bluuebunny/crossref_metadata_2025 huggingface dataset:
A Quantitative Assessment of Comprehensive Ecological Risk for a Loess Erosion Gully: A Case Study of Dujiashi Gully, Northern Shaanxi Province, China
Authors: Liu Di , Liang Xiaoying , Chen Hai , Zhang Hang , Mao Nanzhao
Published: September 2018

As a tool that can effectively support ecosystem management, ecological risk assessment is closely related to the sustainable development of ecosystems and human well-being and has become an active area of research in ecology, geography and other disciplines. Taking Dujiashi Gully for the study of gully loess erosion, a comprehensive risk assessment system for identifying risk probability, sensitivity and impairment was established. The spatial distribution of comprehensive ecological risk was analyzed, the ecological risk management categories were simultaneously delineated based on the risk dominant factor and the risk management strategies were formulated in loess regions. The results were as follows: (1) the spatial differences in comprehensive ecological risk were significantly different in the research area. The regions with extremely high and high risk were mainly located in gully areas and secondary erosion gullies, which are in 28.02% of study area. The extremely low-risk areas covered 1/3 of the study area and were mainly distributed to the northwest and south of the study area, where hills are widely spaced. (2) The combined analysis of ecological risk and terrain found that the elevation decreased first and then rose but the comprehensive ecological risk increased first and then decreased from north to south. Comprehensive ecological risk and terrain generally showed an inverse relationship. (3) The study area was divided into four types of risk management categories. Risk monitoring zones, habitat recovery zones, monitoring and recovery zones and natural regulation zones encompass 14.84%, 12.44%, 26.47% and 46.25% of the study area, respectively. According to four types of risk management categories, different risk reduction measures were designed to improve regional sustainable development capacity. Risk identification and risk management categories based on comprehensive ecological risk model can design a sustainable development path for social ecosystem and local farmers and provide a method for sustainable development for similar gully landscapes.
Multicriteria Assessment of Combined Heat and Power Systems
Authors: Wibowo Santoso , Grandhi Srimannarayana
Published: September 2018

This paper presented a multicriteria assessment model for evaluating the performance of combined heat and power systems. Interval-valued intuitionistic fuzzy numbers were used for representing the subjective and imprecise assessments of the decision maker in evaluating the relative importance of the criteria, and the performance of individual combined heat and power systems. An effective algorithm was developed based on the concept of ideal solutions for calculating the overall performance index, for each combined heat and power system across all criteria. An example was presented to demonstrate the applicability of the multicriteria assessment model, for dealing with real world combined heat and power system performance evaluation problems.
Symmetric Identities for (P,Q)-Analogue of Tangent Zeta Function
Authors: Ryoo Cheon Seoung
Published: September 2018

The goal of this paper is to define the ( p , q ) -analogue of tangent numbers and polynomials by generalizing the tangent numbers and polynomials and Carlitz-type q-tangent numbers and polynomials. We get some explicit formulas and properties in conjunction with ( p , q ) -analogue of tangent numbers and polynomials. We give some new symmetric identities for ( p , q ) -analogue of tangent polynomials by using ( p , q ) -tangent zeta function. Finally, we investigate the distribution and symmetry of the zero of ( p , q ) -analogue of tangent polynomials with numerical methods.
Impacts of Indoxyl Sulfate and p-Cresol Sulfate on Chronic Kidney Disease and Mitigating Effects of AST-120
Authors: Liu Wen-Chih , Tomino Yasuhiko , Lu Kuo-Cheng
Published: September 2018

Uremic toxins, such as indoxyl sulfate (IS) and p-cresol, or p-cresyl sulfate (PCS), are markedly accumulated in the organs of chronic kidney disease (CKD) patients. These toxins can induce inflammatory reactions and enhance oxidative stress, prompting glomerular sclerosis and interstitial fibrosis, to aggravate the decline of renal function. Consequently, uremic toxins play an important role in the worsening of renal and cardiovascular functions. Furthermore, they destroy the quantity and quality of bone. Oral sorbent AST-120 reduces serum levels of uremic toxins in CKD patients by adsorbing the precursors of IS and PCS generated by amino acid metabolism in the intestine. Accordingly, AST-120 decreases the serum IS levels and reduces the production of reactive oxygen species by endothelial cells, to impede the subsequent oxidative stress. This slows the progression of cardiovascular and renal diseases and improves bone metabolism in CKD patients. Although large-scale studies showed no obvious benefits from adding AST-120 to the standard therapy for CKD patients, subsequent sporadic studies may support its use. This article summarizes the mechanisms of the uremic toxins, IS, and PCS, and discusses the multiple effects of AST-120 in CKD patients.
Detection and Characterization of Distinct Alphacoronaviruses in Five Different Bat Species in Denmark
Authors: Lazov Christina M. , Chriél Mariann , Baagøe Hans J. , Fjederholt Esben , Deng Yu , Kooi Engbert A. , Belsham Graham J. , Bøtner Anette , Rasmussen Thomas Bruun
Published: September 2018

Bat populations harbour a multitude of viruses; some of these are pathogenic or potentially pathogenic in other animals or humans. Therefore, it is important to monitor the populations and characterize these viruses. In this study, the presence of coronaviruses (CoVs) in different species of Danish bats was investigated using active surveillance at different geographical locations in Denmark. Faecal samples were screened for the presence of CoVs using pan-CoV real-time RT-PCR assays. The amplicons, obtained from five different species of bats, were sequenced. Phylogenetic analysis revealed a species-specific clustering with the samples from Myotis daubentonii, showing a close resemblance to coronavirus sequences obtained from the same species of bat in Germany and the United Kingdom. Our results show, for the first time, that multiple, distinct alphacoronaviruses are present in the Danish bat populations.
Viruses of Eukaryotic Algae: Diversity, Methods for Detection, and Future Directions
Authors: Coy Samantha R. , Gann Eric R. , Pound Helena L. , Short Steven M. , Wilhelm Steven W.
Published: September 2018

The scope for ecological studies of eukaryotic algal viruses has greatly improved with the development of molecular and bioinformatic approaches that do not require algal cultures. Here, we review the history and perceived future opportunities for research on eukaryotic algal viruses. We begin with a summary of the 65 eukaryotic algal viruses that are presently in culture collections, with emphasis on shared evolutionary traits (e.g., conserved core genes) of each known viral type. We then describe how core genes have been used to enable molecular detection of viruses in the environment, ranging from PCR-based amplification to community scale “-omics” approaches. Special attention is given to recent studies that have employed network-analyses of -omics data to predict virus-host relationships, from which a general bioinformatics pipeline is described for this type of approach. Finally, we conclude with acknowledgement of how the field of aquatic virology is adapting to these advances, and highlight the need to properly characterize new virus-host systems that may be isolated using preliminary molecular surveys. Researchers can approach this work using lessons learned from the Chlorella virus system, which is not only the best characterized algal-virus system, but is also responsible for much of the foundation in the field of aquatic virology.
Functional Scanning of Apple Geminivirus Proteins as Symptom Determinants and Suppressors of Posttranscriptional Gene Silencing
Authors: Zhan Binhui , Zhao Wenyang , Li Shifang , Yang Xiuling , Zhou Xueping
Published: September 2018

Apple geminivirus (AGV) is a recently identified geminivirus which is isolated from the apple tree in China. We carried out functional scanning of apple geminivirus proteins as symptom determinants and suppressors of posttranscriptional gene silencing (PTGS). Our results indicated that AGV V2 is an important virulence factor localized to the nucleus and cytoplasm that suppresses PTGS and induces severe symptoms of crinkling and necrosis. AGV C1 is also a virulence determinant which elicits systemic necrosis when expressed from a PVX-based vector. The AGV C4 is targeted to cytoplasm, plasma membrane, nucleus, and chloroplasts. The inoculation of PVX-C4 on N. benthamiana induced severe upward leaf curling, which implied that AGV C4 also functions as a symptom determinant, and mutation analyses suggested that the acylated residues on Gly2 and Cys8 play important roles in its subcellular localization and symptom development.
Improving Water Distribution Systems Robustness through Optimal Valve Installation
Authors: Choi Young Hwan , Jung Donghwi , Jun Hwandon , Kim Joong Hoon
Published: September 2018

This study proposes a framework for improving the robustness of water distribution systems using the optimal valve installation approach with regard to system reinforcement. To improve the robustness in valve design, this study introduces critical segment selection technique and optimal valve location determination within the concept of segment. Using the segment finding algorithm, the segment and the unintentional isolation can be identified. To select the critical segment, a multicriteria decision technique is applied by considering the hydraulic, social and economic effect. Finally, the optimal valve locations and the number of additional valves is determined by pipe failure analysis through the trade-off relationship with the number of additional valves and the maximum damage under pipe failure situations. To verify the proposed technique, the real-world water distribution systems are applied and compared with the original design.
An Integrated Statistical Method to Generate Potential Future Climate Scenarios to Analyse Droughts
Authors: Collados-Lara Antonio-Juan , Pulido-Velazquez David , Pardo-Igúzquiza Eulogio
Published: September 2018

The objective of this paper is to investigate different methods to generate future potential climatic scenarios at monthly scale considering meteorological droughts. We assume that more reliable scenarios would be generated by using regional climatic models (RCMs) and statistical correction techniques that produce better approximations to the historical basic and drought statistics. A multi-objective analysis is proposed to identify the inferior approaches. Different ensembles (equifeasible and non-equifeasible) solutions are analysed, identifying their pros and cons. A sensitivity analysis of the method to spatial scale is also performed. The proposed methodology is applied in an alpine basin, the Alto Genil (southern Spain). The method requires historical climatic information and simulations provided by multiple RCMs (9 RCMs are considered in the proposed application) for a future period, assuming a potential emission scenario. We generate future series by applying two conceptual approaches, bias correction and delta change, using five statistical transformation techniques for each. The application shows that the method allows improvement of the definition of local climate scenarios from the RCM simulation considering drought statistics. The sensitivity of the results to the applied approach is analysed.
The Efficacy of Prevascularization by Basic FGF for Hepatocyte Transplantation Using Polymer Devices in Rats
Authors: Ogawa Kohei , Asonuma Katsuhiro , Inomata Yukihiro , Kim Ildeok , Ikada Yoshito , Tabata Yasuhiko , Tanaka Koichi
Published: September 2018

This study used polymer devices implanted in rats to investigate the effect of prevascularization by basic fibroblast growth factor (bFGF) on hepatocyte transplantation (HTx). Lewis rats served as both donors and recipients. Polyvinyl alcohol (PVA) sponges with either hydrogel containing bFGF (bFGF group) or distilled water (control group) were implanted between the mesenteric leaves of recipient rats. Hepatotrophic stimulation was induced by a portacaval shunt and a 70% partial hepatectomy. After 1 week of prevascularization, hepatocytes harvested from the donor Lewis rats using a collagenase digestive method were injected into the sponges. Specimens were harvested at 2 weeks, 1 month, and 2 months after HTx. Histologic examination revealed that the control groups contained small numbers of hepatocytes restricted to the peripheral areas of the sponges. However, a large number of hepatocytes, including clusters, was found distributed uniformly in the bFGF group. In the bFGF group at 2 weeks, 1 month, and 2 months, the percentage of the sponge occupied by hepatocytes was 7.21 ± 2.64%, 6.98 ± 2.59%, and 5.58 ± 3.77%, respectively. The corresponding ratios for the control group were 0.40 ± 0.39%, 0.40 ± 0.40%, and 0.87 ± 1.51%. In addition, the mean number of new blood vessels in the bFGF group was significantly greater than that in the control group at 0 days, 2 weeks, and 1 month after HTx. These results suggest that bFGF strongly induced vascularization, which enabled a large number of hepatocytes to survive in the polymer devices.
Islet Cryopreservation Using Intracellular Preservation Solutions
Authors: Lakey Jonathan R. T. , Rajotte Ray V. , Fedorow Christine A. , Taylor Michael J.
Published: September 2018

Cryopreservation of islets adds great flexibility to clinical islet transplant programs. Methods of islet cryopreservation have traditionally utilized permeating cryoprotectants contained within isotonic solutions without specifically addressing issues of ionic balances, buffering capacity, or oxygen free radicals that occur during hypothermic stresses. These factors may become significant issues during low-temperature storage and during the freezing and thawing process. Since its development in the early 1980s, the University of Wisconsin (UW) organ preservation solution has become the standard vascular flush and preservation solution. Recently, Hypothermosol preservation solution (HTS) was developed as a hypothermic blood substitute. The unique characteristics and composition of these preservation solutions may be important when developing solutions specific for the cryopreservation of cells and tissues. It was the aim of this study to evaluate these two hypothermic preservation solutions as the media used in cryopreservation of islets. Groups of canine islets [5000 islet equivalents (IE)/group] were cryopreserved using the standard protocol of stepwise addition of dimethyl sulfoxide (DMSO) to 2 M, controlled nucleation, slow cooling (0.25°C/min), and rapid thawing (200°C/min). The cryopreservation solutions were made with 1) UW solution, 2) HTS solution, or 3) Medium 199 solution with 10% fetal calf serum (FCS). Additional control groups included islets cryopreserved using 4) HTS, 5) UW solution, and 6) Medium 199 alone, without DMSO. Recovery of islets immediately following thawing was equivalent between the groups with the exception of the islets cryopreserved without DMSO (groups 4–6, p < 0.05). After 48 h of postcryopreservation tissue culture, islet recovery was highest in the groups frozen with UW and HTS (mean ± SEM) (79.8 ± 1.9% and 82.5 ± 1.5%, p < 0.05 vs. group 3, 69.1 ± 3.3%, p < 0.05, ANOVA). Less than 15% of the islets were recovered when they were cryopreserved without the cryoprotectant DMSO (groups 4–6). Functional viability was assessed by measuring the glucose-stimulated insulin secretion during static incubation after 48-h culture. The stimulation indexes were 4.6 ± 1.0, 4.2 ± 0.8, 3.6 ± 1.2, 0.6 ± 0.5, and 0.4 ± 0.2 for islets in groups 1–5, respectively. This study demonstrates that postcryopreservation survival can be improved using intracellular-based preservation solutions, including UW or HTS, in conjunction with DMSO.
Correction of Hyperglycemia in Diabetic Mice Transplanted with Reversibly Immortalized Pancreatic β Cells Controlled by the tet-on Regulatory System
Authors: Landesman-Milo Dalit , Surana Manju , Berkovich Irina , Compagni Amelia , Christofori Gerhard , Fleischer Norman , Efrat Shimon
Published: September 2018

Pancreatic β cell lines may offer an abundant source of cells for β-cell replacement in type I diabetes. Using regulatory elements of the bacterial tetracycline (tet) operon for conditional expression of SV40 T antigen oncoprotein in transgenic mouse β cells, we have shown that reversible immortalization is an efficient approach for regulated β-cell expansion, accompanied by enhanced cell differentiation upon growth arrest. The original system employed the tet-off approach, in which the cells proliferate in the absence of tet ligands and undergo growth arrest in their presence. The disadvantage of this system is the need for continuous treatment with the ligand in vivo for maintaining growth arrest. Here we utilized the tet-on regulatory system to generate β cell lines in which proliferation is regulated in reverse: these cells divide in the presence of tet ligands, and undergo growth arrest in their absence, as judged by [3H]thymidine and BrdU incorporation assays. These cell lines were derived from insulinomas, which heritably developed in transgenic mice continuously treated with the tet derivative doxycycline (dox). The cells produce and secrete high amounts of insulin, and can restore and maintain euglycemia in syngeneic streptozotocin-induced diabetic mice in the absence of dox. Such a system is more suitable for transplantation, compared with cells regulated by the tet-off approach, because ligand treatment is limited to cell expansion in culture and is not required for long-term maintenance of growth arrest in vivo.
Grafting of Encapsulated Genetically Modified Cells Secreting GDNF into the Striatum of Parkinsonian Model Rats
Authors: Date Isao , Shingo Tetsuro , Yoshida Hideyuki , Fujiwara Kenjiro , Kobayashi Kazuki , Takeuchi Akira , Ohmoto Takashi
Published: September 2018

In order to deliver glial cell line-derived neurotrophic factor (GDNF) into the brain, we have established a cell line that produces GDNF in a continuous fashion by genetic engineering. These cells were encapsulated and grafted into parkinsonian model rats that had received unilateral intrastriatal injection of 6-hydroxydopamine 2 weeks earlier. Neurochemical analysis showed that GDNF has been produced from the capsule for 6 months after grafting and histological analysis revealed good survival of GDNF-producing cells in the capsule 6 months after grafting. The density of nigrostriatal dopaminergic fibers in the striatum as well as the number of dopaminergic cell bodies in the substantia nigra recovered significantly after GDNF-producing cell grafting. These results suggest the possible application of GDNF-producing cell grafting for the treatment of Parkinson's disease.
Cre/loxP-Based Reversible Immortalization of Human Hepatocytes
Authors: Kobayashi Naoya , Noguchi Hirofumi , Westerman Karen A. , Watanabe Takamasa , Matsumura Toshihisa , Totsugawa Toshinori , Fujiwara Toshiyoshi , Leboulch Philippe , Tanaka Noriaki
Published: September 2018

An ideal alternative to the primary human hepatocytes for hepatocyte transplantation would be to use a clonal cell line that grows economically in culture and exhibits the characteristics of differentiated, nontransformed hepatocytes following transplantation. The purpose of the present studies was to establish a reversibly immortalized human hepatocyte cell line. Human hepatocytes were immortalized with a retroviral vector SSR#69 expressing simian virus 40 large T antigen (SV40Tag) gene flanked by a pair of loxP recombination targets. One of the resulting clones, NKNT-3, showed morphological characteristics of liver parenchymal cells and expressed the genes of differentiated liver functions. NKNT-3 cells offered unlimited availability. After an adenoviral delivery of Cre recombinase and subsequent differential selection, efficient removal of SV40Tag from NKNT-3 cells was performed. Here we represent that elimination of the retrovirally transferred SV40Tag gene can be excised by adenovirus-mediated site-specific recombination.
Development of a Cryopreservation Procedure Employing a Freezer Bag for Pancreatic Islets Using a Newly Developed Cryoprotectant
Authors: Miyamoto Masaaki , Balamurugan A. N. , Nozawa Yuka , Sakurai Tomonori , Xu Baoyou , Yoshimura Shigehiro , Tanaka Tsuneo , Tohyama Toshihiro , Miyakoshi Junji , Inoue Kazutomo
Published: September 2018

One of the most important requirements for success in clinical islet transplantation is the use of a large number of viable donor islets. To achieve this, the ability to cryopreserve islets and to establish an islet bank are critical. Previously, we developed a two-step cryopreservation procedure with freezing tubes utilizing low and high concentrations of dimethyl sulfoxide (DMSO) and using a fully automated cryomachine for human pancreatic islets and porcine islet-like cell clusters (ICCs). Based on these experiments, we developed a simple and efficient cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant consisting of ethylene glycol (EG) instead of DMSO for decreasing injury of the islets by freezing. A 250 ml Cryocyte blood freezer bag and our newly developed cryoprotectant containing ethylene glycol (EG) were used in the freezing procedure. The islets were frozen by a fully automated computer-controlled cryomachine (GE 9,000) with our original program of slow cooling. Nucleation occurred at −8°C, and the frozen islets were stored at −196°C in a liquid nitrogen tank. The frozen-stored islets were subsequently rapidly thawed in a 37°C water bath and cultured before viability testing. In vitro function, the stimulation index of insulin release during the static incubation test for rat islets cryopreserved in a freezer bag vs. nonfrozen islets as control, was 2.13 ± 0.42 and 2.02 ± 0.38 (94.8% compared with control), respectively (n = 5, p = NS). The islet recovery compared with the nonfrozen control group was 85% (n = 5) in insulin content. When 1000 rat islets cryopreserved in a freezer bag were transplanted into the renal capsule of diabetic athymic mice, all the mice became normo-glycemic within 7 days from transplantation. Before nephrectomy, the intravenous glucose torelance test (IVGTT) was performed. The fractional decay constant of the glucose level (K value) of the frozen-thawed group was 0.42 ± 0.06%/min. A histological study of renal subcapsular grafts demonstrated the morphological integrity of the islets. These results demonstrate the utility of our cryopreservation procedure of a freezer bag for isolated islets using a fully automated computer-controlled cryomachine with a newly developed cryoprotectant for the maintenance of viability and function of frozen-stored islets both in culture and after transplantation. Cryopreservation using freezer bags with the new cryoprotectant is an effective and simple method for making an islet bank for clinical trials of islet transplantation.
Molecular Cloning of the Porcine Insulin cDNA Using a Monolayer Culture of Pancreatic Endocrine Cells
Authors: Tsuchiya Mariko , Tsuchiya Ken , Ohgawara Hisako
Published: September 2018

Porcine pancreatic endocrine cells are an attractive candidate for islet cell transplantation in view of the immunological properties and structural similarities of porcine insulin to human insulin. We recently established a method of isolation and a primary monolayer culture of porcine pancreatic endocrine cells. In this study, cloning of the porcine insulin cDNA was performed to clarify the genetic background of the purified isolated cells. A homology-based PCR cloning method was employed to determine the sequence using mRNA extracted from the monolayer-forming cells, and the candidate products were then determined by a homology search on the human insulin cDNA. According to the newly identified sequence, rapid amplification of cDNA ends was applied to the 5′ and 3′ ends, and the entire cDNA sequence was determined. Gene and protein expression was confirmed by Northern blotting, immunohistochemistry, and enzyme assay. To examine the transcriptional level, the cultured cells were incubated in a 20 mM D-glucose medium in the presence or absence of 5 μM forskolin. The porcine insulin cDNA exhibited a high homology to the human cDNA and showed 85% matching with the human amino acid sequence. D-Glucose at 20 mM stimulated the insulin secretion and mRNA expression, and further addition of 5 μM forskolin with the glucose was applied as the strongest stimulus in this culture system.
Development of a New Method to Induce Angiogenesis at Subcutaneous Site of Streptozotocin-Induced Diabetic Rats for Islet Transplantation
Authors: Gu Yuanjun , Tabata Yasuhiko , Kawakami Yoshiyuki , Balamurugan Appakaalai N. , Hori Hiroshi , Nagata Natsuki , Satake Akira , Cui Wanxing , Qi Meirigeng , Misawa Yoko , Toma Maki , Miyamoto Masaaki , Nozawa Masumi , Inoue Kazutomo
Published: September 2018

The subcutaneous space is a potential site for clinical islet transplantation. Even though there are several advantages, poor blood supply at this site mainly causes failure of islet survival. In this study, angiogenesis was induced in advance at the diabetic rats subcutis for islet transplantation by implanting a polyethylene terephthalate (PET) mesh bag containing gelatin microspheres incorporating basic fibroblast growth factor (bFGF) (MS/bFGF) and a collagen sponge. The bFGF was incorporated into gelatin microspheres for controlled release of bFGF. As controls, a PET mesh bag with or without either collagen sponges or MS/bFGF was implanted at the subcutaneous site of diabetic rats. Macroscopic and microscopic examinations revealed the formation of capillary network in and around the PET mesh bag containing MS/bFGF and collagen sponges 7 days after implantation when compare with other control groups. When tissue hemoglobin level was also measured, a significantly high level of hemoglobin amount was observed compared with that of control groups. When allogeneic islets mixed with 5% agarose were transplanted into the prevascularized rat subcutis, normoglycemia was maintained for more than 40 days, while other control groups were ineffective. This study demonstrated that combination of gelatin microspheres incorporating bFGF and collagen sponges enabled the mesh to induce neovascularization even at the subcutaneous site of streptozotocin-induced diabetic rats, resulting in improved function of islet transplantation.
A “Real Time” PCR Assay to Detect Transplanted Human Liver Cells in RAG-1-/- Mice
Authors: Funkhouser Ann W. , Vahed Sabera , Soriano Humberto E.
Published: September 2018

Xenotransplantation of human liver cells is an expanding field in need of new and precise quantitative techniques. “Real time” PCR is a sensitive and accurate method of quantifying picogram quantities of DNA. We used “real time” PCR with primers complementary to the human α-1-antitrypsin gene to assess the efficiency of engraftment of human liver cells transplanted into immunotolerant RAG-1-/- mice. Standard curves were created by mixing known proportions of human and mouse cells. There was a linear relationship between the PCR cycle at which DNA was amplified [threshold cycle (CT)] and the percent human cells (linear regression, p < 0.00009). Results were reliable, with a maximum 1.27-fold variation in the slopes of repeated standard curves. Linearity was maintained from 100% to as low as 0.01%. Therefore, 1 in 10,000 mouse cells could be detected in a 100 ng DNA sample. We measured the percent engraftment of human liver cells transplanted into the spleen of RAG-1-/- mice. By “real time” PCR assay, 0.23% human cells could be detected at 1 day after human cell transplantation. These results show that “real time” PCR assay is highly sensitive, reproducible, and accurate for detecting human cells in xenotransplanted mice.
Delta Opioid Peptide Augments Functional Effects and Intrastriatal Graft Survival of Rat Fetal Ventral Mesencephalic Cells
Authors: Borlongan Cesario V. , Su Tsung-Ping , Wang Yun
Published: September 2018

Delta enkephalin analogue [d-Ala(2),d-Leu(5)]enkephalin (DADLE) has been shown to protect dopamine transporters from methamphetamine-induced neurotoxicity. In the present study, we demonstrate that exposure of embryonic ventral mesencephalic cells to DADLE (0.01 g/ml), prior to intrastriatal transplantation, enhanced functional recovery and graft survival in 6-hydroxydopamine-induced hemiparkinsonian rats. At 6 and 8 weeks posttransplantation, animals that received DADLE-treated cell grafts exhibited significantly higher (near normal) spontaneous locomotor behaviors, as well as trends of greater reversal of motor asymmetrical behaviors compared with animals that received nontreated cell grafts. Histological examination revealed that animals transplanted with DADLE-treated cell grafts exhibited about twice the number of surviving tyrosine hydroxylase-immunoreactive grafted neurons compared with those animals that received nontreated cell grafts. These results suggest that DADLE should be considered as an adjunctive agent for neural transplantation therapy in Parkinson's disease.
Human Pancreatic Ductal Cells: Large-Scale Isolation and Expansion
Authors: Gmyr Valéry , Kerr-Conte Julie , Vandewalle Brigitte , Proye Charles , Lefebvre Jean , Pattou Francçois
Published: September 2018

The in vitro differentiation of pancreatic stem cells has recently been shown to represent a new source of β cells for cell therapy in diabetes. Human ductal cell differentiation, in vitro, has been documented in three-dimensional (3D) culture and recently substantiated. Although encouraging, the optimization of the ductal cell source, expansion and differentiation ex vivo are mandatory for clinical relevance. We compared three sources of human ductal cells (hDC) (method A1-2, B, and C). The classical main duct isolation of hDC by explant (A1), or enzymatic digestion (A2), was compared with two indirect methods: from 3D cultured human islet/duct-enriched fractions (B) and dedifferentiated exocrine fractions (C). Method A: few viable hDC were obtained from the main duct. Method B: embedding islet/duct rich fraction in 3D collagen gels expands the cytokeratin 19 (CK19)-positive ductal component in the form of ductal cysts, as we described previously; monolayers derived from digested cysts were 80% ductal (CK19). Method C: initially adherent amylase-positive exocrine clusters contained 12% (CK19) to 22% (CK7) ductal cells. One-week exocrine cultures were amylase negative and 46% (CK19) to 63% (CK7) ductal. Cell viability varied: <20% (A1), 81 ± 12% (B), 91 ± 2% (C). Extrapolating total yields we obtained (±SEM): 10.5 ± 4.6 × 103 (A1), 36 ± 18 × 103 (A2), 292 ± 50 × 106 (B), 1696 ± 526 × 106 (C) viable hDC per pancreas. A secondary monolayer expansion of cyst-derived hDC (method B) was achieved with NuSerum® (4.2-fold on plastic, 2.6-fold on 804G matrix; p < 0.05 vs. control cells on plastic). First passage exocrine-derived ductal cells also responded to matrix and to growth factors, albeit not significantly. In conclusion, this study demonstrated that an abundant hDC supply can be obtained from islet/duct or exocrine fractions followed by monolayer expansion with NuSerum. If their differentiation capacity is confirmed, in particular exocrine-derived ductal cells may represent a promising abundant source of islets for allogenic and autologous diabetes cell therapy.
Engineering of Human Hepatocyte Lines for Cell Therapies in Humans: Prospects and Remaining Hurdles
Authors: Kobayashi Naoya , Tanaka Noriaki
Published: September 2018

Hepatocyte-based biological therapies are increasingly envisioned for temporary support in acute liver failure and provision of specific-liver functions in liver-based metabolic deficiency. One of the hurdles to develop such therapies is severe shortage of human livers for hepatocyte isolation. To address the issue, we have focused on reversible immortalization of human hepatocytes. Such technology can allow rapid preparation of functional and uniform human hepatocytes. Here we present our strategy to construct transplantable human hepatocyte cell lines.
Role of Intrasplenic Hepatocyte Transplantation in Improving Survival and Liver Regeneration after Hepatic Resection in Cirrhotic Rats
Authors: Ahmad Tarik A. , Eguchi Susumu , Yanaga Katsuhiko , Miyamoto Shungo , Kamohara Yukio , Fujioka Hikaru , Furui Junichiro , Kanematsu Takashi
Published: September 2018

We studied the effect of preoperative hepatocyte transplantation on the prevention of liver failure in cirrhotic rats after hepatic resection. Two groups of Lewis rats were rendered cirrhotic by IP injection of 1% dimethylnitrosamine and were subjected to 33% hepatectomy. Two days before the resection, 36 rats in group I received intrasplenic hepatocyte transplantation, and 25 rats in group II were given intrasplenic injection of normal saline as a control. By the end of the third postoperative day, the rats in group I had better survival and a better biochemical profile than those in group II. The liver growth rate and the labeling index of proliferating cell nuclear antigen (PCNA-LI) showed a steady rise in group I. Compared with group II, group I had a significantly lower transforming growth factor (TGF-β1) level (p < 0.05). We conclude that preoperative intrasplenic hepatocyte transplantation improves survival and facilitates regeneration in cirrhotic rats after hepatic resection.
Transient Hyperproliferation of a Transgenic Human Epidermis Expressing Hepatocyte Growth Factor
Authors: Hamoen Karen E. , Morgan Jeffrey R.
Published: September 2018

Hepatocyte growth factor (HGF) is a fibroblast-derived protein that affects the growth, motility, and differentiation of epithelial cells including epidermal keratinocytes. To investigate the role of HGF in cutaneous biology and to explore the possibility of using it in a tissue engineering approach, we used retroviral-mediated gene transfer to introduce the gene encoding human HGF into diploid human keratinocytes. Modified cells synthesized and secreted significant levels of HGF in vitro and the proliferation of keratinocytes expressing HGF was enhanced compared with control unmodified cells. To investigate the effects of HGF in vivo, we grafted modified keratinocytes expressing HGF onto athymic mice using acellular dermis as a substrate. When compared with controls, HGF-expressing keratinocytes formed a hyperproliferative epidermis. The epidermis was thicker, had more cells per length of basement membrane, and had increased numbers of Ki-67-positive proliferating cells, many of which were suprabasal in location. Hyperproliferation subsided and the epidermis was equivalent to controls by 2 weeks, a time frame that coincides with healing of the graft. Transient hyperproliferation may be linked to the loss of factors present in the wound that activate HGF. These data suggest that genetically modified skin substitutes secreting HGF may have applications in wound closure and the promotion of wound healing.
Enhanced Vascularization and Survival of Neural Transplants with Ex Vivo Angiogenic Gene Transfer
Authors: Casper Diana , Engstrom Samara J. , Mirchandani Gautam R. , Pidel Ann , Palencia David , Cho Paul H. , Brownlee Michael , Edelstein Diane , Federoff Howard J. , Sonstein William J.
Published: September 2018

Restoration of brain function by neural transplants is largely dependent upon the survival of donor neurons. Unfortunately, in both rodent models and human patients with Parkinson's disease the survival rate of transplanted neurons has been poor. We have employed a strategy to increase the availability of nutrients to the transplant by increasing the rate at which blood vessels are formed. Replication-deficient HSV-1 vectors containing the cDNA for human vascular endothelial growth factor (HSVhvegf) and the bacterial β-galacto-sidase gene (HSVlac) have been transduced in parallel into nonadherent neuronal aggregate cultures made of cells from embryonic day 15 rat mesencephalon. Gene expression from HSVlac was confirmed in fixed preparations by staining with X-gal. VEGF expression as determined by sandwich ELISA assay of culture supernatant was up to 322-fold higher in HSVhvegf-infected than HSVlac-infected sister cultures. This peptide was also biologically active, inducing endothelial cell proliferation in vitro. Adult Sprague-Dawley rats received bilateral transplants into the striatum, with HSVlac on one side and HSVhvegf on the other. At defined intervals up to 8 weeks, animals were sacrificed and vibratome sections of the striatum were assessed for various parameters of cell survival and vascularization. Results demonstrate dose-dependent increases in blood vessel density within transplants transduced with HSVhvegf. These transplants were vascularized at a faster rate up to 4 weeks after transplantation. After 8 weeks, the average size of the HSVhvegf-infected transplants was twice that of controls. In particular, the survival of transplanted dopaminergic neurons increased 3.9-fold. Taken together these experiments provide convincing evidence that the rate of vascularization may be a major determinant of neuronal survival that can be manipulated by VEGF gene transduction.
Human Umbilical Cord Blood Cells can be Induced to Express Markers for Neurons and Glia
Authors: Bicknese Alma R. , Goodwin Holly S. , Quinn Cheryl O. , Henderson Verneake C. D. , Chien Shin-Nan , Wall Donna A.
Published: September 2018

Rare cells are present in human umbilical cord blood that do not express the hematopoietic marker CD45 and in culture do not produce cells of hematopoietic lineage. These umbilical cord multipotent stem cells (UC-MC) behave as multilineage progenitor cells (stem cells) and can be expanded in tissue culture. Exposure to basic fibroblast growth factor (bFGF) and human epidermal growth factor (hEGF) for a minimum of 7 days in culture induces expression of neural and glial markers. Western immunoblots demonstrate expression of both β-tubulin III and glial fibrillary acidic protein (GFAP). Immunocytochemistry of the cells showed intense labeling to both compounds on the intracellular cytoskeleton. The oligodendrocyte cell surface marker galactocerebroside (Gal-C) was present on most cells. Many cells show dual labeling, expressing both neuronal and glial markers.
Newly Designed Compliant Hierarchic Hybrid Vascular Graft Wrapped with Microprocessed Elastomeric Film—II: Morphogenesis and Compliance Change upon Implantation
Authors: He Hongbing , Matsuda Takehisa
Published: September 2018

Hierarchic structured hybrid tubular vascular media composed of endothelial cells (ECs), which covered the luminal surface, and smooth muscle cells (SMCs), which resided in the tubular collagen gel, were wrapped with thin segmented polyurethane elastomeric films designed to provide compliance matching with native arteries and transmural tissue permeability using a laser-directed ablation technique to provide different pore densities. Two hybrid grafts with high and low pore densities (inner diameter: 150 μm and length: 4 cm), and exhibiting pressure-dependent distensibility in response to pulsatile pressure, were bilaterally implanted into canine common arteries for up to 6 months. Irrespective of the pore density, high patency was achieved and no dilation and bursting occurred. Maintenance of full endothelialization during the entire course of implantation period was observed for the graft wrapped with the film with higher pore density. On the other hand, the graft wrapped with the film with lower pore density exhibited markedly reduced endothelialization at a later period of implantation, probably due to delamination of neoarterial tissue from the segmented polyurethane (SPU) surface. There were some differences in transmural tissue ingrowth between the two grafts. At anastomotic sites, neoarterial thickness for type A graft was smaller than that for type B graft regardless of the implantation period. Slightly reduced compliance was observed for both types of grafts at the sixth month of the implantation period. This study indicates that a hybrid vascular graft minimally supported with a thin elastomeric film can be used to replace diseased arteries if micropores are well designed for tissue permeability and anchoring.
Striatal Xenotransplantation of Human Retinal Pigment Epithelial Cells Attached to Microcarriers in Hemiparkinsonian Rats Ameliorates Behavioral Deficits without Provoking a Host Immune Response
Authors: Subramanian Thyagarajan , Marchionini Deanna , Potter Elizabeth M. , Cornfeldt Michael L.
Published: September 2018

Attachment of donor cells to microcarriers has been reported to make them more tolerable for transplantation into the brain. Human retinal pigment epithelial (hRPE) cells have been previously reported to contain enzymes for the production of dopa. Therefore, we examined the host immune response and behavioral effects of xenotransplantation of hRPE cells attached to microcarriers (hRPE-M) into the striatum of unilateral dopamine-depleted rats. Thirty-four adult rats were lesioned with 6-OHDA injections into the medial forebrain bundle on the right side. After 5 weeks of testing for apomorphine-induced rotations (AIR), animals were randomized for right striatal surgery into the following four groups: hRPE-M (group 1), hRPE alone (group 2), microcarriers alone (group 3), or needle tract alone (group 4). Following surgery, animals were tested for AIR every 4 weeks for a period of 12–18 weeks and thereafter euthanized. There was a significant reduction in AIR scores posttransplantation in all groups of animals in the initial observation points at 4 weeks and 8 weeks. However, there was a gradual return to baseline scores in groups 2, 3, and 4 animals at 12 weeks and at 18 weeks only group 1 animals had statistically significant (p = 0.001, repeated measures ANOVA, means comparison, predetermined contrasts) reduction in AIR scores. Brain tissue from representative animals from each group was cut into 30-μm coronal sections, stained for cresyl violet, tyrosine hydroxylase (TH), and markers for host immune activation. Sections through the striatum from group 1 animals revealed microcarriers with attached cells resembling RPE cells. No evidence of transplanted hRPE cells could be detected in sections from group 2 animals while those from groups 3 and 4 animals showed microcarriers and a needle tract alone, respectively. There was no host TH-immunoreactive sprouting response in the striatum in any of the groups and the host immune response was minimal. These results suggest that intrastriatal hRPE-M xenotransplantation into rats is well tolerated without systemic immunosuppression and that such transplants may provide behavioral benefit for parkinsonism.
Adeno-Associated Viral Delivery of GDNF Promotes Recovery of Dopaminergic Phenotype following a Unilateral 6-Hydroxydopamine Lesion
Authors: Mcgrath John , Lintz Elishia , Hoffer Barry J. , Gerhardt Greg A. , Quintero E. Matthew , Granholm Ann-Charlotte
Published: September 2018

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for dopamine neurons that has been proposed for use in the treatment of Parkinson's disease (PD). Previous studies using viral vectors to deliver GDNF in rodent models of PD have entailed administering the virus either prior to or immediately after neurotoxin-induced lesions, when the nigrostriatal pathway is largely intact, a paradigm that does not accurately reflect the clinical situation encountered with Parkinson's patients. In this study, recombinant adeno-associated virus carrying the gene encoding GDNF (rAAV-GDNF) was administered to animals bearing a maximal lesion in the nigrostriatal system, more closely resembling fully developed PD. Rats were treated with 6-hydroxydopamine into the medial forebrain bundle and assessed by apomorphine-induced rotational behavior for 5 weeks prior to virus administration. Within 4 weeks of a single intrastriatal injection of rAAV-GDNF, unilaterally lesioned animals exhibited significant behavioral recovery, which correlated with increased expression of dopaminergic markers in the substantia nigra, the medial forebrain bundle, and the striatum. Our findings demonstrate that rAAV-GDNF is capable of rescuing adult dopaminergic neurons from near complete phenotypic loss following a neurotoxic lesion, effectively restoring a functional dopaminergic pathway and diminishing motoric deficits. These data provide further support for the therapeutic potential of rAAV-GDNF-based gene therapy in the treatment of PD.
Muscle Reinnervation with Delayed or Immediate Transplant of Embryonic Ventral Spinal Cord Cells into Adult Rat Peripheral Nerve
Authors: Grumbles Robert M. , Wood Patrick , Rudinsky Michelle , Gomez Anna M. , Thomas Christine K.
Published: September 2018

Muscle denervation is common in various neuromuscular diseases and after trauma. It induces skeletal muscle atrophy. Only muscle reinnervation leads to functional recovery. In previous studies, denervated adult rat muscles were rescued by transplantation of embryonic day 14–15 (E14–15) ventral spinal cord cells into a nearby peripheral nerve. In the present study, changes were made in the environment into which the cells were placed to test whether reinnervation was improved by: 1) prior nerve degeneration, induced by sciatic nerve transection 1 week before cell transplantation; 2) transplantation of 1 million versus 5 million cells; 3) addition of nerve growth factor (NGF) to the transplant. Ten weeks after cell transplantation, axons had grown from all of the transplants. The numbers of myelinated axons that regenerated into the tibial, medial (MG), and lateral gastrocnemius-soleus (LGS) nerves were similar across treatments. The mean diameters of large LGS axons (>6 μm) were significantly larger with nerve degeneration before transplantation. The mean diameters of MG and LGS axons were significantly larger with transplantation of 1 million versus 5 million cells. Silver-stained experimental and control lateral gastronemius (LG) muscles showed axons that terminated at motor end plates. Nodal and terminal sprouts were more common in reinnervated muscles (45–63% of all end plates) than in control muscles (10%). Electrical stimulation of the transplants induced weak contractions in 39 of 47 MG muscles (83%) and 33 of 46 LG muscles (72%) but at higher voltages than needed to excite control muscles. The threshold for MG contraction was lower with transplantation of 1 million cells, while LG thresholds were lower without NGF. The cross-sectional area of whole LG muscles was significantly larger with cell transplantation (immediate or delayed) than with media alone, but all of these muscle areas were reduced significantly compared with control muscle areas. These data suggest that delayed transplantation of fewer cells without NGF assists regeneration of larger diameter axons and prevents some muscle atrophy.