Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation An easy and low cost alternative to radioactive thymidine incorporation

Huong, Phan Le Thanh; Kolk, A. H. J.; Eggelte, Teunis A.; Verstijnen, C.; Gilis, Henk; Hendriks, J.

doi:10.1016/0022-1759(91)90377-r

Cited by 80 publications

(27 citation statements)

References 13 publications

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“…After centrifugation, the supernatant was collected and kept at Ϫ80°C for cytokine production measurement. Cell proliferation was evaluated by a nonradioactive enzyme-linked immunosorbent assay (ELISA) technique using a monoclonal antibody against 5-bromo-2-deoxyuridine (BrdU) (28), which was added for 18 h on day 4, and revealed by an enzyme-conjugated second antibody, according to the manufacturer's instructions (BrdU cell proliferation detection kit III; Boehringer-Mannheim, Germany). The absorbance at 405 nm was read in an ELISA plate reader (Labsystems Multiscan EX).…”

Section: Methodsmentioning

confidence: 99%

The Trypanosoma brucei gambiense Secretome Impairs Lipopolysaccharide-Induced Maturation, Cytokine Production, and Allostimulatory Capacity of Dendritic Cells

et al. 2013

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bTrypanosoma brucei gambiense, a parasitic protozoan belonging to kinetoplastids, is the main etiological agent of human African trypanosomiasis (HAT), or sleeping sickness. One major characteristic of this disease is the dysregulation of the host immune system. The present study demonstrates that the secretome (excreted-secreted proteins) of T. b. gambiense impairs the lipopolysaccharide (LPS)-induced maturation of murine dendritic cells (DCs). The upregulation of major histocompatibility complex class II, CD40, CD80, and CD86 molecules, as well as the secretion of cytokines such as tumor necrosis factor alpha, interleukin-10 (IL-10), and IL-6, which are normally released at high levels by LPS-stimulated DCs, is significantly reduced when these cells are cultured in the presence of the T. b. gambiense secretome. Moreover, the inhibition of DC maturation results in the loss of their allostimulatory capacity, leading to a dramatic decrease in Th1/Th2 cytokine production by cocultured lymphocytes. These results provide new insights into a novel efficient immunosuppressive mechanism directly involving the alteration of DC function which might be used by T. b. gambiense to interfere with the host immune responses in HAT and promote the infectious process.

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Section: Methodsmentioning

confidence: 99%

The Trypanosoma brucei gambiense Secretome Impairs Lipopolysaccharide-Induced Maturation, Cytokine Production, and Allostimulatory Capacity of Dendritic Cells

et al. 2013

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“…100 l (WCS preparation) to 200 l (LB and FKB preparations) of H. pylori-related antigens was added to 60 to 80% confluent Jaws II cells in a 6-well culture plate and incubated at 37°C for 48 h. (8,19). The clonal proliferation of T cells exposed to LB-, FKB-, and WCS-pulsed Jaws II cells was assayed using 5-bromo-2Ј-deoxyuridine (BrdU) (15) and compared to that of T cells exposed to unpulsed Jaws II cells. Briefly, 2 ϫ 10 Ϫ4 mmol of BrdU was added to each well, and the plate was incubated at 37°C for 2 h. The amount of BrdU incorporated into the T cells was measured using the anti-BrdU monoclonal antibody in the ELISA kit (Roche Switzerland).…”

Section: Dcsmentioning

confidence: 99%

Transfer of Antigen-Pulsed Dendritic Cells Induces Specific T-Cell Proliferation and a Therapeutic Effect against Long-TermHelicobacter pyloriInfection in Mice

Otsu¹,

Gotoh²,

Yamashiro

et al. 2006

Infect Immun

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Helicobacter pylori causes persistent infection of the stomach and results in chronic gastritis and peptic ulcers. Jaws II cells, derived from mouse bone marrow, were pulsed with live or formalin-killed or whole-cell sonicates (WCS) of H. pylori. Representative cell surface molecules were expressed at substantial levels on Jaws II cells, indicating that appropriate maturation of the cells was achieved with the three H. pylori antigens without any significant differences. H. pylori WCS-pulsed Jaws II cells secreted a significant amount of tumor necrosis factor alpha into the culture supernatant. The naïve T cells exposed to the WCS-pulsed Jaws II cells showed significant proliferation and gamma interferon (IFN-␥) and interleukin-10 (IL-10) production in vitro. A 2-log reduction in the number of colonizing bacteria was observed in the mice treated with the WCS-pulsed Jaws II cells; however, no significant reductions were achieved in mice treated with Jaws II cells pulsed with other H. pylori antigens. Up-regulated production of IFN-␥ and IL-10 was observed in the stomachs of the mice treated with the WCS-pulsed Jaws II cells, which is consistent with the result obtained in vitro. There were no differences in gastritis scores or H. pylori-specific antibody titers among the mice treated with Jaws II cells pulsed with the three different H. pylori antigens. The results suggest that Th1 cell-mediated immunity in combination with Th2 cell-mediated immunity plays a role in reducing colonizing bacterial numbers in mice with chronic H. pylori infections.

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“…During the last 6 h of stimulation, 20 µl BrdU solution was added and BrdU incorporated into DNA was detected by an ELISA (Huong et al 1991), which was performed as suggested by the manufacturer.…”

Section: Brdu Incorporationmentioning

confidence: 99%

Mechanisms of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in beta(INS-1)-cells

Trümper

K²,

Hörsch³

2002

Journal of Endocrinology

182

101

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Glucose-dependent insulinotropic polypeptide (GIP) acts as a glucose-dependent growth factor for -cells. Here we show that GIP and glucose also act synergistically as anti-apoptotic factors for -cells, using the welldifferentiated -cell line, INS-1. Mitogenic and anti-apoptotic signaling of GIP were dependent upon pleiotropic activation of protein kinase A (PKA)/cAMP regulatory element binder (CREB), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-kinase)/PKB signaling modules. The signaling modules activated by GIP were dependent on glucose metabolism and calcium influx and were tightly linked by multiple activating and inhibiting cross-talk. These interactions included: (i) a central role of tyrosine phosphorylation for stimulation of PKA/CREB, MAPK and PI3-kinase/PKB, (ii) inhibition of PKA/CREB by the MAPK pathway at the level of MAPK kinase-1 or downstream, (iii) activation of MAPK signaling by PI3-kinase and PKA at the level of extracellular-signal regulated kinase 1/2 or upstream, and (iv) activation of PKB by MAPK and PKA signaling at the level of PKB or upstream. Furthermore, we demonstrated inhibition of CREB signaling by Ca 2+ / calmodulin kinase I/IV. These results indicated that GIP acts as a mitogenic and anti-apoptotic factor for -cells by pleiotropic activation of tightly linked signaling pathways in -cells.

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Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation An easy and low cost alternative to radioactive thymidine incorporation

Cited by 80 publications

References 13 publications

The Trypanosoma brucei gambiense Secretome Impairs Lipopolysaccharide-Induced Maturation, Cytokine Production, and Allostimulatory Capacity of Dendritic Cells

The Trypanosoma brucei gambiense Secretome Impairs Lipopolysaccharide-Induced Maturation, Cytokine Production, and Allostimulatory Capacity of Dendritic Cells

Transfer of Antigen-Pulsed Dendritic Cells Induces Specific T-Cell Proliferation and a Therapeutic Effect against Long-TermHelicobacter pyloriInfection in Mice

Mechanisms of mitogenic and anti-apoptotic signaling by glucose-dependent insulinotropic polypeptide in beta(INS-1)-cells

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