scholarly journals Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment

2021 ◽  
Author(s):  
Margaret E Maes ◽  
Gabriele M Wögenstein ◽  
Gloria Colombo ◽  
Raquel Casado-Polanco ◽  
Sandra Siegert
Keyword(s):  
Environmental Changes ◽  
Genetic Material ◽  
Plexiform Layer ◽  
Cell Types ◽  
Transduction Efficiency ◽  
Heparin Binding ◽  
Future Studies ◽  
Distinct Cell ◽  
Transgene Delivery

AbstractAdeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette.Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency.

Heparin stimulates the proliferation of intestinal epithelial cells in primary culture

1994 ◽  
Vol 107 (2) ◽  
pp. 401-411
Author(s):  
N. Flint ◽  
F.L. Cove ◽  
G.S. Evans
Keyword(s):  
Growth Factors ◽  
Heparan Sulphate ◽  
Primary Cultures ◽  
Cell Types ◽  
Heparin Binding ◽  
Epithelial Proliferation ◽  
Inhibition Of Proliferation ◽  
Trophic Effect ◽  
Heparin Binding Growth Factors

Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminoglycans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysaccharides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration.

scholarly journals Pathogenic Stress Induces Human Monocyte to Express an Extracellular Web of Tunneling Nanotubes

2021 ◽  
Vol 12 ◽  
Author(s):  
Michal Shahar ◽  
Auryan Szalat ◽  
Haim Rosen
Keyword(s):  
Intercellular Communication ◽  
Environmental Changes ◽  
Imaging System ◽  
Human Monocyte ◽  
Cell Types ◽  
Similar Response ◽  
Human Monocytes ◽  
Tunneling Nanotubes ◽  
Membrane Components

Actin-based tunneling nanotubes are a means of intercellular communication between remote cells. In the last decade, this type of nanotube was described in a wide variety of cell types and it became widely accepted that communication through these nanotubes is related to response to environmental changes. Few reports, however, are available regarding the expression of similar nanotubes in vivo or in primary cells. Moreover, the functional significance of this intercellular communication for health and disease is largely unknown. In this context, and as a first step in unraveling these questions, we examined the formation of similar nanotubes in primary peripheral human monocytes. To that end, we combined the use of a live cell imaging system along with advanced methods of fluorescent and scanning electron microscopy. This experimental approach reveals for the first time that the bacterial lipopolysaccharide endotoxin induces a transient expression of an unexpected abundance of actin-based tunneling nanotubes associated with vesicles. In addition, it was found that a similar response can be achieved by treating human monocytes with various bacterial and yeast membrane components, as well as with a viral component analog. In all these cases, this response is mediated by distinct complexes of toll-like receptors. Therefore, we suggest that the observed phenomena are related to a broad type of monocyte pathogen response, and raise the possibility that the phenomena described above may be involved in many clinical situations related to inflammation as a new topic of study.

scholarly journals Chemogenetic Tools for Causal Cellular and Neuronal Biology

2018 ◽  
Vol 98 (1) ◽  
pp. 391-418 ◽  
Author(s):  
Deniz Atasoy ◽  
Scott M. Sternson
Keyword(s):  
G Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Cell Populations ◽  
Specific Cell ◽  
Cellular Processes ◽  
Distinct Cell ◽  
G Protein Coupled ◽  
Pharmacological Control

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

scholarly journals Cellular and Synaptic Mechanisms That Differentiate Mitral Cells and Superficial Tufted Cells Into Parallel Output Channels in the Olfactory Bulb

2020 ◽  
Vol 14 ◽  
Author(s):  
Shelly Jones ◽  
Joel Zylberberg ◽  
Nathan Schoppa
Keyword(s):  
Olfactory Bulb ◽  
Plexiform Layer ◽  
Input Resistance ◽  
Cell Types ◽  
Mitral Cells ◽  
Whole Cell ◽  
Wide Range ◽  
Tufted Cells ◽  
Parallel Output

A common feature of the primary processing structures of sensory systems is the presence of parallel output “channels” that convey different information about a stimulus. In the mammalian olfactory bulb, this is reflected in the mitral cells (MCs) and tufted cells (TCs) that have differing sensitivities to odors, with TCs being more sensitive than MCs. In this study, we examined potential mechanisms underlying the different responses of MCs vs. TCs. For TCs, we focused on superficial TCs (sTCs), which are a population of output TCs that reside in the superficial-most portion of the external plexiform layer, along with external tufted cells (eTCs), which are glutamatergic interneurons in the glomerular layer. Using whole-cell patch-clamp recordings in mouse bulb slices, we first measured excitatory currents in MCs, sTCs, and eTCs following olfactory sensory neuron (OSN) stimulation, separating the responses into a fast, monosynaptic component reflecting direct inputs from OSNs and a prolonged component partially reflecting eTC-mediated feedforward excitation. Responses were measured to a wide range of OSN stimulation intensities, simulating the different levels of OSN activity that would be expected to be produced by varying odor concentrations in vivo. Over a range of stimulation intensities, we found that the monosynaptic current varied significantly between the cell types, in the order of eTC > sTC > MC. The prolonged component was smaller in sTCs vs. both MCs and eTCs. sTCs also had much higher whole-cell input resistances than MCs, reflecting their smaller size and greater membrane resistivity. To evaluate how these different electrophysiological aspects contributed to spiking of the output MCs and sTCs, we used computational modeling. By exchanging the different cell properties in our modeled MCs and sTCs, we could evaluate each property's contribution to spiking differences between these cell types. This analysis suggested that the higher sensitivity of spiking in sTCs vs. MCs reflected both their larger monosynaptic OSN signal as well as their higher input resistance, while their smaller prolonged currents had a modest opposing effect. Taken together, our results indicate that both synaptic and intrinsic cellular features contribute to the production of parallel output channels in the olfactory bulb.

scholarly journals Characterization of Tissue Tropism Determinants of Adeno-Associated Virus Type 1

2003 ◽  
Vol 77 (4) ◽  
pp. 2768-2774 ◽  
Author(s):  
Bernd Hauck ◽  
Weidong Xiao
Keyword(s):  
Amino Acids ◽  
Transduction Efficiency ◽  
Antigenic Determinants ◽  
Tissue Tropism ◽  
Heparin Binding ◽  
Adeno Associated Virus ◽  
Heparin Binding Domain

ABSTRACT Muscle is an attractive target for gene delivery because of its mass and because vectors can be delivered in a noninvasive fashion. Adeno-associated virus (AAV) has been shown to be effective for muscle-targeted gene transfer. Recent progress in characterization of AAV serotype 1 (AAV1) and AAV6 demonstrated that these two AAV serotypes are far more efficient in transducing muscle than is the traditionally used AAV2. Since all cis elements are identical in these vectors, the potential determinants for their differences in transducing muscle appear to be located within the AAV capsid proteins. In the present study, a series of AAV capsid mutants were generated to identify the major regions affecting AAV transduction efficiency in muscle. Replacement of amino acids 350 to 736 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector that behaved very similarly to AAV1 in vitro and in vivo in muscle. Characterization of additional mutants carrying smaller regions of the AAV1 VP1 amino acid sequence in the AAV2 capsid protein suggested that amino acids 350 to 430 of VP1 function as a major tissue tropism determinant. Further analysis showed that the heparin binding domain and the major antigenic determinants in the AAV capsid region were not necessary for the efficiency of AAV1 transduction of muscle.

scholarly journals Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

2003 ◽  
Vol 2003 (2) ◽  
pp. 79-91 ◽  
Author(s):  
Lindsay J. Stanbridge ◽  
Vincent Dussupt ◽  
Norman J. Maitland
Keyword(s):  
Prostate Cancer ◽  
Gene Therapy ◽  
Viral Entry ◽  
Mammalian Cells ◽  
Primary Tumour ◽  
Genetic Material ◽  
Cell Types ◽  
Attractive Alternative

Current curative strategies for prostate cancer are restricted to the primary tumour, and the effect of treatments to control metastatic disease is not sustained. Therefore, the application of gene therapy to prostate cancer is an attractive alternative. Baculoviruses are highly restricted insect viruses, which can enter, but not replicate in mammalian cells. Baculoviruses can incorporate large amounts of extra genetic material, and will express transgenes in mammalian cells when under the control of a mammalian or strong viral promoter. Successful gene delivery has been achieved both in vitro and in vivo and into both dividing and nondividing cells, which is important since prostate cancers divide relatively slowly. In addition, the envelope protein gp64 is sufficiently mutable to allow targeted transduction of particular cell types. In this review, the advantages of using baculoviruses for prostate cancer gene therapy are explored, and the mechanisms of viral entry and transgene expression are described.

scholarly journals Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry

2020 ◽  
Author(s):  
Brian Jurgielewicz ◽  
Yao Yao ◽  
Steven L. Stice
Keyword(s):  
Flow Cytometry ◽  
Genetic Material ◽  
Cell Types ◽  
Extracellular Vesicle ◽  
Human Neural Stem Cells ◽  
Imaging Flow Cytometry ◽  
Gene Therapy Vectors ◽  
Time Variables

Abstract Background : Extracellular vesicles (EVs) are nanosized vesicles naturally secreted from cells responsible for intercellular communication and delivery of proteins, lipids, and other genetic material. Ultimately, EVs could provide innate therapeutic contents and loaded therapeutic payloads such as small molecules and gene therapy vectors to recipient cells. However, comparative kinetic measures that can be used to quantify and ultimately optimize delivery and uptake of EV payloads are lacking. We investigated both dose and time effects on EV uptake and evaluated the potential specificity of EV uptake to better understand the kinetics and uptake of human embryonic kidney (HEK293T) derived EVs. Results : Utilizing an imaging flow cytometry platform (IFC), HEK293T EV uptake was analyzed. HEK293T EV uptake was dose and time dependent with a minimum threshold dose of 6,000 EVs per cell at 4 hours of co-culture. HEK293T EV uptake was inhibited when co-cultured with recipient cells at 4°C or with pre-fixed recipient cells. By co-culturing HEK293T EVs with cell lines from various germ layers, HEK293T EVs were taken up at higher quantities by HEK293T cells. Lastly, human neural stem cells (hNSCs) internalized significantly more HEK293T EVs relative to mature neurons. Conclusions : Imaging flow cytometry is a quantitative, high throughput, and versatile platform to quantify the kinetics of EV uptake. Utilizing this platform, dose and time variables have been implicated to affect EV uptake measurements making standardization of in vitro and in vivo assays vital for the translation of EVs into the clinic. In this study, we quantified the selectivity of EV uptake between a variety of cell types in vitro and found that EVs were internalized at higher quantities by cells of the same origin. The characterization of HEK293T EV uptake in vitro, notably specificity, dose response, and kinetic assays should be used to help inform and develop EV based therapeutics.

Prepupal differentiation in Drosophila: distinct cell types elaborate a shared structure, the pupal cuticle, but accumulate transcripts in unique patterns

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 649-656 ◽  
Author(s):  
K. Fechtel ◽  
D.K. Fristrom ◽  
J.W. Fristrom
Keyword(s):  
Cell Types ◽  
Imaginal Discs ◽  
Specific Expression ◽  
Distinct Cell ◽  
Cuticle Proteins ◽  
Shared Structure ◽  
Pupal Cuticle

The components of the pupal cuticle are the main differentiation products synthesized by both the larval and adult epidermis during the prepupal period of Drosophila development. The pupal cuticle is formed in vitro by imaginal discs in response to a 6 h pulse of 20-hydroxyecdysone (20-HE). We previously described the isolation and initial characterization of four ecdysone-dependent genes (EDGs) whose expression in imaginal discs occurs only in response to a pulse of 20-HE. In this report, we demonstrate that the pattern of temporal and tissue-specific expression of these EDGs in vivo is like that expected for genes that encode pupal cuticle proteins. Transcripts of these genes are detected in prepupae only in the epidermis and only when cuticle components are synthesized and secreted. Nonetheless, their temporal and spatial patterns of accumulation differ. EDG-84A-1 transcripts accumulate only in prepupae and only in imaginal cells. EDG-78E and EDG-64CD transcripts accumulate at the same time in both larval and imaginal cells. EDG42-A transcripts appear first in prepupae in imaginal cells and then, after a 2–4 h lag, in larval cells. It is evident that some genes are not restricted in their expression to only larval or imaginal epidermis.

scholarly journals p19ARF Is Dispensable for Oncogenic Stress-Induced p53-Mediated Apoptosis and Tumor Suppression In Vivo

2002 ◽  
Vol 22 (1) ◽  
pp. 370-377 ◽  
Author(s):  
Dawn Tolbert ◽  
Xiangdong Lu ◽  
Chaoying Yin ◽  
Mathew Tantama ◽  
Terry Van Dyke
Keyword(s):  
Tumor Suppressor ◽  
Tumor Suppression ◽  
Tumor Model ◽  
Cell Types ◽  
Negative Regulator ◽  
Suppressor Activity ◽  
Distinct Cell ◽  
Tumor Suppressor Activity ◽  
Oncogenic Stress

ABSTRACT Recent studies have shown the p19ARF tumor suppressor to be involved in the response to oncogenic stress by regulating the activity of p53. This response is mediated by antagonizing the function of Mdm2, a negative regulator of p53, indicating a pathway for tumor suppression that involves numerous genes altered in human tumors. We previously described a transgenic mouse brain tumor model in which oncogenic stress, provided by cell-specific inactivation of the pRb pathway, triggers a p53-dependent apoptotic response. This response suppresses the growth of developing tumors and thus represents a bona fide in vivo tumor suppressor activity. We further showed that E2F1, a transcription factor known to induce p19ARF expression, was required for the response. Here, we use a genetic approach to test whether p19ARF functions to transduce the signal from E2F1 to p53 in this tumor suppression pathway. Contrary to the currently accepted hypothesis, we show that a deficiency in p19ARF has no impact on p53-mediated apoptosis or tumor suppression in this system. All measures of p53 function, including the level of apoptosis induced by pRb inactivation, the expression of p21 (a p53-responsive gene), and the rate of tumor growth, were comparable in mice with and without a functional p19ARF gene. Thus, although p19ARF is required in some cell types to transmit an oncogenic response signal to p53, it is dispensable for this function in an in vivo epithelial system. These results underscore the complexity of p53 tumor suppression and further indicate the existence of distinct cell-specific pathways that respond to similar stimuli.

scholarly journals Murine clusterin: molecular cloning and mRNA localization of a gene associated with epithelial differentiation processes during embryogenesis

1993 ◽  
Vol 122 (5) ◽  
pp. 1119-1130 ◽  
Author(s):  
LE French ◽  
A Chonn ◽  
D Ducrest ◽  
B Baumann ◽  
D Belin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cell Types ◽  
Branching Morphogenesis ◽  
Structural Features ◽  
Heparin Binding ◽  
Binding Domains ◽  
Cell Layers

Clusterin is a broadly distributed glycoprotein constitutively expressed by various tissues and cell types, that has been shown to be involved in cell-cell adhesion and expressed during cellular differentiation in vitro. To assess the suggested participation of clusterin in these processes in vivo, we have cloned the cDNA encoding murine clusterin and studied the cellular distribution of clusterin mRNA during murine embryogenesis. Sequence analysis of the cDNA encoding murine clusterin revealed 92 and 75% sequence identity with the rat and human cDNAs, respectively, and conservation of the predicted structural features which include alpha-helical regions and heparin-binding domains. From 12.5 d of development onwards, the clusterin gene is widely expressed in developing epithelia, and selectively localized within the differentiating cell layers of tissues such as the developing skin, tooth, and duodenum where proliferating and differentiating compartments are readily distinguished. In addition, transient and localized clusterin gene expression was detected in certain morphogenetically active epithelia. In the lung, abundant gene transcripts were detected in cuboidal epithelial cells of the terminal lung buds during branching morphogenesis, and in the kidney, clusterin gene expression in the epithelial cells of comma and S-shaped bodies coincided with the process of polarization. Our results demonstrate the in vivo expression of the clusterin gene by differentiating epithelial cells during murine embryogenesis, and provide novel evidence suggesting that clusterin may be involved in the differentiation and morphogenesis of certain epithelia.

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