scholarly journals In vitro transcribed guide RNAs trigger an innate immune response via the RIG-I pathway

2018 ◽  
Author(s):  
Beeke Wienert ◽  
Jiyung Shin ◽  
Elena Zelin ◽  
Kathleen Pestal ◽  
Jacob E. Corn
Keyword(s):  
Immune Response ◽  
Genome Editing ◽  
Innate Immune ◽  
Kidney Cells ◽  
Primary Cells ◽  
Human Embryonic Kidney ◽  
Hematopoietic Stem ◽  
Human Embryonic Kidney Cells ◽  
Guide Rna

AbstractCRISPR-Cas9 genome editing is revolutionizing fundamental research and has great potential for the treatment of many diseases. While editing of immortalized cell lines has become relatively easy, editing of therapeutically relevant primary cells and tissues can remain challenging. One recent advancement is the delivery of a Cas9 protein and an in vitro transcribed (IVT) guide RNA (gRNA) as a precomplexed ribonucleoprotein (RNP). This approach allows editing of primary cells such as T cells and hematopoietic stem cells, but the consequences beyond genome editing of introducing foreign Cas9 RNPs into mammalian cells are not fully understood. Here we show that the IVT gRNAs commonly used by many laboratories for RNP editing trigger a potent innate immune response that can be several thousand times stronger than benchmark immune stimulating ligands. IVT gRNAs are recognized in the cytosol through the RIG-I pathway but not the MDA5 pathway, thereby triggering a type I interferon response. Removal of the 5’-triphosphate from gRNAs ameliorates inflammatory signaling and prevents the loss of viability associated with genome editing in hematopoietic stem cells. The potential for Cas9 RNP editing to induce a potent antiviral response indicates that care must be taken when designing therapeutic strategies to edit primary cells.AbbreviationsCasCRISPR-associatedCIPcalf intestinal alkaline phosphataseCRISPRclustered, regularly interspaced, short palindromic repeatdCas9nuclease-dead Cas9HEK293Human embryonic kidney cells 293HEK293THuman embryonic kidney cells 293 SV40 large T antigenHeLaHenrietta Lacks cellsHSPCsCD34+ human hematopoietic stem and progenitor cellsIFNAR1Interferon Alpha And Beta Receptor Subunit 1IFNβ/IFNB1Interferon betaISG15Interferon-stimulated gene 15 IVT – in vitro transcribedKOknockoutMAVSmitochondrial activator of virus signalingMDA5/IFIH1melanoma differentiation-associated gene 5/ Interferon Induced with Helicase C Domain 1PAMPpathogen-associated molecular patternRIG-I/DDX58retinoic acid-inducible gene I/ DExD-H-box helicase 58gRNAguide RNASPRIsolid phase reversible immobilizationWTwild type

scholarly journals Induction of Sertoli cells from human fibroblasts by NR5A1 and GATA4

2018 ◽  
Author(s):  
Jianlin Liang ◽  
Nan Wang ◽  
Jing He ◽  
Jian Du ◽  
Yahui Guo ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Lipid Droplets ◽  
Human Fibroblasts ◽  
Seminiferous Tubules ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Reprogramming Factors ◽  
Spermatogonia Cells

AbstractSertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). The induction of human Sertoli cells from fibroblasts could provide cellular sources for fertility and transplantation treatments. Here, we report the in vitro reprogramming of human fibroblasts to Sertoli cells and characterize these human induced Sertoli cells (hiSCs). Initially, five transcriptional factors (NR5A1, GATA4, WT1, SOX9 and DMRT1) and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, i.e., NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles that are similar to those of primary human Sertoli cells. Consistent with the known cellular properties of Sertoli cells, hiSCs attract endothelial cells and exhibit high number of lipid droplets in the cytoplasm. More importantly, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. In addition, hiSCs suppress the production of IL-2 and proliferation of human T lymphocytes. When hiSCs were cotransplanted with human embryonic kidney cells, these xenotransplanted human cells survived longer in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli-only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.

Molecular insight to in vitro biocompatibility of phytofabricated copper oxide nanoparticles with human embryonic kidney cells

Nanomedicine ◽  
2018 ◽  
Vol 13 (19) ◽  
pp. 2415-2433 ◽  
Author(s):  
Puja Kumari ◽  
Pritam Kumar Panda ◽  
Ealisha Jha ◽  
Nandini Pramanik ◽  
Kumari Nisha ◽  
...  
Keyword(s):  
Copper Oxide ◽  
Copper Oxide Nanoparticles ◽  
Oxide Nanoparticles ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
In Vitro Biocompatibility

scholarly journals A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs

2004 ◽  
Vol 384 (2) ◽  
pp. 391-400 ◽  
Author(s):  
Helen McNEILL ◽  
Axel KNEBEL ◽  
J. Simon C. ARTHUR ◽  
Ana CUENDA ◽  
Philip COHEN
Keyword(s):  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Glutathione S Transferase ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Extracellular Signal Regulated Kinase ◽  
Cell Extracts ◽  
Extracellular Signal ◽  
Sb 203580

A widely expressed protein containing UBA (ubiquitin-associated) and UBX (ubiquitin-like) domains was identified as a substrate of SAPKs (stress-activated protein kinases). Termed SAKS1 (SAPK substrate-1), it was phosphorylated efficiently at Ser200in vitro by SAPK3/p38γ, SAPK4/p38δ and JNK (c-Jun N-terminal kinase), but weakly by SAPK2a/p38α, SAPK2b/p38β2 or ERK (extracellular-signal-regulated kinase) 2. Ser200, situated immediately N-terminal to the UBX domain, became phosphorylated in HEK-293 (human embryonic kidney) cells in response to stressors. Phosphorylation was not prevented by SB 203580 (an inhibitor of SAPK2a/p38α and SAPK2b/p38β2) and/or PD 184352 (which inhibits the activation of ERK1 and ERK2), and was similar in fibroblasts lacking both SAPK3/p38γ and SAPK4/p38δ or JNK1 and JNK2. SAKS1 bound ubiquitin tetramers and VCP (valosin-containing protein) in vitro via the UBA and UBX domains respectively. The amount of VCP in cell extracts that bound to immobilized GST (glutathione S-transferase)–SAKS1 was enhanced by elevating the level of polyubiquitinated proteins, while SAKS1 and VCP in extracts were coimmunoprecipitated with an antibody raised against S5a, a component of the 19 S proteasomal subunit that binds polyubiquitinated proteins. PNGase (peptide N-glycanase) formed a 1:1 complex with VCP and, for this reason, also bound to immobilized GST–SAKS1. We suggest that SAKS1 may be an adaptor that directs VCP to polyubiquitinated proteins, and PNGase to misfolded glycoproteins, facilitating their destruction by the proteasome.

Generation of Human Embryonic Kidney Cells with Extended In Vitro Life Span through Viral Oncogene Transfection

1989 ◽  
Vol 7 (9) ◽  
pp. 939-946 ◽  
Author(s):  
Steve Abcouwer ◽  
Polly S. Robinson ◽  
Charles F. Goochee ◽  
Michael T. Crow
Keyword(s):  
Life Span ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Viral Oncogene

Hyperthermia with Magnetic Nanowires for Inactivating Living Cells

2008 ◽  
Vol 8 (5) ◽  
pp. 2323-2327 ◽  
Author(s):  
D. S. Choi ◽  
J. Park ◽  
S. Kim ◽  
D. H. Gracias ◽  
M. K. Cho ◽  
...  
Keyword(s):  
Cell Death ◽  
Magnetic Moments ◽  
Magnetic Hysteresis ◽  
Kidney Cells ◽  
Magnetic Nanowires ◽  
Human Embryonic Kidney ◽  
Lower Field ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells

We describe a method to induce hyperthermia in cells, in-vitro, by remotely heating Ni nanowires (NWs) with radio frequency (RF) electromagnetic fields. Ni NWs were internalized by human embryonic kidney cells (HEK-293). Only cells proximal to NWs or with internalized NWs changed shape on exposure to RF fields indicative of cell death. The cell death occurs as a result of hyperthermia, since the RF field remotely heats the NWs as a result of magnetic hysteresis. This is the first demonstration of hyperthermia induced by NWs; since the NWs have anisotropic and strong magnetic moments, our experiments suggest the possibility of performing hyperthermia at lower field strengths in order to minimize damage to untargeted cells in applications such as the treatment of cancer.

A Role for NFAT in Innate Immunity: Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation Under Cyclosporine Α Treatment

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2495-2495
Author(s):  
Katharina Plein ◽  
Daniel Teschner ◽  
Christian Michel ◽  
Eva-Maria Wagner ◽  
Pamela Stein ◽  
...  
Keyword(s):  
Immune Response ◽  
Innate Immune Response ◽  
Inflammatory Mediators ◽  
Ex Vivo ◽  
Innate Immune ◽  
Hematopoietic Stem ◽  
Effector Functions ◽  
Healthy Donors ◽  
Tnf Α

Abstract Background and Aims: Patients after allogeneic hematopoietic stem cell transplantation (HSCT) suffer from immunodeficiency, in part due to long-term immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA). Additionally, these patients have an increased risk for opportunistic fungal infections like invasive aspergillosis (IA). The nuclear factor of activated T cells (NFAT) is known as an important transcription factor in signaling-pathways downstream of calcineurin in the adaptive immune systems, e.g. in T cells, but also plays an important role in innate immune response as indicated by recent data in rodent models. These studies showed a relevant impact of NFAT-/calcineurin inhibition on the innate immune response by polymorphonuclear neutrophils (PMN) in a candida sepsis model. To clarify whether this is also relevant in humans, we investigated the role of NFAT signaling pathways in PMN activation and effector functions in healthy volunteer donors and patients under immunosuppressive treatment with CsA after HSCT. Methods: Firstly, we performed in vitro experiments using PMN from healthy donors analyzing their effector functions in absence or presence of CsA in titrated doses according to therapeutic levels. In detail, we examined phagocytosis, activation, generation of reactive oxygen species (ROS) and release of inflammatory mediators like IL-8 and TNF-α. After activation with lipopolysaccharide (LPS) and zymosan, phagocytosis and activation-induced shedding of CD62L were measured by flow cytometry using polychromatic microspheres and matching surface markers (CD11b, CD62L, CD66b). In addition, generation of ROS was analyzed by dichlorofluorescein assay (DCF), whereas activation-induced release of inflammatory mediators was measured by enzyme-linked immunosorbent assay (ELISA) and intracellular flow cytometry. In addition, blood samples of patients after HSCT under continuous CsA medication (n=6) and healthy volunteer donors (n=6) were analyzed ex vivo at two different time points after allogeneic HSCT (day 25-35 and 125-135) concerning their PMN effector functions in the same manner as described above. Results: Analysis of healthy donors PMN in vitro showed that CsA had no significant influence on expression of activation markers and shedding of CD62L. Moreover, no substantial influence of CsA on generation of ROS was detected compared to untreated controls (5245 RFU +/- 354 (CsA) vs. 5763 +/- 520 (control) after stimulation with LPS, mean +/- SEM). Furthermore, activation-induced synthesis of IL-8 was not reduced in presence of CsA (519pg/ml +/- 81 vs. 463 +/- 131 (control) after stimulation with LPS). In contrast, CsA rather enhanced phagocytosis after stimulation with LPS (83.5% +/- 1.7 vs. 71.0 +/- 1.5 (control)). Regarding the ex vivo analysis of HSCT patient and healthy donor blood samples, production of ROS was not affected under CsA therapy (35.1% +/- 9.4 vs. 31.1 +/- 6.6 (control) after stimulation with zymosan). Furthermore, CsA medication showed a stimulating effect on PMN phagocytosis which is in line with our in vitro data (58.4% +/- 7.1 vs. 44.3 +/- 2.3 (control) after stimulation with LPS). Interestingly, in several patients an increased production of IL-8 and TNF-α was detectable after stimulation with zymosan (IL-8: 9.1% +/- 3.6 vs. 1.5 +/- 0.3 (control); TNF-α: 16.6% +/- 6.2 vs. 1.0 +/- 0.4 (control)). Conclusions: In contrast to previous results by others in murine model systems, we found an increased phagocytic activity in vitro and ex vivo in human PMN upon NFAT/calcineurin inhibition, whereas other effector mechanisms were unaffected. In addition, HSCT patients under CsA treatment displayed enhanced inflammatory mediators production in PMN. It is currently unclear whether these findings are clinically relevant for the innate immune response after HSCT, e. g. in terms of anti-fungal immunity. Further studies are needed to address whether these enhanced PMN functions in the presence of CsA contribute to a dysregulated innate immune response in humans. Alternatively, our discrepant results may be due to differences in PMN functionality and CsA responsiveness in mice and man. However, while CsA strongly suppresses adaptive immune responses, i.e. T-cell responses in graft-versus-host disease (GvHD), our results so far suggest that CsA does not affect innate immune effector functions by humans PMN in a comparable way. Disclosures Radsak: Celgene: Research Funding.

scholarly journals Cytotoxic Aporphines from Artabotrys Crassifolius

2016 ◽  
Vol 11 (3) ◽  
pp. 1934578X1601100
Author(s):  
Tan Kok Kwan ◽  
Fiona Shipton ◽  
Nadiah Syafiqah Nor Azman ◽  
Shahadat Hossan ◽  
Khoo Ten Jin ◽  
...  
Keyword(s):  
Chloroform Extract ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Cytotoxic Effects ◽  
Human Embryonic Kidney Cells ◽  
Hct 116 ◽  
Ethanol Extracts ◽  
Hct 116 Cells ◽  
Mcf 7

Artabotrys crassifolius Hook. f. & Thomson is a medicinal plant used in Malaysia. The cytotoxic effects of the hexane, chloroform and ethanol extracts of the leaves and bark were examined in vitro against MCF-7, MDA-468 and HCT-116 cells. The chloroform extract of the bark inhibited the growth of all cell lines with GI50 values ranging from 4.2 μg/mL to 9.4 μg/mL. Silica gel column chromatography of this extract yielded artabotrine, liridine, atherospermidine and lysicamine. Artabotrine and lysicamine inhibited the growth of HCT-116 and MCF-7 cells with GI50 values ranging from 3.3 μM to 3.9 μM. These alkaloids were not toxic to human embryonic kidney cells (HEK297) up to a concentration of 50 μg/mL.

scholarly journals Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells

2021 ◽  
Vol 22 (1) ◽  
pp. 397
Author(s):  
Nasir Javaid ◽  
Thuong L. H. Pham ◽  
Sangdun Choi
Keyword(s):  
Protein Level ◽  
Reference Genes ◽  
Mrna Level ◽  
High Specificity ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Sox2 Expression

Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.

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