bluuebunny/crossref_metadata_2025 huggingface dataset:
The moderating effect of higher education on the intergenerational transmission of residing in poverty neighbourhoods
Authors: de Vuijst Elise , van Ham Maarten , Kleinhans Reinout
Published: June 2017

It is well-known that socioeconomic outcomes and (dis)advantage over the life course can be transmitted from parent to child. It is increasingly suggested that these intergenerational effects also have a spatial dimension, although empirical research into this topic remains scarce. Previous research from Sweden and the United States shows that children who grow up in disadvantaged neighbourhoods experience long-term exposure to such neighbourhoods in their adult lives. This study contributes to the literature by examining to what extent educational attainment can break the link between parental neighbourhood disadvantage and the neighbourhood experiences of children as adults up to 12 years after leaving the parental home. We use longitudinal register data from the Netherlands to study a complete cohort of parental home leavers, covering 119,167 individuals who were followed from 1999 to 2012. Using sequence analyses as a visualisation method, and multilevel logit models, we demonstrate that children who lived in deprived neighbourhoods with their parents are more likely to live in similar neighbourhoods later in life than children who grew up in more affluent neighbourhoods. We find that intergenerational neighbourhood patterns of disadvantage can be discontinued when individuals attain higher education over time. Discontinuation is however less prevalent among individuals from ethnic minority groups.
Mild Cognitive Impairment and Changes in Everyday Function Over Time: The Importance of Evaluating Both Speed and Accuracy
Authors: Lassen-Greene Caroline L. , Steward Kayla , Okonkwo Ozioma , Porter Ellen , Crowe Michael , Vance David E. , Griffith H. Randall , Ball Karlene , Marson Daniel C. , Wadley Virginia G.
Published: June 2017

Objectives:

Research estimates that a significant percentage of individuals with mild cognitive impairment (MCI) experience functional difficulties. In addition to reduced accuracy on measures of everyday function, cross-sectional research has demonstrated that speed of performing instrumental activities of daily living (IADLs) is slowed in individuals with MCI. The present study investigated whether baseline and longitudinal changes in speed and accuracy of IADL performance differed between persons with MCI and cognitively normal peers.

Design:

Linear mixed models were used to estimate the group differences in longitudinal performance on measures of IADLs.

Setting:

Assessments were conducted at university and medical research centers.

Participants:

The sample consisted of 80 participants with MCI and 80 control participants who were enrolled in the Alzheimer’s Disease Research Center’s Measuring Independent Living in the Elderly Study.

Measurements:

Instrumental activities of daily living speed and accuracy were directly assessed using selected domains of the Financial Capacity Instrument, the Timed IADL assessment, and driving-related assessments (Useful Field of View, Road Sign Test).

Results:

Individuals with MCI performed worse on speed and accuracy measures of IADLs in comparison to cognitively normal peers and demonstrated significantly steeper rates of decline over three years in either speed or accuracy in all domains assessed.

Conclusion:

Both speed and accuracy of performance on measures of IADL are valuable indices for early detection of functional change in MCI. The performance pattern may reflect a trade-off between speed and accuracy that can guide clinical recommendations for maintaining patient independence.
Evolution of design considerations in complex craniofacial reconstruction using patient-specific implants
Authors: Peel Sean , Bhatia Satyajeet , Eggbeer Dominic , Morris Daniel S , Hayhurst Caroline
Published: June 2017

Previously published evidence has established major clinical benefits from using computer-aided design, computer-aided manufacturing, and additive manufacturing to produce patient-specific devices. These include cutting guides, drilling guides, positioning guides, and implants. However, custom devices produced using these methods are still not in routine use, particularly by the UK National Health Service. Oft-cited reasons for this slow uptake include the following: a higher up-front cost than conventionally fabricated devices, material-choice uncertainty, and a lack of long-term follow-up due to their relatively recent introduction. This article identifies a further gap in current knowledge – that of design rules, or key specification considerations for complex computer-aided design/computer-aided manufacturing/additive manufacturing devices. This research begins to address the gap by combining a detailed review of the literature with first-hand experience of interdisciplinary collaboration on five craniofacial patient case studies. In each patient case, bony lesions in the orbito-temporal region were segmented, excised, and reconstructed in the virtual environment. Three cases translated these digital plans into theatre via polymer surgical guides. Four cases utilised additive manufacturing to fabricate titanium implants. One implant was machined from polyether ether ketone. From the literature, articles with relevant abstracts were analysed to extract design considerations. In all, 19 frequently recurring design considerations were extracted from previous publications. Nine new design considerations were extracted from the case studies – on the basis of subjective clinical evaluation. These were synthesised to produce a design considerations framework to assist clinicians with prescribing and design engineers with modelling. Promising avenues for further research are proposed.
Knee joint kinetics in response to multiple three-dimensional printed, customised foot orthoses for the treatment of medial compartment knee osteoarthritis
Authors: Allan Richard , Woodburn James , Telfer Scott , Abbott Mandy , Steultjens Martijn PM
Published: June 2017

The knee adduction moment is consistently used as a surrogate measure of medial compartment loading. Foot orthoses are designed to reduce knee adduction moment via lateral wedging. The ‘dose’ of wedging required to optimally unload the affected compartment is unknown and variable between individuals. This study explores a personalised approach via three-dimensional printed foot orthotics to assess the biomechanical response when two design variables are altered: orthotic length and lateral wedging. Foot orthoses were created for 10 individuals with symptomatic medial knee osteoarthritis and 10 controls. Computer-aided design software was used to design four full and four three-quarter-length foot orthoses per participant each with lateral posting of 0° ‘neutral’, 5° rearfoot, 10° rearfoot and 5° forefoot/10° rearfoot. Three-dimensional printers were used to manufacture all foot orthoses. Three-dimensional gait analyses were performed and selected knee kinetics were analysed: first peak knee adduction moment, second peak knee adduction moment, first knee flexion moment and knee adduction moment impulse. Full-length foot orthoses provided greater reductions in first peak knee adduction moment (p = 0.038), second peak knee adduction moment (p = 0.018) and knee adduction moment impulse (p = 0.022) compared to three-quarter-length foot orthoses. Dose effect of lateral wedging was found for first peak knee adduction moment (p < 0.001), second peak knee adduction moment (p < 0.001) and knee adduction moment impulse (p < 0.001) indicating greater unloading for higher wedging angles. Significant interaction effects were found for foot orthosis length and participant group in second peak knee adduction moment (p = 0.028) and knee adduction moment impulse (p = 0.036). Significant interaction effects were found between orthotic length and wedging condition for second peak knee adduction moment (p = 0.002). No significant changes in first knee flexion moment were found. Individual heterogeneous responses to foot orthosis conditions were observed for first peak knee adduction moment, second peak knee adduction moment and knee adduction moment impulse. Biomechanical response is highly variable with personalised foot orthoses. Findings indicate that the tailoring of a personalised intervention could provide an additional benefit over standard interventions and that a three-dimensional printing approach to foot orthosis manufacturing is a viable alternative to the standard methods.
A Bayesian approach to linking archaeological, paleoenvironmental and documentary datasets relating to the settlement of Iceland (Landnám)
Authors: Schmid Magdalena ME , Zori Davide , Erlendsson Egill , Batt Cathy , Damiata Brian N , Byock Jesse
Published: June 2017

Icelandic settlement (Landnám) period farmsteads offer opportunities to explore the nature and timing of anthropogenic activities and environmental impacts of the first Holocene farming communities. We employ Bayesian statistical modelling of archaeological, paleoenvironmental and documentary datasets to present a framework for improving chronological robustness of archaeological events. Specifically, we discuss events relevant to the farm Hrísbrú, an initial and complex settlement site in southwest Iceland. We demonstrate that tephra layers are key in constraining reliable chronologies, especially when combined with related datasets and treated in a Bayesian framework. The work presented here confirms earlier interpretations of the chronology of the site while providing increased confidence in the robustness of the chronology. Most importantly, integrated modelling of AMS radiocarbon dates on Hordeum vulgare grains, palynological data, documented evidence from textual records and typologically diagnostic artefacts yield increased dating reliability. The analysis has also shown that AMS radiocarbon dates on bone collagen need further scrutiny. Specifically for the Hrísbrú farm, first anthropogenic footprint palynomorph taxa are estimated to around AD 830–881 (at 95.4% confidence level), most likely before the tephra fall out of AD 877 ± 1 (the Landnám tephra layer), demonstrating the use of arable fields before the first known structures were built at Hrísbrú (AD 874–951) and prior to the conventionally accepted date of the settlement of Iceland. Finally, we highlight the importance of considering multidisciplinary factors for other archaeological and paleoecological studies of early farming communities of previously uninhabited island areas.
In Vitro Characterization of Porcine Hepatocyte Function
Authors: Gregory Paul G. , Connolly Christopher K. , Toner Mehmet , Sullivan Susan J.
Published: June 2017

The clinical consequences of acute liver failure are associated with high mortality. Intensive medical intervention is required to treat the symptoms of liver failure, including coagulopathy, metabolic instability, and encephalopathy. Providing temporary liver support with an extracorporeal liver assist device could stabilize the patient until a donor liver became available or the patient's own liver was able to recover. The use of human hepatocytes as the biologic component of the assist device is precluded by the scarcity of available tissue and the limited proliferative potential of adult hepatocytes in vitro. Consequently, porcine hepatocytes are being evaluated as a cell source for liver assist devices. Maintaining differentiated function in isolated hepatocytes, however, remains a challenge in the development of this technology and is complicated by the fact that the key therapeutic functions for short-term survival have not been well defined. Several approaches have been effective in prolonging rodent hepatocyte function in vitro, including manipulation of extracellular matrix. Here, we have investigated porcine hepatocyte function in vitro with a specific emphasis on the response to exogenous collagen matrix. In control cultures, albumin secretion increased during the first 7–10 days of culture to an average of 50 ± 17 μg/day/106 cells and then decreased over the next 2 weeks. The pattern of urea synthesis was slightly different in that it was highest in the first 1–3 days postisolation (140 ± 19 μg/day/106 cells) and then decreased by about 50% to a plateau level that was stable during the next 3–4 weeks of culture. Cytochrome P450-mediated activities were the most labile with time in culture and were undetectable after the first week in the absence of pharmacological inducers. In contrast to results reported for rat cells, porcine hepatocytes exhibited differentiated function in the absence of any modification of the culture dish surface and function was not increased or prolonged in the presence of exogenous collagen.
Branched Hybrid Vessel: In Vitro Loaded Hydrodynamic Forces Influence the Tissue Architecture
Authors: Kobashi Teisei , Matsuda Takehisa
Published: June 2017

This study was conducted to investigate how a continuous load of hydrodynamic stresses influences the tissue architecture of a branched hybrid vessel in vitro. Tubular hybrid medial tissue of small (3 mm) and large (6 mm) diameters, prepared by thermal gelation of a cold mixed solution of bovine smooth muscle cells (SMCs) and type I collagen in glass molds, was assembled into a branched hybrid medial tissue by end-to-side anastomosis. After a 2-week culture period, bovine endothelial cells (ECs) were seeded onto the luminal surface. The branched hybrid vessel was connected to a mock circulatory loop system and tested for two modes of flow: 1) low flow rate for 24 h, 2) high flow rate for 24 or 72 h. After exposure to a low flow rate for 24 h, cobblestone appearance of the ECs was dominant. After exposure to a high flow rate, EC alignment in the direction of flow was observed in the branch region, except at the region of predicted flow separation where ECs retained their polygonal configuration. Elongation of SMCs with no preferential orientation was observed in the case of vessels exposed to a high flow rate for 24 h, and circumferential orientation was prominent in those exposed to a high flow rate for 72 h. On the other hand, collagen fibrils exhibited no preferential orientation in either case. After injection of Evans blue–albumin conjugate into the circulating medium, the luminal surface of the hybrid vessel exposed to a high flow rate for 24 h was examined by confocal laser scanning microscopy. The fluorescence intensity was low at the high shear zone in the branch region, while at the flow separation region it was very high, indicating the increased albumin permeability at the latter region. These findings reflect region-specific tissue architecture in the branch region, in response to the local flow pattern, and may provide an in vitro atherosclerosis model as well as a fundamental basis for the development of functional branched hybrid grafts.
Improved Vascularization of Planar Membrane Diffusion Devices following Continuous Infusion of Vascular Endothelial Growth Factor
Authors: Trivedi N. , Steil G. M. , Colton C. K. , Bonner-Weir S. , Weir G. C.
Published: June 2017

Improving blood vessel formation around an immunobarrier device should improve the survival of the encapsulated tissue. In the present study we investigated the formation of new blood vessels around a planar membrane diffusion device (the Baxter Theracyte System™) undergoing a continuous infusion of vascular endothelial growth factor through the membranes and into the surrounding tissue. Each device (20 μl) had both an inner immunoisolation membrane and an outer vascularizing membrane. Human recombinant vascular endothelial growth factor-165 was infused at 100 ng/day (low dose: n = 6) and 500 ng/day (high dose: n = 7) for 10 days into devices implanted SC in Sprague-Dawley rats; noninfused devices transplanted for an identical period were used as controls (n = 5). Two days following the termination of VEGF infusion, devices were loaded with 20 μl of Lispro insulin (1 U/kg) and the kinetics of insulin release from the lumen of the device was assessed. Devices were then explanted and the number of blood vessels (capillary and noncapillary) was quantified using morphometry. High-dose vascular endothelial growth factor infusion resulted in two- to threefold more blood vessels around the device than that obtained with the noninfused devices and devices infused with low-dose vascular endothelial growth factor. This increase in the number of blood vessels was accompanied by a modest increase in insulin diffusion from the device in the high-dose vascular endothelial growth factor infusion group. We conclude that vascular endothelial growth factor can be used to improve blood vessel formation adjacent to planar membrane diffusion devices.
The Use of Adenoviral Vectors and Ex Vivo Transduced Neurotransplants: Towards Promotion of Neuroregeneration
Authors: Blits Bas , Dijkhuizen Paul A. , Hermens Wim T. J. M. C. , Van Esseveldt Lisette K. E. , Boer Gerard J. , Verhaagen Joost
Published: June 2017

Regeneration of injured axons following injury depends on a delicate balance between growth-promoting and growth-inhibiting factors. Overexpression of neurotrophin genes seems a promising strategy to promote regeneration. Trophic genes can be overexpressed at the site of injury at the axonal stumps, or at the perikaryal level of the injured neuron. Transduction of the neural cells can be achieved by applying adenoviral vectors, either directly in vivo or—in the case of neurotransplantation—as an ex vivo approach. In both cases it would create a more permissive environment for axonal growth and therefore in functional regeneration. In this article, the feasibility of the use of adenoviral vectors in several neuroregeneration models–-in particularly in spinal cord lesion models and the biological clock transplantation model–-is illustrated. The results show that the adenoviral vectors can be a powerful tool to study the effects of overexpression of genes in an in vivo paradigm of nerve regeneration or nerve outgrowth. The potential use of adenoviral vectors and ex vivo transduced neurotransplants is discussed.
Implication of the Subthalamic Nucleus in the Pathophysiology and Pathogenesis of Parkinson's Disease
Authors: Benazzouz A. , Piallat B. , Ni Z. G. , Koudsie A. , Pollak P. , Benabid A. L.
Published: June 2017

The subthalamic nucleus (STN) has been shown to play an important role in the control of movement and has been considered as a key structure in the functional organization of the basal ganglia. Several studies postulated that the STN plays a critical role in the pathophysiology of Parkinson's disease and that its inhibition or its lesioning can reverse the cardinal motor symptoms. Nevertheless, the beneficial effect was accompanied by dyskinetic abnormal movements. In order to avoid unpleasant and irreversible side effects we used high-frequency stimulation (HFS) of the STN instead of lesions. We have shown that parkinsonian motor symptoms, akinesia, rigidity, and tremor can be alleviated by HFS of the STN in the nonhuman primate model. Side effects were controllable and appeared only at intensities higher than that inducing the improvement of motor symptoms. In severe parkinsonian patients, bilateral STN-HFS greatly improved parkinsonian motor symptoms. Motor fluctuations were attenuated and patients became independent in most activities of daily living. It appears that STN-HFS mimics the effects of lesions by inhibiting its neuronal activity. In a rat model of parkinsonism, we studied the implication of the STN in the excitotoxicity of nigral dopamine cells. We showed that kainic acid lesioning of the STN can protect nigral dopaminergic cells against 6-hydroxydopamine-induced toxicity. The evidence reviewed in the present article clearly demonstrates that the STN is implicated in the pathophysiology and pathogenesis of Parkinson's disease.
Neural Tissue Xenotransplantation: What is Needed Prior to Clinical Trials in Parkinson's Disease?
Authors: Barker Roger A. , Kendall A. Lisa , Widner Håkan ,   Neural Tissue Xenografting Project, Widner H. , Larsson L. , Czech K. A. , Anderson P. , Mirza B. , Dunnett S. B. , Kendall A. L. , Annett L.E. , Barker R. , Torres E. , Rosser A. E. , Watts C. , Zimmer J. , Finsen B. R. , Dahl-Jørgensen A. , Pedersen E. B. , Brevig T. , Meyer M. , Holgersson J. , Korsgren O. , Breimer M. , Gjedde A. , Danielsen E.
Published: June 2017

Embryonic allografted human tissue in patients with Parkinson's disease has been shown to survive and ameliorate many of the symptoms of this disease. Despite this success, the practical problems of using this tissue coupled to the ethical restrictions of using aborted human fetal tissue have lead to an exploration for alternative sources of suitable material for grafting, including xenogeneic embryonic dopaminergic-rich neural tissue. Nevertheless, xenografted neural tissue itself generates a number of practical, ethical, safety, and immunological issues that have to be addressed prior to any clinical xenotransplant program. In this article we review these critical issues and set out the criteria that we consider need to be met in the development of our clinical xenotransplantation research programs. We advocate that these, or similar, criteria should be adopted and made explicit by other centers contemplating similar clinical trials.
Isolation of Adult Porcine Islets of Langerhans
Authors: Toso Christian , Brandhorst Daniel , Oberholzer José , Triponez Frédéric , Bühler Léo , Morel Philippe
Published: June 2017

While human islet allotransplantation is limited due to the shortage of donors, pig islet xenotransplantation appears to be a possible alternative. Enzymatic digestion is the method most commonly used for adult pig islet isolation, but since the description of the semiautomated isolation technique in the late 1980s, yields of pig islet isolations have not increased significantly. This review article is intended to collate published information on adult pig islet isolation to provide the reader with a clear overview of the relevant issues in this field, and to detail the current state of the art.
The Morphology, Integration, and Functional Efficacy of Striatal Grafts Differ between Cell Suspensions and Tissue Pieces
Authors: Watts Colin , Brasted Peter J. , Dunnett Stephen B.
Published: June 2017

In order to develop a surgical protocol for use in clinical trials of striatal transplantation in Huntington's disease (HD), the issues involved in the preparation and implantation of the embryonic striatal tissue must be addressed. Rodent models of HD offer the best experimental paradigm with which to study various aspects of striatal transplantation. In this article we present the results of an investigation of the role of trypsin and the process of trituration in the preparation of cell suspensions compared to the use of solid pieces of tissue. The embryonic material was derived from the lateral ganglionic eminence (LGE) and implanted into the excitotoxically lesioned striatum of the host rats. Twelve weeks following implantation, retrograde tracing of projections from the graft to the globus pallidus was performed. Grafts derived from cell suspensions triturated in the presence of trypsin contained larger quantities of striatal tissue within the graft and more DARPP-32-positive medium spiny neurons than grafts implanted as fragments of tissue. Afferent and efferent connectivity was also better in the trypsinized suspension graft group. Modest recovery in paw reaching was observed contralateral to the grafted side in animals implanted with solid fragments of embryonic striatal tissue. No relationship was observed between functional effect and the graft anatomy. These results suggest that local graft–host interaction may also be involved in graft-mediated functional recovery.
Islet Cell Transplantation: In Vivo and in Vitro Functional Assessment of Nonhuman Primate Pancreatic Islets
Authors: Rammcoli Alessandra , Cautero Nicola , Ricordi Camillo , Masetti Michele , Molano Ruth D. , Inverardi Luca , Alejandro Rodolfo , Kenyon Norma S.
Published: June 2017

Transplantation of pancreatic islets of Langerhans as a therapeutic approach for treatment of type I diabetes offers an alternative to subcutaneous insulin injections. Normalization of blood glucose levels by transplanted islets may prevent the development of diabetes-related complications. Problems related to rejection, recurrence of autoimmunity, and local inflammation upon transplantation of islets into the liver need to be solved before the implementation of islet cell transplantation can be viewed as a justifiable procedure in a large cohort of patients. Islet cell isolation has been quite successful in small animals, but the translation of this approach to nonhuman primates has been less rewarding. One of the main problems encountered in nonhuman primate models is the difficulty of isolating an adequate number of functional islets for transplantation. The aim of the present study was to develop a method for isolating a sufficient number of viable islets from nonhuman primates to allow for reversal of diabetes. By implementing minor modifications in the automated method for human islet isolation we were able to obtain viable, functional islets that responded normally to glucose stimulation in vitro. These islets were also able to reverse diabetes in immunocompromised nude mice, rendered diabetic by streptozotocin. This method of islet cell isolation has enabled us to proceed with protocols of allogeneic islet cell transplantation in preclinical, nonhuman primate models.
Protection from Cell Death in Cultured Human Fetal Pancreatic Cells
Authors: Beattie Gillian M. , Leibowitz Gil , Lopez Ana D. , Levine Fred , Hayek Alberto
Published: June 2017

Endocrine cells from the human fetal pancreas will proliferate in vitro on extracellular matrix but lose hormone expression, and redifferentiation requires removal of the expanded cells from the matrix and reaggregation into cell aggregates. However, extensive cell death occurs during manipulation and culture. The mechanism of cell death was examined at each stage throughout the process under different experimental conditions to determine optimal protocols to increase cell viability. During shipment, the addition of trehalose to the media to prevent necrosis increased yield 17-fold, while during culture as islet-like cell clusters the apoptosis inhibitor Z-VAD increased yield 1.8-fold. Following disruption of cell–matrix interactions and reaggregation, there was marked evidence of apoptotic bodies by the TUNEL assay. Addition of nicotinamide or Z-VAD, or removal of arginine from the media during reaggregation, reduced the number of apoptotic bodies and the effect was additive. However, a combination of treatments was necessary to significantly increase the yield of viable cells. We conclude that cell death of human fetal pancreatic tissue in culture results from both necrosis and apoptosis and that understanding the mechanisms at the cellular level will lead to protocols that will improve cell viability and promote β-cell growth.
The Localization and Functional Contribution of Striatal Aromatic L-Amino Acid Decarboxylase to L-3,4-Dihydroxyphenylalanine Decarboxylation in Rodent Parkinsonian Models
Authors: Nakamura Ken , Ahmed Maqbool , Barr Eliav , Leiden Jeffrey M. , Kang Un Jung
Published: June 2017

L-3,4-Dihydroxyphenylalanine (L-dopa) is the mainstay of therapy for patients with Parkinson's disease (PD), and mediates its primary effects through conversion into dopamine by aromatic L-amino acid decarboxylase (AADC). Given the loss of AADC-containing nigrostriatal dopaminergic neurons in PD, however, the location of residual AADC that converts L-dopa into dopamine remains controversial. The first objective of this study was to establish the presence of AADC expression in striatal neurons and glia using reverse transcriptase and PCR. Transcripts for the neuronal but not nonneuronal forms of AADC were detected in striatal tissue, cultured striatal neurons, and glia. We then examined whether this striatal AADC expression represents a physiologically significant source of dopamine production. No dopamine release was detected following incubation of striatal cultures with L-dopa or transduction with adenovirus expressing tyrosine hydroxylase. Our data establish the presence of AADC expression in the striatum both in vivo and in vitro, but suggest that striatal components do not represent a primary source of L-dopa decarboxylation following nigrostriatal denervation in rats. Understanding the source and localization of AADC is important in understanding the complications of L-dopa therapy and in designing rational therapeutic strategies for PD, including cellular transplantation and gene therapy.
Neurotrophic Factors NGF and FGF-2 Alter Levels of Huntingtin (IT15) in Striatal Neuronal Cell Cultures
Authors: Haque Nadia S. K. , Isacson Ole
Published: June 2017

A mutation of the human IT15 gene is responsible for Huntington's disease (HD) and the causative factor in the major neuronal loss observed in the striatum. The growth factors basic fibroblast growth factor (FGF-2), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) improve survival and promote differentiation of striatal neurons, as well as exert a neuroprotective effect when such neurons are challenged with metabolic toxins or excitatory amino acids. Using Western blotting and striatal cell cultures, we found that FGF-2 increased the level of huntingtin in a dose-dependent fashion, whereas NGF decreased huntingtin expression. The neurotrophic factor-specific, dose-dependent effect on striatal levels of huntingtin may be relevant to understanding the normal role of IT15 and developing new therapies against the disease provoking mutated IT15.
Efficient Cre/loxP Site-Specific Recombination in a HepG2 Human Liver Cell Line
Authors: Kobayashi Naoya , Noguchi Hirofumi , Westerman Karen A. , Matsumura Toshihisa , Watanabe Takamasa , Totsugawa Toshinori , Fujiwara Toshiyoshi , Leboulch Philippe , Tanaka Noriaki
Published: June 2017

A worldwide shortage of donor livers is a limiting factor of the clinical application of hepatocyte transplantation (HTX). To resolve this issue, we focused on a reversible immortalization system that allows temporary expansion of primary hepatocyte populations by transfer of an oncogene that can be subsequently excised. As a preliminary test toward this goal, we examined the efficacy of Cre/loxP site-specific recombination in a transformed human liver cell line, HepG2. The present study utilized retroviral transfer of a prototypical immortalizing gene, simian virus 40 large T antigen (SV40Tag), flanked by a pair of loxP recombination targets and adenovirus-mediated Cre/loxP recombination. Here we report that complete elimination of the retroviral transferred oncogene was achieved by site-specific recombination using a replication-deficient recombinant adenovirus vector producing Cre recombinase (Ad-Cre).
Agarose Enhances the Viability of Intraperitoneally Implanted Microencapsulated L929 Fibroblasts
Authors: Lahooti Shahab , Sefton Michael V.
Published: June 2017

To achieve immunoisolation, mouse L929 fibroblasts were encapsulated in ~400 μm poly(hydroxyethyl methacrylate-co-methyl methacrylate) (HEMA-MMA) microcapsules and were subsequently implanted in the peritoneal cavity of syngeneic C3H mice. As a baseline for the use of genetically engineered cells in cell encapsulation therapy, the L929 cells were transfected to express a secreted form of human alkaline phosphatase (SEAP). Implantation of empty microcapsules in a PBS suspension resulted in deformation, aggregation, and poor retrievability of the microcapsules. Incubation of microcapsules with medium containing xenogeneic horse serum prior to implantation increased the thickness of the fibrous tissue surrounding the microcapsules. However, immobilization of the microcapsules in a 4% (w/v) SeaPlaque® agarose gel prior to implantation allowed complete recovery of the microcapsules and prevented their aggregation and deformation. As a result, ~50% of the encapsulated cells remained viable 21 days postimplantation. Moreover, once the viable cells were released from retrieved microcapsules and regrown as monolayers, they expressed SEAP at a level similar to their encapsulated but nonimplanted counterparts.
SUITO Index for Evaluation of Efficacy of Single Donor Islet Transplantation
Authors: Matsumoto Shinichi , Noguchi Hirofumi , Hatanaka Nobuyo , Shimoda Masayuki , Kobayashi Naoya , Jackson Andrew , Onaca Nicholas , Naziruddin Bashoo , Levy Marlon F.
Published: June 2017

Evaluation of engrafted islets mass is important for clinical care of patients after islet transplantation. Recently, we developed the secretory unit of islet transplant objects (SUITO) index, which reflected engrafted islet mass. In this study, we evaluated the SUITO index for the prediction of clinical outcome after single islet transplantation. Single islet transplantations were performed into six type 1 diabetic patients. Isolated islets were quantitatively assessed at the time of transplantation. The SUITO index was calculated as follows: fasting C-peptide (ng/dl)/[fasting blood glucose (mg/dl) – 63] × 1500. Islet yield/recipient's body weight and SUITO index were evaluated, along with HbA1C, relative insulin dose (insulin dose posttransplant/pretransplant), and M-values. HbA1C improved in all cases, irrespective of the SUITO index score or islet yield/body weight. The average SUITO index from postoperative days 3 to 30 ( R2 = 0.728, p < 0.04), but not islet yield/body weight ( R2 = 0.259, p = 0.303), correlated with relative insulin dose. The daily SUITO index strongly correlated with the daily relative insulin dose ( R2 = 0.558, p < 0.0001) and weakly correlated with the daily M-values ( R2 = 0.207, p < 0.02). A SUITO index score of less than 10 was associated with increasing insulin dose even after islet transplantation. The SUITO index seems to be a better predictor of success of islet transplantations than islet yield/body weight. SUITO index is recommended to assess clinical outcome of islet transplantation.
Comparison of Sendai Virus-Mediated Gene Transfer Efficiency to Adhesive and Floating Adipose Tissue-Derived Stem Cells
Authors: Yukawa Hiroshi , Noguchi Hirofumi , Oishi Koichi , Inoue Makoto , Hasegawa Mamoru , Hamaguchi Michinari , Hamajima Nobuyuki , Hayashi Shuji
Published: June 2017

Sendai virus (SeV) vectors have potential clinical applications because they can efficiently introduce foreign genes without toxicity into various organs. A recent study reported the green fluorescent protein (GFP) gene transfer to adipose tissue-derived stem cells (ASCs) with SeV vectors results in more efficient expression of GFP than AdV and identified the preservation of the multilineage potential of ASCs transfected with SeV vectors. This study assessed the gene transfer efficiency to floating ASCs with SeV vectors. Although a slight cytotoxicity was observed, the efficiency of gene transfer to cells in the floating state was much higher at all times and all concentrations at MOIs of 2, 10, and 20 than in the adhesion state. Moreover, ASCs transfected with SeV vectors in floating state have the same potential for their differentiation into specific tissues, such as adipocytes and osteocytes, as untransfected ASCs. These data suggest that SeV transfection to ASCs in the floating state could therefore be useful for gene transfer technology.
Superagonist CD28 Antibody Preferentially Expanded Foxp3-Expressing nTreg Cells and Prevented Graft-Versus-Host Diseases
Authors: Kitazawa Yusuke , Fujino Masayuki , Li Xiao-Kang , Xie Lin , Ichimaru Naotsugu , Okumi Masayoshi , Nonomura Norio , Tsujimura Akira , Isaka Yoshitaka , Kimura Hiromitsu , Hünig Thomas , Takahara Shiro
Published: June 2017

Regulatory lymphocytes play a pivotal role in preventing organ-specific autoimmune disease and in induction and maintenance of tolerance in various experimental transplantation models. The enhancement of the number and activity of peripheral CD4+CD25+ Treg cells is an obvious goal for the treatment of autoimmunity and for the suppression of alloreactions. The present study demonstrates that naturally occurring CD4+CD25+ Treg (nTreg) cells preferentially proliferate to a fourfold increase within 3 days in response to the administration of a single superagonistic CD28-specific monoclonal antibody (supCD28 mAb). The appearance of increased Foxp3 molecules was accompanied with polarization toward a Th2 cytokine profile with decreased production of IFN-γ and increased production of IL-4 and IL-10 in the expanded Treg subset. Adoptive transfer of supCD28 mAb-expanded cells in a graft-versus-host disease (GvHD) model induced a potent inhibition of lethality. These results suggest that this therapeutic effect is mediated by the in vivo expansion of nTreg cells. Taken together, these data demonstrate that supCD28-mAb may target nTreg cells in vivo and maintain and enhance their potent regulatory functions for the treatment GvHD.
Comparative Study of the Cellular Pharmacodynamics of Calcineurin Inhibitors between Patients with Chronic Renal Failure Awaiting Renal Transplantation and Cirrhosis Patients Awaiting Liver Transplantation
Authors: Kihara Yu , Matsuno Naoto , Mijiti Abuduxukuer , Nagao Takeshi , Takeuchi Hironori , Unezaki Sakae , Hirano Toshihiko
Published: June 2017

The in vitro response of peripheral blood mononuclear cells (PBMCs) to the suppressive effects of calcineurin inhibitors is known to correlate with the clinical efficacy of drugs used in renal transplantations. The present study was conducted to examine the differences of PBMC responses to calcineurin inhibitors between chronic renal failure (CRF) patients awaiting renal transplantation and cirrhosis patients awaiting liver transplantation. The study included 99 CRF patients awaiting renal transplantation and 27 cirrhosis patients awaiting liver transplantation. Twenty milliliters of venous blood was taken 1–7 days before transplantation. The in vitro drug concentrations giving 50% inhibition of PBMC blastogenesis stimulated with concanavalin A (IC50s) were calculated. The suppressive effects of tacrolimus against PBMC blastogenesis were more than 10–100 times stronger than those of cyclosporine. The median IC50value for cyclosporine against the CRF PBMCs was not significantly different from the median IC50value against the cirrhosis PBMCs. In contrast, tacrolimus sensitivity in cirrhosis PBMCs is approximately seven times higher than that in CRF PBMCs. The median IC50value for tacrolimus against cirrhosis PBMCs was significantly lower and therefore the effect was stronger in comparison to the CRF PBMCs ( p < 0.001). These data suggest that the PBMCs of cirrhosis patients, in comparison to those of CRF patients, are highly sensitive to the suppressive effect of tacrolimus. However, PBMC sensitivity to cyclosporine was not significantly different between the CRF and cirrhosis patients. These observations raise the possibility that treatment with tacrolimus, rather than cyclosporine, may therefore be a better choice to reduce the risks of allograft rejection in liver transplantation.
Global Expression Profiles in 1-Hour Biopsy Specimens of Human Kidney Transplantation from Donors after Cardiac Death
Authors: Kusaka Mamoru , Kuroyanagi Yoko , Mori Terumi , Nagaoka Kayuri , Sasaki Hitomi , Maruyama Takahiro , Hayakawa Kunihiro , Shiroki Ryoichi , Kurahashi Hiroki , Hoshinaga Kiyotaka
Published: June 2017

Because of the worldwide shortage of renal grafts, kidney transplantation (KTx) from donors after cardiac death (DCD) is an alternative way to obtain KTx from brain-dead donors. Although the prognosis of DCD KTx is gradually improving, the graft often undergoes delayed graft function (DGF), rendering the control of DGF essential for post-KTx patient care. In an attempt to characterize etiology of DGF, genome-wide gene expression profiling was performed using renal biopsy samples performed at 1 h after KTx from DCD and the data were compared with those of KTx from living donors (LD). A total of 526 genes were differentially expressed between them. Genes involved in acute inflammation were activated, while metabolic pathways were consistently downregulated in DCD. These findings imply the inferior performance of the DCD grafts relative to LD grafts. Several genes were identified where the expression levels were correlated well with parameters indicating short- and long-term prognosis of the DCD patients. In addition, several genes encoding secretory proteins were identified that might reflect the performance of the graft and be potential noninvasive biomarkers. These data provide a good source for candidates of biomarkers that are potentially useful for the control of DGF.
Hepatocytes from Fibrotic Liver Possess High Growth Potential in Vivo
Authors: Nishie Manabu , Tateno Chise , Utoh Rie , Kohashi Toshihiko , Masumoto Norio , Kobayashi Naoya , Itamoto Toshiyuki , Tanaka Noriaki , Asahara Toshimasa , Yoshizato Katsutoshi
Published: June 2017

Hepatocyte transplantation is effective for treating liver failure, but healthy donors as a source of hepatocytes are quite limited. The livers of patients with hepatic fibrosis could be an alternative source; however, few reports have examined the nature of hepatocytes from fibrotic livers (f-hepatocytes). In this study, we compared the growth of f-hepatocytes and hepatocytes from normal livers (n-hepatocytes). Hepatocytes were isolated from normal and CCl4-treated wild-type Fischer rats that express dipeptidyl dipeptidase IV (DPPIV) gene (DPPIV+). The n- and f-hepatocytes proliferated in culture at similar rates. Both types of hepatocytes were transplanted into DPPIV- mutant Fischer rats that had been treated with retrorsine to injure the liver and were partially hepatectomized (PHx) before transplantation. Both n- and f-DPPIV+-hepatocytes proliferated and formed colonies. The colony sizes of f-hepatocytes 21 days posttransplantation were approximately three times those of n-hepatocytes. The hepatocytes were analyzed using a fluorescence activated cell sorter (FACS). The FACS profile differed between f- and n-hepatocytes: f-hepatocytes were less granular, less autofluorescent, and smaller than n-hepatocytes. These characteristics of f-hepatocytes resembled those reported for small-sized n-hepatocytes (SHs), which are highly proliferative and preferentially express a unique set of 10 SH genes. However, f-hepatocytes preferentially expressed only five of the SH genes. The expression profile of f-hepatocytes was rather similar to that of proliferating n-hepatocytes in the regenerating liver after PHx. The f-hepatocytes were morphologically normal and did not show any preneoplastic phenotype. These normal and proliferative natures of f-hepatocytes in vivo suggest the fibrotic liver as a source of hepatocytes for transplantation.
Human Cortical Neuronal Cell Line (Hcn-1): Further in Vitro Characterization and Suitability for Brain Transplantation
Authors: Poltorak Maciej , Isono Mitsuo , Freed William J. , Ronnett Gabriele V. , Snyder Solomon H.
Published: June 2017

The human neuronal cell-1 (HCN-1) line has recently been established. Under favorable conditions, these cells differentiate into mature neuronal phenotypes. Here we report on further characterization of these cells. Cultured HCN-1 cells express fibronectin immunoreactivity and grow well on fibronectin substrate but do not respond to human bFGF. In the undifferentiated state, some HCN-1 cells show MHC class I antigen expression. After differentiation, HCN-1 cells and their processes are MHC class I negative. On the other hand, interferon-γ stimulation enhances MHC class I expression but does not induce MHC class II immunoreactivity. Our in vitro data indicate that HCN-1 cells express mixed characteristics, including both neuronal and mesenchymal markers, and are consistent with the suggestion that the HCN-1 cell line resembles an immature neuroepithelial cell precursor with a complex origin.

One possible application of the use of the HCN-1 cells includes intracerebral transplantation. We also examined the survival of dissociated HCN-1 cells implanted into rat brain parenchyma. The host animals were not immunosuppressed. Despite expression of MHC class I antigens, small clusters of HCN-1 cells survived in the rat brain. These xenografts did not induce distinct immunological responses within the host brain tissue. Surviving HCN-1 cells demonstrated similar features to those observed in culture.

Our preliminary results suggest that the HCN-1 cell line would be suitable for intracerebral transplantation in primates or humans. However, it may be that short-term host immunosuppression or addition of HCN-1 cell differentiation factors would be beneficial for enhanced cell survival.
Polymer-Encapsulated PC12 Cells: Long-Term Survival and Associated Reduction in Lesion-Induced Rotational Behavior
Authors: Tresco Patrick A. , Winn Shelley R. , Tan Sanda , Jaeger Christine B. , Greene Lloyd A. , Aebischer Patrick
Published: June 2017

Intrastriatal implantation of a dopaminergic cell line surrounded by a permeable, thermoplastic membrane was investigated as a method of long-term dopamine (DA) delivery within the central nervous system (CNS). An increase in DA release from PC12 cell-loaded capsules maintained in vitro was associated with an increase in mitotic activity of the encapsulated cell line. A significant reduction in apomorphine-induced rotational behavior was observed after PC12 cell-containing capsules were implanted into unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, which was sustained for 24 wk. Four wk after implantation, micro-dialysis studies revealed the presence of DA near PC12 cell-containing capsules, which was comparable to extracellular striatal levels of unlesioned controls. Extracellular striatal DA was undetectable by microdialysis in lesioned animals near empty polymer capsules. Histological analysis after 24 wk in vivo demonstrated that encapsulated PC12 cells survived, continued to express tyrosine hydroxylase, and that encapsulation prevented tumorigenesis. The data suggested that the release of a diffusible substance, most likely DA, from an implant is sufficient to exert a long-term functional influence upon 6-OHDA unilaterally lesioned rats and that capsules containing DA-secreting cells may be an effective method of long-term DA delivery in the CNS.
A Porcine Model for Adipose Tissue-Derived Endothelial Cell Transplantation
Authors: Young Carlton , Jarrell Bruce E. , Hoying James B. , Williams Stuart K.
Published: June 2017

The transplantation of endothelial cells represents a technology which has been suggested for applications ranging from improvement in function of implanted vascular devices to genetic therapy. The use of microvascular endothelial cell transplantation has seen increased use both in animal studies as well as clinical use. This report describes our techniques for the isolation and establishment of initial cultures of microvascular endothelial cells derived from porcine fat. A variety of anatomic sites within the pig were evaluated to determine the appropriateness of different sources of fat for endothelial cell isolation. The properitoneal fat was determined to be optimal due to the predominance of endothelium in this tissue and the ease of isolation of microvascular endothelium following collagenase digestion. The study of endothelial cell transplantation in the porcine model is now possible using the methods described for adipose tissue-derived micro vessel endothelial cell isolation.