scholarly journals Specific targeting of the IL-23 receptor, using a novel small peptide noncompetitive antagonist, decreases the inflammatory response

2014 ◽  
Vol 307 (10) ◽  
pp. R1216-R1230 ◽  
Author(s):  
Christiane Quiniou ◽  
Maria Domínguez-Punaro ◽  
Frank Cloutier ◽  
Atefeh Erfani ◽  
Jamila Ennaciri ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Inflammatory Diseases ◽  
Jurkat Cells ◽  
Small Peptide ◽  
Proinflammatory Response ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Stat3 Phosphorylation

IL-23 is part of the IL-12 family of cytokines and is composed of the p19 subunit specific to IL-23 and the p40 subunit shared with IL-12. IL-23 specifically contributes to the inflammatory process of multiple chronic inflammatory autoimmune disorders, including psoriasis, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. So far, one antibody targeting the shared p40 subunit of IL-12 and IL-23, Ustekinumab, is approved clinically to treat psoriasis. However, there are no treatments inhibiting specifically the IL-23 proinflammatory response. We have developed small IL-23R-specific antagonists by designing all D-peptides arising from flexible regions of IL-23R. Of these peptides, we selected 2305 (teeeqqly), since in addition to its soluble properties, it inhibited IL-23-induced STAT3 phosphorylation in spleen cells. Peptide 2305 specifically binds to IL-23R/IL-12Rβ1-expressing HEK-293 cells and not to cells devoid of the receptor. Peptide 2305 showed functional selectivity by modulating IL-23-induced gene expression in IL-23R/IL-12Rβ1-expressing cells and in Jurkat cells; 2305 does not inhibit IL-12-induced cytokine expression in IL-12Rβ-IL-12Rβ2-HEK-293 cells. Finally, compared with anti-p40 treatment, 2305 effectively and selectively inhibits IL-23-induced inflammation in three in vivo mouse models: IL-23-induced ear inflammation, anti-CD40-induced systemic inflammatory response, and collagen-induced arthritis. We, hereby, describe the discovery and characterization of a potent IL-23R small-peptide modulator, 2305 (teeeqqly), that is effective in vivo. 2305 may be more convenient, less cumbersome, less costly, and most importantly, more specific than current biologics for the treatment of inflammatory conditions, and conceivably complement the actual therapies for these chronic and debilitating inflammatory diseases.

In Vivo Induction and Delivery of Nerve Growth Factor, Using HEK-293 Cells

2004 ◽  
Vol 10 (9-10) ◽  
pp. 1492-1501 ◽  
Author(s):  
Michael P. McConnell ◽  
Sanjay Dhar ◽  
Sanjay Naran ◽  
Thang Nguyen ◽  
Ralph A. Bradshaw ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Nerve Growth ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
In Vivo Induction

scholarly journals Calcitonin stimulates expression of the rat 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter in HEK-293 cells expressing calcitonin receptor: identification of signaling pathways

2004 ◽  
Vol 32 (1) ◽  
pp. 87-98 ◽  
Author(s):  
XH Gao ◽  
PP Dwivedi ◽  
JL Omdahl ◽  
HA Morris ◽  
BK May
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Basal Expression ◽  
293 Cells ◽  
Gc Box

Regulation of the gene for renal 25-hydroxyvitamin D-24-hydroxylase (CYP24) is important for controlling the level of circulating 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). We report here for the first time that the peptide hormone calcitonin significantly stimulates expression of a rat CYP24 promoter-luciferase construct in both transiently and stably transfected kidney HEK-293 cells. A GC box at -114/-101 and a CCAAT box at -62/-51 have been identified that underlie both basal expression of the CYP24 promoter and the calcitonin inductive response. Data from overexpression studies suggested that Sp1 and NF-Y are the proteins that function through the GC and CCAAT boxes respectively. ERK1/2 signaling pathways were not involved in the calcitonin-mediated response, since stimulation of the promoter was unaffected by the pharmacological ERK1/2 inhibitor PD98059 and by a dominant negative mutant of ERK1/2 (ERK1K71R). In contrast, calcitonin induction but not basal expression was dependent on protein kinase A and protein kinase C (PKC) activities with the inhibitors H89 and calphostin C lowering induction by 50-60%. The atypical PKC, PKCzeta contributes to calcitonin induction, but not to basal expression of the CYP24 promoter, since overexpression of a dominant negative clone PKCzetaK281 M lowered induction by 50%. Cotransfection of a dominant negative form of Ras resulted in calcitonin-mediated induction being reduced also by about 50%. A Ras-PKCzeta signaling pathway for calcitonin action is proposed, which acts through the GC box. The findings have been extrapolated to the in vivo situation where we suggest that induction of renal CYP24 by calcitonin could be important under hypercalcemic conditions thus contributing to the lowering of circulating 1,25(OH)2D3 levels.

scholarly journals Nephroprotective activity of Annona Squamosa leaves against paracetamol-induced nephrotoxicity in rats: in vitro and in vivo experiments

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
S. Neelima ◽  
P. Dwarakanadha Reddy ◽  
Chandra Sekhar Kothapalli Bannoth
Keyword(s):  
Kidney Tissue ◽  
Ethanolic Extract ◽  
Tissue Homogenate ◽  
Annona Squamosa ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
In Vivo Experiments ◽  
293 Cells

Abstract Background Paracetamol (PCM), being extensively adapted analgesic and anti-inflammatory drug all over the world, beyond therapeutic dosages, the oxidative stress-involved nephrotoxicity has been evidenced. However, herbal plants are the windfall for the humankind providing solution for most of the wellness breakdowns. Annona squamosa (AS) is one of such plants with enormous therapeutic and nutraceutical potencies. The main aspiration of the current investigation is to evaluate the nephroprotective ability of ethanolic extract of Annona squamosa (EEAS) leaves against paracetamol-induced nephrotoxicity using in vitro human embryonic kidney (HEK)-293 cells and in vivo experiments in Wistar rats through biochemical parameters, oxidative parameters, and histopathological findings. Results When HEK-293 cells were incubated with PCM, an increased cell death associated with alterations in the morphology of normal cells was observed. At variable concentrations, HEK-293 cells co-treated with PCM and EEAS extracts gave a significant improvement in cell growth on comparing with PCM treatment showing cytoprotective feature of EEAS with an IC50 28.75 μg/mL. In vivo nephroprotective property was assessed from the amount of blood urea nitrogen (BUN) along with creatinine and uric acid which were reduced (P < 0.001) within serum and compact levels of glutathione, catalase, and superoxide dismutase which were termed as GSH, CAT, and SOD, respectively, were increased (P < 0.001) in kidney tissue homogenate in the treated groups than the PCM alone group. Results were additionally supported by histopathological observations. Conclusion The results exhibited that EEAS has impending benefits against PCM-induced nephrotoxicity through in vitro and in vivo experiments.

scholarly journals In vitro 3D Spheroid Culture Developed on the Parafilm Surface Using HEK-293 Cells

2020 ◽  
Vol 3 (1) ◽  
pp. 220-227
Author(s):  
Erdal Eroğlu
Keyword(s):  
Three Dimensional ◽  
Culture Method ◽  
In Vivo Studies ◽  
Preclinical Research ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Hanging Drop Culture

Preclinical research to predict the effects of drugs and chemicals on humans is commonly carried out either by cell culture studies in vitro condition or on animals in vivo condition. While drug studies tested on cells cultured as a monolayer in plastic flasks are incompatible with realistic results, falsifying findings can also be achieved from in vivo studies performed on different species. In recent years, research on drug tests using spheroid cultures formed by growing cells in three-dimensional (3D) in vitro has attracted great interest. 3D spheroid structures are formed by growing the cells in a drop suspended on superhydrophobic surfaces. In this study, HEK-293 cells were investigated on parafilm surfaces displaying superhydrophobic properties by growing in 2 &amp;micro;l volume using hanging drop culture method in terms of spheroid formation. Light microscopy images from spheroid structures were taken on different incubation days and the area of spheroids was measured using the ImageJ program. Our study holds important findings for a chip platform that can be developed for use in vitro drug tests.

334 ADENOVIRAL VECTOR-MEDIATED EXPRESSION OF RECOMBINANT HUMAN GLUCOCEREBROSIDASE IN THE MAMMARY GLAND OF RATS

2013 ◽  
Vol 25 (1) ◽  
pp. 314
Author(s):  
K. C. S. Tavares ◽  
C. Feltrin ◽  
I. S. Carneiro ◽  
A. S. Morais ◽  
C. D. Medeiros ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Mammary Glands ◽  
Santa Cruz ◽  
Cmv Promoter ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Control Milk

Glucocerebrosidase is a lysosomal enzyme that plays a key role in sphingolipid cleavage, an intermediate in glycolipid metabolism. A recessive mutation in the glucocerebrosidase gene leads to the accumulation of glucosylceramide in macrophages (sphingolipidosis), a lysosomal storage disease known in humans as the Gaucher disease. The enzyme replacement treatment with recombinant human glucocerebrosidase (hGCase) dramatically reduces and reverses symptoms, with the need of lifelong treatment for patients to attain a normal life. Currently, hGCase is very costly, being produced through in vitro expression in Chinese hamster ovary cells or in vivo, in plants. The aim of this study was to develop a model for the production of hGCase in the mammary gland of rats transiently transduced with recombinant adenovirus. A replication-defective adenovirus carrying hGCase was generated using the AdEasy™ adenoviral vector system (Stratagene, La Jolla, CA, USA). The hGCase cDNA (NM_001005741) was in vitro-synthesized and ligated in the XhoI site of the pAdTrack-CMV vector (pAdT-hGCase). The resulting plasmid was recombined with the pAdEasy™ vector in BJ5183 electro-competent cells. The purified pAdE-pAdT-hGCase vector was linearized and transfected into HEK-293 cells for the production of a primary viral stock. Further amplifications and the titration assay were done in HEK-293 cells, monitoring the transduction by the qualitative evaluation of green fluorescent protein (GFP) expression. Following transfection, the HEK-293 cells increasingly expressed the GFP reporter, regulated by a CMV promoter, in tandem with the hGCase cDNA, under another CMV promoter. On Day 18 of gestation, a female rat (Rattus norvegicus) was anesthetized and the 2 left caudal mammary glands were infused with 109 GTU mL–1 of the pAdE-pAdT-hGCase in PBS solution supplemented with 36 mM EGTA. The 2 right caudal mammary glands were infused only with PBS-EGTA (control milk). Milk samples collected from Days 2 through 9 post-partum were mixed with separation buffer (10 mM Tris-HCl, pH 8.0; 10 mM CaCl2) and centrifuged, with the supernatant assayed for hGCase by Western blot using a monoclonal anti-human glucocerebrosidase antibody (sc-166407, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Relative quantification of the hGCase expression was done using the FluorChem FC2 system (Alpha Innotech, San Leandro, CA, USA), with hGCase band intensity being normalized against GAPDH expression. The in vivo expression assay confirmed the production of hGCase in the secreted portion of the rat milk, with a specific band between 50 to 60 kDa observed on the Western blot, and no detection of the protein in the control milk. The hGCase peak production occurred in Days 5 and 6 of lactation, with levels being 35 times greater than on Day 9. An ELISA quantification assay and an enzymatic activity assay for the recombinant hGCase are currently in development. In conclusion, the use of the rat for hGCase transient expression in the milk was proven a valid model for testing the potential use of a mammary gland expression system for the production of a functional human glucocerebrosidase protein.

scholarly journals The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo

1999 ◽  
Vol 344 (3) ◽  
pp. 929-936 ◽  
Author(s):  
Alexander GRAY ◽  
Jeroen V AN DER KAAY ◽  
C. Peter DOWNES
Keyword(s):  
Protein Kinase ◽  
Fusion Proteins ◽  
Protein Kinase B ◽  
Pleckstrin Homology ◽  
3T3 Cells ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Swiss 3T3

We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-1), expressed as green fluorescent protein (GFP) fusion proteins [PH(PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin caused a small, but sustained, increase in PtdIns(3,4,5)P3 levels, detected using a radioligand displacement assay, which was mirrored by the translocation of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) and expressing either PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulation of both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to PDGF, but only the latter was present at increased levels in Swiss 3T3 cells 30 min after an oxidative stress (1 mM H2O2). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H2O2. The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger roles of PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

scholarly journals Attenuation of Oxidative Stress in HEK 293 Cells by the TCM Constituents Schisanhenol, Baicalein, Resveratrol or Crocetin and Two Defined Mixtures

2015 ◽  
Vol 18 (4) ◽  
pp. 661 ◽  
Author(s):  
John Richard Bend ◽  
Xue Yan (Iris) Xue Yan Xia ◽  
Daofeng Chen ◽  
Abudi Awaysheh ◽  
Andrea Lo ◽  
...  
Keyword(s):  
Real Time ◽  
Molecular Mechanisms ◽  
Nitrosative Stress ◽  
Fluorescent Protein ◽  
Antioxidant Properties ◽  
Oxidative And Nitrosative Stress ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

PURPOSE:  Our working hypothesis is that single bioactive phytochemicals with antioxidant properties that are important constituents of Traditional Chinese Medicine (TCM) and their defined mixtures have potential as chemoprotective agents for chronic conditions characterized by oxidative and nitrosative stress, including Alzheimer’s. Here we evaluate the ability of baicalein, crocetin, trans-resveratrol or schisanhenol and two defined mixtures of these TCM phytochemicals to attenuate the toxicity resulting from exposure to cell permeant t-butyl hydroperoxide (tBPH) in wild-type and bioengineered (to express choline acetyltransferase) HEK 293 cells. METHODS: Endpoints of tBHP-initiated oxidative and nitrosative stress in both types of HEK 293 cells and its attenuation by TCM constituents and mixtures included cytotoxicity (LDH release); depletion of intracellular glutathione (GSH); formation of S-glutathionylated proteins; oxidative changes to the disulfide proteome; and real-time changes in intracellular redox status. RESULTS: At low µM concentrations, each of the TCM constituents and mixtures effectively attenuated intracellular toxicity due to exposure of HEK 293 cells to 50 or 250 µM tBHP for 30 min to 3 h. Confocal microscopy of HEK 293 cells transfected with mutated green fluorescent protein (roGFP2) showed effective attenuation of tBHP oxidation by baicalein in real time. Three redox-regulated proteins prominent in the disulfide proteome of HEK 293 cells were identified by MALDI-TOF mass spectrometry. CONCLUSIONS: We conclude that single TCM chemicals and their simple mixtures have potential for use in adjunct chemoprotective therapy. Advantages of mixtures compared to single TCM constituents include the ability to combine compounds with varying molecular mechanisms of cytoprotection for enhanced biological activity; and to combine chemicals with complementary pharmacokinetic properties to increase half-life and prolong activity in vivo. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

In Vivo Induction and Delivery of Nerve Growth Factor, Using HEK-293 Cells

2004 ◽  
Vol 10 (9) ◽  
pp. 1492-1501
Author(s):  
Michael P. McConnell ◽  
Sanjay Dhar ◽  
Sanjay Naran ◽  
Thang Nguyen ◽  
Ralph A. Bradshaw ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Nerve Growth ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals N-Formylpeptides Induce Two Distinct Concentration Optima for Mouse Neutrophil Chemotaxis by Differential Interaction with Two N-Formylpeptide Receptor (Fpr) Subtypes

1999 ◽  
Vol 190 (5) ◽  
pp. 741-748 ◽  
Author(s):  
Jennifer K. Hartt ◽  
Grant Barish ◽  
Philip M. Murphy ◽  
Ji-Liang Gao
Keyword(s):  
Neutrophil Migration ◽  
Calcium Flux ◽  
Receptor Subtype ◽  
High Concentration ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Subtype Selective ◽  
Formylpeptide Receptor

The N-formylpeptide receptor (FPR) is a G protein–coupled receptor that mediates mammalian phagocyte chemotactic responses to bacterial N-formylpeptides. Here we show that a mouse gene named Fpr-rs2 encodes a second N-formylpeptide receptor subtype selective for neutrophils which we have provisionally named FPR2. The prototype N-formylpeptide fMLF induced calcium flux and chemotaxis in human embryonic kidney (HEK) 293 cells stably transfected with FPR2. The EC50s, ∼5 μM for calcium flux and chemotaxis, were ∼100-fold greater than the corresponding values for mouse FPR-transfected HEK 293 cells. Consistent with this, fMLF induced two distinct concentration optima for chemotaxis of normal mouse neutrophils, but only the high concentration optimum for chemotaxis of neutrophils from FPR knockout mice. Based on these data, we hypothesize that high- and low-affinity N-formylpeptide receptors, FPR and FPR2, respectively, may function in vivo as a relay mediating neutrophil migration through the high and low concentration portions of N-formylpeptide gradients.

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