bluuebunny/crossref_metadata_2025 huggingface dataset:
Problems Faced by the Small Scale Dairy Owners in Receiving Veterinary Services in Selected Areas of Chittagong
Authors: Imtiaz MA , Rana S
Published: September 2014

This study investigated the problems faced by the small scale dairy owners in receiving veterinary services in selected areas of Chittagong (Chittagong metropolitan area, Sitakunda and Patiya Upazila). A pre-tested structured questionnaire data were collected from a sample of 45 farmers during November 2012 to January 2013. A four (0-3) point rating scale was used to evaluate the constraint as high (3), moderate (2), low (1) and absence of any constraint (0). The highest ranking constraint was unavailability of veterinarian in the farm area with higher service charge on specialist doctor followed by no emergency doorstep services, poor services in local veterinary hospital, ineffectiveness at artificial insemination, high cost for drugs and vaccines and lack of illiteracy among the livestock owners These findings will help to redesign and rectify the condition of present veterinary constraints in selected areas of Chittagong, Bangladesh.DOI: http://dx.doi.org/10.3329/bjvm.v12i1.20465 Bangl. J. Vet. Med. (2014). 12 (1): 63-65
Assessment of Lead Contamination in Different Samples Around the Industrial Vicinity in Selected Areas of Bangladesh
Authors: Hossain MA , Mostofa M , Alam MN , Sultana MR , Rahman MM
Published: September 2014

Lead has long been recognized as a poison to living organisms, with negative effects on general health, reproduction, behavior and mental growth. Drinking water, surface water, industrial waste water, agricultural soil and egg samples were collected to determine the presence of lead. Twenty samples (3-5 g) from each category were collected and detected by Flame Atomic Absorption Spectrophotometer (FAAS) to detect the natural deposition of lead. The analysis of variance of mean lead concentration among different samples in different locations differed significantly (P<0.01). The highest mean values of lead accumulation in drinking water, industrial waste water and agricultural soil samples, collected from Norshindi district was detected as 0.064±0.021ml/kg, 95.735±9.593ml/kg and 119.033±9.853 mg/kg respectively. The highest mean value of lead (0.622±0.096 mg/L) in surface water was significantly (P<0.01) present in Narayangonj district and significantly (P<0.01) higher than other selected areas. Analysis of variance of data on chicken eggs revealed significant (P<0.001) difference in egg albumen, egg yolk and egg shell. The highest mean values of lead were 0.066±0.003mg/kg, 0.089±0.010mg/kg and 0.251±0.032 mg/kg in egg albumen, egg yolk and egg shell in Norshindi. The natural accumulation of lead in Drinking water, egg albumen and egg yolk was found at tolerance level but the cumulative accumulation may bring the serious public health hazards.DOI: http://dx.doi.org/10.3329/bjvm.v12i1.20468 Bangl. J. Vet. Med. (2014). 12 (1): 83-89
jsGraph and jsNMR—Advanced Scientific Charting
Authors: Pellet Norman
Published: September 2014

The jsGraph library is a versatile javascript library that allows advanced charting to be rendered interactively in web browsers without relying on server-side image processing. jsGraph is released under the MIT license and is free of charge. While being highly customizable through an intuitive javascript API, jsGraph is optimized to render a large quantity of data in a short amount of time. jsGraphs can display line, scatter, contour or zone series. Examples can be consulted on the project home page [1]. Customization of the chart, its axis and its series is achieved through simple but comprehensive JSON configurations.
Synthesis, Anticonvulsant, Sedative and Anxiolytic Activities of Novel Annulated Pyrrolo[1,4]benzodiazepines
Authors: Sorra Kumaraswamy , Chen Chien-Shu , Chang Chi-Fen , Pusuluri Srinivas , Mukkanti Khagga , Wu Chi-Rei , Chuang Ta-Hsien
Published: September 2014

Four new pentacyclic benzodiazepine derivatives (PBDTs 13–16) were synthesized by conventional thermal heating and microwave-assisted intramolecular cyclocondensation. Their anticonvulsant, sedative and anxiolytic activities were evaluated by drug-induced convulsion models, a pentobarbital-induced hypnotic model and an elevated plus maze in mice. PBDT 13, a triazolopyrrolo[2,1-c][1,4]benzodiazepin-8-one fused with a thiadiazolone ring, exhibited the best anticonvulsant, sedative and anxiolytic effects in our tests. There was no significant difference in potency between PBDT 13 and diazepam, and we proposed that the action mechanism of PBDT 13 could be similar to that of diazepam via benzodiazepine receptors.
Actual Therapeutic Indication of an Old Drug: Urea for Treatment of Severely Symptomatic and Mild Chronic Hyponatremia Related to SIADH
Authors: Decaux Guy , Kengne Fabrice , Couturier Bruno , Vandergheynst Frédéric , Musch Wim , Soupart Alain
Published: September 2014

Oral urea has been used in the past to treat various diseases like gastric ulcers, liver metastases, sickle cell disease, heart failure, brain oedema, glaucoma, Meniere disease, etc. We have demonstrated for years, the efficacy of urea to treat euvolemic (SIADH) or hypervolemic hyponatremia. We briefly describe the indications of urea use in symptomatic and paucisymptomatic hyponatremic patients. Urea is a non-toxic, cheap product, and protects against osmotic demyelinating syndrome (ODS) in experimental studies. Prospective studies showing the benefit to treat mild chronic hyponatremia due to SIADH and comparing water restriction, urea, high ceiling diuretics, and antivasopressin antagonist antagonist should be done.
Preparation and Characterization of Lignocellulosic Oil Sorbent by Hydrothermal Treatment of Populus Fiber
Authors: Zhang Yue , Yang Sheng , Wu Jian-Quan , Yuan Tong-Qi , Sun Run-Cang
Published: September 2014

This study is aimed at achieving the optimum conditions of hydrothermal treatment and acetylation of Populus fiber to improve its oil sorption capacity (OSC) in an oil-water mixture. The characteristics of the hydrolyzed and acetylated fibers were comparatively investigated by FT-IR, CP-MAS 13C-NMR, SEM and TGA. The optimum conditions of the hydrothermal treatment and acetylation were obtained at170 °C for 1 h and 120 °C for 2 h, respectively. The maximum OSC of the hydrolyzed fiber (16.78 g/g) was slightly lower than that of the acetylated fiber (21.57 g/g), but they were both higher than the maximum OSC of the unmodified fiber (3.94 g/g). In addition, acetylation after hydrothermal treatment for the Populus fiber was unnecessary as the increment of the maximum OSC was only 3.53 g/g. The hydrolyzed and the acetylated Populus fibers both displayed a lumen orifice enabling a high oil entrapment. The thermal stability of the modified fibers was shown to be increased in comparison with that of the raw fiber. The hydrothermal treatment offers a new approach to prepare lignocellulosic oil sorbent.
Effect of Standardized Cranberry Extract on the Activity and Expression of Selected Biotransformation Enzymes in Rat Liver and Intestine
Authors: Bártíková Hana , Boušová Iva , Jedličková Pavla , Lněničková Kateřina , Skálová Lenka , Szotáková Barbora
Published: September 2014

The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this in vivo study, rats were treated with a standardized cranberry extract (CystiCran®) obtained from Vaccinium macrocarpon in two dosage schemes (14 days, 0.5 mg of proanthocyanidins/kg/day; 1 day, 1.5 mg of proanthocyanidins/kg/day). The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 (CYP1A1, CYP1A2, CYP2B and CYP3A), carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious.
A Novel Synthesis of the Oxazolidinone Antithrombotic Agent Rivaroxaban
Authors: Yuan Jianyong , Liu Kai , Li Lun , Yuan Yong , Liu Xuelei , Li Yanwu
Published: September 2014

A facile synthetic route of rivaroxaban has been developed. Using commercially available (R)-epichlorohydrin and bromobenzene as the starting materials, rivaroxaban was obtained in 39% overall yield using a Goldberg coupling as the key step. The synthetic route represents a convenient procedure for the production of rivaroxaban.
Active Disaster Response System for a Smart Building
Authors: Lin Chun-Yen , Chu Edward , Ku Lun-Wei , Liu Jane
Published: September 2014

Disaster warning and surveillance systems have been widely applied to help the public be aware of an emergency. However, existing warning systems are unable to cooperate with household appliances or embedded controllers; that is, they cannot provide enough time for preparedness and evacuation, especially for disasters like earthquakes. In addition, the existing warning and surveillance systems are not responsible for collecting sufficient information inside a building for relief workers to conduct a proper rescue action after a disaster happens. In this paper, we describe the design and implementation of a proof of concept prototype, named the active disaster response system (ADRS), which automatically performs emergency tasks when an earthquake happens. ADRS can interpret Common Alerting Protocol (CAP) messages, published by an official agency, and actuate embedded controllers to perform emergency tasks to respond to the alerts. Examples of emergency tasks include opening doors and windows and cutting off power lines and gas valves. In addition, ADRS can maintain a temporary network by utilizing the embedded controllers; hence, victims trapped inside a building are still able to post emergency messages if the original network is disconnected. We conducted a field trial to evaluate the effectiveness of ADRS after an earthquake happened. Our results show that compared to manually operating emergency tasks, ADRS can reduce the operation time by up to 15 s, which is long enough for people to get under sturdy furniture, or to evacuate from the third floor to the first floor, or to run more than 100 m.
A Lossless Multichannel Bio-Signal Compression Based on Low-Complexity Joint Coding Scheme for Portable Medical Devices
Authors: Kim Dong-Sun , Kwon Jin-San
Published: September 2014

Research on real-time health systems have received great attention during recent years and the needs of high-quality personal multichannel medical signal compression for personal medical product applications are increasing. The international MPEG-4 audio lossless coding (ALS) standard supports a joint channel-coding scheme for improving compression performance of multichannel signals and it is very efficient compression method for multi-channel biosignals. However, the computational complexity of such a multichannel coding scheme is significantly greater than that of other lossless audio encoders. In this paper, we present a multichannel hardware encoder based on a low-complexity joint-coding technique and shared multiplier scheme for portable devices. A joint-coding decision method and a reference channel selection scheme are modified for a low-complexity joint coder. The proposed joint coding decision method determines the optimized joint-coding operation based on the relationship between the cross correlation of residual signals and the compression ratio. The reference channel selection is designed to select a channel for the entropy coding of the joint coding. The hardware encoder operates at a 40 MHz clock frequency and supports two-channel parallel encoding for the multichannel monitoring system. Experimental results show that the compression ratio increases by 0.06%, whereas the computational complexity decreases by 20.72% compared to the MPEG-4 ALS reference software encoder. In addition, the compression ratio increases by about 11.92%, compared to the single channel based bio-signal lossless data compressor.
Toxin ζ Reversible Induces Dormancy and Reduces the UDP-N-Acetylglucosamine Pool as One of the Protective Responses to Cope with Stress
Authors: Tabone Mariangela , Ayora Silvia , Alonso Juan
Published: September 2014

Toxins of the ζ/PezT family, found in the genome of major human pathogens, phosphorylate the peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) leading to unreactive UNAG-3P. Transient over-expression of a PezT variant impairs cell wall biosynthesis and triggers autolysis in Escherichia coli. Conversely, physiological levels of ζ reversibly induce dormancy produce a sub-fraction of membrane-compromised cells, and a minor subpopulation of Bacillus subtilis cells become tolerant of toxin action. We report here that purified ζ is a strong UNAG-dependent ATPase, being GTP a lower competitor. In vitro, ζ toxin phosphorylates a fraction of UNAG. In vivo, ζ-mediated inactivation of UNAG by phosphorylation does not deplete the active UNAG pool, because expression of the toxin enhances the efficacy of genuine cell wall inhibitors (fosfomycin, vancomycin or ampicillin). Transient ζ expression together with fosfomycin treatment halt cell proliferation, but ε2 antitoxin expression facilitates the exit of ζ-induced dormancy, suggesting that there is sufficient UNAG for growth. We propose that ζ induces diverse cellular responses to cope with stress, being the reduction of the UNAG pool one among them. If the action of ζ is not inhibited, e.g., by de novo ε2 antitoxin synthesis, the toxin markedly enhances the efficacy of antimicrobial treatment without massive autolysis in Firmicutes.
The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling
Authors: Van Deun Jan , Mestdagh Pieter , Sormunen Raija , Cocquyt Veronique , Vermaelen Karim , Vandesompele Jo , Bracke Marc , De Wever Olivier , Hendrix An
Published: September 2014

Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63‐positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute‐2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome‐specific functions and biomarkers.
Apoptotic Cell Death and Function of Cryopreserved Porcine Hepatocytes in a Bioartificial Liver
Authors: Matsushita Takakazu , Yagi Toshikazu , Hardin Joseph A. , Cragun Jennifer D. , Crow Frank W. , Bergen H. Robert , Gores Gregory J. , Nyberg Scott L.
Published: September 2014

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN–1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 μmol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 μmol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.
Neural Differentiation and Incorporation of Bone Marrow-Derived Multipotent Adult Progenitor Cells after Single Cell Transplantation into Blastocyst Stage Mouse Embryos
Authors: Keene C. Dirk , Ortiz-Gonzalez Xilma R. , Jiang Yuehua , Largaespada David A. , Verfaillie Catherine M. , Low Walter C.
Published: September 2014

Previously we reported the characterization of multipotent adult progenitor cells (MAPCs) isolated from the bone marrow of rodents. In that study, single murine MAPCs derived from ROSA-26, β-galactosidase (β-Gal)-positive transgenic mice were injected into E3.5 C57/Bl6 mouse blastocysts. The resultant chimeric blastocysts were then implanted into pseudopregnant females and were allowed to develop naturally through birth and into adulthood. Chimeric mice were sacrificed 6 to 20 weeks after birth, and were processed for histological analysis. β-Galactosidase activity was identified in all organs and tissues examined, and tissuespecific differentiation and engraftment was confirmed by colabeling with antibodies that recognize β-Gal and tissue-specific markers. In the present study we have examined neural engraftment derived from the clonal expansion of a single MAPC during rodent development, and characterized the neural phenotype of MAPCs in the resultant chimeric animals. Donor cell-derived β-Gal activity was evident throughout the brain. Double and triple immunofluorescent labeling studies revealed MAPC-derived neurons (NeuN/β-Gal) and astrocytes (GFAP/β-Gal) in the cortex, striatum, medial septal nucleus, hippocampus, cerebellum, substantia nigra, and thalamus. More specifically, donor-derived neurons contributed to each of the cellular layers of the cortex; the pyramidal and granule cell layers, as well as the hilus, of the hippocampus; Purkinje and granule cell layers in the cerebellum; and GABAergic cells in the caudate and putamen. This study haracterizes the potential for MAPCs to differentiate into specific neuronal and glial phenotypes, and to integrate normally during development, after implantation into blastocysts, and provides additional evidence that MAPCs exhibit properties similar to embryonic stem cells.
Mobilized Peripheral Blood Cells Administered Intravenously Produce Functional Recovery in Stroke
Authors: Willing Alison E. , Vendrame Martina , Mallery Jennifer , Cassady C. Jordan , Davis Cyndy D. , Sanchez-Ramos Juan , Sanberg Paul R.
Published: September 2014

Filgratism (granulocyte colony stimulating factor, G-CSF)-mobilized peripheral blood progenitor cells (PBPCs) have replaced bone marrow (BM) as a preferred source of autologous stem cells, in light of the faster hematologic recovery and lesser supportive care requirement exhibited by PBPC transplants. Other hematopoietic stem cells, like the human umbilical cord blood-derived stem cells (hUCBs), and nonhematopoietic stem cells have been shown to improve motor function in rodent models of injury and degenerative disease. In the present study we transplanted either G-CSF-mobilized PBPCs or hUCBs in rats 24 h after permanent middle cerebral artery occlusion (MCAO), and assessed their behavioral abnormalities in spontaneous activity and spontaneous motor asymmetry. In both transplanted groups of rats we observed a significant reduction of the stroke-induced hyperactivity compared with nontransplanted, stroked animals. In addition, transplantation of G-CSF PBPC and hUCB cells prevented the development of extensive motor asymmetry. Our findings raise the possibility that PBPCs could provide a novel transplantation therapy to treat stroke.
The Testicular-Derived Sertoli Cell: Cellular Immunoscience to Enable Transplantation
Authors: Emerich Dwaine F. , Hemendinger Richelle , Halberstadt Craig R.
Published: September 2014

There is a renewed enthusiasm for the potential of cellular transplantation as a therapy for numerous clinical disorders. The revived interest is largely due to the unprecedented success of the “Edmonton protocol,” which produced a 100% cure rate for type I diabetics following the transplantation of human islet allografts together with a modified immunosuppressive regimen. While these data provide a clear and unequivocal demonstration that transplantation is a viable treatment strategy, the shortage of suitable donor tissue together with the debilitating consequences of lifelong immunosuppression necessitate a concerted effort to develop novel means to enable transplantation on a widespread basis. This review outlines the use of Sertoli cells to provide local immunoprotection to cografted discordant cells, including those from xenogeneic sources. Sertoli cells are normally found in the testes where one of their functions is to provide local immunologic protection to developing germ cells. Isolated Sertoli cells 1) engraft and self-protect when transplanted into allogeneic and xenogeneic environments, 2) protect cografted allogeneic and xenogeneic cells from immune destruction, 3) protect islet grafts to reverse diabetes in animal models, 4) enable survival and function of cografted foreign dopaminergic neurons in rodent models of Parkinson's disease (PD), and 5) promote regeneration of damaged striatal dopaminergic circuitry in those same PD models. These benefits are discussed in the context of several potential underlying biological mechanisms. While the majority of work to date has focused on Sertoli cells to facilitate transplantation for diabetes and PD, the generalized ability of these unique cells to potently suppress the local immune environment opens additional clinical possibilities.
Evaluation of Different Types of Alginate Microcapsules as Bioreactors for Producing Endostatin
Authors: Rokstad A. M. , Strand B. , Rian K. , Steinkjer B. , Kulseng B. , Skjåk-Bræk G. , Espevik T.
Published: September 2014

The use of nonautologous cell lines producing a therapeutic substance encapsulated within alginate microcapsules could be an alternative way of treating different diseases in a cost-effective way. Malignant brain tumors have been proposed to be treated locally using engineered cells secreting proteins with therapeutic potential encapsulated within alginate microcapsules. Optimization of the alginate capsule bioreactors is needed before this treatment can be a reality. Recently, we have demonstrated that alginate-poly-L-lysine microcapsules made with high-G alginate and a gelled core disintegrated as cells proliferated. In this study we examined the growth and endostatin secretion of 293-EBNA (293 endo) cells encapsulated in six different alginate microcapsules made with native high-G alginate or enzymatically tailored alginate. Stability studies using an osmotic pressure test showed that alginate-poly-L-lysine-alginate microcapsules made with enzymatically tailored alginate was mechanically stronger than alginate capsules made with native high-G alginate. Growth studies showed that the proliferation of 293 endo cells was diminished in microcapsules made with enzymatically tailored alginate and gelled in a barium solution. Secretion of endostatin was detected in lower amounts from the enzymatically tailored alginate microcapsules compared with the native alginate microcapsules. The stability of the alginate microcapsules diminished as the 293 endo cells grew inside the capsules, while empty alginate microcapsules remained stable. By using microcapsules made of fluorescenamine-labeled alginate it was clearly visualized that cells perforated the alginate microcapsules as they grew, destroying the alginate network. Soluble fluorescence-labeled alginate was taken up by the 293 endo cells, while alginate was not detected in live spheroids within fluorescence-labeled alginate microcapsules. Despite that increased stability was achieved by using enzymatically tailored alginate, the cell proliferation destroyed the alginate microcapsules with time. It is therefore necessary to use cell lines that have properties more suited for alginate encapsulation before this technology can be used for therapy.
The Development of New Immunoisolatory Devices Possessing the Ability to Induce Neovascularization
Authors: Sakurai Tomonori , Satake Akira , Nagata Natsuki , Gu Yuanjun , Hiura Akihito , Doo-Hoon Kim , Hori Hiroshi , Tabata Yasuhiko , Sumi Shoichiro , Inoue Kazutomo
Published: September 2014

The transplantation of a bioartificial pancreas has been regarded as a potential method for successful islet transplantation without any immunosuppressive agents. The subcutaneous site is a very attractive site for transplantation of a bioartificial pancreas because of its advantage of an easy operation site. Our group has been reporting that transplantation of a bioartificial pancreas to the subcutaneous site can reverse hyperglycemia in diabetic recipients. Regarding shapes of a bioartificial pancreas, it is believed that a bag form has an advantage because it is easy to prepare a large quantity. Our group previously reported successful transplantation of a bioartificial pancreas in bag form, a mesh-reinforced polyvinyl alcohol bag (MRPB), implanted in the peritoneal cavity. We also reported that the effect of subcutaneous islet transplantation can be greatly improved with prevascularization treatment. In the present study, we attempted to combine MRPB to our protocol of subcutaneous prevascularization. The main problem of this trial is that the procedure of MRPB implantation injures the prevascularized blood vessel networks. To solve this problem, we made a slight alternation in our protocol, and designed new devices on the basis of MRPB. The new devices, possessing the ability to induce neovascularization, were prepared by collagen coating on the surface of MRPB and were implanted with/without different doses of FGF-2 impregnated in gelatin microspheres. When using 5 μg of FGF-2, more blood vessels were observed on the surface of type I/IV collagen-coated MRPB compared with the original MRPB and type I collagen-coated MRPB. Quite a few blood vessels were observed either around the injection site of 50 μg of FGF-2 impregnated in gelatin microspheres alone or around the implantation site of FGF-2-free gelatin microspheres and type I collagen-coated MRPB or type I/IV collagen-coated MRPB. Here we demonstrated that the combination of both FGF-2 impregnated in gelatin microspheres and collagen-coated MRPB could give an effective system of neovascularization suitable for subcutaneous implantation of a bioartificial pancreas.
Revascularization and Function of Pancreatic Islet Isografts in Diabetic Rats following Transplantation
Authors: Furuya Hajime , Kimura Toshihisa , Murakami Makoto , Katayama Kanji , Hirose Kazuo , Yamaguchi Akio
Published: September 2014

In pancreatic islet transplantation, revascularization is crucial for the graft's survival and function. In this study, the endothelium of isolated islets and revascularization and function of islet isografts in diabetic rat were investigated. Islets were isolated from Lewis rats by collagenase digestion method and were examined using immunohistochemistry (CD31 stain) on days 0, 1, 3, and 7 after isolation. The number of CD31-positive cells in these isolated islets was counted (mean ± SD%). Isografts (freshly isolated islets: group A, and islets cultured for 7 days: group B) transplanted in the renal subcapsule of streptozotocin-induced diabetic Lewis rats were examined using immunohistochemistry (CD31 stain) on days 3, 5, and 7 after transplantation. Intravenous glucose tolerance tests (IVGTT) were performed on days 3 and 7 after transplantation. The number of CD31-positive cells in the isolated islets on days 0, 1, 3, and 7 after isolation were: 17.3 ± 4.1%, 8.2 ± 0.7%, 2.1 ± 0.8%, and 0.8 ± 0.5%, respectively (p < 0.05). On day 5 after transplantation, CD31-positive cells were not detected in group A and B grafts, but were detected in both groups in periphery of the islets. On day 7, CD31-positive microvessels were present throughout the entire graft. IVGTT values in groups A and B on days 3 and 7 after transplantation did not show significant differences. In renal subcapsular isografts in diabetic rats, revascularization into islet grafts occurs from the surrounding host tissue 5 days after transplantation, but has no influence on the response to glucose during this period.
Rapid and Large-Scale Formation of Chondrocyte Aggregates by Rotational Culture
Authors: Furukawa Katsuko S. , Suenaga Hideyuki , Toita Kenshi , Numata Akiko , Tanaka Junzo , Ushida Takashi , Sakai Yasuyuki , Tateishi Tetsuya
Published: September 2014

Chondrocytes in articular cartilage synthesize collagen type II and large sulfated proteoglycans, whereas the same cells cultured in monolayer (2D) dedifferentiate into fibroblastic cells and express collagen type I and small proteoglycans. On the other hand, a pellet culture system was developed as a method for preventing the phenotypic modulation of chondrocytes and promoting the redifferentiation of dedifferentiated ones. Because the pellet culture system forms only one cell aggregate each tube by a centrifugator, the pellet could not be applied to produce a tissue-engineered cartilage. Therefore, we tried to form chondrocyte aggregates by a rotational culture, expecting to form a large number of aggregates at once. In order to increase cell–cell interactions and decrease chondrocyte–material interaction, dishes with low retention of protein adsorption and cell adhesiveness were used. In addition, rotational shaking of the dish including cells was attempted to increase the cell–cell interaction. The shaking speed was set at 80 rpm, so the cells would be distributed in the center of the dish to augment the frequency of cell–cell contact. Under these conditions, bovine articular chondrocytes started aggregating in a few hours. At 24–36 h of rotational culture, aggregates with smooth surfaces were observed. Parameters such as increase of culture time and addition of TGF-β controlled diameters of the aggregates. There were many fusiform cells at the periphery of the aggregates, where the cells tended to form a multilayered zone in cross sections. In addition, lacune-like structure, which was almost the same as pellet culture, was observed. It was found that the internal structure of the aggregates was similar to that of pellets reported previously. Therefore, the aggregates formed by a rotational culture could become an essential component to make tissue-engineered artificial cartilage.
Influence of VEGF on the Viability of Encapsulated Pancreatic Rat Islets after Transplantation in Diabetic Mice
Authors: Sigrist S. , Mechine-Neuville A. , Mandes K. , Calenda V. , Braun S. , Legeay G. , Bellocq J-P. , Pinget M. , Kessler L.
Published: September 2014

After pancreatic islet transplantation, insufficient blood supply is responsible for the loss of islet viability. The aim of our study was: 1) to determine the influence of vascular endothelial growth factor (VEGF) on the survival of encapsulated rat islets transplanted into healthy and diabetic mice and 2) to evaluate the metabolic efficiency of the VEGF-supplemented grafts. Twenty-four hours after culture, 50 rat islets immobilized into collagen in the presence of VEGF (100 ng/ml) and encapsulated (AN69 membrane, HOSPAL) were grafted in the peritoneal cavity of healthy or streptozotocin-induced diabetic mice (n = 6). Seven, 14, and 28 days after implantation, the encapsulation device and tissue surrounding the device were removed and the following parameters were analyzed: the number and the diameter of buds, the distance between devices and buds, the amount of cellular adhesion on the capsule surface, and the level of insulin secreted by encapsulated islet. For reversal of diabetes, 1000 rat islets encapsulated in the presence of VEGF were implanted in the peritoneal cavity of diabetic mice and fasting glycemia was analyzed. After 7 days of islet implantation in the absence of VEGF, the bud diameter was 16.1 ± 6.9 μm in diabetic mice and 34.4 ± 3.9 μm in healthy mice. However, the number of buds increased by a factor 2.5 in the presence of VEGF in both types of mice. Furthermore, when islets were transplanted in the presence of VEGF, the distance between the device and the buds was significantly decreased in both types of mice (p < 0.001) after 7, 14, and 28 days of islet implantation. Capsule analysis showed a decrease in cellular adhesion when the islets were encapsulated in the presence of VEGF. Insulin secretion of the islets was higher in the presence of VEGF compared with islets alone at all steps of the study. When 1000 rat islets were transplanted in the presence of VEGF, the glycemia level decreased to 6.2 ± 0.8 mmol/L after 3 days and remained stable until at least 28 days. In contrast, in the absence of VEGF, the initial decrease in the glucose level was rapidly followed by a relapse in hyperglycemia. In summary, VEGF increased the viability of engrafted encapsulated islets, increasing the duration of a normalized glycemia in diabetic mice following transplantation. Local adjunction of VEGF may therefore improve the clinical outcome of islet transplantation.
Characterization of Long-Term Survival of Syngeneic Hepatocytes in Rat Peritoneum
Authors: Selden Clare , Casbard A. , Themis M. , Hodgson H. J. F.
Published: September 2014

Hepatocyte transplantation is a potential therapy for both acute and chronic hepatic insufficiency and also for treatment of inborn errors of metabolism affecting the liver. The peritoneum is one site for implantation and has several advantages: cells implanted there can be easily identified and observed, and it has a relatively large capacity. Long-term survival using “pure” hepatocytes in the peritoneum have been disappointing. We hypothesized that cotransplantation of hepatocytes with nonparenchymal cells would help maintain differentiated hepatocyte function. Rat liver cells transplanted intraperitoneally into August rats were sacrificed at 7 days, 1, 3, 6, 9, and 12 months and analyzed for presence, basal proliferation, and functionality of hepatocytes. To demonstrate that ectopic hepatocytes remained susceptible to exogenous growth factors affecting cell proliferation, rats 9 and 12 months after transplantation were stimulated with tri-iodothyronine and KGF. Hepatocytes were identified 7 days to >12 months, by H&E and immunohistochemically, as ectopic islands in the omental fat. Functionality was confirmed by glycogen deposition. Basal proliferation in 7-day rats was 28.0 ± 10/1000 hepatocytes in ectopic islands (cf. 5.70 ± 2.7/1000 in recipient liver). Proliferation in ectopic islands was greater than host liver. Growth factor-stimulated proliferation in ectopic islands induced a 70-fold increase in DNA synthesis. In conclusion, hepatocytes transplanted with nonparenchymal cells survive, proliferate, and function in the peritoneum of normal rats, and respond to exogenous growth stimuli. Their survival and proliferation in the presence of a normal functioning liver has implications for the potential use of the peritoneal site clinically for supplementation of liver function in metabolic disorders.
Cryopreservation of Primarily Isolated Porcine Hepatocytes with UW Solution
Authors: Kunieda Takemi , Maruyama Masanobu , Okitsu Teru , Shibata Norikuni , Takesue Michihiko , Totsugawa Toshinori , Kosaka Yoshikazu , Arata Takashi , Kobayashi Kazuya , Ikeda Hideaki , Oshita Mizuko , Nakaji Shuhei , Ohmoto Kenji , Yamamoto Shinichiro , Kurabayashi Yuzuru , Kodama Makoto , Tanaka Noriaki , Kobayashi Naoya
Published: September 2014

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at −80°C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.
The Influence of Donor Age, Nerve Growth Factor, and Cografting with Drosophila Cells on Survival of Peripherally Grafted Embryonic or Fetal Rat Dorsal Root Ganglia
Authors: Pavlova Galina , Enblom Åsa , Revishchin Alexander , Sandelin Martin , Korochkin Leonid , Kozlova Elena N.
Published: September 2014

Previous studies have shown that embryonic rat and human dorsal root ganglion (DRG) cells survive grafting to the cavity of extirpated adult rat DRG. Furthermore, grafted human embryonic neurons were shown to send axons peripherally and into the spinal cord, where they establish functional synaptic connections. This study analyzed the survival of orthotopically allografted rat DRG cells from embryonic stages 15 (E15) and 20 (E20), and the influence on their survival of nerve growth factor (NGF). NGF was delivered to the DRG transplants either by pump infusion or by cotransplantation of cells from Drosophila melanogaster, transgenic for human NGF. Lumbar DRGs of adult rats were removed and a collection of E15 or E20 DRGs placed in the cavity. One month after grafting the total number of DRG cells in the grafts was counted. Differentiation of subpopulations of DRG cells was estimated by counting cells immunostained for calcitonin gene-related peptide (CGRP), Griffonia simplicifolia agglutinin isolectin B4 (GSA), or heavy neurofilament protein (antibody RT97). The results show: i) similar survival of E15 and E20 grafts, with great variability in the survival of different subpopulations in E15 transplants, but a more consistent distribution of different phenotypes in E20 transplants; ii) infusion of NGF for 2 weeks increases the survival of E15 transplants, but has a negative effect on E20 transplants; iii) Drosophila cells transfected with human NGF gene survive peripheral xenografting and have a positive effect on the survival of the GSA- and CGRP-positive populations in E15 and E20 transplants; iv) Drosophila cells without the human NGF gene increase cell survival in E20 transplants. These data suggest that i) the effect of NGF is dependent on the embryonic stage of the transplants, ii) age-dependent sensitivity to NGF influences graft survival, and iii) transgenic Drosophila cells can be cotransplanted with embryonic neural tissue to the mammalian peripheral nervous system with a positive effect on the survival of neural grafts.
Generation of 3D Retina-Like Structures from a Human Retinal Cell Line in a NASA Bioreactor
Authors: Dutt Kamla , Harris-Hooker Sandra , Ellerson Debra , Layne Dione , Kumar Ravindra , Hunt Richard
Published: September 2014

Replacement of damaged cells is a promising approach for treatment of age-related macular degeneration (AMD) and retinitis pigmentosa (RP); however, availability of donor tissue for transplantation remains a major obstacle. Key factors for successful engineering of a tissue include the identification of a neural cell line that is: homogeneous but can be expanded to give rise to multiple cells types; is nontumorigenic, yet capable of secreting neurotrophic factors; and is able to form three-dimensional (3D), differentiated structures. The goal of this study was to test the feasibility of tissue engineering from a multipotential human retinal cell line using a NASA-developed bioreactor. A multipotential human retinal precursor cell line was used to generate 3D structures. In addition, retinal pigment epithelium (RPE) cells were cocultured with neural cells to determine if 3D retinal structures could be generated in the bioreactor with cells grown on laminin-coated cytodex 3 beads. Cell growth, morphology, and differentiation were monitored by light and scanning electron microscopy, Western blot analysis, and analysis of glucose use and lactate production. The neuronal retinal precursor cell line cultured in a bioreactor gave rise to most retinal cell types seen in monolayer culture. They formed composite structures with cell-covered beads associated with one another in a tissue-like array. The beginning of layering and/or separation of cell types was observed. The neuronal cell types previously seen in monolayer cultures were also seen in the bioreactor. Some of the retinal cells differentiate into photoreceptors in the bioreactor with well-developed outer segment-like structures, a process that is critical for retinal function. Moreover, the neuronal cells that were generated resembled their in vivo phenotype more closely than those grown under other conditions. Outer segments were almost never seen in the monolayer cultures, even in the presence of photoreceptor-inducing growth factors such as basic fibroblast growth factor (bFGF) and transforming growth factor (TGF-α). Muller cells were occasionally seen when retinal, RPE cells were cocultured with retinal cells in the bioreactor. These have never been seen in this retinal cell line before. Cells grown in the bioreactor expressed several proteins specific for the retinal cell types: opsin, protein kinase C-α, dopamine receptor D4, tyrosine hydroxylase, and calbindin.
p27Kip1 Inactivation Provides a Proliferative Advantage to Transplanted Hepatocytes in DPPIV/Rag2 Double Knockout Mice after Repeated Host Liver Injury
Authors: Yuan Ray-Hwang , Ogawa Atsushi , Ogawa Emi , Neufeld David , Zhu Liang , Shafritz David A.
Published: September 2014

Studies were conducted to develop a new DPPIV–/–/Rag2–/– mouse model for hepatocyte transplantation by allogeneic and xenogeneic cells and to compare the proliferative capacity of p27 null hepatocytes versus normal hepatocytes in this system. Dipeptidyl peptidase IV (DPPIV) gene knockout mice, in which wild-type (wt) DPPIV+ donor hepatocytes can be readily identified by enzyme histochemistry, were bred with Rag2 null mice to prepare immunotolerant DPPIV–/–/Rag2–/– double knockout mice. DPPIV–/–/Rag–/– mice were transplanted with wt hepatocytes or p27 null mouse hepatocytes, which show enhanced cell cycle activity due to disruption of the Kip1 cyclin kinase inhibitor gene, and liver repopulation was assessed under nonproliferative versus proliferative experimental conditions. After their initial engraftment, transplanted wt hepatocytes did not proliferate in untreated livers or increase significantly in response to an acute liver regenerative stimulus. p27 null hepatocytes engrafted with the same efficiency as wt hepatocytes, but showed a noticeable, although not statistically significant, increase in proliferation in response to partial hepatectomy or acute CCl4 administration. Repeated treatments with CCl4 substantially increased proliferation and liver repopulation by p27 null hepatocytes but not by wt hepatocytes. These results suggest that p27 gene inactivation does not overcome proliferative restrictions imposed on hepatocytes by the normal liver, but that after repeated episodes of toxic liver injury, the augmented proliferative capacity of p27 null hepatocytes leads to significant liver repopulation compared with wt hepatocytes. These properties of p27-deficient hepatocytes could prove useful as a target for liver repopulation in patients with intermittent or a low level of chronic liver injury.
Expression of Neuronal Markers in Differentiated Marrow Stromal Cells and CD133+ Stem-Like Cells
Authors: Padovan Claudio S. , Jahn Klaus , Birnbaum Tobias , Reich Petra , Sostak Petra , Strupp Michael , Straube Andreas
Published: September 2014

Bone marrow stromal cells, which normally give rise to bone, cartilage, adipose tissue, and hematopoiesis-supporting cells, have been shown to differentiate in vitro and in vivo into neural-like cells. In this study, we examined the expression of neuronal and glial markers in human marrow stromal cells under culture conditions appropriate for neural stem cells, and compared the unsorted cell population to bone marrow CD133+ stem-like cells using immunofluorescence, Western blot, and functional patch-clamp analysis. Overall, the expression of the early neuronal marker β3-tubulin was most pronounced in the presence of DMEM/F12 and neurotrophin 3 (NT3) or brain-derived neurotrophic factor (BDNF), when marrow stromal cells were cultured onto fibronectin. Electrophysiological examination, however, could not show fast sodium currents or functional neurotransmitter receptors in differentiated marrow stromal cells. CD133+ mesenchymal stem-like cells, but not CD34+/CD133– cells, generally showed a higher expression of neuronal markers than did unsorted marrow stromal cells, and differentiated CD133+ cells more resembled neuron-like cells.