scholarly journals Poly (D, L-lactide-co-glycolide)/DNA microspheres to facilitate prolonged transgene expression in airway epithelium in vitro, ex vivo and in vivo

Gene Therapy ◽  
2003 ◽  
Vol 10 (16) ◽  
pp. 1282-1288 ◽  
Author(s):  
M Stern ◽  
K Ulrich ◽  
D M Geddes ◽  
E W F W Alton
Keyword(s):  
Airway Epithelium ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Dna Microspheres

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5143-5143
Author(s):  
Liesbeth De Waele ◽  
Kathleen Freson ◽  
Chantal Thys ◽  
Christel Van Geet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Phosphoglycerate Kinase ◽  
Lentiviral Vectors ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

scholarly journals Canine Adenovirus Vectors for Lung-Directed Gene Transfer: Efficacy, Immune Response, and Duration of Transgene Expression Using Helper-Dependent Vectors

2006 ◽  
Vol 80 (3) ◽  
pp. 1487-1496 ◽  
Author(s):  
Anne Keriel ◽  
Céline René ◽  
Chad Galer ◽  
Joseph Zabner ◽  
Eric J. Kremer
Keyword(s):  
Gene Transfer ◽  
Respiratory Tract ◽  
Neutralizing Antibodies ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Adenovirus Type ◽  
Transduction Efficiency

ABSTRACT A major hurdle to the successful clinical use of some viral vectors relates to the innate, adaptive, and memory immune responses that limit the efficiency and duration of transgene expression. Some of these drawbacks may be circumvented by using vectors derived from nonhuman viruses such as canine adenovirus type 2 (CAV-2). Here, we evaluated the potential of CAV-2 vectors for gene transfer to the respiratory tract. We found that CAV-2 transduction was efficient in vivo in the mouse respiratory tract, and ex vivo in well-differentiated human pulmonary epithelia. Notably, the in vivo and ex vivo efficiency was poorly inhibited by sera from mice immunized with a human adenovirus type 5 (HAd5, a ubiquitous human pathogen) vector or by human sera containing HAd5 neutralizing antibodies. Following intranasal instillation in mice, CAV-2 vectors also led to a lower level of inflammatory cytokine secretion and cellular infiltration compared to HAd5 vectors. Moreover, CAV-2 transduction efficiency was increased in vitro in human pulmonary cells and in vivo in the mouse respiratory tract by FK228, a histone deacetylase inhibitor. Finally, by using a helper-dependent CAV-2 vector, we increased the in vivo duration of transgene expression to at least 3 months in immunocompetent mice without immunosuppression. Our data suggest that CAV-2 vectors may be efficient and safe tools for long-term clinical gene transfer to the respiratory tract.

scholarly journals Adeno-Associated Virus Type 2-Mediated Transduction of Human Monocyte-Derived Dendritic Cells: Implications for Ex Vivo Immunotherapy

2001 ◽  
Vol 75 (19) ◽  
pp. 9493-9501 ◽  
Author(s):  
Selvarangan Ponnazhagan ◽  
Gandham Mahendra ◽  
David T. Curiel ◽  
Denise R. Shaw
Keyword(s):  
Dendritic Cells ◽  
Immune Responses ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Adeno Associated Virus ◽  
Dc Vaccine

ABSTRACT Dendritic cells (DCs) are pivotal antigen-presenting cells for regulating immune responses. A major focus of contemporary vaccine research is the genetic modification of DCs to express antigens or immunomodulatory molecules, utilizing a variety of viral and nonviral vectors, to induce antigen-specific immune responses that ameliorate disease states as diverse as malignancy, infection, autoimmunity, and allergy. The present study has evaluated adeno-associated virus (AAV) type 2 as a vector for ex vivo gene transfer to human peripheral blood monocyte (MO)-derived DCs. AAV is a nonpathogenic parvovirus that infects a wide variety of human cell lineages in vivo and in vitro, for long-term transgene expression without requirements for cell proliferation. The presented data demonstrate that recombinant AAV (rAAV) can efficiently transduce MOs as well as DCs generated by MO culture with granulocyte-macrophage colony-stimulating factor plus interleukin in vitro. rAAV transgene expression in MO-derived DCs could be enhanced by etoposide, previously reported to enhance AAV gene expression. rAAV transduction of freshly purified MO followed by 7 days of culture with cytokines to generate DCs, and subsequent sorting for coexpression of DC markers CD1a and CD40, showed robust transgene expression as well as evidence of nuclear localization of the rAAV genome in the DC population. Phenotypic analyses using multiple markers and functional assays of one-way allogeneic mixed leukocyte reactions indicated that rAAV-transduced MO-derived DCs were as equivalent to nontransduced DCs. These results support the utility of rAAV vectors for future human DC vaccine studies.

scholarly journals Cationic Polymer and Lipids Augment Adenovirus-Mediated Gene Transfer to Cerebral Arteries In vivo

2001 ◽  
Vol 21 (9) ◽  
pp. 1125-1131 ◽  
Author(s):  
Kazunori Toyoda ◽  
Hiroshi Nakane ◽  
Donald D. Heistad
Keyword(s):  
Gene Transfer ◽  
Blood Vessels ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Cerebral Arteries ◽  
Cationic Polymer ◽  
Fibroblast Cells ◽  
Intracranial Arteries

Adenovirus-mediated gene transfer to blood vessels is relatively inefficient because binding of adenovirus to vessels is limited. The authors have reported that incorporation of cationic polymer and lipids with adenovirus augments gene transfer to blood vessels ex vivo. In this study, the authors determined whether complexes of adenovirus and cations improve efficiency of gene transfer in vivo. Poly-l-lysine, lipofectamine, or lipofectin was complexed with adenovirus encoding β-galactosidase. Optimum ratios of the cations per adenovirus were determined by gene transfer to fibroblasts. After injection of the adenovirus into the cisterna magna of anesthetized rabbits, transgene activity was greater in the adventitia of intracranial arteries and meninges after injection of the complexes than adenovirus alone. Thirty minutes after application of adenovirus with the cations, binding of adenovirus to fibroblast cells in vitro or the basilar artery in vivo (by Southern blot analysis) was augmented, which suggests that enhanced binding of virus contributes to augmentation of transgene expression. Thus, cationic polymer and lipids improve transgene expression in intracranial arteries, primarily in the adventitia, after adenovirus-mediated gene transfer in vivo. This strategy may be applicable to studies of gene transfer and eventually for gene therapy.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

Significance of Platelet β-Adrenoceptors for Platelet Responses In Vivo and In Vitro

1992 ◽  
Vol 68 (06) ◽  
pp. 687-693 ◽  
Author(s):  
P T Larsson ◽  
N H Wallén ◽  
A Martinsson ◽  
N Egberg ◽  
P Hjemdahl
Keyword(s):  
Ex Vivo ◽  
High Dose ◽  
Platelet Aggregability ◽  
Adrenoceptor Agonist ◽  
Dose Infusion ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Factor Antigen

SummaryThe significance of platelet β-adrenoceptors for platelet responses to adrenergic stimuli in vivo and in vitro was studied in healthy volunteers. Low dose infusion of the β-adrenoceptor agonist isoprenaline decreased platelet aggregability in vivo as measured by ex vivo filtragometry. Infusion of adrenaline, a mixed α- and β-adrenoceptor agonist, increased platelet aggregability in vivo markedly, as measured by ex vivo filtragometry and plasma β-thromboglobulin levels. Adrenaline levels were 3–4 nM in venous plasma during infusion. Both adrenaline and high dose isoprenaline elevated plasma von Willebrand factor antigen levels β-Blockade by propranolol did not alter our measures of platelet aggregability at rest or during adrenaline infusions, but inhibited adrenaline-induced increases in vWf:ag. In a model using filtragometry to assess platelet aggregability in whole blood in vitro, propranolol enhanced the proaggregatory actions of 5 nM, but not of 10 nM adrenaline. The present data suggest that β-adrenoceptor stimulation can inhibit platelet function in vivo but that effects of adrenaline at high physiological concentrations are dominated by an α-adrenoceptor mediated proaggregatory action.

In Vivo Effect of Suloctidil as an Antiplatelet Agent

1979 ◽  
Vol 41 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Marcia R Stelzer ◽  
Thomas S Burns ◽  
Robert N Saunders
Keyword(s):  
Ex Vivo ◽  
Antiplatelet Agent ◽  
Ratio Method ◽  
Platelet Aggregate ◽  
Permanent Effect ◽  
Platelet Aggregates ◽  
5 Ht Uptake ◽  
High Doses

SummaryThe relationship between the effects of suloctidil in vivo as an antiplatelet agent and in vitro as a modifier of platelet serotonin (5-HT) parameters was investigated. Suloctidil was found to be effective in reducing platelet aggregates formation in the retired breeder rat as determined using the platelet aggregate ratio method (PAR) with an ED50 of 16.1 mg/kg 24 hours post administration. In contrast to the hypothesis that 5-HT depletion is involved in the anti-aggregatory mechanism of suloctidil, no correlation was found between platelet 5- HT content and this antiplatelet activity. Reduction of platelet 5-HT content required multiple injections of high doses (100 mg/kg/day) of suloctidil. Suloctidil administration for 8 days at 100 mg/kg/day, which lowered platelet 5-HT content by 50%, resulted in no permanent effect on ex vivo platelet 5-HT uptake or thrombin-induced release, nor alteration in the plasma 5-HT level. However, these platelets exhibited a short-lived, significant increase in percent leakage of 5-HT after 30 minutes of incubation. Therefore, suloctidil treatment at high doses may with time result in platelet 5-HT depletion, however this effect is probably not related to the primary anti-aggregatory activity of the drug.

A Comparative Ex Vivo Study of Plasminogen Activators and Proteases for Fibrinolytic Activity and Side Effects in Rabbits

1977 ◽  
Vol 37 (01) ◽  
pp. 154-161 ◽  
Author(s):  
B. A Janik ◽  
S. E Papaioannou
Keyword(s):  
Side Effects ◽  
Effective Dose ◽  
Fibrinolytic Activity ◽  
Ex Vivo ◽  
Plasminogen Activators ◽  
Assay System ◽  
Coagulation Parameters ◽  
Ex Vivo Assay

SummaryUrokinase, streptokinase, Brinase, trypsin, and SN 687, a bacterial exoprotease, have been evaluated in an ex vivo assay system. These enzymes were injected into rabbits and the fibrinolytic activity as well as other coagulation parameters were measured by in vitro techniques. Dose-response correlations have been made using the euglobulin lysis time as a measure of fibrinolytic activity and the 50% effective dose has been determined for each enzyme. Loading doses, equal to four times the 50% effective dose, were administered to monitor potential toxicity revealing that Brinase, trypsin, and SN 687 were very toxic at this concentration.Having established the 50% effective dose for each enzyme, further testing was conducted where relevant fibrinolytic and coagulation parameters were measured for up to two days following a 50% effective dose bolus injection of each enzyme. Our results have demonstrated that urokinase and streptokinase are plasminogen activators specifically activating the rabbit fibrinolytic system while Brinase, trypsin and SN 687 increase the general proteolytic activity in vivo.The advantages of this ex vivo assay system for evaluating relative fibrinolytic potencies and side effects for plasminogen activators and fibrinolytic proteases have been discussed.

Organische Nitrate und Thrombozytenfunktion

1988 ◽  
Vol 08 (02) ◽  
pp. 90-99 ◽  
Author(s):  
H. Schröder ◽  
K. Schrör
Keyword(s):  
Endothelial Cell ◽  
Angina Pectoris ◽  
Ex Vivo

ZusammenfassungOrganische Nitrate unterschiedlicher chemischer Struktur sowie Nitroprussidnatrium und Molsidomin (bzw. ihre biologisch aktiven Metaboliten) können die (primäre) Aggregation und Sekretion von Humanthrombozyten in vitro und ex vivo hemmen. Eine solche Wirkung wird für Molsidomin (SIN-1) und Nitroprussidnatrium in vitro in Konzentrationen beobachtet, die in der gleichen Größenordnung liegen wie die vasodilatierenden Effekte der Substanzen. Dagegen sind für eine direkte Antiplättchenwirkung organischer Nitrate (Glyzeryltrinitrat, Isosorbiddinitr at, Isosorbidmononitrate, Teopranitol) in vitro Konzentrationen erforderlich, die ca. 100- bis 1000fach höher sind als die Plasmaspiegel der Substanzen nach therapeutischer Dosierung bzw. die Konzentrationen, die isolierte Gefäßstreifen relaxieren. Als gemeinsamer Wirkungsmechanismus der direkten thrombozy-tenfunktionshemmenden und gefäßerweiternden Wirkung all dieser Substanzen kann heute eine Stickoxid-(NO)-vermittelte Stimulation der cGMP-Bildung angenommen werden, das aus organischen Nitraten als »Pro-drug« entsteht. Die Freisetzung von NO, eines »endothelial cell-derived relaxing factors« (EDRF) aus Nitroprussidnatrium und SIN-1 erfolgt spontan. Dagegen erfordert die Freisetzung von NO aus organischen Nitraten einen enzymatischen Stoffwechselweg, der in isolierten Thrombozyten nicht vorhanden ist. Eine Antiplättchenwirkung organischer Nitrate in vivo bzw. ex vivo wird daher über die Stimulation eines endothelialen, thrombozyteninhibitorischen Faktors erklärt. Hierbei sind Prostazyklin sowie ein bisher unbekannter Endothel-zellfaktor neben einer synergistischen Wirkung organischer Nitrate mit endogenem Prostazyklin in Diskussion. Eine thrombozytenfunktionshemmen-de Wirkung organischer Nitrate könnte in Kombination mit ihren hämody-namischen Effekten auch für die an-tianginöse Wirkung in der Klinik bedeutsam sein, insbesondere zur Verhinderung vasospastischer Zustände bei der instabilen Angina pectoris.

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