bluuebunny/crossref_metadata_2025 huggingface dataset:
Deep&Dense Convolutional Neural Network for Hyperspectral Image Classification
Authors: Paoletti Mercedes E. , Haut Juan M. , Plaza Javier , Plaza Antonio
Published: September 2018

Deep neural networks (DNNs) have emerged as a relevant tool for the classification of remotely sensed hyperspectral images (HSIs), with convolutional neural networks (CNNs) being the current state-of-the-art in many classification tasks. However, deep CNNs present several limitations in the context of HSI supervised classification. Although deep models are able to extract better and more abstract features, the number of parameters that must be fine-tuned requires a large amount of training data (using small learning rates) in order to avoid the overfitting and vanishing gradient problems. The acquisition of labeled data is expensive and time-consuming, and small learning rates forces the gradient descent to use many small steps to converge, slowing down the runtime of the model. To mitigate these issues, this paper introduces a new deep CNN framework for spectral-spatial classification of HSIs. Our newly proposed framework introduces shortcut connections between layers, in which the feature maps of inferior layers are used as inputs of the current layer, feeding its own output to the rest of the the upper layers. This leads to the combination of various spectral-spatial features across layers that allows us to enhance the generalization ability of the network with HSIs. Our experimental results with four well-known HSI datasets reveal that the proposed deep&dense CNN model is able to provide competitive advantages in terms of classification accuracy when compared to other state-of-the-methods for HSI classification.
Fecal Volatile Organic Compounds in Preterm Infants Are Influenced by Enteral Feeding Composition
Authors: El Manouni el Hassani Sofia , Niemarkt Hendrik J. , Said Hager , Berkhout Daniel J. C. , Van Kaam Anton H. , Van Lingen Richard A. , Benninga Marc A. , De Boer Nanne K. H. , De Meij Tim G. J.
Published: September 2018

Fecal volatile organic compound (VOC) analysis has shown great potential as a noninvasive diagnostic biomarker for a variety of diseases. Before clinical implementation, the factors influencing the outcome of VOC analysis need to be assessed. Recent studies found that the sampling conditions can influence the outcome of VOC analysis. However, the dietary influences remains unknown, especially in (preterm) infants. Therefore, we assessed the effects of feeding composition on fecal VOC patterns of preterm infants (born at <30 weeks gestation). Two subgroups were defined: (1) daily intake >75% breastmilk (BM) feeding and (2) daily intake >75% formula milk (FM) feeding. Fecal samples, which were collected at 7, 14 and 21 days postnatally, were analyzed by an electronic nose device (Cyranose 320®). In total, 30 preterm infants were included (15 FM, 15 BM). No differences in the fecal VOC patterns were observed at the three predefined time-points. Combining the fecal VOC profiles of these time-points resulted in a statistically significant difference between the two subgroups although this discriminative accuracy was only modest (AUC [95% CI]; p-value; sensitivity; and specificity of 0.64 [0.51–0.77]; 0.04; 68%; and 51%, respectively). Our results suggest that the influence of enteral feeding on the outcome of fecal VOC analysis cannot be ignored in this population. Furthermore, in both subgroups, the fecal VOC patterns showed a stable longitudinal course within the first month of life.
Avoiding Void Holes and Collisions with Reliable and Interference-Aware Routing in Underwater WSNs
Authors: Javaid Nadeem , Majid Abdul , Sher Arshad , Khan Wazir , Aalsalem Mohammed
Published: September 2018

Sparse node deployment and dynamic network topology in underwater wireless sensor networks (UWSNs) result in void hole problem. In this paper, we present two interference-aware routing protocols for UWSNs (Intar: interference-aware routing; and Re-Intar: reliable and interference-aware routing). In proposed protocols, we use sender based approach to avoid the void hole. The beauty of the proposed schemes is that they not only avoid void hole but also reduce the probability of collision. The proposed Re-Intar also uses one-hop backward transmission at the source node to further improve the packet delivery ratio of the network. Simulation results verify the effectiveness of the proposed schemes in terms of end-to-end delay, packet delivery ratio and energy consumption.
Statistic Experience Based Adaptive One-Shot Detector (EAO) for Camera Sensing System
Authors: Zhu Xiaoning , Ding Bojian , Meng Qingyue , Gu Lize , Yang Yixian
Published: September 2018

Object detection in a camera sensing system has been addressed by researchers in the field of image processing. Highly-developed techniques provide researchers with great opportunities to recognize objects by applying different algorithms. This paper proposes an object recognition model, named Statistic Experience-based Adaptive One-shot Detector (EAO), based on convolutional neural network. The proposed model makes use of spectral clustering to make detection dataset, generates prior boxes for object bounding and assigns prior boxes based on multi-resolution. The model is constructed and trained for improving the detection precision and the processing speed. Experiments are conducted on classical images datasets while the results demonstrate the superiority of EAO in terms of effectiveness and efficiency. Working performance of the EAO is verified by comparing it to several state-of-the-art approaches, which makes it a promising method for the development of the camera sensing technique.
Perceptions of Pedestrian and Cyclist Environments, Travel Behaviors, and Social Networks
Authors: Arroyo Rosa , Mars Lidón , Ruiz Tomás
Published: September 2018

The research presented in this paper studied interrelationships within the perceptions of pedestrian and cyclist environments, travel behaviors, and a particular subset of social networks characterized by their being usual trip or activity companions. For this purpose, 404 individuals participated in a web-based survey and provided data related to perceptions of particular elements of pedestrian- and cyclist-specific environments (sidewalks, cycle lanes, and pedestrian crossing), sociodemographics, and characteristics of their usual activity and trip companions. Participants also completed a two-day activity-travel diary. The validity of measurements and reliability of constructs were assessed by mean of Exploratory and Confirmatory Factor Analysis (EFA and CFA) and five models were determined using Structural Equation Modeling (SEM) to identify relations among the variables of the study. Results highlight the importance of how travel behavior influences the formation of perceptions of elements related to pedestrians and cyclist-specific environments.
Topological Gravity Motivated by Renormalization Group
Authors: Mori Taisaku , Nojiri Shin’ichi
Published: September 2018

Recently, we have proposed models of topological field theory including gravity in Mod. Phys. Lett. A 2016, 31, 1650213 and Phys. Rev. D 2017, 96, 024009, in order to solve the problem of the cosmological constant. The Lagrangian densities of the models are BRS (Becchi-Rouet-Stora) exact and therefore the models can be regarded as topological theories. In the models, the coupling constants, including the cosmological constant, look as if they run with the scale of the universe and its behavior is very similar to the renormalization group. Motivated by these models, we propose new models with an the infrared fixed point, which may correspond to the late time universe, and an ultraviolet fixed point, which may correspond to the early universe. In particular, we construct a model with the solutions corresponding to the de Sitter space-time both in the ultraviolet and the infrared fixed points.
Repeated Intrastriatal Botulinum Neurotoxin-A Injection in Hemiparkinsonian Rats Increased the Beneficial Effect on Rotational Behavior
Authors: Hawlitschka Alexander , Holzmann Carsten , Wree Andreas , Antipova Veronica
Published: September 2018

Injection of botulinum neurotoxin-A (BoNT-A) into the striatum of hemiparkinsonian (hemi-PD) rats reduced apomorphine-induced rotation behavior significantly, for at least 3 months. Thereafter, rotation behavior increased again. We injected hemi-PD rats with 1 ng BoNT-A twice, the second injection following 6 months after the first one and tested the rats for apomorphine-induced rotations and spontaneous motor behaviors, i.e., corridor task and stepping test. To test the hypothesis that BoNT-A reduced striatal hypercholinism in hemi-PD rats, the acetylcholinesterase inhibitor donepezil was injected prior to separate apomorphine-induced rotation tests. In hemi-PD rats, the first BoNT-A injection led to a clear reduction of the apomorphine-induced rotations, and the second BoNT-A injection to a more massive and prolonged reaction. In hemi-PD rats whose apomorphine-induced rotation behavior was strongly reduced by an intrastriatal BoNT-A, subsequent donepezil injections led to significant increases of the rotation rate. Concerning corridor task and stepping test, neither first nor second BoNT-A injections changed hemi-PD rats’ behavior significantly. The data give evidence for the possibility of repeated intrastriatal administrations of BoNT-A, for treatment of motor symptoms in experimental hemi-PD over a longer time.
Seroprevalence of Immunoglobulin E Antibodies against Japanese Cedar Pollen Allergens Cry j 1 and Cry j 2 in Dogs Bred in Japan
Authors: Kuribayashi Takashi , Cossu Davide , Momotani Eiichi
Published: September 2018

Levels of Japanese cedar pollen (Cryptomeria japonica) have increased in Japan and cedar pollinosis caused by Japanese cedar pollen has been reported in dogs. Serum levels of immunoglobulin E (IgE) against Cry j 1 and Cry j 2 in dogs raised in institutes and treated at veterinary hospitals in Japan were thus investigated. A total of 71 sera obtained from two institutes and 87 sera obtained from veterinary hospitals in the Hyogo and Kanagawa Prefectures were analyzed in this study. Serum levels of IgE were measured using the enzyme-linked immunosorbent assay with commercial purified Cry j 1 and Cry j 2. IgE against Cry j 1 and Cry j 2 in sera obtained from the two institutes were detected, despite the dogs being bred in enclosed areas. Moreover, significant differences were noted in the serum levels of IgE against Cry j 1 and Cry j 2 between the two institutes. The number of samples showing Cry j 1 or Cry j 2 levels above the cut-off values was greater in the Kanagawa Prefecture than in the Hyogo Prefecture. In total, 14 dogs showed Cry j 1 and Cry j 2 levels greater than the cut-off values in the Hyogo Prefecture, and only three such dogs were seen in the Kanagawa Prefecture. A significant correlation between serum levels against both allergens was observed (r2 = 0.6931, p < 0.0001).
Survival of Macroencapsulated Allogeneic Parathyroid Tissue One Year after Transplantation in Nonimmunosuppressed Humans
Authors: Tibell Annika , Rafael Ehab , Wennberg Lars , Nordenström Jörgen , Bergström Mats , Geller Robin L. , Loudovaris Thomas , Johnson Robert C. , Brauker James H. , Neuenfeldt Steven , Wernerson Annika
Published: September 2018

The use of immunoisolation devices may allow transplantation without need for immunosuppression and could widen the indications for cell transplantation. In this study, we evaluated the survival of encapsulated parathyroid tissue in nonimmunosuppressed humans. Autologous parathyroid implants: Seven patients under-going parathyroidectomy had devices containing small pieces of their own parathyroid tissue implanted SC. These devices were explanted after 2–4 weeks for histological evaluation. Allogeneic parathyroid implants: Four patients with chronic hypoparathyroidism were transplanted with one to three large (40 μl) and one small (4.5 μl) device filled with meshed parathyroid tissue and implanted SC. The small devices were explanted at 4 weeks, while the large ones were explanted 8.5 to 14 months after implantation. In both studies, control implants were placed in nude mice. Autologous study results: At explantation, the grafts consisted of 22 ± 6% endocrine tissue and 63 ± 7% fibrosis, while 15 ± 5% of the grafts were necrotic. Allogeneic study results: In devices explanted from the patients at 4 weeks, fibrosis dominated and only 1%, 5%, and 23% of the grafts consisted of endocrine tissue. A similar histological appearance was found in grafts from nude mice. In devices explanted at 8.5–14 months, histologically intact endocrine tissue was found in all patients. However, nearly all the tissue consisted of fibrosis. There was no detectable increase in the parathormone (PTH) level in all patients. Macroencapsulated human allogeneic parathyroid tissue can survive up to 1 year after transplantation into nonimmunosuppressed patients. However, marked fibroblast overgrowth occurred, especially in the allogeneic implant study, using meshed parathyroid tissue. This was probably not related to the allo-response, because similar findings were observed in the nude mouse implants. In future studies, better tissue preparation and improvements in the physiological milieu inside the device may help to reduce fibroblast overgrowth and increase survival of the parathyroid cells.
In Vitro Organization of Biohybrid Rat Liver Tissue Incorporating Growth Factor- and Hormone-Releasing Biodegradable Polymer Microcapsules
Authors: Sakai Yasuyuki , Furukawa Katsuko , Ushida Takashi , Tateishi Tetsuya , Suzuki Motoyuki
Published: September 2018

To enhance the liver-specific functions of rat hepatocyte aggregates without the addition of exogenous growth factors, polylactic acid-polyglycolic acid (PLGA)/gelatin microcapsules that release insulin, dexamethasone, epidermal growth factors, and glucagon were prepared and incorporated into the hepatocyte aggregates in suspension culture. Precoating the capsules with fibronectin enhanced the incorporation of the microcapsules into the hepatocyte aggregates. In a growth factor- and hormone-free culture medium, these microcapsule-containing aggregates showed a sustained cell number and an ammonium detoxification capacity compared with two types of control culture. One was the culture of microcapsule-free aggregates with albumin-containing control capsules and the other was the culture of capsule-free aggregates that were supplied with the same factors and hormones from the culture medium at concentration levels expected from the release kinetics of the microcapsules. Our new methodology demonstrates that the performance and duration of bioartificial liver systems can be enhanced due to a more efficient maintenance of cell number by using such growth factor- and hormone-releasing microcapsules.
Formation of Human Fibroblast Aggregates (Spheroids) by Rotational Culture
Authors: Furukawa Katsuko S. , Ushida Takashi , Sakai Yasuyuki , Suzuki Motoyuki , Tanaka Junzo , Tateishi Tetsuya
Published: September 2018

In the current study, we attempted to form aggregates of fibroblasts by rotationally shaking, declining fibroblast–material interactions, and augmenting cell–cell interactions. In addition, to promote cell–cell interactions, the medium was supplemented with insulin, dexamethasone, and basic fibroblast growth. Under such improved culture conditions, normal neonatal human dermal fibroblasts formed spheroidal aggregates within 1 day of rotation on a rotational shaker. The aggregates that formed had irregular shapes and were composed from only several cells after 12 h. However, they became nearly spheroidal after 24 h of shaking. The aggregates were approximately 240 μm in diameter. After 36 h of shaking, their shape became more rounded and their surfaces became smoother. No evidence of necrosis in the center of the aggregates was observed, although a small number of dead cells was scattered throughout the aggregates. After 24–36 h, aggregates of normal human fibroblasts were collected and reinoculated onto a scaffold composed of polyglycolic acid, which is used commercially as a scaffold for artificial skin, coated with collagen. The aggregates were successfully trapped to the mesh of polyglycolic acid and became attached within 24 h. Therefore, the aggregates could provide an alternative method for seeding fibroblasts to scaffold for an artificial skin, such as a mesh of polyglycolic acid.
Functional Comparison of the Single-Layer Agarose Microbeads and the Developed Three-Layer Agarose Microbeads as the Bioartificial Pancreas: An In Vitro Study
Authors: Xu Baoyou , Iwata Hiroo , Miyamoto Masaaki , Balamurugan A. N. , Murakami Yoshinobu , Cui Wanxing , Imamura Masayuki , Inoue Kazutomo
Published: September 2018

In this study, the insulin secretory characteristics of the microencapsulated hamster islets were studied during long-term culture. The hamster islets were encapsulated as single-layer agarose microbeads or three-layer agarose microbeads with agarose and agarose containing poly(styrene sulfonic acid) (PSSa), respectively. The influence of PSSa on the function of the rat islets microencapsulted in three-layer microbeads was primarily monitored. The aim of this study was to examine the influence of the PSSa on the in vitro function of the islets encapsulated in the agarose/PSSa microbeads compared with single-layer agarose microbeads during long-term culture. The microbeads were cultured for 30 days in medium of Eagle's MEM at 37°C in 5% CO2and 95% air. The basal insulin secretion into the culture medium was measured daily during the first 12 days and two times per week until 30 days. The microbeads were subjected to static incubation test on the 10th, 20th, and 30th day during culture. The basal insulin secretion level of the agarose/PSSa microbeads was significantly higher than that of single-layer agarose microbeads. The static incubation tests revealed a similar pattern of insulin secretion from both microbeads when they were exposed to high glucose challenge. In the static incubation test, both could significantly increase insulin release to more than 6.61 times (stimulation index) in response to high glucose stimulation and could significantly decrease when glucose concentration returned from high glucose to low glucose on the 10th, 20th, and 30th day of culture. This study demonstrated that the hamster islets enclosed in agarose/PSSa hydrogel not only continuously secreted basal amounts of insulin, but also maintained their response to high glucose stimulation similar to the agarose microbeads. The above results together with those of our previous in vivo study suggest that the three-layer microbeads (agarose/PSSa) are well suitable for xenotransplantation of islets for the clinical application.
Acute Lethal Injury of Lung and Liver in Mice Transplanted with Ex Vivo-Expanded CTLs
Authors: Sato Tadayuki , Ando Kiyoshi , Oki Masayuki , Miyatake Hiroko , Matsuzawa Hideyuki , Hotta Tomomitsu , Kato Shunichi
Published: September 2018

Clinical application of cytotoxic T lymphocytes (CTL) induced in vitro is extensively used for the treatment of viral infection and malignant diseases. We produced anti H-2d CTL in vitro from C57BL/6 (B6) splenocytes presensitized with (B6 × DBA/2) F1 (BDF1) splenocytes to establish a model system of CTL therapy. The specificity and cytotoxic activity were high enough (E/T ratio 1:1 = 38.8%) to induce graft versus host reaction. Though the total number of B6 splenocytes decreased by 0.27 during the 4 days of culture, the number of CD8+ lymphocytes increased 1.3-fold. When more than 5 × 106 cells of H-2d -reactive CTL were transplanted into BDF1 mice, mice died within 2 days postinduction. This lethal effect was not seen in the mice induced with ConA-stimulated T cells. Histological examination of the lungs and liver revealed massive infiltration of neutrophils in alveoli and the necrosis of hepatocytes. Therefore, this protocol was shown to be effective to produce alloantigen-specific CTLs and applicable to in vitro manipulation such as retrovirus-mediated gene transfer.
Neurotrophic Factor-Secreting Cell Grafting for Cerebral Ischemia: Preliminary Report
Authors: Fujiwara K. , Date I. , Shingo T. , Yoshida H. , Kobayashi K. , Takeuchi A. , Tamiya T. , Ohmoto T.
Published: September 2018

In this experiment, we examined a possible protective effect of encapsulated neurotrophic factor-secreting cell grafting for ischemic injury. We established a basic fibroblast growth factor (bFGF)-secreting cell line by genetic manipulation. We enveloped these cells into polymer capsules, which consist of a semipermeable membrane, and implanted them into the right striatum of rats. At 6 days after implantation, these rats received right middle cerebral artery occlusion (MCAO) using interluminal suture technique. At 24 h after MCAO, rats were sacrificed and their cerebral infarction volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining and image analysis. We found approximately 30% reduction in infarct volume in the encapsulated bFGF-secreting cell grafting groups vs. the encapsulated naive BHK cell grafting group or the without implantation group. We measured bFGF secretion from encapsulated bFGF-secreting cells using enzyme-linked immunosorbent assay (ELISA). The retrieved capsules continued to secrete bFGF. There was no significant difference of bFGF secretion between the capsules before and after transplantation. A large number of viable BHK-bFGF cells was observed within the full length of the retrieved capsule. These results indicate that encapsulated bFGF-secreting cell grafting exerts a protective effect on ischemic injury.
In Vitro and In Vivo Tetracycline-Controlled Myogenic Conversion of NIH-3T3 Cells: Evidence of Programmed Cell Death after Muscle Cell Transplantation
Authors: Del Bo Roberto , Torrente Yvan , Corti Stefania , D'angelo Maria Grazia , Comi Giacomo Pietro , Fagiolari Gigliola , Salani Sabrina , Cova Agata , Pisati Federica , Moggio Maurizio , Ausenda Carlo , Scarlato Guglielmo , Bresolin Nereo
Published: September 2018

Ex vivo gene therapy of Duchenne muscular dystrophy based on autologous transplantation of genetically modified myoblasts is limited by their premature senescence. MyoD-converted fibroblasts represent an alternative source of myogenic cells. In this study the forced MyoD-dependent conversion of murine NIH-3T3 fibroblasts into myoblasts under the control of an inducible promoter silent in the presence of tetracycline was evaluated. After tetracycline withdrawal this promoter drives the transcription of MyoD in the engineered fibroblasts, inducing their myogenesis and giving rise to β-galactosidase-positive cells. MyoD-expressing fibroblasts withdrew from the cell cycle, but were unable to fuse in vitro into multinucleated myotubes. Five days following implantation of engineered fibroblasts in muscles of C57BL/10J mice we observed a sevenfold increase of β-galactosidase-positive regenerating myofibers in animals not treated with antibiotic compared with treated animals. After 1 week the number of positive fibers decreased and several apoptotic myonuclei were detected. Three weeks following implantation of MyoD-converted fibroblasts in recipient mice, no positive “blue” fiber was observed. Our results suggest that transactivation by tetracycline of MyoD may drive an in vivo myogenic conversion of NIH-3T3 fibroblasts and that, in this experimental setting, apoptosis plays a relevant role in limiting the efficacy of engineered fibroblast transplantation. This work opens the question whether apoptotic phenomena also play a general role as limiting factors of cellmediated gene therapy of inherited muscle disorders.
Neural Xenotransplantation: Pretreatment of Porcine Embryonic Nigral Tissue with Anti-Gal Antibodies and Complement is not Toxic for the Dopaminergic Neurons
Authors: Brevig Thomas , Meyer Morten , Kristensen Tom , Zimmer Jens
Published: September 2018

The immunogenicity of porcine tissue is a major obstacle to its use as donor material in xenotransplantation for neurodegenerative diseases. We are currently evaluating a novel strategy for reducing the immunogenicity, in which the α-galactosyl epitope (Galα1,3Galß1,4GlcNAc-R) is used as a target for antibody- and complement-mediated removal of microglia. In the present study, our aim was to determine whether a pretreatment with antibodies against the α-galactosyl epitope (anti-Gal) and complement would lyse or other-wise damage dopaminergic neurons in porcine embryonic ventral mesencephalon (VM), the donor tissue for treatment of Parkinson's disease by xenotransplantation. Cell suspensions prepared from VM tissue from 27-day-old pig embryos were incubated with anti-Gal, purified from normal human serum by affinity chromatography, or medium only (control), and subsequently with rabbit complement. After these pretreatments, the cell suspensions were transplanted into the right striatum of 14 adult rats (two groups of 7 animals). The animals were sacrificed 20 days after transplantation, the brains were processed for histology, and the sections were stained for Nissl substance, porcine neurofilament, tyrosine hydroxylase, and rat CD45 to determine graft volume, presence of porcine neurons, content of dopaminergic cells, and leukocyte infiltration, respectively. The VM tissue pretreated with anti-Gal and complement gave rise to dopaminergic grafts that were indistinguishable from those derived from VM tissue given the control pretreatment. In 5 of the 14 animals, the grafts were infiltrated by host leukocytes, but in two of these recipients, the infiltration was only minimal. We conclude that anti-Gal and complement can be applied to porcine embryonic VM tissue without damaging the dopaminergic neurons and their precursors.
A Coculture Model of Intrahepatic Islet Transplantation: Activation of Kupffer Cells by Islets and Acinar Tissue
Authors: Clayton Heather A. , Davies Joanna E. , Sutton Chris D. , Bell Peter R. F. , Dennison Ashley R.
Published: September 2018

Clinical and experimental studies of intrahepatic islet transplantation have allowed histological and systemic observations to be made, but the location of the transplanted islets makes it difficult to assess direct effects on the cells of the liver. An in vitro coculture model of Kupffer cells with islets or pancreatic acinar tissue is described, using porcine tissue and measuring the secretion of thromboxane B2, prostaglandin E2, 6-keto-prostaglandin F1α, and prostaglandin F2α as an indicator of Kupffer cell stimulation. The results have demonstrated activation of Kupffer cells in the presence of acinar or islet tissue, both when the cells were in direct contact and when separated by a membrane. This indicated that the stimulation was due to a soluble factor or factors, and was confirmed by the culture of Kupffer cells with acinar conditioned medium. The degree of stimulation was much greater with acinar tissue than with islets. In subsequent experiments, aprotinin, an enzyme activation inhibitor, was added to the cocultures in an attempt to reduce Kupffer cell activation. This had no effect, possibly due to the fact that the endogenous pancreatic enzymes may already be activated during digestion of the pancreas. Aprotinin alone caused an increase in secretion of eicosanoids from Kupffer cells. The high response to acinar tissue is of particular relevance to islet autotransplantation in which unpurified pancreatic digest is often transplanted. The clinical effectiveness of aprotinin in the light of these results is discussed. In conclusion, although unable to mimic the complex situation following intrahepatic islet transplantation, the coculture model described here allows the opportunity to assess the events relating to specific cell types, and will provide the scope to undertake more detailed studies on the mechanisms involved. The same model could be applied to the coculture of pancreatic tissue with hepatocytes to determine any effects on the normal function of hepatocytes.
Engraftment and Albumin Production of Intrasplenically Transplanted Rat Hepatocytes (Sprague-Dawley), Freshly Isolated versus Cryopreserved, Into Nagase Analbuminemic Rats (NAR)
Authors: David Pascale , Alexandre Eliane , Audet Maxime , Chenard-Neu Marie-Pierre , Wolf Philippe , Jaeck Daniel , Azimzadeh Agnès , Richert Lysiane
Published: September 2018

Banking of cryopreserved hepatocytes is a prerequisite for large-scale hepatocyte transplantation in the clinic. We compared the efficacy of intrasplenic transplantation into Nagase analbuminemic rats (NAR) of freshly isolated (FIH) and cryopreserved (CH) hepatocytes. Hepatocytes were cryopreserved using a controlled rate freezing protocol. Albumin production of thawed CH and FIH was measured in vitro in culture by ELISA and by Western blot. After in vivo intrasplenic transplantation of NAR with either FIH or CH we assessed 1) albumin in the serum of recipients by ELISA and by Western blotting analysis at different time intervals, and 2) hepatocyte engraftment by albumin immunohistochemical staining into spleens and livers at euthanasia. In vitro, albumin was produced up to day 4 of culture in both CH and FIH. In vivo, no intrasplenic engraftment of hepatocytes occurred. Intrahepatic engraftment of CH (cell number/mm2) was significantly (twofold) lower than that of FIH and appeared only as isolated cells and small (<10 cells) clusters, while bigger clusters (>10 cells) were observed with FIH. In the FIH group, serum albumin production was observed up to 32 – 49 days posttransplantation while in the CH group no serum albumin production was detected. Our results emphasize the need to improve 1) hepatocyte transplantation procedures either by repeated hepatocytes injections and/or by transplantation under a regeneration response, and 2) the freeze/thaw protocols of hepatocytes.
Feasibility of Using Early Mesencephalic Neural Plate for Intracerebral Grafting
Authors: Hayashi Takuro , Uchida Koichi , Mine Yutaka , Yamada Motoyuki , Kawase Takeshi
Published: September 2018

The purpose of this study was to elucidate the biological significance and the possibility of intracerebral grafting of neuroepithelial stem cells derived from the mesencephalic neural plate. Immunohistological studies of embryonic day 10.5 (E10.5) Wister rats revealed strong nestin expression in the mesencephalic part of the neural plate. Mesencephalic neural plates removed from E10.5 rats were processed to either tissue or cell dissociation culture. They were cultured in vitro under various conditions and were analyzed 7 days after the primary culture. When they were cultured as a tissue, cell proliferation and differentiation into neurons extending long neurites were obvious in a serum-free medium, in a medium containing 3% serum, and in a medium containing 20 ng/ml epidermal growth factor. On the other hand, in a medium containing 10 ng/ml basic fibroblast growth factor (bFGF), both vigorous cell proliferation and sphere formation were recognized. Furthermore, marked neurite growth was rarely seen in this culture. When they were plated in a dissociation culture, cell proliferation and neurosphere generation were also recognized only in a medium containing bFGF, depending on the initial cell concentration. The spheres, generated 7 days after the primary cell culture, were positively stained by nestin. These data suggested that bFGF was able to amplify the stem cell population present in the mesencephalic neural plate derived from early embryos. This might make it possible to obtain a large number of stem cells as donor material for neural transplantation on demand.
Implantation of BDNF-Producing Packaging Cells into Brain
Authors: Fukumitsu Hidefumi , Takase-Yoden Sayaka , Furukawa Shoei , Nemoto Kiyomitsu , Ikeda Tomio , Watanabe Rihito
Published: September 2018

In order to invent a screening system to check in vivo gene function and the efficiency of gene transfer mediated by a retroviral vector system, we established a novel packaging cell, PacC6/A8, that is transplantable to rat brains. The packaging cell is based on the gene of the neuropatogenic retrovirus, A8-V. For expression in the brain, a vector that expresses brain-derived neurotrophic factor (BDNF) tagged by c-Myc-His6(LxA/bdmh) was constructed. After transfection of LxA/bdmh to PacC6/A8, a cloned cell line, PacC6/ A8/bmh, was established. PacC6/A8/bmh cells stably produced pseudotyped retroviruses carrying LxA/ bdmh. For a control, a retroviral vector that bears the gene that codes enhanced green fluorescent protein (EGFP) tagged by C-Mic-His6was also created and used for the establishment of PacC6/A8/gfmh cells that produce pseudotyped retroviruses carrying LxA/gfmh. PacC6/A8/bmh and PacC6/A8/gfmh cells were injected to the brain of newborn rats. A tumor was formed in all the rats injected that did not exhibit any symptoms until 3–4 weeks after the injection. A histological study of the injected rats revealed that the transferred BDNF gene was expressed in the brain of rats injected with PacC6/A8/bmh cells, but not in rats with PacC6/A8/gfmh cells. Interestingly, many activated microglia had migrated into the tumor induced by PacC6/A8/bmh cells, and expressed a high amount of BDNF.
Transduction of Immortalized Human Hepatocytes with p21 to Enhance Differentiated Phenotypes
Authors: Kunieda Takemi , Kobayashi Naoya , Sakaguchi Masakiyo , Okitsu Teru , Totsugawa Toshinori , Watanabe Takamasa , Matsumura Toshihisa , Maruyama Masanobu , Noguchi Hirofumi , Takesue Michihiko , Shibata Norikuni , Ohmoto Kenji , Fujiwara Toshiyoshi , Yamamoto Shinichiro , Tanaka Noriaki
Published: September 2018

We previously constructed an immortal human hepatocyte line NKNT-3 with a simian virus 40 T antigen (SV40T) to develop cell-based biological therapies. p21 is a molecule that regulates the transition from the G1 phase to the S phase of the cell cycle. Investigators have demonstrated that overexpression of p21 induces differentiation in various cell lines. In the current study we examined the effect of p21 on differentiated phenotypes of SV40T-immortalized NKNT-3 cells. A replication-deficient adenovirus vector expressing a human wild-type p21 cDNA under the control of the CMV promoter (Ad5CMVp21) and a human wild-type p21 protein fused to the protein transduction domain from the human immunodeficiency virus (HIV) TAT protein (TAT/p21) were utilized to achieve efficient delivery of p21 into NKNT-3 cells. Morphological alterations, cell cycle progression, and expression of albumin and p-450 associated enzymes (CYPs) 3A4 and 2C9 were evaluated in NKNT-3 cells treated with Ad5CMVp21 and TAT/p21. Efficient adenovirus-based p21 transfer and TAT-mediated p21 protein delivery were confirmed in NKNT-3 cells in an immuno-fluorescence study and Western blotting analysis. Transduction of NKNT-3 cells with p21 predominantly arrested the cell cycle at the G1 checkpoint, resulting in differentiated hepatic phenotypes in morphology and improvement in protein expression of albumin, CYP 3A4, and CYP C29. We here show that exogenous expression of p21 augments cellular differentiation in immortalized human NKNT-3 cells.
Evidence for Hepatocyte Differentiation from Embryonic Stem Cells In Vitro
Authors: Miyashita Hitoshi , Suzuki Atsushi , Fukao Katashi , Nakauchi Hiromitsu , Taniguchi Hideki
Published: September 2018

We confirmed hepatocyte differentiation from embryonic stem (ES) cells in vitro. RT-PCR analysis revealed that a broad range of hepatic gene expression was observed in ES cells differentiated through formation of embryoid bodies (EBs) and its attachment culture. Quantitative PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of ES cells. The presence of albumin-producing cells in the peripheral region of attached EBs was confirmed by immunocytochemical analysis. Future experiments will reveal the molecules that induce hepatocyte differentiation from ES cells in vitro. This research will provide systems for the investigation of mechanisms in liver development and establish a method of ES cell-based therapy for liver diseases.
In Vitro Study of Encapsulation Therapy for Fabry Disease Using Genetically Engineered CHO Cell Line
Authors: Naganawa Y. , Ohsugi K. , Kase R. , Date I. , Sakuraba H. , Sakuragawa N.
Published: September 2018

Fabry disease is an X-linked recessive disorder caused by a deficiency of the lysosomal hydrolase α-galac-tosidase A (α-gal). The deficiency of this enzyme leads to the systemic deposition of ceramide trihexoside (CTH) in various tissues and organs. Enzyme replacement using IV doses of recombinant human α-gal produced in CHO cells or in human fibroblasts is currently being evaluated in clinical trials as a potential therapy for this disease. However, it requires lifelong therapy involving a large amount of purified α-gal. As a novel approach for treatment of Fabry disease we used polymer encapsulated Chinese hamster ovary (CHO) cells genetically modified to express α-gal. The secreted high levels of α-gal passed through the semipermeable polymeric membrane. Using coculture system with Fabry fibroblasts, the secreted enzyme was taken up in cells, resulting in reduced accumulation of CTH in Fabry fibroblasts. This in vitro study demonstrated that an encapsulated α-gal-secreting cell line can be used to treat Fabry mice by transplantation in vivo. Judging from the protection against immune rejection by a semipermeable synthetic membrane, this novel approach may be applied to treat patients with Fabry disease and other lysosomal storage diseases.
Newly Designed Compliant Hierarchic Hybrid Vascular Grafts Wrapped with a Microprocessed Elastomeric Film—I: Fabrication Procedure and Compliance Matching
Authors: Matsuda Takehisa , He Hongbing
Published: September 2018

The object of this study was to develop a compliant hybrid vascular graft minimally supported by an elastomeric scaffold for arterial replacement. The hybrid vascular grafts designed were composed of three layers: an inner surface lined with endothelial cells (ECs); a hybrid medial tissue composed of a collagenous gel embedded with smooth muscle cells (SMCs); and an outer layer made of a laser-processed micropored segmented polyurethane (SPU) film with the circular pore size (diameter 150 μm) but different film thickness (50–200 μm) and different pore-to-pore distances (1 or 4 mm). The approximate dimensions of the hybrid vascular graft without the SPU film were as follows: inner diameter, 5 mm; length, 5 cm; thickness, 50 μm. The intraluminal pressure–external diameter relationship was measured by infusion of a phosphate buffer solution into the hybrid vascular graft. Canine carotid arteries and commercially available ePTFE grafts served as controls. Decrease in the thickness of the SPU film and increase in the pore density of the SPU film increased the pressure-dependent distensibility of the hybrid vascular grafts. The thinner the film and higher the pore density, the more compliant was the hybrid graft. The pressure-induced distensibility of the designed hybrid graft was found to be close to that of native carotid arteries.
Distinct Characteristics and Features of Allogeneic Chimerism in the NOD Mouse Model of Autoimmune Diabetes
Authors: Zorina Tatiana D. , Subbotin Vladimir M. , Bertera Suzanne , Alexander Angela M. , Haluszczak Catherine , Styche Alexis J. , Trucco Massimo
Published: September 2018

The adaptation of allogeneic chimerism in treatment of autoimmune diabetes has been shown as a promising approach in numerous studies in both experimental and clinical settings. Establishment of hemopoietic chimerism in NOD mice is the most adequate animal model to study mechanisms involved in the multiple aspects of the curative effects of chimerism in autoimmunity-prone individuals. However, there are some discrepancies in the current literature for parameters and criteria used to characterize chimerism in the NOD model. This study was aimed to standardize the criteria for the different pathological stages of diabetogenesis in chimeric versus unmanipulated NOD mice. We report two well-defined scoring systems and a new Index N for the assessment of the pathological characteristics of diabetogenesis and GVHD in chimeric NOD mice. Also, we have demonstrated that, in the NOD model, recipient conditioning resulting in as low as 1% of chimerism is sufficient to promote engraftment of the BM donor-specific islets of Langerhans.
Flt3-L Augments the Engraftment of Donor-Derived Bone Marrow Cells When Combined with Sublethal Irradiation and Costimulatory (CD28/B7 and CD40/CD40L) Blockade
Authors: Woodward Jennifer E. , Salam Abdus , Logar Alison J. , Schaefer Adam T. , Rao Abdul S.
Published: September 2018

T-cell costimulatory blockade as a constituent for recipient conditioning prior to bone marrow transplantation has led to the development of less toxic protocols for the establishment of donor cell chimerism. We therefore hypothesized that the addition of the hematopoietic growth factor, Flt3-ligand (Flt3-L), to the perioperative inhibition of the CD28/B7 and CD40/CD40 ligand costimulatory pathways would enhance the engraftment of allogeneic bone marrow. Recipient BALB/c ByJ (H-2d, Mlsc, Vβ6+/Vβ8+ TCR) received a single sublethal dose of total body irradiation (300 rad) 6 h prior to transplantation IV with unfractionated donor CBA/J (H-2k, Mlsd, Vβ6-/Vβ8+ TCR) bone marrow cells. CTLA4-Ig and/or MR1 were administered at 500 μg IP on days 0, 2, 4, and 6 posttransplantation. Flt3-L was administered at 10 μg IP on days 0–6. Donor cell chimerism was determined on days 30–90 by flow cytometric analysis. Donor-specific tolerance was assessed by skin grafting. In vitro TCR cross-linking assays and flow cytometry were utilized to explore the deletion of donor-reactive T cells. Recipients receiving CTLA4-Ig and MR1 engrafted allogeneic bone marrow cells in the peripheral blood (3/6; 50%) with chimerism being detected at 2–31%. Addition of Flt3-L to this preconditioning regimen enhanced the incidence of engraftment of donor bone marrow cells (10/13; 3–70%). Long-term survival of donor but not third-party-specific skin grafts demonstrated that donor-specific tolerance had been achieved in the chimeric recipients. Deletion of the donor-reactive T cells within the chimeric recipients was also observed. The addition of hematopoietic growth factors and cytokines to the nonmyeloablative regimen of sublethal irradiation and T-cell costimulatory blockade provides a novel strategy for the establishment of donor cell chimerism and for the induction of stable and robust donor-specific tolerance. The deletion of donor-reactive T cells using this protocol suggests the reliability and feasibility of this protocol for clinical transplantation.
Tauroursodeoxycholic Acid Improves the Survival and Function of Nigral Transplants in a Rat Model of Parkinson's Disease
Authors: Duan Wei-Ming , Rodrigures Cecilia M. P. , Zhao Li-Ru , Steer Clifford J. , Low Walter C.
Published: September 2018

There is accumulating evidence showing that the majority of cell death in neural grafts results from apoptosis when cells are implanted into the brain. Tauroursodeoxycholic acid (TUDCA), a taurine-conjugated hydrophilic bile acid, has been found to possess antiapoptotic properties. In the present study we have examined whether the supplementation of TUDCA to cell suspensions prior to transplantation can lead to enhanced survival of nigral grafts. We first conducted an in vitro study to examine the effects of TUDCA on the survival of dopamine neurons in serum-free conditions. The number of tyrosine hydroxylase (TH)-positive neurons in the TUDCA-treated cultures was significantly greater than that of control cultures 7 days in vitro. In addition, a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay showed that the number of apoptotic cells in the TUDCA-treated cultures was dramatically smaller than that in the control cultures. In the transplantation study, a 50 μM concentration of TUDCA was added to the media when nigral tissue from Sprague-Dawley (SD) rats was trypsinized and dissociated. Two microliters of cell suspension containing TUDCA was then stereotaxically injected into the striatum of adult SD rats subjected to an extensive unilateral 6-hydroxydopamine lesion of the nigrastriatal dopamine pathway. At 2 weeks after transplantation, the rats that received a cell suspension with TUDCA exhibited a significant reduction in amphetamine-induced rotation scores when compared with pretransplantation value. There was a significant increase (approximately threefold) in the number of TH-positive cells in the neural grafts for the TUDCA-treated group when compared with the controls 6 weeks postgrafting. The number of apoptotic cells was much smaller in the graft areas in the TUDCA-treated groups than in the control group 4 days after transplantation. These data demonstrate that pretreatment of the cell suspension with TUDCA can reduce apoptosis and increase the survival of grafted cells, resulting in an improvement of behavioral recovery.
Practical tetrafluoroethylene fragment installation through a coupling reaction of (1,1,2,2-tetrafluorobut-3-en-1-yl)zinc bromide with various electrophiles
Authors: Tamamoto Ken , Yamada Shigeyuki , Konno Tsutomu
Published: September 2018

(1,1,2,2-Tetrafluorobut-3-en-1-yl)zinc bromide was prepared by insertion of the zinc–silver couple into the CF2–Br bond of commercially available 4-bromo-3,3,4,4-tetrafluorobut-1-ene in DMF at 0 °C for 0.5 h, The resultant polyfluorinated zinc reagent was found to be thermally stable at ambient temperature and storable for at least 1.5 years in the refrigerator. This CF2CF2-containing organozinc reagent could be easily transmetallated to copper species, which underwent cross-coupling reactions with various aromatic iodides or acyl chlorides to produce a broad range of CF2CF2-containing organic molecules in good-to-excellent yields. Therefore, the zinc reagent could become a new and practical synthetic tool for producing functional molecules with a CF2CF2 fragment.
VEGAS: A VST Early-type Galaxy Survey. III. Mapping the Galaxy Structure, Interactions, and Intragroup Light in the NGC 5018 Group
Authors: Spavone Marilena , Iodice Enrichetta , Capaccioli Massimo , Bettoni Daniela , Rampazzo Roberto , Brosch Noah , Cantiello Michele , Napolitano Nicola R. , Limatola Luca , Grado Aniello , Schipani Pietro
Published: September 2018

Most of the galaxies in the universe today are in groups, which are key to understanding their evolution. In this work we present a new deep mosaic of 1.2 × 1.0 square degrees of the group of galaxies centered on NGC 5018, acquired at the ESO VLT Survey Telescope. We use u, g, r images to analyze the structure of the group members and to estimate the intragroup light. Taking advantage of deep and multiband photometry and of the large field of view of the VST telescope, we studied the structure of the galaxy members and the faint features leading into the intragroup space, and we give an estimate of the intragroup diffuse light in the NGC 5018 group of galaxies. We found that ∼41% of the total g-band luminosity of the group is in the form of intragroup light (IGL). The IGL has a (g – r) color consistent with those of other galaxies in the group, indicating that the stripping leading to the formation of IGL is ongoing. From the study of this group we can infer that there are at least two different interactions involving the group members: one between NGC 5018 and NGC 5022, which generates the tails and ring-like structures detected in the light, and another between NGC 5022 and MCG-03-34-013, which has produced the H i tail. A minor merging event also happened in the formation history of NGC 5018 that perturbed the inner structure of this galaxy.