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Intra-cytoplasmic Cytokine Staining (ICS): Optimizing antigen stimulation for measuring M. tuberculosis-specific T cell response
Published in Ade Gafar Abdullah, Isma Widiaty, Cep Ubad Abdullah, Medical Technology and Environmental Health, 2020
A cytokine secretion inhibitor is often used in most protocols of ICS to trap the cytokine within the cells. The proposed mechanism of Brefeldin A is inhibition some of proteins that activate ADP-ribosilation factors (Arf), which is critical for vesicular transport between endoplasmic reticulum and the Golgi (Chardin & McCormick 1999). Cytokine secretion inhibitors seem to be time and cytokine dependent. For example, another cytokine inhibitor, namely Monesin, in ICS decreased detection of IL-4 within cells (Smith et al. 2015), but the shorter incubation of Monesin improved the detection of IL-10 (Muris et al. 2012). The incubation of Brefeldin A for 3.5 hours (Muflihah et al. 2018) or less than 12 hours is for practicality (Miguel et al. 2012) and avoiding cell toxicity. Our work showed that prolonged incubation of Brefeldin A up to 18 hours improved the frequency (Figure 1) and functionality of T cells secreting IFN-γ, TNF, and IL2 (Figure 2B).
Intracellular Maturation of Acute Phase Proteins
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Erik Fries, E. Mathilda Sjöberg
Recent experiments with the drug brefeldin A have substantiated the idea of recycling of membrane from the GC to the ER. When cells are treated with this drug, protein secretion stops,23 the Golgi stack disassembles,24 and cis- and medial Golgi enzymes appear in the ER.25 Consequently, glycosylated ER proteins become processed by Golgi enzymes;25 for unknown reasons, this is not seen with hepatocytes.26 One explanation for these effects is that in the presence of brefeldin A, the selectivity in the transport from the GC to the ER is lost due to the formation of direct connections.27,28
Progressive multifocal leukoencephalopathy
Published in Avindra Nath, Joseph R. Berger, Clinical Neurovirology, 2020
Eric M. L. Williamson, Joseph R. Berger
Pharmacologic agents that inhibit retrograde transport or endoplasmic reticulum function may decrease JCV infectivity [321,322]. Retro-2cycl inhibits retrograde transport of polyomaviruses to the endoplasmic reticulum and has been demonstrated to inhibit both initial virus infection and infectious spread of virus in cultured cells [323,324]. Retro-2 also is relatively nontoxic in mice and has properties that may allow for blood–brain barrier permeability, which are attractive characteristics. Brefeldin A is an Arf1GTPase inhibitor that inhibits transport to the endoplasmic reticulum and virus disassembly, noting treatment of SVG-A cells with this agent reduced JCV infectivity by 50% in studies [321]. These strategies have yet to be employed in clinical trial.
Schweinfurthin induces ICD without ER stress and caspase activation
Published in OncoImmunology, 2022
Ruoheng Zhang, J.D. Neighbors, T.D. Schell, R.J. Hohl
The last module on the canonical CRT exposure pathway is the translocation module. CRT first anterograde traffics from ER to Golgi, then presents on the cell surface through SNARE dependent exocytosis. To understand if this module is required for the CRT exposed induced by MeSG, we utilized Brefeldin A (BFA). Brefeldin A is a reversible inhibitor of protein translocation from ER to the Golgi apparatus. It inhibits binding of the cytosolic coat protein to Golgi membranes.58UACC903 were pre-treated with 2, 5 or 10 μM BFA for 2 hours then treated with 100 nM MeSG alone for 24 hours. BFA inhibited CRT exposure in a concentration-dependent manner: 2 μM BFA reduced 4-fold, 5 μM BFA reduced 8-fold CRT exposure and 10 μM BFA completely blocked CRT exposure (Figure 10a, b). B16F10 cells were similarly treated. Two hours pretreatment of 2,5,10 μM BFA-pretreatment completely blocked MeSG-induced CRT exposure (Figure 10c). These findings indicate that the source of cell surface CRT before MeSG treatment is the ER and the translocation module is required for CRT exposure.
Oral administration of ammonium metavanadate and potassium dichromate distorts the inflammatory reaction induced by turpentine oil injection in male rats
Published in Drug and Chemical Toxicology, 2021
Marina K. Balabekova, Yekaterina O. Ostapchuk, Yuliya V. Perfilyeva, Aliya N. Tokusheva, Adilman Nurmuhambetov, Rustam R. Tuhvatshin, Vasiliy V. Trubachev, Zhaugashty B. Akhmetov, Nurshat Abdolla, Gulgul K. Kairanbayeva, Koks Sulev, Nikolai N. Belyaev
Splenocytes were resuspended at the concentration of 2 × 106 cells/ml in RPMI-1640 (cat. no. R6504-10X1L, Sigma-Aldrich) supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin (cat. no. P0781, Sigma), and 2 mM L-glutamine (cat. no. G2150, Sigma). Phorbol 12-myristate 13-acetate (PMA, 10 ng/ml) (cat. no. P1585, Sigma) and ionomycin (500 ng/ml) (cat. no. 56092–81-0, Sigma-Aldrich) were added as described previously (Debes et al. 2002) to stimulate cytokine expression. Cells were incubated in 96-well plates for 4 h in a humidified CO2 incubator at 37 °C. Brefeldin A (cat. no. 420601, Biolegend) was added according to the manufacturer’s protocol (1 µl/ml) for the last hour of cultivation to prevent cytokine efflux and the cells were cultured for an additional hour. The intracellular expression of IFN-γ and IL-4 and the surface expression of T helper (Th) markers (CD3, CD4) were assessed by flow cytometry.
Keratinocyte differentiation antigen-specific T cells in immune checkpoint inhibitor-treated NSCLC patients are associated with improved survival
Published in OncoImmunology, 2021
Fiamma Berner, Rebekka Niederer, Jolien J. Luimstra, Oltin Tiberiu Pop, Ann-Kristin Jochum, Mette-Triin Purde, Omar Hasan Ali, David Bomze, Jens Bauer, Lena Katharina Freudenmann, Ana Marcu, Eva-Maria Wolfschmitt, Sebastian Haen, Thorben Gross, Marissa Lisa Dubbelaar, Marie-Therese Abdou, Petra Baumgaertner, Christina Appenzeller, Caroline Cicin-Sain, Tobias Lenz, Daniel E. Speiser, Burkhard Ludewig, Christoph Driessen, Markus Jörger, Martin Früh, Wolfram Jochum, Antonio Cozzio, Hans-Georg Rammensee, Juliane Walz, Jacques Neefjes, Lukas Flatz
Cryopreserved PBMCs of patients with HLA-A 03:01, HLA-A 02:01 and HLA-C 04:01 were plated at a concentration of 1 × 106 cells per well in low-adherence 24-well plates (Sarstedt, ref. 83.3922.500) and resuspended in 2 mL RPMI per well containing 8% human serum (Biowest, ref. S4190-100), 1% penicillin-streptomycin, 1% L-glutamine, 1% non-essential amino acids, 1% sodium pyruvate, 0.1 mg/mL kanamycin and 0.1% β-mercaptoethanol. Cells were kept at 37°C. The following day, cells were stimulated with the predicted epitopes individually at a final concentration of 2ug/mL/peptide. After 48 hours, 1 mL medium was replaced with fresh RPMI (as above) containing IL-2 (final concentration of IL-2 was 150 U/mL). Cells were kept in culture for ten days and 1 mL medium was replaced with fresh IL-2-containing medium every second day. On day ten, cells were washed and transferred to 96-well plates and re-stimulated with each peptide individually for six hours in the absence of IL-2 at a final concentration of 2ug/mL/peptide. Brefeldin A was added at the same time of stimulation at a final concentration of 10ug/mL. After six hours cells were washed and stained for viability, CD3, CD4, CD8, CD45RA and CD14. Cells were then fixed and permeabilized and stained for IFN-γ and TNF. Samples were acquired using the BD LSRFortessa flow cytometer and data was analyzed using the FlowJo software. IFN-γ and TNF production were analyzed to identify antigen-specific T cells. For three patients, the experiment was repeated and CD8+ IFN-γ+ T cells were sorted using the BD FACSMelody flow cytometer.