bluuebunny/crossref_metadata_2025_split huggingface dataset:
Health policy and its implementation in small island developing states in the British West Indies
Authors: McNaught Allan 
Published: December 2003

AbstractHealth policy and its implementation in small island developing states (SIDS) is a neglected area of study, and, seemingly, of little interest. The existing literature is generally characterized by descriptions of failure or incompetence, with little attempt to understand the nature and workings of the policy process in these small, yet complex, societies.The research undertaken in this article was carried out over 6 years in Anguilla (pop. 9000) and the British Virgin Islands (pop. 20 000), two British Overseas Territories in the North East Caribbean. The purpose of the research was to determine to what extent policy theory and the tools of policy analysis could be used to explain the nature and the outcomes of the health policy and implementation process.In trying to analyse and understand the policy process in these small islands it was necessary to understand their socio‐politic character. In addition, the development of a model of the public policy and public administration system in the English‐speaking Caribbean was an essential part of this process.It was found that the conduct and outcome of the policy and implementation process in these islands varied significantly. It is postulated that the variations are anchored in the nature of the local context, the working practices and ideologies of politicians, senior public servants and the local policy elite. Copyright © 2003 John Wiley & Sons, Ltd.
Novel macrolide‐adjustable bidirectional expression modules for coordinated expression of two different transgenes in mice
Authors: Fux Cornelia , Weber Wilfried , Daoud‐El Baba Marie , Heinzen Christoph , Aubel Dominique , Fussenegger Martin 
Published: December 2003

AbstractBackgroundPrecise control of transgene expression is essential for a variety of applications ranging from gene‐function analysis, biopharmaceutical manufacturing to next‐generation molecular interventions in gene therapy and tissue engineering. The regulation of gene expression is currently a key issue for clinical implementation of gene‐therapy‐based treatments since desired transgene expression may need to be maintained within a narrow therapeutic window for successful treatment of a particular human disease.MethodsWe have designed a novel bidirectional expression module that enables adjustable coregulation of two different transgenes in response to clinical doses of macrolide antibiotics. A bidirectional macrolide‐responsive promoter consisting of a central operator module (ETR) specific for the macrolide‐dependent transactivator (ET1) is flanked by two minimal promoters (PhCMVmin; Phsp70min) which drive expression of two divergently oriented transgenes. Macrolide antibiotics modulate the binding affinity of ET1 to ETR and adjust expression of both transgenes to desired levels.ResultsBidirectional expression configurations enabled excellent macrolide‐adjustable coregulation profiles of two secreted reporter genes or one‐vector‐based autoregulated fine‐tuning of a single transgene in various transgenic rodent and human cell lines. Following implantation of microencapsulated CHO‐K1 cell derivatives transgenic for macrolide‐controlled bidirectional expression of erythropoietin (EPO) and the human secreted alkaline phosphatase (SEAP) intraperitoneally into mice, serum EPO and SEAP levels could be coadjusted to desired levels by administration of different erythromycin doses.ConclusionsBased on their in vivo compatibility, the versatile bidirectional and macrolide‐responsive expression modules represent an important advancement on the way to implementing targeted and conditional molecular interventions into a clinical reality. Copyright © 2003 John Wiley & Sons, Ltd.
Increased in vitro and in vivo transgene expression levels mediated through cis‐acting elements
Authors: Johansen Jens , Tornøe Jens , Møller Arne , Johansen Teit E. 
Published: December 2003

AbstractBackgroundGene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down‐regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis‐acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS.MethodsPlasmid vector constructs incorporating Woodchuck hepatitis post‐transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated.ResultsInserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum.ConclusionThe data suggest that incorporating cis‐acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors. Copyright © 2003 John Wiley & Sons, Ltd.
Enhancement of ultrasound‐mediated gene transfection by membrane modification
Authors: Nozaki Tetsuo , Ogawa Ryohei , Feril Loreto B. , Kagiya Go , Fuse Hideki , Kondo Takashi 
Published: December 2003

AbstractBackgroundUltrasound‐mediated gene transfection (USMGT) with an echo contrast agent could be a new promising physical method of triggering localized gene delivery, but the effect is still modest. The aim of this study is to devise a method to improve efficiency of USMGT. We examined the effect of lidocaine and different temperatures on USMGT, each of which is a known membrane modifier, since the plasma membrane can be considered a site of action in USMGT.MethodsWe observed the effect of lidocaine (0.01, 0.1 or 1.0 mM) and different temperatures (7, 20, 37, 42 or 44 °C) on USMGT (1 MHz, 3.6 W/cm2 (ISATA) and 20 s exposure) in the presence of Levovist (10 mg/ml). At 20 h after sonication, transfection efficiency was evaluated by luciferase assay. Membrane fluidity was examined by fluorescence polarization measurement. Cavitational activity was measured by ESR spin trapping with 5,5‐dimethyl‐1‐pyrroline N‐oxide. The number of cells transfected with the GFP gene was counted under a fluorescence microscope.ResultsLidocaine (1 mM) and heat (42–44 °C) significantly increased luciferase expression approximately 18‐fold and 19‐fold higher than Levovist only. Both treatments were shown to increase membrane fluidity; in addition, heat enhanced a cavitational effect. It was confirmed by an experiment using the GFP gene that increase in luciferase expression was due to the increase in number of cells.ConclusionsThis enhancement could be useful for ultrasound‐mediated gene therapy in the future since both treatments for membrane modification could be directly applied to the living body. Copyright © 2003 John Wiley & Sons, Ltd.
Comparison of wild‐type and class I integrase mutant‐FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium
Authors: Loewen Nils , Leske David A. , Chen Yi , Teo Wu‐Lin , Saenz Dyana T. , Peretz Mary , Holmes Jonathan M. , Poeschla Eric M. 
Published: December 2003

AbstractBackgroundIn neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2–5 days post‐natal). In non‐retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol‐encoded functions intact. For HIV‐1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long‐term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector.MethodsWe compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild‐type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV‐G)‐pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post‐injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase‐normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (β‐galactosidase) expression by an observer blinded to vector assignments.ResultsSubretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed β‐galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation.ConclusionsThese results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV‐G‐pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post‐natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy. Copyright © 2003 John Wiley & Sons, Ltd.
Engineering of technetium‐99m‐binding artificial receptors for imaging gene expression
Authors: Simonova Maria , Shtanko Olena , Sergeyev Nikolai , Weissleder Ralph , Bogdanov Alexei 
Published: December 2003

AbstractBackgroundOptimization of gene therapy protocols requires accurate and non‐invasive quantification of vector delivery and gene expression. To facilitate non‐invasive imaging of gene expression, we have genetically engineered ‘artificial receptors’, i.e. membrane proteins that bind 99mTc‐oxotechnetate (99mTcOT) via transchelation from a complex with glucoheptonate. The latter is a component of a widely used clinical imaging kit.MethodsThe engineered marker proteins were designed as type I and II membrane proteins and consisted of (1) an 99mTcOT‐binding domain, metallothionein (MT), and (2) a membrane‐anchoring domain. Engineered constructs were used for transfection of COS‐1 and 293 cells; the expression of mRNA was verified by RT‐PCR.ResultsImmunofluorescent analysis, cell fractionation and immunoblotting revealed expression of marker proteins on plasma membrane. Transfection of cells resulted in strong positive staining of plasma membrane with anti‐His‐tag antibodies. Scintigraphic imaging in vitro confirmed the ability of transfected cells to bind 99mTcOT. The fraction of bound radioactivity reached a peak (3.53%) when 0.93 MBq 99mTcOT was added to transfected COS‐1 cells. The experiment‐to‐control signal ratio was equal to 32 at the same added dose.Conclusions(1) Both types of engineered ‘artificial receptors’ were expressed on the surface of eukaryotic cells; (2) marker proteins were functional in binding 99mTcOT; and (3) type II membrane proteins were more efficient in binding 99mTcOT than type I proteins. We anticipate that the developed approach could be useful for ‘tagging’ transfected cells with 99mTcOT enabling imaging of tracking in vivo transduced cells or cell therapies. Copyright © 2003 John Wiley & Sons, Ltd.
In vivo knockdown of gene expression in brain cancer with intravenous RNAi in adult rats
Authors: Zhang Yun , Boado Ruben J. , Pardridge William M. 
Published: December 2003

AbstractBackgroundGene silencing in mammalian cells is possible with RNA interference (RNAi) with expression plasmids that encode for short hairpin RNAs (shRNA) that hybridize to a specific sequence within a target mRNA. The limiting factor in developing RNAi therapeutics in mammals is the gene delivery system.MethodsThe present studies describe the production of anti‐luciferase shRNA expression plasmids, which are encapsulated in the interior of 85 nm pegylated immunoliposomes (PILs). C6 rat glioma cells, permanently transfected with the luciferase gene, are implanted in the caudate‐putamen nucleus of adult rats, which produces luciferase‐expressing intracranial brain cancer. The PILs are targeted across the blood‐brain barrier and across the tumor cell membrane in vivo with a monoclonal antibody (MAb) to the rat transferrin receptor (TfR). The TfRMAb is tethered to the tips of 1–2% of the poly(ethylene glycol) strands conjugated to the surface of the liposome.ResultsThe TfRMAb‐targeted PILs inhibit luciferase gene expression in the brain cancer by 90%, and this effect persists for at least 5 days after a single intravenous injection of 10 µg/rat of plasmid DNA. RNAi therapy directed against the luciferase gene caused no change in expression of tumor γ‐glutamyl transpeptidase. Targeting the empty expression plasmid with the TfRMAb‐PIL resulted in no change in luciferase activity in the brain cancer in vivo.ConclusionsIn vivo RNAi is enabled with a new form of gene delivery system that encapsulates expression plasmids in PILs, which are targeted to distant sites based on the specificity of a receptor‐specific monoclonal antibody. The combined application of the PIL gene delivery system and RNAi expression plasmids enables gene silencing in remote sites such as brain cancer in mammals after intravenous administration. Copyright © 2003 John Wiley & Sons, Ltd.
Ganciclovir nucleotides accumulate in mitochondria of rat liver cells expressing the herpes simplex virus thymidine kinase gene
Authors: van der Eb Marjolijn M. , Geutskens Sacha B. , van Kuilenburg André B. P. , van Lenthe Henk , van Dierendonck Jan‐Hein , Kuppen Peter J. K. , van Ormondt Hans , van de Velde Cornelis J. H. , Wanders Ronald J. A. , van Gennip Albert H. , Hoeben Rob C. 
Published: December 2003

AbstractBackgroundGanciclovir exhibits broad‐spectrum activity against DNA viruses such as cytomegaloviruses, herpes simplex viruses, varicella‐zoster virus, Epstein‐Barr virus and human herpes virus‐6. Ganciclovir is widely applied for anti‐herpetic treatment, cytomegalovirus prophylaxis after organ transplantation, and, more recently, in experimental gene therapy to eradicate cycling cells that express the herpes simplex virus thymidine kinase gene. Although ganciclovir supposedly acts as a chain terminator, there is compelling evidence demonstrating the presence of ganciclovir, but not of acyclovir, incorporated internally into DNA, leaving the precise mechanism by which ganciclovir inhibits DNA synthesis enigmatic.MethodsTo study the potential involvement of mitochondria in the ganciclovir nucleotide cytotoxicity, we used adenovirus‐mediated gene transfer to express herpes simplex virus thymidine kinase in rat liver and administered ganciclovir 2 days post‐infection. The integrity and function of mitochondria in the rat liver cells were evaluated by several techniques. In addition, we analyzed the nucleotide pools in cellular extracts and in isolated mitochondria.ResultsWe show that ganciclovir nucleotides are abundantly present in the mitochondria of rat livers that express the HSVtk gene. Already 48 h after administration, 10–30% of the total mitochondrial nucleotide pool consists of ganciclovir nucleotides. Their presence is correlated with a lower amount of mitochondrial DNA, a reduced mitochondrial‐membrane potential, morphological abnormalities, and liver dysfunction.ConclusionsThese data provide evidence for the involvement of mitochondria in the hepatotoxicity of the HStk/ganciclovir combination. This may explain the toxicity of the HSVtk/gancilovir combination in some metabolically active but non‐proliferating cells, such as liver cells. This toxicity limits the applicability of this enzyme/prodrug combination. Copyright © 2003 John Wiley & Sons, Ltd.
Mesenchymal stem cells home to injured tissues when co‐infused with hematopoietic cells to treat a radiation‐induced multi‐organ failure syndrome
Authors: Chapel Alain , Bertho Jean Marc , Bensidhoum Morad , Fouillard Loic , Young Randell G. , Frick Johanna , Demarquay Christelle , Cuvelier Frédérique , Mathieu Emilie , Trompier François , Dudoignon Nicolas , Germain Claire , Mazurier Christelle , Aigueperse Jocelyne , Borneman Jade , Gorin Norbert Claude , Gourmelon Patrick , Thierry Dominique 
Published: December 2003

AbstractBackgroundRecent studies have suggested that ex vivo expansion of autologous hematopoietic cells could be a therapy of choice for the treatment of bone marrow failure. We investigated the potential of a combined infusion of autologous ex vivo expanded hematopoietic cells with mesenchymal (MSCs) for the treatment of multi‐organ failure syndrome following irradiation in a non‐human primate model.MethodsHematopoietic cells and MSCs were expanded from bone marrow aspirates. MSCs were transduced with the gene encoding for the green fluorescent protein (e‐GFP), in order to track them following infusion. Twelve animals were studied. Nine animals received total‐body irradiation at 8 Gy from a neutron/gamma source thus resulting in heterogeneous exposure; three animals were sham‐irradiated. The animals were treated with expanded hematopoietic stem cells and MSCs, expanded hematopoietic stem cells alone, or MSCs alone. Unmanipulated bone marrow cell transplants were used as controls.ResultsDepending on the neutron/gamma ratio, an acute radiation sickness of varying severity but of similar nature resulted. GFP‐labeled cells were found in the injured muscle, skin, bone marrow and gut of the treated animals via PCR up to 82 days post‐infusion.ConclusionsThis is the first evidence of expanded MSCs homing in numerous tissues following a severe multi‐organ injury in primates. Localization of the transduced MSCs correlated to the severity and geometry of irradiation. A repair process was observed in various tissues. The plasticity potential of the MSCs and their contribution to the repair process in vivo remains to be studied. Copyright © 2003 John Wiley & Sons, Ltd.
Histology and ultrastructure of the gut epithelium of the neotenic cave salamander, Proteus anguinus (Amphibia, Caudata)
Authors: Bizjak Mali Lilijana , Bulog Boris 
Published: December 2003

AbstractHistological, histochemical, and ultrastructural features of the gut of the European endemic cave salamander Proteus anguinus were studied. The gut is a relatively undifferentiated muscular tube lined with a simple columnar epithelium containing numerous goblet cells. The mucosa and underlying lamina propria/submucosa are elevated into a number of high longitudinal folds projecting into the lumen. The enterocytes are covered apically with uniform microvilli. Irregularity in the arrangement of microvilli was observed. Occasionally, irregular protrusions of the cytoplasm appear between groups of microvilli. Pinocytotic activity occurs at the bases of the intermicrovillous space. Mitochondria are numerous in the apical cytoplasm and basally beneath the nuclei. The supranuclear cytoplasm contains most of the cell organelles. The lateral plasma membranes of adjacent cells interdigitate and are joined by junctional complexes. The periodic acid‐Schiff (PAS) reaction, indicating neutral mucosubstances, is positive only in the apical brush border of enterocytes and in goblet cells. The goblet cells also stained with Alcian blue (AB), at pH 2.5, thus revealing the presence of carboxylated glycosaminoglycans. Compact aggregations of AB‐ and PAS‐negative cells are situated directly below the epithelium. Mitotic figures are present in individual clusters of cells. The fine structure of cells in these clusters indicated that these cells could be responsible for renewal of intestinal epithelium. Numerous endocrine‐like cells could also be seen. The closely packed smooth muscle cells and amorphous extracellular material with collagen fibrils constitute a net‐like structure under the basal lamina that is very closely associated with the epithelium. There are numerous acidophilic granular cells between epithelial cells, in the lamina propria/submucosa, and between cells aggregations. They seem to be associated with nematode infections and possibly constitute a humoral defense mechanism. J. Morphol. 259:82–89, 2004. © 2003 Wiley‐Liss, Inc.
Morphogenesis of parrot jaw muscles: Understanding the development of an evolutionary novelty
Authors: Tokita Masayoshi 
Published: December 2003

AbstractParrots have developed novel head structures in their evolutionary history. The appearance of two new muscles for strong jaw adduction is especially fascinating in developmental and evolutionary contexts. However, jaw muscle development of parrots has not been described, despite its uniqueness. This report first presents the normal developmental stages of the cockatiel (Nymphicus hollandicus), comparable to that of the chick. Next, the peculiar skeletal myogenesis in the first visceral arch of parrots is described, mainly focusing on the development of two new jaw muscles. One of the parrot‐specific muscles, M. ethmomandibularis, was initially detected at Nymphicus Stage 28 (N28) as the rostral budding of M. pterygoideus. After N32, the muscle significantly elongates rostrodorsally toward the interorbital septum, following a course lateral to the palatine bone. Another parrot‐specific muscle, M. pseudomasseter, was first recognized at N36. The muscle branches off from the posteromedial M. adductor mandibulae externus and grows in a dorsolateral direction, almost covering the lateral surface of the jugal bar. The upper tip of the muscle is accompanied by condensed mesenchyme, which seems to be derived from cephalic neural crest cells. J. Morphol. 259:69–81, 2004. © 2003 Wiley‐Liss, Inc.
On the nonlinear isoconversional procedures to evaluate the activation energy of nonisothermal reactions in solids
Authors: Budrugeac P. , Segal E. 
Published: December 2003

AbstractA general procedure for deriving the equations that underlie various isoconversional nonlinear methods for evaluating the activation energy is presented. A new integral isoconversional nonlinear method with integration over a given range of conversion is suggested. This method was applied to simulated nonisothermal data as well as to data for the nonisothermal decomposition of ammonium perchlorate. The obtained dependencies of the activation energy on the degree of conversion were compared with those resulting from other nonlinear and linear methods of analysis. © 2003 Wiley Periodicals, Inc. Int J Chem Kinet 36: 87–93 2004