bluuebunny/crossref_metadata_2025 huggingface dataset:
Accuracy assessment of surgical planning and three-dimensional-printed patient-specific guides for orthopaedic osteotomies
Authors: Sys Gwen , Eykens Hannelore , Lenaerts Gerlinde , Shumelinsky Felix , Robbrecht Cedric , Poffyn Bart
Published: June 2017

This study analyses the accuracy of three-dimensional pre-operative planning and patient-specific guides for orthopaedic osteotomies. To this end, patient-specific guides were compared to the classical freehand method in an experimental setup with saw bones in two phases. In the first phase, the effect of guide design and oscillating versus reciprocating saws was analysed. The difference between target and performed cuts was quantified by the average distance deviation and average angular deviations in the sagittal and coronal planes for the different osteotomies. The results indicated that for one model osteotomy, the use of guides resulted in a more accurate cut when compared to the freehand technique. Reciprocating saws and slot guides improved accuracy in all planes, while oscillating saws and open guides lead to larger deviations from the planned cut. In the second phase, the accuracy of transfer of the planning to the surgical field with slot guides and a reciprocating saw was assessed and compared to the classical planning and freehand cutting method. The pre-operative plan was transferred with high accuracy. Three-dimensional-printed patient-specific guides improve the accuracy of osteotomies and bony resections in an experimental setup compared to conventional freehand methods. The improved accuracy is related to (1) a detailed and qualitative pre-operative plan and (2) an accurate transfer of the planning to the operation room with patient-specific guides by an accurate guidance of the surgical tools to perform the desired cuts.
What does state-subsidized outsourcing of domestic work do for women’s employment? The Belgian service voucher scheme
Authors: Raz-Yurovich Liat , Marx Ive
Published: June 2017

The European Commission, as well as national governments and national lobby groups, have actively promoted the extension of the domestic service sector since the 1990s in response to the prospect of structurally sluggish employment growth among the least skilled and the perceived need for more social investment. The Belgian service voucher scheme, the most heavily subsidized scheme of this type in the European context, yielded growing numbers of domestic service employees, users and employing companies since its enactment in January 2004. The purpose of this study is to identify whether this scheme was successful in increasing employment rates among low-skilled women in Belgium and to assess whether its employment effects have extended beyond this group of women and affected the employment rates of medium-skilled and highly skilled women. Using time-series analyses and difference-in-differences models for the period ranging from the first quarter of 1999 until the second quarter of 2014, our results demonstrate that the scheme had both short-term and long-term positive effects on employment rates of low-skilled women. However, a reversal in their employment rates during the economic recession is also found, which brought their employment rates to a nadir in 2008. We further found that the scheme’s impact extended beyond the employments of low-skilled women to positively affect the employment rates of the highly skilled women.
Optimal Insulin Treatment in Syngeneic Islet Transplantation
Authors: Merino Juan F. , Nacher Victor , Raurell Mercè , Biarnés Montserrat , Soler Joan , Montanya Eduard
Published: June 2017

Insulin-induced normoglycemia has shown to have a beneficial effect on the outcome of pancreatic islets transplanted to diabetic recipients. The aim of the study was to identify the insulin treatment that can maximize its beneficial effect on islet transplants. Six groups of streptozotocin diabetic C57B1/6 mice were transplanted (Tx) with 100 syngeneic islets, an insufficient beta cell mass to restore normoglycemia, and were treated with insulin as follows: group 1 (n = 9): from day 10 before Tx to day 14 after Tx; group 2 (n = 11): from day 6 before Tx to Tx day; group 3 (n = 11): from Tx day to day 6 after Tx; group 4 (n = 7): from Tx day to day 14 after Tx; group 5 (n = 8): from day 10 to day 24 after Tx; group 6 (n = 18): Tx mice were not treated with insulin. Sixty days after Tx, normoglycemia was achieved in 100% of mice in groups 1, 4, and 5, in 73% of mice in group 2, and in only 45% and 33% of mice in groups 3 and 6, respectively (p < 0.01). Intraperitoneal glucose tolerance, determined only in normoglycemic mice, was similar in groups 1, 2, 4, and normal controls. In contrast, normoglycemic mice from groups 3, 5, and 6, exposed to more severe and prolonged hyperglycemia after Tx, showed higher glucose values after glucose injection, suggesting that hyperglycemia had a long-lasting deleterious effect on transplanted beta cell function. The initially transplanted beta cell mass was maintained in the grafts of normoglycemic mice, but was severely reduced in hyperglycemic mice. Transplanted beta cell mass was similar in normoglycemic groups with normal or impaired glucose tolerance, indicating that impaired glucose tolerance was not due to reduced beta cell mass. In summary, the beneficial effect of insulin-induced normoglycemia on transplanted islets was maximal when insulin treatment was maintained the initial 14 days after transplantation. Exposure to sustained hyperglycemia initially after transplantation had a long-lasting deleterious effect on transplanted islets.
Survival, Neuronal Differentiation, and Fiber Outgrowth of Propagated Human Neural Precursor Grafts in an Animal Model of Huntington's Disease
Authors: Armstrong Richard J. E. , Watts Colin , Svendsen Clive N. , Dunnett Stephen B. , Rosser Anne E.
Published: June 2017

Expanded neural precursor cells provide an attractive alternative to primary fetal tissue for cell replacement therapies in neurodegenerative diseases. In this study we transplanted epigenetically propagated human neural precursor cells into a rat model of Huntington's disease. Neural precursors survived transplantation and large numbers differentiated to express neuronal antigens, including some that expressed DARPP-32, indicating a mature striatal phenotype had been adopted. Neuronal fibers from the grafts projected diffusely throughout the host brain, although there was no evidence that outgrowth was specifically target directed. This study supports the contention that propagated human neural precursors may ultimately be of use in therapeutic neural transplantation paradigms for diseases such as Huntington's disease.
Improving the Survival of Grafted Dopaminergic Neurons: A Review over Current Approaches
Authors: Brundin Patrik , Karlsson Jenny , Emgård Mia , Schierle Gabriele S. Kaminski , Hansson Oskar , Petersén Åsa , Castilho Roger F.
Published: June 2017

Neural transplantation is developing into a therapeutic alternative in Parkinson's disease. A major limiting factor is that only 3–20% of grafted dopamine neurons survive the procedure. Recent advances regarding how and when the neurons die indicate that events preceding actual tissue implantation and during the first week thereafter are crucial, and that apoptosis plays a pivotal role. Triggers that may initiate neuronal death in grafts include donor tissue hypoxia and hypoglycemia, mechanical trauma, free radicals, growth factor deprivation, and excessive extracellular concentrations of excitatory amino acids in the host brain. Four distinct phases during grafting that can involve cell death have been identified: retrieval of the embryo; dissection and preparation of the donor tissue; implantation procedure followed by the immediate period after graft injection; and later stages of graft maturation. During these phases, cell death processes involving free radicals and caspase activation (leading to apoptosis) may be triggered, possibly involving an increase in intracellular calcium. We review different approaches that reduce cell death and increase survival of grafted neurons, typically by a factor of 2–4. For example, changes in transplantation procedure such as improved media and implantation technique can be beneficial. Calcium channel antagonists such as nimodipine and flunarizine improve nigral graft survival. Agents that counteract oxidative stress and its consequences, such as superoxide dismutase overexpression, and lazaroids can significantly increase the survival of transplanted dopamine neurons. Also, the inhibition of apoptosis by a caspase inhibitor has marked positive effects. Finally, basic fibroblast growth factor and members of the transforming growth factor-beta superfamily, such as glial cell line-derived neurotrophic factor, significantly improve the outcome of nigral transplants. These recent advances provide hope for improved survival of transplanted neurons in patients with Parkinson's disease, reducing the need for human embryonic donor tissue and increasing the likelihood of a successful outcome.
Complex Sensorimotor Behavioral Changes after Terminal Striatal 6-OHDA Lesion and Transplantation of Dopaminergic Embryonic Micrografts
Authors: Rödter Alexandra , Winkler Christian , Samii Madjid , Nikkhah Guido
Published: June 2017

In this study sensorimotor behavioral changes were monitored in rats following bilateral 6-hydroxydopamine (6-OHDA) axon terminal lesion and uni- or bilateral implantation of embryonic dopaminergic (DA) micrografts. A total of 28 μg of 6-OHDA was distributed over four injection tracts in the dorsolateral part of the caudate-putamen (CPU) bilaterally followed 4 months later by the implantation of DA micrografts spread over seven implantation tracts placed within the denervated area. Bilaterally 6-OHDA-lesioned animals exhibited significantly reduced behavioral performance scores in tests of explorational and stepping behavior as well as in skilled forelimb use. However, in contrast to the established medial forebrain bundle (MFB) lesion model of PD, these animals showed a spontaneous recovery in the side falling and skilled forelimb behavior and no deficits in overnight locomotor activity at 6 months after the lesion. Unilateral DA micrografts elicited a substantial amphetamine-induced rotational bias contralateral to the graft, but led to a significant impairment of contralateral skilled forelimb use and reduced scores in overnight locomotor activity. Bilateral DA micrografts caused a significant, though partial, increase in explorational and backhand stepping behavior, but resulted also in a significant decrease in performance levels in overnight locomotor activity and skilled forelimb use on both paws. In conclusion, DA grafts placed ectopically in the CPU in the partial lesion model of PD result in a double innervation of the GABAergic striatal neurons, arising from the residual nigrostriatal DA projections of the host and from the graft-derived DA efferent fibers. These two DA fiber systems may indeed function in a cooperative and competitive manner depending on their respective and different afferent and efferent connections, which, in turn, may lead to positive or negative influences on basal ganglia function and behavioral performances. The different patterns of 6-OHDA lesion and transplant-induced behavioral changes demonstrated in the present study compared to the “classical” MFB lesion model of PD may thus provide further insights in the complex functional organization of the basal ganglia and, thereby, may help to further optimize restorative strategies for neurodegenerative diseases, such as Parkinson's disease.
In Vivo Persistence of Donor Cells following Adoptive Transfer of Allogeneic Dendritic Cells in HIV-Infected Patients
Authors: Shapero Michael H. , Kundu Smriti K. , Engleman Edgar , Laus Reiner , Van Schooten Wim C. A. , Merigan Thomas C.
Published: June 2017

Peripheral blood samples from HIV-seropositive individuals enrolled in a pilot clinical trial investigating the use of allogeneic dendritic cell therapy were evaluated for mixed chimerism. In this study, dendritic cells from HLA-identical, HIV-seronegative siblings were used. Patients received an infusion of dendritic cells pulsed with HIV MN gp160 protein or with peptides from HLA-A2 restricted epitopes of env, gag, and pol proteins every month for 6–9 months. Of the five allogeneic dendritic cell recipients, two showed increases in HIV antigen-specific immune responses. Allele-specific polymorphisms were identified in three sib-pairs that allowed infused donor cells to be detected using sensitive PCR-based molecular methods. Analysis of blood samples from patients showed similar patterns of donor cell persistence after the first infusion, in that cells were detectable for at least 1 week. Also, differences were observed in the kinetics of cell survival between the first and subsequent infusion cycles in all three patients. This suggests variation in HIV-specific immune responses detected among these three patients was not due to differences in persistence of infused donor cells.
In Vivo Expression of Human Growth Hormone by Genetically Modified Murine Bone Marrow Stromal Cells and its Effect on the Cells in Vitro
Authors: Suzuki K. , Oyama M. , Faulcon L. , Robbins P. D. , Niyibizi C.
Published: June 2017

Human growth hormone (hGH) is frequently used clinically for growth abnormalities in children and also in adults with growth hormone deficiency. The hormone is usually administered to the individuals by frequent injections. In the present study we investigated the potential of bone marrow stromal cells as vehicles to deliver the GH in vivo by infusion of cells transduced with hGH cDNA into mice femurs. The effect of the hormone on the transduced cells in vitro was also assessed. Bone marrow stromal cells established from a mouse model of human osteogenesis imperfecta mice (oim) were transduced with a retrovirus containing hGH and neomycin resistance genes. The hGH-expressing cells were selected in a medium containing G418 and were then assessed for the hGH expression in vitro. The selected cells synthesized 15 ng/106 cells of hGH per 24 h in vitro and exhibited alkaline phosphatase activity when they were treated with the human recombinant bone morphogenetic protein 2 (rhBMP-2). The transduced cells also proliferated faster than the LacZ transduced cells but they did not exhibit a higher rate of matrix synthesis. When 2 × 106 hGH+ cells were injected into the femurs of mice, hGH was detected in the serum of the recipient mice up to 10 days after injection. The highest level of growth hormone expression, 750 pg/ml, was detected in the serum of the recipient mice 1 day after injection of the transduced cells. hGH was also detected in the medium conditioned by cells that were flushed from the femurs of the recipient mice at 1, 3, and 6 days after cell injection. These data indicate that bone marrow stromal cells could potentially be used therapeutically for the delivery of GH or any other therapeutic proteins targeted for bone. The data also suggest that GH may exert its effects on bone marrow stromal cells by increasing their rate of proliferation.
Comparison of Benefits on Myocardial Performance of Cellular Cardiomyoplasty with Skeletal Myoblasts and Fibroblasts
Authors: Hutcheson Kelley A. , Atkins B. Zane , Hueman Matthew T. , Hopkins M. Benjamin , Glower Donald D. , Taylor Doris A.
Published: June 2017

Cellular cardiomyoplasty (CCM), or introduction of immature cells into terminally injured heart, can mediate repair of chronically injured myocardium. Several different cell types, ranging from embryonic stem cells to autologous skeletal myoblasts, have been successfully propagated within damaged heart and shown to improve myocardial performance. However, it is unclear if the functional advantages associated with CCM depend upon the use of myogenic cells or if similar results can be seen with other cell types. Thus, we compared indices of regional contractile (systolic) and diastolic myocardial performance following transplantation of either autologous skeletal myoblasts (Mb) or dermal fibroblasts (Fb) into chronically injured rabbit heart. In vivo left ventricular (LV) pressure (P) and regional segment length (SL) were determined in 15 rabbits by micromanometry and sonomicrometry 1 week following LV cryoinjury (CRYO) and again 3 weeks after autologous skeletal Mb or dermal Fb transplantation. Quantification of systolic performance was based on the linear regression of regional stroke work and end-diastolic (ED) SL. Regional diastolic properties were assessed using the curvilinear relationships between LVEDP and strain (ε) as well as LVEDP and EDSL. At study termination, cellular engraftment was characterized histologically in a blinded fashion. Indices of diastolic performance were improved following CCM with either Mb or Fb. However, only Mb transplantation improved systolic performance; Fb transfer actually resulted in a significant decline in systolic performance. These data suggest that both contractile and noncontractile cells can improve regional material properties or structural integrity of terminally injured heart, as reflected by improvements in diastolic performance. However, only Mb improved systolic performance in the damaged region, supporting the role of myogenic cells in augmenting contraction. Further studies are needed to define the mechanism by which these effects occur and to evaluate the long-term safety and efficacy of CCM with any cell type.
The Influence of Muscle Fiber Type in Myoblast-Mediated Gene Transfer to Skeletal Muscles
Authors: Petersen Zhuqing Qu , Huard Johnny
Published: June 2017

Myoblast transplantation has been hindered by immune rejection problems, as well as the poor survival and spread of transplanted cells. Our recent study has shown that the poor survival of the injected cells can be totally overcome by the use of specific populations of muscle-derived cells. In the present study, we have investigated whether a relationship exists between the fate of transplanted cells and the muscle fiber types. Four kinds of myogenic cells [primary myoblasts at a high purity (PMb), myoblasts isolated from fast single fibers (FMb), mdx (MCL), and MtMd-1 cell lines] were infected with an adenoviral vector carrying a LacZ reporter gene and injected into mdx hindlimb muscle. The LacZ transduced myofibers formed by the fusion of the injected myoblasts at 2–10 days postinjection were colocalized with MyHC stainings. The PMb cells, which expressed both slow and fast MyHCs in vitro, displayed the same phenotypes when injected into the m. soleus and m. gastrocnemius (white) muscles, which contained 70% and 0% of slow myofibers, respectively, and showed a high degree of fusion with host muscle fibers. In contrast, the FMb cells only expressed fast MyHCs in vitro and fused exclusively with each other or with host fast muscle fibers when injected in the m. gastrocnemius. Injected MCL and MtMd-1 fused predominantly with each other and displayed a similar expression of MyHCs to those they expressed in vitro. Just a few host myofibers were found to express the reporter gene product following implantation of both cell lines, indicating that these myogenic cell lines display an intrinsic potential to fuse together rather than with host myofibers. Based on the data, we concluded that 1) the essential key to survival is the ability of the donor cells to fuse with the host myofibers, and 2) the most successful combination is achieved between donor primary muscle cells that express both fast and slow MyHC and a host muscle type that facilitates fusion.
A Factor Implicated in the Myogenic Conversion of Nonmuscle Cells Derived from the Mouse Dermis
Authors: Goldring K. , Jones G. E. , Watt D. J.
Published: June 2017

Using the mdx mouse model for human Duchenne muscular dystrophy we have shown that a cell population residing in the dermis of C57B1/10ScSn mouse skin is capable of converting to a myogenic lineage when implanted into the mdx muscle environment. It was important to determine the characteristics of the converting cell. A previous in vitro study indicated that 10% of cells underwent conversion but only when the cells were grown in medium previously harvested from a myogenic culture. In the present study we cloned cells derived from the dermis to identify the converting cells. Clones grown in normal growth medium showed no conversion, but when grown in medium conditioned by muscle cells around 40% conversion was achieved in several individual clones. We investigated whether the protein β-galactoside binding protein (βGBP), which is secreted by myoblasts and acts as a cell growth regulator of fibroblasts, could be a candidate factor responsible for conversion. Medium harvested from COS-1 cells infected with a construct containing βGBP has been used for this investigation. Growth of dermal fibroblasts in medium enriched with this factor showed a high rate of conversion to cells expressing muscle-specific factors.
Deflazacort Increases Laminin Expression and Myogenic Repair, and Induces Early Persistent Functional Gain in mdx Mouse Muscular Dystrophy
Authors: Anderson J. E. , Weber M. , Vargas C.
Published: June 2017

Deflazacort slows the progress of Duchenne muscular dystrophy (DMD) with fewer side effects than prednisone. In mdx mice, deflazacort treatment augments repair and growth of new muscle fibers. We tested the hypothesis that deflazacort improves muscle function and promotes repair by increasing myogenic cell proliferation and fiber differentiation. mdx mice (3.5 weeks old) were treated with deflazacort (1.2 mg/kg) or vehicle for 4 weeks. Forelimb grip strength was measured. After 4 weeks, the right tibialis anterior muscle (TA) was crush injured to induce synchronous regeneration. DNA was labeled using different markers 24 and 2 h before collecting tissues 4 days after injury. The expression of creatine kinase (CK) isoforms, laminin-2 (merosin) mRNA and protein, and proliferation by myogenic cells were measured and satellite cells were identified by immunolocalization of c-met receptor. Peak grip strength increased 15% within 10 days of treatment, and was maintained up to 6 weeks after the end of treatment in a second experiment. Expression of CK MM in the regenerating TA rose from 46% to 55% of total CK activity after deflazacort treatment. Satellite cells were more numerous and appeared earlier on new fibers, in concert with a threefold increase in proliferation by myogenin+ (but not MyoD+) myoblasts. α2-Laminin mRNA expression and protein increased 1.3–5.5-fold relative to MM CK in regenerating and dystrophic TA, respectively. These studies support the hypothesis that deflazacort promotes functional gains, myogenic differentiation, myoblast fusion, and laminin expression in regenerating dystrophic muscle. The potential to augment precursor specification, strength, and possible membrane stability may be useful in directing long-term benefits for DMD patients and short-term amplification of precursors prior to myoblast transfer.
Distribution of AAV-TK following Intracranial Convection-Enhanced Delivery into Rats
Authors: Cunningham Janet , Oiwa Yoshitsugu , Nagy Dea , Podsakoff Greg , Colosi Peter , Bankiewicz Krys S.
Published: June 2017

Adeno-associated virus (AAV)-based vectors are being tested in animal models as viable treatments for glioma and neurodegenerative disease and could potentially be employed to target a variety of central nervous system disorders. The relationship between dose of injected vector and its resulting distribution in brain tissue has not been previously reported nor has the most efficient method of delivery been determined. Here we report that convection-enhanced delivery (CED) of 2.5 × 108, 2.5 × 109, or 2.5 × 1010 particles of AAV-thymidine kinase (AAV-TK) into rat brain revealed a clear dose response. In the high-dose group, a volume of 300 mm3 of brain tissue was partially transduced. Results showed that infusion pump and subcutaneous osmotic pumps were both capable of delivering vector via CED and that total particle number was the most important determining factor in obtaining efficient expression. Results further showed differences in histopathology between the delivery groups. While administration of vector using infusion pump had relatively benign effects, the use of osmotic pumps resulted in notable toxicity to the surrounding brain tissue. To determine tissue distribution of vector following intracranial delivery, PCR analysis was performed on tissues from rats that received high doses of AAV-TK. Three weeks following CED, vector could be detected in both hemispheres of the brain, spinal cord, spleen, and kidney.
Intraparenchymal NGF Infusions Rescue Degenerating Cholinergic Neurons
Authors: Tuszynski Mark H.
Published: June 2017

Nerve growth factor (NGF) exerts both trophic (cell survival) and tropic (axonal growth-promoting) effects on several neuronal populations. In particular, its robust ability to prevent lesion-induced and spontaneous age-related basal forebrain cholinergic neuronal degeneration, and to promote mnemonic recovery, has suggested its potential use as a therapeutic agent in Alzheimer's disease. When infused intracerebroventricularly, however, NGF is associated with several adverse effects that make this delivery route impractical. The present study examined whether intraparenchymal infusions of NGF adjacent to cholinergic neuronal soma are an effective and well-tolerated means of providing NGF to degenerating cholinergic neurons. Cholinergic neuronal rescue together with axonal sprouting responses and local tissue damage in the brain were assessed in adult rats that underwent complete unilateral fornix transections, followed by intraparenchymal infusions of recombinant human NGF for a 2-week period. Intraparenchymal NGF infusions prevented the degeneration of 94.7 ± 6.6% of basal forebrain cholinergic neurons compared to 21.7 ± 2.6% in vehicle-infused animals (p < 0.0001). Cholinergic axons sprouted toward the intraparenchymal NGF source in an apparent gradient-dependent manner. Glial responses to intraparenchymal infusions were minimal, and no apparent toxic effects of the infusions were observed. Thus, when infused intraparenchymally, NGF rescues basal forebrain cholinergic neurons, alters the topography of axonal sprouting responses, and does not induce adverse affects over a 2-week infusion period. Intraparenchymal NGF delivery merits further study at longer term time points as a means of treating the cholinergic component of neuronal loss in Alzheimer's disease.
Article Commentary: The X-gal Caution in Neural Transplantation Studies
Authors: Sanchez-Ramos J. , Song S. , Dailey M. , Cardozo-Pelaez F. , Hazzi C. , Stedeford T. , Willing A. , Freeman T. B. , Saporta S. , Zigova T. , Sanberg P. R. , Snyder E. Y.
Published: June 2017

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme β-galactosidase (β-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, “X-gal,” in an oxidation reaction. LacZ is the “reporter gene” most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully—with adequate optimization and individualization of various parameters (e.g., pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls—then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial β-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have emphasized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli β-gal activity.
Phenotype Correction in Murine Mucopolysaccharidosis Type VII by Transplantation of Human Amniotic Epithelial Cells after Adenovirus-Mediated Gene Transfer
Authors: Kosuga Motomichi , Takahashi Satori , Sasaki Kyoko , Enosawa Shin , Li Xiao-Kang , Okuyama Sanae , Fujino Masayuki , Suzuki Seiichi , Yamada Masao , Matsuo Nobutake , Sakuragawa Norio , Okuyama Torayuki
Published: June 2017

Cell therapy with human amniotic epithelial (HAE) cells was developed as an alternative method for enzyme replacement therapy in congenital lysosomal storage disorders, but only limited therapeutic efficacy has been reported. A major drawback is insufficient production and secretion of lysosomal enzymes from HAE cells. In this study, we infected HAE cells with an E1-deleted adenoviral vector expressing human β-glucuronidase (GUSB), and generated cells overexpressing GUSB by a hundred times as much as endogenous GUSB in untreated HAE cells. GUSB secreted from the gene-transferred HAE cells were efficiently transported to murine fibroblasts with endocytosis mediated by mannose-6-phosphate receptors. The cells were administered into the spleen of the mice with the lysosomal storage disease mucopolysaccharidosis type VII (B6/MPSVII). Approximately 10–15% of the normal GUSB activity was detected in both liver and spleen 7 days after the cell administration. Histopathological examination showed that lysosomal enlargement in tissue macrophages in the liver and the spleen had disappeared by day 14. These results suggest that transplantation of the HAE cells transduced with adenoviral vectors can be employed for the treatment of congenital lysosomal storage disorders.
Neurite Growth Capability of Rat Fetal Neuronal Cells against Matured CNS Myelin in Vitro
Authors: Hara Koichi , Uchida Koichi , Fukunaga Atsushi , Kuroshima Yoshiaki , Yamada Motoyuki , Kawase Takeshi
Published: June 2017

Reconstruction of neurocircuits by transplanted cells is expected to become an effective therapy for brain damage. In order to establish the transplantation therapy, it is necessary to find transplantable cells capable of reconstructing the lesioned neurocircuitry. We have reported that the younger neuronal cells such as neural stem cells are useful transplant materials because of their vigorous capacity for forming abundant neurites. On the other hand, it was reported that myelin-associated neurite growth inhibitor prevents neurite regeneration. In this study, we used rat fetal neuronal cells to examine the neurite growth capacity in the presence of mature CNS myelin. Crude CNS myelin was prepared from the brains of adult Wistar rats using previously described procedures. Testing wells were precoated with poly-L-lysine and additionally by overnight drying of a suspension containing 0, 5, 10, 15, or 20 μg/cm2 of the crude myelin protein. On embryonic days 10, 12, 15, and 17 (E10, E12, E15, and E17) embryos were surgically removed, mesencephalic neural plates were dissected out from the E10 embryos, and midbrain cells were taken from the E12, E15, and E17 embryos. The neural plates and midbrain cells were placed on the myelin-coated wells. After 24 h of culture (72 h in the case of neural plates), the number of surviving cells and the length of the neurites were examined immunocytochemically using anti-neurofilament (NF) antibody. Neurite length was measured by image analyzer Luzex-F. The mesencephalic neural plate was able to grow neurites even on 20 μg/cm2 central myelin. Almost the same number of midbrain cells attached themselves to the wells without myelin in every culture obtained from various stages of embryos. The number of cells attached on the myelin-coated wells decreased with the concentration of myelin. The number of NF-positive cells was higher in cultures of materials obtained from older embryos than in cultures obtained from younger embryos. The younger cells grew longer neurites than the older cells in the myelin noncoated wells. Neurite growth was inhibited strongly when the concentration of the central myelin was 10 μg/cm2 or greater, but on the 5 μg/cm2 myelin, the younger the cells were, the longer neurites they had. When the length of the longest neurites in one field of the image analyzer was further examined in the same way, the younger the cells were, the longer their axons grew on 0 and 5 μg/cm2 myelin. Thus, CNS myelin was seen to be a significant inhibitor of the recovery of injured neural tissue of the adult CNS. Younger cells grew longer neurites than older cells on CNS myelin, and so it was suggested that neural stem cells or younger neurons may serve as tissue for transplantation therapy.
Treatment of Surgically Induced Acute Liver Failure with Transplantation of Highly Differentiated Immortalized Human Hepatocytes
Authors: Kobayashi Naoya , Miyazaki Masahiro , Fukaya Kenichi , Inoue Yusuke , Sakaguchi Masakiyo , Noguchi Hirofumi , Matsumura Toshihisa , Watanabe Takamasa , Totsugawa Toshinori , Tanaka Noriaki , Namba Masayoshi
Published: June 2017

Primary human hepatocytes are an ideal source of hepatic function in bioartficial liver (BAL), but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, primary human fetal hepatocytes were immortalized using simian virus 40 large T antigen. One of the immortal cell lines, OUMS-29, showed highly differentiated liver functions. Intrasplenic transplantation of OUMS-29 cells protected 90% hepatectomized rats from hyperammonemia and significantly prolonged their survival. Essentially unlimited availability of OUMS-29 cells supports their clinical use for BAL treatment.
ICAM-1 Antisense Oligodeoxynucleotide Improves Islet Allograft Survival and Function
Authors: Katz Stephen M. , Bennett Frank , Stecker Kim , Clark James H. , Phan Tommy , Wang Mou-Er , Kahan Barry D. , Stepkowski Stanislaw M.
Published: June 2017

Expression of intercellular adhesion molecule-1 (ICAM-1) and its ligand, leukocyte function antigen-1 (LFA-1), after pancreatic islet transplantation may affect both nonspecific and alloantigen-specific phases of graft destruction. We examined the effects of ICAM-1/LFA-1 blockade on the survival of islet allografts. Fresh C57BL/10 (H2b) pancreatic islets were transplanted under the renal subcapsular space (KC) or embolized into the liver after portal vein (PV) injection to C3H (H2k) mice. Recipients remained untreated or were treated for 7 days by IP administration of: ICAM-1 antisense phosphorothioate oligodeoxynucleotide (oligo) alone; anti-ICAM-1 (αICAM-1) monoclonal antibody (mAb) alone; αLFA-1 mAb alone; ICAM-1 oligo/αLFA mAb combination; αICAM-1 mAb/αLFA-1 mAb combination; or control oligo IP-8997 or IP-1082. In some experiments, donors were pretreated with ICAM-1 oligo. Inhibition of single ligand with 5.0 mg/kg ICAM-1 oligo (25.1 ± 10.3), 100 μg/daily αICAM-1 mAb (24.2 ± 8.0 days), or 50 μg/daily αLFA-1 mAb (42.8 ± 25.9 days) prolonged the survivals of KC islet allografts in comparison with untreated controls (11.9 ± 1.0 days; all p < 0.01). However, dual ICAM-1/LFA-1 blockade with either ICAM-1 oligo/αLFA-1 mAb (78.3 ± 16.5 days) or αICAM-1 mAb/αLFA-1 mAb (65.2 ±31.3 days) was the most effective therapy. Although pretreatment of donors with ICAM-1 oligo alone was ineffective (12.2 ± 0.8 days; NS), a combination of donor pretreatment and recipient treatment started 1 day prior to grafting with ICAM-1 oligo (39.2 ± 14.0 days) was more effective than the recipient treatment alone (24.6 ± 8.8 days). Furthermore, ICAM-1/LFA-1 blockade improved islet function as evaluated by glucose tolerance test, and decreased inflammation in comparison with untreated controls. Similar in vivo results were obtained following PV administration of islet allografts. Thus, ICAM-1/LFA-1 blockade prolongs the survival of pancreatic islet allografts and improves their early function.
Development and Characterization of Pituitary GH3 Cell Clones Stably Transfected with a Human Proinsulin cDNA
Authors: Meoni Cesare , Bertuzzi Federico , Pontiroli Antonio E. , Falqui Luca , Monaco Lucia , Soria Marco , Arcelloni Cinzia , Paroni Rita , Foglieni Chiara , Polastri Luca , Galbiati Francesca , Folli Franco , Davalli Alberto M.
Published: June 2017

Successful β-cell replacement therapy in insulin-dependent (type I) diabetes is hindered by the scarcity of human donor tissue and by the recurrence of autoimmune destruction of transplanted β cells. Availability of non-β cells, capable of releasing insulin and escaping autoimmune recognition, would therefore be important for diabetes cell therapy. We developed rat pituitary GH3 cells stably transfected with a furin-cleavable human proinsulin cDNA linked to the rat PRL promoter. Two clones (InsGH3/clone 1 and 7) were characterized in vitro with regard to basal and stimulated insulin release and proinsulin transgene expression. Mature insulin secretion was obtained in both clones, accounting for about 40% of total released (pro)insulin-like products. Immunocytochemistry of InsGH3 cells showed a cytoplasmic granular insulin staining that colocalized with secretogranin II (SGII) immunoreactivity. InsGH3 cells/clone 7 contained and released in vitro significantly more insulin than clone 1. Secretagogue-stimulated insulin secretion was observed in both InsGH3 clones either under static or dynamic conditions, indicating that insulin was targeted also to the regulated secretory pathway. Proinsulin mRNA levels were elevated in InsGH3 cells, being significantly higher than in PTC3 cells. Moreover, proinsulin gene expression increased in response to various stimuli, thereby showing the regulation of the transfected gene at the transcriptional level. In conclusion, these data point to InsGH3 cells as a potential β-cell surrogate even though additional engineering is required to instruct them to release insulin in response to physiologic stimulations.
Induction of Allogeneic Islet Tolerance in a Large-Animal Model
Authors: Horton Peter J. , Hawthorne Wayne J. , Walters Stacey N. , Patel Anita T. , O'connell Philip J. , Chapman Jeremy R. , Allen Richard D. M.
Published: June 2017

For islet allotransplantation to become a therapy widely applicable to patients with insulin-dependent diabetes, it will be important to avoid conventional immunosuppression and yet maintain long-term rejection-free islet survival. This possibility was tested in a large-animal model using mixed allogeneic chimeras established using total lymphoid irradiation (TLI) and donor-specific bone marrow transplantation (BMTX). Four recipient sex-mismatched and DLA class II-matched English springer spaniels became chimeric after TLI and donor-specific BMTX. Subsequent donor-specific renal allografts survived for more than a year. Acceptance of a donor-specific skin graft and rejection of a third-party graft demonstrated tolerance with maintenance of immunocompetence. Pancreatic microfragments containing islets were refluxed into the splenic vein of the recipient. Purified islets were placed under the capsule of spleen and liver. After 75 days, recipients underwent total native pancreatectomy. All four chimeric pancreatectomized dogs had functioning islet grafts 75 days after transplantation, evidenced by a prompt rise in serum insulin levels following an IVGTT and histological demonstration of islet tissue at the site of transplantation. After removal of the transplanted islet tissue, no insulin was released after IVGTT. In summary, intrasplenic allogeneic canine islets transplanted into chimeric dogs rendered tolerant to donor MHC survive and function for greater than 75 days in the absence of immunosuppression. This study represents proof of the concept that allogeneic islet transplants have the potential to reverse diabetes without the use of conventional immunosuppression.
Beneficial Effect of Pancreas and Kidney Transplantation on Advanced Diabetic Retinopathy
Authors: KožNarová R. , Saudek F. , Sosna T. , Adamec M. , Jedináková T. , BoučEk P. , Bartoš V. , Lánská V.
Published: June 2017

In pancreas recipients with advanced diabetic eye disease, conflicting ophthalmologic results over different follow-up periods have been reported. In the present prospective study we performed ophthalmologic evaluation groups of type I diabetic patients: 1) normoglycemic recipients of pancreas and kidney grafts (group SPK, n = 43, follow-up 44.9 ± 35.1 months), 2) pancreas and kidney graft recipients with nonfunctioning pancreatic graft, and recipients of isolated kidney graft (group K, n = 45, follow-up 60.3 ± 34.2 months). The examinations were performed before transplantation, at the end of follow-up (at least 1 year), and in 63 recipients also at 3 years posttransplant. Visual acuity results at baseline and at the end of follow-up were 0.48 ± 0.39 vs. 0.50 ± 0.39 in the SPK group, and 0.46 ± 0.38 vs. 0.40 ± 0.39 in the K group. While intragroup changes were not significant, the changes were significantly different between the groups (p < 0.05). Fundoscopic findings at the end of follow-up were improved, stabilized, or deteriorated in the SPK group in 21.3%, 61.7%, and 17.0%, respectively. The respective figures for the K group were 6.1%, 48.8%, and 45.1% (p < 0.001). Similar results were obtained when evaluating findings at 3 years posttransplant. Before transplantation, 78% of the SPK group and 81% of the K group had been treated by laser. The need for additional posttransplant laser therapy was significantly lower in the SPK (31%) than in the K group (58%; p < 0.001). In conclusion, pancreas transplant exerts a beneficial effect on the course of diabetic retinopathy even in its late stage.
Sustained Improvements in Cardiac Geometry and Function following Kidney–Pancreas Transplantation
Authors: Gaber A. Osama , Wicks Mona N. , Hathaway Donna K. , Burlew Brad S.
Published: June 2017

Kidney–pancreas (KP) transplantation has been shown to improve left ventricular (LV) geometry and function 6–24 months after the procedure, yet whether these improvements are sustained in long-term survivors has not been demonstrated. This study examined whether early improvements in LV geometry and function were sustained 3–5 years after KP transplantation. Left ventricular function and geometry were prospectively evaluated prior to, and at 1, 2, and 3–5 years posttransplant using two-dimensional, M-mode, echocardiography with Doppler interrogation in the parasternal and apical views. The sample included 21 KP and a comparison group of 12 diabetic kidney-alone (KA) recipients. Long-term (3–5 years) data were obtained for KP recipients only. Although KA recipients had a longer duration of dialysis and worse diastolic function pretransplant, the groups were similar on other baseline measures. KA recipients experienced minimal improvements while KP recipients had significant improvements in cardiac function and geometry, both in terms of mean values and the percentage of KP recipients who experienced normalization posttransplant (p < 0.05). KP recipient improvements were also sustained at 3–5 years posttransplant on three of five measures, with 75% of long-term KP recipients achieving normal LV mass posttransplant compared with 31% pretransplant. Data indicate that significant impairments in cardiac geometry and function occur in diabetic KA and KP recipients. Though both groups experienced early improvements posttransplant, KP recipients achieved more dramatic and clinically significant improvements at 1, 2, and 3–5 years posttransplant. Additional studies are needed to examine the relevance of these findings with regard to the cardiac morbidity and mortality of these patients.
Regenerative Medicine for Diabetes Mellitus
Authors: Kobayashi Naoya , Yuasa Takeshi , Okitsu Teru
Published: June 2017

In diabetes, a loss of pancreatic β-cells causes insulin dependency. When insulin dependency is caused by type 1 diabetes or pancreatic diabetes, for example, pancreatic β-cells need to be regenerated for definitive treatment. The methods for generating pancreatic β-cells include a method of creating pancreatic β-cells in vitro and implanting them into the body and a method of regenerating pancreatic β-cells in the body via gene introduction or the administration of differential proliferation factors to the body. Moreover, the number of pancreatic β-cells is also low in type 2 diabetes, caused by the compounding factors of insulin secretory failure and insulin resistance; therefore, if pancreatic β-cells can be regenerated in a living body, then a further amelioration of the pathology can be expected. The development of pancreatic β-cell-targeting regenerative medicine can lead to the next generation of diabetes treatment.
Study on the Preservation with CO (PCO = 200–2,000 hPa), Resuscitation, and Heterotopic Transplantation of an Isolated Rat Heart
Authors: Yoshida Yu , Hatayama Naoyuki , Seki Kunihiro
Published: June 2017

In this experiment, CO was used as a gas mixture in a reversible relationship with O2. CO was added in a gas form mixed with O2. An isolated donor rat heart was obtained, exposed to a gas mixture such as PO2 = 1,800 hPa and PCO = 200 hPa, and PO2 = 1,000 hPa and PCO = 1,000 hPa in a 2 ATA high-pressure chamber and preserved in a refrigerator at 4°C. This report demonstrates that significant reproducibility has been verified. The heart was removed from the refrigerator 24 h later and heterotopic heart transplantation was performed in the right neck of a recipient rat and the pulsating of the transplanted heart was detected by an electrocardiogram.
Comparison of Trypsin Inhibitors in Preservation Solution for Islet Isolation
Authors: Noguchi Hirofumi , Ueda Michiko , Hayashi Shuji , Kobayashi Naoya , Okitsu Teru , Iwanaga Yasuhiro , Nagata Hideo , Liu Xiaoling , Kamiya Hiroki , Levy Marlon F. , Matsumoto Shinichi
Published: June 2017

Islet transplantation has recently emerged as an effective therapy and potential cure for type 1 diabetes mellitus. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and University of Wisconsin (UW) solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. Moreover, we recently reported that islet yield was significantly higher in the ET-Kyoto solution with ulinastatin (MK)/PFC preservation solution compared with the UW/PFC preservation solution in the porcine model and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with another trypsin inhibitor, Pefabloc, in preservation solution for islet isolation. Islet yield before purification was higher in the MK/PFC group compared with the ET-Kyoto with Pefabloc (PK)/PFC group. The stimulation index was higher for the MK/PFC group than for the PK/PFC group. These data suggest that ET-Kyoto with ulinastatin was the better combination for pancreas preservation than ET-Kyoto with Pefabloc. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.