The Use of Adenoviral Vectors and Ex Vivo Transduced Neurotransplants: Towards Promotion of Neuroregeneration

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

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Unraveling the In Vivo Protein Corona

Cells ◽

10.3390/cells10010132 ◽

2021 ◽

Vol 10 (1) ◽

pp. 132

Author(s):

Johanna Simon ◽

Gabor Kuhn ◽

Michael Fichter ◽

Stephan Gehring ◽

Katharina Landfester ◽

...

Keyword(s):

Plasma Proteins ◽

Ex Vivo ◽

Protein Corona ◽

Blood Proteins ◽

Therapeutic Application ◽

Complex Situation ◽

Ex Vivo Approach ◽

In Vivo Administration

Understanding the behavior of nanoparticles upon contact with a physiological environment is of urgent need in order to improve their properties for a successful therapeutic application. Most commonly, the interaction of nanoparticles with plasma proteins are studied under in vitro conditions. However, this has been shown to not reflect the complex situation after in vivo administration. Therefore, here we focused on the investigation of magnetic nanoparticles with blood proteins under in vivo conditions. Importantly, we observed a radically different proteome in vivo in comparison to the in vitro situation underlining the significance of in vivo protein corona studies. Next to this, we found that the in vivo corona profile does not significantly change over time. To mimic the in vivo situation, we established an approach, which we termed “ex vivo” as it uses whole blood freshly prepared from an animal. Overall, we present a comprehensive analysis focusing on the interaction between nanoparticles and blood proteins under in vivo conditions and how to mimic this situation with our ex vivo approach. This knowledge is needed to characterize the true biological identity of nanoparticles.

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Development and Evaluation of Alginate Membranes with Curcumin-Loaded Nanoparticles for Potential Wound-Healing Applications

Pharmaceutics ◽

10.3390/pharmaceutics11080389 ◽

2019 ◽

Vol 11 (8) ◽

pp. 389 ◽

Cited By ~ 10

Author(s):

Mónica C. Guadarrama-Acevedo ◽

Raisa A. Mendoza-Flores ◽

María L. Del Prado-Audelo ◽

Zaida Urbán-Morlán ◽

David M. Giraldo-Gomez ◽

...

Keyword(s):

Wound Healing ◽

Wound Dressing ◽

Clinical Performance ◽

Ex Vivo ◽

High Capacity ◽

Wound Dressings ◽

In Vivo Studies ◽

Potential Use

Non-biodegradable materials with a low swelling capacity and which are opaque and occlusive are the main problems associated with the clinical performance of some commercially available wound dressings. In this work, a novel biodegradable wound dressing was developed by means of alginate membrane and polycaprolactone nanoparticles loaded with curcumin for potential use in wound healing. Curcumin was employed as a model drug due to its important properties in wound healing, including antimicrobial, antifungal, and anti-inflammatory effects. To determine the potential use of wound dressing, in vitro, ex vivo, and in vivo studies were carried out. The novel membrane exhibited the diverse functional characteristics required to perform as a substitute for synthetic skin, such as a high capacity for swelling and adherence to the skin, evidence of pores to regulate the loss of transepidermal water, transparency for monitoring the wound, and drug-controlled release by the incorporation of nanoparticles. The incorporation of the nanocarriers aids the drug in permeating into different skin layers, solving the solubility problems of curcumin. The clinical application of this system would cover extensive areas of mixed first- and second-degree wounds, without the need for removal, thus decreasing the patient’s discomfort and the risk of altering the formation of the new epithelium.

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A Simple in vivo Assay Using Amphipods for the Evaluation of Potential Biocompatible Metal-Organic Frameworks

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.584115 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ines Sifaoui ◽

Idaira Pacheco-Fernández ◽

José E. Piñero ◽

Verónica Pino ◽

Jacob Lorenzo-Morales

Keyword(s):

Ex Vivo ◽

Colorimetric Assay ◽

Metal Organic Frameworks ◽

Screening Method ◽

Toxicity Tests ◽

Toxicity Screening ◽

Metal Organic ◽

Potential Use

In this study, the application of amphipods in vivo assays was evaluated. The main aim of this work was to check the potential use of this model in biocompatibility assessments of metal-organic frameworks (MOFs). Hence, six different MOFs were synthesized and the in vitro and ex vivo cytotoxicity was first assessed using a colorimetric assay and a macrophage cell line. Obtained results were compared to validate the in vivo toxicity tests carried out using amphipods and increasing concentrations of the different MOFs. Amphipods do not require the need of ethics approval and also are less expensive to keep than conventional in vivo models, showing its potential as a fast and reliable platform in toxicity studies. The obtained results showed that the amphipods based-assay was simple, easy to replicate and yielded toxicity data corresponding to the type of MOFs tested. In addition, it was observed that only CIM-80(Al) and CIM-84(Zr) did not show any toxicity to the animals at the different tested concentrations. Therefore, the developed in vivo model could be applied as a high-throughput toxicity screening method to evaluate the toxicity of numerous materials, chemicals and therapeutic agents among others.

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Platelet signaling to procoagulant activity and heterogeneity in thrombus formation : an in vivo and ex vivo approach

10.26481/dis.20070615im ◽

2007 ◽

Author(s):

I.C.A. Munnix

Keyword(s):

Ex Vivo ◽

Thrombus Formation ◽

Procoagulant Activity ◽

Platelet Signaling ◽

Ex Vivo Approach

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Red cell-derived microparticles (RMP) as haemostatic agent

Thrombosis and Haemostasis ◽

10.1160/th12-12-0941 ◽

2013 ◽

Vol 110 (10) ◽

pp. 751-760 ◽

Cited By ~ 34

Author(s):

Max Johansen ◽

Carlos Bidot ◽

Lawrence Horstman ◽

Yeon Ahn ◽

Wenche Jy

Keyword(s):

Flow Cytometry ◽

Blood Loss ◽

Bleeding Time ◽

Ex Vivo ◽

Annexin V ◽

Haemostatic Agent ◽

Platelet Aggregometry ◽

Primary Platelet ◽

Potential Use

SummaryAmong circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential haemostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-pressure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribution was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggregometry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 μm and 50% of them exhibit annexin V binding, a proxy for procoagulant phospholipids (PL). No TF could be detected by flow cytometry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP enhanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP reduced bleeding time and blood loss in thrombocytopenic rabbits (busulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and secondary (coagulation) haemostasis, suggesting potential use as haemostatic agent for treatment of bleeding.

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529 Cancer cells educate natural killer cells to a metastasis promoting cell state

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0529 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A565-A565

Author(s):

Isaac Chan ◽

Hildur Knútsdóttir ◽

Gayathri Ramakrishnan ◽

Veena Padmanaban ◽

Manisha Warrier ◽

...

Keyword(s):

Breast Cancer ◽

Nk Cells ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Nk Cell ◽

Ex Vivo ◽

Tumor Bearing ◽

Growth Promoting ◽

Tumor Organoids

BackgroundMetastatic disease drives breast cancer mortality. We recently discovered that leading cells at the invasive edge of mammary tumor organoids retain a conserved basal epithelial program defined by their expression of keratin-14 (K14), establishing K14 as a good marker of invasive breast cancer cells. K14-positive invasive cells also exhibit characteristics that make them targets of immunosurveillance by natural killer (NK) cells. While NK cells are key immune mediators in the control of metastasis, our understanding of the specific mechanisms behind this regulation and its eventual evasion by metastatic cells remains incomplete.MethodsWe have developed a novel preclinical 3D co-culture assay to discover mechanisms behind interactions between K14+ invasive breast cancer cells and NK cells. Combined with in vivo assays of metastasis, we are able to determine how NK cells limit the early stages of metastasis and also how tumor cells can influence key NK cell properties.ResultsIn ex vivo co-culture assays of NK cells isolated from healthy mouse donors and mammary tumor organoids from MMTV-PyMT and C31T mouse models of breast cancer, we demonstrate that NK cells limit the early stages of metastasis. Antibodies to invasive K14+ cells were able to enhance the ability of NK cells to limit colony formation, suggesting antibody-dependent cell mediated cytotoxicity. Surprisingly, when isolated from tumor bearing mice, NK cells did not limit invasion and instead promoted colony formation. The in vivo adoptive transfer of NK cells from healthy donors prevents the progression of early lung metastatic seeds to macrometastases, while the adoptive transfer of cells isolated from tumor bearing donors promotes macrometastatic development. Transcriptomic analysis of reprogrammed NK cells demonstrate they have similar profiles to resting NK cells. This growth promoting phenotype can be reversed with antibodies targeting inhibitory cell surface receptors or the epigenome.ConclusionsOur ex vivo and in vivo data demonstrate that healthy donor NK cells can limit metastasis through the directed cytotoxicity against pioneering K14+ invasive cells. However, prolonged exposure to tumors reprogram NK cells from tumor killing to tumor promoting, specifically in promoting the outgrowth of macrometastases. Further, we can neutralize this effect using NK cell specific inhibitory antibodies and epigenetic modifiers. This is the first time inhibitory signaling on NK cells have been linked with a growth promoting phenotype. These data can provide insight into when the use of NK cell directed therapies can be used to treat or prevent clinically relevant metastatic disease.

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Gene Expression of Mouse Hippocampal Stem Cells Grown in a Galactose-Derived Molecular Gel Compared to In Vivo and Neurospheres

Processes ◽

10.3390/pr9040716 ◽

2021 ◽

Vol 9 (4) ◽

pp. 716

Author(s):

Keziban Korkmaz Bayram ◽

Juliette Fitremann ◽

Arslan Bayram ◽

Zeynep Yılmaz ◽

Ecmel Mehmetbeyoğlu ◽

...

Keyword(s):

Neural Progenitor Cells ◽

Ex Vivo ◽

Neural Cells ◽

Transcript Analysis ◽

Supramolecular Hydrogel ◽

Carbohydrate Derivative ◽

In Vitro Investigation ◽

Synthetic Carbohydrate

Background: N-heptyl-D-galactonamide (GalC7) is a small synthetic carbohydrate derivative that forms a biocompatible supramolecular hydrogel. In this study, the objective was to analyze more in-depth how neural cells differentiate in contact with GalC7. Method: Direct (ex vivo) cells of the fresh hippocampus and culture (In vitro) of the primary cells were investigated. In vitro, investigation performed under three conditions: on culture in neurospheres for 19 days, on culture in GalC7 gel for 7 days, and on culture in both neurospheres and GalC7 gel. Total RNA was isolated with TRIzol from each group, Sox8, Sox9, Sox10, Dcx, and Neurod1 expression levels were measured by qPCR. Result: Sox8 and Sox10, oligodendrocyte markers, and Sox9, an astrocyte marker, were expressed at a much higher level after 7 days of culture in GalC7 hydrogel compared to all other conditions. Dcx, a marker of neurogenesis, and Neurod1, a marker of neuronal differentiation, were expressed at better levels in the GalC7 gel culture compared to the neurosphere. Conclusions: These results show that the GalC7 hydrogel brings different and interesting conditions for inducing the differentiation and maturation of neural progenitor cells compared with polymer-based scaffolds or cell-only conditions. The differences observed open new perspectives in tissue engineering, induction, and transcript analysis.

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ET-CORM Mediated Vasorelaxation of Small Mesenteric Arteries: Involvement of Kv7 Potassium Channels

Frontiers in Pharmacology ◽

10.3389/fphar.2021.702392 ◽

2021 ◽

Vol 12 ◽

Author(s):

Danfeng Zhang ◽

Bernhard M. Krause ◽

Hans-Günther Schmalz ◽

Paulus Wohlfart ◽

Benito A. Yard ◽

...

Keyword(s):

Soluble Guanylate Cyclase ◽

Ex Vivo ◽

Peripheral Resistance ◽

Mesenteric Arteries ◽

Kv7 Channels ◽

Pre Treatment ◽

Functional Relevance ◽

Potential Use

Although the vasoactive properties of carbon monoxide (CO) have been extensively studied, the mechanism by which CO mediates vasodilation is not completely understood. Through-out published studies on CO mediated vasodilation there is inconsistency on the type of K+-channels that are activated by CO releasing molecules (CORMs). Since the vasorelaxation properties of enzyme triggered CORMs (ET-CORMs) have not been studied thus far, we first assessed if ET-CORMs can mediate vasodilation of small mesenteric arteries and subsequently addressed the role of soluble guanylate cyclase (sGC) and that of K-channels herein. To this end, 3 different types of ET-CORMs that either contain acetate (rac-1 and rac-4) or pivalate (rac-8) as ester functionality, were tested ex vivo on methoxamine pre-contracted small rat mesenteric arteries in a myograph setting. Pre-contracted mesenteric arteries strongly dilated upon treatment with both types of acetate containing ET-CORMs (rac-1 and rac-4), while treatment with the pivalate containing ET-CORM (rac-8) resulted in no vasodilation. Pre-treatment of mesenteric arteries with the sGC inhibitor ODQ abolished rac-4 mediated vasodilation, similar as for the known sGC activator SNP. Likewise, rac-4 mediated vasodilation did not occur in KCL pretreated mesenteric arteries. Although mesenteric arteries abundantly expressed a variety of K+-channels only Kv7 channels were found to be of functional relevance for rac-4 mediated vasodilation. In conclusion the current results identified Kv7 channels as the main channel by which rac-4 mediates vasodilation. In keeping with the central role of Kv7 in the control of vascular tone and peripheral resistance these promising ex-vivo data warrant further in vivo studies, particularly in models of primary hypertension or cardiac diseases, to assess the potential use of ET-CORMs in these diseases.

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High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of DMD Gene Repair Tools and Strategies in Human Cells

Cells ◽

10.3390/cells9040869 ◽

2020 ◽

Vol 9 (4) ◽

pp. 869 ◽

Cited By ~ 3

Author(s):

Marcella Brescia ◽

Josephine M. Janssen ◽

Jin Liu ◽

Manuel A. F. V. Gonçalves

Keyword(s):

Muscle Cell ◽

Ex Vivo ◽

High Capacity ◽

High Specificity ◽

Adenoviral Vectors ◽

Human Cells ◽

Cell Populations ◽

Gene Repair ◽

Dmd Gene

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo DMD-correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective DMD alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in E1 and E2A. Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% ± 13% of defective DMD alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both DMD gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of β-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of DMD gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of DMD-correcting agents.

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