bluuebunny/crossref_metadata_2025 huggingface dataset:
Islet Cell Mass and the Longevity of Islet Grafts in Diabetic Rats
Authors: Beattie Gillian M. , Hayek Alberto
Published: June 2017

In an attempt to demonstrate that a limited β-cell mass is able to maintain normoglycemia in streptozocin-diabetic rats, two groups of inbred rats received transplants of 500 or 1000 neonatal islets (corresponding to 10 and 20% of total islet cell mass, respectively) into the spleen. A close correlation was found between the functional longevity of the islet graft, and the total β-cell mass transplanted into the diabetic animals. Although euglycemia was maintained for 4-12 mo, the islet transplants were insufficient to produce glucose tolerance tests equal to those observed in normal rats. Relapse of diabetes correlated with histological findings of distorted islet architecture containing vacuolated β-cells in the grafts. This condition was not reversed after a month of normoglycemia. Spontaneous recovery of β-cell function in native islets was documented in 50% of the diabetic rats with prolonged normoglycemia following islet transplantation. This report shows that transplantation of as few as 500 neonatal islets, the equivalent of 10% of total islet cell mass, is sufficient to maintain normoglycemia in diabetic rats, albeit temporarily. A close correlation was found between the transplanted islet cell mass and the functional longevity of the islet graft.
Establishment of a Stable Glutamate Decarboxylase (Gad) Expressing Cell-Line by Transfection
Authors: Zhou Feng C. , Cheng Christine , Bledsoe Sharon
Published: June 2017

We have constructed a recombinant DNA clone containing the gene encoding glutamic acid decarboxylase (GAD), which catalyzes the synthesis of γ-amino-butyric acid (GABA). This recombinant DNA was then transfected into mouse NIH-3T3 fibroblast cells for transplantation into Swiss-Web mice. In order to construct a plasmid capable of transcribing the DNA insert in the eucaryotic cells, the GAD gene was removed from pSP65-13, and was ligated into the vector pSV2neo, which contains the SV40 early promoter, and the neomycin resistance gene. The pSV2GAD was then transfected into NIH-3T3 fibroblasts by calcium phosphate precipitation, or by electroporation. The transfected fibroblasts were then selected with antibiotic G418 for amplification. The transient expression of GAD in the transfected fibroblasts was detected by immunocytochemical staining using anti-GAD antibody. A small population of GAD immunoreactive cells were clearly stained, and were easily distinguished from the majority of unstained background cells. These GAD-immunoreactive cells were not seen in either mock-transfected, or pSV2neo-transfected cells (vector-alone control). The transfected fibroblasts were continuously selected with antibiotic G418. Six out of 35 subcultures that had GAD-positive immunostaining in the cell lines were selected. Granular GAD-positive staining was observed in the cytoplasm and fiber extensions of the transfected cell lines in varying densities. The GAD-mRNA was also detected in the subcultures by in situ hybridization using a 35S-labeled 369-nucleotide riboprobe in pBluescript. The GAD-transfected NIH-3T3 cells were then transplanted into Swiss-Web mice. Fifteen to 30 days later, transplanted animals were perfused for identification. These cells were first identified with anti-fibronectin antibody, and the adjacent sections with anti-GAD or anti-GABA antibodies. All the transplants are fibronectin-positive. Both GAD- and GABA-positive cells were observed in the transplant.
Human Midsized Neurofilament Expression in Transgenic Mouse-Derived Grafts Facilitates Study of Graft-Host Interactions in Hypogonadal Mice
Authors: Gibson Marie J. , Friedrich Victor L. , Elder Gregory , Lazzarini Robert A. , Silverman Ann-Judith
Published: June 2017

Our previous studies established that targeted axonal outgrowth into the host median eminence (ME) from grafted gonadotropin-releasing hormone (GnRH) neurons is essential for stimulation of reproductive function in hypogonadal (HPG) mice homozygous for a deletion in the GnRH gene. In the current experiments transgenic mice expressing the human midsized neurofilament NF(M) were used as sources of grafts to clarify the extent of transplant-derived innervation of the host that accompanies this dramatic recovery process. Preoptic area (POA) tissue from 1- or 2-day-old transgenic pups was implanted in the third ventricle of adult male HPG mice. Polymerase chain reaction (PCR) analysis verified that each donor bore the transgene. Immunohistochemistry using a monoclonal antibody specific for human NF(M) revealed intensely immunoreactive parvicellular soma and proximal dendrites in the grafts. Interestingly, human NF(M)-positive neurons also migrated out of the graft into the host hypothalamus. In the animal with the most dramatic increase in testicular and seminal vesicle weight, human NF(M)-positive cells were observed within the host ME. In some cases, human NF(M)-positive axonal bundles were present within the wall of the third ventricle of the host as well as in the host ME. These were GnRH negative. More extensive anatomical studies will delineate the degree and patterning of axonal outgrowth. In these early studies, however, it is apparent that neuronal fiber outgrowth into the host brain is not exclusive to GnRH fibers from the preoptic area grafts. This is important information in regard to our studies of the specificity of the signals derived from the ME that may be involved in GnRH fiber targeting.
Method for the Quantification of Alginate in Microcapsules
Authors: Hallé Jean-Pierre , Landry Danielle , Fournier Alain , Beaudry Michèle , Leblond Francois A.
Published: June 2017

Alginate is a key reagent in the preparation of microcapsules for cell transplantation. To address the question of the intracapsular alginate concentration, a sensitive assay has been developed to quantify the alginate content of microcapsules. The method is based on the metachromatic change induced by alginate binding to the dye, 1,9-dimethyl methylene blue (DMMB). The assay has a high sensitivity and precision. It covers a wide concentration range enabling the measurement of alginate in dilute supernatants as well as in microcapsules. For the latter, the membrane is initially dissolved by incubating the microcapsules in an alkaline medium. The effect of potentially interfering substances (poly-l-lysine (PLL), citrate, chloride, sodium) and of pH has been studied. Poly-l-lysine interfered with the assay at pH 6.5 but not at pH 13. Interference by sodium augmented with increasing sodium concentration and reached a plateau at 200 mM. This problem was overcome by routinely adjusting all samples to 500 mM sodium. The other substances tested had a negligible effect on the assay. The reliable measurement of alginate with this new assay will allow the optimization of the intracapsular alginate concentration.
Human Embryonic Retinal Cell Transplants in Athymic Immunodeficient Rat Hosts
Authors: Aramant Robert B. , Seiler Magdalene J.
Published: June 2017

This study investigates the possibility to use the athymic “nude” rat as a host for the transplantation of human embryonic retinal cells without immunosuppression. The long-term development of such transplants is compared with results from our earlier study that used immunosuppressed rats, and showed transplant immunoreactivity for S-antigen. Several additional cell markers have been included: rhodopsin, rod (α-transducin, neuron-specific enolase (NSE), synaptophysin (SYN), cone-specific opsins, vimentin, cellular retinaldehyde binding protein (CRALBP), glial fibrillary acidic protein (GFAP), rat major histocompatibility antigen class II (MHC-II) and a rat macrophage marker (Ox-42). Human retinal cells (9-13 wk postconception) were transplanted to the eyes of 28 athymic rats. Host rats were kept in microisolator cages for up to 48 wk after surgery. Host immune response and the development of the transplants were studied using histology, immunohistochemistry and electron microscopy. When using retinas of donors 9-11 wk postconception, transplants grew to 2-3 mm in diameter with many rosettes, in 31 of 35 eyes. Transplants derived from donors 12-13 wk postconception did not survive as well (8 out of 11 eyes), were smaller and less organized. All transplants fused well with the host retina, better than corresponding transplants to immunosuppressed rat hosts. Most transplants appeared to be healthy, even after long survival times, and only occasionally were MHC-II positive macrophages observed in transplants or host retinas. All retinal layers were observed, except for an inner limiting membrane on the vitreous surface. The oldest transplants (34-57 wk total age = donor age + time after surgery) exhibited well developed photoreceptors, rods and cones, with inner and outer segments. SYN-staining showed the development of inner and outer plexiform layers. Although many cones stained for SYN and NSE, few were immunoreactive for red-green or blue opsin. Most rods became immunoreactive for S-antigen and rhodopsin. Transplant Möller cells stained for vimentin and CRALBP. Immunoreactivity for GFAP developed slowly and was not completely expressed in all transplant Möller cells until 44 wk total age. Nude rats offer an excellent model for the study of human retinal xenografts without the negative effects of immunosuppression. Compared to immunosuppressed rats, transplantation to nude rats gives consistent results and superior long-term survival of hosts and transplants.
Tracer Technique to Measure in Vivo Chemical Transport Rates within an Implantable Cell Transplantation Device
Authors: Sarver Jeffrey G. , Fournier Ronald L. , Goldblatt Peter J. , Phares Tamara L. , Mertz Sara E. , Baker Andrew R. , Mellon Robert J. , Horner James M. , Selman Steven H.
Published: June 2017

An in vivo tracer technique that uses radiolabeled inulin as the tracer molecule has been developed to assess the rate of chemical transport between the cell transplantation chamber of an implantable bioartificial device and the host's circulatory system. The device considered here employs site-directed neovascularization of a porous matrix to induce capillary growth adjacent to an immunoisolated cell implantation chamber. This device design is being investigated as a vehicle for therapeutic cell transplantation, with the advantages that it allows the cells to perform their therapeutic function without the danger of immune rejection and it avoids damaging contact of blood flow with artificial surfaces. A pharmacokinetic model of the mass transport between the implantation chamber, the vascularized matrix, and the body has been devised to allow proper analysis and understanding of the experimental tracer results. Experiments performed in this study have been principally directed at evaluation of the tracer model parameters, but results also provide a quantitative measure of the progression of capillary growth into a porous matrix. Measured plasma tracer levels demonstrate that chemical transport rates within the implanted device increase with the progression of matrix vascular ingrowth. Agreement between the fitted model curves and the corresponding measured concentrations at different levels of capillary ingrowth demonstrate that the model provides a realistic representation of the actual capillary-mediated transport phenomena occurring within the device.
Porcine Repeat Element DNA: In Situ Detection of Xenotransplanted Cells
Authors: Oettinger Henry F. , Rodrigue-Way Amelie , Bousquet Joyce J. , Edge Albert S.B.
Published: June 2017

Using a digoxygenin-labelled DNA probe derived from the porcine repeat element PRE-1, we have developed a protocol for the detection of transplanted porcine islets and hepatocytes against a background of murine host tissue. Analysis of this probe by Southern blotting indicated that PRE-1 hybridizes to pig genomic DNA but not to human or mouse DNA. On tissue sections, hybridizing probe was detected using alkaline phosphatase-conjugated anti-digoxygenin antibody visualized with 5-bromo-4-chloro-3-indolyl-phosphate/4-nitro-blue tetrazolium chloride (BCIP/ NBT) substrate. We have demonstrated sensitive and highly specific staining of porcine nuclei in fixed, paraffin embedded tissue sections, and have applied the technique to detect porcine pancreatic islets and hepatocytes transplanted into murine kidney and spleen. Applications of this technique include detection of transplanted cells or organs across a variety of xenogeneic barriers.
Site for Unpurified Islet Transplantation is an Important Parameter for Determination of the Outcome of Graft Survival and Function
Authors: Al-Abdullah Ismail H. , Kumar M.S. Anil , Kelly-Sullivan Dawn , Abouna George M.
Published: June 2017

Transplantation of unpurified islets into the liver, unlike that of purified islets, causes portal hypertension and coagulopathy. The aim of this project was to determine the most suitable alternative site for transplantation of unpurified pancreatic islets in autotransplanted dogs. Twenty-five female mongrel dogs were divided into 5 groups depending on the site of islet transplantation: liver (3), spleen (7), skeletal muscle (5), omental pouch (6), and renal subcapsule (4). Pancreatic digestion of the total pancreatectomized specimen was carried out by distension of the pancreas with 1.5 mg/mL collagenase suspended in 250 mL Hanks' balanced salt solution using a semiautomatic method. The total number of islets equivalent isolated from 25 dogs was 90948 ± 6053. Only islets > 60 μm in diameter were counted, and the mean islet equivalent transplanted per kg body wt was 6762 ± 429. Islet function was achieved with transplantation into spleen in 71%, omental pouch in 50%, and muscle in 20%, but none in the renal subcapsule or liver groups. Glucose tolerance test at 30 d showed a mean K Value (decline in glucose, %/min) of 1.94 ± 0.73,0.79 ± 0.15 and 1.02 in the splenic, omental pouch and muscle groups, respectively. All animals in the liver group, 2 from the splenic group, and 2 from the renal subcapsule group died of diffuse bleeding. Four out of 5 dogs in the muscle group developed necrosis at the site of transplantation and the islets never functioned. This study demonstrates that in dogs, spleen and omental pouch appear to be suitable sites for transplantation of unpurified islets.
Osmotic Separation of Pancreatic Exocrine Cells from Crude Islet Cell Preparations
Authors: Liu Chi , Mcgann Locksley E. , Gao Dayong , Haag Brian W. , Critser John K.
Published: June 2017

A novel approach is introduced here to selectively lyse exocrine cells in an islet preparation by hypoosmotic treatment. Time to hypotonic cell lysis required for the islet cells was much longer than that for the exocrine cells, which permits a possibility of selectively killing the exocrine cells by hypotonic treatment. The first set of experiments was designed to select an appropriate osmolality for the hypotonic treatment. Kinetic changes in cell volume in response to extracellular anisosmolalities (30 to 90 mOsm/kg) were recorded using an electronic particle counter. The results indicated that, when exposed to a 30 mOsm/kg solution, islet cells swelled slowly to reach volumetric equilibrium in approximately 3 min. There was no significant hypotonic cell lysis observed even at the end of 4 min (n = 4). In contrast, pancreatic exocrine cells, when exposed to the same solution, expanded rapidly to the lytic volume and burst within 30 s. Significant exocrine cell lysis was invariably achieved within 30 s when cells were exposed to the osmolalities below 60 mOsm/kg. For osmolalities between 70 to 80 mOsm/kg, exocrine cell lysis was highly variable. When cells were exposed to 80 to 90 mOsm/kg, no significant cell lysis was observed. Thus, an osmolality of 50 mOsm/kg is recommended for hypotonic treatment, as it maximizes the lysis of exocrine cells without unnecessarily stressing (osmotically) the islet cells. The second set of experiments (time-course experiments, 20 to 120 s) was designed to determine the length of exposure time for which the exocrine cells were irreversibly damaged but the islet cells had only swollen to such a degree that cell function is restored upon returning to an isotonic condition. Viability of the hypotonic treated cells was evaluated at two different levels: membrane integrity, measured by combined fluorescent dye staining with propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), and mitochondrial function, measured by colorimetric MTT assay. The results showed that hypotonic treatment in a 50 mOsm/kg solution for 30 s resulted in over 85% loss of the membrane integrity for the exocrine cells. About 90% of these membrane lysed cells lost mitochondrial function (n = 3). By contrast, under the same treatment, less than 15% of the islet cells lost membrane integrity and mitochondrial function (n = 3). In conclusion, hypotonic treatment with a 50 mOsm/kg solution for 20 to 30 s at room temperature is sufficient to lyse the majority of the contaminating exocrine cells in an islet cell preparation, while maintaining function in the islet cells.
Transfer and Expression of the Interferon Gamma Gene in Human Endothelial Cells Inhibits Vascular Smooth Muscle Cell Growth in Vitro
Authors: Stopeck Alison T. , Vahedian Mahmood , Williams Stuart K.
Published: June 2017

Intimal hyperplasia in blood vessels is primarily caused by the migration and proliferation of vascular smooth muscle cells. Excessive intimal thickening characterizes atherosclerosis as well as bypass graft and angioplasty failures. Endothelial cell-smooth muscle cell interactions and local cytokine production are important regulators of smooth muscle cell growth. Interferon gamma (γ-IFN), a product of T lymphocytes found in atherosclerotic lesions, inhibits smooth muscle cell proliferation in vitro. To determine if local delivery of γ-IFN may be useful in the treatment or prevention of vascular proliferative diseases, we transferred the human γ-IFN gene into endothelial cells isolated from human arteries and microvessels using a retroviral vector. Biologically active γ-IFN was produced and secreted by γ-IFN transduced endothelial cells, but not by control, nontransduced cells, or cells identically transduced with E. coli beta galactosidase (β-gal). To more closely approximate the microenvironment of blood vessels, subconfluent smooth muscle cells were plated in coculture with control, nontransduced endothelial cells, γ-IFN transduced endothelial cells, or β-gal transduced endothelial cells. Smooth muscle cell growth was inhibited 30-70% by coculture with γ-IFN transduced endothelial cells compared to coculture with β-gal transduced or control endothelial cells (p < 0.05). Our results suggest endothelial cells modified to produce γ-IFN may be a useful therapy in proliferative vascular diseases. Copyright © 1997 Elsevier Science Inc.
Control of Inflammatory Damage by Anti-Lfa-1: Increase Success of Myoblast Transplantation
Authors: Guérette Benoit , Asselin Isabelle , Skuk Daniel , Entman Mark , Tremblay Jacques P.
Published: June 2017

Myoblast transplantation is a potential treatment for Duchenne Muscular Dystrophy. This article confirms by experiments in mice that one problem that has limited the success of clinical trials of this procedure is a rapid (within 3 days) inflammatory reaction which kills most of the injected myoblasts. The death of the transplanted myoblasts can be prevented by treating the host with a mAb against LFA-1. This led to a 27-fold increase in the number of muscle fibers expressing a reporter gene present in the donor myoblasts when the host is also adequately immunosuppressed with FK506. Therefore, both the nonspecific inflammatory reaction and the specific immune response should be adequately controlled following myoblast transplantation.
Metabolic Energy Capacity of Dopaminergic Grafts and the Implanted Striatum in Parkinsonian Nonhuman Primates as Visualized with Cytochrome Oxidase Histochemistry
Authors: Collier Timothy J. , Redmond D. Eugene , Roth Robert H. , Elsworth John D. , Taylor Jane R. , Sladek John R.
Published: June 2017

Histochemistry for visualization of the mitochondrial enzyme cytochrome oxidase has been used to detect cellular and regional differences in brain energy metabolism. We have examined the pattern of cytochrome oxidase (CO) staining in grafts of embryonic ventral mesencephalic tissue, and in the implanted striatum, of MPTP-treated monkeys as one index of the functional activity of grafted tissue and its influence on the host brain. Four monkeys were selected for study based on interesting variations in dopamine (DA) neuron content of their bilateral grafts as demonstrated with tyrosine-hydroxylase (TH) immunocytochemistry. The results suggest that grafts rich in DA neurons increase the metabolic activity of the implanted striatum of DA-depleted monkeys, and that this improvement of local energy metabolism is greater in the vicinity of grafts containing greater numbers of DA neurons. In addition, the pattern of CO staining within tissue transplants indicates that DA neurons exhibit the highest rate of metabolic activity among all cell types contained in the ventral mesencephalic grafts, and that the transplants receive metabolically active innervation from outside or within the grafted tissue.
The Age of Striatum Determines the Pattern and Extent of Dopaminergic Innervation: a Nigrostriatal Double Graft Study
Authors: Stromberg Ingrid , Björklund Lars , Forander Petter
Published: June 2017

In animal models of Parkinson's disease, transplanted fetal mesencephalic dopaminergic neurons can innervate the dopamine-depleted host brain, but it is unclear why large portions of the host striatum are left uninnervated. During normal development, the dopaminergic innervation first occurs in the form of a dense patchy pattern in the striatum, followed by a widespread nerve fiber network. Using intraocular double grafts we have investigated dopaminergic growth patterns initiated when ventral mesencephalic grafts innervate striatal targets. The fetal lateral ganglionic eminence was implanted into the anterior eye chamber. After maturation in oculo, fetal ventral mesencephalon was implanted and placed in contact with the first graft. In other animals the two pieces of tissue were implanted simultaneously. Tyrosine hydroxylase (TH) immunohistochemistry revealed a pattern of dense TH-positive patches throughout the total volume of the striatal grafts in simultaneously transplanted cografts, while a widespread, less dense, pattern was found when mature striatal transplants were innervated by fetal dopaminergic grafts. To investigate which type or types of growth patterns that developed after grafting to striatum in situ of an adult host, fetal ventral mesencephalic tissue was implanted into the lateral ventricle adjacent to the dopamine-lesioned striatum. After maturation of the mesencephalic graft, the fetal lateral ganglionic eminence was implanted into the reinnervated part of the host striatum. TH immunohistochemistry revealed a few nerve fibers within the striatal graft and the growth pattern was of the widespread type. In conclusion, grafted dopaminergic neurons preferably innervate mature striatum with a widespread sparse nerve fiber network, while the innervation of the immature striatum occurs in the form of dense patches. Furthermore, when the patchy pattern is formed, the total volume of the striatal target is innervated while growth of the widespread type terminates prior to reaching distal striatal parts. Thus, the growth pattern seems essential to the final volume that is innervated. Once the widespread growth pattern is initiated, the presence of immature striatum does not change the dopaminergic growth pattern.
Histochemical Analysis of the Role of Class I and Class II Clostridium Histolyticum Collagenase in the Degradation of Rat Pancreatic Extracellular Matrix for Islet Isolation
Authors: Vos-Scheperkeuter Greetje H. , Van Suylichem Paul T.R. , Vonk Maike W.A. , Wolters Gerrit H.J. , Van Schilfgaarde Reinout
Published: June 2017

To understand why class II Clostridium histolyticum collagenase is much more effective than class I in the isolation of rat pancreatic islets, we analyzed the role of these collagenases in pancreatic tissue dissociation. Crude collagenase was purified and then fractionated into class I and II with different enzyme activities and protein compositions. Pancreatic tissue was incubated with either class I, class II, or class I + II, with or without added protease, under conditions that eliminated endogenous proteolytic activity. The degradation of pancreatic extracellular matrix was monitored by selective histochemical staining of tissue samples. Class I and II showed similar capacities to degrade glycoproteins and degraded about one-third of the glycoproteins during 120 min of incubation. The degradation of collagens by class I and II was relatively more effective, 80 to 95% of the collagens being removed in 120 min, and also class dependent. Both in the presence and absence of protease, class II was more effective at degrading collagens than class I, but this difference in efficacy was less apparent than with islet isolation. Class I + II degraded collagens faster and more complete than did the individual classes, indicating a synergistic effect of class I and II. Evaluation of collagen degradation at various pancreatic locations did not show a selective degradation of collagens by any of the collagenase classes. The present data offer a partial explanation for the major role of class II in islet isolation.
Polymer-Encapsulated PC-12 Cells Demonstrate High-Affinity Uptake of Dopamine in Vitro and 18F-DOPA Uptake and Metabolism after Intracerebral Implantation in Nonhuman Primates
Authors: Subramanian Thyagarajan , Emerich Dwaine F. , Bakay Roy A. E. , Hoffman John M. , Goodman Mark M. , Shoup Timothy M. , Miller Gary W. , Levey Allan I. , Hubert George W. , Batchelor Scott , Winn Shelly R. , Saydoff Joel A. , Watts Ray L.
Published: June 2017

Intracranial implantation of polymer-encapsulated PC-12 cells has been shown to improve motor behavioral performance in animal models of Parkinson's disease. The purpose of this blinded study was to examine whether such improvement is associated with the active uptake and metabolism of dopamine precursors by intracerebrally implanted polymer-encapsulated PC-12 cells. In an in vitro experiment we demonstrate that 3H-dopamine uptake by PC-12 cells was 108 fmol/min × 106 cells, and that this uptake can be specifically blocked 88% by the addition of 10 nM of nomifensine. In the in vivo experiments, polymer-encapsulated PC-12 cells were implanted in four MPTP-treated monkeys into the left deep parietal white matter (R1) or left striatum (R2-4). A fifth MPTP-treated monkey (R5) served as a control and received left striatal implants of empty capsules. 18F-Dopa Positron Emission Tomography (PET) imaging was performed on each monkey before and after implantation surgery by blinded investigators. PET images obtained 5-13 wk after implantation demonstrated well delineated focal areas of high 18F-dopa uptake in R1, R2, and R4. The focal area of high 18F-dopa uptake in R1 precisely coregistered on a brain magnetic resonance image to the site of implantation. R3 (in whom the polymer-encapsulated PC-12 cells demonstrated poor cell survival upon explantation) and R5 (empty capsules) failed to demonstrate any area of increased 18F-dopa uptake in their PET images. Histological examination of the host brain revealed no sprouting of dopaminergic nerve terminals around the implantation sites of the polymer-encapsulated PC-12 cells. These results indicate that the previously noted behavioral improvement after intrastriatal implantation of polymer encapsulated PC-12 cells is at least in part due to their highly specific uptake and metabolism of dopamine precursors. Furthermore, these data suggest that polymer-encapsulated PC-12 cells can store, reuptake, and functionally replenish dopamine and therefore, may be an effective treatment for Parkinson's disease.
Grafts of Genetically Modified Schwann Cells to the Spinal Cord: Survival, Axon Growth, and Myelination
Authors: Tuszynski Mark H. , Weidner Norbert , Mccormack Melissa , Miller Ian , Powell Henry , Conner James
Published: June 2017

Schwann cells naturally support axonal regeneration after injury in the peripheral nervous system, and have also shown a significant, albeit limited, ability to support axonal growth and remyelination after grafting to the central nervous system (CNS). It is possible that Schwann cell-induced axonal growth in the CNS could be substantially increased by genetic manipulation to secrete augmented amounts of neurotrophic factors. To test this hypothesis, cultured primary adult rat Schwann cells were genetically modified using retroviral vectors to produce and secrete high levels of human nerve growth factor (NGF). These cells were then grafted to the midthoracic spinal cords of adult rats. Findings were compared to animals that received grafts of nontransduced Schwann cells. Spinal cord lesions were not placed prior to grafting because the primary aim of this study was to examine features of grafted Schwann cell survival, growth, and effects on host axons. In vitro prior to grafting, Schwann cells secreted 1.5 + 0.1 ng human NGF/ml/106cells/day. Schwann cell transplants readily survived for 2 wk to 1 yr after in vivo placement. Some NGF-transduced grafts slowly increased in size over time compared to nontransduced grafts; the latter remained stable in size. NGF-transduced transplants were densely penetrated by primary sensory nociceptive axons originating from the dorsolateral fasciculus of the spinal cord, whereas control grafts showed significantly fewer penetrating sensory axons. Over time, Schwann cell grafts also became penetrated by TH- and DBH-labeled axons of putative coerulospinal origin, unlike control cell grafts. Ultrastructurally, axons in both graft types were extensively myelinated by Schwann cells. Grafted animals showed no changes in gross locomotor function. In vivo expression of the human NGF transgene was demonstrated for periods of at least 6 m. These findings demonstrate that primary adult Schwann cells 1) can be transduced to secrete augmented levels of neurotrophic factors, 2) survive grafting to the CNS for prolonged time periods, 3) elicit robust growth of host neurotrophin-responsive axons, 4) myelinate CNS axons, and 5) express the transgene for prolonged time periods in vivo. Some grafts slowly enlarge over time, a feature that may be attributable to the propensity of Schwann cells to immortalize after multiple passages. Transduced Schwann cells merit further study as tools for promoting CNS regeneration.
Pig Fetal Septal Neurons Implanted into the Hippocampus of Aged or Cholinergic Deafferented Rats Grow Axons and Form Cross-Species Synapses in Appropriate Target Regions
Authors: Deacon Terrence , Whatley Brandi , Leblanc Celeste , Lin Ling , Isacson Ole
Published: June 2017

The anatomical specificity of axon growth from fetal pig septal xenografts was studied by transplanting septal cells from E30–35 pig fetuses into cholinergic deafferented (192-IgG-saporin-infused) rats or into aged rats (> 18 months). Cell suspensions (100,000 cells/μl) were injected bilaterally into the dorsal and ventral hippocampus of immunosuppresed rats (10 mg/kg/day cyclosporine A). To assess axonal growth and synapse formation, acetylcholinesterase histochemistry, an antibody to choline acetyltransferase (ChAT), and three pig-positive/rat-negative antibodies: bovine 70kD neurofilament (NF70), human low-affinity NGF receptor (hNGFr), and human synaptobrevin (hSB) were used. In rats with surviving grafts at 6 months, NF70 axonal labeling was more extensive than either ChAT or hNGFr labeling. All three markers demonstrated graft axons extending selectively through the hippocampal CA fields and the molecular layer of the dentate gyrus. Graft axons did not extend into adjacent entorhinal cortex or neocortex. The distribution of pig hSB-positive synapses correlated with AChE-positive fiber outgrowth in to the host. Electron microscopic analysis of hSB-immunostained hippocampal sections revealed pig presynaptic terminals in contact with normal rat postsynaptic structures in the CA fields and the dentate gyrus. These data demonstrate target-appropriate growth of pig cholinergic axons and the formation of cross-species synapses in the deafferented or aged rat hippocampus.
Morris Water Maze Analysis of 192-IgG-Saporin-Lesioned Rats and Porcine Cholinergic Transplants to the Hippocampus
Authors: Leblanc C. J. , Deacon T. W. , Whatley B. R. , Dinsmore J. , Lin L. , Isacson O.
Published: June 2017

Adults rats were lesioned with 192-IgG-saporin, an immunotoxin that targets cholinergic neurons in the basal forebrain expressing the low-affinity nerve growth factor receptor (p75). One month later, rats received E30–35 porcine cholinergic neurons bilaterally into the hippocampus, and were tested in the Morris water maze and the passive avoidance task 4.5–6 months after transplantation (in two experiments, rats were retested in the water maze) followed by histological and cellular analyses. The 192-IgG-saporin-lesioned animals displayed clear cognitive deficits in the Morris water maze. In all experiments the lesioned animals had spatial probe deficits on day 5 testing. A large variance was found among the transplanted animals, with individual animals exhibiting improved performance, but little overall improvement when compared to lesion-alone animals as a group. The relationships between behavioral performance and graft cholinergic factors were established by histological analyses. Grafted animals exhibited an increase in cholinergic innervation of the dentate gyrus (DG) region of the dorsal hippocampus when compared to lesion-alone animals. There was a significant correlation between the level of cholinergic innervation in the dentate gyrus and spatial navigation performance (latency and spatial probe) in the Morris water maze task. These data provide evidence of memory and spatial deficits following cholinergic denervation, and of target-specific growth of xenogeneic cholinergic neurons into the hippocampus. The lack of a clear treatment (transplant) effect in the behavioral measures leads us to believe that functional restoration of cognitive function would require cholinergic reinnervation of both the hippocampus and the neocortex in this 192-IgG-saporin animal model.
Development of Poly(Propylene Fumarate-co-Ethylene Glycol) as an Injectable Carrier for Endothelial Cells
Authors: Suggs Laura J. , Mikos Antonios G.
Published: June 2017

Poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)] hydrogels were examined as in situ polymerizable carriers for endothelial cells. The temperature increase from 37°C during cross-linking was measured. The maximum temperature did not increase above 38.3°C for any copolymer formulation. The temperature profiles also appeared to be independent of the amount or molecular weight of poly(ethylene glycol). These materials were polymerized in situ in a subcutaneous rat model and evaluated for initial biocompatibility. A normal wound-healing response was seen with formation and subsequent maturity of a fibrous capsule. Endothelial cells were embedded in vitro during the cross-linking process and their proliferation was assessed over the first 24 h. There was significant DNA synthesis by the embedded endothelial cells during this time period. These data suggest that P(PF-co-EG) hydrogels could be developed for use as injectable cell carriers.
Effectiveness of Acidic Oxidative Potential Water in Preventing Bacterial Infection in Islet Transplantation
Authors: Miyamoto Masaaki , Inoue Kazutomo , Gu Yuanjun , Hoki Masanori , Haji Seiji , Ohyanagi Harumasa
Published: June 2017

At a number of points in the current procedures of islet isolation and islet culture after the harvesting of donor pancreata, microorganisms could potentially infect the islet preparation. Furthermore, the use of islets from multiple donors can compound the risks of contamination of individual recipients. Acidic oxidative potential water (also termed electrolyzed strong acid solution, function water, or acqua oxidation water), which was developed in Japan, is a strong acid formed on the anode in the electrolysis of water containing a small amount of sodium chloride. It has these physical properties: pH, from 2.3 to 2.7; oxidative-reduction potential, from 1,000 to 1,100 mV; dissolved chlorine, from 30 to 40 ppm; and dissolved oxygen, from 10 to 30 ppm. Because of these properties, acidic oxidative potential water has strong bactericidal effects on all bacteria including methicillin-resistant Staphylococcus aureus (MRSA), viruses including HIV, HBV, HCV, CMV, and fungi as a result of the action of the active oxygen and active chlorine that it contains. We conducted this study to evaluate the effect of acidic oxidative potential water irrigation on bacterial contamination on the harvesting of porcine pancreata from slaughterhouses for islet xenotransplantation by counting the number of pancreatic surface bacteria using the Dip-slide method, and on the results of islet culture; and to evaluate the direct effect on isolated islets when it is used to prevent bacterial contamination by the static incubation test and by morphorogical examination. Direct irrigation of the pancreas by acidic oxidative potential water was found to be very effective in preventing bacterial contamination, but direct irrigation of isolated islets slightly decreased their viability and function.
Revisiting the Basics
Authors: Hooper Amy , Spann Crystin , McCray Tiyahri , Kimberly Claire
Published: June 2017

It has been found that the presence of negative communicative techniques and emotional flooding in marital relationships negatively relates to marital satisfaction. However, current literature has not consistently included known demographical differences (e.g., race, gender, etc.) to truly test how these variables impact marital satisfaction. This study investigated the specific impact that both emotional flooding and negative communicative techniques had on 591 married individuals’ marital satisfaction while controlling for gender, race, and length of relationship. The results indicated that emotional flooding tends to decrease as marriages go on, and refusing to talk with the partner had the greatest negative impact on relationship satisfaction regardless of demographical differences. Suggestions are provided for how therapists and practitioners can use these findings to assist couples who are using negative communication techniques and are, subsequently, experiencing marital dissatisfaction.
A Software Application for Mining and Presenting Relevant Cancer Clinical Trials per Cancer Mutation
Authors: Gandy Lisa M , Gumm Jordan , Blackford Amanda L , Fertig Elana J , Diaz Luis A
Published: June 2017

ClinicalTrials.org is a popular portal which physicians use to find clinical trials for their patients. However, the current setup of ClinicalTrials.org makes it difficult for oncologists to locate clinical trials for patients based on mutational status. We present CTMine, a system that mines ClinicalTrials.org for clinical trials per cancer mutation and displays the trials in a user-friendly Web application. The system currently lists clinical trials for 6 common genes (ALK, BRAF, ERBB2, EGFR, KIT, and KRAS). The current machine learning model used to identify relevant clinical trials focusing on the above gene mutations had an average 88% precision/recall. As part of this analysis, we compared human versus machine and found that oncologists were unable to reach a consensus on whether a clinical trial mined by CTMine was “relevant” per gene mutation, a finding that highlights an important topic which deems future exploration.
Clinical commentary on ‘Recurrent natalizumab-related aseptic meningitis in a patient with multiple sclerosis’, by Foley et al.
Authors: Chan Andrew
Published: June 2017

This clinical commentary discusses the occurrence of drug-induced aseptic meningitis in patients undergoing natalizumab treatment and other immunotherapies.
Benefits of Long Versus Short Thrombolysis Times for Acutely Thrombosed Hemodialysis Native Fistulas
Authors: Regus Susanne , Lang Werner , Heinz Marco , Uder Michael , Schmid Axel
Published: June 2017

Introduction:

Local thrombolysis with a time of exposure to recombinant tissue plasminogen activator of 15 to 150 minutes is commonly used to declot acutely thrombosed hemodialysis fistulas. The duration of thrombolysis for the restoration of arteriovenous blood flow remains controversial. The aim of this study was to investigate the outcomes of long thrombolysis treatment (LTT, 3 hours or more) and short thrombolysis treatment (STT, less than 3 hours) in our institution.

Methods:

We retrospectively analyzed 86 interventional declotting procedures (28 STT and 58 LTT) applied to 86 acutely thrombosed hemodialysis fistulas. The intervention time (IT) following thrombolysis (from the initial fistulography to the end of the angioplasty maneuvers), the time of day of the intervention (ie, during working hours vs off-hours), and the need for temporary catheter placement (TCP) were assessed. Success was defined as complete access recanalization, and major adverse events were defined as ischemia, bleeding, and access rupture.

Results:

The ITs were reduced after LTT (63.3 [9.3] minutes) compared to STT (106.7 [24.7], P = .01), but there was no difference in success rate (85.7% STT, 89.7% LTT, P = .722). While all (100%, 58/58) of the angioplasty maneuvers after LTT were performed during regular working hours, 75% (21/28) of those following STT were managed during off-hours ( P < .001). Despite the longer treatment, the need for TCP was not increased after LTT (10.7%) compared to STT (12.1%, P = .515), and the major complication rate was reduced (3.4% after LTT and 28.6% after STT, P = .004).

Conclusion:

Long thrombolysis treatment results in shorter and less complicated percutaneous stenosis treatments during regular working hours. Despite the LTT of up to 25 hours until access for dialysis was achieved, no increase in the risks of TCP or major adverse events were observed following LTT.
European employment policies
Authors: Weiss Manfred
Published: June 2017

This article provides a critical analysis of the EU Employment Policies legal framework. The need to focus on both the quantitative and qualitative dimensions of labour is highlighted in the first part. The second part deals with the quantitative aspects, addressing the Employment chapter, State aid, and the freedom of movement for workers.

The third part discusses the qualitative side – providing an overview on the EU labour law – and addresses the limitation of the EU’s legislative powers and the need for minimum labour standards. The conclusion underlines the difficulties to get further hard law and the soft law inadequacy.
What role for solopreneurs in the labour market?
Authors: Waas Bernd
Published: June 2017

The article discusses the legal position of ‘solopreneurs’ under German law. After a look at questions of definition (Section 2), it is explained why we are likely to see a growing number of such persons in the labour market (Section3). The basic categories (self-employed, employees, ‘quasi-salaried persons, and home workers) are introduced (Section 4), before taking a closer look at the provisions that can be applied to ‘solopreneurs’ (Section 5). Different hypotheses are examined with the aim of understanding ways to provide ‘solopreneurs’ with more protection (Section 6). The conclusion highlights possible ways to achieve this, but freedom of contract and fair competition must not be compromised.
The Location of Anterior Cruciate Ligament Tears: A Prevalence Study Using Magnetic Resonance Imaging
Authors: van der List Jelle P. , Mintz Douglas N. , DiFelice Gregory S.
Published: June 2017

Background:

Over the past decade, there has been a resurgence of interest in anterior cruciate ligament (ACL) preservation. Proximal and distal avulsion tears have been treated with arthroscopic primary repair, while augmented repair, remnant tensioning, primary repair with biological scaffold, and remnant preservation have been proposed for different types of midsubstance tears. Currently, the incidence of these different tear types is unknown.

Purpose:

To propose a magnetic resonance imaging (MRI) classification system for different tear types based on clinical relevance and to assess the distribution of these different ACL tear types.

Study Design:

Case series; Level of evidence, 4.

Methods:

A retrospective search in an institutional radiographic database was performed for patients who underwent knee MRI at our institution between June 2014 and June 2016. Patients younger than 18 years and those with reports of chronic tears, partial tears, multiligamentous injuries, were excluded. Tear types were graded as proximal avulsion (distal remnant length >90% of total ligament length, type I), proximal (75%-90%, type II), midsubstance (25%-75%, type III), distal (10%-25%, type IV), and distal avulsion (<10%, type V). An orthopaedic surgeon, a radiologist, and a research fellow graded the tear type on 30 MRIs to determine reliability, and the research fellow graded all MRIs. Inter- and intraobserver reliability were measured using kappa statistics.

Results:

A total of 353 patients (57% male; mean age, 37.1 years; range, 18.1-81.2 years) were included. Interobserver reliability was 0.670 (95% confidence interval, 0.505-0.836), and intraobserver reliability ranged from 0.741 to 0.934. Incidence of type I tears was 16%, type II tears 27%, type III tears 52%, type IV tears 1%, and type V tears 3% (2.5% with bony avulsion). Type I tears were more common in patients older than 35 years compared with those younger than 35 years (23% vs 8%; P < .001).

Conclusion:

This classification system was reliable in assessing tear location in acute ACL injuries. Type I tears were seen in 16%, type II in 27%, and type III in 52% of patients in our cohort. These data suggest that there may be greater potential application for ACL preservation techniques.
Intravitreal injections service: a patient experience evaluation
Authors: Hasan Hani , Flockhart Sarah , Qureshi Waseem , Khan Selina , Ahmed Shahina , Shah Nimish
Published: June 2017

Introduction:

This survey has been conducted following the introduction of nurse-led intravitreal injections clinics at the Great Western Hospital. A team of 5 nurses regularly carry out an average of 90 injections per week.

Methods:

A series of 169 consecutive injection patients have been offered the study questionnaire between 28 January 2016 and 28 February 2016; patients with no previous experience with a nurse injector were excluded.

Results:

76.9% strongly agree that the nurse is more friendly and sensitive to their needs. Only 12.4% strongly agree that a doctor will be more suitable to deliver the injections. Seventy-five percent stated that they were very happy with the care, while 0.4% thought a nurse would be unsafe; 69.2% feel more comfortable asking questions when with a nurse injector. Anxiety and nervousness were found to be the main two symptoms experienced by patients around the time of injections (27.8% and 34.9%, respectively).

Discussion:

The questionnaire was designed based on the feedback from the local friends and family test. A clear majority of patients gave highly positive feedback about nurse injectors. Considering patients' individual needs at the time of injections, such as allergies or points that needs clarification, helps in overcoming some of the psychological complications of treatment.