state for (amplitude [A], period [T]) = (20 mm, 2 s); and 0.2 mm and 29.4 mm for (A, T) = (20 mm, 4 s); and 0.0 mm and 11.2 mm for (A, T) = (10 mm, 2 s), respectively. In the geometric accuracy testing, 95th percentile of the tracking error was 0.5 mm in left-right, 1.0 mm in superior-inferior, and 0.5 mm in anterior-posterior direction. CONCLUSIONS: We demonstrated that Vero4DRT substantially reduced the blurring effects on dose distribution with high tracking accuracy, and confirmed the safety of the DTT irradiation for a clinical application. This research was supported by the Japan Society for the Promotion of Science (JSPS) through its Funding Program for World-Leading Innovation R&D on Science and Technology (FIRST Program), and sponsored in part by Mitsubishi Heavy Industries, Ltd.

The geometric accuracy of frameless stereotactic radiosurgery using a 6D robotic couch system.

The aim of this paper is to assess the overall geometric accuracy of the Novalis system using the Robotic Tilt Module in terms of the uncertainty in frameless stereotactic radiotherapy. We analyzed the following three metrics: (1) the correction accuracy of the robotic couch, (2) the uncertainty of the isocenter position with gantry and couch rotation, and (3) the shift in position between the isocenter and central point detected with the ExacTrac x-ray system. Based on the concept of uncertainty, the overall accuracy was calculated from these values. The accuracy in positional correction with the robotic couch was 0.07 +/- 0.22 mm, the positional shift of the isocenter associated with gantry rotation was 0.35 mm, the positional shift of the isocenter associated with couch rotation was 0.38 mm and the difference in position between the isocenter and the ExacTrac x-ray system was 0.30 mm. The accuracy of intracranial stereotactic radiosurgery with the Novalis system in our clinic was 0.31 +/- 0.77 mm. The overall geometric accuracy based on the concept of uncertainty was 0.31 +/- 0.77 mm, which is within the tolerance given in the American Association of Physicists in Medicine report no. 54.

An integrated Monte Carlo dosimetric verification system for radiotherapy treatment planning.

An integrated Monte Carlo (MC) dose calculation system, MCRTV (Monte Carlo for radiotherapy treatment plan verification), has been developed for clinical treatment plan verification, especially for routine quality assurance (QA) of intensity-modulated radiotherapy (IMRT) plans. The MCRTV system consists of the EGS4/PRESTA MC codes originally written for particle transport through the accelerator, the multileaf collimator (MLC), and the patient/phantom, which run on a 28-CPU Linux cluster, and the associated software developed for the clinical implementation. MCRTV has an interface with a commercial treatment planning system (TPS) (Eclipse, Varian Medical Systems, Palo Alto, CA, USA) and reads the information needed for MC computation transferred in DICOM-RT format. The key features of MCRTV have been presented in detail in this paper. The phase-space data of our 15 MV photon beam from a Varian Clinac 2300C/D have been developed and several benchmarks have been performed under homogeneous and several inhomogeneous conditions (including water, aluminium, lung and bone media). The MC results agreed with the ionization chamber measurements to within 1% and 2% for homogeneous and inhomogeneous conditions, respectively. The MC calculation for a clinical prostate IMRT treatment plan validated the implementation of the beams and the patient/phantom configuration in MCRTV.

Immunolocalization of voltage-gated potassium channel Kv3.1b subunit in the cochlea.

Immunohistochemical localization of voltage-gated potassium channel Kv3.1b subunit was studied in the cochlea. Intense Kv3.1b-like immunoreactivity was present in the type I, type III, type IV and suprastrial fibrocytes of the cochlear lateral wall. Immunostaining was also found in the interdental cells and the fibrocytes of the spiral limbus and in the supralimbal dark cells. K+ ions, which play a pivotal role in the mechanosensory transduction process in the inner ear, are recycled via gap junctional networks in the cochlea. These results suggest that the voltage-gated potassium channel, containing Kv3.1b, in the cochlear lateral wall fibrocytes may control the intracellular potential and play an important role in regulating the potassium ion recycling mechanism via gap junctions in the inner ear.

Adenomyomatous polyp mimicking hydatidiform mole on ultrasonography.

The authors present a case report of a 62-year-old woman who experienced irregular genital bleeding. Although the serum hCG level was extremely low, transvaginal ultrasonography revealed vesicle pattern in the uterine cavity, suggesting trophoblastic disease. Hysterectomy was performed and histological diagnosis was adenomyomatous polyp. To our knowledge, this is the first report that the adnomyomatous polyp can demonstrate the vesicle pattern on ultrasonography.

Potassium ion recycling pathway via gap junction systems in the mammalian cochlea and its interruption in hereditary nonsyndromic deafness.

In the mammalian cochlea, there are two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. Thus far, four different connexin molecules, including connexin 26, 30, 31, and 43, have been reported in the cochlea. The two networks of gap junctions form the route by which K+ ions that pass through the sensory cells during mechanosensory transduction can be recycled back to the endolymphatic space, from which they reenter the sensory cells. Activation of hair cells by acoustic stimuli induces influx of K+ ions from the endolymph to sensory hair cells. These K+ ions are released basolaterally to the extracellular space of the organ of Corti, from which they enter the cochlear supporting cells. Once inside the supporting cells they move via the epithelial cell gap junction system laterally to the lower part of the spiral ligament. The K+ ions are released into the extracellular space of the spiral ligament by root cells and taken up by type II fibrocytes. This uptake incorporates K+ into the connective tissue gap junction system. Within this system, the K+ ions pass through the tight junctional barrier of the stria vascularis and are released within the intrastrial extracellular space. The marginal cells of the stria vascularis then take up K+ and return it to the endolymphatic space, where it can be used again in sensory transduction. It is highly probable that mutations of connexin genes that result in human nonsyndromic deafness cause dysfunction of cochlear gap junctions and thereby interrupt K+ ion recirculation pathways. In addition to connexin mutations, other conditions may disrupt gap junctions within the ear. For example, mice with a functionally significant mutation of Brain-4, which is expressed in the connective tissue cells within the cochlea, show marked depression of the endolymphatic potential and profound sensorineural hearing loss. It seems likely that disruption of connective tissue cells by this mutation disrupts K+ ion entry into the stria vascularis and thereby results in loss of endolymphatic potential. The association of sensorineural hearing loss with these genetic disorders provides strong evidence for the necessity of gap junction systems for the normal functioning of the cochlea.

A case of thrombopoietin-producing ovarian carcinoma confirmed by immunohistochemistry.

A case of ovarian carcinoma with thrombopoietin production is reported. A 49-year-old Japanese woman had serous cystadenocarcinoma of the right ovary with peritoneal spread. The platelet count was elevated to 432 x 10(9)/L. Enzyme-linked immunosorbent assay of the serum demonstrated remarkably high levels of thrombopoietin (2.96 fmol/ml). Immunohistochemical examination using an antibody specific for thrombopoietin revealed positive staining in the carcinoma cells, confirming thrombopoietin production.

Comparison of complications of vaginal hysterectomy in patients with leiomyomas and in patients with adenomyosis.

We reviewed 1246 vaginal hysterectomies performed at Handa City Hospital between January 1984 and December 1996. We divided the patients into 2 groups: those with leiomyomas (n = 893) and those with adenomyosis (n = 353). There was no difference in operative time and estimated blood loss between the 2 groups when analyzed by uterine weight. However, adenomyosis was associated with an increased risk of bladder injury.

Dual effects of anti-inflammatory 2-arylpropionic acid derivatives on a major isoform of human liver 3alpha-hydroxysteroid dehydrogenase.

Nonsteroidal anti-inflammatory drugs have been shown to be potent inhibitors of mammalian 3alpha-hydroxysteroid dehydrogenase. Here, we report that the drugs of the 2-arylpropionic acid class act as both activators and inhibitors for a predominant isoform of the human liver enzyme which is involved in the metabolism of steroid hormones, bile acids, drug ketones and xenobiotic aromatic hydrocarbons. Flurbiprofen, fenoprofen, ibuprofen, naproxen, ketoprofen and suprofen stimulated the activity of the human enzyme (1.5-2.4-fold) at low concentrations of less than 20-100 microM, whereas at higher concentrations they inhibited the activity. Comparison of the effects of the structurally related compounds with the drugs revealed that the essential structure required as the activator molecule is 2-phenylpropionic acid with a hydrophobic substituent on the aromatic ring. In addition, an R-enantiomer of ibuprofen showed higher activation (3-fold) than its S-enantiomer. Kinetic analysis with respect to NADP+ concentration indicated that R- and S-ibuprofens are nonessential activators showing binding constants of 23 and 34 microM, respectively. Neither enantiomers activated, but rather inhibited the enzyme, with Met replacing Arg-276 which has been shown to interact with the 2'-phosphate of NADP+. The inhibitions of the mutant enzyme by R- and S-ibuprofens were competitive with respect to the substrate, giving Ki values of 95 and 18 microM, respectively. The results suggest that the human liver 3alpha-hydroxysteroid dehydrogenase isoform possesses the two distinct sites, activator and inhibitor sites, to which anti-inflammatory 2-arylpropionates stereoselectively bind.

Activation of human liver 3alpha-hydroxysteroid dehydrogenase by clofibrate derivatives.

The NADP+-dependent dehydrogenase activity of a predominant isoenzyme of human liver 3alpha-hydroxysteroid dehydrogenase was activated by antihyperlipidemic drugs, such as bezafibrate and clinofibrate, and by clofibric acid and fenofibric acid (active metabolites of clofibrate and fenofibrate, respectively). The optimal pH of the activation by the drugs was about 7.5, and the concentrations giving maximum stimulation (1.8- to 2.4-fold) were 100, 50, 400 and 50 microM for bezafibrate, clinofibrate, clofibric acid and fenofibric acid, respectively. Clofibrate and fenofibrate acted as weak inhibitors, and the clofibric acid derivatives that lack the chloro group, methyl group on the alpha-carbon or carboxyl group greatly decreased the stimulatory effects. The activation by the drugs increased both Km and kcat (turnover number) values for the coenzyme and substrates. Kinetic analysis with respect to NADP+ showed that bezafibrate, clinofibrate, clofibric acid and fenofibric acid were nonessential activators showing dissociation constants of 32, 6, 103 and 11 microM, respectively. The combined activators experiments with one of the above drugs and sulfobromophthalein, a known activator specific for this enzyme, and comparison of their effects on the activities of mutant enzymes (with Met replacing Lys-270 or Arg-276) indicated that sulfobromophthalein and the drugs bind to an identical site on the enzyme. These results suggest that the long-term therapy with the antihyperlipidemic drugs influences the metabolism of steroid hormones, bile acids and several ketone-containing drugs mediated by the enzyme.

Magnetic resonance imaging with gadolinium-diethylenetriamine pentaacetic acid is useful in assessment of tubal patency in a patient with iodine-induced hypothyroidism.

A 32-year-old woman presented for evaluation of primary infertility. Because she had a history of iodine-induced hypothyroidism, conventional hysterosalpingography was contraindicated. Tubal patency was assessed by magnetic resonance imaging (MRI) after infusion of gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). Visualization of the contrast medium in the peritoneal cavity revealed tubal patency. Our case indicates that MRI with gadolinium-diethylenetriamine pentaacetic acid is a safe, simple, and easy way to confirm that at least one tube is patent when a patient is at risk for hysterosalpingography. To our knowledge, this is the first report that tubal patency was diagnosed on MRI.

Gonadotropin-releasing hormone-induced elevation of serum hCG in choriocarcinoma: a case report.

We evaluated the effect of GnRH on the serum hCG level in gestational trophoblastic disease (GTD). Five patients with GTD were studied. Three patients had hydatidiform mole (two complete and one partial mole) and two had choriocarcinoma. Blood samples were collected immediately before and 30, 60 min after the 100 microg GnRH iv injection, followed by hCG assay. Only one case of choriocarcinoma demonstrated an hCG increase after intravenous administration of GnRH (positive GnRH test). In that case, the hCG level dropped to the normal range after eight cycles of chemotherapy but the GnRH test was still positive, suggesting the existence of viable cancer cells. Since the GnRH test became negative, no increase in hCG has been observed, indicating that the patient achieved complete remission. Although a positive GnRH test is not common in GTD, GnRH test before treatment might be useful to find a positive case where the test can be repeated to determine complete remission and the time when the chemotherapy may be discontinued.

[A sensitive fluorometric assay for dihydrodiol dehydrogenase].

Dihydrodiol dehydrogenase (DD) oxidizes naphthalene dihydrodiol to 1,2-dihydroxynaphthalene, which is immediately autoxidized to 1,2-naphthoquinone. Here we established a fluorometric assay for the enzyme, which is based on the conversion of 1,2-naphthoquinone to fluorescent compounds by reacting with ethylenediamine. The formed fluorescent compounds were synthetically identified as 6-(2-aminoethylamino)benzo[f]quinoxaline and 2,6- or 3,6-bis(2-aminoethylamino)benzo[f]quinoxaline, which showed the same fluorescence at 550 nm at an excitation wavelength of 420 nm. This method provides a 9000-fold increase in sensitivity over a currently available assay which measures the change in the absorbance of a cofactor, NADPH. Since this simple and sensitive method allowed many samples to be assayed simultaneously, we applied it to the analysis of multiple forms of DD, separated by an anion-exchange chromatography, from six human liver specimens.

Activation of human liver 3 alpha-hydroxysteroid dehydrogenase by sulphobromophthalein.

Human liver contains at least two isoenzymes (DD2 and DD4) of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase. The NADP(H)-linked oxidoreductase activities of DD4 were activated more than 4-fold by sulphobromophthalein at concentrations above 20 microM and under physiological pH conditions. Sulphobromophthalein did not stimulate the activities of DD2 and human liver aldehyde reductase, which are functionally and/or structurally related to DD4. No stimulatory effect on the activity of DD4 was observed with other organic anions such as Indocyanine Green, haematin and Rose Bengal. The binding of sulphobromophthalein to DD4 was instantaneous and reversible, and was detected by fluorescence and ultrafiltration assays. The activation by sulphobromophthalein decreased the activation energy in the dehydrogenation reaction for the enzyme, and increased both kcat, and Km values for the coenzymes and substrates. Kinetic analyses with respect to concentrations of NADP+ and (S)-(+)-indan-1-ol indicated that sulphobromophthalein was a non-essential activator of mixed type showing a dissociation constant of 2.6 microM. Thus, the human 3 alpha-hydroxysteroid dehydrogenase isoenzyme has a binding site specific to sulphobromophthalein, and the hepatic metabolism mediated by this isoenzyme may be influenced when this drug is administered.

Relationship of human liver dihydrodiol dehydrogenases to hepatic bile-acid-binding protein and an oxidoreductase of human colon cells.

We previously isolated three monomeric dihydrodiol dehydrogenases, DD1, DD2 and DD4, from human liver, and cloned a cDNA (C9) thought to encode DD2, which is identical with those for human bile-acid-binding protein and an oxidoreductase of human colon carcinoma HT29 cells. In the present study we have provided evidence that the C9 cDNA clone encodes DD1, not DD2. A recombinant enzyme expressed from the cDNA in a bacterial system was purified, and its catalytic properties, bile-acid-binding ability and primary sequence were compared with those of the hepatic dihydrodiol dehydrogenases. The results show that DD1 encoded by C9 possesses prostaglandin F synthase activity but low affinity for lithocholic acid, whereas DD2, showing differences of six amino acid residues from the DD1 sequence, exhibited high-affinity binding for the bile acid. Refined relationship between dihydrodiol dehydrogenases and their related proteins of human tissues is proposed.