scholarly journals Glycoprotein targeting signals influence the distribution of measles virus envelope proteins and virus spread in lymphocytes

2008 ◽  
Vol 89 (3) ◽  
pp. 687-696 ◽  
Author(s):  
Nicole Runkler ◽  
Erik Dietzel ◽  
Markus Moll ◽  
Hans-Dieter Klenk ◽  
Andrea Maisner
Keyword(s):  
Epithelial Cells ◽  
Measles Virus ◽  
M Protein ◽  
Target Cells ◽  
Virus Spread ◽  
Virus Propagation ◽  
Virus Envelope ◽  
Polarized Epithelial Cells

We previously demonstrated the presence of tyrosine-dependent motifs for specific sorting of two measles virus (MV) glycoproteins, H and F, to the basolateral surface in polarized epithelial cells. Targeted expression of the glycoproteins was found to be required for virus spread in epithelia via cell-to-cell fusion in vitro and in vivo. In the present study, recombinant MVs (rMVs) with substitutions of the critical tyrosines in the H and F cytoplasmic domains were used to determine whether the sorting signals also play a crucial role for MV replication and spread within lymphocytes, the main target cells of acute MV infection. Immunolocalization revealed that only standard glycoproteins are targeted specifically to the uropod of polarized lymphocytes and cluster on the surface of non-polarized lymphocytes. H and F proteins with tyrosine mutations did not accumulate in uropods, but were distributed homogeneously on the surface and did not colocalize markedly with the matrix (M) protein. Due to the defective interaction with the M protein, all mutant rMVs showed an enhanced fusion capacity, but only rMVs harbouring two mutated glycoproteins showed a marked decrease in virus release from infected lymphocytes. These results demonstrate clearly that the tyrosine-based targeting motifs in the MV glycoproteins are not only important in polarized epithelial cells, but are also active in lymphocytes, thus playing an important role in virus propagation in different key target cells during acute MV infection.

scholarly journals Human Cytomegalovirus Cell Tropism and Host Cell Receptors

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 70 ◽  
Author(s):  
Gerna ◽  
Kabanova ◽  
Lilleri
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Epithelial Cells ◽  
Human Cytomegalovirus ◽  
Current Data ◽  
Immunosuppressed Patient ◽  
Cell Tropism ◽  
Virus Envelope

In the 1970s–1980s, a striking increase in the number of disseminated human cytomegalovirus (HCMV) infections occurred in immunosuppressed patient populations. Autopsy findings documented the in vivo disseminated infection (besides fibroblasts) of epithelial cells, endothelial cells, and polymorphonuclear leukocytes. As a result, multiple diagnostic assays, such as quantification of HCMV antigenemia (pp65), viremia (infectious virus), and DNAemia (HCMV DNA) in patient blood, were developed. In vitro experiments showed that only low passage or endothelial cell-passaged clinical isolates, and not laboratory-adapted strains, could reproduce both HCMV leuko- and endothelial cell-tropism, which were found through genetic analysis to require the three viral genes UL128, UL130, and UL131 of the HCMV UL128 locus (UL128L). Products of this locus, together with gH/gL, were shown to form the gH/gL/pUL128L pentamer complex (PC) required for infection of epithelial cells/endothelial cells, whereas gH/gL and gO form the gH/gL/gO trimer complex (TC) required for infection of all cell types. In 2016, following previous work, a receptor for the TC that mediates entry into fibroblasts was identified as PDGFRα, while in 2018, a receptor for the PC that mediates entry into endothelial/epithelial cells was identified as neuropilin2 (Nrp2). Furthermore, the olfactory receptor family member OR14I1 was recently identified as a possible additional receptor for the PC in epithelial cells. Thus, current data support two models of viral entry: (i) in fibroblasts, following interaction of PDGFRα with TC, the latter activates gB to fuse the virus envelope with the cell membrane, whereas (ii) in epithelial cells/endothelial cells, interaction of Nrp2 (and OR14I1) with PC promotes endocytosis of virus particles, followed by gB activation by gH/gL/gO (or gH/gL) and final low-pH entry into the cell.

scholarly journals Live-Attenuated Measles Virus Vaccine Targets Dendritic Cells and Macrophages in Muscle of Nonhuman Primates

2014 ◽  
Vol 89 (4) ◽  
pp. 2192-2200 ◽  
Author(s):  
Linda J. Rennick ◽  
Rory D. de Vries ◽  
Thomas J. Carsillo ◽  
Ken Lemon ◽  
Geert van Amerongen ◽  
...  
Keyword(s):  
Measles Virus ◽  
Nonhuman Primates ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Vaccine Virus ◽  
Target Cells ◽  
Recombinant Viruses ◽  
Green Fluorescent

ABSTRACTAlthough live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replicationin vivoare still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MVEZ. Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMVEZEGFP(1), rMVEZEGFP(3), and rMVEZEGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were comparedin vitroby growth curves, which indicated that rMVEZEGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMVEZEGFP(1) and rMVEZEGFP(6) did not induce satisfactory serum antibody responses, whereas bothin vitroandin vivorMVEZEGFP(3) was functionally equivalent to the commercial MVEZ-containing vaccine. Intramuscular vaccination of macaques with rMVEZEGFP(3) resulted in the identification of EGFP+cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV.IMPORTANCEEven though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicatesin vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

scholarly journals In Vitro and In Vivo Effects of Soluble, Monovalent Globotriose on Bacterial Attachment and Colonization

2005 ◽  
Vol 49 (9) ◽  
pp. 3842-3846 ◽  
Author(s):  
James L. Leach ◽  
Stacey A. Garber ◽  
Andrea A. Marcon ◽  
Pedro A. Prieto
Keyword(s):  
Urinary Tract ◽  
Epithelial Cells ◽  
Alternative Therapy ◽  
Bacterial Attachment ◽  
Target Cells ◽  
Cell Agglutination ◽  
Core Motif ◽  
Tract Infections

ABSTRACT Epithelial cells lining the urinary tract are rich in globo series glycolipids, structurally defined by a Galα1,4Gal motif in the oligosaccharide moiety of this glycolipid family. This Galα1,4Gal motif is the attachment target for the P-fimbrial adhesin of uropathogenic Escherichia coli. We investigated the ability of a trisaccharide analog of this core motif, globotriose (Galα1,4Galβ1,4Glc), to interfere with uropathogen attachment and colonization in vitro and in vivo. We assessed the ability of globotriose to inhibit and reverse the binding and agglutination of a P-fimbriated strain of E. coli (JR1) using human erythrocytes and immortalized human colonic epithelial cells as targets. Globotriose (5 mg/ml) completely inhibited and reversed cell agglutination and caused a 10- to 100-fold reduction in JR1 binding to target cells, as determined by flow cytometry. In preparation for an in vivo efficacy study, we investigated the distribution and pharmacokinetics of globotriose in the BALB/c mouse. Globotriose was administered via the tail vein, targeting an instantaneous plasma concentration of 5 mg/ml, and in a different experiment, animals were gavaged at 10 times the intravenous (i.v.) dose. Globotriose was rapidly cleared from plasma (half-life [t 1/2], 6 min) and slowly excreted via the kidney (t 1/2, 4 h). Urine levels of >5 mg/ml were maintained from 4 to 12 h after the i.v. bolus dose, which resulted in a 1-log reduction in established bladder colonization by JR1. These results suggest that free, soluble globotriose is a feasible alternative therapy for urinary tract infections.

scholarly journals Extracellular Vesicle-Mediated siRNA Delivery, Protein Delivery, and CFTR Complementation in Well-Differentiated Human Airway Epithelial Cells

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 351 ◽  
Author(s):  
Brajesh K. Singh ◽  
Ashley L. Cooney ◽  
Sateesh Krishnamurthy ◽  
Patrick L. Sinn
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelial Cells ◽  
Target Cells ◽  
Airway Epithelial ◽  
Long Distance ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelial Cells

Extracellular vesicles (EVs) are a class of naturally occurring secreted cellular bodies that are involved in long distance cell-to-cell communication. Proteins, lipids, mRNA, and miRNA can be packaged into these vesicles and released from the cell. This information is then delivered to target cells. Since EVs are naturally adapted molecular messengers, they have emerged as an innovative, inexpensive, and robust method to deliver therapeutic cargo in vitro and in vivo. Well-differentiated primary cultures of human airway epithelial cells (HAE) are refractory to standard transfection techniques. Indeed, common strategies used to overexpress or knockdown gene expression in immortalized cell lines simply have no detectable effect in HAE. Here we use EVs to efficiently deliver siRNA or protein to HAE. Furthermore, EVs can deliver CFTR protein to cystic fibrosis donor cells and functionally correct the Cl− channel defect in vitro. EV-mediated delivery of siRNA or proteins to HAE provides a powerful genetic tool in a model system that closely recapitulates the in vivo airways.

Expression of neuropilin-1 by human glomerular epithelial cells in vitro and in vivo

2001 ◽  
Vol 101 (4) ◽  
pp. 439-446 ◽  
Author(s):  
Steven J. HARPER ◽  
Chang Ying XING ◽  
Cathy WHITTLE ◽  
Robin PARRY ◽  
David GILLATT ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Reverse Transcription ◽  
Target Cells ◽  
Normal Kidney ◽  
Autocrine Loop ◽  
Reverse Transcription Pcr ◽  
Neuropilin 1 ◽  
Glomerular Epithelial Cells

Vascular endothelial growth factor (VEGF) is a potent promoter of endothelial mitogenesis and of endothelial permeability. Within the kidney it is synthesized primarily in the visceral glomerular epithelial cells (vGECs); however, the role of VEGF in the glomerulus remains unknown, as does the target cell upon which it acts. Although the target cells may be those of the glomerular endothelium, there are micro-anatomical reasons why this might not be the case. This, therefore, led us to consider the possibility that glomerular VEGF may bind to the vGECs themselves. Since it has been shown that vGECs do not express the main tyrosine kinase VEGF receptors, we chose to study vGEC expression of the more recently described VEGF isoform-specific receptors, the neuropilins. The expression of mRNAs for neuropilin-1, neuropilin-2 and soluble neuropilin was studied in whole kidney, sieved glomeruli and cultured podocytes by reverse transcription-PCR, and neuropilin-1 mRNA expression in isolated single glomeruli was analysed by nested reverse transcription-PCR. The expression of neuropilin-1 protein was investigated in cultured vGECs by Western blotting and immunocytochemistry, and in normal kidney sections by immunohistochemistry. Neuropilin-1 mRNA was detected in whole kidney, single and sieved glomeruli and cultured vGECs. Neuropilin-1 protein was detected in cultured vGECs and in vGECs in normal kidney sections by immunohistochemistry. Thus the present study suggests that vGECs may have the potential to bind the VEGF that they secrete. Functional studies will be required to address the potential significance of this finding in terms of an autocrine loop or VEGF sequestration.

scholarly journals Targeting of the SF/HGF receptor to the basolateral domain of polarized epithelial cells.

1994 ◽  
Vol 125 (2) ◽  
pp. 313-320 ◽  
Author(s):  
T Crepaldi ◽  
A L Pollack ◽  
M Prat ◽  
A Zborek ◽  
K Mostov ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Cell Contact ◽  
Apical Surface ◽  
Surface Distribution ◽  
Polarized Epithelial Cells ◽  
Cell Cell

Scatter Factor, also known as Hepatocyte Growth Factor (SF/HGF), has pleiotropic functions including direct control of cell-cell and cell-substrate adhesion in epithelia. The subcellular localization of the SF/HGF receptor is controversial. In this work, the cell surface distribution of the SF/HGF receptor was studied in vivo in epithelial tissues and in vitro in polarized MDCK monolayers. A panel of monoclonal antibodies against the beta chain of the SF/HGF receptor stained the basolateral but not the apical surface of epithelia lining the lumen of human organs. Radiolabeled or fluorescent-tagged anti-receptor antibodies selectively bound the basolateral cell surface of MDCK cells, which form a polarized monolayer sealed by intercellular junctions, when grown on polycarbonate filters in a two-chamber culture system. The receptor was concentrated around the cell-cell contact zone, showing a distribution pattern overlapping with that of the cell adhesion molecule E-cadherin. The basolateral localization of the SF/HGF receptor was confirmed by immunoprecipitation after domain selective cell surface biotinylation. When cells were fully polarized the SF/HGF receptor became resistant to non-ionic detergents, indicating interaction with insoluble component(s). In pulse-chase labeling and surface biotinylation experiments, the newly synthesized receptor was found exclusively at the basolateral surface. We conclude that the SF/HGF receptor is selectively exposed at the basolateral plasma membrane domain of polarized epithelial cells and is targeted after synthesis to that surface by direct delivery from the trans-Golgi network.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing

1993 ◽  
Vol 21 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Knut-Jan Andersen ◽  
Erik Ilsø Christensen ◽  
Hogne Vik
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Toxicity Testing ◽  
Renal Tissue ◽  
Epithelial Cell Line ◽  
Multicellular Spheroids ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cells

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.

scholarly journals Combined In Vitro and In Vivo Approaches to Propose a Putative Adverse Outcome Pathway for Acute Lung Inflammation Induced by Nanoparticles: A Study on Carbon Dots

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 180
Author(s):  
Maud Weiss ◽  
Jiahui Fan ◽  
Mickaël Claudel ◽  
Luc Lebeau ◽  
Françoise Pons ◽  
...  
Keyword(s):  
Carbon Dots ◽  
Lung Inflammation ◽  
Adverse Outcome ◽  
Cellular Responses ◽  
Target Cells ◽  
Inflammasome Activation ◽  
Adverse Outcome Pathway ◽  
Acute Lung Inflammation

With the growth of nanotechnologies, concerns raised regarding the potential adverse effects of nanoparticles (NPs), especially on the respiratory tract. Adverse outcome pathways (AOP) have become recently the subject of intensive studies in order to get a better understanding of the mechanisms of NP toxicity, and hence hopefully predict the health risks associated with NP exposure. Herein, we propose a putative AOP for the lung toxicity of NPs using emerging nanomaterials called carbon dots (CDs), and in vivo and in vitro experimental approaches. We first investigated the effect of a single administration of CDs on mouse airways. We showed that CDs induce an acute lung inflammation and identified airway macrophages as target cells of CDs. Then, we studied the cellular responses induced by CDs in an in vitro model of macrophages. We observed that CDs are internalized by these cells (molecular initial event) and induce a series of key events, including loss of lysosomal integrity and mitochondrial disruption (organelle responses), as well as oxidative stress, inflammasome activation, inflammatory cytokine upregulation and macrophage death (cellular responses). All these effects triggering lung inflammation as tissular response may lead to acute lung injury.

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