Enhanced angiogenic properties of umbilical cord blood primed by OP9 stromal cells ameliorates neurological deficits in cerebral infarction mouse model.

Umbilical cord blood (UCB) transplantation shows proangiogenic effects and contributes to symptom amelioration in animal models of cerebral infarction. However, the effect of specific cell types within a heterogeneous UCB population are still controversial. OP9 is a stromal cell line used as feeder cells to promote the hematoendothelial differentiation of embryonic stem cells. Hence, we investigated the changes in angiogenic properties, underlying mechanisms, and impact on behavioral deficiencies caused by cerebral infarction in UCB co-cultured with OP9 for up to 24 h. In the network formation assay, only OP9 pre-conditioned UCB formed network structures. Single-cell RNA sequencing and flow cytometry analysis showed a prominent phenotypic shift toward M2 in the monocytic fraction of OP9 pre-conditioned UCB. Further, OP9 pre-conditioned UCB transplantation in mice models of cerebral infarction facilitated angiogenesis in the peri-infarct lesions and ameliorated the associated symptoms. In this study, we developed a strong, fast, and feasible method to augment the M2, tissue-protecting, pro-angiogenic features of UCB using OP9. The ameliorative effect of OP9-pre-conditioned UCB in vivo could be partly due to promotion of innate angiogenesis in peri-infarct lesions.

Identification of Novel Multipotent Stem Cells in Mouse Spinal Cord Following Traumatic Injury.

We showed that injury-induced multipotent stem cells (iSCs) emerge in the brain after stroke. These brain-derived iSCs (B-iSCs) can differentiate into various lineages, including neurons. This study aimed to determine whether similar stem cells can be induced even after nonischemic injuries, such as trauma to the spinal cord. We characterized these cells, mainly focusing on their stemness, multipotency, and neuronal differentiation activities. Spinal cord injury (SCI) was produced using forceps in adult mice. On day 3 after SCI, samples were obtained from the injured areas. Spinal cord sections were subjected to histological analyses. Cells were isolated and assessed for proliferative activities, immunohistochemistry, reverse transcriptase-polymerase chain reaction, fluorescence-activated cell sorter, and microarray analysis. Although nerve cell morphology was disrupted within the injured spinal cord, our histological observations revealed the presence of cells expressing stem cells, such as nestin and Sox2 in these areas. In addition, cells extracted from injured areas exhibited high proliferative abilities. These cells also expressed markers of both neural stem cells (eg, nestin, Sox2) and multipotent stem cells (eg, Sox2, c-myc, Klf4). They differentiated into adipocytes, osteocytes, and chondrocytes, as well as neuronal cells. Microarray analysis further identified similar properties between spinal cord (SC)-derived iSCs and B-iSCs. However, SC-iSCs revealed specific genes related to the regulation of stemness and neurogenesis. We identified similar features related to multipotency in SC-iSCs compared with B-iSCs, including neuronal differentiation potential. Although the differences between SC-iSCs and B-iSCs remain largely undetermined, this study shows that iSCs can develop even after nonischemic injuries such as trauma. This phenomenon can occur outside the brain within the central nervous system.

Intravenous Administration of Human Amniotic Mesenchymal Stem Cells in the Subacute Phase of Cerebral Infarction in a Mouse Model Ameliorates Neurological Disturbance by Suppressing Blood Brain Barrier Disruption and Apoptosis via Immunomodulation.

Neuro-inflammation plays a key role in the pathophysiology of brain infarction. Cell therapy offers a novel therapeutic option due to its effect on immunomodulatory effects. Amniotic stem cells, in particular, show promise owing to their low immunogenicity, tumorigenicity, and easy availability from amniotic membranes discarded following birth. We have successfully isolated and expanded human amniotic mesenchymal stem cells (hAMSCs). Herein, we evaluated the therapeutic effect of hAMSCs on neurological deficits after brain infarction as well as their immunomodulatory effects in a mouse model in order to understand their mechanisms of action. One day after permanent occlusion of the middle cerebral artery (MCAO), hAMSCs were intravenously administered. RT-qPCR for TNFα, iNOS, MMP2, and MMP9, immunofluorescence staining for iNOS and CD11b/c, and a TUNEL assay were performed 8 days following MCAO. An Evans Blue assay and behavioral tests were performed 2 days and several months following MCAO, respectively. The results suggest that the neurological deficits caused by cerebral infarction are improved in dose-dependent manner by the administration of hAMSCs. The mechanism appears to be through a reduction in disruption of the blood brain barrier and apoptosis in the peri-infarct region through the suppression of pro-inflammatory cytokines and the M2-to-M1 phenotype shift.

Usefulness of a New Electronic Conductivity Device with a Pedicle Probe and a Multi-axis Angiography Unit for Inserting a C1 Lateral Mass Screw Safely and Tightly: A Technical Note.

The C1 lateral mass screw (LMS) is widely used as one of the screws for atlantoaxial fixation. Tight bicortical screwing from the posterior to anterior cortical margin of the atlas is recommended. However, important structures, such as the internal carotid artery, are located around this area so precision is required to avoid injuring them. We describe the usefulness of a new electronic conductivity device (ECD) with a pedicle probe and a multi-axis angiography unit for inserting the C1 LMS. Four consecutive patients who were treated with C1 and C2 posterior fixation using an ECD and a multi-axis angiography unit in the hybrid operating room were included. All patients were treated successfully. Seven of eight bicortical screws could be inserted into the perfectly ideal location. The median (interquartile range) distance from the anterior margin of the atlas to the tip of the screw was 0.81 mm (0.43, 1.21 mm). This study suggested that the ECD and multi-axis angiography unit are useful for inserting the C1 LMS safely and tightly.

Basic Gene Expression Characteristics of Glioma Stem Cells and Human Glioblastoma.

BACKGROUND: Glioma stem cells (GSCs) play important roles in the tumorigenesis of glioblastoma multiforme (GBM). Using a novel cellular bioinformatics pipeline, we aimed to characterize the differences in gene-expression profiles among GSCs, U251 (glioma cell line), and a human GBM tissue sample. MATERIALS AND METHODS: Total RNA was extracted from GSCs, U251 and GBM and microarray analysis was performed; the data were then applied to the bioinformatics pipeline consisting of a principal component analysis (PCA) with factor loadings, an intracellular pathway analysis, and an immunopathway analysis. RESULTS: The PCA clearly distinguished the three groups. The factor loadings of the PCA suggested that v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), dipeptidyl-peptidase 4 (DPP4), and macrophage migration-inhibitory factor (MIF) contribute to the stemness of GSCs. The intracellular pathway and immunopathway analyses provided relevant information about the functions of representative genes in GSCs. CONCLUSION: The newly-developed cellular bioinformatics pipeline was a useful method to clarify the similarities and differences among samples.

Grading of astrocytomas using the PRESTO (principles of echo-shifting with a train of observations) magnetic resonance imaging sequence.

OBJECTIVE: Changes in brain tissue can be detected sensitively using PRESTO (principles of echo-shifting with a train of observations) magnetic resonance imaging (MRI). The aim of this study was to evaluate the correlation between the proliferative ability of astrocytoma and intratumoral spotty signal voids seen as hypo-intense dots on PRESTO MRI. PATIENTS AND METHODS: Fifty-seven astrocytic tumors, comprising 14 astrocytomas, 12 anaplastic astrocytomas, and 31 glioblastomas, were included in this retrospective study. The tumors were classified independently by blinded radiologists according to the number of spotty signal voids detected on PRESTO-MRI as follows: spot-free (grade 0), less than 3 spots (grade 1), or more than 3 spots or a large spot (grade 2). RESULTS: Thirteen patients (92.9%) with astrocytoma were classified as PRESTO grade 0 and 1 patient (7.1%) was classified as grade 1. Seven patients (58.3%) with anaplastic astrocytoma were classified as PRESTO grade 0, 1 (8.3%) as grade 1, and 4 as grade 2 (33.3%). Three patients (9.7%) with glioblastoma were classified as grade 0, 6 (19.4%) as grade 1, and 22 (70.9%) as grade 2. There was a strong correlation between PRESTO tumor grade and the mean MIB-1 index. CONCLUSIONS: These results indicate that a grading system based on the number of spotty signal voids detected on PRESTO images would be useful for the diagnosis of astrocytic tumors and predicting their proliferative ability.

[Two Cases of Ruptured Blood Blister-like Aneurysm Treated with X-ray Angiography Perfusion(XAP)Analysis].

Two cases of ruptured blood blister-like internal carotid artery aneurysms for which low flow bypass was sufficient to attain successful treatment of trapping are reported. In the acute stage of rupture, it is troublesome to perform accurate examinations of tolerance to ischemia like balloon occlusion test(BOT)for estimating the required amount of bypass flow. In our cases, X-ray angiography perfusion(XAP)analysis was introduced, which could be performed in a couple dozen seconds without room-to-room transfer of patients, following the ordinary examination of diagnostic digital subtraction angiography. The perfusion index(PI)ratio measured in this analysis is equivalent to the laterality of cerebral blood flow between the right and left hemispheres. The PI ratio of 0.85 approximately corresponds to the mean stump pressure(MSTP)of 40mmHg, on the basis of the correlation diagram between the PI ratio and MSTP(approximate straight line:PI ratio%=0.6×MSTP+60). Even though the PI ratio of the cases was superior to this threshold of tolerance for parent artery occlusion, complementary low flow bypass was added in the acute case for the overwhelming succeeding vasospasm and for securing the flow to peripheral perforators, which resulted in a successful treatment without any ischemic events.

Protodioscin, Isolated from the Rhizome of Dioscorea tokoro Collected in Northern Japan is the Major Antiproliferative Compound to HL-60 Leukemic Cells.

BACKGROUND: The rhizome of Oni-dokoro (a wild yam, Dioscorea tokoro) has extremely bitter taste and is not generally regarded edible;, however, in northern part of Japan, such as Iwate and a part of Aomori, it is used as health promoting food. To clarify the reason, we examined the biologically active compounds in the rhizome collected at Iwate and compared them from the other area in literature. METHODS: The acetonitrile extract from northern part of Japan was purified by bioassay-guided separation using antiproliferative activity to human leukemia HL-60 cell, and protodioscin (PD) was isolated and identified by instrumental analyses as the major active compound. RESULTS: PD known as a saponin with four sugar moieties, an inhibitor for platelet aggregation, and a low density lipoprotein (LPL) lowering agent, displayed strong growth inhibitory effect to HL-60. The literature search suggested that the rhizome from other area contained dioscin and other saponins with three sugar moieties as their major component. We assume that the edible and health promoting effect of the rhizome in the particular area is partially derived from these different components. CONCLUSION: We were interested in the differences of utilization in the rhizome of wild yam Dioscorea tokoro, and examined the chemical composition in the rhizome to find protodioscin as antiproliferative compound to HL-60. In the report from other area, the rhizome exhibited dioscin as the major compound. Our study indicated that the protodioscin/dioscin composition varied regionally, although the reason is still needs to be investigated.

[Twenty-year History and Future Challenges in Transparency Enhancement of Review Process for Approval: Focus on Public Release of Review Reports regarding New Drugs and Medical Devices].

For 20 years, the Ministry of Health, Labour and Welfare (MHLW, formerly Ministry of Health and Welfare (MHW)) has been trying to increase transparency of the review process for approving reports in order to promote the rational use of newly approved drugs and medical devices. The first Summary Basis of Approval (SBA) was published by MHW in 1994. In 1999, evaluation reports were prepared by MHW and the Pharmaceuticals and Medical Devices Evaluation Center to make them available to the public. In 2005, a notice from the Chief Executive of the Pharmaceuticals and Medical Devices Agency (PMDA) made procedures for public release of information on reviewing applications for new drugs. In 2006, 90 review reports of newly approved drugs and eight medical devices were revealed on PMDA websites. The dissemination of information by the United States Food and Drug Administration (FDA) and that of the European Medicines Agency (EMA) were studied and compared with that of the MHLW and PMDA. While common technical documents (CTD) for new drugs and summary technical documents (STED) for new medical devices have been released by PMDA, such documents are not released by the FDA and EMA. The European Public Assessment Report (EAPR) summary for the public is an interesting questionnaire approach that uses the "What," "How" and "Why" format. Finally, future proposals for the next decade are also outlined.

Transport mechanisms of trans-1-amino-3-fluoro[1-(14)C]cyclobutanecarboxylic acid in prostate cancer cells.

INTRODUCTION: We investigated the mechanisms of trans-1-amino-3-fluoro[1-(14)C]cyclobutanecarboxylic acid (anti-[(14)C]FACBC) transport by human-derived prostate cancer (PCa) cells and normal human prostatic epithelial cells (PrECs). METHODS: Using PCa cells (DU145, PC-3, LNCaP) and PrECs, we performed the following in vitro experiments: time-course, kinetics, competitive inhibition by synthetic/naturally occurring amino acids (AAs), exchange transport with synthetic/naturally occurring AAs and pH-dependency of anti-[(14)C]FACBC uptake. We also examined the amino acid transporter (AAT) expression using flow cytometry. RESULTS: The uptake of anti-[(14)C]FACBC by LNCaP and DU145 cells was higher than that by PC-3 and PrECs. The K(m) values for anti-[(14)C]FACBC were 64.4 and 191.7 µmol/L in the DU145 cells and PrECs, respectively. Total levels of anti-[(14)C]FACBC uptake were positively correlated with the expression level of system ASC in PCa cells. The contributions of Na(+)-dependent AATs to anti-[(14)C]FACBC uptake were greater than those of Na(+)-independent AATs, especially in PCa cells. In the presence of Na(+), glutamine and serine showed the strongest inhibitory effect against anti-[(14)C]FACBC uptake, suggesting that system ASC, especially ASCT2, is an important AAT for anti-[(14)C]FACBC. In contrast, phenylalanine and 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid, but not N-ethylmaleimide, almost completely inhibited the anti-[(14)C]FACBC uptake in the absence of Na(+), indicating the contribution of LAT1. In the exchange transport experiments, glutamine showed the strongest transstimulation of intracellular anti-[(14)C]FACBC efflux in DU145 cells. Furthermore, the contributions of Na(+)-independent AATs to the uptake of anti-[(14)C]FACBC in DU145 and PrECs were greater under acidic pH conditions than under neutral or alkaline pH conditions. CONCLUSIONS: Total uptake of anti-[(14)C]FACBC by PCa cells correlates with the expression level of system ASC in PCa cells. Furthermore, LAT1 is an important transport system for anti-[(14)C]FACBC uptake, especially in an acidic environment, such as the intra-tumoural environment.

A preliminary study of anti-1-amino-3-18F-fluorocyclobutyl-1-carboxylic acid for the detection of prostate cancer.

UNLABELLED: We evaluated the feasibility of anti-1-amino-3-(18)F-fluorocyclobutyl-1-carboxylic acid (anti-(18)F-FACBC) in diagnosing prostate cancer (PCa), using a rat orthotopic prostate cancer transplantation (OPCT) model. Furthermore, using in vivo experiments, we examined the potential of anti-(18)F-FACBC for differentiating between PCa and inflammation and between PCa and benign prostatic hyperplasia (BPH). METHODS: The OPCT model was developed by transplanting DU145, a human PCa cell line, into the ventral prostate of athymic F344 rats. To develop a dual PCa and inflammation (DPCI) model, MAT-Ly-Lu-B2--a rat PCa cell line--was transplanted subcutaneously into male Copenhagen rats. Streptozotocin was injected into the hind footpad of these rats for inducing popliteal lymphadenitis. For inducing the BPH, normal F344 rats were castrated and injected subcutaneously with testosterone propionate. In biodistribution studies, the rats were injected with anti-(18)F-FACBC or (18)F-FDG and sacrificed at 15 or 60 min after injection. We performed dynamic small-animal PET of the abdominal portion of the OPCT rats for 60 min after the injection of anti-(18)F-FACBC or (18)F-FDG. RESULTS: The biodistribution in the OPCT rats at 60 min after injection showed that the uptake of anti-(18)F-FACBC and (18)F-FDG into the PCa tissue was 1.58 +/- 0.40 %ID/cm(3) (percentage injected dose per cm(3)) and 1.48 +/- 0.90 %ID/cm(3), respectively (P > 0.05). The accumulation of anti-(18)F-FACBC in the urinary bladder at 60 min after injection was 3.09 +/- 1.43 %ID/cm(3), whereas that of (18)F-FDG was 69.31 +/- 16.55 %ID/cm(3) (P < 0.05). Consequently, small-animal imaging with anti-(18)F-FACBC facilitated the visualization of the PCa tissue of the OPCT rats with higher contrast than (18)F-FDG. Furthermore, in comparison with (18)F-FDG, apparently higher ratios of PCa to inflammation and PCa to BPH accumulation of anti-(18)F-FACBC were demonstrated in the animal models. CONCLUSION: FACBC PET is believed to be useful not only for the visualization of human PCa but also for differentiating between PCa and inflammation and between PCa and BHP.

Living copolymerization of ethylene with norbornene catalyzed by bis(pyrrolide-imine) titanium complexes with MAO.

Bis(pyrrolide-imine) Ti complexes in conjunction with methylalumoxane (MAO) were found to work as efficient catalysts for the copolymerization of ethylene and norbornene to afford unique copolymers via an addition-type polymerization mechanism. The catalysts exhibited very high norbornene incorporation, superior to that obtained with Me(2)Si(Me(4)Cp)(N-tert-Bu)TiCl(2) (CGC). The sterically open and highly electrophilic nature of the catalysts is probably responsible for the excellent norbornene incorporation. The catalysts displayed a marked tendency to produce alternating copolymers, which have stereoirregular structures despite the C(2) symmetric nature of the catalysts. The norbornene/ethylene molar ratio in the polymerization medium had a profound influence on the molecular weight distribution of the resulting copolymer. At norbornene/ethylene ratios larger than ca. 1, the catalysts mediated room-temperature living copolymerization of ethylene and norbornene to form high molecular weight monodisperse copolymers (M(n) > 500,000, M(w)/M(n) < 1.20). (13)C NMR spectroscopic analysis of a copolymer, produced under conditions that gave low molecular weight, demonstrated that the copolymerization is initiated by norbornene insertion and that the catalyst mostly exists as a norbornene-last-inserted species under living conditions. Polymerization behavior coupled with DFT calculations suggested that the highly controlled living polymerization stems from the fact that the catalysts possess high affinity and high incorporation ability for norbornene as well as the characteristics of a living ethylene polymerization though under limited conditions (M(n) 225,000, M(w)/M(n) 1.15, 10-s polymerization, 25 degrees C). With the catalyst, unique block copolymers [i.e., poly(ethylene-co-norbornene)(1)-b-poly(ethylene-co-norbornene)(2), PE-b-poly(ethylene-co-norbornene)] were successfully synthesized from ethylene and norbornene. Transmission electron microscopy (TEM) indicated that the PE-b-poly(ethylene-co-norbornene) possesses high potential as a new material consisting of crystalline and amorphous segments which are chemically linked.

Living ethylene/norbornene copolymerisation catalyzed by titanium complexes having two pyrrolide-imine chelate ligands.

Ethylene/norbornene copolymerisation behaviour of titanium complexes with two pyrrolide-imine chelate ligands is described.

Living polymerization of ethylene catalyzed by titanium complexes having fluorine-containing phenoxy-imine chelate ligands.

Seven titanium complexes bearing fluorine-containing phenoxy-imine chelate ligands, TiCl(2)[eta(2)-1-[C(H)=NR]-2-O-3-(t)Bu-C(6)H(3)](2) [R = 2,3,4,5,6-pentafluorophenyl (1), R = 2,4,6-trifluorophenyl (2), R = 2,6-difluorophenyl (3), R = 2-fluorophenyl (4), R = 3,4,5-trifluorophenyl (5), R = 3,5-difluorophenyl (6), R = 4-fluorophenyl (7)], were synthesized from the lithium salt of the requisite ligand and TiCl(4) in good yields (22%-76%). X-ray analysis revealed that the complexes 1 and 3 adopt a distorted octahedral structure in which the two phenoxy oxygens are situated in the trans-position while the two imine nitrogens and the two chlorine atoms are located cis to one another, the same spatial disposition as that for the corresponding nonfluorinated complex. Although the Ti-O, Ti-N, and Ti-Cl bond distances for complexes 1 and 3 are very similar to those for the nonfluorinated complex, the bond angles between the ligands (e.g., O-Ti-O, N-Ti-N, and Cl-Ti-Cl) and the Ti-N-C-C torsion angles involving the phenyl on the imine nitrogen are different from those for the nonfluorinated complex, as a result of the introduction of fluorine atoms. Complex 1/methylalumoxane (MAO) catalyst system promoted living ethylene polymerization to produce high molecular weight polyethylenes (M(n) > 400 000) with extremely narrow polydispersities (M(w)/M(n) < 1.20). Very high activities (TOF > 20 000 min(-1) atm(-1)) were observed that are comparable to those of Cp(2)ZrCl(2)/MAO at high polymerization temperatures (25, 50 degrees C). Complexes 2-4, which have a fluorine atom adjacent to the imine nitrogen, behaved as living ethylene polymerization catalysts at 50 degrees C, whereas complexes 5-7, possessing no fluorine adjacent to the imine nitrogen, produced polyethylenes having M(w)/M(n) values of ca. 2 with beta-hydrogen transfer as the main termination pathway. These results together with DFT calculations suggested that the presence of a fluorine atom adjacent to the imine nitrogen is a requirement for the high-temperature living polymerization, and the fluorine of the active species for ethylene polymerization interacts with a beta-hydrogen of a polymer chain, resulting in the prevention of beta-hydrogen transfer. This catalyst system was used for the synthesis of a number of unique block copolymers such as polyethylene-b-poly(ethylene-co-propylene) diblock copolymer and polyethylene-b-poly(ethylene-co-propylene)-b-syndiotactic polypropylene triblock copolymer from ethylene and propylene.