Metaelectric multiphase transitions in a highly polarizable molecular crystal.

Metaelectric transition, i.e. an abrupt increase in polarization with an electric field is just a phase change phenomenon in dielectrics and attracts increasing interest for practical applications such as electrical energy storage and highly deformable transducers. Here we demonstrate that both field-induced metaelectric transitions and temperature-induced phase transitions occur successively on a crystal of highly polarizable bis-(1H-benzimidazol-2-yl)-methane (BI2C) molecules. In each molecule, two switchable polar subunits are covalently linked with each other. By changing the NH hydrogen location, the low- and high-dipole states of each molecule can be interconverted, turning on and off the polarization of hydrogen-bonded molecular ribbons. In the low-temperature phase III, the tetragonal crystal lattice comprises orthogonally crossed arrays of polar ribbons made up of a ladder-like hydrogen-bond network of fully polarized molecules. The single-step metaelectric transition from this phase III corresponds to the forced alignment of antiparallel dipoles typical of antiferroelectrics. By the transition to the intermediate-temperature phase II, the polarity is turned off for half of the ribbons so that the nonpolar and polar ribbons are orthogonal to each other. Considering also the ferroelastic-like crystal twinning, the doubled steps of metaelectric transitions observed in the phase II can be explained by the additional switching at different critical fields, by which the nonpolar ribbons undergo "metadielectric" molecular transformation restoring the strong polarization. This mechanism inevitably brings about exotic phase change phenomena transforming the multi-domain state of a homogeneous phase into an inhomogeneous (phase mixture) state.

Creation of nano eye-drops and effective drug delivery to the interior of the eye.

Nano eye-drops are a new type of ophthalmic treatment with increased potency and reduced side effects. Compounds in conventional eye-drops barely penetrate into the eye because the cornea, located at the surface of eye, has a strong barrier function for preventing invasion of hydrophilic or large-sized materials from the outside. In this work, we describe the utility of nano eye-drops utilising brinzolamide, a commercially available glaucoma treatment drug, as a target compound. Fabrication of the nanoparticles of brinzolamide prodrug increases the eye penetration rate and results in high drug efficacy, compared with that of commercially available brinzolamide eye-drops formulated as micro-sized structures. In addition, the resulting nano eye-drops were not toxic to the corneal epithelium after repeated administration for 1 week. The nano eye-drops may have applications as a next-generation ophthalmic treatment.

[A Case of Laparoscopic Total Gastrectomy for Gastric Cancer with Diffuse Cystic Malformation].

A 75-years-old man was diagnosed with a cystic submucosal tumor of the middle body of the stomach and diffuse cystic malformation. Laparoscopic total gastrectomy was performed since a type II c gastric cancer was found at the lower body of the stomach after 1-year follow-up. Histopathological examination revealed mucosal adenocarcinoma with diffuse cystic malformation. Strict observation and appropriate treatment are needed for diffuse cystic malformation because of its significant carcinogenic rate.

[Study of CT Automatic Exposure Control System (CT-AEC) Optimization in CT Angiography of Lower Extremity Artery by Considering Contrast-to-Noise Ratio].

PURPOSE: To evaluate the image quality and effect of radiation dose reduction by setting for computed tomography automatic exposure control system (CT-AEC) in computed tomographic angiography (CTA) of lower extremity artery. METHODS: Two methods of setting were compared for CT-AEC [conventional and contrast-to-noise ratio (CNR) methods]. Conventional method was set noise index (NI): 14and tube current threshold: 10-750 mA. CNR method was set NI: 18, minimum tube current: (X+Y)/2 mA (X, Y: maximum X (Y)-axis tube current value of leg in NI: 14), and maximum tube current: 750 mA. The image quality was evaluated by CNR, and radiation dose reduction was evaluated by dose-length-product (DLP). RESULTS: In conventional method, mean CNRs for pelvis, femur, and leg were 19.9±4.8, 20.4±5.4, and 16.2±4.3, respectively. There was a significant difference between the CNRs of pelvis and leg (P<0.001), and between femur and leg (P<0.001). In CNR method, mean CNRs for pelvis, femur, and leg were 15.2±3.3, 15.3±3.2, and 15.3±3.1, respectively; no significant difference between pelvis, femur, and leg (P=0.973) in CNR method was observed. Mean DLPs were 1457±434 mGy⋅cm in conventional method, and 1049±434 mGy·cm in CNR method. There was a significant difference in the DLPs of conventional method and CNR method (P<0.001). CONCLUSION: CNR method gave equal CNRs for pelvis, femur, and leg, and was beneficial for radiation dose reduction in CTA of lower extremity artery.

[Usefulness of subtraction computed tomography angiography employing orbital synchronized helical scanning for diagnosis of lower extremity arteries with vessel wall calcification].

PURPOSE: Massive calcification complicates the diagnosis of the blood vessel lumen in computed tomography angiography (CTA) of the arteries of the lower extremities. The purpose of this study was to evaluate subtraction CTA with the use of orbital synchronized helical scanning (OS-SCTA). METHOD: Phantom study: We performed OS-SCTA and non-OSCTA of a calcified vessel phantom (ψ2.5 mm), and compared them with a non-calcified vessel phantom as the reference by full width at half maximum (FWHM) and full width at tenth maximum (FWTM) of maximum intensity projection (MIP) images. Clinical study: 58 patients with peripheral artery disease who were referred for angiography also underwent OS-SCTA. OS-SCTA was produced using MIP images. Findings were graded according to three categories: (1) stenosis greater than 50% or occluded; (2) stenosis less than 50%; (3) not detected due to insufficient image quality. OS-SCTA findings were compared with the angiographic findings for each arterial segment. RESULTS: In the phantom study, FWHM showed no significant difference between OS-SCTA and the reference (P=0.135), whereas FWTM showed a significant difference (P<0.001). FWHM and FWTM showed a significant difference between non-OS-SCTA and the reference (P<0.001), due to misregistration with helical artifacts. In a clinical study comparing OS-SCTA with angiography, the sensitivity and specificity were 93.3% and 95.1% in calcified segments, 91.8% and 93.9% in non-calcified segments, and 92.2% and 94.6% in all segments. There was no significant difference between calcified segments and non-calcified segments (sensitivity: P=0.568, specificity: P=0.549). CONCLUSION: OS-SCTA is beneficial for the diagnosis of lower extremity arteries with vessel wall calcification, since it shows detection accuracy comparable to that of angiography.

[Optimization of the chest computed tomography scan by varying the position of the arms].

Computed tomography automatic exposure control (CT-AEC) technique is calculated from a localizer radiograph. When we perform neck and chest CT examination, at first, we acquire localizer radiograph and neck images by placing the arm in a lowered position. Next, the arm is raised for the chest scan. Therefore, the localizer radiograph and subject information are different in the chest scan. In this situation, the chest scan with the use of the CT-AEC causes radiation over-dose. The purpose of this study is to optimize the CT-AEC by controlling noise index (NI), and make a chest CT scan condition considering the position of the arms. We measured the image noise (SD) in the phantom by using CT-AEC. In addition, dose length product (DLP) was recorded. Moreover, we examined the correlation with the clinical images. The results of our experiments show that radiation dose can be reduced with the image quality kept by controlling NI.

[The comparison between dose rates at the interventional reference point of the angiography systems in many facilities].

The management of the radiation dose is very important in interventional radiology (IVR), especially in percutaneous coronary intervention (PCI). Therefore, we measured entrance surface doses at the interventional reference point of 27 cardiac intervention procedures in 22 cardiac catheterization laboratories around Hiroshima, and compared these doses. Recently, for cardiac interventional radiology, the X-ray machines using flat-panel detectors (FPD) instead of image intensifiers (I.I.) is increasing; 13 systems used FPD and 14 systems used I.I. For fluoroscopy rate, the difference between laboratories was 9 times. For cineangiography rate, the difference between laboratories was 7 times. In addition, between both devices, the I.I. group is bigger than the FPD group. When comparing by the same condition, for the dose at the interventional reference point, no significant difference was detected between the FPD group and the I.I. group. This study shows that FPD is not available for reducing the radiation dose simply. Therefore, it is necessary that we think of the balance with image quality and radiation dose. The optimization of the devices and cardiac intervention procedures becomes very important.

Tracking of green fluorescent protein (GFP)-labeled LAK cells in mice carrying B16 melanoma metastases.

In adoptive immunotherapy, in vivo trafficking of adoptively transferred cells, including their accumulation at tumor sites, remains to be further investigated. Tracking of these cells by visualization is useful to clarify antitumor mechanisms and develop new modalities to enhance antitumor capacities. In the present study, an in vivo tracking study was performed using an adoptive transfer model of lymphokine-activated killer (LAK) cells induced from green mice into C57/BL6 mice with B16 melanoma metastases. Green mice are green fluorescent protein (GFP) transgenic mice originating from C57/BL6 mice. All of the tissues, except for erythrocytes and hair, express green fluorescence under excitation light. Although LAK cells in combination with IL-2 potently suppressed pulmonary metastases with survival prolongation, very few LAK cells accumulated in tumor tissues compared to those localized in the spleen, as visualized by fluorescent microscopy and quantitated by flow cytometry. The present method using transfer of green mice-derived cells into parental tumor-bearing mice is simple because there is no need for in vitro labeling and is feasible for the in vivo tracking of effector cells in an adoptive immunotherapy model.

Successful gene transfer into dendritic cells with cationized gelatin and plasmid DNA complexes via a phagocytosis-dependent mechanism.

The use of gene-modified dendritic cells (DC) is a powerful tool to enhance antitumor immune responses stimulated by these cells in cancer immunotherapy. Cationized gelatin is preferably incorporated via phagocytosis and is gradually degraded by proteolysis while buffering lysosomal activity. This may be appropriate for gene transfer into phagocytic cells, such as immature DC. In the present study, successful transfection into monocyte-derived immature DC was demonstrated using cationized gelatin and plasmid DNA complexes. A high transfection efficiency, approaching 16%, was obtained upon transfection of the enhanced green fluorescent protein (EGFP) gene as evaluated by flow cytometry. Transgene expression of EGFP and murine interleukin 12 were also detected by RT-PCR. The antigen-presenting capacity of the transfected DC was equal to that of untransfected DC as evaluated by the allogeneic mixed lymphocyte reaction. Cationized gelatin has the potential to be a unique non-viral vector for gene transfer into DC.

[An antitumor effect of oncolytic adenovirus capable of selectively replicating in CEA-expressing cancer cells and its enhancement by 5-FU].

In recent years, many have focused on the use of oncolytic virus capable of lysing cancer cells by a cancer-selective replication for a new treatment modality of cancer. In the present study, we used oncolytic adenovirus, AdCEAp/Rep, genetically modified to selectively replicate in CEA-expressing cancer cells, and investigated whether AdCEAp/Rep could induce selective cytotoxicity to CEA expressing cancer cells, and where the AdCEAp/Rep induced cytotoxic effect could be enhanced by 5 FU in using human gastric cancer cell lines, MKN45 (CEA-positive) and MKN74 (CEA-negative). The results showed that AdCEAp/Rep showed cytotoxicity against MKN45 in a dose-dependent manner, while it did not against MKN74. Furthermore, 5-FU remarkably enhanced AdCEAp/Rep-induced cytotoxicity against MKN45 by being added 3 days after adenovirus infection. These findings strongly suggest that AdCEAp/Rep may be applicable as a new therapeutic agent for clinical cancers expressing CEA in combination with chemotherapeutic agents such as 5-FU.

Possible inhibition of cancer cell adhesion to the extracellular matrix in NK4-induced suppression of peritoneal implantation.

Milky spots (MS), peritoneal lymphoid tissues, expose the extracellular matrix (ECM) due to a defect of mesothelial cells on their surface, which may explain why peritoneal implantation of cancer cells preferentially takes place at MS. We recently reported that adenovirus vector-mediated intraperitoneal production of NK4 strongly suppressed MS-selective implantation of cancer cells and subsequent peritoneal dissemination, without histological evidence of angiogenesis inhibition. The present study was conducted to clarify the mechanisms underlying the suppressive effects of NK4 on peritoneal implantation. In mice intraperitoneally injected with CT26 cells that were genetically modified to produce NK4 (CT26-NK4), peritoneal dissemination was significantly suppressed with survival prolongation. A decreased cell implantation to omental MS was also detected and evaluated by green fluorescence protein (GFP) imaging. In an in vitro adhesion assay, hepatocyte growth factor-stimulated adhesion to ECM components, such as fibronectin and collagen, was inhibited in CT26-NK4 compared to control cells. These results strongly suggest an inhibition of cancer cell adhesion to the ECM in the suppression of peritoneal implantation by NK4.

Suppression of peritoneal implantation of gastric cancer cells by adenovirus vector-mediated NK4 expression.

Peritoneal dissemination is the most common mode of metastasis in gastric cancer. We previously reported the importance of milky spots (MS), peritoneal lymphoid tissues, as selective sites of cancer implantation in peritoneal dissemination. In the present study, we first demonstrated that intraperitoneal injection of adenovirus vector encoding the GFP gene into tumor-free nude mice resulted in GFP expression at omental and mesenteric MS; MS macrophages were target cells for adenovirus infection. We confirmed that intraperitoneal injection of adenovirus vector encoding the NK4 gene (AdNK4) resulted in NK4 production localized to the peritoneal cavity, especially the omentum. Adenovirus vector-mediated MS-selective transgene expression was markedly impaired in tumor-bearing mice whose MS had already been replaced by infiltrating cancer cells. However, prior injection of AdNK4 successfully inhibited MS-selective cancer cell implantation, resulting in suppression of peritoneal dissemination and prolongation of survival. Adenovirus vector-mediated MS-selective delivery of a therapeutic gene may prevent peritoneal dissemination of gastric cancer.

[Suppression of peritoneal implantation by NK4 and its mechanisms].

NK4 suppresses invasion and metastasis of tumor cells by means of dual actions as HGF antagonist and angiogenesis inhibitor. Our previous studies showed that NK4 suppresses the implantation of tumor cells to the peritoneal milky spots (MS) by intraperitoneal injection (i.p.) of adenovirus vector expressing NK4 (Ad-NK4) or NK4 gene-transfected tumor cells. In the present study, we investigated the antitumor mechanisms of NK4 in the suppression of peritoneal implantation. When evaluated by a fluorescent microscopy, a prior injection of Ad-NK4 suppressed peritoneal implantation immediately after the injection of GFP-expressing tumor cells. DNA microarray analyses also demonstrated a reduced expression of some adhesion molecules in NK4 gene-transfected tumor cells as compared to neomycin gene-trasfected cells (control). In the in vitro adhesion assay, the adhesion to some types of the extra cellular matrixs (ECM) was significantly decreased in NK4 gene-transfected cells as compared to the control. These results suggest that NK4 may suppress peritoneal implantation by inhibiting adhesion of tumor cells to ECM around MS.

[Nonviral gene transfection into human dendritic cells by using biodegradable cationized gelatin and plasmid DNA complex].

Dendritic cells (DC) are potent antigen-presenting cells capable of stimulating T cell mediated immunity. Gene transfer of tumor specific antigens or cytokines into DC would be a useful strategy for immunotherapeutical purposes. In the present study, in vitro transfection of human DCs (hDC) with the complex of biodegradable cationized gelatin and an EGFP gene was performed. Flow cytometric analyses revealed that approximately 14% of DC was positively expressed for EGFP, and the mRNA expression of EGFP gene in transfected hDC was detected by RT-PCR. Additionally, when evaluated by allogeneic MLR, the antigen-presenting capacity of transfected DC was equal to that of control DC. Cationized gelatin is a promising nonviral vector for gene transfer into DC.

[Biodistribution of immune cells in adoptive immunotherapy by using GFP transgenic mice].

All of the cells and tissues from GFP transgenic mice, with the exception of erythrocytes and hair, express green fluorescent protein. Additionally, lymphokine activated killer cells induced from splenocytes of the mice (GFP-LAK) express green fluorescence under the observation by fluorescent microscopy. In the present study, we studied the biodistribution of LAK in the two adoptive immunotherapy models by injecting GFP-LAK into non-GFP expressing syngeneic mice. In peritoneal dissemination model of B16 melanoma cells, intraperitoneally injected GFP-LAK accumulated densely on the tumor. On the other hand, in the lung metastases model, intravenously injected GFP-LAK stayed scattered around the tumor, although they were observed abundantly in the spleen. Our adoptive transfer model using GFP transgenic mice is useful for understanding antitumor mechanisms induced by adoptively transferred immune cells, without any troublesome marking procedures.

Reduced HGF expression in subcutaneous CT26 tumor genetically modified to secrete NK4 and its possible relation with antitumor effects.

Tumor-stromal interactions, which are regulated by stromal-derived HGF and tumor-derived HGF inducers, are essential for tumor cell acquisition of such malignant properties as invasion and metastasis. NK4, a proteolytic cleavage product of HGF, has antitumor activities as both an HGF antagonist and an angiogenesis inhibitor. In this study, we examined the in vitro and in vivo behaviors of mouse colon adenocarcinoma C T26 cells modified by gene transfer to secrete NK4, and investigated the influence of NK4 on expression of HGF and HGF inducers associated with tumor-stromal interactions. In vitro cell proliferation rates of NK4 transfectant (C T26-NK4) and mock transfectant (C T26-NEO) were essentially the same, and scattering and invasion were stimulated by HGF in C T26-NEO, but not in C T26-NK4. In syngeneic BALB/c female mice, subcutaneous tumor growth of C T26-NK4 was potently suppressed, and the survival was prolonged significantly. Immunohistochemistry showed significantly decreased microvessels and increased apoptotic cells in C T26-NK4 tumor compared with control. Interestingly, HGF, strongly expressed in C T26-NEO tumor stroma, was reduced in C T26-NK4. In vitro, conditioned medium of C T26-NK4 inhibited fibroblast-derived HGF production, which was increased by that of C T26-NEO. Moreover, although similar constitutive expression levels of PDGF and TGF-alpha (both HGF inducers) were detected in C T26-NK4 and C T26-NEO in semiquantitative RT-PCR analyses, the expression was up-regulated by HGF in C T26-NEO, but not C T26-NK4. These results suggest that NK4 may exert antitumor activities not only by antagonizing HGF, but also by inhibiting HGF amplification via tumor-stromal interactions. Continuous, abundant NK4 production induced at a tumor site by gene transfer should show multiple antitumor activities with potential therapeutic benefit.

[Growth suppression of subcutaneous tumor by CT26 expressing NK4 in syngeneic mice].

An HGF antagonist, NK4, inhibits not only invasion and metastasis of tumor cells driven by HGF-Met receptor binding, but also tumor angiogenesis. To address the antitumor activities of NK4, we investigated the biological behaviors of CT26 transfected with the NK4 gene (CT26-NK4) in vitro and in vivo. In the in vitro assay, the invasion in MOCK transfected cells (control) was stimulated by HGF; however, in CT26-NK4 cells, these effects were completely inhibited. In the in vivo assay, the tumor growth of CT26-NK4 was strongly suppressed and the survival of CT26-NK4 tumor-bearing mice was significantly prolonged. Immunohistochemical analysis revealed that while proliferating cells (PCNA immunostaining) of CT26-NK4 tumors were weakly suppressed, the micro-vessel number (CD31/PECAM-1 immunostaining) in those tumors was significantly suppressed as compared with the control tumors. In conclusion, NK4 exerts potent antitumor effects via anti-angiogenesis rather than inhibition of biological events of tumor cells stimulated by HGF.

[Functional analysis of peritoneal lymphoid tissues by GFP expression in mice--possible application for targeting gene therapy against peritoneal dissemination].

It is important to develop an efficient adjuvant therapy for the prevention of the postoperative peritoneal recurrence of gastrointestinal cancers such as gastric cancer or pancreatic cancer. Milky sports (MS) are peritoneal lymphoid tissues broadly distributed on the peritoneal tissues such as the omentum, mesentery, or Douglas pouch, and are the selective implantation sites of disseminated cancer cells. In this study, we introduced GFP gene into various cancer cell lines and host-derived cells such as peritoneal macrophages and dendritic cells by adenovirus vetor and injected them i.p. into mice. We then investigated the sites of GFP expression on the peritoneum by fluorescence microscopy. The results showed that green fluorescence was detected specifically for the MS sites that stained black with activated carbon particles (CH40), despite the differences in the cell type injected. These findings indicate that 1) the physioanatomical characteristics of MS may play an essential role in the formation of the initial disseminated lesions at MS, and 2) the host-derived cells accumulating near MS may be available as carriers of a therapeutic gene in intraperitoneal gene therapy against peritoneal dissemination.