bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Amnion-Isolated Cells Normalize Blood Glucose in Streptozotocin-Induced Diabetic Mice
Authors: Wei Jun Ping , Zhang Tian Shu , Kawa Shigeyuki , Aizawa Toru , Ota Masao , Akaike Toshihiro , Kato Kiyoshi , Konishi Ikuo , Nikaido Toshio
Published: September 2014

Whole pancreas or β-cell transplantation has opened the way for the treatment of advanced stage of diabetes mellitus. However, it is always limited by the scarcity of transplantation materials. The amniotic membrane is part of the fetal membrane and is composed of amniotic epithelium (HAE) and mesenchymal (HAM) cells that are derived from the inner cell mass in the blastocyst. Thus, HAE and HAM cells may have the potential to differentiate into various organs. The aim of our study was to assess the possibility of HAE cells differentiating into insulin-producing cells. In vitro, HAE cells stimulated with nicotinamide induced insulin mRNA in the culture cells. In vivo, HAE cells were capable of normalizing the blood glucose level of diabetic mice after several weeks of implantation into streptozotocin-induced diabetic mice. The distribution of human cells and human insulin secretion in mouse tissue studied by immunohistochemistry for anti-human-specific β-2-microglobulin and anti-human-specific insulin shows the same location in mouse tissue. These studies suggest that HAE cells have the potential to differentiate into β-cells in vivo, and hence that HAE cells have therapeutic potential for the treatment of type I diabetes mellitus.
Development of Highly Functional Long-Term Culture Method of Liver Slice Embedded in Agarose Gel for Bioartificial Liver
Authors: Nonaka Hideki , Ise Hirohiko , Sugihara Nobuhiro , Hirose Shinichi , Negishi Naoki , Kondo Yoshiyuki , Akaike Toshihiro
Published: September 2014

It is difficult to a produce highly functional bioartificial liver (BAL) using only hepatocytes, because it is believed that liver-specific three-dimensional structure is necessary to maintain high function for BAL. But it is difficult to construct a culture system with liver-specific three-dimensional structure in vitro. To realize a highly functional culture system with liver-specific three-dimensional structure, we developed a culture system using liver slices that keep liver-specific architecture, such as liver lobule and hepatic microvascular system. Liver slices were embedded in agarose gel to maintain them under a moist and three-dimensional environment. We examined the viability and function of liver slices by using various shapes of agarose gel. Liver slices were cultured 1) under stationary condition (control), 2) directly embedded in gel, and 3) embedded in cylindrical gel for good drainage of medium and ventilation of air. The viability and function of the incubated liver slices were evaluated by LDH leakage, histomorphology, and immunohistochemistry. At 10 days, the morphological condition and function of liver slices embedded in cylindrical gel were maintained better than liver slices directly embedded in gel or in the stationary condition. We suggest that high functionality and morphological condition of liver slices could be maintained by embedding in cylindrical gel. In the future, it is possible that this method could be used to develop a highly functional bioartificial liver.
Activated Porcine Embryonic Brain Endothelial Cells Induce a Proliferative Human T-Lymphocyte Response
Authors: Sumitran-Holgersson Suchitra , Brevig Thomas , Widner Håkan , Holgersson Jan
Published: September 2014

Transplantation of allogeneic embryonic neural tissue is a potential treatment for patients with Parkinson's and Huntington's diseases. The supply of human donor tissue is limited, and alternatives such as the use of animal (e.g., porcine) donor tissue are currently being evaluated. Before porcine grafts can be used clinically, strategies to prevent neural xenograft rejection must be developed. Knowledge on how human T lymphocytes recognize porcine embryonic neural tissue would facilitate the development of such strategies. To investigate the ability of porcine embryonic brain microvascular endothelial cells (PBMEC) to stimulate human T-cell proliferation, PBMEC were immuno-magnetically isolated and cocultured with purified human CD4 or CD8 single-positive T cells. PBMEC had a cobblestone-like growth pattern and expressed the endothelial cell markers CD31 and CD106. PBMEC stimulated with the supernatant of phytohemagglutinin-activated porcine peripheral blood mononuclear cells or porcine IFN-γ, but not nonstimulated PBMEC, induced proliferation of both CD8 and CD4 T cells as assessed by [3H]thymidine incorporation. Flow cytometric analyses showed that the degree of CD8 and CD4 T cell proliferation correlated with the expression levels of class I and II major histocompatibility complex (MHC) antigens, respectively. PBMEC expressed a CTLA-4/Fc-reactive molecule, most likely CD86, suggesting that these cells are able to deliver a costimulatory signal to the T cells. Human TNF-α, but not human IFN-γ, induced class I, but not class II, MHC expression on PBMEC. Within a neural graft or the regional lymph nodes, PBMEC might stimulate human T cells via the direct pathway, and should therefore be removed from the donor tissue prior to transplantation.
The Bag Method for Islet Cell Infusion
Authors: Baidal David A. , Froud Tatiana , Ferreira Jacqueline V. , Khan Aisha , Alejandro Rodolfo , Ricordi Camillo
Published: September 2014

As islet cell transplantation gains increasing interest following results published by the Edmonton group, results that have been successfully reproduced by several centers nationwide and abroad, the need of guidelines to standardize the procedure becomes highly important. We detail the key steps of the infusion procedure utilizing a closed gravity fed bag system utilized at our institution since 1990, which consists of a 600-ml transfer bag and a 150-ml rinse bag connected via sterile tubing. The use of gravity allows for a control rate of infusion as well as providing a safety mechanism through natural reduction of flow that parallels any increase in portal pressure, therefore allowing the operator to prevent precipitous pressure rises. Reports on significant rise in portal pressures during islet cell infusion as well as portal vein thrombosis have been published. Infusion at these centers was carried out using a syringe method. Using our technique, portal vein thrombosis (partial or complete) was not detected in any of the infusions performed at our institution. This method may be of assistance to minimize some of the observed complications associated with islet transplant procedures and has now been adapted by most centers performing clinical islet transplantation.
Transplantation of Embryonic Porcine Neocortical Tissue into Newborn Rats
Authors: Castro Anthony J. , Meyer Morten , Møller-Dall Annette , Zimmer Jens
Published: September 2014

Several previous studies, suggesting the potential use of embryonic xenografts in the treatment of neurological disorders, indicate that neural growth and axonal guidance factors may function across species. In this light, blocks of fetal porcine neocortex were grafted into small cortical lesion cavities made in newborn rats. Sacrifice at 3–12.5 weeks posttransplantation revealed healthy looking grafts in several animals. Apparent graft rejection evidenced by areas of necrosis and OX1 reactivity was observed in some of the older transplants. Treatment of nursing mothers or of postweaning newborns with cyclosporin A did not appear to promote graft survival. Some transplants grew to extremely large proportions and were characterized by bands of cells and bundles of axons as observed using immunohistochemical staining for pig neurofilament. Neurofilament-positive axons projected from several of the grafts to course through the corpus callosum to the contralateral cortex or to course ipsilaterally within the subcortical white matter, where labeled fibers could be traced to the midbrain crus cerebri in older transplants. Bundles of axons were also observed coursing within the ipsilateral caudate putamen where terminal branching was apparent. The normal course of transplant efferents within the host brain indicates that growing pig axons can respond to rodent axonal guidance factors.
Improved Efficacy of Stem Cell Labeling for Magnetic Resonance Imaging Studies by the Use of Cationic Liposomes
Authors: Van Den Bos Ewout J. , Wagner Anja , Mahrholdt Heiko , Thompson Richard B. , Morimoto Yoshihisa , Sutton Brad S. , Judd Robert M. , Taylor Doris A.
Published: September 2014

Labeling stem cells with FDA-approved superparamagnetic iron oxide particles makes it possible to track cells in vivo with magnetic resonance imaging (MRI), but high intracellular levels of iron can cause free radical formation and cytotoxicity. We hypothesized that the use of cationic liposomes would increase labeling efficiency without toxic effects. Rabbit skeletal myoblasts were labeled with iron oxide by: 1) uptake of iron oxide incorporated into cationic transfection liposomes (group I) or 2) customary endocytosis (group II). In both groups, cell proliferation and differentiation were measured and toxicity was assayed using trypan blue and ratio fluorescence microscopy with BODIPY® 581/591 C11. The effects of the intracellular iron oxide on magnetic resonance image intensities were assessed in vitro and in vivo. Both methods resulted in uptake of iron intracellularly, yielding contrast-inducing properties on MRI images. In group II, however, incubation with iron oxide at high concentrations required for endocytosis caused generation of free radicals, a decrease in proliferation, and cell death. Cytotoxic effects in the remaining cells were still visible 24 h after incubation. Conversely, in group I, sufficient intracellular uptake for detection in vivo by MRI could be achieved at 100-fold lower concentrations of iron oxide, which resulted in a high percentage of labeled cells, high retention of the label, and no cytotoxic effects even after stressing the cells with a hypoxia–reoxygenation insult. The use of cationic liposomes for iron oxide stem cell labeling increases labeling efficiency approximately 100-fold without toxic effects. This technique results in high-contrast-inducing properties on MRI images both in vitro and in vivo and could thus be a valuable tool for tracking stem cells noninvasively.
Long-Term Cell Survival and Hemodynamic Improvements after Neonatal Cardiomyocyte and Satellite Cell Transplantation into Healed Myocardial Cryoinfarcted Lesions in Rats
Authors: Huwer Hanno , Winning Johannes , Vollmar Brigitte , Welter Cornelius , Löhbach Christoph , Menger Michael D. , Schäfers Hans-Joachim
Published: September 2014

Cell engraftment is a new strategy for the repair of ischemic myocardial lesions. The hemodynamic effectiveness of this strategy, however, is not completely elucidated yet. In a rat model of cryothermia-induced myocardial dysfunction, we investigated whether syngeneic transplantation of neonatal cardiomyocytes or satellite cells is able to improve left ventricular performance. Myocardial infarction was induced in female Lewis rats by a standardized cryolesion to the obtuse margin of the left ventricle. After 4 weeks, 5 × 106 genetically male neonatal cardiomyocytes (n= 16) or satellite cells (n = 16) were engrafted into the myocardial scar. Sham-transplanted animals (n = 15) received injections with cell-free medium. Sham-operated animals (n = 15) served as controls. Left ventricular performance was analyzed 4 months after cell engraftment. Chimerism after this sex-mismatched transplantation was evaluated by detection of PCR-amplified DNA of the Y chromosome. The average heart weight of the infarcted animals significantly exceeded that of controls (p < 0.05). In sham-transplanted animals, mean aortic pressure, left ventricular systolic pressure, aortic flow (indicator of cardiac output), and left ventricular systolic reserve were significantly lower (p < 0.05) compared with sham-operated controls. This was associated with deterioration of ventricular diastolic function (maximal negative dP/dt, time constants of isovolumic relaxation; p < 0.05). Transplantation of satellite cells was found more effective than transplantation of neonatal cardiomyocytes, resulting in i) normalization of mean aortic pressure compared with sham-operated controls, and ii) significantly improved left ventricular systolic pressure and aortic flow (p < 0.05) compared with sham-transplanted animals. Left ventricular systolic reserve and diastolic function, however, were improved by neither satellite cell nor neonatal cardiomyocyte transplantation. Analysis of male genomic DNA revealed 3.98 ± 2.70 ng in hearts after neonatal cardiomyocyte engraftment and 6.16 ± 4.05 ng in hearts after satellite cell engraftment, representing approximately 103 viable engrafted cells per heart. Our study demonstrates i) long-term survival of both neonatal cardiomyocytes and satellite cells after transplantation into cryoinfarcted rat hearts, ii) slight superiority of satellite cells over neonatal cardiomyocytes in improving global left ventricular pump performance, and iii) no effect of both transplant procedures on diastolic dysfunction.
Hepatic Cells via Cava Vein Can Influence Allogenic Islet Rat Transplantation
Authors: Jara-Albarrán Antonino , Soto-Montenegro M. Luisa , Zugasti Ana , Rollán Eduardo , Alvarez Ysmael , Alvarez Granada
Published: September 2014

We have reported, previously, some effect of allogenic hepatic cells for islet tolerance when they are injected mixed (hepatic cells and islets) in different proportions via portal vein, in diabetic Wistar rats. Now we have studied the role of allogenic hepatic cells injected sequentially 15 min before islets, comparing via the portal vein (A and B groups) and via the cava vein (C and D groups) with a control group of islets alone. The allogenic islets were always injected via portal vein, in similar conditions, while the ratio of hepatic cells/islets was 100:1 (A, C groups) or 200:1 (B, D groups). Islets and hepatic cells were obtained from several different rats. The transplanted rats were observed during 30 days and results compared among the different rat groups: porta-porta (P/P), cava-porta (C/P), and control group. Statistically, a significant interaction between type of transplant and proportion of hepatic cells was observed. Also, C plus D groups showed statistical difference with the control group (p < 0.017) and also all the groups together (p < 0.047). These results suggest that hepatic cells can induce, in some cases, islet graft prolongation in Wistar rats. Better results were obtained when hepatic cells were injected via the cava vein than via the portal vein. Because we used a liver cell suspension integrated for several kinds of cells, the study does not clarify if this effect can be related to some specific hepatic cell subpopulation. To confirm the results and to determine if the hypothetical mechanism can be attributed to a block of the immune system or to some factor secreted by hepatic cells, more studies must be performed.
Response of Encapsulated Rat Pancreatic Islets to Hypoxia
Authors: de Groot M. , Schuurs T. A. , Keizer P. P. M. , Fekken S. , Leuvenink H. G. D. , Van Schilfgaarde R.
Published: September 2014

Hypoxia contributes to encapsulated pancreatic islet graft failure. To gain insight into the mechanisms that lead to hypoxia-induced graft failure, encapsulated islet function, vitality, and cell replication were assessed after 2 and 5 days of hypoxic (1% O2) and normoxic (20% O2) culture. The mRNA expression levels of Bcl-2, Bax, inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein 1 (MCP-1) were assessed, as well as the amount of nitrite and MCP-1 in the culture medium. Hypoxia was associated with loss of encapsulated islet function and vitality, but not with an increase in islet cell replication. Loss of vitality was due to necrosis, and only modestly due to apoptosis. Hypoxia was not associated with changes in the Bcl-2/Bax mRNA ratio, but it did increase the expression of iNOS and MCP-1 mRNA. The increased mRNA levels were, however, not associated with elevated concentrations of nitrite nor with elevated levels of MCP-1 protein. The increased iNOS mRNA levels imply a role for NO in the completion of cell death by hypoxia. The increased MCP-1 mRNA levels suggest that encapsulated islets in vivo contribute to their own graft failure by attracting cytokine-producing macrophages. The discrepancy between iNOS mRNA and nitrite is explained by the longer half-life of NO during hypoxia. MCP-1 protein levels are underestimated as a consequence of the lower number of vital cells in combination with a higher proteolytic activity due to necrosis. Thus, strategies to eliminate hypoxia may not only improve islet function and vitality, but may also reduce the attraction of macrophages by encapsulated islets.
Cryopreservation of Alginate-Encapsulated Recombinant Cells for Antiangiogenic Therapy
Authors: Stensvaag V. , Furmanek T. , Lønning K. , Terzis A. J. A. , Bjerkvig R. , Visted T.
Published: September 2014

The potential benefit of continuous local administration of antiangiogenic proteins to CNS tumors in vivo has recently been demonstrated using endostatin-producing recombinant cells encapsulated in alginate beads. Due to the treatment potential of transplanted alginate-encapsulated cells producing therapeutic proteins, we describe a successful method of cryopreservation (CP) of such beads, in which cellular viability, alginate structure, and protein secretion were maintained. Alginate beads containing human embryonic kidney cells (HEK 293 cells) stably transfected with the gene encoding for endostatin were cryopreserved in dimethyl sulfoxide (DMSO) using a slow freezing procedure. Briefly, the DMSO concentration was gradually increased prior to the freezing procedure. The cryotubes were further supercooled to −7.5°C and nucleated. Thereafter, the samples were cooled at a rate of 0.25°C/min and stored in liquid nitrogen. The viability of the encapsulated cells was assessed using confocal microscopy quantification (CLSM) technique and a MTS assay. The cell cycle distribution inside the beads was assessed by DNA flow cytometry and endostatin production was determined by an endostatin-specific ELISA assay, both prior to and after CP. CLSM measurements showed sustained esterase activity in the beads after thawing, with only a slight transient decrease 24 h after CP. The MTS assay verified these findings by displaying similar variations of intracellular dehydrogenase activity. Flow cytometric analyses revealed no cryorelated disturbances in cellular ploidy. Furthermore, ELISA measurements showed a well-preserved endostatin production after CP. In conclusion, this work describes the successful CP of alginate-encapsulated recombinant cells secreting a therapeutic protein. Together with previous published reports, these results further substantiate the feasibility and potential of cell encapsulation therapy in the treatment of malignant tumors.
Polyphenol, an Extract of Green Tea, Increases Culture Recovery Rates of Isolated Islets from Nonhuman Primate Pancreata and Marginal Grade Human Pancreata
Authors: Zhang Guangming , Matsumoto Shinichi , Hyon Suong-Hyu , Qualley Sabrina A. , Upshaw Lisa , Strong D. Michael , Reems Jo-Anna
Published: September 2014

Investigations indicate that an extract of green tea, polyphenol, can significantly increase the culture survival rate of rat islets without deteriorating their functionality. In this study, we examined the effect of adding polyphenol to islets isolated from human pancreata and nonhuman primate pancreata. Islets were isolated from human pancreata that did not meet criteria for clinical transplantation (n = 6) and from nonhuman primate pancreata (n = 5). The islets were cultured in CMRL-1066 + 10% FCS with the addition of 0, 30, 60, 125, 250, or 500 μg/ml of polyphenol. After 24 or 48 h of culture, islet yield, viability, purity, morphology, and stimulation index was assessed. RT-PCR and Western blot analysis were also performed to assess the expression levels of the apoptotic related genes, Bcl-2 and BAX. After 24 h of culture, islet yields were significantly higher in cultures supplemented with 30–250 μg/ml of polyphenol than in cultures without polyphenol. After 48 h of culture, significant differences in islet numbers were observed with polyphenol concentrations of 125 μg/ml (p < 0.01) and 250 μg/ml (p < 0.01). However, no significant differences were noted in islet viability, purity, morphology, and stimulation index at each time point with or without polyphenol. RT-PCR and Western blot analysis of the islets indicated that Bcl-2 levels increased by 2.5-fold and BAX levels decreased by twofold in cultures supplemented with polyphenol. This resulted in BAX/Bcl-2 ratios that were lower in polyphenol-supplemented cultures than with control cultures. Polyphenol increases culture recovery rates by precluding islet apoptosis.
Tissue-Engineered Grafts Matured in the Right Ventricular Outflow Tract
Authors: Ozawa Tsukasa , Mickle Donald A. G. , Weisel Richard D. , Matsubayashi Keiji , Fujii Takeshiro , Fedak Paul W. M. , Koyama Nobuya , Ikada Yoshito , Li Ren-Ke
Published: September 2014

Autologous smooth muscle cell (SMC)-seeded biodegradable scaffolds could be a suitable material to repair some pediatric right ventricular outflow tract (RVOT) cardiac anomalies. Adult syngenic Lewis rat SMCs (2 × 106) were seeded onto a new biodegradable copolymer sponge made of ∊-caprolactone-co-L-lactide reinforced with poly-L-lactide fabric (PCLA). Two weeks after seeding, the patch was used to repair a surgically created RVOT defect in an adult rat. At 8 weeks after implantation the spongy copolymer component was biodegraded, and SM tissue and extracellular matrices containing elastin fibers were present in the scaffolds. By 22 weeks more fibroblasts and collagen were present (p < 0.05). The number of capillaries in the grafts also increased (p < 0.001) between 8 and 22 weeks. The fibrous poly-L-lactide component of the PCLA scaffold remained. The 22-week grafts maintained their thickness and surface area in the RVOT. The SMCs prior to implantation were in a synthetic phenotype and developed in vivo into a more contractile phenotype. By 8 weeks the patches were endothelialized on their endocardial surfaces. Future work to increase the SM tissue and elastin content in the patch will be necessary before implantation into a pediatric large-animal model is tested.
Long-Term Maintenance of the Drug Transport Activity in Cryopreservation of Microencapsulated Rat Hepatocytes
Authors: Koizumi Tomotake , Aoki Takeshi , Kobayashi Yasuna , Yasuda Daisuke , Izumida Yoshihiko , Jin Zhenghao , Nishino Nobukazu , Shimizu Yoshinori , Kato Hirohisa , Murai Noriyuki , Niiya Takashi , Enami Yuta , Mitamura Keitaro , Yamamoto Toshinori , Kusano Mitsuo
Published: September 2014

Transplantation of isolated hepatocytes has been proposed to compensate for essential functions lacking in liver failure or for genetic defects that alter a specific liver metabolic pathway. Hepatocyte utilization for these purposes would be facilitated with a reliable, reproducible, and effective method of long-term hepatocyte storage. We have recently developed a simple new system for cryopreservation of hepatocytes that encapsulates alginate microspheres and maintains liver-specific function. The aim of this study was to elucidate the transport and drug-metabolizing enzyme activities of cryopreserved microencapsulated hepatocytes stored for a long time. Morphological examinations showed there is no apparent injury of the hepatocytes during cryopreservation processes. A drug-metabolizing enzyme (testosterone 6β-hydroxylase, a specific probe for CYP3A2) and drug transport activities [salicylate, allopurinol, and prostaglandin E2 (PGE2), typical substrates of rOat2] in cryopreserved microencapsulated hepatocytes were maintained up to 120 days. Our results thus demonstrate for the first time that cryopreservation of primary rat hepatocytes by the encapsulation technique allows long-term retention of drug metabolism and drug transport activities.
Detection of Microbial Contamination during Human Islet Isolation
Authors: Kin Tatsuya , Rosichuk Shawn , Shapiro A. M. James , Lakey Jonathan R. T.
Published: September 2014

Current good manufacturing practice (cGMP) islet processing facilities provide an ultraclean environment for the safe production of clinical grade islets for transplantation into immunosuppressed diabetic recipients. The objective of this study was to monitor the rate of microbial contamination in islet products after implementation of good manufacturing practice conditions. Fluid samples for microbial contamination were collected at the following steps: from the pancreas transport solution upon arrival of the organ (n = 157), after surface decontamination of the pancreas with antiseptic agents (n = 89), from islet supernatant at the end of the isolation (n = 104), and from islet supernatant as a final transplantable product after culture (n = 53). Bacterial, fungal, and mycoplasma cultures were conducted for 2, 2, and 3 weeks, respectively. Microbial contamination was detected in 31% of transport solution. The contamination was not associated with the presence of the duodenum during the preservation, cold ischemia time, or procurement team (local vs. distant). Surface decontamination of the pancreas resulted in clearance of 92% of the microbial contamination. Six preparations at the end of the isolation revealed microbial growth. All were de novo contamination during the processing. Fifty-three preparations that met our release criteria in terms of product sterility were transplanted into type 1 diabetic patients. In two instances, positive culture of the islet preparation was reported after transplantation had occurred. No patient showed any clinical findings suggestive of infection or any radiological abnormalities suggestive of abscess; a single dose of antibiotic coverage was given routinely to recipients prior to islet infusion. Although transport solution carries a high risk of microbial contamination, most contaminants become undetectable during islet processing. Microbial contamination in final products is rare, but de novo contamination still occurs during processing even under cGMP conditions.
Transplantation of Cultured Adult Porcine Full-Thickness Retina
Authors: Engelsberg Karl , Ghosh Fredrik
Published: September 2014

In this study we wanted to examine how an adult neuroretina from an animal with an eye similar to the human one survives in vitro. We also wanted to investigate how the culture process affects the adult retina when used in a transplantation paradigm. Full-thickness neuroretinal sheets from adult porcine eyes were dissected into pieces measuring 3 mm in diameter. These were kept in culture for 1–3 days. After this time, the explants were fixed or transplanted subretinally to adult pigs, which were killed after 72–74 days. Transplanted eyes, as well as tissue kept in culture only, were processed for hematoxylin and eosin staining and immunohistochemistry. Explants kept 1 day in vitro (DIV) displayed the normal morphology. In these specimens, single pyknotic cells were evident in the outer nuclear layer (ONL) and ganglion cell layer, but were more frequent in the inner nuclear layer (INL). After longer times in vitro, severe degenerative changes appeared. Transplanted explants kept 1 DIV prior to transplantation exhibited normal retinal lamination in two out of four specimens. Transducin and recoverin labeling revealed photoreceptors with inner segments in these grafts. Rod bipolar cells displayed a normal morphology. Vertically arranged Mùller cells were also seen in the laminated grafts. Two of the three transplants kept 2 DIV displayed minimal lamination. Eyes with transplants kept 3 DIV prior to transplantation displayed degenerated grafts in all eyes. This study shows that adult porcine neuroretinal explants kept in culture for 1 day display a normal morphology in their major part. Additionally, 1-day explants can survive transplantation with retained morphology even after several months. This indicates the possibility of storing adult donor tissue between harvest and transplantation. The culture system may also be used in the future as a tool for manipulating retinal donor tissue prior to transplantation.
Huntington's Disease: Pathological Mechanisms and Therapeutic Strategies
Authors: Ramaswamy Shilpa , Shannon Kathleen M. , Kordower Jeffrey H.
Published: September 2014

Huntington's disease (HD) is a devastating neurodegenerative disorder that occurs in patients with a mutation in the huntingtin or IT15 gene. Patients are plagued by early cognitive signs, motor deficits, and psychiatric disturbances. Symptoms are attributed to cell death in the striatum and disruption of cortical–striatal circuitry. Mechanisms of cell death are unclear, but processes involving mitochondrial abnormalities, excitotoxicity, and abnormal protein degradation have been implicated. Many factors likely contribute to neuron death and dysfunction, and this has made it difficult to systematically address the pathology in HD. Pharmaceutical therapies are commonly used in patients to treat disease symptoms. These have limited benefit and do not address the inexorable disease progression. Several neuroprotective therapies are being evaluated in animal models of HD as well as in clinical trials. Similarly, cell replacement strategies such as fetal transplantation have been used in the clinic with minimal success, making future cell replacement strategies such as stem cell therapy uncertain. This review describes the disease pathology in HD and addresses many of the past and emerging therapeutic strategies.
Differential Loss of Presynaptic Dopaminergic Markers in Parkinsonian Monkeys
Authors: Stephenson Diane T. , Childs Mary Abigail , Li Qiu , Carvajal-Gonzalez Santos , Opsahl Alan , Tengowski Mark , Meglasson Martin D. , Merchant Kalpana , Emborg Marina E.
Published: September 2014

Assessment of dopamine nerve terminal function and integrity is a strategy employed to monitor deficits in Parkinson's disease (PD) patients and in preclinical models of PD. Dopamine replacement therapies effectively replenish the diminished supply of endogenous dopamine and provide symptomatic benefit to patients. Tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), and amino acid decarboxylase (AADC) are widely used markers of dopaminergic neurons and terminals. The present studies were initiated to: (a) assess alterations in all four markers in the MPTP primate model of dopaminergic degeneration and (b) to determine whether L-DOPA treatment may itself modulate the expression of these markers. MPTP treatment induced a significant decline of dopaminergic immunoreactive fiber and terminal density in the basal ganglia. The amount of reduction varied between markers. The rank order of presynaptic marker loss, from most to least profound reduction, was TH > VMAT2 > DAT > AADC. Semiquantitative image analysis of relative dopaminergic presynaptic fiber and terminal density illustrated region-specific reduction of all four markers. Double immunofluorescence colocalization of two presynaptic markers on the same tissue section confirmed there was a more dramatic loss of TH than of VMAT2 or of DAT following MPTP treatment. L-DOPA treatment was associated with a significantly higher level of AADC and VMAT2 immunoreactivity in the caudate nucleus compared to placebo. These results illustrate that neurotoxic injury of the dopamine system in primates leads to altered and differential expression of presynaptic dopaminergic markers in the basal ganglia and that expression of such markers may be modulated by L-DOPA therapy. These findings have implications for the use of biomarkers of disease progression as well as for the assessment of neurorestorative strategies, such as cell replacement, for the treatment of PD.
Schwann Cell Transplantation Improves Reticulospinal Axon Growth and Forelimb Strength after Severe Cervical Spinal Cord Contusion
Authors: Schaal S. M. , Kitay B. M. , Cho K. S. , Lo T. P. , Barakat D. J. , Marcillo A. E. , Sanchez A. R. , Andrade C. M. , Pearse D. D.
Published: September 2014

Schwann cell (SC) implantation alone has been shown to promote the growth of propriospinal and sensory axons, but not long-tract descending axons, after thoracic spinal cord injury (SCI). In the current study, we examined if an axotomy close to the cell body of origin (so as to enhance the intrinsic growth response) could permit supraspinal axons to grow onto SC grafts. Adult female Fischer rats received a severe (C5) cervical contusion (1.1 mm displacement, 3 KDyn). At 1 week postinjury, 2 million SCs ex vivo transduced with lentiviral vector encoding enhanced green fluorescent protein (EGFP) were implanted within media into the injury epicenter; injury-only animals served as controls. Animals were tested weekly using the BBB score for 7 weeks postimplantation and received at end point tests for upper body strength: self-supported forelimb hanging, forearm grip force, and the incline plane. Following behavioral assessment, animals were anterogradely traced bilaterally from the reticular formation using BDA-Texas Red. Stereological quantification revealed a twofold increase in the numbers of preserved NeuN+ neurons rostral and caudal to the injury/graft site in SC implanted animals, corroborating previous reports of their neuroprotective efficacy. Examination of labeled reticulospinal axon growth revealed that while rarely an axon was present within the lesion site of injury-only controls, numerous reticulospinal axons had penetrated the SC implant/lesion milieu. This has not been observed following implantation of SCs alone into the injured thoracic spinal cord. Significant behavioral improvements over injury-only controls in upper limb strength, including an enhanced grip strength (a 296% increase) and an increased self-supported forelimb hanging, accompanied SC-mediated neuroprotection and reticulospinal axon growth. The current study further supports the neuroprotective efficacy of SC implants after SCI and demonstrates that SCs alone are capable of supporting modest supraspinal axon growth when the site of axon injury is closer to the cell body of the axotomized neuron.
Stem Cell-Based Cell Therapy for Spinal Cord Injury
Authors: Kim Byung Gon , Hwang Dong Hoon , Lee Seung Im , Kim Eun Jeong , Kim Seung U.
Published: September 2014

Traumatic injuries to the spinal cord lead to severe and permanent neurological deficits. Although no effective therapeutic option is currently available, recent animal studies have shown that cellular transplantation strategies hold promise to enhance functional recovery after spinal cord injury (SCI). This review is to analyze the experiments where transplantation of stem/progenitor cells produced successful functional outcome in animal models of SCI. There is no consensus yet on what kind of stem/progenitor cells is an ideal source for cellular grafts. Three kinds of stem/progenitor cells have been utilized in cell therapy in animal models of SCI: embryonic stem cells, bone marrow mesenchymal stem cells, and neural stem cells. Neural stem cells or fate-restricted neuronal or glial progenitor cells were preferably used because they have clear capacity to become neurons or glial cells after transplantation into the injured spinal cord. At least a part of functional deficits after SCI is attributable to chronic progressive demyelination. Therefore, several studies transplanted glial-restricted progenitors or oligodendrocyte precursors to target the demyelination process. Directed differentiation of stem/progenitor cells to oligodendrocyte lineage prior to transplantation or modulation of microenvironment in the injured spinal cord to promote oligodendroglial differentiation seems to be an effective strategy to increase the extent of remyelination. Transplanted stem/progenitor cells can also contribute to promoting axonal regeneration by functioning as cellular scaffolds for growing axons. Combinatorial approaches using polymer scaffolds to fill the lesion cavity or introducing regeneration-promoting genes will greatly increase the efficacy of cellular transplantation strategies for SCI.
Time Course and Quantification of Pancreatic Islet Revasculariztion following Intraportal Transplantation
Authors: Jones Gareth L. , Juszczak Maciej T. , Hughes Stephen J. , Kooner Paul , Powis Stephen H. , Press Martin
Published: September 2014

A large proportion of islets are lost after transplantation partly due to a lack of functional vasculature. Islets revascularize from host tissue but the process takes up to 2 weeks and has been suggested to result in reduced vascular density in engrafted islets. We describe a method for observing and quantifying the revascularization of intraportally transplanted islets that includes number, density, and branching of islet capillaries. Syngeneic islets were transplanted selectively into the two right posterior lobes of the liver of adult Lewis rats. Sections of the livers were dual stained for insulin and Bandeiraea simplicifolia and analyzed for islet morphology, area, and vascular density from day 0 to day 14 posttransplant and compared to native islets. Vascular density was 1431 ± 75.7 vessels/mm2 in native islets and fell to 325.3 ± 30.8 vessels/mm2 (p < 0.001) by day 1 posttransplant and subsequently increased until day 14 when it was significantly higher than in native islets (2612.5 ± 107.8 vessels/mm2, p < 0.001). The percentage of islet area occupied by vascular space was 9.1 ± 0.9% in native islets. After falling to 2.3 ± 0.3% (p < 0.001) 1 day posttransplant this rose to supranormal levels (21.5 ± 0.8%, p < 0.001) by day 14. The index of capillary branching was 0.771 ± 0.017 in native islets and fell to 0.465 ± 0.02 (p = 0.001) by day 3 but returned to native values by day 7 posttransplantation (0.726 ± 0.03). This technique provides a robust method for tracking and quantifying the revascularization of intraportally transplanted islets, which should enable the comparison of different strategies aimed at accelerating islet revascularization.
Cryopreservation-Induced Nonattachment of Human Hepatocytes: Role of Adhesion Molecules
Authors: Terry Claire , Hughes Robin D. , Mitry Ragai R. , Lehec Sharon C. , Dhawan Anil
Published: September 2014

Good quality cryopreserved human hepatocytes are becoming an important source for clinical hepatocyte transplantation. However, the process of cryopreservation leads to both structural and functional impairment of hepatocytes. The aim of this study was to investigate the mechanisms of cryopreservation-induced nonattachment in human hepatocytes. Hepatocytes were cryopreserved after isolation from unused donor liver tissue. Cell attachment to collagen-coated plates was measured. A cDNA gene array system for 96 cell adhesion-related molecules was used to determine mRNA expression in fresh and cryopreserved hepatocytes. Two cell adhesion molecule proteins were investigated further: β1-integrin, a cell-matrix adhesion molecule, and E-cadherin, a cell–cell adhesion molecule. Attachment efficiency was significantly decreased after cryopreservation of human hepatocytes. Twenty-two genes were downregulated after cryopreservation including integrins, cadherins, catenins, and matrix metalloproteinases (MMPs). β1-Integrin gene and protein expression were significantly decreased in cultured cryopreserved hepatocytes compared to fresh hepatocytes. There was a significant correlation between loss of β1-integrin and attachment in cryopreserved cells. Degradation of E-cadherin was increased in cryopreserved hepatocytes. The process of cryopreservation leads to downregulation of cell adhesion molecules at the gene and the cellular level. New cryopreservation protocols are needed to prevent these effects on cell attachment.
Toward Maximizing the Success Rates of Human Islet Isolation: Influence of Donor and Isolation Factors
Authors: Ponte Gaston M. , Pileggi Antonello , Messinger Shari , Alejandro Angel , Ichii Hirohito , Baidal David A. , Khan Aisha , Ricordi Camillo , Goss John A. , Alejandro Rodolfo
Published: September 2014

In order to make islet transplantation a therapeutic option for patients with diabetes there is an urgent need for more efficient islet cell processing to maximize islet recovery. Improved donor management, organ recovery techniques, implementation of more stringent donor criteria, and improved islet cell processing techniques may contribute to enhance organ utilization for transplantation. We have analyzed the effects of donor and islet processing factors on the success rate of human islet cell processing for transplantation performed at a single islet cell processing center. Islet isolation outcomes improved when vasopressors, and in particular pitressin, and steroids were used for the management of multiorgan donors. Higher islet yields were obtained from adult male donors, BMI >25 kg/m2, adequate glycemic control during hospital stay, and when the pancreas was retrieved by a local surgical team. Successful isolations were obtained in 58% of the cases when ≥ 4 donor criteria were met, and even higher success rates (69%) were observed when considering ≥ 5 criteria. Our data suggest that a sequential, integrated approach is highly desirable to improve the success rate of islet cell processing.
Aging Reduces the Neuroprotective Capacity, VEGF Secretion, and Metabolic Activity of Rat Choroid Plexus Epithelial Cells
Authors: Emerich Dwaine F. , Schneider Patricia , Bintz Briannan , Hudak Jebecka , Thanos Christopher G.
Published: September 2014

Delivery of neurotrophic molecules to the brain has potential for preventing neuronal loss in neurodegenerative disorders. Choroid plexus (CP) epithelial cells secrete numerous neurotrophic factors, and encapsulated CP transplants are neuroprotective in models of stroke and Huntington's disease (HD). To date, all studies examining the neuroprotective potential of CP transplants have used cells isolated from young donor animals. Because the aging process significantly impacts the cytoarchitecture and function of the CP the following studies determined whether age-related impairments occur in its neuroprotective capacity. CP was isolated from either young (3–4 months) or aged (24 months) rats. In vitro, young CP epithelial cells secreted more VEGF and were metabolically more active than aged CP epithelial cells. Additionally, conditioned medium from cultured aged CP was less potent than young CP at enhancing the survival of serum-deprived neurons. Finally, encapsulated CP was tested in an animal model of HD. Cell-loaded or empty alginate capsules (control group) were transplanted unilaterally into the rat striatum. Seven days later, the animals received an injection of quinolinic acid (QA; 225 nmol) adjacent to the implant site. Animals were tested for motor function 28 days later. In the control group, QA lesions severely impaired function of the contralateral forelimb. Implants of young CP were potently neuroprotective as rats receiving CP transplants were not significantly impaired when tested for motor function. In contrast, implants of CP from aged rats were only modestly effective and were much less potent than young CP transplants. These data are the first to directly link aging with diminished neuroprotective capacity of CP epithelial cells.
Donor–acceptor graphene-based hybrid materials facilitating photo-induced electron-transfer reactions
Authors: Stergiou Anastasios , Pagona Georgia , Tagmatarchis Nikos
Published: September 2014

Graphene research and in particular the topic of chemical functionalization of graphene has exploded in the last decade. The main aim is to increase the solubility and thereby enhance the processability of the material, which is otherwise insoluble and inapplicable for technological applications when stacked in the form of graphite. To this end, initially, graphite was oxidized under harsh conditions to yield exfoliated graphene oxide sheets that are soluble in aqueous media and amenable to chemical modifications due to the presence of carboxylic acid groups at the edges of the lattice. However, it was obvious that the high-defect framework of graphene oxide cannot be readily utilized in applications that are governed by charge-transfer processes, for example, in solar cells. Alternatively, exfoliated graphene has been applied toward the realization of some donor–acceptor hybrid materials with photo- and/or electro-active components. The main body of research regarding obtaining donor–acceptor hybrid materials based on graphene to facilitate charge-transfer phenomena, which is reviewed here, concerns the incorporation of porphyrins and phthalocyanines onto graphene sheets. Through illustrative schemes, the preparation and most importantly the photophysical properties of such graphene-based ensembles will be described. Important parameters, such as the generation of the charge-separated state upon photoexcitation of the organic electron donor, the lifetimes of the charge-separation and charge-recombination as well as the incident-photon-to-current efficiency value for some donor–acceptor graphene-based hybrids, will be discussed.
Photochemical approach to functionalized benzobicyclo[3.2.1]octene structures via fused oxazoline derivatives from 4- and 5-(o-vinylstyryl)oxazoles
Authors: Šagud Ivana , Božić Simona , Marinić Željko , Šindler-Kulyk Marija
Published: September 2014

Novel cis/trans-4- and cis/trans-5-(2-vinylstyryl)oxazoles have been synthesized by Wittig reactions from the diphosphonium salt of α,α’-o-xylene dibromide, formaldehyde and 4- and 5-oxazolecarbaldehydes, respectively. In contrast, trans-5-(2-vinylstyryl)oxazole has been synthesized by the van Leusen reaction from trans-3-(2-vinylphenyl)acrylaldehyde which is prepared from o-vinylbenzaldehyde and (formylmethylene)triphenylphosphorane. The 4- and 5-(2-vinylstyryl)oxazoles afford, by photochemical intramolecular cycloaddition, diverse fused oxazoline-benzobicyclo[3.2.1]octadienes, which are identified and characterized by spectroscopic methods. The photoproducts formed are relatively unstable and spontaneously or on silica gel undergo oxazoline ring opening followed by formation of formiato- or formamido-benzobicyclo[3.2.1]octenone derivatives. On irradiation of 4-(2-vinylstyryl)oxazole small quantities of electrocyclization product, 4-(1,2-dihydronaphthalen-2-yl)oxazole, are isolated and spectroscopically characterized.
UK CONSTRUCTION COMPANIES’ STRATEGIES IN THE FACE OF BUSINESS CYCLES
Authors: Ruddock Les , Kheir Amir , Ruddock Steven
Published: September 2014

Firms in the construction industry have always had to deal with the challenges of the economic cycle and develop strategies to deal with the resulting fluctuations in their business environment. In the context of the 2008–2011 double-dip recession in the UK, the results of a survey targeting the top one hundred construction companies in the UK are reported here. This research is particularly intended to assess whether the strategies of large companies in the construction sector, when faced with the issues associated with the variation in the economic cycle, have changed since the previous business cycle (i.e. the 1986–1990 boom followed by the 1990–1991 recession). The survey reveals the challenges that companies have faced, reports on company behaviour and on the policies adopted. While there are many similarities between policies adopted during the recessionary periods of the two cycles, the research found notable changes in attitudes towards diversification, human resource management and price bidding.