Effects of Thymoglobuline on the hypoxia-reoxigenation injury in HEK-293 cells inculture

Antitimocitni globulini su poliklona protutijela koja dovode do smanjenja broja T stanica. Često se koriste za imunosupresiju primatelja tijekom transplantacije bubrega. Timoglobulin su IgG protutijela dobivena imunizacijom zečeva timocitima ljudskog fetusa. Timoglobulin dovodi do smanjenja broja T-limfocita putem razgradnje stanica mehanizmom ovisnim o komplementu te apoptozom. Da bismo dokazali direktan učinak timoglobulina na stanice, koristili smo ljudske embrionalne stanice porijeklom iz bubrega (HEK-293) u staničnoj kulturi. Mjerili smo membranski potencijal stanice metodom prikovanih potencijala. Depolarizacijski učinak timoglobulina na membranski potencijal HEK-293 stanica je ovisan o koncentraciji timoglobulina te o početnom membranskom potencijalu stanica. Učinak timoglobulina u hipoksično-reoksigenacijskih uvjetima utvrđen je određivanjem stanične smrtnosti te stanične migracije testom grebanja. Timoglobulin je smanjio staničnu smrtnost u hipoksijskim uvjetima nakon kojih je slijedila reoksigenacija. Ovo je prvo istraživanje koje pokazuje direktan učinak timoglobulina na HEK-293 stanice. Rezultati ovog istraživanja potiču nova istraživanja o djelovanju timoglobulina na stanice transplantiranih bubrega.Antithymocyte globulines are polyclonal T cell-depleting immunoglobulins used in induction of immunosuppression in kidney transplant recipients. Thymoglobuline, is purified rabbit IgG, obtained by immunization of rabbits with fetal human thymi, which depletes T lymphocytes by complement-dependent lysis and apoptosis. To determine possible direct effects of Thymoglobuline on the cells, we used Human Embryonic Kidney cell line (HEK-293) in culture. We measured membrane potential of the cells using slow whole patch clamp technique. Depolarizations of HEK-293 cells caused by Thymoglobuline were concentration- and membrane potential- dependent, showing direct effect of Thymoglobuline on the HEK-293 cells. The effects of Thymoglobuline in hypoxia/reoxigenation were detected by calculating cell death and determining the cell migration using scratch test. Thymoglobuline prevented the cell death induced by hypoxia and reoxigenation conditions. This is first study showing direct effect of thymoglobulin on HEK-293 cells. The results of this study encourage the research on epithelial cells in transplanted kidneys

Effects of Thymoglobuline on the hypoxia-reoxigenation injury in HEK-293 cells inculture

Antitimocitni globulini su poliklona protutijela koja dovode do smanjenja broja T stanica. Često se koriste za imunosupresiju primatelja tijekom transplantacije bubrega. Timoglobulin su IgG protutijela dobivena imunizacijom zečeva timocitima ljudskog fetusa. Timoglobulin dovodi do smanjenja broja T-limfocita putem razgradnje stanica mehanizmom ovisnim o komplementu te apoptozom. Da bismo dokazali direktan učinak timoglobulina na stanice, koristili smo ljudske embrionalne stanice porijeklom iz bubrega (HEK-293) u staničnoj kulturi. Mjerili smo membranski potencijal stanice metodom prikovanih potencijala. Depolarizacijski učinak timoglobulina na membranski potencijal HEK-293 stanica je ovisan o koncentraciji timoglobulina te o početnom membranskom potencijalu stanica. Učinak timoglobulina u hipoksično-reoksigenacijskih uvjetima utvrđen je određivanjem stanične smrtnosti te stanične migracije testom grebanja. Timoglobulin je smanjio staničnu smrtnost u hipoksijskim uvjetima nakon kojih je slijedila reoksigenacija. Ovo je prvo istraživanje koje pokazuje direktan učinak timoglobulina na HEK-293 stanice. Rezultati ovog istraživanja potiču nova istraživanja o djelovanju timoglobulina na stanice transplantiranih bubrega.Antithymocyte globulines are polyclonal T cell-depleting immunoglobulins used in induction of immunosuppression in kidney transplant recipients. Thymoglobuline, is purified rabbit IgG, obtained by immunization of rabbits with fetal human thymi, which depletes T lymphocytes by complement-dependent lysis and apoptosis. To determine possible direct effects of Thymoglobuline on the cells, we used Human Embryonic Kidney cell line (HEK-293) in culture. We measured membrane potential of the cells using slow whole patch clamp technique. Depolarizations of HEK-293 cells caused by Thymoglobuline were concentration- and membrane potential- dependent, showing direct effect of Thymoglobuline on the HEK-293 cells. The effects of Thymoglobuline in hypoxia/reoxigenation were detected by calculating cell death and determining the cell migration using scratch test. Thymoglobuline prevented the cell death induced by hypoxia and reoxigenation conditions. This is first study showing direct effect of thymoglobulin on HEK-293 cells. The results of this study encourage the research on epithelial cells in transplanted kidneys

Effects of Thymoglobuline on the hypoxia-reoxigenation injury in HEK-293 cells inculture

Antitimocitni globulini su poliklona protutijela koja dovode do smanjenja broja T stanica. Često se koriste za imunosupresiju primatelja tijekom transplantacije bubrega. Timoglobulin su IgG protutijela dobivena imunizacijom zečeva timocitima ljudskog fetusa. Timoglobulin dovodi do smanjenja broja T-limfocita putem razgradnje stanica mehanizmom ovisnim o komplementu te apoptozom. Da bismo dokazali direktan učinak timoglobulina na stanice, koristili smo ljudske embrionalne stanice porijeklom iz bubrega (HEK-293) u staničnoj kulturi. Mjerili smo membranski potencijal stanice metodom prikovanih potencijala. Depolarizacijski učinak timoglobulina na membranski potencijal HEK-293 stanica je ovisan o koncentraciji timoglobulina te o početnom membranskom potencijalu stanica. Učinak timoglobulina u hipoksično-reoksigenacijskih uvjetima utvrđen je određivanjem stanične smrtnosti te stanične migracije testom grebanja. Timoglobulin je smanjio staničnu smrtnost u hipoksijskim uvjetima nakon kojih je slijedila reoksigenacija. Ovo je prvo istraživanje koje pokazuje direktan učinak timoglobulina na HEK-293 stanice. Rezultati ovog istraživanja potiču nova istraživanja o djelovanju timoglobulina na stanice transplantiranih bubrega.Antithymocyte globulines are polyclonal T cell-depleting immunoglobulins used in induction of immunosuppression in kidney transplant recipients. Thymoglobuline, is purified rabbit IgG, obtained by immunization of rabbits with fetal human thymi, which depletes T lymphocytes by complement-dependent lysis and apoptosis. To determine possible direct effects of Thymoglobuline on the cells, we used Human Embryonic Kidney cell line (HEK-293) in culture. We measured membrane potential of the cells using slow whole patch clamp technique. Depolarizations of HEK-293 cells caused by Thymoglobuline were concentration- and membrane potential- dependent, showing direct effect of Thymoglobuline on the HEK-293 cells. The effects of Thymoglobuline in hypoxia/reoxigenation were detected by calculating cell death and determining the cell migration using scratch test. Thymoglobuline prevented the cell death induced by hypoxia and reoxigenation conditions. This is first study showing direct effect of thymoglobulin on HEK-293 cells. The results of this study encourage the research on epithelial cells in transplanted kidneys

Attenuation of oxidative stress in HEK 293 cells by the TCM constituents schisanhenol, baicalein, resveratrol or crocetin and two defined mixtures

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

The Antibody Targeting the E314 Peptide of Human Kv1.3 Pore Region Serves as a Novel, Potent and Specific Channel Blocker

Selective blockade of Kv1.3 channels in effector memory T (TEM) cells was validated to ameliorate autoimmune or autoimmune-associated diseases. We generated the antibody directed against one peptide of human Kv1.3 (hKv1.3) extracellular loop as a novel and possible Kv1.3 blocker. One peptide of hKv1.3 extracellular loop E3 containing 14 amino acids (E314) was chosen as an antigenic determinant to generate the E314 antibody. The E314 antibody specifically recognized 63.8KD protein stably expressed in hKv1.3-HEK 293 cell lines, whereas it did not recognize or cross-react to human Kv1.1(hKv1.1), Kv1.2(hKv1.2), Kv1.4(hKv1.4), Kv1.5(hKv1.5), KCa3.1(hKCa3.1), HERG, hKCNQ1/hKCNE1, Nav1.5 and Cav1.2 proteins stably expressed in HEK 293 cell lines or in human atrial or ventricular myocytes by Western blotting analysis and immunostaining detection. By the technique of whole-cell patch clamp, the E314 antibody was shown to have a directly inhibitory effect on hKv1.3 currents expressed in HEK 293 or Jurkat T cells and the inhibition showed a concentration-dependence. However, it exerted no significant difference on hKv1.1, hKv1.2, hKv1.4, hKv1.5, hKCa3.1, HERG, hKCNQ1/hKCNE1, L-type Ca2+ or voltage-gated Na+ currents. The present study demonstrates that the antibody targeting the E314 peptide of hKv1.3 pore region could be a novel, potent and specific hKv1.3 blocker without affecting a variety of closely related Kv1 channels, KCa3.1 channels and functional cardiac ion channels underlying central nervous systerm (CNS) disorders or drug-acquired arrhythmias, which is required as a safe clinic-promising channel blocker

Biosynthesis of ternary NiCoFe2_2O4_4 nanoflowers: investigating their 3D structure and potential use in gene delivery

Multicomponent nanoparticle systems are known for their varied properties and functions, and have shown potential as gene nanocarriers. This study aims to synthesize and characterize ternary nickel–cobalt-ferrite (NiCoFe$_2$O$_4$) nanoparticles with the potential to serve as gene nanocarriers for cancer/gene therapy. The biogenic nanocarriers were prepared using a simple and eco-friendly method following green chemistry principles. The physicochemical properties of the nanoparticles were analyzed by X-ray diffraction, vibrating sample magnetometer, X-ray photoelectron spectroscopy, and Brunauer–Emmett–Teller. To evaluate the morphology of the nanoparticles, the field emission scanning electron microscopy with energy dispersive X-Ray spectroscopy, high-resolution transmission electron microscopy imaging, and electron tomography were conducted. Results indicate the nanoparticles have a nanoflower morphology with a mesoporous nature and a cubic spinel structure, where the rod and spherical nanoparticles became rose-like with a specific orientation. These nanoparticles were found to have minimal toxicity in human embryonic kidney 293 (HEK-293 T) cells at concentrations of 1 to 250 µg·mL–1. We also demonstrated that the nanoparticles could be used as gene nanocarriers for delivering genes to HEK-293 T cells using an external magnetic field, with optimal transfection efficiency achieved at an N/P ratio of 2.5. The study suggests that biogenic multicomponent nanocarriers show potential for safe and efficient gene delivery in cancer/gene therapy

Effects of receptor tyrosine kinase inhibitors on VEGF165a- and VEGF165b-stimulated gene transcription in HEK-293 cells expressing human VEGFR2

BACKGROUND AND PURPOSE: Receptor tyrosine kinase inhibitors (RTKIs) targeted at VEGF receptor 2 (VEGFR2) have proved to be attractive approaches to cancer therapy based on their ability to reduce angiogenesis. Here we have undertaken a quantitative analysis of the interaction of RTKIs and two VEGF splice variants, VEGF165a and VEGF165b, with VEGFR2 by studying nuclear factor of activated T-cells (NFAT) reporter gene activity in live HEK-293 cells. EXPERIMENTAL APPROACH: HEK-293 cells expressing the human VEGFR2 and a firefly luciferase reporter gene regulated by an NFAT response element were used for quantitative analysis of the effect of RTKIs on VEGF165a- and VEGF165b-stimulated luciferase gene expression. KEY RESULTS: VEGF165a produced a concentration-dependent activation of the NFAT-luciferase reporter gene in living cells that was inhibited in a non-competitive fashion by four different RTKIs (cediranib, pazopanib, sorafenib and vandetanib). The potency obtained for each RTKI from this analysis was similar to those obtained in binding studies using purified VEGFR2 kinase domains. VEGF165b was a lower-efficacy agonist of the NFAT-luciferase response when compared with VEGF165a. Analysis of the concentration–response data using the operational model of agonism indicated that both VEGF165 isoforms had similar affinity for VEGFR2. CONCLUSIONS AND IMPLICATIONS: Quantitative pharmacological analysis of the interaction of VEGF165 isoforms and RTKIs with VEGFR2 in intact living cells has provided important insights into the relative affinity and efficacy of VEGF165a and VEGF165b for activation of the calcineurinNFAT signalling pathway by this tyrosine kinase receptor

Effects of receptor tyrosine kinase inhibitors on VEGF165a- and VEGF165b-stimulated gene transcription in HEK-293 cells expressing human VEGFR2

BACKGROUND AND PURPOSE: Receptor tyrosine kinase inhibitors (RTKIs) targeted at VEGF receptor 2 (VEGFR2) have proved to be attractive approaches to cancer therapy based on their ability to reduce angiogenesis. Here we have undertaken a quantitative analysis of the interaction of RTKIs and two VEGF splice variants, VEGF165a and VEGF165b, with VEGFR2 by studying nuclear factor of activated T-cells (NFAT) reporter gene activity in live HEK-293 cells. EXPERIMENTAL APPROACH: HEK-293 cells expressing the human VEGFR2 and a firefly luciferase reporter gene regulated by an NFAT response element were used for quantitative analysis of the effect of RTKIs on VEGF165a- and VEGF165b-stimulated luciferase gene expression. KEY RESULTS: VEGF165a produced a concentration-dependent activation of the NFAT-luciferase reporter gene in living cells that was inhibited in a non-competitive fashion by four different RTKIs (cediranib, pazopanib, sorafenib and vandetanib). The potency obtained for each RTKI from this analysis was similar to those obtained in binding studies using purified VEGFR2 kinase domains. VEGF165b was a lower-efficacy agonist of the NFAT-luciferase response when compared with VEGF165a. Analysis of the concentration–response data using the operational model of agonism indicated that both VEGF165 isoforms had similar affinity for VEGFR2. CONCLUSIONS AND IMPLICATIONS: Quantitative pharmacological analysis of the interaction of VEGF165 isoforms and RTKIs with VEGFR2 in intact living cells has provided important insights into the relative affinity and efficacy of VEGF165a and VEGF165b for activation of the calcineurinNFAT signalling pathway by this tyrosine kinase receptor

Predict cells viability, proliferation, and metabolic status, based in one unique and simple assay

A new method to simultaneously predict cells viability, proliferation and metabolic status, in a rapid, simple but also specific and sensitive mode was developed. The method is based on mid-infrared (MIR) spectroscopic analysis of cells. As model system were used Human embryonic kidney (HEK) 293 cells and T lymphocytes. After submitting cells to different environments as the toxic dimethyl sulfoxide, or metabolic activation, cells viability was analyzed by optical microscopy after coloration with trypan blue, and the cell count was determined with a Neubauer hemocytometer. The principal component analysis (PCA) of the cells second derivative spectra enabled to discriminate the cells viability and the cells proliferation as assayed by conventional methods, while spectra PCA and Hierarchical Cluster Analysis (HCA) enabled to discriminate T cells metabolic activation. The new methods, based on MIR spectroscopy, present the advantages of being applicable in automatic, simple and high-throughput mode in relation to the onventional methods.info:eu-repo/semantics/publishedVersio

Validation of a miniaturised near infrared spectrometer for contaminant assessment in biodiesel

A new method to simultaneously predict cells viability, proliferation and metabolic status, in a rapid, simple but also specific and sensitive mode was developed. The method is based on mid-infrared (MIR) spectroscopic analysis of cells. As model system were used Human embryonic kidney (HEK) 293 cells and T lymphocytes. After submitting cells to different environments as the toxic dimethyl sulfoxide, or metabolic activation, cells viability was analyzed by optical microscopy after coloration with trypan blue, and the cell count was determined with a Neubauer hemocytometer. The principal component analysis (PCA) of the cells second derivative spectra enabled to discriminate the cells viability and the cells proliferation as assayed by conventional methods, while spectra PCA and Hierarchical Cluster Analysis (HCA) enabled to discriminate T cells metabolic activation. The new methods, based on MIR spectroscopy, present the advantages of being applicable in automatic, simple and high-throughput mode in relation to the conventional methods.info:eu-repo/semantics/publishedVersio