scholarly journals EFFECT OF DNA CONSTRUCTIONS ELECTROPORATION ON DENDRITIC CELLS

2021 ◽  
Vol 23 (4) ◽  
pp. 653-658
Author(s):  
A. S. Bulygin ◽  
V. P. Tereshchenko ◽  
R. Yu. Zavodskii ◽  
I. A. Obleukhova ◽  
S. V. Sennikov ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Cell Cultures ◽  
Culture Medium ◽  
Bone Marrow Cells ◽  
Culture Media ◽  
Pcr Analysis ◽  
Electric Impulse ◽  
Plasmacytoid Dcs ◽  
Conventional Type

Today transfection of mammalian cell with DNA or RNA construction is the only method for delivering programmed information into the cell nucleus. Electroporation is most commonly used method of transfection in experiments with dendritic cell. The aim of electroporation is to permeabilize the membrane by passing electric impulse through the cell. Due to the increase permeability of the membrane chance DNA or RNA construction getting inside into the cell is increased, wherein survival of the cells is decreased.In the study male mice C57Bl/6 line 2-4 months old were used. From femur bones was isolated 20 × 106 bone marrow cells, which were cultured in 20 mL of complete RPMI-1640 for 7 days. To generate dendritic cells from BM cells, 100 ng/mL of rmFlt3-L was added to culture media at day 0. After 7 days of cultivation, the cell cultures were electroporated with control noncoding plasmids p5 (EP P5) or pmaxCCR9 encoding mouse chemokine receptor CCR9 (EP CCR9). The controls were cell cultures electroporated without any plasmids (mock EP) and cell cultures without electroporation (none EP). 5 × 105 cells were electroporated and resting for 10 minutes. After 10 minutes, cells were harvested and seeded into 24-well plates in 1 mL of culture medium and conditioning medium (1:1). Then, 50 ng/mL of Flt3-L was added to each well. The next day, transfected cells were collected and used for flow cytometry, qRT-PCR analysis.It was found that after electroporation in the groups mock EP, EP P5, EP CCR9 relative amount of live CD11c+ dendritic cells was significantly less than in the non EP group. Moreover, in the EP P5 and EP CCR9 groups the frequency of live CD11c+ dendritic cells was significantly less than in the mock EP group. Expression of the CD86 marker in the EP P5 and EP CCR9 groups was significantly higher than in the non EP and mock EP groups. Expression of the I-AB(MHCII) in the EP CCR9 group on cDC2s was significantly higher than in the non EP group. On plasmacytoid DCs (pDCs) and conventional type 2 DCs (cDC2s) in the EP CCR9 group expression of CCR9 was significantly higher than in the non EP group. Therefore, in this study, we demonstrated the effectiveness of electroporation, accompanied by the decrease in the survival rate and maturation of DCs. 

Mouse CD11c+ B220+ Gr1+plasmacytoid dendritic cells develop independently of the T-cell lineage

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2852-2857 ◽  
Author(s):  
Isabel Ferrero ◽  
Werner Held ◽  
Anne Wilson ◽  
Fabienne Tacchini-Cottier ◽  
Freddy Radtke ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Bone Marrow Cells ◽  
T Cell Development ◽  
Cell Lineage ◽  
Notch 1 ◽  
T Cell Factor ◽  
Plasmacytoid Dcs ◽  
Lineage Markers ◽  
Mixed Chimeras

The developmental origin of dendritic cells (DCs) is controversial. In the mouse CD8α+ and CD8α− DC subsets are often considered to be of lymphoid and myeloid origin respectively, although evidence on this point is conflicting. Very recently a novel CD11c+ B220+ DC subset has been identified that appears to be the murine counterpart to interferon alpha (IFNα)–producing human plasmacytoid DCs (PDCs). We show here that CD11c+ B220+ mouse PDCs, like human PDCs, are present in the thymus and express T lineage markers such as CD8α and CD4. However, the intrathymic development of PDCs can be completely dissociated from immature T lineage cells in mixed chimeras established with bone marrow cells from mice deficient for either Notch-1 or T-cell factor 1, two independent mutations that severely block early T-cell development. Our data indicate that thymic PDCs do not arise from a bipotential T/DC precursor.

scholarly journals IRF4 Expression Is Required for the Immunoregulatory Activity of Conventional Type 2 Dendritic Cells in Settings of Chronic Bacterial Infection and Cancer

2020 ◽  
Vol 205 (7) ◽  
pp. 1933-1943
Author(s):  
Xiaozhou Zhang ◽  
Mariela Artola-Boran ◽  
Angela Fallegger ◽  
Isabelle C. Arnold ◽  
Achim Weber ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Bacterial Infection ◽  
Conventional Type

scholarly journals Inhibition of IRF4 in dendritic cells by PRR-independent and -dependent signals inhibit Th2 and promote Th17 responses

2019 ◽  
Author(s):  
Jihyung Lee ◽  
Junyan Zhang ◽  
Young-Jun Chung ◽  
Jun Hwan Kim ◽  
Chae Min Kook ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
Dendritic Cells ◽  
Molecular Basis ◽  
Camp Signaling ◽  
Biological Processes ◽  
The Novel ◽  
Microbial Product ◽  
Conventional Type ◽  
Recognition Receptor

AbstractCyclic AMP (cAMP) is involved in multiple biological processes. However, little is known about its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent) regulates conventional type-2 Dendritic Cells (cDC2s), but not cDC1s and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors (TFs) for Th2 induction. Genetic loss of IRF4 phenocopies the effects of cAMP signaling on Th17-induction, indicating that the cAMP effect is secondary to repression of IRF4. Moreover, signaling in cDC2s by a PRR-dependent microbial product, curdlan, represses IRF4 and KLF4, resulting in a pro-Th17 phenotype. These results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the novel cDC2 and cDC17 classification. In addition, the data reveal that cAMP signaling can alter DCs function and fate by repressing IRF4 and KLF4, a pathway that can be harnessed for immuno-regulation.

scholarly journals A Discrete Subset of Monocyte-Derived Cells among Typical Conventional Type 2 Dendritic Cells Can Efficiently Cross-Present

Cell Reports ◽  
2017 ◽  
Vol 21 (5) ◽  
pp. 1203-1214 ◽  
Author(s):  
Jianpeng Sheng ◽  
Qi Chen ◽  
Irene Soncin ◽  
See Liang Ng ◽  
Klaus Karjalainen ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Discrete Subset ◽  
Conventional Type

scholarly journals Inhibition of IRF4 in dendritic cells by PRR-independent and -dependent signals inhibit Th2 and promote Th17 responses

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jihyung Lee ◽  
Junyan Zhang ◽  
Young-Jun Chung ◽  
Jun Hwan Kim ◽  
Chae Min Kook ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Biological Processes ◽  
Microbial Product ◽  
Independent Mechanism ◽  
Conventional Type ◽  
Recognition Receptor

Cyclic AMP (cAMP) is involved in many biological processes but little is known regarding its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent mechanism) regulates conventional type-2 Dendritic Cells (cDC2s) in mice and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors essential for cDC2-mediated Th2 induction. In mice, genetic loss of IRF4 phenocopies the effects of cAMP on Th17 induction and restoration of IRF4 prevents the cAMP effect. Moreover, curdlan, a PRR-dependent microbial product, activates CREB and represses IRF4 and KLF4, resulting in a pro-Th17 phenotype of cDC2s. These in vitro and in vivo results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the classification of novel cDC2 and cDC17 subsets. The findings also reveal that repressing IRF4 and KLF4 pathway can be harnessed for immuno-regulation.

scholarly journals Myocarditis Elicits Dendritic Cell and Monocyte Infiltration in the Heart and Self-Antigen Presentation by Conventional Type 2 Dendritic Cells

2018 ◽  
Vol 9 ◽  
Author(s):  
Katrien Van der Borght ◽  
Charlotte L. Scott ◽  
Liesbet Martens ◽  
Dorine Sichien ◽  
Gert Van Isterdael ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Dendritic Cell ◽  
Antigen Presentation ◽  
Self Antigen ◽  
Conventional Type

scholarly journals TARM1 contributes to development of arthritis by activating dendritic cells through recognition of collagens

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rikio Yabe ◽  
Soo-Hyun Chung ◽  
Masanori A. Murayama ◽  
Sachiko Kubo ◽  
Kenji Shimizu ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Bone Marrow Cells ◽  
Dendritic Cell Maturation ◽  
Mouse Bone Marrow ◽  
Gm Csf ◽  
Good Target ◽  
Antigen Presenting ◽  
Dc Maturation

AbstractTARM1 is a member of the leukocyte immunoglobulin-like receptor family and stimulates macrophages and neutrophils in vitro by associating with FcRγ. However, the function of this molecule in the regulation of the immune system is unclear. Here, we show that Tarm1 expression is elevated in the joints of rheumatoid arthritis mouse models, and the development of collagen-induced arthritis (CIA) is suppressed in Tarm1–/– mice. T cell priming against type 2 collagen is suppressed in Tarm1–/– mice and antigen-presenting ability of GM-CSF-induced dendritic cells (GM-DCs) from Tarm1–/– mouse bone marrow cells is impaired. We show that type 2 collagen is a functional ligand for TARM1 on GM-DCs and promotes DC maturation. Furthermore, soluble TARM1-Fc and TARM1-Flag inhibit DC maturation and administration of TARM1-Fc blocks the progression of CIA in mice. These results indicate that TARM1 is an important stimulating factor of dendritic cell maturation and could be a good target for the treatment of autoimmune diseases.

scholarly journals THE PROBLEM OF CELL CULTURES CONTAMINATION WITH MAMMALIAN ORTHOREOVIRUSES

2018 ◽  
pp. 104-107
Author(s):  
E. B. Faisuloev ◽  
E. P. Korchevaya ◽  
D. V. Markov ◽  
O. A. Petrusha ◽  
V. V. Zverev
Keyword(s):  
Cell Cultures ◽  
Culture Medium ◽  
Fetal Bovine Serum ◽  
Animal Origin ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Mandatory Requirement ◽  
Mammalian Orthoreovirus ◽  
Fetal Bovine ◽  
Pharmaceutical Production

A mandatory requirement for cell cultures used in scientific researches and biomanufacturing is the absence of their contamination by viruses. We have described the case of adventitious isolation of mammalian orthoreovirus in the rhesus macaque embryo kidney cells MA-104. PCR analysis for the presence of reovirus RNA of all probable sources of reovirus (trypsin, fetal bovine serum, clinical samples, cell culture) revealed no viral RNA in any of the samples. An important condition for the activation of the reovirus reproduction in the MA-104 cells was the presence of trypsin in the culture medium. The obtained results underscore the urgency of control for the reovirus contamination of chemicals of animal origin and cell cultures. Since reoviruses are associated with human diseases, such control in pharmaceutical production is mandatory.

scholarly journals Serum-free medium increases the replication rate of the avian coronavirus infectious bronchitis virus in chicken embryo kidney cells

2021 ◽  
Author(s):  
José A. Quinteros ◽  
Glenn F. Browning ◽  
Amir H. Noormohammadi ◽  
Mark A. Stevenson ◽  
Mauricio J. C. Coppo ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Infectious Bronchitis Virus ◽  
Chicken Embryo ◽  
Culture Medium ◽  
Culture Media ◽  
Cell Culture Medium ◽  
Cell Culture Media ◽  
Infectious Bronchitis ◽  
Porcine Epidemic Diarrhoea Virus

AbstractInfectious bronchitis virus (IBV), an avian coronavirus, can be isolated and cultured in tracheal organ cultures (TOCs), embryonated eggs and cell cultures. TOCs and embryonated eggs are commonly used for viral isolation but use of these is laborious and expensive. Cell cultures have been used only with IBV strains that have previously been adapted to grow under laboratory conditions, and not for primary isolation. Previous studies using the coronavirus porcine epidemic diarrhoea virus (PEDV) have suggested that foetal bovine serum (FBS), a common component of cell culture media, can inhibit the adsorption of coronaviruses onto the host cell membrane receptors. In the present study, the replication of IBV in primary chicken embryo kidney (CEK) cell cultures and the Leghorn hepatocellular carcinoma (LMH) cell line was examined using two different cell culture media, one containing FBS and the other containing yeast extract (YE). A reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay was used to quantify viral RNA copies in cell lysates. The highest concentrations of viral genomes were observed when the cell culture medium did not contain FBS. Examination of the infectivity of virus grown in CEK cell cultures was examined by titration in embryonated chicken eggs, demonstrating that the cell lysate from CEK cell cultures in medium without FBS contained a higher median embryo infectious dose (EID50) than that from CEK cell cultures in medium containing FBS. These results suggest that improved replication of IBV in cell cultures can be achieved by the omission of FBS from the cell culture medium. This may enhance the potential for production of vaccines in cell culture and facilitate the isolation of emergent IBV strains in cell cultures.

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