Evaluation of lysozyme-HCl for the treatment of chalkbrood disease in honey bee colonies.

Chalkbrood, caused by Ascosphaera apis (Maassen and Claussen) Olive and Spiltor, is a cosmopolitan fungal disease of honey bee larvae (Apis mellifera L.) for which there is no chemotherapeutic control. We evaluated the efficacy of lysozyme-HCl, an inexpensive food-grade antimicrobial extracted from hen egg white, for the treatment of chalkbrood disease in honey bee colonies. Our study compared three doses of lysozyme-HCl in sugar syrup (600, 3,000, and 6,000 mg) administered weekly for 3 wk among chalkbrood-inoculated colonies, colonies that were inoculated but remained untreated, and colonies neither inoculated or treated. Lysozyme-HCl at the highest dose evaluated was found to suppress development of chalkbrood disease in inoculated colonies to levels observed in uninoculated, untreated colonies, and did not adversely affect adult bee survival or brood production. Honey production was significantly negatively correlated with increased disease severity but there were no significant differences in winter survival among treatment groups. Based on our results, lysozyme-HCl appears to be a promising, safe therapeutic agent for the control of chalkbrood in honey bee colonies.

SU-E-J-203: Determination of PTV Margin for Lung Tumor Using In-Treatment 4D CBCT.

PURPOSE: To determine a planning target volume (PTV) margin for lung cancer patients using a four-dimensional cone-beam CT (4D CBCT) acquired during volumetric modulated arc therapy (VMAT) treatment. METHODS: A VMAT plan for lung cancer patients was created by Pinnacle v9.0 (Philips) treatment planning system (TPS), where the gross target volume (GTVs) in each breathing phase was delineated by using 4D-planning CT scan (TOSHIBA and ANZAI). The VMAT treatment was performed with a stereotactic body frame after the registration using Elekta X-ray volume imaging (XVI) unit. Simultaneous cone-beam projection images were acquired for 3 or 4 fractions of 10 patients. The in-treatment 4D CBCT was reconstructed by dividing into four breathing phase bins. A total of 38 in-treatment 4D-CBCT sets were exported to Pinnacle TPS. The isocenter of in-treatment 4D CBCT was matched with that of 4D-planning CT. The tumor motion during treatment was manually tracked on in-treatment 4D CBCT, and the center-of-mass (COM) location of the tumor was estimated. Analyzing the tumor regions observed by in-treatment 4D CBCT, a PTV margin in our system was derived. RESULTS: The average difference in COM location of the tumor was less than 1mm for all directions, while the standard deviations (SD's) were about 1.3mm, 1.6mm, and 2.1mm for the lateral, the vertical, and the longitudinal directions, respectively. The large discrepancy more than 3mm was observed for one patient. The required PTV margin was about 3-4mm for the lateral and the vertical directions, whereas it was about 5mm for the longitudinal direction. CONCLUSIONS: The uncertainties of the tumor motion caused by respiration were observed by in-treatment 4D CBCT images. It was feasible to determine the PTV margin from 4D volume images. K. Nakagawa receives research funding from Elekta.

SU-E-J-183: Dose Distribution Generated from 131 I Radionuclide Using SPECT-CT.

PURPOSE: 131 I radionuclide therapy is widely performed in a thyroid cancer treatment, but there has been almost no evaluation of the dose distribution. The aim of this work is to develop the calculation system using the data of SPECT-CT and to examine the effects of their image resolutions on the dose distribution. METHODS: We designed and constructed an acrylic phantom for measurement. A radioactive iodine capsule and glass dosimeters can be set in the layer structure of the phantom. We put iodine capsules (37MBq, 111MBq, 185MBq) in the middle of the phantom and acquired SPECT-CT (Infinia Hawkeye4 (GE)) images. Both the CT value data (image resolution: 1.1 mm) and the intensity map data of SPECT (image resolution: 4.4 mm) were independently used for the estimation of the cumulative dose distribution generated from the radioactive iodine in the phantom. We adopted Monte Carlo program PHITS2.0 as the simulation of the dose calculation. The absolute dose was measured by glass dosimeters. RESULTS: The measurement result by glass dosimeters was very similar to the Monte Carlo simulation result, in which the difference was about 0.3 %. We obtained the dose distributions reconstructed by the radioactive iodine distribution using CT value data and SPECT data, respectively. The iodine distribution from CT could be finer than that of SPECT data because of its higher image resolution. As a result, the difference was found to be factor two in the middle of the iodine distribution. On the other hand, both of the dose distribution was almost same above 2.2 cm distance from the center. CONCLUSIONS: We can reconstruct the 131I dose distribution using SPECT-CT data. For more accurate calculation of the dose distribution, it would be crucial to increase the resolution of SPECT data.

SU-E-J-33: Geometric Agreement Check for Imaging System with Radiation Beam by KV and MV-CBCT.

PURPOSE: The verification method of the geometry agreement between a light field and/or a laser coordinate and treatment beam should be easy and quick. In this presentation, we propose a novel QA method by using both kV- and MV-CBCT for kV-IGRT system. This method confirms the temporal unchanging the agreement of geometry in the kV-IGRT system with the treatment beam geometry. METHODS: 1) MV-flexmap: Sequential MV-projection images were acquired during gantry rotation by iViewGT (Elekta) and MV-CBCT was reconstructed by in-house software with a flexmap correction. The flexmap is displacement of gantry and detector panel related with gantry sag. The geometric change affects the deranging reconstructed image. To evaluate how much displacement of EPID panel and gantry was detectable, the images of 8mm diameter ball-bearing (BB) located at the radiation isocenter were reconstructed with improper Flexmap.2) A comparison between the kV-CBCT and the MV-CBCT: The kV-CBCT was provided by X-ray Volume image (XVI) system (Elekta). To confirm the agreement for the geometry between kV-IGRT system and treatment beam, the kV-CBCTs of BB are compared with that of MV-CBCTs. RESULTS: The flexmaps were modified to (b)1mm / (c)3mm shifted to the rotation direction and (d)3mm to the rotation axis. The MV-CBCT were reconstructed with the correct flexmap and with incorrect flexmap (b), (c) and (d).　The geometric confirmation for MV-CBCT was done by comparison of the width and center of the BB on the MV-CBCT. The discrepancy of center between kV-CBCT and MV-CBCT was less than 1mm. CONCLUSIONS: Less than 1mm of the geometrical changing to rotation direction for MV-detector panel could be recognized by reconstructed images of BB. Using kV- and MV-CBCT enable us to perform the simple comparison for geometrical non-idealities between the kV-IGRT system and the treatment beam. Dr. K. Nakagawa received research grant from Elekta.

Electron gun using carbon-nanofiber field emitter.

An electron gun constructed using carbon-nanofiber (CNF) emitters and an electrostatic Einzel lens system has been characterized for the development of a high-resolution x-ray source. The CNFs used were grown on tungsten and palladium tips by plasma-enhanced chemical-vapor deposition. Electron beams with the energies of 10

MCG measurement in the environment of active magnetic shield.

MCG (Magnetocardiography) measurement by a SQUID gradiometer was attempted with only active magnetic shielding (active shielding). A three-axis-canceling-coil active shielding system, where three 16-10-16 turns-coil sets were put in the orthogonal directions, produces a homogeneous magnetic field in a considerable volume surrounding the center. Fluxgate sensors were used as the reference sensors of the system. The system can reduce environmental magnetic noise at low frequencies of less than a few Hz, at 50 Hz and at 150 Hz. Reducing such disturbances stabilizes biomagnetic measurement conditions for SQUIDs in the absence of magnetically shielded rooms (MSR). After filtering and averaging the measured MCG data by a first-order SQUID gradiometer with only the active shielding during the daytime, the QRS complex and T wave was clearly presented.

Active magnetic compensation composed of shielding panels.

Magnetically shielded rooms (MSRs) with materials of high permeability and active shield systems have been used to shield magnetic noise for biomagnetic measurements up to now. However, these techniques have various disadvantages. Therefore, we have developed a new shielding system composed of shielding panels using an active compensation technique. In this study, we evaluated the shielding performance of several unit panels attached together. Numerical and experimental approaches indicated that the shielding factor of a cubic model composed of 24 panels was 17 for uniform fields, and 7 for disturbances due to car movement. Furthermore, the compensation space is larger than that of an ordinary active system using large coils rather than panels. Moreover, the new active compensation system has the important advantage that panels of any shape can be assembled for occasional use because the unit panels are small and light.

Regulation of cell motility via high and low affinity autocrine motility factor (AMF) receptor in human oral squamous carcinoma cells.

A tumour-secreted cytokine autocrine motility factor (AMF) induces in vivo invasion and metastasis, and in vitro tumour cell motility by a signal transduction through interaction with its cell surface receptor gp78. In this report, we investigated the characterization of a high-metastatic human oral squamous cell carcinoma (SCC) cell line LMF4 and low-metastatic HSC-3 in comparison with non-metastatic HSC-2 and HSC-4. Morphological and motility analyses revealed LMF4 cells to have the highest motile activity among those cells. However, LMF4 cells shared the similar features with HSC-3: high level secretion of AMF, enhancement of gp78 expression, co-expression of vimentin and cytokeratin, although LMF4 cells showed twice as high motile reactivity as HSC-3. The only difference was that LMF4 had twice as high amount of low-affinity receptor(s) as HSC-3, shown by Scatchard analysis.

Pricing and reimbursement of pharmaceuticals in Norway.

Although not a member of the EU, Norway participates in the European-wide regulatory framework for granting marketing authorization to pharmaceutical products. Maximum prices for prescription medicines are determined by the Norwegian Medicines Agency that sets pharmacy purchase prices (based on prices in other Northern European countries) and the Ministry of Health that sets pharmacy margins. A "discount sharing model" encourages pharmacies to perform parallel import and generic switching by allowing them to keep up to 50% of the difference between maximum price and actual price. The costs of pharmaceuticals in Norway are covered in part by the public budget and in part directly by the patient. Over one-half of pharmaceutical costs are borne by the Norwegian National Insurance Administration through the reimbursement scheme; membership in this program is mandatory, and costs are covered through taxes from employers and employees. Over 90% of reimbursed drug sales are accounted for by the established product list for general reimbursement, but supplementary reimbursement can be granted on the basis of individual patient applications and also to ensure that all patients with serious communicable diseases are given adequate treatment without costs to the patient (e.g., HIV/AIDS, tuberculosis). Patient copayment currently amounts to 36% of the total amount of prescriptions; the maximum per prescription is 48 euros, and total within a single calendar year is 180 euros. Copayments for physician visits, radiology examinations, and laboratory tests, can be included in this amount. The overall system is now undergoing reevaluation, as it has been criticized for being complicated and difficult to comprehend for the users.

Neuromyelitis optica.

Neuromyelitis optica disease is characterized by simultaneous or successive attacks involving both the optics nerves and spinal cord without any evidence of the disease elsewhere. We report a 22-year-old Saudi woman with relapsing neuromyelitis optica disease. She had all the clinical, cerebrospinal fluid, and radiological features that differ from primary demyelinating disease. However, our patient responded well to long-term corticosteroid therapy and azathioprine with improvement in her expanded disability status scale, and ambulation. In addition, no acute relapses occurred with significant improvement on magnetic resonance imaging lesions and favorable outcome.

Development and validation of chemiluminescent immunoassay for vitellogenin in five salmonid species.

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of vitellogenin (Vg) in five salmonids. The CLIA for salmon Vg was performed using the two-site method, with anti-masu salmon beta'-component as primary antibody and chemiluminescent acridinium-labeled anti-rainbow trout lipovitellin F(ab)'(2) as the second antibody. Using cutthroat trout Vg as the standard, the working range of the CLIA was from 60 pg to 500 ng Vg/ml. Intra- and inter-assay coefficients of variation ranged from 3.04 to 6.67% and 3.23 to 5.86%, respectively. For the various salmonid species, serially diluted samples of serum from vitellogenic fish ran parallel to their purified Vg standard curve in the CLIA. In male cutthroat trout maturing during the 4 months before spawning, serum Vg levels ranged from 1.56 to 8000 ng/ml. High levels of Vg in some individuals may have resulted from temporary elevation of estradiol-17beta levels in the same fish during December or January (1-2 months before spawning). This is the first report on changes in serum Vg levels in maturing male trout using CLIA, the most sensitive assay for Vg yet developed.

Tumor autocrine motility factor is an angiogenic factor that stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine which primarily stimulates tumor cell motility via receptor-mediated signaling pathways, and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for antiangiogenic treatment.

Tumor autocrine motility factor is an angiogenic factor hat stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine that primarily stimulates tumor cell motility via receptor-mediated signaling pathways and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for anti-angiogenic treatment.

Quantification of cell motility : gold colloidal phagokinetic track assay and wound healing assay.

Cellular migration is an integral aspect in response to extracellular stimuli, which is fundamental to numerous biological processes such as embryogenesis, inflammation, wound healing, tissue regeneration, and tumor invasion and metastasis (1,2). Abundant studies centered on the identification and characterization of factors that regulate and direct cell movement have shown that host serum components and extracellular matrix breakdown products exert a chemotactic effect on various tumor cells (3) and that basement membrane and extracellular matrix components promote cellular haptotaxis (4). Furthermore, host growth factors influence recipient cells by modulating growth and motility independently or in a coordinated manner (2). Moreover, cellular migration in vitro has been reported to be correlated with tumor invasion and metastasis in vivo. A group of motility factors has been described, the primary function of which is thought to be the regulation of cellular kinesis. Motility factors have been originally distinguished by their ability to induce the random (chemoki-netic) and directional (chemotactic) migration of the cells (5). Therefore, quantitating the cell motility is one of the most important clues to comprehend the cellular characteristics of malignancy and/or the effect and activities of motility inducing properties. Gold colloidal method was invented to measure the random motility (chemokinesis) by Albrecht-Buehler, in which area of phagokinetic track cleared by a single cell is measured (6). The Boyden chamber method, described in Chapter 5 by Brown and Bicknell, was invented to quantitate the directional motility (chemotaxis) and was modified in various ways to.

Phosphohexose isomerase/autocrine motility factor/neuroleukin/maturation factor is a multifunctional phosphoprotein.

Phosphohexose isomerase (PHI) is a member of the ectoenzyme/exoenzyme family and plays a key role in both glycolysis and gluconeogenesis pathways. Upon secretion PHI acts as a cytokine with tumor autocrine motility factor (AMF), neuroleukin (NLK) and maturation factor (MF) functions. Signaling is initiated by its binding to a cell surface 78 kDa glycoprotein (gp78). However, since PHI protein is a 'leaderless' secretory protein, released from cells via a non-classical route(s), we questioned whether the molecule undergoes post-translation modification while retaining proper folding and maintaining intact enzymatic and motogenic activities. To address this, we have generated, expressed and isolated a recombinant human AMF (rhAMF). The rhAMF retained the biological activities of the native AMF, i.e., catalyzes phosphohexose isomerization and stimulated cell motility. Additionally, we show here that human PHI is phosphorylated at serine 185 by casein kinase II (CK II) and we provide experimental evidence suggesting that this phosphorylation is associated with secretion, thus providing insights for elucidating the intracellular signal transmission of cell response to stimulation by AMF/NLK/MF.

Structure of insect chitin isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia.

Chitin samples in a alpha-form structure were isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia by treatment with 1 N HCl and 1 N NaOH. Chitosan was prepared by treating them in 40% NaOH containing NaBH(4). Chitin and chitosan were analyzed by X-ray, [13C]CP/MAS NMR, [13C]FT-NMR, and scanning electron microscopy (SEM) methods. Insect chitin degraded more readily than shrimp chitin when treated with 6 N HCl and the enzyme-chitinase. After treatment with 2 N HCl at 100 degrees C, the insect chitin crystallinity increased. N-deacetylation of insect chitin was easier than that of crustaceous chitin, and about 94% of the N-acetyl groups were removed in one treatment with 40% NaOH for 4 h at 110 degrees C. After treatment with 2 N HCl, 55% of the N-acetyl groups of silkworm chitin were removed under the same conditions. Beetle chitin showed a higher affinity for chitinase than shrimp chitin.

A clinicocytopathologic study of adenoma malignum of the uterine cervix.

OBJECTIVES: To investigate the nature and the clinical course of adenoma malignum (the so-called minimal deviation adenocarcinoma) of the uterine cervix by conducting a retrospective study of 6 cases consecutively treated in a single institute. METHODS: The pathologic classification of adenoma malignum was performed according to the WHO classification (1994). RESULTS: These tumors accounted for only 1.32% (6/453) of invasive cervical adenocarcinomas. All the cases showed either a watery discharge or atypical genital bleeding, or both, at the time of diagnosis. The preoperative cytologic diagnosis of adenoma malignum was made in 83.3% (5/6) of cases. The preoperative punch biopsy, on the other hand, failed to confirm the diagnosis of adenoma malignum in all cases, although the presence of the disease was suspected in 2 cases (33%). The 5-year survival rate and 5-year disease-free survival rate were 100 and 83.3%, respectively. CONCLUSIONS: This series demonstrates that cytologic examination is a potent screening method to detect this rare disease. When the presence of this disease is suspected by the cytologic examination, a deep biopsy is necessary to make an accurate diagnosis. An ordinary cervical biopsy is usually insufficient to detect deeply positioned tumor glands. The prognosis of the disease may be better than that for ordinary cervical adenocarcinoma.

Expression and secretion of neuroleukin/phosphohexose isomerase/maturation factor as autocrine motility factor by tumor cells.

The results obtained from fragmented protein microsequencing have suggested that autocrine motility factor (AMF), a tumor-secreted Mr 55,000 cytokine that regulates cell motility in vitro as well as invasion and metastasis in vivo, is the neuroleukin (NLK)/phosphohexose isomerase (PHI)/maturation factor (MF) polypeptide. Here, we cloned, sequenced, and studied the expression, secretion, and distribution of AMF/NLK/PHI/MF in neoplastic and their normal counterpart cells. Although both normal and neoplastic cells express the gene product, overexpression associated with selective secretion of the protein was observed only in tumor cells. The cDNA sequences of AMF/NLK/PHI/MF found in both human cancer and normal cells were found to be identical, suggesting that its secretion by neoplastic cells is independent of mutation or alternative splicing. Immunohistochemical visualization has depicted AMF/NLK/PHI/MF to be localized into tubular-like vesicles, diffusely distributed throughout the cytoplasm and not colocalized with any particular cytoskeletal network. Confocal microscopic imaging had shown a partial colocalization between AMF and its receptor (Mr 78,000 glycoprotein), especially on the malignant cell surface periphery. The results suggest that extracellular AMF activity may be a result of the product of intracellular cleavage of a precursor polypeptide, which is overexpressed and selectively secreted through a nonclassical secretory mechanism by neoplastic cells.

Inhibitory effect of delphinidin from Solanum melongena on human fibrosarcoma HT-1080 invasiveness in vitro.

We investigated the inhibitory effect of eggplant (Solanum melongena var. marunasu) extract on human fibrosarcoma HT-1080 cell invasion of reconstituted basement membrane [Matrigel (MG)]. We found that the effective component of the plant extract was delphinidin, a flavonoid pigment contained in the peel. The extract and delphinidin did not affect tumor cell adhesion to MG or haptotactic migration to MG. HT-1080 secretes matrix metalloproteinase(MMP)-2 and MMP-9, which degrade extracellular matrix as part of the invasive process. Delphinidin slightly inhibited the activity of MMPs, which may have been responsible, in part, for the inhibition of tumor cell invasiveness.

Inhibition effect of propargylglycine on human fibrosarcoma HT-1080 cell invasiveness.

Effect of propargylglycine (2-Amino-4-pentynoic acid, PPG) on invasive property of human fibrosarcoma HT-1080 cell was investigated. PPG treatment of HT-1080 significantly reduced the total cellular metallothioneins (MTs) contents, and the resistance of HT-1080 against heavy metals toxicity decreased with the decrease of the MTs contents. The HT-1080 cell invasion to reconstituted basement membrane Matrigel (MG) was inhibited by the PPG treatment in a PPG concentration-dependent fashion. The inhibition was due to the lowering of HT-1080 cells attachment to MG and degradation activity of matrix metalloproteinases (MMPs) secreted from HT-1080 by the PPG treatment. However, the chemotactic ability of the PPG treated HT-1080 was enhanced. Our results suggest that MTs concentration levels in a malignant tumor cell are closely related to its invasiveness, and if MTs level of tumor cell can be controlled, cancer metastasis may be able to be controlled.